Cellular Stress Responses In Renal Diseases (Contributions to Nephrology)

Cellular Stress Responses in Renal Diseases Contributions to Nephrology Vol. 148 Series Editor Claudio Ronco Vicen...

Author: Mohammed S. Razzaque | Takashi Taguchi

34 downloads 789 Views 2MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Cellular Stress Responses in Renal Diseases

Contributions to Nephrology Vol. 148

Series Editor

Claudio Ronco

Vicenza

Cellular Stress Responses in Renal Diseases

Volume Editors

Mohammed S. Razzaque Boston, Mass. Takashi Taguchi Nagasaki

24 figures, 5 in color, and 9 tables, 2005

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Contributions to Nephrology (Founded 1975 by Geoffrey M. Berlyne)

Mohammed S. Razzaque

Takashi Taguchi

Department of Oral and Department of Pathology Developmental Biology Nagasaki University Graduate Harvard School of Dental Medicine School of Biomedical Sciences 188 Longwood Avenue 1–12–4, Sakamoto Boston, MA 02115 (USA) Nagasaki 852–8523 (Japan) E-Mail [email protected] E-Mail [email protected] and Department of Pathology Nagasaki University Graduate School of Biomedical Sciences 1–12–4, Sakamoto, Nagasaki 852–8523 (Japan) E-Mail [email protected] Library of Congress Cataloging-in-Publication Data Cellular stress responses in renal diseases / volume editors, Mohammed S. Razzaque, Takashi Taguchi. p. ; cm. – (Contributions to nephrology, ISSN 0302-5144 ; v. 148) Includes bibliographical references and index. ISBN 3-8055-7858-X (hard cover : alk. paper) 1. Kidneys–Pathophysiology. 2. Heat shock proteins–Pathophysiology. 3. Stress (Physiology) [DNLM: 1. Kidney Diseases–physiopathology. 2. Heat-Shock Proteins–physiology. 3. Heat-Shock Response–physiology. WJ 300 C3925 2005] I. Razzaque, Mohammed S. II. Taguchi, Takashi. III. Series. RC903.9.C44 2005 616.6⬘1–dc22 2004024870 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2005 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0302–5144 ISBN 3–8055–7858–X

Contents

VII Preface Razzaque, M.S. (Boston, Mass.); Taguchi , T. (Nagasaki) 1 Involvement of Stress Proteins in Renal Diseases Razzaque, M.S. (Boston, Mass./Nagasaki); Taguchi, T. (Nagasaki) 8 Stress Proteins in Glomerular Epithelial Cell Injury Bijian, K.; Cybulsky, A.V. (Montreal) 21 Response of Renal Medullary Cells to Osmotic Stress Neuhofer, W.; Beck, F.-X. (Munich) 35 Heat Shock Proteins in Renal Cell Carcinomas Atkins, D.(Mainz/Wuppertal); Lichtenfels, R.; Seliger, B. (Mainz/Halle) 57 Heat Shock Protein 47 and Renal Fibrogenesis Razzaque, M.S. (Boston, Mass./Nagasaki); Le, V.T.; Taguchi, T. (Nagasaki) 70 Cytoprotective Effects of Heme Oxygenase in Acute Renal Failure Akagi, R.; Takahashi, T. (Okayama); Sassa, S. (New York, N.Y.) 86 Heat Shock (Stress Response) Proteins and Renal Ischemia/ Reperfusion Injury Kelly, K.J. (Indianapolis, Ind.) 107 Cisplatin-Associated Nephrotoxicity and Pathological Events Taguchi, T.; Nazneen, A. (Nagasaki); Abid, M.R.; Razzaque, M.S. (Boston, Mass./Nagasaki)

V

122 Heat Shock Proteins and Allograft Rejection Pockley, A.G.; Muthana, M. (Sheffield) 135 Oxidant Stress in Renal Pathophysiology Abid, M.R. (Boston, Mass.); Razzaque, M.S. (Boston, Mass./Nagasaki); Taguchi, T. (Nagasaki) 154 Author Index 155 Subject Index

Contents

VI

Preface

Studies on heat shock proteins (HSPs) and stress responses following an injury have made remarkable advances in recent years, benefiting mostly from scientific research and the greater availability of technology. Both HSPs and stress responses are involved in pathophysiology of various renal injuries, and the studies thereon offer prospects of new therapeutic options. The purpose of this book is to present an overview of contemporary thoughts on the clinical significance of stress responses following renal injury. Chapters are arranged and topics are selected to provide the readers integral and up to date information concerning the involvement of HSPs in acute and chronic progressive renal diseases. The effects of osmotic stress on renal medullary cells, the protective role of HSP27 during glomerular epithelial cell injury, the cytoprotective effects of HSP32 in acute renal failure, and the involvement of various stress proteins in renal ischemia and reperfusion injury are discussed by authors who are actively involved in the related fields of research. Consideration is also given to a number of selected articles dealing with the fibrogenic role of HSP47 in chronic renal diseases, the role of oxidative stress in various renal diseases, and the possible involvement of HSPs in renal cell carcinoma and their potential role during allograft rejection. The wide range of topics that are covered in this book will provide the reader with a fundamental understanding of stress responses during various renal diseases. This will be particularly helpful for scientists and clinicians who, in their research and practice, need a quick update on HSPs and stress responses following injury. The clinical importance, and significance of stress responses in various renal diseases inspired us to edit this book. We are confident that it will help to promote awareness of the fact that stress response is a major determinant factor

VII

following renal injury. Our utmost hope is that the reader will be inspired by the content of the book to take up the challenge of further research to enhance understanding of stress responses, a noble endeavor that will lead to the development of new therapeutic approaches to treat some of the fatal untreatable renal diseases. This will be an important reference book for clinical and basic researchers devoted to define various stress responses following tissue injury. We extend our sincere thanks to the contributing authors for their expert contributions. Finally, we wish to acknowledge the help, support and encouragement provided by our families (Rafi, Yuki, Ai, Naoko and Kaneko). We hope that this book will help scientists and clinicians in the fields of cell biology, pathology and nephrology to appreciate the unique relationship between stress responses following an injury and the subsequent progression of the illness. Mohammed S. Razzaque, Boston, Mass. Takashi Taguchi, Nagasaki

Preface

VIII

Razzaque MS, Taguchi T (eds): Cellular Stress Responses in Renal Diseases. Contrib Nephrol. Basel, Karger, 2005, vol 148, pp 1–7

Involvement of Stress Proteins in Renal Diseases Mohammed S. Razzaquea,b, Takashi Taguchib a Department of Oral and Developmental Biology, Harvard School of Dental Medicine, Boston, Mass., USA; b Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Abstract Heat shock proteins (HSPs) are a distinctive class of proteins that have evolved to cope with stress to provide cellular defence against a wide range of cell injuries. HSPs play an important role in the assembly and folding of intracellular polypeptides, and help in restoring the biological activities of abnormal proteins. Cellular stress responses include a transient rearrangement of functional activities, in order to protect and maintain essential cellular functions, possibly by inducing HSPs. HSPs help in restoring protein homeostasis and assist in cellular recovery from stress, either by repairing damaged proteins through refolding or by degrading them. Recent studies have documented the important roles of stress proteins in renal cell survival and matrix remodeling in a number of acute and chronic renal diseases. This brief review summarizes some of the important aspects of HSPs and their relevance to various renal diseases. Copyright © 2005 S. Karger AG, Basel

Introduction

The heat shock response, first observed by Ritossa in Drosophila in 1962 [1], is now widely accepted as one of the universally conserved cellular defence systems. In early 1970s, the heat shock response was found to coincide with synthesis of a number of new proteins [2]. The genes and protein products quickly gained much attention and a number of heat shock proteins (HSPs) have since been identified and characterized. Subsequent research work found that in addition to heat stress, a wide range of other stressful conditions could induce the heat shock responses. The heat shock response is mediated by a

group of HSPs, a response that has been observed both in eukaryotic and prokaryotic cells. Some HSPs are strictly stress induced, whereas others could be constitutively expressed, developmentally regulated or induced by stress. In addition to heat shock, a variety of other stresses, including metabolic, toxic, and oxidative injuries can elicit similar stress responses. The primary structure of the stress proteins is highly conserved [3, 4], and the expression of HSP is not always limited to cells undergoing acute stress; a number of HSPs are constitutively expressed and actively involved in maintaining cellular homeostasis, by acting as molecular chaperones [5–7]. The HSPs regulate folding and assembly of nascent and unfolded peptides, help in transporting proteins to a particular subcellular compartment and assist in the degradation of misfolded proteins [8]. As molecular chaperones, HSPs do not only assist in the folding of nascent polypeptide chains but also help in preventing aggregation of surfaceexposed hydrophobic portions of proteins, and ultimately enhance their folding. Certain HSPs exert anti-inflammatory effects by modulating the transcriptional activation of proinflammatory cytokines and adhesion molecules [9], while others including HSP-60 and -70 can induce proinflammatory cytokines, including interleukin (IL)-1␤, IL-6, IL-12, IL-15 and tumor necrosis factor-␣ from human monocytes [10]. Several HSPs are involved in antigen presentation, steroid receptor function, nuclear receptor binding, and apoptosis [11, 12]. HSPs also exert important roles in signal transduction by maintaining and stabilizing intracellular microenvironments.

Regulation of HSPs

The stress responses in mammalian cells are thought to be transcriptionally regulated by the heat shock transcription factor (HSF), which specifically binds to the heat shock promoter element (HSE) that contains palindromic sequences rich in repetitive purine and pyrimidine motifs [13]. The HSF family consists of four members (HSF1, HSF2, HSF3, and HSF4) in higher eukaryotes [14–18]. HSF is present in normal, unstressed cells as a monomer in the cytoplasm, but exposure of such cells to stress conditions results in conversion of HSF from an inactive monomeric form to an active trimeric DNA-binding form, which then translocates to the nucleus and interacts with HSE to induce transcription of HSP genes (fig. 1) [19, 20]. Oligomerization of the HSF and its interaction with the HSE are the hallmark of active transcriptional response to a variety of stresses that include physical and chemical stresses. All members of the HSF family share common structural features, including a conserved DNA-binding domain, an extended hydrophobic repeat involved in trimerization, and a transactivation domain [21, 22]. With the exception of those from

Razzaque/Taguchi

2

Stress HSP HSF

HSP

HSF

HSF P HSF P HSF P

HSF P

HSF HSE

HSP

HSP

Fig. 1. Simplified schematic diagram showing the transcriptional regulation of heat shock proteins (HSPs). Heat shock transcription factor (HSF) is normally bound to HSPs and present as an inactive molecule in the cytosol. Upon exposure to stressors, HSFs are phosphorylated (P) by protein kinases, rapidly form trimers, and translocate to the nucleus where HSFs interact with heat shock promoter element (HSE) to induce the transcription of HSPs, which are then transcribed and relocated to the cytosol.

budding yeasts, HSFs also have a carboxyl-terminal hydrophobic repeat, which is thought to suppress trimer formation by interaction with the amino-terminal hydrophobic repeats [14, 23]. Most of our understanding of protein folding is based on in vitro studies, which needs careful interpretation for their relevance to the complex in vivo system, because the in vitro experimental solvents do not always mimic the in vivo complex microenvironments. One of the major differences is that most of the in vitro experiments deal with a single unfolded protein, which usually does not interact with other components, while in the in vivo situation, complex interactions among various proteins occur during protein folding. In the native in vivo microenvironment, chaperone-assisted folding of certain proteins is an essential phenomenon. Moreover, in vitro studies provide the opportunity to examine the properties of proteins, by chemical or physical denaturing, which may not always replicate the in vivo circumstances of protein folding. Despite experimental limitations, both in vivo and in vitro studies have documented the important roles of HSPs in the pathogenesis of various diseases, ranging from autoimmune diseases (arthritis and diabetes) to tumors and renal diseases.

Stress Proteins in Renal Diseases

3

HSPs in Renal Diseases

In the accompanying chapters, the authors have elaborated on the important roles of several stress proteins in various renal pathophysiological conditions, ranging from hypoxic injury to renal fibrotic diseases and malignancies. These chapters provide comprehensive information on the involvement of HSPs in the pathophysiology of a wide range of renal injuries, including acute and chronic progressive renal diseases. Neuhofer and Beck [24] summarize the effects of osmotic stress on renal medullary cells, and how these cells adapt to high salt and urea-rich microenvironments, not only to survive, but also to achieve their organ-specific functions. It appears likely that the high expression of HSP70 in the hyperosmotic renal medulla is cytoprotective for medullary cells. Similarly, Bijian and Cybulsky [25], in their chapter, discuss the protective role of HSP27 in glomerular epithelial cells injury, and postulate that the manipulation of HSP27 expression could be potentially beneficial by modulating glomerular epithelial cell injury and subsequent proteinuria. In a separate chapter, Kelly [26] describes the diverse effects of various stress proteins in renal ischemia and reperfusion injury. HSP32, also known as heme oxygenase, oxidizes the heme portion of hemoglobin to bilirubin. The generated bilirubin regulates the NADPH concentration in the cell, and provides an antioxidant defence system to the cell. In another chapter, Akagi et al. [27] provide details on the cytoprotective roles of HSP32 and its function in acute renal failure. The relevance of HSPs in the pathomechanisms of renal cell carcinoma is outlined by Atkins et al. [28]; the differential expression of certain HSPs, including HSP27, HSP70 and HSP72 in renal cell carcinoma and in cell lines generated from renal tumors suggests the involvement of HSPs in tumor progression, possibly by regulating the rate of tumor cell proliferation and apoptosis. The synthesis and post-translational modifications of collagens are rather complex processes and require the help of numerous enzymes and chaperones for correct conformation. HSP47, found in the endoplasmic reticulum of collagen-producing cells, helps in the correct formation of quaternary structure of collagen [29]. In many fibroproliferative diseases, the expression of HSP47 mostly parallels the extent of collagen accumulation [30–34]. In the accompanying chapter, we explain the fibrogenic role of HSP47 in chronic renal diseases, and discuss its potential as a target for the development of novel antifibrotic therapeutic agents [35]. Pockley and Muthana [36] discuss the potential role of HSPs during allograft rejection. Although direct relevance of HSPs in transplant rejection needs further studies, selective induction of HSPs during allograft rejection suggests their possible involvement in the complex process of rejection. There are still inconsistencies in human and experimental studies, which needed to be resolved. Furthermore, some of the HSPs might

Razzaque/Taguchi

4

exert dual effects in allograft rejection process, both as protective and aggravating factors. In view of the fact that some of the immunoinflammatory features of allograft rejection appear to be similar irrespective of the organ involved, our knowledge of transplant rejection, and exact role of HSPs in such complex process, in general, will enhance our understanding of transplant rejection responses involving kidney. The balance between the activities of the pro- and antioxidant enzymes tightly regulates oxidative homeostasis, and this delicate balance seems to be disrupted in various renal diseases [37]. Since oxidative stress-induced renal injuries are involved in a wide range of acute and chronic renal diseases, we also included chapters that briefly summarize the involvement of oxidative stress in renal diseases [38, 39].

Conclusion

Extensive research work in the last couple of years has significantly improved our understanding of the crucial roles of stress proteins in various acute and chronic renal diseases. It has been convincingly demonstrated that constitutively expressed HSPs, by acting as molecular chaperones, help in folding and conformation of nascent polypeptides through binding to their C-terminal domain, while inducible HSPs are mostly responsible for inhibiting denaturation and incorrect or abnormal aggregation of proteins following cell injury, and may have determinant effect on overall cell survival. However, the transcriptional and translational regulation of the involved stress proteins, and their signaling events in various renal diseases, need further studies to elucidate their molecular and cellular interactions, and most importantly to determine their exact role in various disease processes. The availability of such large-scale gene expression studies such as microarray and proteomics may yield useful information that will not only help in determining novel pathways but also help in focusing studies of relevant molecules. Such focused research studies will help in developing disease-specific therapeutic strategies for the treatment of various acute and chronic renal diseases.

References 1 2 3

Ritossa F: A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962;18:571–573. Tissie‘res A, Mitchell HK, Tracy UM: Protein synthesis in salivary glands of Drosophila melanogaster: Relation to chromosome puffs. J Mol Biol 1974;84:389–398. Lindquist S: The heat shock response. Annu Rev Biochem 1986;55:1151–1191.


5

4 5 6 7 8 9 10 11 12 13 14 15

16 17 18

19 20 21 22 23

24 25 26 27 28 29 30

Georgopoulos C, Welch WJ: Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol 1993;9:601–634. Gething MJ, Sambrook J: Protein folding in the cell. Nature 1992;355:33–45. Hendrick JP, Hartl FU: Molecular chaperone functions of the heat-shock proteins. Annu Rev Biochem 1993;62:349–384. Becker J, Craig EA: Heat-shock proteins as molecular chaperones. Eur J Biochem 1994;219:11–23. Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996;381:571–579. Cahill CM, Lin HS, Price BD, Bruce JL, Calderwood SK: Potential role of heat shock transcription factor in the expression of inflammatory cytokines. Adv Exp Med Biol 1997;400B:625–630. Pockley AG: Heat shock proteins in health and disease: Therapeutic targets or therapeutic agents? Expert Rev Mol Med 2001;3:1–21. Sharp FR, Massa SM, Swanson RA: Heat-shock protein protection. Trends Neurosci 1999;22:97–99. Kiang JG, Tsokos GC: Heat shock protein 70 kDa: Molecular biology, biochemistry, and physiology. Pharmacol Ther 1998;80:183–201. Perisic O, Xiao H, Lis JT: Stable binding of Drosophila heat shock factor to head-to-head and tailto-tail repeats of a conserved 5 bp recognition unit. Cell 1989;59:797–806. Rabindran SK, Giorgi G, Clos J, Wu C: Molecular cloning and expression of a human heat shock factor, HSF1. Proc Natl Acad Sci USA 1991;88:6906–6910. Sarge KD, Zimarino V, Holm K, Wu C, Morimoto RI: Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Gene Dev 1991;5:1902–1911. Schuetz TJ, Gallo GJ, Sheldon L, Tempst P, Kingston RE: Isolation of a cDNA for HSF2: Evidence for two heat shock factor genes in human. Proc Natl Acad Sci USA 1991;88:6911–6915. Nakai A, Morimoto RI: Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggest a new regulatory pathway. Mol Cell Biol 1993;13:1983–1997. Nakai A, Tanabe M, Kawazoe Y, Inazawa J, Morimoto RI, Nagata K: HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol Cell Biol 1997;17:469–481. Kroeger PE, Morimoto RI: Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity. Mol Cell Biol 1994;14:7592–7603. Bonner JJ, Ballou C, Fackenthal DL: Interactions between DNA-bound trimers of the yeast heat shock factor. Mol Cell Biol 1994;14:501–508. Harrison CJ, Bohm AA, Nelson HCM: Crystal structure of the DNA binding domain of the heat shock transcription factor. Science 1994;263:224–227. Peteranderl R, Nelson HCM: Trimerization of the heat shock transcription factor by a triplestranded alpha-helical coiled-coil. Biochemistry 1992;31:12272–12276. Zuo J, Baler R, Dahl G, Voellmy R: Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure. Mol Cell Biol 1994;14:7557–7568. Neuhofer W, Beck F-X: Response of renal medullary cells to osmotic stress. Contrib Nephrol 2005;148:21–34. Bijian K, Cybulsky AV: Stress Proteins in glomerular epithelial cell injury. Contrib Nephrol 2005;148:8–20. Kelly KJ: Heat shock (stress response) proteins and renal ischemia/reperfusion injury. Contrib Nephrol 2005;148:86–106. Akagi R, Takahashi T, Sassa S: Cytoprotective effects of heme oxygenase in acute renal failure. Contrib Nephrol 2005;148:70–85. Atkins D, Lichtenfels R, Seliger B: Heat shock proteins in renal cell carcinomas. Contrib Nephrol 2005;148:35–56. Nagata K: HSP47 as a collagen-specific molecular chaperone: Function and expression in normal mouse development. Semin Cell Dev Biol 2003;14:275–282. Razzaque MS, Foster CS, Ahmed AR: Role of collagen-binding heat shock protein 47 and transforming growth factor beta 1 in conjunctival scarring in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44:1616–1621.

Razzaque/Taguchi

6

31 32 33

34

35 36 37 38 39

Razzaque MS, Taguchi T: Collagen-binding heat shock protein (HSP) 47 expression in antithymocyte serum (ATS)-induced glomerulonephritis. J Pathol 1997;183:24–29. Razzaque MS, Nazneen A, Taguchi T: Immunolocalization of collagen and collagen-binding heat shock protein 47 in fibrotic lung diseases. Mod Pathol 1998;11:1183–1188. Razzaque MS, Taguchi T: The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999;14: 1199–1212. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T: Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47. Cell Tissue Res 1998;293:471–478. Razzaque MS, Taguchi T: Heat shock protein 47 and renal fibrogenesis. Contrib Nephrol 2005;148:57–69. Pockley MR, Muthana M: Heat shock proteins and allograft rejection. Contrib Nephrol 2004;148: 2005;148:122–134. Cochrane AL, Ricardo SD: Oxidant stress and regulation of chemokines in the development of renal interstitial fibrosis. Contrib Nephrol 2003;139:102–119. Abid MR, Razzaque MS, Taguchi T: Oxidant stress in renal pathophysiology. Contrib Nephrol 2005;148:135–153. Taguchi T, Nazneen A, Abid MR, Razzaque MS: Cisplatin-associated nephrotoxicity and pathological events. Contrib Nephrol 2005;148:107–121.

Mohammed S. Razzaque, MBBS, PhD Department of Oral and Developmental Biology Harvard School of Dental Medicine 188 Longwood Avenue, Boston, MA 02115 (USA) Tel. ⫹1 617 432 5768, Fax ⫹1 617 432 5767, E-Mail [email protected]


7


Stress Proteins in Glomerular Epithelial Cell Injury Krikor Bijian, Andrey V. Cybulsky Department of Medicine, McGill University Health Centre, McGill University, Montreal, Quebec, Canada

Abstract Glomerular visceral epithelial cells (GEC) or podocytes are highly differentiated, specialized cells that play a key role in the maintenance of glomerular permselectivity. Injury of GEC, leading to proteinuria, contributes to the pathogenesis of human and experimental glomerulopathies. Recent studies have demonstrated that stress proteins may be induced and may be involved in the modulation of GEC injury. The C5b-9 membrane attack complex of complement induces GEC injury and proteinuria in the passive Heymann nephritis (PHN) model of membranous nephropathy. C5b-9 induces upregulation of the endoplasmic reticulum (ER) stress proteins, bip and grp94, in part, via activation of cytosolic phospholipase A2. These ER stress proteins limit complement-mediated GEC injury. In experimental nephropathy associated with hyperlipidemia, and in experimental diabetic nephropathy, there is an upregulation of the ER heat shock protein (Hsp) 47, a chaperone protein involved in the synthesis or assembly of collagens. Hsp47 expression in GEC is associated with increased deposition of collagen, and glomerulosclerosis. Hsp27, a stress protein involved in actin polymerization, is localized in GEC, and its expression and activation are increased in the rat puromycin aminonucleoside model of focal segmental glomerulosclerosis, and in PHN. Hsp27 may preserve actin structure, and facilitates survival in the context of injury. Development of methods to induce expression/activation of stress proteins may eventually lead to novel approaches to the therapy of GEC injury and proteinuria. Copyright © 2005 S. Karger AG, Basel

Introduction

Stress proteins are involved in diverse cellular processes, including assembly and correct folding of proteins, intracellular transport of proteins to specific organelles, degradation of proteins, maintenance of cytoskeletal structure, etc.

Stress proteins have been classified into families according to their molecular mass, and/or subcellular distribution. Many stress proteins are expressed constitutively, but most can be upregulated by metabolic or physical stresses. A number of stress proteins may be involved in kidney physiology, including adaptation of cells to high osmolality, volume regulation, and maturation of steroid receptors, and certain stress proteins may be involved in renal pathophysiology [reviewed in ref. 1]. Glomerular visceral epithelial cells (GEC) or podocytes are highly differentiated, specialized cells that are intrinsic components of the kidney glomerulus, and play a key role in the maintenance of glomerular permselectivity [2–4]. Injury of GEC, leading to proteinuria, contributes to the pathogenesis of various types of human and experimental glomerulopathies. There is presently only limited information on the role of stress proteins in GEC pathophysiology. This review will focus on the role of the glucose-regulated proteins (grp) in the endoplasmic reticulum (ER), as well as the heat shock proteins (Hsp) 47 and Hsp27 in GEC injury, and proteinuria.

ER Stress Proteins

The ER serves as a site for folding, assembly and degradation of proteins [5–7]. Membrane and secreted proteins are translocated into the lumen of the ER shortly after initiation of synthesis, and resident ER lumenal proteins, including grp78 (bip) and grp94, mediate protein folding. Moreover, bip and grp94 are believed to bind to misfolded or abnormal proteins and prevent their aggregation, either by rescuing such proteins from irreversible damage, or by increasing their susceptibility to proteolytic attack. Other ER proteins, including ERp72, may participate in disulfide isomerization. Perturbation of the ER may lead to ER stress responses [5–8]. These may include the ‘unfolded protein response’ (UPR), which in part consists of upregulating the capacity of the ER to process abnormal proteins. On accumulation of unfolded proteins in the ER, activating transcription factor 6 moves to the Golgi, where it is cleaved by S1P and S2P proteases to yield a cytosolic fragment. This fragment migrates to the nucleus to activate transcription of UPR-responsive genes, e.g., bip and grp94. In parallel, ‘inositol requiring 1’ dimerizes and activates its endoribonuclease activity. Inositol requiring 1 cleaves X-box binding protein 1 mRNA and changes the reading frame to yield a potent transcriptional activator. At the same time, pancreatic ER kinase is activated to phosphorylate the ␣ subunit of eukaryotic translation initiation factor 2, which reduces AUG codon recognition. The general rate of translation is reduced (which aims at decreasing the protein load on a damaged

Stress Proteins in Glomerular Epithelial Cell Injury

9

ER), but selective mRNAs can be preferentially translated under these conditions. Induction of the UPR may allow cells to recover from ER stress; however, prolonged or more substantial ER stress may lead to cell death via apoptosis. There are several pathophysiological stimuli that can trigger ER stress. Tunicamycin, a nucleoside antibiotic that blocks N-linked glycosylation, or the Ca2⫹ ionophore, ionomycin, which can deplete Ca2⫹ from intracellular stores, can induce accumulation of unfolded proteins in the ER. Other perturbations that may lead to induction of ER stress proteins include ischemia-reperfusion injury, hyperhomocystinemia, viral infections, and genetic mutations that impair protein folding.

Hsp27

Although Hsps were originally shown to be induced in cells exposed to high temperatures, it is now well appreciated that Hsps also participate in stress responses caused by metabolic stresses, such as accumulation of misfolded proteins in the cytosol. Hsp27 (and the mouse homolog, Hsp25) is a member of the small Hsp family of proteins, which have been shown to participate in the actin polymerization/depolymerization processes [1, 9]. Hsp27 may exist in phosphorylated and dephosphorylated forms, and phosphorylation may occur through the p38 mitogen-activated protein kinase (MAPK) pathway in response to diverse stresses [10]. Hsp27 phosphorylation may regulate its oligomerization, and leads to the dissociation of Hsp27 from the barbed ends of actin filaments, thus removing its inhibitory effect and promoting actin polymerization and stress fiber formation [11]. This process may allow for remodeling of the actin cytoskeleton following stress [12]. Small Hsps, including Hsp27 in their multimeric forms may act as molecular chaperones, e.g., preventing protein aggregation, and facilitating protein refolding after stressful stimuli [13].

Hsp47

Hsp47 is an ER resident glycoprotein that is heat-inducible, and may function as a molecular chaperone for collagens. Hsp47 has been shown to interact with collagens I–V and procollagens, and may participate in the proper processing of procollagens in the ER and/or secretion [1, 14]. Thus, Hsp47 may play an important role in a variety of diseases where sclerotic or fibrotic changes are associated with increased collagen production.

Bijian/Cybulsky

10

Role of ER Stress Proteins in Complement-Mediated GEC Injury

Activation of the complement cascade near a cell surface leads to assembly of terminal components, exposure of hydrophobic domains, and insertion of the C5b-9 membrane attack complex into the lipid bilayer of the plasma membrane [15, 16]. Assembly of C5b-9 results in formation of transmembrane channels or rearrangement of membrane lipids with loss of membrane integrity. Nucleated cells require multiple C5b-9 lesions for lysis, but at lower doses, C5b-9 induces sublethal (sublytic) injury, and various metabolic effects. An example of sublytic C5b-9-mediated cell injury in vivo is passive Heymann nephritis (PHN) in the rat, a widely accepted model of human membranous nephropathy [17]. In PHN, nephritogenic antibody binds to GEC antigens, and leads to the in situ formation of subepithelial immune complexes. C5b-9 assembles in GEC plasma membranes, ‘activates’ GEC, and leads to proteinuria and sublytic GEC injury [18]. Based on studies in GEC culture and in vivo, C5b-9 assembly induces transactivation of receptor tyrosine kinases, an increase in cytosolic free Ca2⫹ concentration, and activation of protein kinase C, as well as cytosolic phospholipase A2-␣ (cPLA2) [19–21]. cPLA2 is an important mediator of C5b-9-dependent GEC injury. Complement enhances cPLA2 phosphorylation and catalytic activity [19–21]. cPLA2 localizes and hydrolyzes phospholipids principally at the membrane of the ER, as well as the plasma membrane and the nuclear envelope, but not at the mitochondria or Golgi [22]. The arachidonic acid released by cPLA2 is metabolized in GEC via cyclooxygenases-1 and -2 to prostaglandin E2 and thromboxane A2, and inhibition of prostanoid production reduces proteinuria in PHN and in human membranous nephropathy [18]. cPLA2 may also mediate GEC injury more directly [19]. C5b-9-induced sublethal cell injury may lead to a decline in cellular ATP, mitochondrial lipid perturbation, or loss of mitochondrial membrane potential, whereas at high doses, C5b-9 can induce mitochondrial damage and cell necrosis. Based on biochemical and morphological observations, it is likely that during complement-dependent GEC injury, integral membrane and secretory proteins are altered. Such proteins may include integrins, transporters, and/or cell junctional proteins, and these alterations may contribute to the permselectivity defect of the glomerular capillary wall in PHN. On the other hand, complement attack may also activate pathways that restrict injury or facilitate recovery. For example, one mechanism of protection from complement attack is ‘ectocytosis’ (shedding) of C5b-9 complexes from cell membranes [15, 16]. We examined if C5b-9-mediated activation of cPLA2 and induction of GEC injury are associated with induction of the UPR [23]. Resting GEC constitutively


11

NS, 24h

HIS, 24h

NS, 6 h

HIS, 6h

NS, 4 h

HIS, 4h

Tunic

Untr

NS, 24h

NS, 6 h

HIS, 24h

cPLA2 HIS, 6h

HIS, 4h

Tunic

Untr

NS, 4 h

neo

a

bip

grp94

2.2

bip

grp94

2.0 1.8 1.6 1.4

grp94

NS ⫹ MAFP

Tunic

6h

24h

4h Fold increase

bip

HIS

NS

1.4 1.0

Tunic

6h

4h

Tunic

24h

24h

cPLA2

1.8

grp94

grp94

neo

d

bip

bip

1.0

cPLA2

Fold increase

C8DS

c

C8DS ⫹ C8

neo

6h

4h

24h

Tunic

1.2 6h

2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0

4h

Fold increase

b

1.8 1.6 1.4 1.2 1.0 1.8 1.6 1.4 1.2 1.0

bip

grp94

C M

Fig. 1. Effect of complement and cPLA2 on expression of ER stress proteins in GEC. Anti-GEC antibody-sensitized neo (control) GEC, or GEC that overexpress cPLA2 (at a level ~5-fold of neo) were incubated with 2.5% normal serum (NS; to form sublytic C5b-9), or heat-inactivated serum (HIS) in controls, for 4, 6 or 24 h. Cell lysates were immunoblotted with antibodies to bip or grp94. a Representative immunoblot. b Densitometric quantification of immunoblots (NS/HIS). Complement increased expression of bip and grp94 significantly in GEC that overexpress cPLA2 (nine experiments), while upward trends in bip and grp94 expression occurred in neo GEC (six experiments). Bip: p ⬍ 0.002 NS versus HIS and p ⫽ 0.05 cPLA2 versus neo (at all time points); grp94: p ⬍ 0.001 NS versus HIS and p ⬍ 0.035 cPLA2 versus neo (at all time points). The effect of tunicamycin (Tunic) is shown for comparison (positive control). c Assembly of C5b-9 is required for increased

Bijian/Cybulsky

12

express the ER stress proteins, bip and grp94. Brief incubation of GEC with sublytic complement induced leakage of bip and grp94 from the ER into the cytosol, and this leakage was dependent on the activation of cPLA2. This result is in keeping with the observation that complement-induced activation of cPLA2 leads to phospholipid hydrolysis at the membrane of the ER [22], and suggests that activation of cPLA2 by complement perturbed the ER membrane sufficiently to allow a small portion of ER lumenal proteins, including bip or grp94, to leak into the cytosol. Exposure of GEC to chronic complement attack (4–24 h) resulted in increased expression of bip and grp94 mRNAs and proteins, and these increases were, at least in part, mediated via activation of cPLA2 (fig. 1) [23]. Complement, however, had no effect on the expression of the cytosolic stress protein, Hsp70. Similar to C5b-9, ER stress protein expression was increased after incubation of GEC with the Ca2⫹ ionophore, ionomycin (24 h), and these changes were also mediated via activation of cPLA2. The increases in the expression of ER stress proteins were a direct result of cPLA2-mediated phospholipid hydrolysis (and presumably membrane injury), and were not due to products of phospholipid hydrolysis (e.g., arachidonic acid, lysophosphatidylcholine) or their metabolites (i.e., prostanoids). To determine if C5b-9-mediated induction of ER stress proteins is functionally important, GEC were stably transfected with bip antisense cDNA [23]. The bip antisense clones and neo (control) GEC were incubated with serially increasing doses of complement that induced minimal-to-moderate cell lysis at 18 h. This protocol allowed for C5b-9 to increase ER stress protein expression, but with increasing incubation time and complement dose, a portion of the cells underwent lysis. After 18 h of incubation, cytolysis was consistently greater in the GEC clones that express bip antisense mRNA (fig. 2), indicating that induction of bip plays a functionally important role in limiting the amount of expression of ER stress proteins. Antibody-sensitized GEC that overexpress cPLA2 were incubated with 2.5% C8-deficient serum (C8DS) or 2.5% C8-deficient serum reconstituted with purified C8 (C8DS ⫹ C8) for 24 h. Cell lysates were immunoblotted with antibodies to bip or grp94. A representative immunoblot and densitometric quantification of immunoblots are presented. C8DS ⫹ C8 increased expression of bip and grp94 significantly, as compared with C8DS. Bip: p ⬍ 0.0001 C8DS ⫹ C8 versus C8DS; grp94: p ⬍ 0.005 C8DS ⫹ C8 (six incubations). d Antibody-sensitized neo GEC were incubated with 4.0% normal serum (to form sublytic C5b-9), 4.0% normal serum plus the cPLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP; 25 ␮M), or heat-inactivated serum in controls, for 24 h. A representative immunoblot and densitometric quantification of immunoblots are presented. In control GEC (c), complement increased bip and grp94 expression significantly (p ⬍ 0.01), whereas MAFP (M) inhibited the complement-induced increases (five experiments). Reprinted from [23].


13

LDH release (%)

40

bipAS-3

30

bipAS-1 bipAS-2

20

neo

10

0 2

4

6

8

10

Normal serum (%)

Fig. 2. Complement-mediated cytotoxicity in GEC that express bip antisense mRNA. Neo (control) GEC and three clones of GEC that express bip antisense mRNA (bipAS) were incubated with anti-GEC antibody, and normal serum for 18 h. Complement-mediated cytotoxicity was determined by measuring release of lactate dehydrogenase (LDH) into cell supernatants. Complement induced greater cytotoxicity in the bipAS-1 and bipAS-3 GEC (p ⬍ 0.015 bipAS-1 vs. neo, p ⬍ 0.003 bipAS-3 vs. neo), while an upward trend in cytotoxicity was evident in bipAS-2 (p ⫽ 0.078 bipAS-2 vs. neo; 5 experiments). Reprinted from [23].

complement-dependent injury. Thus, induction of ER stress proteins is an important novel mechanism of protection from sustained complement attack. The capability of the GEC to recover or limit the severity of complement attack may depend on its capacity to resynthesize or reassemble integral membrane proteins, which may require the presence of bip or grp94. Induction of these ER stress proteins during complement attack may also limit accumulation of abnormal proteins and help sustain physiological functions and viability [6, 7]. In another series of experiments, we assessed whether other stimuli that induce ER stress protein expression would affect complement-mediated GEC injury [23]. GEC are particularly sensitive to the cytotoxic effect of puromycin aminonucleoside [24]. Incubation of cultured GEC with puromycin aminonucleoside increased expression of bip. When these treated GEC were subsequently exposed to complement, cytolysis was attenuated, as compared with untreated cells. Unlike puromycin aminonucleoside, preincubation of GEC with tunicamycin (a potent inducer of ER stress proteins) enhanced complementmediated lysis. To model ischemia-reperfusion injury in vitro, GEC were incubated with 2-deoxyglucose plus antimycin A for 90 min (to inhibit glycolytic and/or oxidative metabolism), followed by re-exposure to glucose-replete cell culture medium for 24 h. This protocol resulted in increased expression of bip

Bijian/Cybulsky

14

grp94

bip

Ctrl Tun

ⴙ

bip

**

0.8

Ctrl ADR

0.4

Ctrl PHN

0.6

Ctrl PHN

Densitometry (arbitrary units)

*

grp94

Ctrl Tun

bip

Ctrl ADR

grp94 1.0

Fig. 3. Expression of ER stress proteins in vivo. Glomeruli were isolated from normal (control) rats (Ctrl) and from rats with PHN on day 14, and lysates were immunoblotted with antibodies to bip or grp94. Bip and grp94 expression was increased in glomeruli isolated from rats with PHN, as compared with control (densitometric quantification; *p ⬍ 0.002, ⫹ p ⬍ 0.005 PHN vs. control; 7–9 rats per group). Glomerular grp94 and bip expression was altered in rats injected with subnephritogenic adriamycin, 6 mg/kg intravenously (ADR, day 14, **p ⬍ 0.001 adriamycin vs. control; 9–12 rats per group), as well as in rats injected with tunicamycin, 1 mg/kg intraperitoneally (Tun, 24 h, the dots represent the values of 2 individual rats in each group). Reprinted from [23].

and grp94; however, it did not protect, but rather enhanced complement-mediated injury. Thus, certain stimuli that enhance ER stress protein expression in GEC provided protective effects, while others enhanced complement lysis. These results are in keeping with some observations in other cells, i.e., exposure of cells to mild stress, sufficient to induce upregulation of ER stress proteins, may be protective to additional insults, although progression to cell death may occur if the stress is more intense or prolonged [25]. To determine if changes in ER stress protein expression occur in C5b9-mediated GEC injury in vivo, we assessed levels of bip and grp94 expression in PHN, where GEC injury is due to C5b-9 assembly, and is associated with cPLA2 activation and production of prostanoids [23]. In our model of PHN, proteinuria begins to appear at approximately day 7, and is well established at day 13–14. On day 14, expression of glomerular bip and grp94 was increased in rats with PHN, as compared with control (fig. 3), although increases in levels of bip and grp94 proteins were not detected consistently on days 3 and 7. GEC in vivo are sensitive to the cytotoxic effects of adriamycin, and injection of rats with adriamycin may lead to GEC injury, in association with proteinuria [26]. Glomeruli of rats that had been injected with a subnephritogenic dose of adriamycin (i.e., a dose that did not induce proteinuria for up to 14 days) showed increases in grp94 and bip expression (fig. 3). Injection of rats with


15

PHN-ADR

Proteinuria (mg/24h)

1,500

PHN-Tun PHN

1,000

500

0 Day 0

Day 7

Day 9

Day 13

Fig. 4. Pretreatment of rats with a subnephritogenic dose of adriamycin or tunicamycin reduces proteinuria in PHN. Rats were untreated, or were injected with adriamycin, 6 mg/kg intravenously (ADR), or tunicamycin, 1 mg/kg intraperitoneally (Tun) to upregulate ER stress proteins (see fig. 3). Four days later, rats were injected with the nephritogenic antibody, anti-Fx1A, to induce PHN (day 0). Urine protein was measured on days 0, 7, 9 and 13. Compared with the PHN-untreated group (4 rats), proteinuria was significantly lower in the adriamycin (p ⬍ 0.005, 3 rats) and tunicamycin groups (p ⬍ 0.025, 3 rats). Reprinted from [23].

tunicamycin also enhanced glomerular expression of bip and grp94 without inducing proteinuria (fig. 3). Based on these results, rats were treated with a subnephritogenic dose of adriamycin, or tunicamycin, and PHN was then induced in these pretreated rats and in untreated animals. Substantial proteinuria developed in untreated rats with PHN (on days 7, 9 and 13), whereas the amount of proteinuria was significantly lower in the rats with PHN that had been pretreated with adriamycin or tunicamycin (fig. 4) [23]. Thus, increased ER stress protein expression can reduce C5b-9-mediated GEC injury in vivo. Adriamycin and tunicamycin did not decrease proteinuria by reducing the amount of glomerular antibody deposition or complement activation, and serum creatinine was not significantly different among the three groups of rats, indicating that most likely there were no significant differences in renal function. These results provide a rationale for developing nontoxic methods to induce expression of ER stress proteins in vivo, which may eventually have applications to therapy of glomerular disease. The importance of these results extends beyond complement-induced GEC injury. For example, preinduction of ER stress proteins prior to xenotransplantation may potentially be a means of reducing hyperacute xenograft rejection, which is complement dependent [27].

Bijian/Cybulsky

16

Hsp47 in Experimental Nephropathy Induced by Hyperlipidemia and in Diabetic Nephropathy

The ability of Hsp47 to bind to newly synthesized procollagen in the ER and aid in its proper folding and assembly has associated Hsp47 with the pathogenesis of several sclerotic/fibrotic diseases. Previous studies have demonstrated increased expression of Hsp47 in the CCl4-induced liver cirrhosis, bleomycin-induced pulmonary fibrosis and antithymocyte serum-induced glomerulosclerosis rat models. Recent studies have analyzed the expression of Hsp47 in experimental glomerular diseases. Razzaque and Taguchi. [28] studied the role of Hsp47 in high cholesterol diet-induced glomerulosclerosis in rats. Normal male Wistar rats were fed a high fat diet, which consisted of 2% cholesterol, 5% sugar, 0.2% propyl thiouracil, 0.5% cholic acid, and 10% lard, for 4 months. Glomerular hypercellularity and expansion of mesangial matrix with glomerular infiltration of foam cells and inflammatory cells were among the many morphological changes observed. Despite the significant increase in serum cholesterol in the rats exposed to high fat diet, as compared with controls, there were no significant differences in serum creatinine levels between the two groups. Immunohistochemical staining revealed increased deposition of collagens and increased expression of Hsp47 in rat kidneys derived from hypercholesterolemic rats, as compared with controls. Double immunostaining for Hsp47 and desmin (a marker of phenotypically altered GEC), ED-1 (marker of infiltrating monocytes/macrophages), or ␣-smooth muscle actin (marker of phenotypically altered mesangial cells) confirmed that increased Hsp47 was present specifically in GEC. Another study addressed the renal expression of Hsp47 and collagens during the acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) phases of streptozotocin-induced diabetic nephropathy in rats [29]. Morphological analysis of kidney sections obtained from acute diabetic rats revealed no significant development of fibrosis, as compared with untreated controls. However, kidney sections obtained from chronic diabetic rats demonstrated progressive thickening of the glomerular basement membrane, glomerulosclerosis, tubular damage, and interstitial fibrosis. Immunohistochemical analysis of these kidney sections revealed increased deposition of collagen III and IV only in the samples obtained from chronic diabetic rats, consistent with the aforementioned morphological changes. Furthermore, immunocytochemical studies demonstrated an increase in Hsp47 expression in kidneys of chronic diabetic rats, as compared with control or acute diabetic rats. Upregulation of Hsp47 coincided with the initiation of collagen deposition, and was localized to GEC, mesangial cells, and tubular epithelial cells.


17

These results implicate the GEC as collagen- and Hsp47-producing cells in both nephropathies associated with hyperlipidemia and diabetes. Moreover, these studies highlight Hsp47 as a potential target for clinical therapy in order to limit collagen production, and prevent renal scarring and glomerulosclerosis.

Hsp27 and the GEC Actin Cytoskeleton in Glomerulopathies

Hsp27 has been implicated in the actin polymerization/depolymerization process. Electron microscopic evaluation of normal kidney sections has localized Hsp27 exclusively within GEC, suggesting a possible role for Hsp27 in altering the actin architecture within the actin-rich GEC foot processes. Nephrotic syndrome is characterized by marked effacement of GEC foot processes, leading to proteinuria, hypoalbuminemia, and edema. In contrast to the well-established morphological changes observed in GEC during the development of nephrotic syndrome, little is known about the chemical or molecular events that occur. Smoyer et al. [30] analyzed the expression and phosphorylation of glomerular Hsp27 in the rat puromycin aminonucleoside model of focal segmental glomerulosclerosis, which features GEC injury and heavy proteinuria. Using combined immunofluorescence and electron microscopic studies, the authors showed significant increases in glomerular Hsp27 protein expression and phosphorylation (both on day 10), exclusively in GEC. Renal cortical Hsp27 mRNA expression was also found to be increased in rats with nephrosis (day 10). Another study by these authors [31] demonstrated the importance of Hsp27 in the cytoskeletal rearrangement process in GEC. Murine GEC were stably transfected with Hsp27 sense, antisense or empty vector control cDNAs, and were treated with puromycin aminonucleoside. GEC expressing Hsp27 antisense mRNA demonstrated significant decreases in cell survival, polymerized actin content and cell area after treatment, whereas sense mRNA-expressing cells had increased cell survival and cell area. Protection against puromycin aminonucleoside-induced microfilament disruption was also evident in GEC that express or overexpress Hsp27 (empty vector and sense transfections), as compared with antisense mRNA-expressing cells. To ascertain the signal transduction pathway leading to Hsp27 phosphorylation, Aoudjit et al. [32] monitored the p38 MAPK activity in the PHN model of membranous nephropathy. Glomeruli isolated from rats with PHN demonstrated significant increases in p38 MAPK activity as compared with controls. Furthermore, treatment of these rats with p38 MAPK inhibitors increased the levels of proteinuria, implying that the p38 MAPK pathway limits complement-mediated GEC injury in vivo. In cultured GEC, complement significantly increased the phosphorylation of MAPK-associated protein kinase-2 (MAPKAPK-2), a kinase downstream of p38. MAPKAPK-2 has also been shown to phosphorylate Hsp27,

Bijian/Cybulsky

18

thus suggesting a signaling pathway involving p38, MAPKAPK-2, and Hsp27. Moreover, GEC overexpressing wild-type Hsp27 showed increased resistance to complement-mediated injury. Phosphorylation of Hsp27 was required to protect GEC from complement-mediated injury, since a phosphorylation-deficient mutant of Hsp27 was unable to provide a protective effect. These studies propose a protective or reparative role for Hsp27 in GEC injury. Thus, Hsp27 expression in the normal kidney may serve to maintain the architecture of the GEC foot processes, while cytoskeletal changes incurred during GEC injury may trigger increases in Hsp27 expression and/or activation. Development of methods to modulate Hsp27 may eventually prove to be a fruitful approach to the therapy of GEC injury and proteinuria. Acknowledgements This work was supported by Research Grants from the Canadian Institutes of Health Research. A.V. Cybulsky holds a scholarship from the Fonds de la Recherche en Santé du Québec. K. Bijian was awarded a fellowship from the McGill University Health Centre Research Institute.

References 1 2 3 4 5 6 7 8 9 10

11

12

Beck FX, Neuhofer W, Muller E: Molecular chaperones in the kidney: Distribution, putative roles, and regulation. Am J Physiol 2000;279:F203–F215. Kerjaschki D: Caught flat-footed: Podocyte damage and the molecular bases of focal glomerulosclerosis. J Clin Invest 2001;108:1583–1587. Mundel P, Shankland SJ: Podocyte biology and response to injury. J Am Soc Nephrol 2002;13:3005–3015. Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 2003;83:253–307. Lee A: Mammalian stress response: Induction of the glucose-regulated protein family. Curr Opin Cell Biol 1992;4:267–273. Pahl HL: Signal transduction from the endoplasmic reticulum to the cell nucleus. Physiol Rev 1999;79:683–701. Kaufman RJ: Stress signaling from the lumen of the endoplasmic reticulum: Coordination of gene transcriptional and translational controls. Genes Dev 1999;13:1211–1233. Kaufman RJ, Scheuner D, Schroder M, Shen X, Lee K, Liu CY, Arnold SM: The unfolded protein response in nutrient sensing and differentiation. Nature Rev Mol Cell Biol 2002;3:411–421. Arrigo AP, Welch WJ: Characterization and purification of the small 28,000-dalton mammalian heat shock protein. J Biol Chem 1987;262:15359–15369. Zhou M, Lambert H, Landry J: Transient activation of a distinct serine protein kinase is responsible for 27-kDa heat shock protein phosphorylation in mitogen-stimulated and heat-shocked cells. J Biol Chem 1993;268:35–43. Huot J, Houle F, Marceau F, Landry J: Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 1997;80:383–392. Jakob U, Gaestel M, Engel K, Buchner J: Small heat shock proteins are molecular chaperones. J Biol Chem 1993;268:1517–1520.


19

13

14

15 16 17

18

19 20 21 22

23

24 25 26 27 28 29 30 31 32

Ciocca DR, Oesterreich S, Chamness GC, McGuire WL, Fuqua SAW: Biological and clinical implications of heat shock protein 27000 (Hsp27): A review. J Natl Cancer Inst 1993;85: 1558–1570. Nagata K, Satoh M, Hosokawa N, Miller AD: Involvement of Hsp47 in the folding and processing of procollagen in the endoplasmic reticulum; in Fink AL, Goto Y (eds): Molecular Chaperones in the Life Cycle of Proteins. New York, Dekker, 1998, pp 225–240. Morgan BP: Effects of the membrane attack complex of complement on nucleated cells. Curr Topics Microbiol Immunol 1992;178:115–140. Nicholson-Weller A, Halperin JA: Membrane signaling by complement C5b-9, the membrane attack complex. Immunol Res 1993;12:244–257. Tischer CG, Couser WG: Milestones in nephrology. [Heymann W, Hackel DB, Harwood S, Wilson SGF, Hunter JLP: Production of nephrotic syndrome in rats by Freund’s adjuvants and rat kidney suspensions (24736). Proc Soc Exp Biol Med 1951;180:660–664.] J Am Soc Nephrol 2000;10: 183–188. Cybulsky AV, Foster MH, Quigg RJ, Salant DJ: Immunologic mechanisms of glomerular disease; in Seldin DW, Giebisch G (eds): The Kidney: Physiology and Pathophysiology, ed 3. Philadelphia, Lippincott-Raven Publishers, 2000, pp 2645–2697. Cybulsky AV, Monge JC, Papillon J, McTavish AJ: Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells. Am J Physiol 1995;269:F739–F749. Panesar M, Papillon J, McTavish AJ, Cybulsky AV: Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells. J Immunol 1997;159:3584–3594. Cybulsky AV, Papillon J, McTavish AJ: Complement activates phospholipases and protein kinases in glomerular epithelial cells. Kidney Int 1998;54:360–372. Liu J, Takano T, Papillon J, Khadir A, Cybulsky AV: Cytosolic phospholipase A2-␣ associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells. Biochem J 2001;353:79–90. Cybulsky AV, Takano T, Papillon J, Khadir A, Liu J, Peng H: Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells. J Biol Chem 2002;277:41342–41351. Quigg RJ, Cybulsky AV, Jacobs JB, Salant DJ: Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells. Kidney Int 1988;34:43–52. Maag RS, Hicks SW, Machamer CE: Death from within: Apoptosis and the secretory pathway. Curr Opin Cell Biol 2003;15:456–461. Grond JG, Weening JJ, Elema JD: Glomerular sclerosis in nephrotic rats. Comparison of the longterm effects of adriamycin and aminonucleoside. Lab Invest 1984;51:277–285. Samstein B, Platt JL: Physiologic and immunologic hurdles to xenotransplantation. J Am Soc Nephrol 2001;12:182–193. Razzaque MS, Taguchi T: Role of glomerular epithelial cell-derived heat shock protein 47 in experimental lipid nephropathy. Kidney Int 1999;71:S256–S259. Liu D, Razzaque MS, Cheng M, Taguchi T: The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats. Histochem J 2001;33:621–628. Smoyer WE, Gupta A, Mundel P, Ballew JD, Welsh MJ: Altered expression of glomerular heat shock protein 27 in experimental nephrotic syndrome. J Clin Invest 1996;97:2697–2704. Smoyer WE, Ransom RF: Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction. FASEB J 2002;16:315–326. Aoudjit L, Stanciu M, Li H, Lemay S, Takano T: p38 mitogen-activated protein kinase protects glomerular epithelial cells from complement-mediated injury. Am J Physiol 2003;285: F765–F774.

Andrey V. Cybulsky, MD Division of Nephrology, Royal Victoria Hospital 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1 Tel. ⫹1 514 398 8148, Fax ⫹1 514 843 2815, E-Mail [email protected].

Bijian/Cybulsky

20


Response of Renal Medullary Cells to Osmotic Stress Wolfgang Neuhofer, Franz-X. Beck Physiologisches Institut der Universität, München, Germany

Abstract In antidiuresis renal medullary cells are exposed to high NaCl and urea concentrations. Long-term adaptation of renal medullary cells to high extracellular NaCl concentrations is accomplished by intracellular accumulation of organic osmolytes. The underlying mechanisms include enhanced uptake from the extracellular space (betaine, myo-inositol and amino acids), increased intracellular production [sorbitol and glycerophosphorylcholine (GPC)] and reduced intracellular degradation (GPC). Apart from osmotically balancing the high extracellular NaCl concentration, betaine and GPC also contribute to protecting medullary cells against the adverse effects of high urea concentrations. A similar function has been demonstrated for HSP70, which is expressed abundantly in the inner medulla. The functional significance of osmolyte accumulation and HSP70 expression for medullary cells is highlighted by observations showing that inappropriately low rates of intracellular osmolyte accumulation or HSP70 expression are associated with an increased incidence of apoptotic cell death. Copyright © 2005 S. Karger AG, Basel

Both the shape and the function of cells are influenced profoundly by hypertonic stress: after acute exposure to hypertonic fluids, cells shrink due to osmotically induced water efflux and the concentration of intracellular solutes, i.e., ions, cytosolic proteins etc., rises. Such changes, which may entail a number of adverse consequences such as DNA damage, growth arrest and inhibition of protein synthesis [1, 2], are most prominent in cells of the renal medulla. As a result of the renal countercurrent system, extracellular NaCl concentrations more than 2-fold higher than those in other organs may be attained in the inner medulla. In addition to high NaCl concentrations, the cells of the renal medulla are also challenged with high urea concentrations. In contrast to NaCl, which by virtue of the Na/K-ATPase resides primarily in the extracellular

compartment, urea penetrates most cell membranes readily. This infers that in antidiuresis the stability of intracellular proteins will be endangered in the inner medulla, in which urea concentrations in the molar range are reached in many mammals. The following is a brief summary of the present knowledge of the pertinent strategies adopted by medullary cells allowing them to survive and fulfill their organ-specific functions in the NaCl- and urea-rich milieu of the renal medulla.

Adaptation to High NaCl Concentrations

Short-Term Adaptation When after prolonged diuresis, i.e., in a situation when interstitial tonicity in the inner medulla is severely reduced, the renal urine concentrating mechanism is activated and interstitial NaCl concentrations rise rapidly, medullary cells shrink [3]. Initially, entry of Na⫹ and Cl⫺, presumably via parallel Na⫹/H⫹- and Cl⫺/HCO3-exchange, is enhanced and, due to osmotically induced water influx, cell volume recovers [4, 5]. Both Na⫹ entry and cell shrinkage cause the intracellular Na⫹ concentration to rise initially and, as a consequence, stimulation of Na⫹/K⫹-exchange via the Na⫹/K⫹-ATPase. The major effect of Na⫹ and Cl⫺ entry eventually is a significant increase of intracellular K⫹ and Cl⫺ concentrations and hence of intracellular ionic strength (i.e., the sum of intracellular Na⫹, Cl⫺ and K⫹ concentrations) [3, 6]. As already briefly mentioned, this has a number of adverse effects on cell function: the activity of enzymes is modulated by changes in ionic strength, both DNA and protein synthesis are reduced by increased ionic strength, DNA damage may occur and, finally, apoptotic cell death ensue [1, 7]. DNA double-strand breaks caused by high NaCl concentrations are associated with a p53-dependent inhibition of cell cycle progression [1, 8–10]. This kind of DNA damage, which may occur also physiologically during DNA replication and transcription, leads to the activation of DNA repair systems. However, after exposure to high NaCl concentrations, DNA repair systems are also impeded, entailing the accumulation of DNA damage. Specifically, the Mre11 exonuclease complex, which normally resides in the nucleus and is part of this DNA repair system, is excluded from the nucleus following NaCl-mediated hypertonic stress [10]. In cells exposed to high NaCl concentrations, adequate repair of DNA damage occurring during DNA replication and transcription is thus hindered and DNA double-strand breaks accumulate. Of interest, high urea concentrations do not cause comparable DNA damage or Mre11 translocation [1, 10]. It is conceivable that the NaCl-induced arrest of cell cycle progression provides the time necessary for restoration of cell volume and accumulation of

Neuhofer/Beck

22

organic osmolytes paralleled by normalization of intracellular ionic strength. This would allow reactivation of DNA repair systems and elimination of DNA damage, thus preventing replication or transcription of damaged DNA and apoptosis [9]. The adverse effects of high intracellular ionic strength on protein synthesis are presumably due to impairment of both translation initiation and chain elongation by high Na⫹ concentrations. Of interest is that high concentrations of both betaine and myo-inositol increase rather than decrease protein synthesis, underscoring their role of metabolically neutral ‘compatible’ osmolytes (see below). Neutral amino acids have no major effect in this context [2]. Long-Term Adaptation The adverse effects provoked by elevated intracellular concentrations of inorganic electrolytes make it plausible that renal medullary cells avoid prolonged periods of high intracellular ionic strength: This is achieved by accumulation of small organic osmoeffectors (‘organic osmolytes’) that are metabolically neutral and, even at high concentrations, do not perturb cell function. These organic osmolytes include the trimethylamines betaine and glycerophosphorylcholine (GPC), the polyols myo-inositol and sorbitol and, to a lesser extent, free amino acids. Accumulation of organic osmolytes takes several hours (to days) and proceeds either by uptake from the extracellular compartment (betaine, myo-inositol and taurine) or by intracellular production (GPC and sorbitol). In antidiuresis the individual organic osmolytes show a characteristic intrarenal distribution (fig. 1a) [6, 11, 12]. GPC content is low in the cortex, intermediate in the outer medulla and highest in the papilla. A similar distribution pattern is observed for sorbitol, which, however, usually is below detection limit in the cortex. The betaine concentration gradient between the corticomedullary boundary and the tip of the papilla is less steep than that of either GPC or sorbitol. In that part of the inner medulla adjacent to the outer medulla, betaine contents may even be lower than in the neighboring outer medulla. Myo-inositol contents are usually comparable in the outer medulla and inner medulla/papilla. GPC. Elevated intracellular concentrations of GPC in response to acute osmotic stress are achieved mainly by reduced degradation and less by enhanced production of GPC. Uptake from the extracellular compartment does not contribute appreciably to the intracellular accumulation of this trimethylamine compound. The production of GPC entails the removal of two fatty acid residues from phosphatidylcholine by phospholipase A1, phospholipase A2 and lysophospholipase [13, 14]. Choline, an important precursor for the synthesis of phosphatidylcholine, is taken up by inner medullary collecting duct cells via a Na⫹-independent transport pathway that is not induced by osmotic stress

Osmoadaptation in the Renal Medulla

23

1,200

6 5 800 4 600

3

0

0

m

GPC

Inositol

Betaine

Amino acids

C

C

a

or te x O m u ed te ul r in la ne rm T ed ota ul l la

1

T pa ip pi of lla

200

I ed nne ul r la Ba s pa e pi of lla

2

x m Ou ed te ul r la

400

HSP70, ng/µg protein

1,000

or te

Organic osmolytes, mmol/kg protein

1,400

b

Sorbitol

Fig. 1. a Intrarenal distribution of organic osmolytes in hydropenic rats (urine osmolality 3,364 mosm/kg H2O) [96]. Amino acids analyzed include: asparate, glutamate, glutamine, glycine, taurine, alanine; GPC ⫽ glycerophosphorylcholine. b Intrarenal distribution of HSP70 in control rats [58].

[15]. The degradation of GPC to choline and glycerol-3-phosphate is accomplished by GPC:choline phosphodiesterase [13, 14]. Following an acute rise in extracellular osmolality by increasing NaCl and/or urea concentrations, GPC in renal epithelial cells accumulates primarily because of reduced activity of GPC:choline phosophodiesterase and less because of an increased phospholipase activity [16, 17]. After prolonged exposure to high NaCl (but not urea) concentrations, the activity of GPC:choline phosphodiesterase, i.e., GPC degradation, recovers and phospholipase A2, i.e., GPC production, is enhanced [16, 18]. Hence, a combination of reduced rates of degradation and enhanced rates of production may contribute to the intracellular accumulation of GPC in medullary cells exposed to high NaCl and urea concentrations during longterm water deprivation. Betaine. The second trimethylamine, betaine, is taken up via the betaine/GABA transporter (BGT1) from the extracellular compartment [19]. Both the human and canine BGT1 genes encode a protein of 614 amino acids with sorting signals and basolateral retention motifs in the cytosolic C-terminal

Neuhofer/Beck

24

domain [19–22]. In Madin-Darby canine kidney cells, BGT1 is located preferentially in the basolateral membrane [23, 24] coupling the uphill transport of betaine to the entry of three Na⫹ and one or two Cl⫺ [20, 25]. In cells derived from the thick ascending limb of the loop of Henle, however, osmotically induced betaine uptake proceeds primarily across the apical membrane [26]. Betaine is produced from choline via betaine aldehyde by the sequential action of choline dehydrogenase and betaine aldehyde dehydrogenase. The oxidation of betaine aldehyde to betaine may be accomplished also by choline dehydrogenase [27, 28]. The proximal tubule, notably the pars recta of the proximal tubule, is the most prominent site of intrarenal betaine synthesis, although betaine production has been demonstrated also in the outer and inner medulla [27, 29, 30]. Betaine accumulated by medullary cells appears to stem mainly from the renal cortex; the contribution of extrarenal sources is minimal [31]. Hence, betaine synthesized by and released from proximal tubule cells may reach the medulla by the tubular and/or vascular route and be taken up by medullary cells. Interestingly, osmotic stress stimulates betaine synthesis substantially in the cortex but not in the inner medulla [27, 31] implying that, under this condition, betaine synthesis in the cortex is well adjusted to the needs of medullary cells for high intracellular betaine concentrations. Upregulation of BGT1 in response to hypertonic stress is due mostly to enhanced transcription of the BGT1 gene leading to increased abundance of BGT1 mRNA in the renal medulla [32–35]. Apart from transcriptional regulation of BGT1 expression, cytoskeletal elements (microfilament network and microtubules) may play a role in the control of betaine uptake by modulating BGT1 insertion into the cell membrane [36, 37]. Sorbitol. Enhanced conversion of glucose to sorbitol by aldose reductase (AR) is the principal process leading to the intracellular accumulation of sorbitol in response to hypertonic stress [38]. In antidiuresis, expression of both AR mRNA and protein increases steeply between the cortico-medullary boundary and the tip of the papilla reflecting the intrarenal distribution of sorbitol [3, 35, 39, 40]. During diuresis, sorbitol, AR mRNA, AR and AR activity decrease sharply in the inner medulla and papilla [6, 11, 35, 39, 41, 42]. In contrast, the enzyme responsible for the conversion of sorbitol to fructose, sorbitol dehydrogenase, is far less subject to osmotic regulation than AR [35, 41, 42]. The ability to produce highly concentrated urine develops only gradually after birth. This development is accompanied by a concomitant rise in AR mRNA abundance and AR immunoreactivity in the renal medulla [40, 43]. During this period, immature papillary thick ascending limbs are transformed into thin ascending limbs by a process involving apoptosis of thick ascending limb cells. Since AR immunoreactivity is observed only in the remaining cells that differentiate into mature ascending thin limb cells, AR may protect these


25

cells from apoptosis and play a role in the differentiation of this nephron segment [40]. Myo-inositol. High intracellular myo-inositol concentrations in response to hypertonic stress are accomplished by uptake from the extracellular space via the sodium/myo-inositol cotransporter (SMIT) [44]. Sequencing of cDNAs encoding the human or canine SMIT predicts a protein of 718 amino acids with 12–13 transmembrane domains [45, 46]. This transporter couples the uptake of one myo-inositol molecule to the entry of two Na⫹ [47]. While in Madin-Darby canine kidney cells Na⫹-dependent myo-inositol uptake is localized primarily in the basolateral cell membrane, cells derived from the medullary thick ascending limb display both basolateral and apical Na⫹/myo-inositol cotransporter activity [24, 26]. SMIT mRNA abundance, which, in antidiuresis is highest in the inner medulla, decreases significantly in both outer and inner medulla after induction of diuresis [35, 48, 49]. Free amino acids. Accumulation of free amino acids, including the sulfonic ␤-amino acid taurine, in response to hypertonic stress is widespread in the animal kingdom. Indeed, uptake of free amino acids by renal epithelial cells via system A and the Na⫹- and Cl⫺-dependent taurine transporter is stimulated after hypertonic stress [50–52]. The finding of rapid activation of amino acid uptake in Madin-Darby canine kidney cells after hypertonic stress in conjunction with a rapid rise in medullary levels of ninhydrin-positive substances (i.e., free amino compounds) led to the assumption that accumulation of free amino acids may be an early adaptive response to hypertonic stress [51, 53]. A role for taurine in the osmotic adaptation of medullary cells is corroborated by the observation of increased taurine transporter mRNA abundance, which is highest in the outer medulla, during antidiuresis in both the outer medulla and papilla of the rat kidney [54]. Compared with trimethylamines or polyols, however, the quantitative contribution of free amino acids to the adaptation of medullary cells to longterm hypertonic stress appears to be minor [55] (fig. 1a). Adaptation of medullary cells to falling extracellular tonicity, i.e., during the transition from antidiuresis to diuresis, is accomplished in the short term by release of organic osmolytes through transmembrane pathway(s), the molecular identity of which remains unknown to date [55]. Eventually, transcription of genes encoding osmolyte-accumulating proteins is downregulated, making allowance for the reduced needs of medullary cells for organic osmolytes.

Adaptation to High Urea Concentrations

As already mentioned, high urea concentrations reduce the activity of various enzymes and even may induce apoptosis [56–58]. Trimethylamine osmolytes

Neuhofer/Beck

26

(e.g., betaine and GPC) are believed to contribute to the amelioration of the deleterious effects of high urea concentrations on protein structure and function [57], and the loss in enzyme activity in the presence of high urea concentrations is attenuated in the presence of betaine or GPC [59]. In the past few years it has become increasingly clear that accumulation of organic osmolytes is only one component of the adaptation of medullary cells to high solute concentrations. The expression of heat shock protein 70 (HSP70) is much higher in the hyperosmotic renal medulla than in the iso-osmotic cortex (fig. 1b). In the renal papilla, HSP70 is expressed at 5 ng/␮g protein, representing 0.5% of total cellular protein [58, 60]. Experiments involving forced overexpression or downregulation of HSP70 have demonstrated that HSP70 protects medullary cells from the detrimental effects of high urea concentrations by counteracting urea-mediated inhibition of enzymes and protection from urea-induced apoptosis [58, 59, 61–63]. The underlying mechanism that prevents urea-induced loss in enzyme activity is most likely due to the chaperoning function of HSP70: urea is a potent protein-unfolding agent that, in the concentrations reached in the renal papilla of many mammals, may lead to destabilization of protein structure and deterioration of protein function [57]. HSP70, by binding reversibly to hydrophobic side chains exposed by unfolded or partially unfolded proteins, promotes re-establishment of the correct conformation, thus preventing incorrect intramolecular interactions, intermolecular aggregation and irreversible loss of function [64]. This protective function of HSP70 is underscored by the observation that targeted disruption of the tonicity-inducible HSP70 gene in mice results in increased apoptosis in the renal medulla upon salt-loading [65]. Other stress proteins that are much more abundant in the inner medulla than in the cortex include HSP27 and ␣B-crystallin which share structural similarities, osmotic stress protein 94 and HSP110 [66–68]. Since these chaperones are induced by ‘tonicity stress’ both in cultured renal medullary cells in vitro [66, 68, 69] and in the renal papilla in vivo after water deprivation [68, 70, 71], it seems likely that they exert significant biological functions in the hyperosmotic renal papilla. However, experimental evidence for a protective role against osmotic stress is available only for ␣B-crystallin, which protects glial cells from acute hypertonic stress [72].

Signaling by Papillary Solutes and Regulation of Tonicity-Responsive Enhancer Binding Protein (TonEBP) Transcriptional Activator

In general, the papillary concentrations of NaCl and urea change in parallel. Each of these solutes activates unique signaling pathways and thereby regulates


27

downstream expression of solute-specific effector gene products [73, 74]. Urea signaling exhibits hallmarks of a peptide mitogen-like signaling pathway in cells of the renal medulla, including transcription and expression of immediateearly genes and activation of the signaling intermediates and receptor tyrosine kinase effectors ERK and Elk-1, Shc, Grb2, SOS, Ras, phospholipase-C␥, PI3K, Akt, and p70 S6 kinase [74, 75]. Hypertonic NaCl induces three mammalian mitogen-activated protein kinase pathways, i.e., the p38-, JNK- and ERK-signaling cascades [73]. Experimental evidence indicates that at least the p38-mitogen-activated protein cascade is essential for the NaCl-induced upregulation of HSP70 and thus for the protective effect observed against high urea concentrations in cells preconditioned with hypertonic NaCl [62, 76, 77]. While the molecular details of p38-mediated upregulation of HSP70 expression are not clear, the role of the tonicity-responsive enhancer binding protein (TonEBP) is far better understood. This rel-like transcription factor binds to tonicity-responsive enhancers located in the 5⬘-region of BGT1, SMIT, AR and HSP70.1 genes [78–81] and stimulates transcription of the respective genes in response to hypertonic stress. TonEBP, however, not only plays a central role in the cellular accumulation of compatible organic osmolytes and HSP70 but also stimulates transcription of the vasopressin-regulated papillary UT-A urea transporters, which are essential for generating high interstitial urea concentrations during antidiuresis [82]. The 5⬘-region of the above-mentioned TonEBP target genes contain multiple tonicity-responsive enhancers to which TonEBP binds, stimulating transcription and thus regulating a functional network of genes essential for the proper function of the renal medulla during antidiuresis (fig. 2). Thus, the high concentration of NaCl (hypertonicity) in the renal medulla plays a central role in the generation of high papillary urea concentrations and in initiating the expression of gene products essential for protecting renal medullary cells against the adverse effects of both high NaCl and urea concentrations. Tonicity is considered the key modulator of TonEBP transcriptional activity. Hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP and, more slowly, an increase in TonEBP abundance, resulting in enhanced transcriptional activity of TonEBP [79]. The C-terminus of TonEBP contains a transactivation domain, the activity of which varies with extracellular NaCl concentration, i.e., increased activity in cells exposed to hypertonic medium and repressed activity in those challenged with hypotonic medium [83]. Tonicity-dependent regulation of the TonEBP-transactivation domain is apparently mediated by phosphorylation by protein kinase A in a cAMP-independent manner [84]. Furthermore, nuclear translocation of TonEBP requires proteasome activity, suggesting the existence of an inhibitory subunit that must be removed to render TonEBP active upon osmotic challenge [85]. The complete signal transduction pathway by which cells sense changes in ambient tonicity are not fully

Neuhofer/Beck

28

Hypertonicity

TonEBP

AR, BGT1, SMIT

UT-A

HSP70

Compatible osmolyte accumulation

Urea accumulation

Protection from hypertonicity

Urea-induced cell death

Fig. 2. Network of genes regulated by TonEBP and their function in the renal medulla. AR ⫽ Aldose reductase; BGT1 ⫽ betaine/GABA transporter 1; SMIT ⫽ sodium/myo-inositol cotransporter; TonEBP ⫽ tonicity-responsive enhancer binding protein; UT-A ⫽ vasopressinregulated urea transporters.

understood so far. However, recent studies have demonstrated that the transcriptional activity of TonEBP may be modulated by changes in cell volume or/and by alterations in intracellular molecular crowding. Under conditions causing increased intracellular ionic strength, stimulation of TonEBP transcriptional activity is hampered by preventing cell shrinkage [86]. Cells may detect such alterations in cell volume by cell-cell and/or cell-matrix interactions or changes in cytoskeletal architecture that occur during cell swelling and shrinkage [87–89].

Implications for Pathophysiology

Recent studies indicate that interference with the adaptation of renal medullary cells to high NaCl or urea concentrations may cause injury to the renal medulla, in particular, during dehydration when the renal concentrating mechanism is stimulated and medullary cells have to adapt to increased solute concentrations. The functional significance of Na⫹-dependent myo-inositol uptake can be deduced from the occurrence of extensive injury of thick ascending limbs associated with acute renal failure after inhibition of this transport pathway [90].


29

In this context, the observation that long-term use of nonsteroidal antiinflammatory drugs is associated occasionally with papillary injury and deterioration of renal function [91] is of interest. Selective cyclooxygenase-2 (COX-2) inhibitors are beneficial with regard to gastrointestinal complications after long-term use [92], but renal side effects have been reported. In particular, in dehydrated subjects, the use of COX-2 inhibitors may lead to deterioration of renal function or even to acute renal failure [93]. In contrast to many other tissues, COX-2 is expressed constitutively in the renal inner medulla and is induced by dehydration [94]. Interestingly, COX-2-derived cyclopentenone prostaglandins induce HSP70 and thus confer resistance against high urea concentrations in inner medullary collecting duct cells. COX-2 inhibition both in vivo and in vitro is associated with reduced HSP70 levels and with death of papillary collecting duct cells [63]. In agreement, COX-2 inhibition also interferes with osmoadaptation of papillary interstitial cells, thereby reducing osmotic tolerance and leading to apoptosis [95].

Acknowledgements Work in the authors’ laboratory was supported by grants from the Deutsche Forschungsgemeinschaft.

References 1 2

3 4 5 6 7 8 9

10

Kültz D, Chakravarty D: Hyperosmolality in the form of elevated NaCl but not urea causes DNA damage in murine kidney cells. Proc Natl Acad Sci USA 2001;98:1999–2004. Brigotti M, Petronini PG, Carnicelli D, Alfieri RR, Bonelli MA, Borghetti AF, Wheeler KP: Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. Biochem J 2003;369:369–374. Sone M, Albrecht GJ, Dörge A, Thurau K, Beck FX: Osmotic adaptation of renal medullary cells during transition from chronic diuresis to antidiuresis. Am J Physiol 1993;264:F722–F729. Sun A, Hebert SC: Rapid hypertonic cell volume regulation in the perfused inner medullary collecting duct. Kidney Int 1989;36:831–842. Fu WJ, Kuwahara M, Cragoe EJ Jr, Marumo F: Mechanisms of regulatory volume increase in collecting duct cells. Jpn J Physiol 1993;43:745–757. Beck FX, Schmolke M, Guder WG, Dörge A, Thurau K: Osmolytes in renal medulla during rapid changes in papillary tonicity. Am J Physiol 1992;262:F849–F856. Petronini PG, Tramacere M, Mazzini A, Kay JE, Borghetti AF: Control of protein synthesis by extracellular Na⫹ in cultured fibroblasts. J Cell Physiol 1989;140:202–211. Dmitrieva N, Michea L, Burg M: p53 protects renal inner medullary cells from hypertonic stress by restricting DNA replication. Am J Physiol 2001;281:F522–F530. Dmitrieva N, Kültz D, Michea L, Ferraris J, Burg M: Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation. J Biol Chem 2000;275: 18243–18247. Dmitrieva NI, Bulavin DV, Burg MB: High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair. Am J Physiol 2003;285:F266–F274.

Neuhofer/Beck

30

11 12 13

14

15 16

17

18

19

20 21

22

23

24 25

26 27 28 29

30 31 32

Yancey PH, Burg MB: Distribution of major organic osmolytes in rabbit kidneys in diuresis and antidiuresis. Am J Physiol 1989;257:F602–F607. Yancey PH: Osmotic effectors in kidneys of xeric and mesic rodents: Corticomedullary distributions and changes with water availability. J Comp Physiol 1988;158:369–380. Bauernschmitt HG, Kinne RKH: Metabolism of the ‘organic osmolyte’ glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. I. Pathways for synthesis and degradation. Biochim Biophys Acta 1993;1148:331–341. Zablocki K, Miller SPF, Garcia-Perez A, Burg MB: Accumulation of glycerophosphocholine (GPC) by renal cells: Osmotic regulation of GPC: choline phosphodiesterase. Proc Natl Acad Sci USA 1991;88:7820–7824. Bevan C, Kinne R: Choline transport in collecting duct cells isolated from the rat renal inner medulla. Pflügers Arch 1990;417:324–328. Kwon ED, Zablocki K, Jung KY, Peters EM, Garcia-Perez A, Burg MB: Osmoregulation of GPC:Choline phosphodiesterase in MDCK cells: Different effects of urea and NaCl. Am J Physiol 1995;269:C35–C41. Bauernschmitt HG, Kinne RKH: Metabolism of the ‘organic osmolyte’ glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. II. Regulation by extracellular osmolality. Biochim Biophys Acta 1993;1150:25–34. Kwon ED, Jung KY, Edsall LC, Kim HY, Garcia-Perez A, Burg MB: Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells. Am J Physiol 1995;268:C402–C412. Yamauchi A, Uchida S, Kwon HM, Preston AS, Robey RB, Garcia-Perez A, Burg MB, Handler JS: Cloning of a Na⫹- and Cl⫺-dependent betaine transporter that is regulated by hypertonicity. J Biol Chem 1992;267:649–652. Rasola A, Galietta LJV, Barone V, Romeo G, Bagnasco S: Molecular cloning and functional characterization of a GABA/betaine transporter from human kidney. FEBS Lett 1995;373:229–233. Perego C, Bulbarelli A, Longhi R, Caimi M, Villa A, Caplan MJ, Pietrini G: Sorting of two polytopic proteins, the ␥-aminobutyric acid and betaine transporters, in polarized epithelial cells. J Biol Chem 1997;272:6584–6592. Perego C, Vanoni C, Villa A, Longhi R, Kaech SM, Fröhli E, Hajnal A, Kim SK, Pietrini G: PDZmediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells. EMBO J 1999;18:2384–2393. Ahn J, Mundigl O, Muth TR, Rudnick G, Caplan MJ: Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons. J Biol Chem 1996;271:6917–6924. Yamauchi A, Kwon HM, Uchida S, Preston AS, Handler JS: Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells. Am J Physiol 1991;261:F197–F202. Matskevitch I, Wagner CA, Stegen C, Bröer S, Noll B, Risler T, Kwon HM, Handler JS, Waldegger S, Busch AE, Lang F: Functional characterization of the betaine/␥-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes. J Biol Chem 1999;274:16709–16716. Grunewald RW, Oppermann M, Schettler V, Fiedler GM, Jehle PM, Schuettert JB: Polarized function of thick ascending limbs of Henle cells in osmoregulation. Kidney Int 2001;60:2290–2298. Grossman EB, Hebert SC: Renal inner medullary choline dehydrogenase activity: Characterization and modulation. Am J Physiol 1989;256:F107–F112. Beck FX, Schmolke M, Guder WG: Osmolytes. Curr Opin Nephrol Hypertens 1992;1:43–52. Wirthensohn G, Guder WG: Studies on renal choline metabolism and phosphatidylcholine synthesis; in Morel F (ed): Biochemistry of Kidney Function. Amsterdam, Elsevier, 1982, pp 119–128. Lohr J, Acara M: Effect of dimethylaminoethanol, an inhibitor of betaine production, on the disposition of choline in the rat kidney. J Pharmacol Exp Ther 1990;252:154–158. Moeckel GW, Lien YH: Distribution of de novo synthesized betaine in rat kidney: Role of renal synthesis on medullary betaine accumulation. Am J Physiol 1997;272:F94–F99. Uchida S, Yamauchi A, Preston AS, Kwon HM, Handler JS: Medium tonicity regulates expression of the Na⫹- and Cl⫺-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene. J Clin Invest 1993;91:1604–1607.


31

33

34 35

36 37 38

39 40 41 42

43 44 45

46

47

48

49 50

51 52 53 54

Kaneko T, Takenaka M, Okabe M, Yoshimura Y, Yamauchi A, Horio M, Kwon HM, Handler JS, Imai E: Osmolarity in renal medulla of transgenic mice regulates transcription via 5⬘-flanking region of canine BGT1 gene. Am J Physiol 1997;272:F610–F616. Moeckel GW, Lai L, Guder WG, Kwon HM, Lien YH: Kinetics and osmoregulation of Na⫹- and Cl⫺-dependent betaine transporter in rat renal medulla. Am J Physiol 1997;272:F100–F106. Burger-Kentischer A, Müller E, Neuhofer W, März J, Thurau K, Beck FX: Expression of aldose reductase, sorbitol dehydrogenase and Na⫹/myo-inositol and Na⫹/Cl⫺/betaine transporter mRNAs in individual cells of the kidney during changes in the diuretic state. Pflügers Arch 1999;437: 248–254. Basham JC, Chabrerie A, Kempson SA: Hypertonic activation of the renal betaine/GABA transporter is microtubule dependent. Kidney Int 2001;59:2182–2191. Bricker JL, Chu S, Kempson SA: Disruption of F-actin stimulates hypertonic activation of the BGT1 transporter in MDCK cells. Am J Physiol 2003;284:F930–F937. Bagnasco S, Uchida S, Balaban RS, Kador PF, Burg MB: Induction of aldose reductase and sorbitol in renal inner medullary cells by elevated extracellular NaCl. Proc Natl Acad Sci USA 1987;84:1718–1720. Cowley BD Jr, Ferraris JD, Carper D, Burg MB: In vivo osmoregulation of aldose reductase mRNA, protein, and sorbitol in renal medulla. Am J Physiol 1990;258:F154–F161. Jung JY, Kim JH, Cha JH, Han KH, Kim MK, Madsen KM, Kim J: Expression of aldose reductase in developing rat kidney. Am J Physiol 2002;283:F481–F491. Sands JM, Schrader DC: Coordinated response of renal medullary enzymes regulating net sorbitol production in diuresis and antidiuresis. J Am Soc Nephrol 1990;1:58–65. Boulanger Y, Legault P, Tejedor A, Vinay P, Theriault Y: Biochemical characterization and osmolytes in papillary collecting ducts from pig and dog kidneys. Can J Physiol Pharmacol 1988;66:1282–1290. Bondy CA, Lightman SL, Lightman SL: Developmental and physiological regulation of aldose reductase mRNA expression in renal medulla. Mol Endocrinol 1989;3:1409–1416. Nakanishi T, Turner RJ, Burg MB: Osmoregulatory changes in myo-inositol transport by renal cells. Proc Natl Acad Sci USA 1989;86:6002–6006. Kwon HM, Yamauchi A, Uchida S, Preston AS, Garcia-Perez A, Burg MB, Handler JS: Cloning of the cDNA for a Na⫹/myo-inositol cotransporter, a hypertonicity stress protein. J Biol Chem 1992;267:6297–6301. Berry GT, Mallee JJ, Kwon HM, Rim JS, Mulla WR, Muenke M, Spinner NB: The human osmoregulatory Na⫹/myo-inositol cotransporter gene (SLC5A3): Molecular cloning and localization to chromosome 21. Genomics 1995;25:507–513. Hager K, Hazama A, Kwon HM, Loo DD, Handler JS, Wright EM: Kinetics and specificity of the renal Na⫹/myo-inositol cotransporter expressed in Xenopus oocytes. J Membr Biol 1995;143: 103–113. Yamauchi A, Nakanishi T, Takamitsu Y, Sugita M, Imai M, Noguchi T, Fujiwara Y, Kamada T, Ueda N: In vivo osmoregulation of Na/myo-inositol cotransporter mRNA in rat kidney medulla. J Am Soc Nephrol 1994;5:62–67. Wiese TJ, Matsushita K, Lowe WL, Stokes JB, Yorek MA: Localization and regulation of renal Na⫹/myo-inositol cotransporter in diabetic rats. Kidney Int 1996;50:1202–1211. Uchida S, Nakanishi T, Kwon HM, Preston AS, Handler JS: Taurine behaves as an osmolyte in Madin-Darby canine kidney cells. Protection by polarized, regulated transport of taurine. J Clin Invest 1991;88:656–662. Chen JG, Coe M, McAteer JA, Kempson SA: Hypertonic activation and recovery of system A amino acid transport in renal MDCK cells. Am J Physiol 1996;270:F419–F424. Kempson SA: Differential activation of system A and betaine/GABA transport in MDCK cell membranes by hypertonic stress. Biochim Biophys Acta 1998;1372:117–123. Law RO: Alterations in renal inner medullary levels of amino nitrogen during acute water diuresis and hypovolaemic oliguria in rats. Pflügers Arch 1991;418:442–446. Bitoun M, Levillain O, Tappaz M: Gene expression of the taurine transporter and taurine biosynthetic enzymes in rat kidney after antidiuresis and salt loading. Pflügers Arch 2001;442: 87–95.

Neuhofer/Beck

32

55 56 57 58 59 60 61

62

63

64 65

66 67

68

69

70

71 72 73 74 75

76

77

Beck FX, Burger-Kentischer A, Müller E: Cellular response to osmotic stress in the renal medulla. Pflügers Arch 1998;436:814–827. Colmont C, Michelet S, Guivarc’h D, Rousselet G: Urea sensitizes mIMCD3 cells to heat shockinduced apoptosis: Protection by NaCl. Am J Physiol 2001;280:C614–C620. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN: Living with water stress: Evolution of osmolyte systems. Science 1982;217:1214–1222. Neuhofer W, Fraek ML, Beck FX: Heat shock protein 72, a chaperone abundant in renal papilla, counteracts urea-mediated inhibition of enzymes. Pflügers Arch 2002;445:67–73. Burg MB, Kwon ED, Peters EM: Glycerophosphocholine and betaine counteract the effect of urea on pyruvate kinase. Kidney Int 1996;50 (suppl 57):S100–S104. Smoyer WE, Ransom R, Harris RC, Welsh MJ, Lutsch G, Benndorf R: Ischemic acute renal failure induces differential expression of small heat shock proteins. J Am Soc Nephrol 2000;11:211–221. Neuhofer W, Lugmayr K, Fraek ML, Beck FX: Regulated overexpression of heat shock protein 72 protects Madin-Darby canine kidney cells from the detrimental effects of high urea concentrations. J Am Soc Nephrol 2001;12:2565–2571. Neuhofer W, Müller E, Burger-Kentischer A, Fraek ML, Thurau K, Beck FX: Inhibition of NaClinduced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations. Pflügers Arch 1999;437:611–616. Neuhofer W, Holzapfel K, Fraek ML, Ouyang N, Lutz J, Beck FX: Chronic COX-2 inhibition reduces medullary HSP70 expression and induces papillary apoptosis in dehydrated rats. Kidney Int 2004;65:431–441. Fink AL: Chaperone-mediated protein folding. Physiol Rev 1999;79:425–449. Shim EH, Kim JI, Bang ES, Heo JS, Lee JS, Kim EY, Lee JE, Park WY, Kim SH, Smithies O, Jang JJ, Jin DI, Seo JS: Targeted disruption of hsp70.1 sensitizes to osmotic stress. EMBO Rep 2002;3:857–861. Santos BC, Chevaile A, Kojima R, Gullans SR: Characterization of the Hsp110/SSE gene family response to hyperosmolality and other stresses. Am J Physiol 1998;274:F1054–F1061. Müller E, Neuhofer W, Ohno A, Rucker S, Thurau K, Beck FX: Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney. Pflügers Arch 1996;431:608–617. Takenaka M, Imai E, Nagasawa Y, Matsuoka Y, Moriyama T, Kaneko T, Hori M, Kawamoto S, Okubo K: Gene expression profiles of the collecting duct in the mouse renal inner medulla. Kidney Int 2000;57:19–24. Neuhofer W, Müller E, Burger-Kentischer A, Fraek ML, Thurau K, Beck FX: Pretreatment with hypertonic NaCl protects MDCK cells against high urea concentrations. Pflügers Arch 1998;435: 407–414. Müller E, Neuhofer W, Burger-Kentischer A, Ohno A, Thurau K, Beck FX: Effects of long-term changes in medullary osmolality on heat shock proteins HSP25, HSP60, HSP72 and HSP73 in the rat kidney. Pflügers Arch 1998;435:705–712. Medina R, Cantley L, Spokes K, Epstein FH: Effect of water diuresis and water restriction on expression of HSPs-27, -60 and -70 in rat kidney. Kidney Int 1996;50:1191–1194. Kegel KB, Iwaki A, Iwaki T, Goldman JE: ␣B-crystallin protects glial cells from hypertonic stress. Am J Physiol 1996;270:C903–C909. Burg MB, Kwon ED, Kültz D: Regulation of gene expression by hypertonicity. Annu Rev Physiol 1997;59:437–455. Tian W, Cohen DM: Signaling and gene regulation by urea in cells of the mammalian kidney medulla. Comp Biochem Physiol 2001;130:429–436. Cohen DM, Gullans SR, Chin WW: Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase. J Clin Invest 1996;97:1884–1889. Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts BA, Rouse D: p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J Biol Chem 1998;273:1832–1837. Zhang Z, Cohen DM: NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells. Am J Physiol 1996;271:F1234–F1238.


33

78

79

80

81 82 83

84

85 86

87 88 89

90

91 92

93 94 95

96

Rim JS, Atta MG, Dahl SC, Berry GT, Handler JS, Kwon HM: Transcription of the sodium/myoinositol cotransporter gene is regulated by multiple tonicity-responsive enhancers spread over 50 kilobase pairs in the 5⬘-flanking region. J Biol Chem 1998;273:20615–20621. Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM: Tonicity-responsive enhancer binding protein, a Rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 1999;96:2538–2542. Ko BCB, Ruepp B, Bohren KM, Gabbay KH, Chung SSM: Identification and characterization of multiple osmotic response sequences in the human aldose reductase gene. J Biol Chem 1997;272: 16431–16437. Woo SK, Lee SD, Na KY, Park WK, Kwon HM: TonEBP/NFAT5 stimulates transcription of HSP70 response to hypertonicity. Mol Cell Biol 2002;22:5753–5760. Nakayama Y, Peng T, Sands JM, Bagnasco S: The TonE/TonEBP pathway mediates tonicityresponsive regulation of UT-A urea transporter expression. J Biol Chem 2001;275:38275–38280. Ferraris JD, Williams CK, Persaud P, Chen Y, Burg MB: Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration. Proc Natl Acad Sci USA 2002;99:739–744. Ferraris JD, Persaud P, Williams CK, Chen Y, Burg MB: cAMP-independent role of PKA in tonicityinduced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. Proc Natl Acad Sci USA 2002;99:16800–16805. Woo SK, Maouyo D, Handler JS, Kwon HM: Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity. Am J Physiol 2000;278:C323–C330. Neuhofer W, Woo SK, Na KY, Grünbein R, Park WK, Beck FX, Kwon HM: Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity. Am J Physiol 2002;283:C1604–C1611. Zhao H, Tian W, Tai C, Cohen DM: Hypertonic induction of COX-2 expression in renal medullary epithelial cells requires transactivation of the EGF receptor. Am J Physiol 2003;285:F281–F288. Zhang Z, Avraham H, Cohen DM: Urea and NaCl differentially regulate FAK and RAFTK/PYK2 in mIMCD3 renal medullary cells. Am J Physiol 1998;275:F447–F451. Sheikh-Hamad D, Yourker K, Truong LD, Nielsen S, Entman ML: Osmotically relevant membrane signaling complex: Association between HB-EGF, ␤1-integrin, and CD9 in mTAL. Am J Physiol 2000;279:C136–C146. Kitamura H, Yamauchi A, Sugiura T, Matsuoka Y, Horio M, Tohyama M, Shimada S, Imai E, Hori M: Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat. Kidney Int 1998;53:146–153. Schlöndorff D: Renal complications of nonsteroidal anti-inflammatory drugs. Kidney Int 1993;44:643–653. Laine L, Connors LG, Reicin A, Hawkey CJ, Burgos-Vargas R, Schnitzer TJ, Yu Q, Bombardier C: Serious lower gastrointestinal clinical events with nonselective NSAID or coxib use. Gastroenterology 2003;124:288–292. Brater DC, Harris C, Redfern JS, Gertz BJ: Renal effects of COX-2-selective inhibitors. Am J Nephrol 2001;21:1–15. Yang T, Schnermann JB, Briggs JP: Regulation of cyclooxygenase-2 expression in renal medulla by tonicity in vivo and in vitro. Am J Physiol 1999;277:F1–F9. Moeckel GW, Zhang L, Fogo AB, Hao C-M, Pozzi A, Breyer MD: COX-2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress. J Biol Chem 2003;278:19352–19357. Sone M, Ohno A, Albrecht GJ, Thurau K, Beck FX: Restoration of urine concentrating ability and accumulation of medullary osmolytes after chronic diuresis. Am J Physiol 1995;269:F480–F490.

Franz-X. Beck, MD Physiologisches Institut der Universität Pettenkoferstrasse 12, DE–80336 München (Germany) Tel. ⫹49 0 89218075534, Fax ⫹49 0 89218075512 E-Mail [email protected]

Neuhofer/Beck

34


Heat Shock Proteins in Renal Cell Carcinomas Derek Atkinsa,b, Rudolf Lichtenfelsa,c, Barbara Seligera,c a

IIIrd Department of Internal Medicine, Johannes Gutenberg University, Mainz, Bayer Health Care AG, PH-R&D-EU Enabling Technologies, Wuppertal, c Institute of Medical Immunology, Martin Luther University, Halle, Germany b

Abstract Renal cell carcinoma (RCC) represents one of the most common cancer types in the Western World. One third of the RCC patients had metastasis at presentation with a poor 5-year survival. Nephrectomy is the most important treatment modality of this disease, since most of the RCCs are resistant to cytotoxic chemotherapy and radiation therapy. Recent immunotherapeutic approaches have been shown to improve the survival rate of RCC patients. Thus, RCC appears to have an immunogenic basis, and therefore represents an attractive target for immunotherapies. So far, only a few RCC-associated antigens have been characterized. However, with the implementation of ‘ome’-based technologies, an increasing number of tumor-associated antigens and tumor markers has been identified that includes various heat shock proteins (HSPs). RCC lesions demonstrate heterogeneous expression patterns for HSPs. In most cases overexpression of certain HSPs, such as HSP27, HSP70 and HSP72, has been detected both in RCC cell lines as well as in the tumor lesions when compared to normal kidney epithelium. Furthermore, HSPs play an important role in apoptotic cell death, in the regulation of cell proliferation and in the augmentation of lysis of RCC by HLA class I-restricted cytotoxic T lymphocytes. In this context, it is noteworthy that wild-type and mutated HSPs have been identified to act as tumor-associated antigens, which consequently resulted in the first clinical phase I and II trials using HSP for vaccination of RCC patients. In this chapter we will briefly present the relevance of HSPs in the pathomechanisms of RCC. Copyright © 2005 S. Karger AG, Basel

Introduction

Renal epithelial tumors are amongst the most common types of cancer in the Western World. In the United States renal tumors were estimated to cause 29,900 new cancer cases in 1998 [1]. It is the tenth leading cause of cancer in

Table 1. Classification of epithelial renal tumors WHO (1981)

UICC/AJCC (1997)

WHO (1998)

Renal cell adenoma (RCA)

RCA, metanephric type RCA, papillary type RCA, oncocytic type

metanephric adenoma tubulo-papillary adenoma oncocytic adenoma

Renal cell carcinoma (RCC)

RCC, clear cell type RCC, papillary type RCC, chromophobic type RCC, collecting duct type RCC, neuroendocrine type RCC, unclassified

clear cell carcinoma papillary carcinoma chromophobic carcinoma collecting duct carcinoma granular cell carcinoma spindle cell carcinoma cyst associated carcinoma

man and the fourteenth in women, respectively, with an increasing incidence [1, 2]. The prognosis is blurred by the high frequency of tumor relapse and/or metastases during the course of disease, and nearly one third of the patients had metastases at presentation [3]. The classification of epithelial renal tumors has been under debate for many years. In 1986, Thoenes et al. [4] established a new pathomorphological classification which was confirmed by cytogenetic and molecular genetic studies [4, 5] resulting in the current classification of renal epithelial tumors as proposed by the UICC and AJCC [6] (table 1). According to this classification, renal epithelial tumors are roughly divided into the benign renal cell adenomas (RCA), malignant tumors, the renal cell carcinomas (RCCs). RCCs are further subclassified into the most common clear cell RCC accounting for more than 70% of all RCCs, the papillary (chromophilic) RCC (15%) and chromophobic RCC (5%), whereas all other RCC subtypes are rare with a frequency of ⬍1%. The benign RCAs are mainly represented by the oncocytic subtype (oncocytoma) which account for approximately 5% of all renal epithelial tumors. During the last decade molecular studies on hereditary renal epithelial neoplasms revealed new insights into the phenotypic and/or genotypic correlation and sequential genetic progression of these tumors [7] leading to a hypothetical genetic model of the evolution of renal epithelial tumors [8] (fig. 1). So far, for nearly every sporadic renal epithelial tumor entity, a hereditary counterpart is known. The prototype of hereditary RCCs is represented by the von-Hippel-Lindau syndrome. Despite the fact that a substantial progress in the understanding of the underlying genetic disorders of renal epithelial tumors has been made, the exact

Atkins/Lichtenfels/Seliger

36

Initiation

Phenotypic association

Promotion

Adenoma

VHl-Mutation ⫺3p (Proximal) tubule system

⫺2p13 c-Met-Mutation

Clear cell type Metanephric

Papillary

⫹7, ⫹17, ⫺Y ? (Distal) tubule system Collecting duct system

t (5;11) (q35;q13) ⫺1, ⫺Y ⫺1, ⫺4, ⫺6, ⫺8, ⫺9, ⫺13, ⫺14, ⫺15, ⫺22

Oncocytic Hybrid/ chromophobic Tubular mucinous tumor ?

Progression ⫺8p, ⫺9p, ⫺11p, ⫹5q, ⫺17p, ⫺14p, ⫹12, ⫹20 t(Xp11.2)

Carcinoma

Clear cell type

⫹7,⫹17

⫺3p21 ⫺9p, ⫺11q, ⫺14q, ⫹20

Papillary type

⫺X ⫺10, ⫺13, ⫺21, ⫺6 ⫺2, ⫺17, ⫺3, ⫺9

Chromophobic type

? ⫺18, ⫺1q, ⫺6p, ⫺8p, Collecting duct type ⫺13q, ⫺21q

Fig. 1. Hypothetical model of the evolution of renal epithelial tumors [8]. Phenotypic–genotypic correlation of renal epithelial tumors provide evidence for an adenoma-carcinoma sequence. According to the current histological classification and cytogenetic data renal cell adenomas (RCAs) of the clear cell type as well as of the papillary subtype develop from the epithelium of the proximal tubule system. In RCAs of the clear cell type this is accompanied by mutations of the von-Hippel-Lindau gene and/or partial/complete loss of chromosome 3, whereas RCAs of the papillary type are characterized by trisomy 7 and/or 17. With the ongoing acquisition of further chromosomal aberrations, RCAs give rise to the corresponding RCCs. In contrast, RCAs of the oncocytic type or oncocytic/chromophobic type and their corresponding malignant counterparts originate in the epithelium of the distal tubule system which are accompanied by the chromosomal aberrations indicated.

molecular mechanisms leading to the initiation as well as to the progression of this disease are still not well defined. So far, no validated molecular markers for the diagnosis, prognosis and/or monitoring of this disease during therapy is available and TNM is still the only indicator of patient’s prognosis which gained widespread acceptance among pathologists and urologists. Nephrectomy is the most important therapeutic modality, whereas RCCs are resistant to cytotoxic chemotherapy and radiation. Well-documented reports on spontaneous, partial or complete remissions, high levels of tumor-infiltrating T lymphocytes and the fact that immunotherapeutic approaches, such as treatment with interleukin-2 and interferon (IFN)-␣ alone or in combination, appear to improve the overall patient survival rate suggests that RCC represents an

Heat Shock Proteins in RCC

37

‘immunogenic’ tumor and is therefore an attractive target for vaccine strategies. However, only a few RCC-associated antigens have yet been characterized. With the advent of ‘ome’-based technologies like genomics, transcriptomics, proteomics and serological screening approaches such as SEREX, the serological analysis of recombinant DNA expression or PROTEOMEX, which is a combination of proteomics and Western blot analysis using sera from RCC patients and healthy donors to detect immunoreactive proteins in tumor and/or control tissue lysates, a relatively large number of tumor-associated antigens (TAA)/tumor markers have been identified in RCCs including different heat shock proteins (HSPs) [9].

Characteristics and Function of HSPs

HSPs are ubiquitously expressed and highly conserved throughout all organisms from bacteria to humans. HSPs were first discovered in 1962 by Ritossa [10] as a set of proteins whose expression was induced by heat shock. In addition, HSPs play an important role in a number of physiological cellular processes as well as in pathophysiological situations involving both systemic and cellular stress, such as cellular homeostasis, apoptosis and tumor immunogenicity [11–13]. Furthermore, HSPs have been described as molecular chaperones for other cellular proteins which regulate protein synthesis, translocation of proteins into various intracellular organelles and protein degradation [14]. HSP Families and Their Main Function During the last decades HSPs have been extensively studied in particular with regard to their cellular localization, regulation and function. HSPs are present in both prokaryotic and eukaryotic cells. Due to their high level of conservation, HSPs appear to play an important role in fundamental cellular processes. According to their molecular weight, HSPs are categorized into six major families: the small HSPs (sHSP) HSP27, HSP40, HSP60, HSP70, HSP90 and HSP100 (table 2). Each family consists of members which are either constitutively expressed, inducible, regulated and/or targeted to different compartments. The main function of HSPs is not solely restricted to cellular stress situations, but also to the chaperoning of native and aberrantly folded proteins. HSPs interact with a number of protein substrates to assist in folding, and therefore play a critical role during cell stress to prevent the appearance of misfolded or otherwise damaged molecules. Consequently, HSPs assist in the recovery from stress by protein refolding or by their degradation which restores protein homeostasis and promotes cell survival. In addition, they are involved


38

Table 2. Characteristics of human heat shock protein families HSP family

Cellular localization

Proposed function

HSP27 (sHSP)

cytosol, nucleus

HSP60

mitochondria

HSP70 family: HSP72 (Hsp70) HSP73 (Hsc70) HSP78/BIP (GRP78)

cytosol, mitochondria, nucleus cytosol, nucleus cytosol, nucleus ER

microfilament stabilization, anti-apoptotic activity, suppression of aggregation and heat native hot refolds proteins and prevents aggregation of denatured proteins, pro-apoptotic anti-apoptotic effects

HSP90

cytosol

GRP94, gp96

ER

HSP110/104

cytosol

protein folding, cytoprotection molecular chaperones cytoprotection, molecular chaperones role in signal transduction, cell proliferation, protein translocation molecular chaperone, antigen processing protein folding

HSP ⫽ Heat shock protein; sHSP ⫽ small HSP; ER ⫽ endoplasmic reticulum.

in the regulation of transcription factors and kinases as well as in signal transduction, antigen recognition and inhibition of apoptosis. The members of the sHSP with molecular masses ranging from 16 to 30 KD include hemeoxygenase, HSP32, HSP27, ␣␤-crystallin and HSP20. sHSPs exhibit tissue-specific expression and represent cytoplasmic chaperones participating in stress resistance, cell growth and differentiation, microfilament organization and assembly of polypeptides. In contrast, the HSP60 family belongs to the mitochondrial matrix chaperones, which are involved in refolding and prevention of aggregation of denatured proteins in vitro and may further facilitate protein degradation by acting as a cofactor in proteolytic systems [15]. The HSP40 family exhibits an essential cochaperone activity for the HSP70 proteins and is thus involved in the folding or assembly of important proteins for the maintenance of protein integrity. The HSP70 family consists of four distinct proteins: HSP72, HSP73, HSP75 and HSP78 and represents the most temperature-sensitive HSP family. Although the precise function of the members of the HSP70 family has not been completely delineated, they are involved in correct folding, in assembly with newly synthesized proteins, in the refolding of denatured or misfolded proteins and in the participation in the


39

disassembly of protein aggregates [16, 17]. The HSP90 molecular chaperone family, including the cytosolic HSP90 and the endoplasmic reticulum (ER) resident glucose-regulated protein 94 (GRP94), is involved in signal transduction, in microfilament organization, different nuclear functions as well as in cytoplasmic trafficking and particle migration [15, 18]. Regulation of HSP Expression HSP expression is highly controlled by the metabolic status of the cell. This includes cell cycle progression, development and differentiation. Besides constitutive expression, HSP synthesis is increased in cells during periods of cell stress due to their exposure to environmental stress, including sudden increase in temperature, oxidative stress, exposure to heavy metals, or due to pathophysiological conditions like ischemia, infection, inflammation, tissue damage and neoplastic transformation. Constitutive and induced HSP expression is mainly regulated by heat shock transcription factors (HSF), which bind to the heat shock element in the promoter regions of all HSP genes [19]. Until now at least four different HSF members have been characterized. Three of them HSF1, 2 and 4 were found in humans [20]. HSF1 is ubiquitously expressed and activated upon heat shock and other physiological stress situations. In contrast, HSF2 is activated during specific stages of the development and by inhibition of the proteasome function, whereas HSF4 is expressed in a tissue-specific manner and displays constitutive DNA-binding capacity. So far, two isoforms of HSF4 have been identified, which are derived by alternative RNA splicing. HSF4a acts as an inhibitor of the constitutive HSP expression, HSF4b as a transcriptional activator [21]. Under physiological conditions HSFs are localized in the cytoplasm. Upon heat shock exposure the HSF1 monomer is directed to the nucleus where it assembles to a homotrimer and binds with high affinity to heat shock elements. In contrast, HSF4 forms trimers in the absence of stress but to acquire transactivating activity, the DNA-binding form of HSF4b has to undergo subsequent modifications [21]. However, its transition from a monomer via dimer to trimer formation is modulated by regulatory elements, including HSP90 and HSP70, both known to suppress HSF activation [22–25]. HSP Expression in Human RCC and Its Clinical Relevance Until now, there exists only limited information about the role of HSPs in normal kidney epithelium and in the chemotherapy-resistant, highly heterogeneic RCC lesions. Most information available is based on in vitro experimental data using RCC lines or murine systems using different methods (table 3).


40

Table 3. HSP expression in RCC analyzed by various methods Method

Number of RCC lesions/ cell lines

IHC

Number of normal kidney lesions/cell lines

Frequency of upregulation (%)

Literature

15

n.d.

31

24

Takashi et al. [29] (1998) Seliger et al. [9] (2003, unpubl.) Lichtenfels et al. [32] (2002) Lichtenfels et al. [32] (2002) Lichtenfels et al. [32] (2002)

Western blot

3

1

n.d.

Proteomics

3

1

n.d.*

PROTEOMEX

3

3

n.d.

n.d. ⫽ Not determined. *modification of HSP27.

The major HSPs are mainly localized in the tubular epithelial cells in the kidney [26]. Normal human kidney tissue exhibits a uniform granular cytoplasmic HSP72/73-specific staining of the visceral glomerula, epithelia of distal convoluted tubules and collecting ducts, but lacks expression in the proximal tubules [27]. Moreover, induction of HSP72 expression was reported in renal tubular cells by stress conditions, such as heat shock, hyper-osmotic stress and exposure to hormones or cytotoxic agents. Recently, Komatsuda et al. [28] demonstrated that HSP72 overexpression plays a direct role in protecting renal tubular cells against oxidative injury and cisplatin toxicity. So far, RCC lesions and few RCC cell cultures have been monitored for the expression of some HSPs using immunohistochemistry and commercially available anti-HSP-specific antibodies. A significant HSP72 overexpression was found in some of the RCC specimens analyzed when compared to corresponding normal kidney cells, although the frequency of HSP72 overexpression was not determined [29]. Moreover, the percentage of HSP72-positive tumor cells significantly correlated with a shorter disease-free survival. The RCC lesions of patients who relapsed within a medium time of 13 months had a significantly lower percentage of HSP72-positive cells than RCC lesions obtained from patients remaining tumor free over a medium period of 72 months. In this context, it is noteworthy that HSP72 expression was increased following IFN-␥ treatment for 48 h. Thus, HSP72 may represent a favorable prognostic factor independent of tumor staging and grading which is upregulated by IFN-␥ [30].


41

In contrast to HSP70, Kohler et al. [31] described low expression of HSP70 in primary RCC cell cultures based on immunohistochemical staining. However, these data were not correlated to HSP70 expression in normal kidney epithelium. Regarding HSP27, its expression in RCC lesions and/or RCC cell lines and autologous normal kidney epithelium was evaluated by different experimental strategies. These include immunohistochemistry, Western blot analysis, proteomics and PROTEOMEX. Immunohistochemical staining with an antiHSP27-specific antibody revealed HSP27 overexpression in 15–30% of RCC lesions compared to normal kidney epithelium [29; Atkins and Seliger, pers. commun.] (fig. 2). Interestingly, there exists also a RCC subtype-specific HSP27 expression pattern: HSP27 overexpression occurs at a higher frequency in clear cell carcinoma than in RCC of the chromophobic subtype (table 3). The constitutive and IFN-␥-inducible expression of various HSPs has been recently investigated in a number of RCC cell lines as well as cell cultures obtained from normal kidney epithelium. As shown in figure 2, a heterogeneous HSP expression pattern was found in both RCC cell lines and cell lines representing normal kidney epithelium, in particular for HSP27, HSP70 and HSP90 family members. The level of expression significantly differed between the RCC cell lines analyzed. However, it is noteworthy that HSP27 expression was significantly downregulated in normal kidney epithelium cells when compared to the RCC cell lines. In addition to the variable constitutive HSP expression pattern, treatment with IFN-␥, known to induce most of the components of the MHC class I antigen processing and presentation pathway, only marginally influenced the respective HSP expression levels. HSP27, GRP75 and GRP78 expression was merely unaltered in the IFN-␥-treated RCC cell lines, whereas HSP60, HSP70 and HSP90 expression was slightly upregulated by this cytokine in some of the RCC cell lines. The differential HSP expression pattern demonstrated by Western blot analysis was further confirmed by proteome-based technologies. Using conventional proteomics consisting of 2D gel electrophoresis followed by mass spectrometry of the differentially expressed protein spots, different HSP proteins were detected. In case of HSP27, some variants of this protein were defined in RCC [32]. Although the type of these modifications have not been determined, phosphorylations at the prominent serine residues sites can be excluded. In addition, HSP serum levels in tumor patients appear to be of prognostic value in some malignancies. Using the PROTEOMEX approach it has been further investigated whether HSPs react with the IgG molecules in serum obtained from RCC patients and healthy donors. As shown in table 4, HSPs induce antibody responses in RCC patients which varied between the different RCC cell lines. Moreover, the IgG responses against various HSP families do not necessarily reflect the HSP expression pattern obtained by Western blot and classical proteome analysis [32].


42

Expression of HSP27 in RCC of clear cell subtype

33%

5%

Strong positive Intermediate Weak Negative

19%

43%

a

b Expression of HSP27 in RCC of chromophobic subtype

0%

Strong positive Intermediate Weak Negative

10%

50% 40%

c

d

e Fig. 2. Expression of HSP27 in normal kidney epithelium and RCCs of different subtypes. Panel a and IHC staining b: In RCCs of the clear cell subtype only 5% and 19% display strong or intermediate positive staining for HSP27, respectively, whereas 43% of the tumors stain only weak positive and 33% are negative for this antigen. IHC staining c: In contrast, normal kidney tissue displays strong positive staining for HSP27 in the epithelium of the collecting duct system, urothelial cells and smooth muscle cells of blood vessels. Panel d and IHC staining e: Only 10% of RCCs of the chromophobic subtype show intermediate positive staining for HSP27, whereas 40% of these tumors reveal weak positive staining, whereas 50% stain negative for this antigen.


43

Table 4. Heat shock protein recognition by PROTEOMEX Atkins/Lichtenfels/Seliger

HSP family member

HSP27 variant A HSP27 variant B HSP27 variant C HSP60 HSP70 HSP75 GRP78 HSP90 GRP94

Immunoreactivity of patient sera (n ⫽ 3) with

Immunoreactivity of control sera (n ⫽ 3) with

RCC line 1

RCC line 2

RCC line 3

NN* line 3

RCC line 1

RCC line 2

RCC line 3

NN* line 3

none

2 out of 3

none

2 out of 3

none

none

2 out of 3

none

none

2 out of 3

none

none

1 out of 3

1 out of 3

1 out of 3

none

none

1 out of 3

none

1 out of 3

2 out of 3

none

2 out of 3

none

all all all 2 out of 3 2 out of 3 none

all 2 out of 3 2 out of 3 1 out of 3 2 out of 3 none

all all 1 out of 3 2 out of 3 2 out of 3 1 out of 3

all 2 out of 3 all none 2 out of 3 none

1 out of 3 1 out of 3 1 out of 3 2 out of 3 none none

all 2 out of 3 1 out of 3 2 out of 3 2 out of 3 none

2 out of 3 all 1 out of 3 none 1 out of 3 none

1 out of 3 2 out of 3 2 out of 3 none 2 out of 3 1 out of 3

*NN ⫽ Cell line derived from normal renal tissue. Differences in the recognition pattern are indicated in bold.

44

Membrane receptor

Mitochondria

Cytochrome C

Apaf-1/ Caspase 9

HSP70 HSP27

Caspase 8

Caspase 3

Pro-caspase 3

Apoptosis

Fig. 3. Inhibition of apoptotic cell death by HSP70 and HSP27. HSPs interact with different members of the mitochondrial apoptotic pathway and are able to modulate pro- as well as anti-apoptotic signaling. HSP27 and HSP70 have been shown to inhibit the cytochrome C-dependent activation of pro-caspase 9 resulting in prevention of apoptotic cell death. Moreover, overexpression of HSP27 has been reported in RCCs of the clear cell as well as chromophobic subtype, and therefore might function as an inhibitor of apoptosis in these RCCs.

The Role of HSP Overexpression in Apoptotic Cell Death of RCC

During the last decades substantial efforts have been made to gain further insights into the biology of RCCs regarding the inhibition of apoptotic tumor cell death. Apoptotic cell death representing a fundamental process during embryonic development, differentiation and tissue homeostasis is mediated either by a direct receptor-mediated or mitochondrial pathway both of which converge at the activation of the caspase cascade (fig. 3). HSPs interact with different members of the mitochondrial apoptotic pathway, and therefore they are able to modulate pro- as well as anti-apoptotic signaling. HSP60 and HSP10 are positive regulators of caspases during apoptosis [11, 33, 34] (table 2). In contrast, HSP27 and HSP70 have been shown to inhibit the cytochrome C-dependent activation of procaspase 9 resulting in prevention of apoptotic cell death [35, 36]. Deregulation or inhibition of apoptosis is present in almost all human cancers [37–39]. Since HSP27 overexpression was reported in RCCs of clear cell as well as chromophobic subtype [9, 29], it thus might function as an inhibitor of apoptosis in these RCC subtypes. However, this topic needs


45

further investigation, because its precise underlying mechanism is as yet not understood. Furthermore, HSP70 has been shown to interact with mutant, but not wildtype p53 protein, a key molecule of the cell cycle regulation [13, 40]. HSP70 stabilizes mutant p53 protein which abrogates normal wild-type p53 function thereby blocking G0/1 cell cycle arrest and apoptosis. Although losses of the whole or the short arm of chromosome 17 have been detected in RCCs of clear cell and chromophobic subtype, the relevance of p53 mutations and/or loss of p53 function in RCCs in context with HSP70 expression has not yet been defined [41]. In contrast to HSP60 and HSP70, HSP90 appears to have a more substratespecific folding activity. Sato et al. [42] demonstrated that Akt, a downstream target of PI3K, binds to HSP90 thereby preserving phosphorylated Akt levels and Akt kinase activity. Activated Akt is known to phosphorylate Bad and caspase 9 leading to their inactivation and subsequent apoptosis inhibition. Moreover, Akt has been shown to activate I␬B kinase by phosphorylation, which results in activation of NF␬B-mediated apoptosis inhibition. In contrast, inhibition of the Akt-HSP90 complex formation resulted in dephosphorylation and Akt inactivation followed by apoptotic cell death of the human embryonic kidney cell line 293T. Since Akt is known to be overexpressed in certain human carcinomas, these data provide evidence that HSPs could be targeted in immunotherapeutic approaches thus counteracting the anti-apoptotic Akt signaling pathway.

HSP-Mediated Modulation of Growth Factor Receptor Signaling: A Negative Mode of Regulation of RCC Cell Proliferation

Growth factors regulate cellular proliferation, differentiation, migration and cell survival by stimulating specific intracellular signaling cascades via receptor tyrosine kinases. Binding to their specific extracellular receptor domains results in receptor tyrosine kinase activation and subsequent autophosphorylation of tyrosine residues located at the cytoplasmic tails, followed by binding and activation of intracellular signal transduction molecules. The ErbB subfamily of receptor tyrosine kinases include the receptors ErbB1 (epidermal growth factor receptor), ErbB2 (Her2/neu), ErbB3 and ErbB4. Overexpression, amplification and/or rearrangements of erbB genes have been implicated in the initiation and progression of human malignancies, including RCCs [43, 44]. A high level of EFG-R and HER-2/neu expression due to amplification and/or overexpression has been found in RCC lesions and cell lines [43; 45; Seliger et al., pers. commun.].


46

Downstream signaling by ErbB receptors involves the Ras/PI3K/Akt as well as the Raf/Mek/ERK pathways [46]. Signaling through the Ras/Raf-1 pathway has an important impact on cell proliferation, differentiation and growth arrest and cell death [47, 48]. HSP90 and HSP70 interact with the Ras/Raf-1 pathway resulting in Raf-1 activation [49, 50]. Prolonged exposure to the HSP90 inhibitor geldanamycin (GA) caused the dissociation of HSP90Raf-1 complexes and subsequent proteasomal degradation of the Raf-1 protein [51] which also modulates the immune response. Moreover, GA downregulates both mature and nascent ErbB2 proteins as well as nascent, but not mature ErbB1/EGF-R proteins [52]. Since HSP90 stabilizes ErbB2 protein due to its binding to the kinase domain, GA stimulates ErbB2 degradation secondary to disruption of the ErbB2-HSP90 complex followed by subsequent ErbB2 ubi-quitination and proteasomal degradation which also modulates immune responses [53]. Furthermore, GA or its analog 17-AAG-induced downregulation of the hypoxia-inducible factor 1-␣, a transcription factor essential for tumor vascularization and metabolic adaptation, suggesting subsequent inhibition of vascular endothelial growth factor expression, thereby implicating that HSP90 antagonists have anti-angiogenic activities.

HSPs and HLA Class I Antigen Processing in RCC

HLA class I molecules are expressed by virtually all human cells and consist of a polymorphic ␣-chain non-covalently bound to ␤2-microglobulin. Endogenously synthesized proteins have access to the complex HLA class I antigen-processing pathway. This is mediated by four major steps: (1) peptide generation by proteasomal degradation of proteins; (2) peptide transport from the cytosol to the ER by the heterodimer transporter associated with antigen processing (TAP); (3) MHC class I assembly which is assisted by various chaperones and (4) transport of the trimeric complex to the cell surface and MHC class I antigen presentation to CD8⫹ cytotoxic T lymphocyte (CTL) [54]. A number of human cancers, including RCCs, exhibit abnormalities in the presentation of endogenously derived antigens due to impaired expression of different components of the HLA class I antigen processing and presentation machinery [55]. Deficiencies in the antigen processing machinery are frequently linked to the aberrant expression of the IFN-␥-inducible proteasome subunits LMP2 and LMP7, of the peptide transporter TAP1 and of tapasin. The expression pattern strongly varies between the different RCC subtypes analyzed, but appears to be independent of tumor staging, grading and metastatic spread [9, 56].


47

It has been postulated that HSPs are involved in the augmentation and priming of HLA class I-restricted CTL [57–59]. Besides targeting ubiquitinated proteins to proteasomal degradation, HSP90 can influence the activity of this multicatalytic enzyme complex [60, 61]. The constitutive subunits of the proteasome X, Y and Z are replaced by IFN-␥-inducible molecules LMP2, LMP7 and LMP10 as well as PA28, thereby forming the so-called immunoproteasome [62]. Recently, two distinct pathways responsible for the generation of MHC class I ligands have been described: one being HSP90 dependent and profoundly inhibited by GA treatment, whereas the other being PA28 dependent. Both pathways redundantly contribute to the generation of some antigenic peptides. Since HSP90 does not enhance the peptide-hydrolyzing activity of the proteasome, but binds to the 20S proteasome, HSP90 might function as a postproteasomal carrier, chaperoning antigenic peptides to the TAP system, thereby preventing further degeneration by cytosolic peptidases [60, 63, 64]. In consequence, HSP90 appears to be linked to the constitutive processing pathway, whereas PA28 may be regarded as an adaptive pathway in antigen processing during an ongoing immune response where IFN-␥ shifts the functional balance from HSP90 towards PA28. Besides HSP90, HSP72 has been implicated in the trafficking and processing of antigenic peptides, thereby enhancing HLA class I surface expression on various cell types including tumor cells [65, 66]. Upregulation of HLA class I surface expression by HSP72 is TAP dependent suggesting that HSP72 may deliver antigenic peptides to TAP heterodimers or may influence the processing of cytosolic proteins into antigenic peptides by enhancing the proteasome activity [67]. However, the potential contribution of HSP loss of function to such an immune escape mechanism in RCCs has not yet been investigated. Therefore, the role and functioning of HSPs in the context of HLA class I antigen processing in RCCs is still to be defined.

HSP Mutants Represent TAAs of RCC

The identification of TAAs and the specificity of the cellular immune responses to cancer has led to the development of T cell-based immunotherapeutic strategies. Tumor-specific CTLs recognize and eliminate cells displaying antigenic peptides in the context of HLA molecules [68–71]. Most of the TAAs identified so far are expressed by a broad range of tumors and in a high percentage of each tumor type. They have been categorized into four major groups: (1) tumor differentiation antigens which represent remnants of the cellular lineage of origin [72–74]; (2) cancer-testis antigens normally expressed


48

in a variety of tumors, but not in normal adult tissues except testis [75–78]; (3) antigens caused by point mutations that are present in individual tumors [79–81] and (4) ubiquitously overexpressed genes [82, 83]. In the case of RCCs, only a small number of different TAA [84], including RAGE-1 [85, 86], PRAME, gp75 [86], MAGE [87], SART3 [88], RU2S [89], MUC1 [90], G250 [91] and HER2-neu [45, 92] have been identified. Interestingly, Gaudin et al. [93] reported a new antigen encoded by a stressinduced mutated form of the hsp70-2 gene found in RCC tumor cells, but not in autologous peripheral blood lymphocytes. The peptide derived from the mutated hsp70-2 is presented by HLA-A2 and can be efficiently recognized by an RCC-specific HLA class I-restricted CTL clone which was generated by clonal expansion of tumor infiltrating lymphocytes. Characterization of the antigenic peptide revealed a decamer with a mutated residue at amino acid position 8. Data from target sensitization and peptide-binding assays revealed half-maximal lysis with 5 ⫻ 10⫺11 M of the mutant decapeptide compared to 5 ⫻ 10⫺8 M for the wild-type decapeptide. The difference in recognition was not due to different binding to HLA-A*0201-presenting molecules. Thus, the data presented by Gaudin et al. [93] demonstrated for the first time that peptides derived from mutated HSPs are directly immunogenic. It is noteworthy that other HSPs have also been shown to elicit efficient CD8⫹ T cell responses and HSP70 has been shown to induce efficient protective antiviral immunity due to the generation of peptide-specific CTLs [94]. In addition, PROTEOMEX analyses performed on the basis of a limited number of patient and control sera revealed immunoreactivity of several HSPs [32]. However, the nature of this immunoreactivity, which could be attributable to distinct peptides associated with the various HSPs recognized or to HSP variants recognized, still remains to be elucidated. Nevertheless, the observed immunoreactivity indicates that HSPs may play a role in the process of tumor recognition and/or manifestation.

Vaccination with HSPs: An Alternative Approach for Immunotherapies

An alternative approach to tumor vaccination originates from early reports by Srivastava and Das [95] as well as Ullrich et al. [96] during the 1980s when the connection of HSPs to tumor immunity was discovered. The observation that structurally unaltered HSPs purified from tumor cells, but not from normal tissues can immunize animals and generate tumor-specific immunity, led to further investigations demonstrating that immunogenicity of tumor-derived HSPs is dependent on the associated peptides complexed with the respective HSP and not against the HSP molecules [97–99].


49

Among the HSPs demonstrated to display prophylactic as well as therapeutic immune responses are HSP70, HSP90, HSP110 as well as the ER-resident chaperones gp96 and calreticulin [57, 95, 96, 100–104]. Using HSPs as cancer vaccines offers several advantages: (1) HSPs bind potentially the whole cellular peptide repertoire of a given tumor cell; (2) there is no need for characterization and purification of a defined TAA; (3) due to binding to a plethora of potentially antigenic peptides, HSPs can induce multifactorial immune responses and (4) this approach delivers an individually/personalized antigenic ‘peptide fingerprint’ of the tumor to be treated thereby theoretically reducing the risk of undesirable side effects [105]. However, effective immunization with HSPs is dependent on the quantity, extent of necrosis and the location of the tumor, since immunizations of mice with the sub- or supra-optimal HSP concentrations either have no effect or suppress rather than induce immune responses [100, 106, 107]. Tumor-derived HSPs, in particular HSP70 and gp96, have been shown to be effective cancer vaccines in multiple preclinical studies including primary and metastatic murine tumor models [97, 108–110]. Since phase I and phase II studies have yielded preliminary evidence of the safety and feasibility as well as immunological and clinical activity of HSP preparations in cancer patients, multiple randomized phase III clinical trials are currently being performed [109, 111–114]. Up to date, two phase II studies have been described using HSP96-peptide complexes from RCC patients for vaccination [115] for 4 weeks beginning 4–6 weeks after nephrectomy. In accordance to the results obtained from studies on murine tumor models, the HSP96-peptide complex vaccine appears to be more effective in the adjuvant setting, but less so in the situation of progressive tumors [108, 115]. Currently there is an ongoing phase III trial for the treatment of stage III RCCs in the adjuvant setting. Conclusions

During the last two decades, substantial progress has been made in the understanding of HSP functioning under physiological as well as pathophysiological conditions, especially in the field of tumor immunity. However, despite the promising preliminary results obtained from preclinical studies and several phase I and II clinical trials, the data reviewed here emphasize the need for further investigations addressing the effects of immunization and the adaptation of the host immune system within the tumor microenvironment as tumor-mediated immune suppression is one of the main obstacles for vaccine therapy. Moreover, the role of HSPs in immunological tolerance, memory and tumor surveillance is still not fully understood.


50

References 1 2 3 4

5 6 7

8 9

10 11

12 13 14 15 16 17 18 19 20

21

22 23

24

Landis SH, Murray T, Bolden S, Wingo PA: Cancer statistics 1998. CA Cancer J Clin 1998;48 6–29. Robert Koch – Institut: Cancer in Germany, ed 2, 1999. Figlin RA: Renal cell carcinoma: Management of advanced disease. J Urol1999;161:381–387. Thoenes W, Störkel S, Rumpelt HJ: Histopathology and classification of renal tumors (adenomas, oncocytomas and carcinomas): The basic cytological and histopathological elements and their use for diagnostics. Pathol Res Pract 1986;181:125–143. Störkel S, van den Berg E: Morphological classification of renal cancer. World J Urol 1995;13:153–158. Störkel S, Eble JN, Adlakha K, Mahul A, Blute ML, Bostwick DG, Darson M, Delahunt B, Iczkowski K: Classification of renal cell carcinoma. Cancer 1997;80:987–991. Zambrano NR, Lubensky IA, Merino MJ, Linehan WM, McClellan MW: Histopathology and molecular genetics of renal tumors: Toward unification of a classification system. J Urol 1999;162:1246–1258. Störkel S: Molecular genetic classification of renal cell carcinoma. Verh Dtsch Ges Pathol 2002;86:28–39. Seliger B, Menig M, Lichtenfels R, Atkins D, Bukur J, Halder TM, Kersten M, Harder A, Ackermann A, Brenner W, Melchior S, Kellner R, Lottspeich F: Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy. Proteomics 2003;3:979–990. Ritossa F: A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962;18:571–573. Samali A, Cai J, Zhivotovsky B, Jones DP, Orrenius S: Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of Jurkat cells. EMBO J 1999;18:2040–2048. Zügel U, Kaufmann SHE: Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin Microbiol Rev 1999;12:19–39. Jolly C, Morimoto RI: Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst 2000;92:1564–1572. Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996;381:571–579. Fink AL: Chaperone-mediated protein folding. Physiol Rev 1999;79:425–449. Liang P, MacRae TH: Molecular chaperones and the cytoskeleton. J Cell Sci 1997;110: 1431–1440. Kregel KC: Heat shock proteins: Modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 2002;92:2177–2186. Caplan AJ: Hsp90’s secrets unfold: New insights from structural and functional studies. Trends Cell Biol 1999;9:262–268. Wu C: Heat shock transcription factors: Structure and regulation. Annu Rev Cell Dev Biol 1995;11:441–469. Morimoto RI: Regulation of the heat shock transcriptional response: Cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998;12: 3788–3796. Tanabe M, Sasai N, Nagata K, Liu XD, Liu PCC, Thiele DJ, Nakai A: The mammalian HSF 4 gene generates both an activator and a repressor of heat shock genes by alternative splicing. JBC 1999;274:27845–27856. Mosser DD, Duchaine J, Massie B: The DNA-binding activity of the human heat shock transcription factor is regulated in vivo by hsp70. Mol Cell Biol 1993;13:5427–5438. Zuo J, Baler R, Dahl G, Voellmy R: Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure. Mol Cell Biol 1994;14:7557–7568. Duina AA, Kalton HM, Gaber RF: Requirement for Hsp90 and a CyP-40-type cyclophilin in negative regulation of the heat shock response. J Biol Chem 1998;273:18974–18978.


51

25 26 27 28

29

30

31

32

33 34

35 36 37 38 39 40

41

42 43

44 45

46

Pirkkala L, Nykänen P, Sistonen L: Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J 2001;15:1118–1131. Wakui H, Komatsuda A, Miura AB: Heat-shock proteins in animal models for acute renal failure. Ren Fail 1995;17:641–649. Venkataseshan VS, Marquet E: Heat shock protein 72/73 in normal and diseased kidneys. Nephron 1996;73:442–449. Komatsuda A, Wakui H, Oyama Y, Imai H, Miura AB, Itoh H, Tashima Y: Overexpression of the human 72 kDa heat shock protein in renal tubular cells confers resistance against oxidative injury and cisplatin toxicity. Nephrol Dial Transplant 1999;14:1385–1390. Takashi M, Katsumo S, Sakata T, Ohshima S, Kato K: Different concentrations of two small stress proteins, ␣B crystallin and HSP27 in human urological tumor tissues. Urol Res 1998;26: 395–399. Santarosa M, Favaro D, Quaia M, Galligioni E: Expression of heat shock protein 72 in renal cell carcinoma: Possible role and prognostic implications in cancer patients. Eur J Cancer 1997;33: 873–877. Kohler G, Veelken H, Rosenthal F, Mackensen A, Lindemann A, Schaefer HE, Lahn M: Oncogen and HSP-70 expression in primary tumor cell cultures of renal cell carcinoma compared to their corresponding cell line. Anticancer Res 1997;17:3225–3231. Lichtenfels R, Kellner R, Bukur J, Beck J, Brenner W, Ackermann A, Seliger B: Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma. Proteomics 2002;2:561–570. Bukau B, Horwich AL: The Hsp70 and Hsp60 chaperone machines. Cell 1998;92:351–366. Xanthoudakis S, Roy S, Rasper D, Hennessey T, Aubin Y, Cassady R, Tawa P, Ruel R, Rosen A, Nicholson D: Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis. EMBO J 1999;18:2049–2056. Saleh A, Srinivasula SM, Balkir L, Robbins PD, Alnemri ES: Negative regulation of the Apaf-1 apoptosome by Hsp70. Nat Cell Biol 2000;2:476–483. Garrido C, Bruey JM, Fromentin A, Hammann A, Arrigo AP, Solary E: HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J 1999;13:2061–2070. Jacobsen MD, Weil M, Raff MC: Programmed cell death in animal development. Cell 1997;88:347–354. Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 1995;267: 1456–1462. Nylandsted J, Brand K, Jaattela M: Heat shock protein 70 is required for the survival of cancer cells. Ann NY Acad Sci 2000;926:122–125. Finlay CA, Hinds PW, Tan TH, Eliyahu D, Oren M, Levine AJ: Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life. Mol Cell Biol 1988;8:531–539. Srigley JR, Hutter RV, Gelb AB, Henson DE, Kenney G, King BF, Raziuddin S, Pisansky TM: Current prognostic factors – Renal cell carcinoma: Workgroup No. 4. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80: 994–996. Sato S, Fujita N, Tsuruo T: Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA 2000;97:10832–10837. Moch H, Sauter G, Gasser TC, Bubendorf L, Richter J, Presti JC Jr, Waldman FM, Mihatsch MJ: EGF-R gene copy number changes in renal cell carcinoma detected by fluorescence in situ hybridization. J Pathol 1998;184:424–429. Moch H, Mihatsch MJ: Genetic progression of renal cell carcinoma. Virchows Arch 2002;441: 320–327. Seliger B, Rongcun Y, Atkins D, Hammers S, Huber C, Storkel S, Kiessling R: HER-2/neu is expressed in human renal cell carcinoma at heterogeneous levels independently of tumor grading and staging and can be recognized by HLA-A2.1-restricted cytotoxic T lymphocytes. Int J Cancer 2000;87:349–359. Semenza GL: HIF-1 and human disease: One highly involved factor. Genes Dev 2000;14: 1983–1991.


52

47 48 49 50 51

52

53

54

55 56 57 58 59

60

61

62 63 64

65 66 67 68 69 70

Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ: Increasing complexity of Ras signaling. Oncogene 1998;17:1395–1413. Kolch W: Meaningful relationships: The regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 2000;351:289–305. Cutforth T, Rubin GM: Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila. Cell 1994;77:1027–1036. Van der Straten A, Rommel C, Dickson B, Hafen E: The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J 1997;16:1961–1969. Schneider C, Sepp-Lorenzino L, Nimmesgern E, Ouerfelli O, Danishefsky S, Rosen N, Hartl FU: Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc Natl Acad Sci USA 1996;93:14536–14541. Xu W, Mimnaugh E, Rosser MFN, Nicchitta C, Marcu M, Yarden Y, Neckers L: Sensitivity of mature ErbB2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90. J Biol Chem 2001;276:3702–3708. Mimnaugh E, Chavany C, Neckers L: Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin. J Biol Chem 1996;271: 22796–22801. Zinkernagel RM, Doherty PC: MHC restricted cytotoxic T cells: Studies on the biological role of polymorphic major transplantation antigens determining T cell restriction specifity, function and reponsiveness. Adv Immunol 1979;27:51–77. Seliger B, Cabrera T, Garrido F, Ferrone S: HLA class I antigen abnormalities and immune escape by malignant cells. Semin Cancer Biol 2002;12:3–13. Atkins D, Ferrone S, Schmahl GE, Störkel S, Seliger B: Downregulation of HLA class I antigen processing molecules – An immune escape mechanism of renal cell carcinomas? J Urol, in press Udono H, Srivastava PK: Heat shock protein70-associated peptides elicit specific cancer immunity. J Exp Med 1993;178:1391–1396. Udono H, Srivastava PK: Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol 1994;152:5398–5403. Tamura Y, Tsuboi N, Sato N, Kikuchi K : 70 kDa heat shock cognate protein is a transformationassociated antigen and a possible target for the host’s anti-tumor immunity. J Immunol 1993;151:5516–5524. Wagner BJ, Margolis JW: Age-dependent association of isolated bovine lens multicatalytic proteinase complex (proteasome) with heat-shock protein 90, an endogenous inhibitor. Arch Biochem Biophys 1995;323:455–462. Connell P, Ballinger CA, Jiang J, Wu Y, Thompson LJ, Hohfeld J, Patterson C: The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat Cell Biol 2001;3:93–96. Kloetzel PM: Antigen processing by the proteasome. Nat Rev Mol Cell Biol 2001;2:179–187. Rechsteiner M, Realini C, Ustrell V: The proteasome activator 11 S REG (PA28) and class I antigen presentation. Biochem J 2000;345:1–15. Binder RJ, Blachere NE, Srivastava PK: Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules. J Biol Chem 2001;276:17163–17171. Srivastava PK, Maki RG: Stress-induced proteins in immune response to cancer. Curr Top Microbiol Immunol 1991;167:109–123. Srivastava PK, Udono H, Blachere NE, Li Z: Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 1994;39:93–98. Wells AD, Rai SK, Salvato MS, Band H, Malkovsky M: Hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int Immunol 1998;10:609–617. Boon T, van der Bruggen P: Human tumour antigens recognized by T lymphocytes. J Exp Med 1996;183:725–729. Rosenberg SA: Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens. J Natl Cancer Inst 1996;88:1635–1644. Lehner PJ, Cresswell P: Processing and delivery of peptides presented by MHC class I molecules. Curr Opin Immunol 1996;8:59–67.


53

71 72

73

74

75

76

77

78

79 80

81

82

83

84

85

86

87 88 89

Groetrupp M, Soza A, Kuckelhorn U, Kloetzel PM: Peptide antigen production by the proteasome: Complexity provides efficiency. Immunol Today 1996;17:429–435. Brichard V, Van Pel A, Wolfel T, Wolfel C, De Plaen E, Lethe B, Coulie P, Boon T: The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 1993;178:489–495. Wölfel T, Schneider J, Meyer Zum Buschenfelde KH, Rammensee HG, Rotzschke O, Falk K: Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1. Int J Cancer 1994;57:413–418. Kawakami Y, Eliyahu S, Delgado CH, Robbins PF, Rivoltini L, Topalian SL, Miki T, Rosenberg SA: Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci USA 1994;91:3515–3519. Van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A, Boon T: A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643–1647. Gaugler B, Van den Eynde B, van der Bruggen P, Romero P, Gaforio JJ, De Plaen E, Lethe B, Brasseur F, Boon T: Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med 1994;179:921–930. Boel P, Wildmann C, Sensi ML, Brasseur R, Renauld JC, Coulie P, Boon T, van der Bruggen P: BAGE: A new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity 1995;2:167–175. Van den Eynde B, Gaugler B, van der Bruggen P, Coulie P, Brichard V, Boon T: Human tumour antigens recognized by T-cells: Perspectives for new cancer vaccines. Biochem Soc Trans 1995;23:681–686. Robbins PF, Kawakami Y: Human tumor antigens recognized by T cells. Curr Opin Immunol 1996;8:628–636. Coulie PG, Lehmann F, Lethe B, Herman J, Lurquin C, Andrawiss M, Boon T: A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA 1995;92:7976–7980. Wölfel T, Hauer M, Schneider J, Serrano M, Wolfel C, Klehmann-Hieb E, De Plaen E, Hankeln T, Meyer zum Buschenfelde KH, Beach DA: p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 1995;269:1281–1284. Theobald M, Ruppert T, Kuckelkorn U, Hernandez J, Haussler A, Ferreira EA, Liewer U, Biggs J, Levine AJ, Huber C, Koszinowski UH, Kloetzel PM, Sherman LA: The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J Exp Med 1998;188:1017–1028. Stanislawski T, Voss RH, Lotz C, Sadovnikova E, Willemsen RA, Kuball J, Ruppert T, Bolhuis RL, Melief CJ, Huber C, Stauss HJ, Theobald M: Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer. Nat Immunol 2001;2:962–970. Brouwenstijn N, Gaugler B, Kruse KM, van der Spek CW, Mulder A, Osanto S, van den Eynde BJ, Schrier PI: Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes. Int J Cancer 1996;68:177–182. Gaugler B, Brouwenstijn N, Vantomme V, Szikora JP, Van der Spek CW, Patard JJ, Boon T, Schrier P, Van den Eynde BJ: A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma. Immunogenetics 1996;44:323–330. Neumann E, Engelsberg A, Decker J, Storkel S, Jaeger E, Huber C, Seliger B: Heterogeneous expression of the tumor-associated antigens RAGE-1, PRAME, and glycoprotein 75 in human renal cell carcinoma: Candidates for T-cell-based immunotherapies? Cancer Res 1998;58: 4090–4095. Yamanaka K, Miyake H, Hara I, Gohji K, Arakawa S, Kamidono S: Expression of MAGE genes in renal cell carcinoma. Int J Mol Med 1998;2:57–60. Kawagoe N, Shintaku I, Yutani S, Etoh H, Matuoka K, Noda S, Itoh K: Expression of the SART3 tumor rejection antigen in renal cell carcinoma. J Urol 2000;164:2090–2095. Van Den Eynde BJ, Gaugler B, Probst-Kepper M, Michaux L, Devuyst O, Lorge F, Weynants P, Boon T: A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription. J Exp Med 1999;190:1793–1800.


54

90 Brossart P, Heinrich KS, Stuhler G, Behnke L, Reichardt VL, Stevanovic S, Muhm A, Rammensee HG, Kanz L, Brugger W: Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies. Blood 1999;93:4309–4317. 91 Grabmaier K, Vissers JL, De Weijert MC, Oosterwijk-Wakka JC, Van Bokhoven A, Brakenhoff RH, Noessner E, Mulders PA, Merkx G, Figdor CG, Adema GJ, Oosterwijk E: Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250. Int J Cancer 2000;85: 865–870. 92 Brossart P, Stuhler G, Flad T, Stevanovic S, Rammensee HG, Kanz L, Brugger W: Her-2/neuderived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes. Cancer Res 1998;58:732–736. 93 Gaudin C, Kremer F, Angevin E, Scott V, Triebel F: A hsp70–2 mutation recognized by CTL on a human renal cell carcinoma. J Immunol 1999;162:1730–1738. 94 Ciupitu AM, Petersson M, O’Donnell CL, Williams K, Jindal S, Kiessling R, Welsh RM: Immunization with alymphocytic choriomeningitis virus peptide mixed with heat shock protein70 results in protective antiviral immunity and specific cytotoxic T lymphocytes. J Exp Med 1998;187:685–691. 95 Srivastava PK, Das MR: The serologically unique cell surface antigen of Zajdela ascitic hepatoma is also its tumor-associated transplantation antigen. Int J Cancer 1984;33:417–422. 96 Ullrich SJ, Robinson EA, Law LW, Willingham M, Appella E: A mouse tumor-specific transplantation antigen is a heat shock-related protein. Proc Natl Acad Sci USA 1986;83:3121–3125. 97 Li Z: Priming T cells by heat shock protein-peptide complexes as the basis of tumor vaccines. Semin Immunol 1997;9:315–322. 98 Li Z: The roles of heat shock proteins in tumor immunity. Cancer Chemother Biol Response Modif 2001;19:371–383. 99 Liu B, DeFilippo AM, Li Z: Overcoming immune tolerance to cancer by heat shock protein vaccines. Mol Cancer Ther 2002;1:1147–1151. 100 Srivastava PK, DeLeo AB, Old LJ: Tumor rejection antigens of chemically induced sarcomas of inbred mice. Proc Natl Acad Sci USA 1986;83:3407–3411. 101 Udono H, Levey Dl, Srivastava PK: Cellular requirements for tumor-specific immunity elicited by heat shock proteins: Tumor rejection antigen gp96 primes CD8⫹ T cells in vivo. Proc Natl Acad Sci USA 1994;91:3077–3081. 102 Basu S, Srivastava PK: Calreticulin, a peptide-binding chaperone of the endoplasmic reticulum, elicits tumour- and peptide-specific immunity. J Exp Med 1999;189:797–802. 103 Nair S, Wearsch PA, Mitchell DA, Wassenberg JJ, Gilboa E, Nicchitta CV: Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides. J Immunol 1999;162:6426–6432. 104 Wang XY, Kazim L, Repasky EA, Subjeck JR: Characterization of heat shock protein 110 and glucose-regulated protein 170 as cancer vaccines and the effect of fever-range hyperthermia on vaccine activity. J Immunol 2001;166:490–497. 105 Srivastava PK, Amato RJ: Heat shock proteins: The ‘Swiss Army knife’ vaccines against cancers and infectious agents. Vaccine 2001;19:2590–2597. 106 Chandawarkar RY, Wagh MS, Srivastava PK: The dual nature of specific immunological activity of tumor-derived gp96 preparations. J Exp Med 1999;189:1437–1442. 107 Srivastava PK: Hypothesis: Controlled necrosis as a tool for immunotherapy of human cancer. Cancer Immun 2003;3:48. 108 Tamura Y, Peng P, Liu K, Daou M, Srivastava PK: Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997;278:117–120. 109 Srivastava PK: Immunotherapy of human cancer: Lessons from mice. Nat Immunol 2000;1: 363–366. 110 Li Z, Menoret A, Srivastava PK: Roles of heat-shock proteins in antigen presentation and crosspriming. Curr Opin Immunol 2002;14:45–51. 111 Janetzki S, Palla D, Rosenhauer V, Lochs H, Lewis JJ, Srivastava PK: Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: A pilot study. Int J Cancer 2000;88:232–238.


55

112 Caudill MM, Li Z: HSPPC-96: A personalised cancer vaccine. Expert Opin Biol Ther 2001;1:539–547. 113 Belli F, Tetori A, Rivotini L, Maio M, Andreola G, Sertoli MR, Gallino G, Piris A, Cattelan A, Lazzari I, Carrabba M, Scita G, Santantonio C, Pilla L, Tragni G, Lombardo C, Arienti F, Marchiano A, Bertolini F, Cova A, Lamaj E, Ascani L, Camerini R, Corsi M, Cascinell N, Lewis JJ, Srivastava PK, Parmiani G: Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes: Clinical and immunologic findings. J Clin Oncol 2002;20:4169–4180. 114 Mazzaferro V, Coppa J, Carrabba MG, Rivoltini L, Schiavo M, Regalia E, Mariani L, Camerini T, Marchiano A, Andreola S, Camerini R, Corsi M, Lewis JJ, Srivastava PK, Parmiani G: Vaccination with autologous tumor-derived heat-shock protein gp96 after liver resection for metastatic colorectal cancer. Clin Cancer Res 2003;9:3235–3245. 115 Amato RJ: Vaccine Therapy for Renal Cell carcinoma. Rev Urol 2003;5:65–71.

Barbara Seliger, PhD Martin Luther University Halle – Wittenberg Institute of Medical Immunology Magdeburger Str. 2, DE–06112 Halle (Germany) Tel. ⫹49 345 557 4054, Fax ⫹49 345 557 4055 E-Mail [email protected]


56


Heat Shock Protein 47 and Renal Fibrogenesis Mohammed S. Razzaquea,b, Viet Thang Leb, Takashi Taguchib a Department of Oral and Developmental Biology, Harvard School of Dental Medicine, Boston, Mass., USA; bDepartment of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Abstract Recent research has greatly increased our knowledge regarding the molecular mechanisms of collagen synthesis and processing. Heat specific shock protein (HSP47) is a collagenspecific molecular chaperone, and helps in post-translational modifications of procollagens, during biosynthesis of collagen. Both in vivo and in vitro studies have convincingly demonstrated that HSP47 is localized in the endoplasmic reticulum of collagen-producing cells, and that its synthesis is closely associated with the rate of procollagen assembly. Recent studies are directed towards the pathological relevance of HSP47 in tissue scarring, a process that is characterized by excessive accumulation of collagens. It appears likely that increased levels of HSP47 in fibrotic diseases assist in increased assembly of procollagen, and thereby help in excessive accumulation of collagens in the fibrotic mass. Such profibrotic effects of HSP47 suggest that modulation of HSP47 expression in scarring diseases might alter the course of fibrotic diseases. In this brief article, we review the role of HSP47 in renal fibrotic diseases and its relevance to other scarring diseases. Copyright © 2005 S. Karger AG, Basel

Introduction

Most living tissues are capable of regenerative repair in response to an injury. In normal wound healing, this regenerative process is self-limiting. However, in tissue scarring, excessive accumulation of matrix proteins, partly due to their uncontrolled metabolism, alters the structural integrity of the involved tissues or organs, and thereby affects their functional activities. The pathogenesis of late-stage tissue scarring appears to be independent of early initiating events or diseases, and a final common pathway for scarring is

believed to exist for tissue scarring for all affected organs, including kidneys, heart, lungs, and liver. Our understanding of the underlying molecular mechanisms of renal fibrosis may therefore reap benefits in fibrotic diseases in general. The multistep, multifactorial chronic scarring diseases usually progress to irreversible end-stage organ failure, resulting in increased disability and eventual death. Extensive research has been performed to elucidate the underlying mechanisms of tissue scarring, and one of the molecules that have been comprehensively studied in the scarring process is collagen. Collagen is an abundant extracellular matrix protein, and is involved in the maintenance of the structural integrity of cells and tissues. In certain pathological states, proliferation of matrix-producing cells with subsequent overproduction, and excessive accumulation of essentially insoluble collagen fibers, lead to the development of tissue scarring. On the other hand, defects in the genes encoding for different types of collagens are associated with various debilitating diseases, including Ehlers-Danlos syndrome, osteogenesis imperfecta, Alport syndrome, epidermolysis bullosa, Schmid’s metaplasia chondrodysplasia (SMCD), and Bethlem myopathy. Furthermore, autoantibodies against different types of collagens have been detected in a number of autoimmune diseases.

Collagen

Collagen is a widely distributed structural protein that is rich in three nonessential amino acids: glycine, proline and hydroxyproline. About one third of the all-mammalian protein is collagen, and it is essential for providing the necessary mechanical support for a variety of hard and soft tissues. So far, more than 40 distinct polypeptide chains, forming more than 25 different types of collagens, have been identified. The typical collagen molecule consists of three collagen polypeptide chains, called ␣-chains, which are wrapped around one another to form a triple-stranded helical structure; every third amino acid within the helix is glycine, while the remaining positions in the chain are filled with proline and hydroxyproline. Thus, the sequence of the ␣-chain can be expressed as (Gly-X-Y)n, where X and Y represent amino acids, proline and hydroxyproline respectively [1]. Hydroxyproline is vital for the stability of the collagen molecules; and vitamin C is required to convert proline to hydroxyproline. A number of complex cotranslational and post-translational modifications are required for collagen biosynthesis. The synthesis of the ␣-polypeptide chains, their hydroxylation, and formation of stable triple-helical procollagen molecules are important intracellular events in collagen synthesis; the intracellular processing requires a number of specific enzymes, including prolyl 4-hydroxylase, prolyl

Razzaque/Le/Taguchi

58

3-hydroxylase, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosyl-hydroxylysyl glucosyltransferase, while the extracellular modifications require procollagen N-proteinase, procollagen C-proteinase and lysyl oxidase [2–7]. In addition to the above-mentioned enzymes, recent studies have documented the essential roles of a number of chaperones and folding proteins during procollagen synthesis, including protein disulphide isomerase, and heat shock protein 47 (HSP47) [8–10]. Protein disulphide isomerase is believed to bind with C-propeptides of procollagen, and forms intra- and inter-chain disulphide bonds, resulting in the formation of a trimer of procollagen at the C-terminus. Upon assembly of C-propetides into a correctly aligned trimer, triplehelix formation proceeds from the C-terminus to the N-terminus [11]. Recently, HSP47, a collagen-binding heat shock protein, has also been found to be involved in the post-translational modifications, and triple-helix formation of procollagen molecules (fig. 1). We will be briefly presenting the pathogenic relevance of HSP47 in human and experimental fibrotic diseases.

HSP47

HSP47 is a 47-kDa glycoprotein protein (two potential N-glycosylation sites), resides in the endoplasmic reticulum of collagen-producing cells, and is involved in the post-translational modifications of procollagen molecule [10]. At the N-terminus, HSP47 has a signal sequence that targets the molecule to the endoplasmic reticulum, while at the C-terminus it has RDEL, the endoplasmic reticulum retention signal [12]. Once the RDEL signal is removed from the C-terminus, the mutant protein is rapidly secreted out of cells [13]. HSP47 has a unique collagen binding ability and specifically binds to various types of collagens [10]. The binding abilities of HSP47 to procollagens have been demonstrated by co-immunoprecipitation studies [14]. Both native HSP47 (from chick embryos) and synthetic HSP47 (produced in E. coli) has been shown to bind various collagens (types I to V), as demonstrated by in vitro pull-down studies by using surface plasmon resonance [15, 16]. Recent in vitro binding analysis of a synthetic peptide model of collagen resulted in the identification of a specific HSP47 binding sequence; binding of HSP47 to typical collagen model peptides (Pro-Pro-Gly)n occurred when n was more than 8 [17]. The probability of HSP47 binding to the triplet repeats was higher with greater numbers of repeats. HSP47 was initially identified by Kurkinen et al. [18], from murine parietal endoderm cells. Thereafter, species-specific collagen-binding proteins were characterized in human and rat as gp46 [19], in the mouse as J6 [20] and in the chick, Zebrafish and rabbit as HSP47 [12, 21, 22]. All these proteins

Role of HSP47 in Renal Fibrogenesis

59

Synthesis of ␣-chains

Hydroxylation Association

Triple helix formation

HSP47

Dissociation Secretion

Propeptide cleavage

Fibril formation

Fig. 1. Simplified schematic diagram showing involvement of HSP47 during biosynthesis of collagen.

were later found to be the same group of molecules with common collagen binding abilities. There is ample evidence for the involvement of HSP47 in the folding, assembly and/or post-translational modification of procollagen [10]. The essential role of HSP47 in collagen biosynthesis has been documented in various in vivo and in vitro studies; HSP47 disruption resulted in embryonic lethality in mice. The HSP47 null mice died at 11.5 days postcoitus, and these mice showed abnormal collagen formation and impaired organogenesis [23]. Having identified the essential role of HSP47 in the synthesis of collagen, recent studies have focused on its role during tissue scarring. A number of experimental studies have found a close association between overexpression of

Razzaque/Le/Taguchi

60

HSP47 and increased deposition of collagens in various human and experimental fibrotic diseases [24–27].

Renal Fibroproliferative Diseases

Most renal diseases, irrespective of glomerular, tubulointerstitial or vascular involvements, develop renal fibrosis in chronic and advanced stages. These diverse groups include, but are not limited to, diabetic nephropathy, lupus nephritis, hypertensive nephropathy, renal scleroderma, IgA nephropathy, sickle cell nephropathy, glomerulonephritis, interstitial nephritis, toxic- and drug-induced nephropathy, and chronic allograft nephropathy. The extent of renal fibrosis is a prognostic indicator of most of the above-mentioned renal diseases. One common feature of renal fibrosis is excessive accumulation of matrix proteins due to uncontrolled synthesis and/or degradation. Most of the renal fibrotic diseases are progressive, and gradual expansion of scarring tissues eventually leads to the destruction of normal renal tissues. The degree of renal fibrosis correlates with a progressive loss of renal function, and eventual end-stage renal disease. Although the exact molecular mechanisms of renal fibroproliferative diseases are not yet clear, increased synthesis and deposition of types I, III IV, and VI collagens have been detected in chronic progressive fibrotic renal diseases including IgA nephropathy, diabetic nephropathy (fig. 2) and hypertensive nephrosclerosis [28–30]. Morphological changes in various stages of renal diseases range from activation and proliferation of intrarenal cells to severe glomerulosclerosis and tubulointerstitial fibrosis. Although numerous studies have convincingly demonstrated that increased synthesis with excessive deposition of collagens are mainly responsible for the initiation and progression of renal fibrotic diseases, our knowledge of intracellular processing of the collagen molecules during fibrotic diseases is still very limited. Recent studies have shown a correlation between the expression of HSP47, a collagen-specific molecular chaperone, and increased deposition of collagens in human and experimental fibrotic renal diseases [31–33].

HSP47 in Experimental Models of Nephritis

Dysregulated activation and proliferation of resident glomerular cells produce in due course increased levels of collagen, and facilitate the development of progressive glomerular scarring, an important cause of irreversible end-stage renal failure [34]. Anti-thymocyte serum-induced nephritis is a widely used experimental model characterized by mesangial cell proliferation


61

a

b

c

d Fig. 2. Expression of type III and type IV collagens in renal tissues collected from a control subject (a, b) and from a patient with diabetic nephropathy (c, d). Type III collagen (a) is mostly present in the interstitium and absent from the glomeruli, while type IV collagen (b) is present in the mesangium, and along the glomerular and tubular basement membranes in control kidney. In contrast to the control, increased accumulation of type III collagen (c) and type IV collagen (d) is detected in the sclerotic glomeruli (arrow) and fibrotic interstitium (open arrow) in diabetic nephropathy.

and sclerotic changes in the glomeruli [35]; increased glomerular expression of HSP47 in this experimental model was associated with excessive accumulation of collagens in the scleroproliferative glomeruli [36]. Furthermore, phenotypically altered collagen producing glomerular myofibroblasts (␣-smooth muscle actin positive), and glomerular epithelial cells (desmin-positive) are the main HSP47-producing cells in the scleroproliferative glomeruli [35–37]. All these HSP47-expressing glomerular cells are collagen-producing cells, as well. Since

Razzaque/Le/Taguchi

62

HSP47 is a molecular chaperone intimately involved in the synthesis of procollagens, it is likely that high levels of glomerular HSP47 might help in enhancing the production rate of collagens, and thus contribute to the glomerular sclerotic process. A similar increase in the expression of HSP47, and excessive accumulation of collagens in the glomeruli was also noted in other experimental models of glomerulosclerosis, including diabetic nephropathy [26].

HSP47 in Experimental Models of Tubulointerstitial Fibrosis

Tubulointerstitial fibrosis is characterized by interstitial accumulation of collagens, produced by phenotypically altered interstitial cells and tubular epithelial cells. Expression of ␣-smooth muscle actin in renal interstitial cells is indicative of acquiring myofibroblastic phenotype [38], while expression of intermediate filament vimentin in tubular epithelial cells is suggestive of phenotypical alteration of renal tubular epithelial cells [39]. Earlier studies demonstrated that increased synthesis of collagens by phenotypically altered interstitial myofibroblasts and tubular epithelial cells plays an important role in the initiation and progression of tubulointerstitial fibrotic process. As expected, in various experimental models of tubulointerstitial fibrosis, including in cisplatin nephropathy, aged F-344 rats and hypertensive nephrosclerosis, increased expression of HSP47 was always detected in collagen-producing interstitial myofibroblasts and tubular epithelial cells (fig. 3) [24, 31–33, 40, 41]. Elevated expression of HSP47 was associated with excessive accumulation of collagens, and mostly detected in and around interstitial fibrotic areas. No such expression of HSP47 was detected in infiltrating monocytes/macrophages in the interstitium.

HSP47 in Human Scarring Renal Diseases

Until now, only a few studies have examined the role and expression of HSP47 in human fibrotic diseases [42]. The first such human study was conducted using renal biopsy tissues of IgA nephropathy, and diabetic nephropathy [42]. In adult human kidneys, a weak expression level of HSP47 was detected in glomerular cells, tubular epithelial cells and interstitial cells. In contrast, enhanced expression of HSP47 was detected in the early sclerotic glomeruli of IgA nephropathy, diabetic nephropathy and crescentic nephritis. HSP47 was also expressed in tubulointerstitial cells in areas around interstitial fibrosis [42]. The glomerular and tubulointerstitial expression of HSP47 in renal biopsy tissues was closely associated with glomerular accumulation of


63

a

b

c

d Fig. 3. Expression of HSP47 in renal tissues collected from an age-matched control rat (a) and from rats with unilateral ureteral obstruction [(UUO); after 21 days] (b). HSP47 is weakly expressed in the glomeruli and in the interstitial cells in control rat kidney and absent from the tubules (a). In contrast to the control, an increased expression of HSP47 is detected in UUO-induced fibrotic rat kidney (b). Double staining shows HSP47 expressing cells (black) are ␣-smooth muscle actin-positive (red) myofibroblasts (c, open arrow), and vimentin-positive (red) tubular epithelial cells (d, arrow) in UUO-induced fibrotic rat kidney.

type IV collagen, and interstitial accumulation of types I and III collagens, respectively. Although further studies are needed, it appears likely at this stage that irrespective of primary diseases, upregulation of HSP47 is a common phenomenon during collagenization of glomeruli and tubulointerstitium.

HSP47 in Non-Renal Scarring Diseases

Similar to the renal fibrotic diseases, HSP47 is also upregulated in various fibrotic diseases involving the lung, liver, heart, eye and skin. Upregulated

Razzaque/Le/Taguchi

64

expression of HSP47 with accumulation of collagen was detected in bleomycininduced pulmonary fibrosis [26]. Consistent with the experimental data, a similar correlation in the expression of HSP47 and excessive accumulation of collagen has been detected in human pulmonary fibrotic diseases [43]. In human dermal fibrosis of patients with keloid [44], and cicatricial pemphigoid [25], increased dermal expression of HSP47 is believed to be partly responsible for enhanced accumulation of interstitial collagens in the scarring tissues. In a separate study, the correlation of HSP47 expression and collagen accumulation was also documented in human conjunctival scarring diseases in patients with ocular cicatricial pemphigoid [45], and Stevens-Johnson syndrome (unpubl data). Similar profibrogenic role of HSP47 has been proposed in the development of fibrotic lesions involving the liver and heart [46, 47]. Regulatory Mechanisms of HSP47 Expression

Several studies have recently documented the possible regulatory roles of known fibrogenic molecules, and the expression of HSP47. For instance, stimulation of mouse osteoblast MC3T3-E1 cells by transforming growth factor-␤1 (TGF-␤1) resulted in a dose-dependent induction of both HSP47 and type I collagen [48]. Similar in vitro induction of HSP47 by TGF-␤1 was also noted in various human cells, including conjunctival fibroblasts, dermal fibroblasts, and aortic smooth muscle cells [25, 45, 49]. Using human embryonic lung fibroblasts, it has been shown that both TGF-␤1 and IL-␤1 could induce trimer formation of heat shock transcription factor 1, which then bound to heat shock element of HSP47, resulting in increased expression of HSP47 [50]. In addition, certain other profibrogenic cytokines, including IL-4, IL-13, and connective tissue growth factor have shown to induce the expression of HSP47 [51–53]. Human conjunctival fibroblasts, treated with various concentrations of IL-4 [51] and IL-13 [52], could induce the expression of both HSP47 and collagens. Recent in vitro studies and in vivo experimental model of diabetic nephropathy, reported that advanced glycation end-products could induce the expression of HSP47 in association with collagens, and thereby could play a role in diabetic nephrosclerosis. [54]. Conclusion

Fibrosis accounts for considerable chronic morbidity in various renal diseases and could be a potential target of therapy. Recent studies have identified a number of important molecules that are involved in the regulation of chronic fibrotic processes, and some of these molecules are potential therapeutic candidates


65

for modulating renal fibroproliferative diseases. However, because of complex molecular interactions, despite identifying crucial molecules involved in fibrogenesis, there are difficulties in developing therapeutic strategies for the treatment of these chronic progressive diseases. Moreover, regardless of the in vitro efficacy of targeting a number of these identified fibrogenic molecules, it is not always easy to predict the in vivo effects of similar targeting, because of the complex in vivo microenvironment. Taking into consideration the altered microenvironment of the affected tissues, the design of therapeutic strategies that include targeting relevant molecules involved in the regulation of excessive accumulation of collagens in the fibrotic mass, may be a more practical and effective approach. Since excessive accumulation of collagens is one of the main pathological events of scarring diseases [28–30, 42, 55–58], therapies should be designed to prevent excessive synthesis and accumulation of such matrix molecules. In view of the fact that HSP47 is involved in the biosynthesis of collagen molecules, selective blockage of this molecule in fibrotic diseases, not only offers an attractive therapeutic strategy, but also provides a functional explanation of why this therapeutic approach might be successful. In fact recent in vivo studies have documented the beneficial effects of interfering with the expression of HSP47 in fibrotic renal diseases [59, 60]. In an experimental model of nephritis, interference with the enhanced expression of HSP47 by the administration of antisense oligodeoxynucleotides resulted in relatively less glomerular accumulation of collagens [59]. Furthermore, improvement of age-related renal scarring was achieved by prolonged caloric restriction, possibly by modulating renal expression of HSP47 and accumulation of collagens [60]. These preliminary studies provide the basis for the development of antifibrotic therapies that control chronic fibroproliferative diseases by targeting HSP47. Needless to say, pharmacological amelioration of renal fibrosis may require stage-specific modulation of multiple factors involved in a certain stage of the fibrotic process; since HSP47 appears to be involved in nearly all stages of the fibrotic process, by facilitating accumulation of collagens, a targeted modulation of its activities might be a useful approach to control the progression of fibrotic diseases. Furthermore, as HSP47 plays a role in renal fibrogenesis, monitoring the expression of HSP47 may help in defining those patients at risk for developing fibrotic complications, and in assessing the response to conventional and selective therapies.

Acknowledgments Our apology goes to the authors whose primary works could not be cited due to limitation of space. Part of this chapter is modified from the review articles published in Histol Histopathol 1999;14:1199–1212, and Nephron 2000; 86:339–341.

Razzaque/Le/Taguchi

66

References 1 2 3 4 5 6 7 8 9 10 11

12

13

14 15

16 17 18 19 20

21

22 23

Myllyharju J, Kivirikko KI: Collagens and collagen-related diseases. Ann Med 2001;33:7–21. Kivirikko KI, Pihlajaniemi T: Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases. Adv Enzymol Relat Areas Mol Biol 1998;72:325–398. Prockop DJ, Fertala A: The collagen fibril: The almost crystalline structure. J Struct Biol 1998;122:111–118. Smith-Mungo LI, Kagan HM: Lysyl oxidase: Properties, regulation and multiple functions in biology. Matrix Biol 1998;16:387–398. Myllyharju J: Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis. Matrix Biol 2003;22:15–24. Kagan HM, Li W: Lysyl oxidase: Properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem 2003;88:660–672. Kivirikko KI, Myllyharju J: Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. Matrix Biol 1998;16:357–368. Lamande SR, Bateman JF: Procollagen folding and assembly: The role of endoplasmic reticulum enzymes and molecular chaperones. Semin Cell Dev Biol 1999;10:455–464. Hendershot LM, Bulleid NJ: Protein-specific chaperones: The role of hsp47 begins to gel. Curr Biol 2000;10:R912–R15. Nagata K: Expression and function of heat shock protein 47: A collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 1998;16:379–386. Bulleid NJ, Dalley JA, Lees JF: The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis. EMBO J 1997;16:6694–6701. Hirayoshi K, Kudo H, Takechi H, Nakai A, Iwamatsu A, Yamada KM, Nagata K: HSP47: A tissuespecific transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts. Mol Cell Biol 1991;11:4036–4044. Satoh M, Hirayoshi K, Yokota S, Hosokawa N, Nagata K: Intracellular interaction of collagenspecific stress protein HSP47 with newly synthesized procollagen. J Cell Biol 1996;133: 469–483. Nakai A, Satoh M, Hirayoshi K, Nagata K: Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J Cell Biol 1992;117:903–914. Natsume T, Koide T, Yokota S, Hirayoshi K, Nagata K: Interactions between collagen-binding stress protein HSP47 and collagen. Analysis of kinetic parameters by surface plasmon resonance biosensor. J Biol Chem 1994;269:31224–31228. Sauk JJ, Smith T, Norris K, Ferreira L: Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen. J Biol Chem 1994;269:3941–3946. Koide T, Asada S, Nagata K: Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation. J Biol Chem 1999;274:34523–34526. Kurkinen M, Taylor A, Garrels JI, Hogan BL: Cell surface-associated proteins which bind native type IV collagen or gelatin. J Biol Chem 1984;259:5915–5922. Clarke EP, Sanwal BD: Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins. Biochim Biophys Acta 1992;1129:246–248. Takechi H, Hirayoshi K, Nakai A, Kudo H, Saga S, Nagata K: Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells. Eur J Biochem 1992;206:323–329. Hart DA, Reno C, Hellio Le Graverand MP, Hoffman L, Kulyk W: Expression of heat shock protein 47(Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy. Biochem Cell Biol 2000;78:511–518. Pearson DS, Kulyk WM, Kelly GM, Krone PH: Cloning and characterization of a cDNA encoding the collagen-binding stress protein hsp47 in zebrafish. DNA Cell Biol 1996;15:263–272. Nagai N, Hosokawa M, Itohara S, Adachi E, Matsushita T, Hosokawa N, Nagata K: Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol 2000;150:1499–1506.


67

24 25

26

27 28 29

30

31 32 33 34 35 36

37 38 39 40

41 42

43 44

45

Razzaque MS, Taguchi T: Localization of HSP47 in cisplatn-treated rat kidney: Possible role in tubulointerstitial damage. Clin Exp Nephrol 1999;3:222–228. Razzaque MS, Ahmed AR: Collagens, collagen-binding heat shock protein 47 and transforming growth factor ␤1 are induced in cicatricial pemphigoid: Possible role(s) in dermal fibrosis. Cytokine 2002;17:311–316. Razzaque MS, Hossain MA, Kohno S, Taguchi T: Bleomycin-induced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat shock protein (HSP) 47. Virchows Arch 1998;432:455–460. Liu D, Razzaque MS, Cheng M, Taguchi T: The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats. Histochem J 2001;33:621–628. Razzaque MS, Koji T, Taguchi T, Harada T, Nakane PK: In situ localization of type III and type IV collagen-expressing cells in human diabetic nephropathy. J Pathol 1994;174:131–138. Kamimura H, Honda K, Nitta K, Horita S, Kobayashi H, Uchida K, Yamaguchi Y, Yumura W, Nihei H: Glomerular expression of alpha2(IV) and alpha5(IV) chains of type IV collagen in patients with IgA nephropathy. Nephron 2002;91:43–50. Razzaque MS, Koji T, Kawano H, Harada T, Nakane PK, Taguchi T: Glomerular expression of type III and type IV collagens in benign nephrosclerosis: Immunohistochemical and in situ hybridization study. Pathol Res Pract 1994;190:493–499. Razzaque MS, Ahsan N, Taguchi T: Heat shock protein 47 in renal scarring. Nephron 2000;86: 339–341. Liu D, Razzaque MS, Nazneen A, Naito T, Taguchi T: Role of heat shock protein 47 on tubulointerstitium in experimental radiation nephropathy. Pathol Int 2002;52:340–347. Cheng M, Razzaque MS, Nazneen A, Taguchi T: Expression of the heat shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 1998;79:125–132. Razzaque MS, Taguchi T: Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc 2002;35:68–80. Razzaque MS, Taguchi T: Collagen-binding heat shock protein (HSP) 47 expression in antithymocyte serum (ATS)-induced glomerulonephritis. J Pathol 1997;183:24–29. Razzaque MS, Taguchi T: The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999;14: 1199–1212. Razzaque MS, Taguchi T: Possible role of glomerular epithelial cell-derived HSP47 in experimental lipid nephropathy. Kidney Int 1999;56:S256–S259. Nagle RB, Kneiser MR, Bulger RE, Benditt EP: Induction of smooth muscle characteristics in renal interstitial fibroblasts during obstructive nephropathy. Lab Invest 1973;29:422–427. Grone HJ, Weber K, Grone E, Helmchen U, Osborn M: Coexpression of keratin and vimentin in damaged and regenerating tubular epithelia of the kidney. Am J Pathol 1987;129:1–8. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T: Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47. Cell Tissue Res 1998;293:471–478. Razzaque MS, Azouz A, Shinagawa T, Taguchi T: Factors regulating the progression of hypertensive nephrosclerosis. Contrib Nephrol 2003;139:173–186. Razzaque MS, Kumatori A, Harada T, Taguchi T: Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 1998;80: 434–443. Razzaque MS, Nazneen A, Taguchi T: Immunolocalization of collagen and collagen-binding heat shock protein 47 in fibrotic lung diseases. Mod Pathol 1998;11:1183–1188. Naitoh M, Hosokawa N, Kubota H, Tanaka T, Shirane H, Sawada M, Nishimura Y, Nagata K: Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid. Biochem Biophys Res Commun 2001;280:1316–1322. Razzaque MS, Foster CS, Ahmed AR: Role of collagen-binding heat shock protein 47 and transforming growth factor-beta1 in conjunctival scarring in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44:1616–1621.

Razzaque/Le/Taguchi

68

46

47

48

49

50

51 52

53

54

55

56

57 58 59

60

Masuda H, Fukumoto M, Hirayoshi K, Nagata K: Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride induced rat liver fibrosis. J Clin Invest 1994;94:2481–2488. Takeda K, Kusachi S, Ohnishi H, Nakahama M, Murakami M, Komatsubara I, Oka T, Doi M, Ninomiya Y, Tsuji T: Greater than normal expression of the collagen-binding stress protein heat-shock protein-47 in the infarct zone in rats after experimentally-induced myocardial infarction. Coron Artery Dis 2000;11:57–68. Yamamura I, Hirata H, Hosokawa N, Nagata K: Transcriptional activation of the mouse HSP47 gene in mouse osteoblast MC3T3-E1 cells by TGF-beta 1. Biochem Biophys Res Commun 1998;244:68–74. Murakami S, Toda Y, Seki T, Munetomo E, Kondo Y, Sakurai T, Furukawa Y, Matsuyama M, Nagate T, Hosokawa N, Nagata K: Heat shock protein (HSP) 47 and collagen are upregulated during neointimal formation in the balloon-injured rat carotid artery. Atherosclerosis. 2001;157: 361–368. Sasaki H, Sato T, Yamauchi N, Okamoto T, Kobayashi D, Iyama S, Kato J, Matsunaga T, Takimoto R, Takayama T, Kogawa K, Watanabe N, Niitsu Y: Induction of heat shock protein 47 synthesis by TGF-beta and IL-1 beta via enhancement of the heat shock element binding activity of heat shock transcription factor 1. J Immunol 2002;168:5178–5183. Razzaque MS, Ahmed BS, Foster CS, Ahmed AR: Effects of IL-4 on conjunctival fibroblasts: Possible role in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44:3417–3423. Ahmed AR, Razzaque MS, Daoud YJ, Rashid KA, Foster CS: Effects of IL–13 on fibroblasts isolated from conjunctivae of control and cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2004;45:E-Abstract 1490. Wang JF, Olson ME, Ma L, Brigstock DR, Hart DA: Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts. Wound Repair Regen 2004;12:205–216. Ohashi S, Abe H, Takahashi T, Yamamoto Y, Takeuchi M, Arai H, Nagata K, Kita T, Okamoto H, Yamamoto H, Doi T: Advanced glycation end products increase collagen-specific chaperone protein in mouse diabetic nephropathy. J Biol Chem 2004;279:19816–19823. Razzaque MS, Koji T, Harada T, Taguchi T: Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections. Histochem Cell Biol 1999;111:1–6. Razzaque MS, Taguchi T: Expression of type III collagen mRNA in renal biopsy specimens of patients with idiopathic membranous glomerulonephritis. J Clin Pathol (Mol Pathol) 1996:49: M40–M42. Razzaque MS, Taguchi T: Pulmonary fibrosis: Cellular and molecular events. Pathol Int 2003;53:133–145. Azouz A, Razzaque MS, El-Hallak M, Taguchi T: Immunoinflammatory responses and fibrogenesis. Med Electron Microsc 2004;37:141–148. Sunamoto M, Kuze K, Tsuji H, Ohishi N, Yagi K, Nagata K, Kita T, Doi T: Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis. Lab Invest 1998;78:967–972. Razzaque MS, Shimokawa I, Nazneen A, Liu D, Naito T, Higami Y, Taguchi T: Life-long dietary restriction modulates the expression of collagens and collagen-binding heat shock protein 47 in aged Fischer 344 rat kidney. Histochem J 1999;31:123–132.

Mohammed S. Razzaque, MD, PhD Department of Oral and Developmental Biology, Harvard School of Dental Medicine 188 Longwood Avenue, Boston, MA 02115 (USA) Tel. ⫹1 617 432 5768, Fax ⫹1 617 432 5767, E-Mail [email protected]


69


Cytoprotective Effects of Heme Oxygenase in Acute Renal Failure Reiko Akagia, Toru Takahashib, Shigeru Sassac a

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja-shi, Okayama-ken, Japan and bDepartment of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama City, Japan; cLaboratory of Biochemical Hematology, The Rockefeller University, New York, N.Y., USA

Abstract Following ischemia, superoxide is produced during the reperfusion phase in various organs. In renal pathophysiology, excess of free heme, which is released from hemeproteins under these conditions, catalyzes the formation of further reactive oxygen species to accelerate cellular injuries. There is accumulating evidence suggesting that transcriptional activation of heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation as well as the 32-kDa heat shock protein, participates in the defense against oxidative tissue injuries. Ischemia followed by reperfusion of the rat kidney accompanies significant induction of HO-1 mRNA, protein and enzyme activity, which is in part mediated through a rapid and transient increase in microsomal heme concentration. Inhibition of HO activity by tin mesoporphyrin results in a sustained and enhanced increase in microsomal heme content, and significantly exacerbates renal function. In contrast, SnCl2 treatment, which specifically induces HO-1 mRNA and protein in the proximal tubular epithelial cells, prevents the ischemia-reperfusionmediated increase in microsomal heme concentration, and ameliorates the ischemic renal injury. In addition to these findings, recent evidence on the role of HO-1 in the kidney pathophysiology is summarized, with a particular emphasis on its protective role in the ischemic acute renal failure. Copyright © 2005 S. Karger AG, Basel

Introduction

Heme is widely present in cells, playing the essential role as the prosthetic group of hemeproteins such as hemoglobin, myoglobin, and cytochromes, and other enzymes involved in cellular oxidative metabolism [1]. The linkage of

Ischemia/reperfusion heat shock, inflammation, UV heavy metals, NO, heme

Oxidative stress

ROS

Lipid peroxidation Protein modification ER

Hemeproteins (CYP) V

M

M

V N

N

HO

Fe2+ N

Heme

N

H N

P M

V M

O

N

M P

H N

H N

O

P

V

M NADPH NADP CO O2 Iron

M

M

P

Biliverdin IX␣ NADPH/NADH BVR NADP/NAD

Ferritin

M; -CH3 P; -CH2-CH2-COOH V; -CH2=CH2

O

H N

H N

V M M P Bilirubin IX␣

H N

H N

P M

V M

O

Cytosol

Fig. 1. Oxidative catabolism of heme induced by oxidative stress. In the series of heme catabolism reactions catalyzed by NADPH-cytochrome P450 reductase, heme oxygenase (HO) and biliverdin reductase (BVR), all electrons (at least seven) are provided by NADPH-cytochrome P450 reductase. For BVR, an electron provided by NADH-b5 reductase may additionally serve as a reducing equivalent. Heme released from hemeproteins such as cytochrome P450 (CYP) is cleaved by HO bound to the endoplasmic reticulum (ER), and yields an equimolar amount of iron, CO and biliverdin IX␣. Biliverdin IX␣ is then reduced to bilirubin IX␣, by BVR, which is present in the cytosol.

heme with an appropriate protein moiety is not only essential for the functional activity of hemeproteins, but also restrains free heme from exerting its toxic effects. In injured cells, destabilization of hemeproteins is found because of the generation of reactive oxygen species (ROS). Heme is also a lipophilic prooxidant that can further augment ROS generation, and damage lipid bilayers and organelles as well as a number of enzyme proteins [2–5] (fig. 1). When heme was inadvertently administered in inordinate amounts to patients with

Role of HO-1 in Acute Renal Failure

71

acute intermittent porphyria for treatment of acute relapse [4], it was found to result in marked nephrotoxicity. In injured cells, heme oxygenase (HO) is induced, and degrades free heme released from intracellular hemeproteins, safeguarding against oxidative cytotoxity due to free heme. The degradation of heme [6] is catalyzed by a sequence of three enzymatic reactions, i.e., NADPH-cytochrome P450 reductase, HO and biliverdin reductase (BVR), and HO is the rate-limiting enzyme in this sequence. HO converts heme to an equimolar amount of biliverdin (BV) IX␣, carbon monoxide (CO), and iron [7, 8] (fig. 1). Among the three HO isoforms known, HO-1 and -2 have heme-cleaving activity, while HO-3 is a poor heme catalyst. HO-1, which is also known as the heat shock protein 32 [9, 10], is highly inducible by a number of stimuli, including oxidative stress [7, 11]. In contrast, HO-2 and -3 are both expressed in constitutive fashion, and appear to function as heme-binding molecules in normal cells [12, 13]. Metabolites of heme produced by HO-1 or -2 were thought to be useless waste or toxic products, but recent data suggest that they may have significant biological properties, such as anti-oxidant, antiinflammatory, or anti-apoptotic activities. They are also implicated in signal transduction, immune modulation and adhesion molecule expression [14–18]. The strong adaptive response of HO-1 [19] to various stimuli also suggests that HO-1 may play a significant role in the protection of inflammatory processes and oxidative tissue injuries, and HO-1 is now recognized as a new therapeutic target for a wide variety of oxidative tissue injuries. HO-1 has recently been identified as a graft survival gene. For example, hearts transplanted into HO-1 transgenic mice showed decreased lymphocyte infiltration and diminished immune activation, resulted in a significant blunting of host immune activation [20]. HO or its products as a protective factor against transplant rejection have been reported in heart, lung [21], kidney [22], liver [23] and pancreatic ␤ cells [24]. In this chapter, recent findings on the role of HO-1 in renal pathophysiology are reviewed, and discussed with particular emphasis on its relevance to the ischemic acute renal failure (IARF).

HO-1 in Oxidative Tissue Injuries

Cellular protective responses to oxidative stress are essential to cell survival since they must rely on the use of molecular oxygen for various enzymatic reactions. It has been shown that HO-1 is the major 32-kDa stress protein induced in skin fibroblasts by UVA radiation, hydrogen peroxide and sodium arsenite [9]. It was proposed that the induction of HO-1 is a general response to oxidative stress. In addition to oxidants, the induction of the ho-1 gene also occurs following cellular exposure to agents, such as heme [25], proinflammatory

Akagi/Takahashi/Sassa

72

cytokines [26], bacterial lipopolysaccharide (LPS) [27, 28], growth factors [29, 30], nitric oxide (NO) [31] and tumor promoters [32, 33]. These agents share the ability to directly or indirectly generate intracellular ROS and/or moderate intracellular redox equilibrium. Thus, HO-1 activation appears to be a general indicator of oxidative stress in various tissues including kidney (fig. 1). Following ischemia, superoxide is produced during the reperfusion phase, and it rapidly reacts with NO and forms peroxynitrite. It has been demonstrated in various organs, including kidney, that NO production increases through NO synthase (NOS) activation during a reperfusion phase. Under these conditions, prevention of peroxynitrite generation by inhibition of NO biosynthesis markedly reduces reperfusion injury [34]. Since NOS is a hemeprotein, regulatory interactions between NOS and HO-1 mediated by heme is of interest. It was reported that induction of HO-1 resulted in NOS inhibition due to the degradation of the essential prosthetic group for NOS [35, 36], while both exogenously or inducible NOS-derived NO enhances HO-1 expression in mesangial cells [37]. Thus, HO-1 activation may also defend against NOmediated toxicity in oxidative tissue injury.

HO-1 Deficiency Causes Renal Failure

The physiological importance of HO-1 was unequivocally provided by studies of HO-1-deficient mice [38, 39]. The adult HO-1-deficient mice developed anemia associated with low serum iron levels but increased serum ferritin levels. Non-heme iron accumulated in both renal proximal cortical tubules and in Kupffer cells and hepatocytes. Such iron deposits contributed to oxidative damage by ROS production through Fenton reaction, resulting in chronic inflammation and tissue injury. In fact, the extent of lipid peroxidation and protein carbonyls in the kidney of HO-1-deficient mice was significantly higher than that in control mice [38], and renal artery clipping led to increased ischemic damages in the kidney in uninephrectomized HO-1-deficient mice [40]. Moreover, adult HO-1-deficient mice were more vulnerable to endotoxin challenge than control mice [39]. Thus, the induction of HO-1 represents a defense mechanism to protect cells from oxidative damage. In contrast to HO-1-deficient mice, HO-2-deficient mice showed a milder phenotype, and they were fertile and survived normally for at least one year [39]. There were no noticeable organ injuries in HO-2-deficient mice, except for increased susceptibility to hyperoxic lung damage [41]. The milder phenotype of HO-2-deficient mice suggests that HO-2 plays a lesser role than HO-1 in the overall rate of heme breakdown, and in the protective function against oxidative stress.


73

In 1999, a case of human HO-1 deficiency was reported [42]. The patient was a 26-month-old Japanese boy, who suffered from recurrent fever and severe growth retardation. Low serum bilirubin (BR) levels associated with persistent hemolytic anemia suggested a defect in heme catabolism. Nucleotide sequence analysis of the patient’s ho-1 gene revealed a deletion of exon 2 in the maternal allele and a two-base deletion within exon 3 in the paternal allele. Both mutant alleles encoded truncated HO-1 proteins because of the frame shift, resulting in no functional HO-1 protein in the patient. Blood samples showed hyperlipidemia, increased haptoglobin levels, and extremely high concentration of heme (490 ␮M), associated with undetectable levels of hemopexin. The patient suffered from persistent hemolytic anemia characterized by marked erythrocyte fragmentation. Electron microscopy of renal glomeruli revealed detachment of endothelium with subendothelial deposition of an unidentified material. Iron deposition was noted in renal and hepatic tissue. The patient had persistent proteinuria and hematuria due to renal tubular injury [43]. Most of his symptoms were similar to those observed in HO-1-deficient mice [38, 39]. The unique feature of the patient was that he was associated with asplenism, which may have, in part, accounted for the fact that he was able to live up to 6 years of age. Marked hepatomegaly was present which was presumably involved in compensatory heme catabolism, in the absence of the spleen. These findings suggest that the HO-1 plays an essential role in the host defense against oxidative stress and also in iron metabolism, which includes the kidney.

HO-1 Induction in the Experimental Model of Renal Failure

In intensive care units, ARF is still a major problem with respect to its high mortality and morbidity [44, 45]. IARF is the major form of ARF, and accompanies acute tubular epithelial cell injury [46]. The IARF injury is thought to be due to ROS generated by reperfusion, producing peroxynitrite through reaction with NO. Moreover, ROS is thought to occur as a result of a rapid release of heme from microsomal cytochrome P450 [47]. As the experimental model of IARF, rats with the ligation of a contralateral renal artery following unilateral nephrectomy, or rats with bilateral ligation followed by reperfusion, have been used. The renal function in IARF critically depends on the length of the ischemic treatment, i.e., partial restoration of renal function takes place if the hypoxic challenge is less than 60 min, and an irreversible renal damage occurs if ischemia is longer than 60 min [48]. There were discordant findings between two laboratories, however, on the expression of HO-1 mRNA in different types of reversible IARF models, while both groups reported similar induction of heat shock protein 70 [49, 50]. Namely, in one


74

study, both ho-1 gene expression and its enzyme activity were significantly increased in the reversible IARF model [49, 51], while in another study, HO-1 was not increased [50]. In the former study, there was a rapid and significant increase in microsomal heme concentration in the ischemic kidney prior to HO-1 induction [49, 51] (fig. 2), suggesting that an increased intracellular heme concentration may have contributed to the induction of HO-1 mRNA [25]. In both types of IARF models, there were concomitant increases of microsomal heme concentration and HO-1 mRNA levels in the kidney of rats after reperfusion [49, 51] (fig. 2), suggesting that heme-mediated induction of ho-1 also occurs in these models of IARF. The involvement of heme in the induction of HO-1 mRNA was also reported in the rat models of glycerol-induced ARF [52] and the cisplatin-induced toxic renal injury [53]. Various animal models of ARF and treatment used to influence HO activity are summarized in table 1. It should be noted that HO-1 induction was observed in all ARF models.

HO Inhibition Worsens,While Its Induction Alleviates IARF

Certain metalloporphyrins have been shown to act as competitive inhibitors of HO activity [54]. For example, Sn-protoporphyrin (Sn-PP), or Sn-mesoporphyrin (Sn-MP), has been shown to be a clinically useful inhibitor of HO activity. Sn-MP is a stronger inhibitor of HO activity than Sn-PP, presumably due to its greater water solubility [55], and is used for the treatment of neonatal jaundice [56]. Inhibition of HO activity by Sn-MP [57] was found to result both in a marked increase in intracellular heme content, and in the aggravation of renal function as assessed by serum creatinine concentration (fig. 2). HO-1 induction thus plays an important role via eliminating free heme in the protection of renal dysfunction [51]. In contrast to Sn-PP or Sn-MP, tin chloride (SnCl2) treatment has been shown to potently induce HO-1 exclusively in the renal tubular epithelial cells in the kidney [58, 59]. The effect of SnCl2 treatment prior to ischemia/reperfusion was found to restore renal dysfunction, as shown by a marked decrease in serum creatinine concentration in the IARF animals [60] (fig. 2). While there were significant damages in proximal tubular cells in control animals with IARF, there were hardly any morphologically affected cells in SnCl2-pretreated animals [60]. Following SnCl2 treatment, a marked elevation of renal HO-1 mRNA was also observed, followed by increases in HO-1 protein expression and HO activity [60] (fig. 2). HO-1 protein also accumulated specifically in the renal tubular epithelial cells following SnCl2 treatment [60] (fig. 2). Consistent with HO activity, the increase in intracellular heme concentration was only marginal in SnCl2-treated animals, and the renal dysfunction by IARF was


75

4 Control level

SnCl2⫹IARF

SnMP⫹IARF

3

1.0 0.8

2

0.6 0.4

1

0.2 0

Serum creatinine (mg/dl)

1.2

IARF SnCl2⫹IARF SnMP ⫹IARF

HO-1 immunostaining Control

0 0

6

12

18

24

(h)

0 HO activity (pmol bilirubin/60 min/mg prot)

IARF

Heme concentration (nmol/mg prot)

1.4

IARF

50 100 150

SnCl2 ⫹IARF

200 250 300

Fig. 2. Effect of SnCl2 or Sn-MP administration on kidney function and renal heme contents in rats with IARF. Rats were uninephrectomized and subjected to unilateral ischemia for 40 min to produce IARF. SnCl2 (10 mg/100 g body weight) was administered subcutaneously, and Sn-MP (1 ␮mol/kg body weight) was administered intravenously 24 h prior to the uninephrectomy. After the initiation of reperfusion, whole blood was collected for the determination of serum creatinine concentrations. The kidney was removed for histological examination and measurement of microsomal heme concentration and heme oxygenase (HO) activity. Measurements are shown as the mean ⫾ SEM (n ⫽ 6). SnCl2 pretreatment produced a decrease in microsomal heme concentration associated with the marked increase in HO activity. In contrast, inhibition of HO activity by treatment with Sn-MP resulted in an increase in microsomal heme concentration, which are in agreement with the aggravation of renal function as determined by serum creatinine level. Shown on the right side are the sections of renal cortex from IARF rats following various treatments, stained immunohistochemically using anti-rat HO-1 as a primary antibody. Positive HO-1 staining was observed as brown color. Sections were counterstained with methyl green. The bar represents 100 ␮m. Top ⫽ Untreated kidney section. Middle ⫽ Kidney section from 8 h after reperfusion of IARF model. Bottom ⫽ Kidney section from 8 h after reperfusion of IARF model, pretreated by SnCl2 (10 mg/100 g body weight) 24 h before ischemia treatment. HO-1 protein was slightly induced in tubular epithelial cells in IARF animal, while the induction became obvious in IARF with SnCl2 pretreatment.


76

Table 1. HO expression in experimental acute renal failure Procedure

Animal

HO induction/inhibition

Reference

Ischemia/reperfusion

Rats, Sprague-Dawley Rats, Sprague-Dawley Rats, Sprague-Dawley Rats, Sprague-Dawley Rats, Sprague-Dawley Mice, HO-1 knockout Rats, Sprague-Dawley Rats, Sprague-Dawley

– – Induction by endotoxin Induction by nephrotoxic serum Inhibition by Sn-MP – Induction by SnCl2 Induction by SnCl2 Inhibition by Sn-MP Induction by BSO Induction by hemolysate

[49] [50] [63] [83] [51] [40] [60] [84]

Rats, Wistar Rats, Wistar Glycerol-induced

Rats, Inbred strains Rats, Sprague-Dawley Rats, Sprague-Dawley Rats, Wistar Mice, Inbred C57BL Mice, Mutant Strains Rats, Sprague-Dawley

[85] [86]

Induction by hemoglobin Inhibition by Zn-PP Induction by endotoxin Induction by nephrotoxic serum – Induction by hemoglobin

[52] [63] [83] [87] [88]

Induction by bile duct ligation

[89]

Cisplatin-induced

Rats, Sprague-Dawley

Inhibition by Sn-PP

[53]

Hemoglobin-induced

Rats, Sprague-Dawley Mice, Inbred C57BL Mice, Mutant strains

Induction by endotoxin Induction by hemoglobin

[63] [88]

Gentamicin-induced

Mice, Inbred C57BL Mice, Mutant strains

Induction by hemoglobin

[88]

Cyclosporine A-, Gold sodium thiomalate-induced

Mice, Inbred C57BL Mice, Mutant strains

Induction by hemoglobin

[88]

FeSO4–, Ca2⫹ ionophore-, cytochalasin D-, PLA2-induced

Rats, Sprague-Dawley

Induction by glycerol

[90]

much improved as measured by serum creatinine levels [60] (fig. 2). Thus, the tissue-specific upregulation of HO-1 in the kidney by SnCl2 treatment prior to ischemia is effective to protect the renal tubular cells from an oxidative injury due to IARF. These findings clearly suggest that HO-1 induction plays an important role in the protection of animals from renal dysfunction, presumably by removing an excess amount of free heme.


77

Metabolites of HO Reaction Also Function as Cytoprotective Factors

HO breaks down the pro-oxidant heme into three elements, i.e., iron, CO and BV, which is rapidly converted by BVR to BR IX␣, which is an antioxidant [14] (fig. 1). Albumin-bound BR is oxidized by hydroxyl, hydroperoxyl, and superoxide anion radicals, and BR also strongly inhibits hydroxyl-mediated formation of protein carbonyls [61]. The potent physiologic antioxidant actions of BR are reflected in an amplification cycle, whereby BR is oxidized to BV and then recycled by BVR back to BR [62]. Iron, which is an oxidant, is directly sequestered and inactivated by coinduced ferritin in the cell [63]. HO-1 has also been reported to prevent cell death by exporting intracellular iron both in vitro [64] and in vivo [38]. Recently, CO attracted special attention as a novel gaseous modulator similar to NO [65]. For example, exogenous CO is able to suppress rejection of mouse-to-rat cardiac transplants and restores long-term graft survival [66]. Presumably CO suppresses graft rejection by inhibiting platelet aggregation, myocardial infarction and suppressing endothelial cell apoptosis [66]. Furthermore, CO produced from heme by HO can suppress apoptosis of endothelial cells via the activation of p38 MAPK [16]. Thus, all these metabolites of the HO reaction act as a member of the important protective response against oxidative stimuli and contribute to the suppression of tissue injury. In addition to its key role in heme catabolism, the immediate and adaptive response of HO-1 to the wide variety of oxidative injuries suggests an important role of HO-1 in the protection of oxidative stress. The absence of HO-1 was also associated with an abnormally elevated serum heme concentration (0.5 mM), and resulted in various oxidative and inflammatory complications [39, 42], indicating the importance of HO-1 in the protection from oxidative injuries associated with inflammation. Exposure of cells to heme stimulates the expression of adhesion molecules ICAM-1, VCAM-1, and E-selectin on endothelial cells in vitro, probably through heme-mediated generation of ROS, which may underscore reactive inflammatory changes [17]. Mechanism of ho-1 Gene Expression

HO-1 is characterized by its remarkable property being induced by its substrate, hemin, as well as by a number of nonheme substances, such as insulin, epinephrine, endotoxin, heavy metals, hydrogen peroxide, ultraviolet, or sulfhydryl reagents [9, 67]. It should be noted that the 5⬘-flanking regions of the human and the rat ho-1 gene contain several potential cis-regulatory elements such as heat shock element (HSE) and IL-6-responsive element [9, 68] (fig. 3). HSE is the cis-acting element responsible for transcriptional activation of heat


78

1,000 b MARE

5'

CdRE

AP-1 ⫺4,000 b

5'

HSE ⫺400 b

IL- 6

3' Exon 1

Exon 2

E-box IL- 6

Exon 3

Exon 1

Exon 4

Exon 5

3'

Fig. 3. Structural organization of the human ho-1 gene. The composite enhancer and the proximal cis-acting elements of the ho-1 gene are schematically shown. Human ho-1 gene contains a potential heat shock responsive element (HSE), but it appears to be silent in most human cells. It also contains two copies of the IL-6-responsive element in the promoter region, and it is known that IL-6 treatment induces ho-1 gene expression. The upstream cis-acting element of the human ho-1 gene, located at minus 4 kb, consists of the overlapping of a Maf recognition element (MARE)/stress-responsive element (StRE), including an AP-1-binding site, and a cadmium-responsive element (CdRE). A transcriptional repressor, Bach 1/Maf binds with MARE, to keep ho-1 gene repressed in normal cells. Its repression is released when Bach 1 binds heme, and then Nrf2/Maf binds with MARE instead of Bach 1/Maf to activate the gene expression of ho-1.

shock protein genes by heat shock. In fact, heat shock treatment induces HO-1 in rat cells at the transcriptional level [10, 69]. In contrast to rat cells, HO activity in tissue cultures of human, monkey, pig, and mouse cells was not necessarily induced in all cells by heat shock, suggesting that there may be interspecies difference in the regulation of HO-1 expression [7]. The HSE of the human ho-1 gene is also potentially functional [70, 71], but HO-1 is not inducible in most human cells. Perhaps a sequence flanking the HSE may act as a silencer and prevent the heat-mediated activation of the ho-1 gene [70]. The proximal promoter region of the human ho-1 gene also contains two copies of the IL-6-responsive element and two functional CANNTG motifs, known as an E-box [26, 72, 73] (fig. 3). Each IL-6-responsive element overlaps with the HSE or the E-box. IL-6, an inducer of the acute phase reaction, increases the expression of HO-1 and haptoglobin in human hepatoma cells [26]. Thus, HO-1 is a positive acutephase reactant in human hepatoma cells. Recent studies have revealed that the induction of ho-1 expression involves the interplay of various basic leucine zipper transcription factors on critical enhancer elements including stress responsive element (StRE) [74] or Maf recognition element (MARE) [75]. StRE and MARE are similar to each other and override with a binding motif for AP-1 transcription factors [75] (fig. 3). The regulation of the ho-1 gene expression through MARE/StRE has been shown to


79

involve the small Maf proteins, either with Nrf or Bach 1. The small Maf proteins form heterodimers with Nrf proteins to activate MARE-dependent ho-1 gene expression [76, 77]. Bach 1 forms heterodimers with the small Maf proteins that bind to MARE similar to the Nrf/small Maf heterodimers [78, 79]. However, Bach 1 heterodimers function as transcription repressors [79]. There is a competitive interplay between the Bach 1-containing repressor dimers and Nrf-containing activator dimers, which effectively regulates ho-1 gene expression in dynamic fashion. The well-known ho-1 induction by heme is now accounted for by the competitive regulation system. The heme molecule regulates the DNAbinding activity of Bach 1 through a direct interaction [80, 81], mediated by an evolutionarily conserved multiple heme regulatory motif including the cysteineproline dipeptide sequence in Bach 1. Increased levels of heme abrogate the repressor function of Bach 1, by binding to the heme regulatory motif of Bach 1, which then result in the inhibition of its DNA-binding activity. Consequent detachment of Bach1 from the MARE sequence allows binding of Nrf2 and other small Maf proteins to the MARE sequence, resulting in transcriptional activation of target genes that include HO-1, thioredoxin, and keratinocyte growth factor [80–82]. Thus, the regulation of ho-1 gene expression involves a direct sensing of intracellular free heme levels by Bach 1, generating a simple feedback loop whereby the substrate affects the repressor. In the IARF model, a rapid increase of microsomal heme concentration in the kidney immediately following reperfusion [51] may contribute to the ho-1 gene expression through Bach 1. Conclusion

In this review, recent evidence concerning the protective role of HO-1 in ARF induced by ischemia followed by reperfusion is summarized. Both inhibition of HO activity, and suppression of ho-1 gene expression, lead to aggravation of oxidative tissue injuries in the kidney. In contrast, induction of HO-1 prior to ischemia confers significant protection. These findings thus suggest that HO-1 may be a major player in the defense against the oxidative tissue injury. Even SnCl2, which has been thought to be toxic, may offer a new mode of treatment of IARF, because of their highly kidney-specific HO-1-inducing property. Acknowledgement This study was in part supported by grants from Grant-in-Aid for Scientific Research (08877240, 09671564, 10671416, 11671499, 12877246, 13671582, 14571440 and 15590252) from the Ministry of Education, Science and Culture of Japan, and from USPHS DK32890. We are grateful to Drs. H. Shimizu, H. Fujii, K. Nakahira, H. Hirakawa and Ms. E. Ohmori for their assistance in this work.


80

References 1 2 3

4

5 6

7 8 9

10 11 12 13 14 15

16

17

18

19

20

21

Sassa S: Recent progress in heme synthesis and metabolism. Regulation of Heme Protein Synthesis, ed. F. H. 1994, Dayton: Alpha Med Press, 1–10. Hebbel RP, Eaton JW: Pathobiology of heme interaction with the erythrocyte membrane. Semin Hematol 1989;26:136–149. Balla G, Vercellotti GM, Muller-Eberhard U, Eaton J, Jacob HS: Exposure of endothelial cells to free heme potentiates damage mediated by granulocytes and toxic oxygen species. Lab Invest 1991;64:648–655. Dhar GJ, Bossenmaier I, Cardinal R, Petryka ZJ, Watson CJ: Transitory renal failure following rapid administration of a relativelylarge amount of hematin in a patient with acute intermittent porphyria in clinical remission. Acta Med Scand 1978;203:437–443. Nath KA, Grande JP, Croatt AJ, Likely S, Hebbel RP, Enright H: Intracellular targets in heme protein-induced renal injury. Kidney Int 1998;53:100–111. Yoshinaga T, Sassa S, Kappas A: A comparative study of heme degradation by NADPHcytochrome c reductase alone and by the complete heme oxygenase system: Distinctive aspects of heme degradation by NADPH-cytochrome c reductase. J Biol Chem 1982;257:7794–7802. Shibahara S: Regulation of heme oxygenase gene expression. Semin Hematol 1988;25:370–376. Tenhunen R, Marver HS, Schmid R: The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 1968:61:748–755. Keyse SM, Tyrrell RM: Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA 1989;86:99–103. Shibahara S, Muller RM, Taguchi H: Transcriptional control of rat heme oxygenase by heat shock. J Biol Chem 1987;262:12889–12892. Otterbein LE, Choi AM: Heme oxygenase: Colors of defense against cellular stress. Am J Physiol Lung Cell Mol Physiol 2000;279:L1029-L1037. Maines MD: Heme oxygenase: Cunction, multiplicity, regulatory mechanisms, and clinical applications. Faseb J 1988:2:2557–2568. McCoubrey WK Jr, Huang TJ, Maines MD: Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 1997;247:725–732. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN: Bilirubin is an antioxidant of possible physiological importance. Science 1987;235:1043–1046. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, Wysk M, Davis RJ, Flavell RA, Choi AM: Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422–428. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, Soares MP: Carbon monoxide generated by heme oxygenase 1 suppress esendothelial cell apoptosis. J Exp Med 2000;192: 1015–1026. Wagener FA, Eggert A, Boerman OC, Oyen WJ, Verhofstad A, Abraham NG, Adema G, van Kooyk Y, de Witte T, Figdor CG: Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase. Blood 2001;98:1802–1811. DeBruyne LA, Magee JC, Buelow R, Bromberg JS: Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival. Transplantation 2000;69:120–128. Sassa S, Kappas A, Bernstein SE, Alvares AP: Heme biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. Heme oxygenase induction as an adaptive response for maintaining cytochrome P-450 in chronic hemolysis. J Biol Chem 1979;254:729–735. Araujo JA, Meng L, Tward AD, Hancock WW, Zhai Y, Lee A, Ishikawa K, Iyer S, Buelow R, Busuttil RW, Shih DM, Lusis AJ, Kupiec-Weglinski JW: Systemic rather than local heme oxygenase-1 overexpression improves cardiac allograft outcomes in a new transgenic mouse. J Immunol 2003;171:1572–1580. Song R, Kubo M, Morse D, Zhou Z, Zhang X, Dauber JH, Fabisiak J, Alber SM, Watkins SC, Zuckerbraun BS, Otterbein LE, Ning W, Oury TD, Lee PJ, McCurry KR, Choi AM: Carbon


81

22

23

24

25 26 27

28

29

30

31 32

33 34 35

36

37 38 39 40

41

monoxide induces cytoprotection in rat orthotopic lung transplantation via anti-inflammatory and anti-apoptotic effects. Am J Pathol 2003;163:231–242. Reutzel-Selke A, Zschockelt T, Denecke C, Bachmann U, Jurisch A, Pratschke J, Schmidbauer G, Volk HD, Neuhaus P, Tullius SG: Short-term immunosuppressive treatment of the donor ameliorates consequences of ischemia/reperfusion injury and long-term graft function in renal allografts from older donors. Transplantation 2003;75:1786–1792. Tsuchihashi S, Tamaki T, Tanaka M, Kawamura A, Kaizu T, Ikeda A, Kakita A: Pyrrolidine dithiocarbamate provides protection against hypothermic preservation and transplantation injury in the rat liver: The role of heme oxygenase-1. Surgery 2003;133:556–567. Ribeiro MM, Klein D, Pileggi A, Molano RD, Fraker C, Ricordi C, Inverardi L, Pastori RL: Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic beta-cells. Biochem Biophys Res Commun 2003;305:876–881. Alam J, Shibahara S, Smith A: Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells. J Biol Chem 1989;264:6371–6375. Mitani K, Fujita H, Kappas A, Sassa S: Heme oxygenase is a positive acute-phase reactant in human Hep3B hepatoma cells. Blood 1992;79:1255–1259. Suzuki T, Takahashi T, Yamasaki A, Fujiwara T, Hirakawa M, Akagi R: Tissue-specific gene expression of heme oxygenase-1 (HO-1) and non-specific delta-aminolevulinate synthase (ALAS-N) in a rat model of septic multiple organ dysfunction syndrome. Biochem Pharmacol 2000;60:275–283. Camhi SL, Alam J, Wiegand GW, Chin BY, Choi AM: Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5⬘ distal enhancers: Role of reactive oxygen intermediates and AP-1. Am J Respir Cell Mol Biol 1998;18:226–234. Ning W, Song R, Li C, Park E, Mohsenin A, Choi AM, Choi ME: TGF-beta1 stimulates HO-1 via the p38 mitogen-activated protein kinase in A549 pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2002;283:L1094-L1102. Durante W, Peyton KJ, Schafer AI: Platelet-derived growth factor stimulates heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1999;19:2666–2672. Motterlini R, Green CJ, Foresti R: Regulation of heme oxygenase-1 by redox signals involving nitric oxide. Antioxid Redox Signal 2002:4:615–624. Kageyama H, Hiwasa T, Tokunaga K, Sakiyama S: Isolation and characterization of a complementary DNA clone for a Mr 32,000 protein which isinduced with tumor promoters in BALB/c 3T3 cells. Cancer Res 1988;48:4795–4798. Hiwasa T, Fujimura S, Sakiyama S: Tumor promoters increase the synthesis of a 32,000-dalton protein in BALB/c 3T3 cells. Proc Natl Acad Sci USA 1982;79:1800–1804. Yu L, Gengaro PE, Niederberger M, Burke TJ, Schrier RW: Nitric oxide: A mediator in rat tubular hypoxia/reoxygenation injury. Proc Natl Acad Sci USA 1994;91:1691–1695. Turcanu V, Dhouib M, Poindron P: Heme oxygenase inhibits nitric oxide synthase by degrading heme: A negative feedback regulation mechanism for nitric oxide production. Transplant Proc 1998;30:4184–4185. Datta PK, Koukouritaki SB, Hopp KA, Lianos EA: Heme oxygenase-1 induction attenuates inducible nitric oxide synthase expression and proteinuria in glomerulonephritis. J Am Soc Nephrol 1999;10:2540–2550. Datta PK, Lianos EA: Nitric oxide induces heme oxygenase-1 gene expression in mesangial cells. Kidney Int 1999;55:1734–1739. Poss KD, Tonegawa S: Heme oxygenase 1 is required for mammalian iron reutilization. Proc Natl Acad Sci USA 1997;94:10919–10924. Poss KD, Tonegawa S: Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci USA 1997;94:10925–10930. Wiesel P, Patel AP, Carvajal IM, Wang ZY, Pellacani A, Maemura K, DiFonzo N, Rennke HG, Layne MD, Yet SF, Lee ME, Perrella MA: Exacerbation of chronic renovascular hypertension and acute renal failure in heme oxygenase-1-deficient mice. Circ Res 2001;88:1088–1094. Dennery PA, Spitz DR, Yang G, Tatarov A, Lee CS, Shegog ML, Poss KD: Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2. J Clin Invest 1998;101: 1001–1011.


82

42

43

44

45

46

47 48 49

50 51

52 53 54 55

56 57 58 59 60

61 62 63 64

65

Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, Koizumi S: Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest 1999;103:129–135. Ohta K, Yachie A, Fujimoto K, Kaneda H, Wada T, Toma T, Seno A, Kasahara Y, Yokoyama H, Seki H, Koizumi S: Tubular injury as a cardinal pathologic feature in human heme oxygenase-1 deficiency. Am J Kidney Dis 2000;35:863–870. Cole L, Bellomo R, Silvester W, Reeves JH: A prospective, multicenter study of the epidemiology, management, and outcome of severe acute renal failure in a ‘closed’ ICU system. Am J Respir Crit Care Med 2000;162:191–196. Brivet FG, Kleinknecht DJ, Loirat P, Landais PJ: Acute renal failure in intensive care units – causes, outcome, and prognostic factors of hospital mortality; a prospective, multicenter study. French Study Group on Acute Renal Failure. Crit Care Med 1996;24:192–198. Liano F, Junco E, Pascual J, Madero R, Verde E: The spectrum of acute renal failure in the intensive care unit compared with that seen in other settings. The Madrid Acute Renal Failure Study Group. Kidney Int Suppl 1998;66:S16-S24. Paller MS, Hoidal JR, Ferris TF: Oxygen free radicals in ischemic acuterenal failure in the rat. J Clin Invest 1984;74:1156–1164. Finn WF, Chevalier RL: Recovery from postischemic acute renal failure in the rat. Kidney Int 1979;16:113–123. Maines MD, Mayer RD, Ewing JF, McCoubrey WK Jr: Induction of kidney hemeoxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: Possible role of heme as both promotor of tissue damage and regulator of HSP32. J Pharmacol Exp Ther 1993;264:457–462. Paller MS, Nath KA, Rosenberg ME: Heme oxygenase is not expressed as a stress protein after renal ischemia. J Lab Clin Med 1993:122:341–345. Shimizu H, Takahashi T, Suzuki T, Yamasaki A, Fujiwara T, Odaka Y, Hirakawa M, Fujita H, Akagi R: Protective effect of heme oxygenase induction in ischemic acute renal failure. Crit Care Med 2000;28:809–817. Nath KA, Balla G, Vercellotti GM, Balla J, Jacob HS, Levitt MD, Rosenberg ME: Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest 1992;90:267–270. Agarwal A, Balla J, Alam J, Croatt AJ, Nath KA: Induction of heme oxygenase in toxic renal injury: A protective role in cisplatin nephrotoxicity in the rat. Kidney Int 1995;48:1298–1307. Drummond GS: Control of heme metabolism by synthetic metalloporphyrins. Ann NY Acad Sci 1987;514:87–95. Drummond GS, Galbraith RA, Sardana MK, Kappas A: Reduction of the C2 and C4 vinyl groups of Sn-protoporphyrin to form Sn-mesoporphyrin markedly enhances the ability of the metalloporphyrin to inhibit in vivo heme catabolism. Arch Biochem Biophys 1987;255:64–74. Valaes T, Petmezaki S, Henschke C, Drummond GS, Kappas A: Control of jaundice in preterm newborns by an inhibitor of bilirubin production: Studies with tin-mesoporphyrin. Pediatrics 1994;93:1–11. Kappas A, Drummond GS: Control of heme metabolism with synthetic metalloporphyrins. J Clin Invest 1986;77:335–339. Kappas A, Maines MD: Tin: A potent inducer of heme oxygenase in kidney. Science 1976;192:60–62. Sunderman FW Jr: Metal induction of heme oxygenase. Ann NY Acad Sci 1987;514:65–80. Toda N, Takahashi T, Mizobuchi S, Fujii H, Nakahira K, Takahashi S, Yamashita M, Morita K, Hirakawa M, Akagi R: Tin chloride pretreatment prevents renal injury in rats with ischemic acute renal failure. Crit Care Med 2002;30:1512–1522. Neuzil J, Stocker R: Bilirubin attenuates radical-mediated damage to serum albumin. FEBS Lett 1993;331:281–284. Baranano DE, Rao M, Ferris CD, Snyder SH: Biliverdin reductase: A major physiologic cytoprotectant. Proc Natl Acad Sci USA 2002;99:16093–16098. Vogt BA, Alam J, Croatt AJ, Vercellotti GM, Nath KA: Acquired resistance to acute oxidative stress. Possible role of heme oxygenase and ferritin. Lab Invest 1995;72:474–483. Ferris CD, Jaffrey SR, Sawa A, Takahashi M, Brady SD, Barrow RK, Tysoe SA, Wolosker H, Baranano DE, Dore S, Poss KD, Snyder SH: Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol 1999;1:152–157. Toxic gas protects rodent hearts. Lab Anim (NY) 2003;32:12–13.


83

66

67 68 69

70 71 72

73 74

75 76 77

78

79

80

81

82

83

84 85

Sato K, Balla J, Otterbein L, Smith RN, Brouard S, Lin Y, Csizmadia E, Sevigny J, Robson SC, Vercellotti G, Choi AM, Bach FH, Soares MP: Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J Immunol 2001;166:4185–4194. Maines MD, Kappas A: Studies on the mechanism of induction of haem oxygenase by cobalt and other metal ions. Biochem J 1976;154:125–131. Muller RM, Taguchi H, Shibahara S: Nucleotide sequence and organization of the rat heme oxygenase gene. J Biol Chem 1987;262:6795–6802. Okinaga S, Shibahara S: Identification of a nuclear protein that constitutively recognizes the sequence containing a heat-shock element. Its binding properties and possible function modulating heat-shock induction of the rat heme oxygenase gene. Eur J Biochem 1993;212:167–175. Okinaga S, Takahashi K, Takeda K, Yoshizawa M, Fujita H, Sasaki H, Shibahara S: Regulation of human heme oxygenase-1 gene expression under thermal stress. Blood 1996;87:5074–5084. Mitani K, Fujita H, Sassa S, Kappas A: A heat-inducible nuclear factor that binds to the heatshock element of the human haem oxygenase gene. Biochem J 1991;277:895–897. Muraosa Y, Shibahara S: Identification of a cis-regulatory element and putative transacting factors responsible for 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of heme oxygenase expression in myelomonocytic cell lines. Mol Cell Biol 1993;13:7881–7891. Sato M, Ishizawa S, Yoshida T, Shibahara S: Interaction of upstream stimulatory factor with the human heme oxygenase gene promoter. Eur J Biochem 1990;188:231–237. Inamdar NM, Ahn YI, Alam J: The heme-responsive element of the mouse heme oxygenase-1 gene isan extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors. Biochem Biophys Res Commun 1996;221:570–576. Kataoka K, Noda M, Nishizawa M: Maf nuclear oncoprotein recognizes sequences related to an AP-1 site and forms heterodimers with both Fos and Jun. Mol Cell Biol 1994;14:700–712. Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL: Nrf2, a Cap’n’Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 1999;274:26071–26078. Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S, Yamamoto M: Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 2000;275:16023–16029. Oyake T, Itoh K, Motohashi H, Hayashi N, Hoshino H, Nishizawa M, Yamamoto M, Igarashi K: Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site. Mol Cell Biol 1996;16: 6083–6095. Igarashi K, Hoshino H, Muto A, Suwabe N, Nishikawa S, Nakauchi H, Yamamoto M: Multivalent DNA binding complex generated by small Maf and Bach1 as a possible biochemical basis for beta-globin locus control region complex. J Biol Chem 1998;273:11783–11790. Sun J, Hoshino H, Takaku K, Nakajima O, Muto A, Suzuki H, Tashiro S, Takahashi S, Shibahara S, Alam J, Taketo MM, Yamamoto M, Igarashi K: Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. Embo J 2002;21:5216–5224. Ogawa K, Sun J, Taketani S, Nakajima O, Nishitani C, Sassa S, Hayashi N, Yamamoto M, Shibahara S, Fujita H, Igarashi K: Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1. Embo J 2001;20:2835–2843. Braun S, Hanselmann C, Gassmann MG, auf dem Keller U, Born-Berclaz C, Chan K, Kan YW, Werner S: Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound. Mol Cell Biol 2002;22: 5492–5505. Vogt BA, Shanley TP, Croatt A, Alam J, Johnson KJ, Nath KA: Glomerular inflammation induces resistance to tubular injury in the rat. A novel form of acquired, heme oxygenase-dependent resistance to renal injury. J Clin Invest 1996;98:2139–2145. Akagi R, Takahashi T, Sassa S: Fundamental role of heme oxygenase in the protection against ischemic acute renal failure. Jpn J Pharmacol 2002;88:127–132. Horikawa S, Yoneya R, Nagashima Y, Hagiwara K, Ozasa H: Prior induction of heme oxygenase-1 with glutathione depletor ameliorates the renal ischemia and reperfusion injury in the rat. FEBS Lett 2002;510:221–224.


84

86 87

88

89 90

Yoneya R, Nagashima Y, Sakaki K, Hagiwara K, Teraoka H, Ozasa H, Horikawa S: Hemolysate pretreatment ameliorates ischemic acute renal injury in rats. Nephron 2002;92:407–413. Ishizuka S, Nagashima Y, Numata M, Yano T, Hagiwara K, Ozasa H, Sone M, Nihei H, Horikawa S: Regulation and immunohistochemical analysis of stress protein heme oxygenase-1 in rat kidney with myoglobinuric acute renal failure. Biochem Biophys Res Commun 1997;240:93–98. Nath KA, Haggard JJ, Croatt AJ, Grande JP, Poss KD, Alam J: The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am J Pathol, 2000;156:1527–1535. Leung N, Croatt AJ, Haggard JJ, Grande JP, Nath KA: Acute cholestatic liver disease protects against glycerol-induced acute renal failure in the rat. Kidney Int 2001;60:1047–1057. Zager RA: Heme protein-induced tubular cytoresistance: Expression at the plasma membrane level. Kidney Int 1995;47:1336–1345.

Reiko Akagi, PhD Department of Nutritional Science, Faculty of Health and Welfare Science Okayama Prefectural University 111 Kuboki, Soja-shi, Okayama-ken, 719–1197 (Japan) Tel. ⫹81 866 94 2156, Fax 81 8669 94 2156, E-Mail [email protected]


85


Heat Shock (Stress Response) Proteins and Renal Ischemia/Reperfusion Injury Katherine J. Kelly Indiana University School of Medicine, Department of Medicine, Division of Nephrology, Indianapolis, Ind., USA

Abstract Acute renal failure occurs frequently, may be increasing, carries an unacceptably high mortality, yet there is no specific treatment. The induction of stress response (heat shock) proteins (HSPs) is a highly conserved response that protects many cell types from diverse physiological and environmental stressors. HSP families of different sizes function as molecular chaperones that facilitate the folding of enzymes and other proteins into functional conformations. After injury, HSPs are believed to facilitate the restoration of normal function by assisting in the refolding of denatured proteins and degradation of irreparably damaged proteins and toxic metabolites, limitation of aggregation of damaged peptides and aiding appropriate folding of newly synthesized essential polypeptides. HSPs may also regulate apoptosis and immune functions. We have demonstrated protection from the functional deficits and histological evidence of experimental ischemic renal injury with heat stress 6 but not 48 h prior to ischemia. Limitation of the induction of HSPs (either with a short period of hyperthermia or pharmacologically) attenuated the protection observed. Other investigators have demonstrated a correlation between the levels of HSP25 and renal ischemic preconditioning in the mouse. Several pharmacological agents have been shown to increase HSP expression. Enhancement of these endogenous protective mechanisms has potential benefit in human disease. Copyright © 2005 S. Karger AG, Basel

Acute renal ischemia injury is common in hospitalized patients. The mortality remains ⬎50% in many series and has not improved significantly in several decades [1, 2]. The incidence may be increasing [3]. In a recent study, the median survival of patients with acute renal failure (ARF) requiring dialysis

was 32 days [4]. There is no specific treatment that improves outcome, and dialysis may actually exacerbate renal ischemia [5]. Ischemia is also critical in renal dysfunction in allograft recipients [6]. A more complete understanding of endogenous protective mechanisms could improve clinical care.

Ischemic Preconditioning

Ischemic preconditioning is a remarkably effective experimental procedure that consistently protects the heart and other organs from additional episodes of ischemia [7]. In the kidney, varying results have been seen with ischemic preconditioning. Zager et al. [8, 9] found increased susceptibility to ischemia 30 min but not 3.5 or 24 h following an initial 15-minute period of bilateral ischemia in female rats. In contrast, these investigators [10] found a higher glomerular filtration rate after ischemia in rats with prior reduction in renal mass. Yoshioka et al. [11] demonstrated better inulin clearances in animals after daily ischemia for 6 but not 3 days. Islam et al. [12] found no protection from 20 or 40 min of unilateral renal ischemia in the rat after ischemic preconditioning [12]. Prior ischemia resulted in less lactate dehydrogenase release from proximal tubule suspensions subjected to subsequent hypoxia [13]. Human proximal tubular cells are more resistant to subsequent hypoxic injury after an initial period of hypoxia [14]. Park et al. [15] demonstrated remarkable protection (lower mean serum creatinine, absence of medullary congestion, less histological evidence of injury) from ischemic renal injury in mice with preconditioning 8 or 15 days prior to a second ischemic insult. Partial protection in this model was seen as long as 12 weeks after the initial insult [16]. Preconditioning when the initial and subsequent insults are different has been recognized in the kidney for many years [17]. Vogt et al. [18] found that administration of nephrotoxic serum to rats prior to the induction of ARF was associated with a lower mean serum creatinine. The protection could be inhibited with tin protoporphyrin, a specific inhibitor of heat shock protein (HSP)32 (hemoxygenase-1). Prior ureteral obstruction (with an increase in HSP25 for at least 6 days) also protects against renal ischemia [19]. In addition to protection of the same organ, preconditioning can result in protection of other organs. For example, Pell et al. [20] have shown protection from myocardial ischemia in the rabbit with prior renal ischemia. Mean infarct volume was reduced by 59% after 10 min of renal artery occlusion prior to 30 min of left coronary artery occlusion in vivo. In another study, Leung et al. [21] found that rats were protected from glycerol-induced ARF with ligation of the common bile duct. At the time of initiation of ARF, there was induction of HSP32 in the kidney.

HSPs and Renal Ischemia

87

Table 1. Stress response (heat shock) protein functions Functions in normal physiological state Accurate protein folding Translocation of proteins across organelle membranes Formation of multimeric complexes Degradation of abnormal proteins Functions in stress states Prevention of aggregation of damaged proteins Repair of denatured proteins Folding and transport of newly synthesized (replacement) proteins Degradation of irreparably damaged proteins Inhibition of apoptosis Stabilization of cytoskeleton Immune functions Cytokine-like functions Target for autoantibody formation Apoptosis regulator

Stress Response Proteins and Endogenous Protective Mechanisms (table 1)

One mechanism of ischemic preconditioning is thought to involve the induction of stress response or heat shock proteins (HSPs). HSPs are induced, activated and/or redistributed in response to many stressors including hyperthermia, hypothermia, hypoxia, ischemia, oxidative and osmotic stress and exposure to toxins. The regulated synthesis of HSPs is also thought important in normal physiological processes including growth and development. The role of HSPs in human disease is illustrated by the ability of constitutive HSP73 to facilitate a functional conformation of a mutant (⌬ F503) of the cystic fibrosis transmembrane conductance regulator [22]. Diverse signals are thought to activate the stress-inducible heat shock factor-1 (HSF-1), which oligomerizes, translocates to the nucleus and binds to heat shock element in the promoters of HSPs [23]. Hyperphosphorylation of HSF-1 may be needed for full activation [24]. HSF-1 is activated with renal ischemia in proportion to the decrease in ATP levels [25]. Studies in cultured renal tubular (LLC-PK1) cells suggest that nucleotide depletion and not hypoxia is responsible for HSF-1 activation with ischemia [26]. Stress response proteins are highly conserved and are found in diverse organisms from plants to Drosophila to mammals [27, 28]. The ubiquity and phylogenetic conservation suggest that the stress response is a basic protective mechanism. Alterations in protein folding and location occur in many forms of

Kelly

88

DNA

Denatured protein RNA Stress

Polypeptide

Stress

HSPs

HSPs

Irreparably damaged protein

Correctly folded, functional protein

HSPs

Aggregated protein

Stress

HSPs Degradation

HSPs Correct intracellular location

Fig. 1. Stress response (heat shock) protein function. Many stress response or heat shock proteins serve as ‘molecular chaperones’, insuring the correct conformation and intracellular location of newly synthesized or damaged proteins. After injury, HSPs assist in refolding of damaged proteins and degradation of irreparably denatured proteins, limit aggregation of polypeptides and facilitate the correct conformation and function of newly synthesized essential proteins.

renal injury and are fundamental mechanisms of ischemic damage. The potential functions for HSPs in the injured kidney include (fig. 1) [27–33]: (1) Refolding of denatured proteins and restoration of their function (2) Correct folding of newly synthesized essential polypeptides (3) Limitation of detrimental interactions among damaged peptides (e.g., aggregation) (4) Transport of irreparably damaged proteins, non-native proteins and potentially toxic metabolites for timely degradation by the proteasome (5) Assembly/stabilization of multiprotein complexes (6) Translocation of proteins to the appropriate intracellular location (for example, across organelle membranes) (7) Prevention of apoptosis (for example, via mitochondrial HSP60 binding to cytochrome c and/or HSP70 binding to cytosolic targets) (8) Suppression of proinflammatory cytokines (9) Protection of mitochondria from reactive oxygen species and cytokines (10) Stabilization of the cytoskeleton (11) Suppression of NADPH oxidase and the oxidative burst (12) Repair of DNA damage (13) Prevention of calcium redistribution within the cell


89

Following stresses such as ischemia/reperfusion or hyperthermia, overall protein synthesis is inhibited but HSPs are efficiently translated and may rapidly reach 15–25% of cellular protein content [34, 35]. In addition, some cytosolic HSPs translocate to the nucleus in response to stress [36]. Genetic studies have provided convincing evidence that stress response proteins can protect against ischemia and other injuries in many experimental systems. HSP70 expression is inversely correlated with myocardial infarct size in animal models [37] and overexpression of HSP70 in transgenic mice reduces infarct size [38] and improves cardiac function after myocardial ischemia [39, 40]. Overexpression of HSP70 in neural cells also protects from hypoxia [41, 42]. In the kidney, postischemic HSP70 induction correlates with cellular ATP levels [43] but correlates poorly with protection of isolated rat proximal tubule segments from hypoxic injury [44]. In cultured opossum renal epithelial cells, Wang and Borkan [45] have demonstrated a correlation between HSP72 content after heat stress and cell survival after nucleotide depletion. Members of the HSP70 family are induced in the rat kidney at early time points following ischemia and reperfusion in vivo [46, 47]. Emami et al. [47] found increases in HSP72 3 h after unilateral renal ischemia in the rat. Van Why et al. [46] showed that HSP72 localized to the apical membrane of the proximal tubule 15 min postischemia and was dispersed through the cytosol in a vesicular pattern by 2 h after ischemia. Cortical HSP72 remains elevated 14 days after 60 min of renal ischemia in the rat [48]. The protection of mice deficient in inducible nitric oxide synthase from renal ischemic injury may be due to the upregulation of cortical HSP72 [49].

HSP Families (table 2)

Specific HSPs are essential for cell function under physiological conditions (‘constitutive’) but most are inducible. Members of the HSP70 family are the most widely studied and abundant group of stress response proteins in eukaryotic cells and often act in concert with cochaperones, for example, HSP40, HSP60 or HSP90 [50]. Other stress response proteins play critical roles in both physiological and pathophysiological states. Small HSPs are abundant in the papilla with less in the cortex at baseline (in an animal with unrestricted access to water), possibly as a consequence of differences in tonicity [51]. Small HSPs form large multimeric complexes and can be phosphorylated at several sites [52]. HSP22 (␣B-crystallin), a major structural protein of the ocular lens, is expressed at high levels in kidney and muscle, tissues with high rates of oxidative metabolism. In the unstressed kidney, HSP22 is expressed primarily in the loop of Henle and collecting ducts [53]. HSP22 is increased in the renal cortex for at least 5 days after ischemia and its pattern of labeling changes from homogeneous to

Kelly

90

Table 2. Stress response (heat shock) protein families HSP family

Subcellular localization (Prokaryotic homolog)

Small HSPs

Form large multimeric structures; restricted to specific tissues

HSP32 HSP25/27

Cytosol Cytosol/nuclear

HSP22 HSP10 (GroES) P20 MKBD

Cytosol Mitochondria Cytosol Cytosol

HSP60 (GroEL; chaperonins) HSP 60 TCP-1

Antioxidant Inhibits actin polymerization; may limit apoptosis Binds denatured proteins Regulates HSP60 Interacts with HSP25/27 Maintains myofibril integrity; increased in myotonic dystrophy Large oligomers

Mitochondria Cytosol

HPS70 (DnaK) HSP72/HSP70

Cytosol/nuclear with stress HSP73/HSC70 (Ssa) Cytosol/peroxisome GRP75 Mitochondria GRP78/BiP (Kar2) Endoplasmic reticulum

HSP90 (HtpG) HSP90␣ (HSP86)

Postulated significance

Cytosol

Molecular chaperone; limits protein aggregation and facilitates folding Facilitates folding of cytoskeletal proteins Stress-inducible chaperone; limits protein aggregation; inhibits apoptosis Constitutive/CFTR binding Molecular chaperone Molecular chaperone Part of steroid hormone receptor complex; molecular chaperone

HSP90␤ (HSP84) GRP94

Cytosol Endoplasmic reticulum

Calcium-binding chaperone

HSP 110 (C1p/SSE families) HSP110 HSP105␣/␤ OSP94/APG-1

Cytosol/nucleus Cytosol Cytosol (renal medulla)

Proteolysis Solubilizes aggregated proteins Induced by hyperosmotic stress

Other GRP170 HSP56

Cytosol

Calnexin Calreticulin Ubiquitin (only eukaryotes)

Part of steroid receptor complex; binds FK 506 Endoplasmic reticulum Glycoprotein folding Endoplasmic reticulum Glycoprotein folding Cytosol/nuclear/membrane Protein degradation


91

Table 2 (continued) HSP family

Subcellular localization (Prokaryotic homolog)

Postulated significance

HSP47 HSP40 (DnaJ)

Endoplasmic reticulum Cytosol

HSP10 (GroES)

Mitochondria

Processing of procollagen Regulates HSP70; minimizes protein aggregation Binds HSP60

*GRP ⫽ Glucose-regulated protein; TCP ⫽ T complex polypeptide; MKBD ⫽ myotonic dystrophy protein kinase-binding protein; CFTR ⫽ cystic fibrosis transmembrane conductance regulator.

inhomogeneous in S3 segments of proximal tubules in the outer medulla [54]. Cardiac muscle HSP22 is phosphorylated [55] and rapidly translocates from the cytosolic fraction to the Z bands of the cytoskeleton in response to ischemia [56]. Overexpression of HSP22 in neonatal and adult cardiomyocytes results in decreased lactate dehydrogenase and creatinine phosphokinase release following simulated ischemia [57]. ␣-crystallins are also thought to inhibit tubulin aggregation [58] and regulate intermediate filament assembly [59]. Another small HSP (HSP25) is induced and, by immunohistochemistry, its location in the proximal tubule changes from diffuse and subapical to punctuate and cytoplasmic after ischemia. Its distribution also changes from the soluble to cytoskeletal fraction of cell lysates [60]. HSP25 may also be induced in vascular structures postischemia [61]. Park et al. [15] found that the levels of HSP25 correlated well with the extent of protection in renal ischemic preconditioning in the mouse. Overexpression of HSP25 in cultured renal epithelial (LLC-PK1) cells decreases the injury seen after chemical anoxia [19]. Phosphorylated HSP25 is thought to regulate microfilament dynamics [62] and protect the actin cytoskeleton after ischemia [63]. HSP25 and HSP70 are also thought to stabilize the cytoskeletal anchorage of Na⫹/K⫹ ATPase with ischemic preconditioning [64]. HSP32 is the rate-limiting enzyme in the degradation of heme to biliverdin (a potential antioxidant) and iron and is induced by diverse stresses including hypoxia and ischemia. Its expression is regulated by hypoxia-inducible factor-1 (HIF-1). Overexpression of HSP32 protects against oxidant-mediated injury in several cell types [65, 66]. In HSP32-deficient mice, mortality is increased and glycerol-induced ARF is more severe than in mice with normal HSP32 expression [67]. Mitochondrial HSP60 and HSP10 are important in protein folding and assembly in that organelle [68]. Overexpression of both HSP60 and HSP10

Kelly

92

(but not individually) protects cultured myocytes from simulated ischemia [69]. In the unstressed kidney, the expression of HSP60 parallels mitochondrial abundance [51] and increases in areas of severe damage in mercury-induced tubular necrosis [70]. Members of the HSP70 family contain a peptide-binding site, a linking region and an ATPase domain and thus display weak ATPase activity [71]. Aufricht et al. [72] have demonstrated that HSP72 colocalizes with Na⫹, K⫹ ATPase postischemia and may facilitate its transport to the correct intracellular location in an ATP-dependent manner. HSP73 (HSC70) is induced immediately after renal ischemia in the S3 segment and remains elevated for 7 days [73]. HSP73 is also induced after gentamicin and accumulates in a pattern consistent with lysosomes, suggesting that it participates in the lysosomal degradation of abnormal proteins [73]. The induction of HSP73 is protective in intestinal ischemia-reperfusion injury [74]. HSP73 is also increased in the ischemiaresistant and tolerance-acquired neurons in the gerbil brain [75]. HSP90, with other chaperones, assists in the maturation of steroid hormone receptors and protein kinases [76]. In the unstressed kidney, HSP90 is expressed in the distal convoluted tubule and collecting ducts, paralleling that of mineralocorticoid receptors [77]. HSP90 is induced in the S3 segment of the proximal tubule and the loop of Henle after ischemia [73], and may stabilize Na⫹, K⫹ ATPase along with HSP25 and HSP70 [78]. Endoplasmic reticulum (ER) stress response proteins may be especially important following ischemic injury since damaged polypeptides are repaired or degraded in the ER and enzymes and membrane proteins (such as Na⫹/K⫹ ATPase [79] and tight junction components [80, 81]) critical to recovery after injury are synthesized in this organelle. Increases in mRNA levels of the ER chaperones GRP78 (BiP), GRP94 and ERP72 have been demonstrated in the kidney following ischemia in vivo and nucleotide depletion in cultured tubular cells [82]. Increases in the ER chaperone HSP47, which participates in collagen maturation, have been shown in gentamicin-mediated tubular injury [83]. Induction of GRP78 in Madin-Darby canine kidney cells protects against cell death following nucleotide depletion with antimycin A [84]. Overexpression of the calcium-binding ER chaperone calreticulin prevents death of LLC-PK1 cells after toxicant exposure [32]. Recently, Omi, a novel ER stress response protein regulated by renal ischemia has been described [85].


The induction of HSPs prior to renal ischemia in vivo has had variable efficacy [86–88]. In the rat, we have demonstrated protection from the functional


93

Urea nitrogen (mg/dl)

50

40

30 6h 12h 48h Sham

20

10 0

2

4 Time (days)

6

Fig. 2. Effect of heat stress prior to renal ischemia on postischemic renal function. Rats were subjected to sham hyperthermia (37⬚C) or hyperthermia (42⬚C) 6, 12 or 48 h prior to bilateral renal ischemia. Mean urea nitrogen values were significantly lower in the group subjected to heat 6 h prior to ischemia than in that subjected to sham hyperthermia. Reproduced from Kidney International [89] with permission.

deficits and histological evidence of ischemic renal injury with heat stress (8 min) 6 h but not 48 h prior to the ischemic insult [89]. Partial protection was observed with heat stress 12 h before ischemia (fig. 2). Heat stress and quercetin (which blocked the induction of HSP70 and HSP84 but not HSP22) was associated with renal failure postischemia but recovery was more rapid. HSP22 (␣B-crystallin) and other small HSPs are thought to be important in maintaining cytoskeletal integrity after ischemia and other stresses [56]. Marked disruption of the actin cytoskeleton in renal tubular and vascular cells after renal ischemia is believed to be critical in the impaired function as well as abnormal intrarenal blood flow postischemia [90, 91]. Levels of HSP22 in the kidney increase from 16 days of gestation to 5 weeks of age in the rat [53] suggesting that this protein is important in organogenesis and thus may be important in repair following injury. HSP22 is induced in Bowman’s capsule postischemia [54], recapitulating kidney development. Induction of HSP32 is also protective in experimental ischemia/reperfusion with improvement in function and decreased expression of intercellular adhesion molecule-1, renal leukocyte infiltration and apoptosis [92]. In the injured kidney, the role of HSPs in repair may depend on the exact nature and timing of the initial and subsequent insults [28]. Chatson et al. [87] found protection with 8–11 but not 12–15 min of hyperthermia prior to renal ischemia. Joannidis et al. [86] also found no protection with 15 min of heat. Resistance to simulated ischemia, hyperthermia and exposure to cyclosporin but increased sensitivity to cadmium was shown in inner medulla collecting

Kelly

94

duct cells after induction of HSP70, OSP94 and HSP110 mRNA [93]. LLC-PK1 cells are protected from toxicant-mediated injury by reductive stress (with induction of the stress response proteins Gadd153 and GRP78 but not HSP70) but not oxidative stress (which results in increases in HSP70 and slight increases in Gadd153 and GRP78) [94]. Henle et al. [95] showed increases in HSP expression in proximal tubule cells immediately after isolation with no further increase with heat stress. If the cells were incubated with continuous motion of culture media, HSP expression was undetectable at 3 h. It remained elevated if the incubation was carried out without media motion. HSP27 increases in cultured human proximal tubule cells with acute exposure to cadmium but decreases with chronic exposure [96]. Gaudio et al. [97] have demonstrated a greater increase in HSP72 mRNA levels in tubules from 8- to 10-day-old rat than in those from adult rats following hyperthermia, oxygen stress and anoxia [97]. Greater increases in HSP72 protein and preservation of renal function have also been demonstrated after ischemia in the kidneys of 10-day-old rats as compared to mature rats [98]. The increased expression of this stress response protein may be one explanation for the resistance of the immature kidney to ischemia. Gender differences in the expression of HSP72 and HSP60 in the kidney have also been demonstrated [99]. The protection we observed after renal ischemia in the rat (fig. 2) was dependent on the timing of ischemia after heat. A lack of protection at 48 h after hyperthermia is consistent with the observations of Joannidis et al. [86]. In a rat model of lung transplantation, hyperthermia 6 but not 12 h prior to organ harvest results in protection from ischemia/reperfusion injury [100].

HSPs and Apoptosis after Ischemia [33]

Protection from ischemic injury with HSP induction most likely occurs via multiple mechanisms. Different postulated factors – for example, maintenance of ATP levels [25], decreases in oxidative stress [101], decreases in cytokine levels [102, 103], maintenance of cytoskeletal and tight junction [104] integrity – may be critical in different systems. Evidence suggests that induction of several different HSPs results in decreased apoptosis [105–108] and necrosis [33]. Apoptosis has been demonstrated in human ARF [109] and limiting apoptosis in experimental models preserves renal function [110, 111]. Increased HSP70/HSP72 expression results in inhibition of apoptosis in several cell types [112, 113]. Decreased apoptosis and decreased tumor necrosis factor ␣ production have been demonstrated in cultured renal tubular cells after simulated ischemia [114]. In vivo, induction of HSP70 with erythropoietin is associated with increased expression of the antiapoptotic bcl-2 and decreased


95

caspase-3 proteolytic activity after ischemia [115]. Interestingly, a recent study found widespread caspase 3 cleavage without cell death in preconditioned neural tissue [116]. Wang et al. [105] demonstrated decreased apoptosis in opossum kidney renal tubular cells with heat stress or overexpression of HSP72 [117] and nucleotide depletion with cyanide and 2-deoxyglucose. HSP70 can prevent the release of cytochrome c from mitochondria and the processing of procaspase 9 and 3 [106] as well as the assembly of the apoptosome [118]. HSP27 [108] and HSP70 [106] have been shown to inhibit the activation of c-Jun-N-terminal kinase, an early component of the stress-induced apoptotic signaling pathway. HSP72 induction in opossum kidney renal tubular cells decreases mitochondrial membrane injury and caspase-3 activation [117, 119] and inhibits degradation of focal adhesion kinase, an antiapoptotic protein [120], after simulated ischemia. HSP25/27 and ␣B-crystallin expression are also associated with resistance to apoptosis and increases cellular glutathione (which can also limit oxidative stress) [121]. However, as in other systems, the role of HSPs may depend on the exact conditions. Membrane (vs. cytoplasmic) expression of HSPs is positively associated with apoptosis in lymphocytes [122, 123].

HSPs and Postischemic Inflammation

The presence of inflammatory cells is a characteristic feature of human ischemic ARF [124]. Leukocytes generate reactive oxygen species and other mediators, which can damage cells, increase vascular permeability and decrease regional blood flow [125]. The induction of HSPs may also afford protection from subsequent insults by decreasing leukocyte infiltration of postischemia tissue [100]. Javadpour et al. [126] demonstrated fewer pulmonary neutrophils and less myeloperoxidase activity after aortic occlusion in rats with prior induction of HSPs via hyperthermia or pharmacological means [127]. These investigators also demonstrated that hyperthermia prevents decreased leukocyte rolling velocity seen with mesenteric ischemia/reperfusion [128]. Hyperthermia also results in decreased activity of the granulocyte enzyme myeloperoxidase in transplanted lung tissue [100] and intestinal neutrophil infiltration and mucosal injury after intestinal ischemia [129]. Induction of HSP72 via hyperthermia or exposure to sodium arsenite results in attenuation of neutrophil-mediated necrosis of endothelial cells in vitro [130]. Ischemiainduced increases in neutrophil products in the skin are markedly attenuated with induction of the stress response [131]. We have demonstrated decreased renal myeloperoxidase activity with hyperthermia prior to renal ischemia [89]. Decreases in ischemia-induced myeloperoxidase activity with prior hyperthermia have also been demonstrated by Stokes et al. [132]. In addition, decreases

Kelly

96

in the production of tumor necrosis factor ␣ have been demonstrated in the heart with the induction of HSP72 [133]. Decreases in tumor necrosis factor and interleukin-8 production have been found in cultured bronchial epithelial cells with heat shock [102]. Decreases in cytokines may also decrease tissue leukocyte infiltration. In contrast, it has been shown that HSP70, when extracellular, can act as a cytokine and increase the expression of proinflammatory mediators in leukocytes [134] and cytotoxic T-lymphocyte responses [135].

Negative Effects

Although the induction of stress response proteins is protective in many experimental models, the stress response also has potentially deleterious effects. In Drosophila, large increases in HSP70 decrease thermotolerance [136]. In the kidney, HSPs induced after renal injury may be antigenic targets. T-lymphocytes from rejected human renal allografts have been shown to proliferate in response to HSP72 [137]. In human acute and chronic allograft rejection, a marked increase in the stress response proteins HSP60 and HDJ2 (a member of the HSP40 family of chaperones) has been found [138]. AntiHSP60 antibodies are found in patients with ischemic heart disease and correlate with severity [139]. HSP47 is induced and is correlated with interstitial fibrosis and infiltrating macrophages in chronic allograft rejection [140]. In mice, cadmium chloride results in induction of HSP70 on renal tubular epithelia in vivo and in vitro. Isolated HSP-reactive T cells from these mice are cytotoxic to stressed renal tubular cells and can affect the passive transfer of interstitial nephritis in mice [141]. Although heat certainly has many effects in addition to the induction of HSPs, Chatson et al. [87] found that heat stress was lethal in 6% of rats and induced a coagulopathy in 40% of heat-stressed animals.

Therapeutic Strategies

Presently available supportive therapies for ARF are clearly inadequate. Recurrent episodes of ischemia occur in patients, although preconditioning has not been investigated in the human kidney. Ischemic preconditioning has been demonstrated in other organs. In a prospective, randomized trial, ischemic preconditioning resulted in less liver injury (lower aspartate aminotransferase and alanine aminotransferase) in patients undergoing liver resection [142]. Increases in HSPs are found in patients with myocardial ischemia [143]. Angina immediately prior to myocardial infarction is associated with smaller infarct


97

size and better survival [144, 145]. In both the liver and heart, ischemic preconditioning is limited to younger patients [142, 146], consistent with animal studies. In a retrospective analysis of 2,379 patients, those with prior transient ischemic attacks were less likely to have an altered level of consciousness on admission and more likely to have minimal functional deficits one month after a cerebral infarction [147]. In humans, less endothelial dysfunction and less leukocyte activation after forearm ischemia have been demonstrated with ischemic preconditioning [148]. Pharmacological strategies to increase stress protein expression have potential merit to prevent ischemic injury to the kidney and other organs. Proteasome inhibitors transiently elevate the level of unfolded proteins inside cells, increase the expression of HSPs and have been shown to confer thermotolerance to Madin-Darby canine kidney cells [149]. Arachidonate [150] and indomethacin [151] induce HSF-1 DNA-binding activity, induce HSP transcription and lower the temperature threshold for HSF activation. Bimoclomol ([2-hydroxy-3-(1-piperidinyl) propoxy]-3-pyridinecarboximidoilchloride maleate; BRPL-42) increases the expression of HSPs in stressful (but not physiological) conditions by prolonging activation of HSF-1 [152]. Addition of bimoclomol to cultured myogenic cells 16 h prior to heat stress increases the level of HSP60, HSP70, HSP90 and GRP94 to levels above that observed with hyperthermia alone and increases resistance of HeLa cells to hyperthermia. In isolated perfused hearts, bimoclomol alone did not increase HSP70 mRNA or protein levels but the levels following ischemia and bimoclomol were significantly higher than those seen with ischemia alone. Hearts perfused with bimoclomol had less evidence of ischemic injury [153]. Oral bimoclomol decreases myocardial infarct size in rats when administered 6 h but not 3 or 18 h prior to ischemia. In this study infarct size was correlated with HSP70 expression [154]. In dogs, bimoclomol was shown to decrease ST elevations secondary to coronary occlusion when given only 5 min prior to ischemia [155]. Clinical trials, however, have not yielded significant results [156]. Similar inducers of HSPs have been developed [157]. Tunicamycin, herbimycin and geldanamycin can also result in increased expression of stress response proteins [149]. Hypothermia induces HSP70 expression in mouse kidney and other tissues [158], suggesting that the stress response may have a role in organ preservation. In recent animal studies, preconditioning consisted of raising core temperature 1⬚C for 15 min each day for 5 consecutive days. This resulted in protection in aortic cross-clamping and mesenteric ischemia models, suggesting that thermal preconditioning may be possible in the clinical situation [159, 160]. Gene therapy also has the potential to increase the expression of HSPs or HSF-1 and confer protection from ischemia. In the kidney, effective therapies will require early diagnosis and treatment [125]. Although many agents are protective in experimental renal ischemia,

Kelly

98

studies in human ARF have yielded disappointing results. There are many considerations in designing potential therapies, but one consideration is early diagnosis. In the synthetic atrial natriuretic peptide trial, the mean serum creatinine at enrollment was approximately 4.5 mg/dl [161]. In the trial of insulin-like growth factor-1, the mean serum creatinine at enrollment was ⬎6 mg/dl and iothalamate clearance ⬍8 ml/min [162]. Urinary HSP72 has been found after renal ischemia in recipients of allografts within 12 h but not in chronic renal disease, stable transplant patients or in rats with renal induction of HSP72 after heat, suggesting that urinary HSP72 is a potential clinically relevant marker of renal ischemia [163]. The kidney, unlike many other organs, has the potential for complete recovery from an ischemic insult [164]. There is some evidence that HSPs have a role in renal regeneration [73]. Studies in vitro and in animal models have shown that protection with induction of stress response proteins is dependent on many factors, including the specific stress response proteins induced and the nature and timing of the insults examined. In addition, it is possible that protective mechanisms may have negative effects if induced to an extreme degree. Further studies will hopefully establish the means and parameters in which induction of endogenous protective mechanisms will improve clinical outcomes. In addition, it is possible that HSP induction may serve as one biomarker of renal injury in the future.

Acknowledgments The work from the author’s laboratory was supported in part by an award from the National Institutes of Health (DK02364) and portions have been published in Kidney International 59: 1798–1802, 2001. The author is grateful to Drs. B. Molitoris, P Dagher, T. Sutton and J. Bonventre for their helpful discussions and E. Caldwell and N. Ray for their technical assistance.

References 1 2 3 4

5

Nolan C, Anderson R: Hospital-acquired acute renal failure. J Am Soc Nephrol 1998;9:710–718. Corwin HL, Bonventre JV: Factors influencing survival in acute renal failure. Semin Dial 1989;2:220–225. Chertow G: On the design and analysis of multicenter trials in acute renal failure. Am J Kidney Dis 1997;30(suppl 4):S96–S101. Hamel M, Phillips R, Davis R, et al: Outcomes and cost-effectiveness of initiating dialysis and continuing aggressive care in seriously ill hospitalized adults. SUPPORT Investigators. Study to understand prognoses and preferences for outcomes and risks of treatments. Ann Intern Med 1997;127:195–202. Hakim RM, Wingard RL, Parker RA: Effect of dialysis membrane in the treatment of patients with acute renal failure. N Engl J Med 1994;331:1343–1346.


99

6 7 8 9 10 11 12 13 14 15

16

17 18

19

20 21 22

23 24

25

26 27 28 29

Cecka M: Clinical outcome of renal transplantation. Factors influencing patient and graft survival. Surg Clin North Am 1998;78:133–148. Murry C, Jennings R, Reimer K: Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–1136. Zager R, Jurkowitz M, Merola A: Responses of the normal rat kidney to sequential ischemic events. Am J Physiol 1985;249:F148–F159. Zager R, Baltes L, Sharma H, Jurkowitz M: Responses of the ischemic acute renal failure kidney to additional ischemic events. Kidney Int 1984;26:689–700. Zager R, Baltes L: Progressive renal insufficiency induces increasing protection against ischemic acute renal failure. J Lab Clin Med 1985;103:511–523. Yoshioka T, Bills T, Moore-Jarrett T, Greene H, Burr I, Ichikawa I: Role of intrinsic antioxidant enzymes in renal oxidant injury. Kidney Int 1990;38:282–288. Islam C, Mathie R, Dinneen M, Kiely E, Peters A, Grace P: Ischaemia-reperfusion injury in the rat kidney: The effect of preconditioning. Br J Urol 1999;79:842–847. Zager R, Iwata M, Burkhart K, Schimpf B: Post-ischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia. Kidney Int 1994;45:1760–1768. Turman M, Bates C: Susceptibility of human proximal tubular cells to hypoxia: Effect of hypoxic preconditioning and comparison to glomerular cells. Ren Fail 1997;19:47–60. Park KM, Chen A, Bonventre JV: Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J Biol Chem 2001;276:11870–11876. Park KM, Byun JY, Kramers C, Kim JI, Huang PL, Bonventre JV: Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney. J Biol Chem 2003;278:27256–27266. MacNider WD: A pathological and physiological study of the naturally nephropathic kidney of the dog, rendered acutely nephropathic by uranium or by an anesthetic. J Med Res 1916;34:199–230. Vogt BA, Shanley TP, Croatt A, Alam J, Johnson KJ, Nath KA: Glomerular inflammation induces resistance to tubular injury in the rat. A novel form of acquired, heme oxygenase-dependent resistance to renal injury. J Clin Invest 1996;98:2139–2145. Park KM, Kramers C, Vayssier-Taussat M, Chen A, Bonventre JV: Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J Biol Chem 2002;277:2040–2049. Pell TJ, Baxter GF, Yellon DM, Drew GM: Renal ischemia preconditions myocardium: Role of adenosine receptors and ATP-sensitive potassium channels. Am J Physiol 1998;275:H1542–H1547. Leung N, Croatt AJ, Haggard JJ, Grande JP, Nath KA: Acute cholestatic liver disease protects against glycerol-induced acute renal failure in the rat. Kidney Int 2001;60:1047–1057. Strickland E, Qu BH, Millen L, Thomas PJ: The molecular chaperone Hsc70 assists the in vitro folding of the N-terminal nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 1997;272:25421–25424. Amin J, Ananthan J, Voellmy R: Key features of heat shock regulatory elements. Mol Cell Biol 1988;8:3761–3769. Xia W, Voellmy R: Hyperphosphorylation of heat shock transcription factor 1 is correlated with transcriptional competence and slow dissociation of active factor trimers. J Biol Chem 1997;14: 4094–4102. Van Why SK, Mann AS, Thulin G, Zhu XH, Kashgarian M, Siegel NJ: Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat. J Clin Invest 1994;94: 1518–1523. Eickelberg O, Seebach F, Riordan M, et al: Functional activation of heat shock factor and hypoxiainducible factor in the kidney. J Am Soc Nephrol 2002;13:2094–2101. Benjamin I, McMillan D: Stress (heat shock) proteins: Molecular chaperones in cardiovascular biology and disease. Circ Res 1998;83:117–132. Van Why SK, Siegel NJ: Heat shock proteins in renal injury and recovery. Curr Opin Nephrol Hypertens 1998;7:407–412. Welch WJ, Suhan JP: Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol 1986;103:2035–2052.

Kelly

100

30 31 32

33 34 35 36 37

38 39

40 41

42 43 44 45 46 47 48 49 50 51

52 53 54 55

Hout J, Houle F, Spitz D, Landry J: HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 1996;56:273–279. Lewis MJ, Pelham HR: Involvement of ATP in the nuclear and nucleolar functions of the 70 kD heat shock protein. EMBO J 1985;4:3137–3143. Liu H, Bowes RC 3rd, van de Water B, Sillence C, Nagelkerke JF, Stevens JL: Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative, stress, Ca2⫹ disturbances, and cell death in renal epithelial cells. J Biol Chem 1997;272:21751–21759. Takayama S, Reed JC, Homma S: Heat-shock proteins as regulators of apoptosis. Oncogene 2003;22:9041–9047. Gething MJ, Sambrook J: Protein folding in the cell. Nature 1992;355:33–45. Lindquist S, Craig E: The heat-shock proteins. Ann Rev Genet 1988;22:631–677. Arrigo AP, Suhan JP, Welch WJ: Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein. Mol Cell Biol 1988;8:5059–5071. Hutter M, Sievers R, Barbosa V, Wolfe C: Heat-shock protein induction in rat hearts. A direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection. Circulation 1994;89:355–360. Hutter J, Mestril R, Tam E, Sievers R, Dillmann W, Wolfe C: Overexpression of heat shock protein 72 in transgenic mice decreases infarct size in vivo. Circulation 1996;94:1408–1411. Marber M, Mestril R, Chi S, Sayen M, Yellon D, Dillmann W: Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 1995;95:1446–1456. Plumier J, Ross B, Currie R, et al: Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest 1995;95:1854–1860. Amin V, Cumming DV, Latchman DS: Over-expression of heat shock protein 70 protects neuronal cells against both thermal and ischaemic stress but with different efficiencies. Neurosci Lett 1996;206:45–48. Papadopoulos M, Sun X, Cao J, Mivechi N, Giffard R: Over-expression of HSP-70 protects astrocytes from combined oxygen-glucose deprivation. Neuroreport 1996;7:429–432. Akcetin Z, Pregla R, Darmer D, et al: Differential expression of heat shock proteins 70–1 and 70–2 mRNA after ischemia-reperfusion injury of rat kidney. Urol Res 1999;27:306–311. Zager RA, Iwata M, Burkhart KM, Schimpf BA: Post-ischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia. Kidney Int 1994;45:1760–1768. Wang YH, Borkan SC: Prior heat stress enhances survival of renal epithelial cells after ATP depletion. Am J Physiol 1996;270:F1057–F1065. Van Why SK, Hildebrandt F, Ardito T, Mann AS, Siegel NJ, Kashgarian M: Induction and intracellular localization of HSP-72 after renal ischemia. Am J Physiol 1992;263:F769–F775. Emami A, Schwartz JH, Borkan SC: Transient ischemia or heat stress induces a cytoprotectant protein in rat kidney. Am J Physiol 1991;260:F479–F485. Schober A, Müller E, Thurau K, Beck FX: The response of heat shock proteins 25 and 72 to ischaemia in different kidney zones. Pflügers Arch 1997;434:292–299. Ling H, Edelstein C, Gengaro P, et al: Attenuation of renal ischemia-reperfusion injury in inducible nitric oxide synthase knockout mice. Am J Physiol 1999;277:F383–F390. Fink AL: Chaperone-mediated protein folding. Physiol Rev 1999;79:425–449. Muller E, Neuhofer W, Ohno A, Rucker S, Thurau K, Beck F-X: Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney. Pflügers Arch 1996;431:608–617. Arrigo AP, Welch WJ: Characterization and purification of the small 28,000-Da mammalian heat shock protein. J Biol Chem 1987;262:15359–15369. Iwaki T, Iwaki A, Liem RKH, Goldman JE: Expression of ␣B-crystallin in the developing rat kidney. Kidney Int 1991;40:52–56. Smoyer WE, Ransom R, Harris RC, Welsh MJ, Lutsch G, Bendorf R: Ischemic acute renal failure induces differential expression of small heat shock proteins. J Am Soc Neph 2000;11:211–221. Golenhofen N, Ness W, Koob R, Htun P, Schaper W, Drenckhahn D: Ischemia-induced phosphorylation and translocation of stress protein ␣B-crystallin to Z lines of myocardium. Am J Physiol 1998;274:H1457–H1464.


101

56 57 58 59 60

61 62

63 64

65

66

67

68 69

70

71 72 73 74

75

76 77 78

Bennardini F, Wrzosek A, Chiesi M: Alpha B-crystallin in cardiac tissue. Association with actin and desmin filaments. Circ Res 1992;71:288–294. Martin J, Mestril R, Hilal-Dandan R, Bruton L, Dillmann W: Small heat shock proteins and protection against ischemic injury in cardiac myocytes. Circulation 1997;96:4343–4348. Arai H, Atomi Y: Chaperone activity of alpha B-crystallin suppresses tubulin aggregation through complex formation. Cell Struct Funct 1997;22:539–544. Nicholl ID, Quinlan RA: Chaperone activity of ␣-crystallins modulates intermediate filament assembly. EMBO J 1994;13:945–953. Aufricht C, Ardito T, Thulin G, Kashgarian M, Siegel NJ, Van Why SK: Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia. Am J Physiol 1998;274:F215–F222. Schober A, Burger-Kentischer A, Muller E, Beck F: Effect of ischemia on localization of heat shock protein 25 in kidney. Kidney Int 1998;54 (suppl 67):S174–S176. Lavoie JN, Hickey E, Weber LA, Landry J: Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J Biol Chem 1993;268: 24210–24214. Loktionova S, Ilyinskaya O, Kabakov A: Early and delayed tolerance to simulated ischemia in heatpreconditioned endothelial cells: A role for HSP27. Am J Physiol 1998;275: H2147–H2158. Aufricht C, Bidmon B, Ruffingshofer D, et al: Ischemic conditioning prevents Na,K-ATPase dissociation from the cytoskeletal cellular fraction after repeat renal ischemia in rats. Pediatr Res 2002;51:722–727. Motterlini R, Foresti R, Intaglietta M, Winslow RM: NO-mediated activation of heme oxygenase: Endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol 1996;270: H107–H114. Lee PJ, Alam J, Wiegand GW, Choi AM: Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc Natl Acad Sci USA 1996;93:10393–10398. Nath KA, Haggard JJ, Croatt AJ, Grande JP, Poss KD, Alam J: The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am J Pathol 2000;156:1485–1488. Hartl F: Molecular chaperones in cellular protein folding. Nature 1996;381:571–579. Lau S, Patnaik N, Sayen MR, Mestril R: Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia-induced injury. Circulation 1997;96:2287–2294. Hernadez-Pando R, Pedraza-Chaverri J, Orozco-Estevez H, et al: Histological and subcellular distribution of 65 and 70 kD heat shock proteins in experimental nephrotoxic injury. Exp Toxicol Pathol 1995;47:501–508. Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996;381:571–579. Aufricht C, Lu E, Thulin G, Kashgarian M, Siegel NJ, Van Why SK: ATP releases HSP-72 from protein aggregates after renal ischemia. Am J Physiol 1998;274:F268–F274. Morita K, Wakui H, Komatsuda A, et al: Induction of heat-shock proteins HSP73 and HSP90 in rat kidneys after ischemia. Ren Fail 1995;17:405–419. Tsuruma T, Yagihashi A, Tarumi K, Sasaki K, Wananabe N, Hirata K: Induction of heat shock protein-73 reduces ischemia-reperfusion injury in rat small intestine. Transplant Proc 1998;30: 3349–3351. Tanaka S, Kitagawa K, Ohtsuki T, et al: Synergistic induction of HSP40 and HSC70 in the mouse hippocampal neurons after cerebral ischemia and ischemic tolerance in gerbil hippocampus. J Neurosci Res 2002;67:37–47. Hutchison KA, Dittmar KD, Czar MJ, Pratt WB: Proof that hsp 70 is required for assembly of the glucocorticoid receptor into a heterocomplex with hsp 90. J Biol Chem 1994;269:5043–5049. Farman N, Oblin ME, Lombes M, et al: Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney. Am J Physiol 1991;260:C226–C233. Bidmon B, Endemann M, Muller T, Arbeiter K, Herkner K, Aufricht C: HSP-25 and HSP-90 stabilize Na,K-ATPase in cytoskeletal fractions of ischemic rat renal cortex. Kidney Int 2002;62: 1620–1627.

Kelly

102

79

80 81 82

83 84

85

86 87 88 89 90 91 92 93

94

95 96

97 98 99

100

101

Molitoris B, Geerdes A, McIntosh J: Dissociation and redistribution of Na⫹,K⫹-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion. J Clin Invest 1991;88:462–469. Molitoris BA, Dahl RH, Falk SA: Ischemic-induced loss of epithelial polarity. Role of the tight junction. J Clin Invest 1989;84:1334–1339. Kwon O, Nelson W, Sibley R, et al: Backleak, tight junctions, and cell-cell adhesion in postischemic injury to the renal allograft. J Clin Invest 1998;101:2054–2064. Kuznetsov G, Bush KT, Zhang PL, Nigam SK: Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia. Proc Natl Acad Sci USA 1996;93:8584–8589. Cheng M, Razzaque MS, Nazneen A, Taguchi T: Expression of the heat shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 1998;79:125–132. Bush KT, George SK, Zhang PL, Nigam SK: Pretreatment with inducers of ER molecular chaperones protects epithelial cells subjected to ATP depletion. Am J Physiol 1999;277: F211–F218. Faccio L, Fusco C, Chen A, Martinotti S, Bonventre JV, Zervos AS: Characterization of a novel human serine protease that has extensive homology to bacterial heat shock endoprotease HtrA and is regulated by kidney ischemia. J Biol Chem 2000;275:2581–2588. Joannidis M, Cantley L, Spokes K, et al: Induction of heat-shock proteins does not prevent renal tubular injury following ischemia. Kidney Int 1995;47:1752–1759. Chatson G, Perdrizet G, Anderson C, Pleau M, Berman M, Schweizer R: Heat shock protects kidneys against warm ischemic injury. Curr Surg 1990;47:420–423. Perdrizet G, Kaneko H, Buckley T, Fishman M, Schweizer R: Heat shock protects pig kidneys against warm ischemic injury. Transplant Proc 1990;22:460–461. Kelly KJ, Baird NR, Greene AL: Induction of stress response proteins and experimental renal ischemia/reperfusion. Kidney Int 2001;59:1798–1802. Kwon O, Phillips CL, Molitoris BA: Ischemia induces alterations in actin filaments in renal vascular smooth muscle cells. Am J Physiol 2002;282:F1012–F1019. Molitoris BA: Putting the actin cytoskeleton into perspective: Pathophysiology of ischemic alterations. Am J Physiol 1997;272:F430-F433. Kaizu T, Tamaki T, Tanaka M, et al: Preconditioning with tin-protoporphyrin IX attenuates ischemia/reperfusion injury in the rat kidney. Kidney Int 2003;63:1393–1403. Santos BC, Pullman JM, Chevaile A, Welch WJ, Gullans SR: Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury. Am J Physiol 2003;284:F564–F574. Halleck M, Liu H, North J, Stevens J: Reduction of trans-4,5-dihydroxy-1,2-dithiane by cellular oxidoreductases activates gadd153/chop and grp78 transcription and induces cellular tolerance in kidney epithelial cells. J Biol Chem 1997;272:21760–21766. Henle KJ, Jethmalani SM, Nolen GT, Wang SY, Nowak G, Schnellmann RG: Stress response in a leporine renal cell model. Nephron 1998;78:54–62. Somji S, Sens DA, Garrett SH, Sens MA, Todd JH. Heat shock protein 27 expression in human proximal tubule cells exposed to lethal and sublethal concentrations of CdCl2. Environ Health Perspect 1999;107:545–552. Gaudio KM, Thulin G, Mann A, Kashgarian M, Siegel NJ: Role of heat stress response in the tolerance of immature renal tubules to anoxia. Am J Physiol 1998;274:1029–1036. Vicencio A, Bidmon B, Ryu J, et al: Developmental expression of HSP-72 and ischemic tolerance of the immature kidney. Pediatr Nephrol 2003;18:85–91. Voss MR, Stallone JN, Li M, Cornelussen RN, Knuefermann P, Knowlton AA: Gender differences in the expression of heat shock proteins: The effect of estrogen. Am J Physiol 2003; 285:H687–H692. Hiratsuka M, Yano M, Mora B, Nagahiro I, Cooper J, Patterson G: Heat shock pretreatment protects pulmonary isografts from subsequent ischemia-reperfusion injury. J Heart Lung Transplant 1998;17:1238–1246. Karmazyn M, Mailer K, Currie R: Acquisition and decay of heat-shock-enhanced postischemic ventricular recovery. Am J Physiol 1990;259:H424-H431.


103

102 Yoo CG, Lee S, Lee CT, Kim YW, Han SK, Shim YS: Anti-inflammatory effect of heat shock protein induction is related to stabilization of I kappa B alpha through preventing I kappa B kinase activation in respiratory epithelial cells. J Immunol 2000;164:5416–5423. 103 Kusher D, Ware C, Gooding L: Induction of heat shock response protects cells from lysis by tumor necrosis factor. J Immunol 1990;145:2925–2931. 104 Borkan SC, Wang YH, Lieberthal W, Burke PR, Schwartz JH: Heat stress ameliorates ATP depletion-induced sublethal injury in mouse proximal tubule cells. Am J Physiol 1997;272: F347-F355. 105 Wang Y, Knowlton AA, Christensen TG, Shih T, Borkan SC: Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells. Kidney Int 1999;55:2224–2235. 106 Mosser DD, Caron AW, Bourget L, et al: The chaperone function of hsp70 is required for protection against stress-induced apoptosis. Mol Cell Biol 2000;20:7146–7159. 107 Mehlen P, Schulze-Osthoff K, Arrigo A: Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1 and staurosporine-induced cell death. J Biol Chem 1996;271:16510–16514. 108 Meriin AB, Gabai VL, Yaglom J, Shifrin VI, Sherman MY: Proteasome inhibitors activate stress kinases and induce Hsp72. Diverse effects on apoptosis. J Biol Chem 1998;273:6373–6379. 109 Solez K, Racusen LC: Role of the renal biopsy in acute renal failure. Contrib Nephrol 2001; 132:68–75. 110 Daemen MA, van’t Veer C, Denecker G, et al: Inhibition of apoptosis induced by ischemiareperfusion prevents inflammation. J Clin Invest 1999;104:541–549. 111 Kelly KJ, Plotkin Z, Dagher PC: Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury. J Clin Invest 2001;108:1291–1298. 112 Mosser D, Caron A, Bourget L, Denis-Larose C, Massie B: Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol 1997;17:5317–5327. 113 DeMeester S, Buchman T, Qiu Y, et al: Heat shock induces IkappaB-alpha and prevents stressinduced endothelial cell apoptosis. Arch Surg 1997;132:1283–1287. 114 Meldrum KK, Burnett AL, Meng X, et al: Liposomal delivery of heat shock protein 72 into renal tubular cells blocks nuclear factor-kappaB activation, tumor necrosis factor-alpha production, and subsequent ischemia-induced apoptosis. Circ Res 2003;92:293–299. 115 Yang CW, Li C, Jung JY, et al: Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney. FASEB J 2003;17:1754–1755. 116 McLaughlin B, Hartnett KA, Erhardt JA, et al: Caspase 3 activation is essential for neuroprotection in preconditioning. Proc Natl Acad Sci USA 2003;100:715–720. 117 Wang YH, Knowlton AA, Borkan SC: Hsp 72 expression enhances survival in adenosine triphosphate-depleted renal epithelial cells. Cell Stress Chaperones 2002;7:137–145. 118 Beere HM, Wolf BB, Cain K, et al: Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2000;2:469–475. 119 Li F, Mao HP, Ruchalski KL, et al: Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells. Am J Physiol 2002;283:C917–C926. 120 Mao H, Li F, Ruchalski K, et al: Hsp72 inhibits focal adhesion kinase degradation in ATP-depleted renal epithelial cells. J Biol Chem 2003;278:18214–18220. 121 Mehlen P, Kretz-Remy C, Preville X, Arrigo AP: Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death. EMBO J 1996;15:2695– 2706. 122 Poccia F, Piselli P, Vendetti S, et al: Heat-shock protein expression on the membrane of T cells undergoing apoptosis. Immunology 1996;88:6–12. 123 Sapozhnikov AM, Ponomarev ED, Tarasenko TN, Telford WG: Spontaneous apoptosis and expression of cell surface heat-shock proteins in cultured EL-4 lymphoma cells. Cell Prolif 1999;32: 363–378. 124 Solez K, Kramer EC, Heptinstall RH: The pathology of acute renal failure (ARF): Leukocyte accumulation in the vasa recta. Am J Pathol 1974;74:31a. 125 Kelly KJ, Molitoris BA: Acute renal failure in the new millennium: Time to consider combination therapy. Semin Nephrol 2000;20:4–19.

Kelly

104

126 Javadpour M, Kelly C, Chen G, Stokes K, Leahy A, Bouchier-Hayes D: Thermotolerance induces heat shock protein 72 expression and protects against ischaemia-reperfusion lung injury. Br J Surg 1998;85:943–946. 127 Javadpour M, Kelly C, Chen G, Bouchier-Hayes D: Herbimycin-A attenuates ischaemia-reperfusion induced pulmonary neutrophil infiltration. Eur J Vasc Endovasc Surg 1998;16:377–382. 128 Chen G, Kelly C, Stokes K, Wang J, Leahy A, Bouchier-Hayes D: Induction of heat shock protein 72 kDa expression is associated with attenuation of ischaemia-reperfusion induced microvascular injury. J Surg Res 1997;69:435–439. 129 Stojadinovic A, Kiang J, Smallridge R, Galloway R, Shea-Donohue T: Induction of heat-shock protein 72 protects against ischemia/reperfusion in rat small intestine. Gastroenterology 1995;109:505–515. 130 Wang J, Redmond H, Watson R, Condron C, Bouchier-Hayes D: Induction of heat shock protein 72 prevents neutrophil-mediated human endothelial cell necrosis. Arch Surg 1995;130:1260–1265. 131 Rees R, Punch J, Shaheen K, Cashmer B, Guice K, Smith DJ Jr: The stress response in skin: The role of neutrophil products in preconditioning. Plast Reconstr Surg 1993;92:110–117. 132 Stokes K, Abdih H, Kelly C, Redmond H, Bouchier-Hayes D: Thermotolerance attentuates ischemia-reperfusion induced renal injury and increased expression of ICAM-1. Transplantation 1996;62:1143–1149. 133 Meng X, Banerjee A, Ao L, et al: Inhibition of myocardial TNF-alpha production by heat shock. A potential mechanism of stress-induced cardioprotection against postischemic dysfunction. Ann NY Acad Sci 1999;874:69–82. 134 Asea A, Rehli M, Kabingu E, et al: Novel signal transduction pathway utilized by extracellular HSP70: Role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem 2002;277:15028–15034. 135 Millar DG, Garza KM, Odermatt B, et al: Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo. Nat Med 2003;9:1469–1476. 136 Krebs RA, Feder ME: Hsp70 and larval thermotolerance in Drosophila melanogaster: How much is enough and when is more too much? J Insect Physiol 1998;44:1091–1101. 137 Trieb K, Grubeck-Loebenstein B, Eberl T, Margreiter R: T cells from rejected human kidney allografts respond to heat shock protein 72. Transpl Immunol 1996;4:43–45. 138 Alevy YG, Brennan D, Durriya S, Howard T, Mohanakumar T: Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidney. Transplantation 1996;61:963–967. 139 Zhu J, Quyyumi AA, Rott D, et al: Antibodies to human heat-shock protein 60 are associated with the presence and severity of coronary artery disease: Evidence for an autoimmune component of atherogenesis. Circulation 2001;103:1071–1075. 140 Abe K, Ozono Y, Miyazaki M, et al: Interstitial expression of heat shock protein 47 and alphasmooth muscle actin in renal allograft failure. Nephrol Dial Transplant 2000;15:529–535. 141 Weiss RA, Madaio MP, Tomaszewski JE, Kelly CJ: T cells reactive to an inducible heat shock protein induce disease in toxin-induced interstitial nephritis. J Exp Med 1994;180:2239–2250. 142 Clavien PA, Selzner M, Rudiger HA, et al: A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning. Ann Surg 2003;238:843–852. 143 Snoeckx LH, Cornelussen RN, Van Nieuwenhoven FA, Reneman RS, Van Der Vusse GJ: Heat shock proteins and cardiovascular pathophysiology. Physiol Rev 2001;81:1461–1497. 144 Bahr RD, Leino EV, Christenson RH: Prodromal unstable angina in acute myocardial infarction: Prognostic value of short- and long-term outcome and predictor of infarct size. Am Heart J 2000;140:126–133. 145 Kloner RA, Shook T, Antman EM, et al: Prospective temporal analysis of the onset of preinfarction angina versus outcome: An ancillary study in TIMI-9B. Circulation 1998;97:1042–1045. 146 Ishihara M, Sato H, Tateishi H, et al: Beneficial effect of prodromal angina pectoris is lost in elderly patients with acute myocardial infarction. Am Heart J 2000;139:881–888. 147 Moncayo J, de Freitas GR, Bogousslavsky J, Altieri M, van Melle G: Do transient ischemic attacks have a neuroprotective effect? Neurology 2000;54:2089–2094. 148 Kharbanda RK, Peters M, Walton B, et al: Ischemic preconditioning prevents endothelial injury and systemic neutrophil activation during ischemia-reperfusion in humans in vivo. Circulation 2001;103:1624–1630.


105

149 150 151

152 153 154 155 156

157

158 159 160 161 162 163 164

Bush KT, Goldberg AL, Nigam SK: Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. J Biol Chem 1997;272:9086–9092. Jurivich D, Sistonen L, Sarge K, Morimoto R: Arachidonate is a potent modulator of human heat shock gene transcription. Proc Natl Acad Sci USA 1994;91:2280–2284. Lee B, Chen J, Angelidis C, Jurivich D, Morimoto R: Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage. Proc Natl Acad Sci USA 1995;92:7207–7211. Hargitai J, Lewis H, Boros I, et al: Bimoclomol, a heat shock protein co-inducer, acts by the prolonged activation of heat shock factor-1. Biochem Biophys Res Commun 2003;307:689–695. Vigh L, Literati P, Horvath I, et al: Bimoclomol: A nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects. Nature Med 1997;3:1150–1154. Lubbers NL, Polakowski JS, Wegner CD, et al: Oral bimoclomol elevates heat shock protein 70 and reduces myocardial infarct size in rats. Eur J Pharmacol 2002;18:79–83. Jednakovits A, Ferdinandy P, Jaszlits L, et al: In vivo and in vitro acute cardiovascular effects of bimoclomol. Gen Pharmacol 2000;34:363–369. Nanasi PP, Jednakovits A: Multilateral in vivo and in vitro protective effects of the novel heat shock protein coinducer, bimoclomol: Results of preclinical studies. Cardiovasc Drug Rev 2001;19:133–151. Torok Z, Tsvetkova NM, Balogh G, et al: Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase. Proc Natl Acad Sci USA 2003;100: 3131–3136. Cullen K, Sarge K: Characterization of hypothermia-induced cellular stress response in mouse tissues. J Biol Chem 1997;272:1742–1746. McCormick PH, Chen G, Tlerney S, Kelly CJ, Bouchier-Hayes DJ: Clinically relevant thermal preconditioning attenuates ischemia-reperfusion injury. J Surg Res 2003;109:24–30. McCormick PH, Chen G, Tierney S, Kelly CJ, Bouchier-Hayes DJ: Clinically applicable thermal preconditioning attenuates leukocyte-endothelial interactions. J Am Coll Surg 2003;197:71–78. Allegren RL, Marbury TC, Rahman SN, et al: Anaritide in acute tubular necrosis. New Engl J Med 1997;336:828–834. Hirschberg R, Kopple J, Lipsett P, et al: Multicenter clinical trial of recombinant human insulinlike growth factor I in patients with acute renal failure. Kidney Int 1999;55:2423–2432. Mueller T, Bidmon B, Pichler P, et al: Urinary heat shock protein-72 excretion in clinical and experimental renal ischemia. Pediatr Nephrol 2003;18:97–99. Pawar S, Kartha S, Toback FG: Differential gene expression in migrating renal epithelial cells after wounding. J Cell Physiol 1995;165:556–565.

Katherine J. Kelly, MD, MSc Indiana University School of Medicine, Division of Nephrology 950 West Walnut Street, RII 201 Indianapolis, IN 46202 (USA) Tel. ⫹1 (317) 274–7453, Fax ⫹1 317 274 8575, E-Mail [email protected]

Kelly

106


Cisplatin-Associated Nephrotoxicity and Pathological Events Takashi Taguchia, Arifa Nazneena, M. Ruhul Abidb, Mohammed S. Razzaquea,c a

Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; bDivision of Molecular and Vascular Medicine, Department of Medicine, and Vascular Biology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass., USA; cDepartment of Oral and Developmental Biology, Harvard School of Dental Medicine, Boston, Mass., USA

Abstract Cisplatin (cis-diamminedichloroplatinum(II)) is an effective chemotherapeutic agent, and is successfully used in the treatment of a wide range of tumors. Despite its effectiveness as an anti-tumor drug, nephrotoxic side effects have significantly restricted its clinical use. Tubular epithelial cell deletion following cisplatin treatment is a major cause of renal injury. Oxidative stress significantly contributes to cisplatin-associated cytotoxicity, and use of antioxidants could counteract such cytotoxic effects of cisplatin. The renal microenvironmental changes following cisplatin treatment is a complex process and could be broadly categorized into three main pathological events, which at times might overlap: initial cytotoxic events, inflammatory events and fibroproliferative events. Stress responses and heat shock proteins generated following cisplatin treatment are actively involved in the initiation and progression of these events. In this article, we will briefly summarize factors involved in various phases of cisplatin-induced renal injuries. Copyright © 2005 S. Karger AG, Basel

Introduction

Cis-dichlorodiaminoplatinum (II), cisplatin, is one of the most widely used antineoplastic drugs. Cisplatin is an inorganic complex formed by an atom of platinum surrounded by chlorine and ammonia atoms in the cis position of a horizontal plane. One of the possible mechanisms by which cisplatin accumulates in the cells is by a carrier-mediated processes, through probenecid-sensitive organic anion transporters; the chloride ions are displaced by hydrolysis, resulting in

Table 1. Partial list of tumors where cisplatin has been used as an antitumor drug

Adrenocortical tumor Bladder tumor Brain tumor Breast tumor Cervical tumor Endometrial cancer Gastrointestinal tumor Germ cell tumor Gynecological sarcoma Head and neck tumor Hepatoblastoma Lung cancer, small cell Malignant melanoma Neuroblastoma Non-Hodgkin’s lymphoma Osteosarcoma Ovarian tumor Testicular tumor Thyroid tumor

the formation of highly reactive, charged platinum complexes. Probenecid restricts renal secretion of anionic drugs through inhibition of the organic anion transport system(s). Coadministration of probenecid has shown to decrease renal excretion of various drugs including cidofovir, ciprofloxacin and cisplatin [1]. Probenecid could interfere with tubular secretion of cisplatin, and thereby could increase cisplatin toxicity. On entry into the cell, the platinum compounds cross-link with DNA; this binding of platinum to complexes of DNA apparently disrupts and unwinds the double helix, especially in the case of intrastrand cross-links to G-rich sequences such as GG and AG [2, 3]. Cisplatin also inflicts mitochondrial damage, induces cell cycle arrest in the G2 phase, reduces ATPase activity, alters cellular transport system, eventually leading to apoptotic and/or necrotic cell death. Cisplatin is the single most active antitumor agent against testicular, bladder, ovarian, lung, head and neck tumors (table 1). The use of cisplatin in combination with drugs such as bleomycin, vinblastine, cyclophosphamide, fluorouracil and doxorubicin has resulted not only in higher effectiveness in treating various tumors, but has also increased the risk of secondary morbidity. Although cisplatin was first synthesized in 1845, the side effects associated with cisplatin treatment were not adequately described until 1965. Cisplatin entered into clinical trials in and around 1971. Despite its effectiveness as an antitumor drug, various side effects (table 2), especially nephrotoxicity, has restricted its clinical use. The nephrotoxic effect of cisplatin is dose limiting [4, 5], and is manifested by a decrease in creatinine clearance and electrolyte

Taguchi/Nazneen/Abid/Razzaque

108

Table 2. Partial list of side effects of cisplatin

Acute encephalopathy Anaphylactic reactions Elevated liver function tests Hair loss Hearing loss Hemolytic anemia Infertility Mucositis Myelosuppression Nausea and vomiting Optic neuropathy Peripheral neuropathy Raynaud’s syndrome Retinopathy Tinnitus Nephrotoxicity

imbalances, particularly hypomagnesemia, mainly due to the acute cytotoxic effect of cisplatin on proximal and distal tubules, and on loop of Henle [6]. Severe magnesium deficiency following cisplatin treatment could result in seizures [7]. Cisplatin-induced excessive urinary loss of magnesium and potassium [8] could be partly restored by supplementation [9, 10]. In addition, both human and experimental studies have shown that the use of diuretics and hydration can substantially reduce cisplatin-associated nephrotoxicity [11, 12]. A detailed and comprehensive review of all aspects of cisplatin-associated toxicity is beyond the scope of this article, which will thus be restricted to various pathological events of cisplatin-associated nephrotoxicity. Cisplatin and Nephrotoxicity

Cisplatin-induced nephrotoxicity is a complex process that comprises of acute cytotoxic effects on tubular epithelial cells, resulting in loss of tubular epithelial cells by necrosis and apoptosis, followed by inflammatory cell infiltration and fibroproliferative changes [13]. From in vivo experimental studies, the progression of cisplatin-induced renal damages can be tentatively divided into three main events, which at times may overlap: initial cytotoxic, inflammatory and fibroproliferative events. Initial Cytotoxic Events It has been convincingly demonstrated that renal tubular dysfunction is the immediate effect of cisplatin treatment. Higher doses of cisplatin induce

Cisplatin-Associated Nephrotoxicity

109

necrosis of tubular epithelial cells, while lower doses remove tubular epithelial cells via apoptosis [14–16]. Cisplatin exerts its cytotoxic effects partly by inhibiting protein synthesis of tubular epithelial cells. Besides, cisplatin disrupts the cellular oxidant defense system (i.e., glutathione, GSH), leading to lipid peroxidation and DNA damage. Cisplatin-associated cytotoxicity and generation of reactive oxygen species (ROS) could be counteracted by using antioxidants such as alpha-tocopherol, vitamin C and N-acetylcysteine [17, 18]. Nephrotoxicity induced by high-doses of cisplatin therapy could be altered by GSH administration [19–22]. GSH treatment could also protect nerve injury following cisplatin therapy, without reducing its antitumor activities [23–25]. A protective role of metallothionein, a scavenger of hydroxyl radicals, against a number of oxidative stress-associated xenobiotics, including cisplatin, has been reported by Bauman et al. [26]. Renal proximal tubular epithelial cells (LLC-PK1), stably transfected with human HSP72 gene, have shown to be resistant to both hydrogen peroxide and cisplatin-induced cellular damage, implicating a protective role of heat shock protein 72 (HSP72) against oxidative injury and cisplatin toxicity [27]. Cisplatin could also activate various proapoptotic molecules including caspase-3 and -9, Bax and Fas system [14, 28, 29]. In vitro studies have shown that cisplatin-induced apoptosis in LLC-PK1 is mediated through activation of mitochondrial signaling pathways, possibly by activating Bax-induced mitochondrial permeability, with release of cytochrome c and activation of caspase-9. A role of caspase-3 has also been reported in cisplatin-induced apoptosis in LLC-PK1 cells, and shown to be prevented by bcl-2 [30]. Moreover, a relationship between loss of cytoskeletal F-actin stress fibers and cisplatin-induced apoptosis has been shown in renal epithelial cells (within 4–6 h), and prevention of F-actin damage by phalloidin has shown to prevent nuclear fragmentation of these cells [31]. van de Water et al. [32] reported that decreased phosphorylation of focal adhesion kinase was related to loss of focal adhesions and F-actin stress fibers, leading to the onset of apoptosis in renal tubular epithelial cells caused by nephrotoxicants. In addition, involvement of Fas/Fas ligand system has been demonstrated in cisplatin-induced apoptosis in various cells lines [33–37]. Cisplatin- induced apoptosis in human proximal tubular epithelial cells was associated with an increased expression of Fas and its ligand [37]. Similar Fasmediated cisplatin-induced apoptosis has been reported in neuroblastoma [36], leukemia [35] and hepatoma cells [34] and thymocytes [33]; in contrast a Fasindependent cisplatin-induced apoptosis has also been reported in various tumors cell lines [38, 39] including lung cancer cells. It appears likely that cisplatininduced apoptosis does not always take a uniform pathway, and there might be a cell-specific mode of apoptosis. Early cytotoxic events following cisplatin treatment are usually associated with inflammatory changes in the kidneys.


110

Inflammatory Events Detailed inflammatory phenotypes of infiltrating cells in kidney of cisplatintreated patients are not well studied, but data from animal experiments have shown that by day 7, a single dose of cisplatin injection (6 mg/kg body weight) to rats lead to the accumulation of a maximum number of ED-1-positive macrophages in the cortico-medullary junction of the kidneys (fig. 1). The number of accumulated macrophages declined on day 14 and 28 [40–42]. Macrophages, through generation of ROS, could intensify cytotoxic effects encountered following cisplatin treatment. It is well accepted that cytokines and chemokines play a major role in the inflammatory events of various human and experimental diseases. Cisplatin has been reported to induce the expression of inflammatory cytokines, such as interleukin (IL)-1 and IL-6 by endothelial cells isolated from a human umbilical vein [43]. Increased renal expression of tumor necrosis factor-␣, transforming growth factor (TGF)-␤, RANTES, macrophage inflammatory protein-2, macrophage chemoattractant protein-1, thymus-derived chemotactic agent 3, IL-1␤ and intercellular adhesion molecule-1 has been detected in kidneys of cisplatin-treated animals [44]. Recently, salicylate has been shown to reduce experimental cisplatin nephrotoxicity, by inhibition of tumor necrosis factor-␣ production through stabilization of I ␬ B [45]. Moreover, increased interstitial expression of osteopontin has been detected in the kidneys of cisplatin-treated rats [46]. It is likely that tubular epithelial cell-derived chemokines and ROS following cisplatin treatment serve to recruit inflammatory cells, which can contribute to the development of subsequent fibroproliferative lesions by releasing mitogenic and fibrogenic factors, which then act on matrix-producing cells to regulate abnormal matrix remodeling. Fibroproliferative Events Development of irreversible tubulointerstitial fibrosis is a relatively late change found in the kidneys of cisplatin-treated experimental animals. Excessive production of matrix proteins by the activated and phenotypically altered resident cells gradually help in the development of tubulointerstitial fibrosis. An increased expression and deposition of collagens (types I, III and IV) were detected in cisplatin-induced tubulointerstitial fibrosis in rats [47], a pattern that is similar to other experimental models of tubulointerstitial fibrosis [48–51]. Fibrogenic factors, released by the activated and phenotypically altered resident cells (fig. 2) and infiltrating inflammatory cells, such as TGF-␤1 and HSP47, have the potential to mediate both human and experimental fibrotic diseases by regulating increased production of collagens, and thereby matrix remodeling [51–54]. TGF-␤1 affects formation of connective tissue by


111

a

b Fig. 1. Infiltration of ED-1-positive macrophages (arrows) in control (a) and cisplatintreated rat kidneys (b). Note a significantly increased accumulation of macrophages (arrows) in cisplatin-treated rat kidney (b).

stimulating the transcription of genes encoding extra cellular matrix proteins. Studies have convincingly demonstrated that blocking TGF-␤1 results in the suppression of collagen production and subsequent modulation of fibrotic processes [55, 56]. A fibrogenic role for TGF-␤1 has been reported in kidneys of patients with various renal diseases [54, 55, 57]. In the kidneys of cisplatintreated rats, an increased expression of TGF-␤1 has been detected in tubular


112

G

G

G

G

a

b

c

d Fig. 2. Immunostaining of ␣-smooth muscle actin in a control rat kidney (a), showing positive staining mainly in the vessel walls (arrows); increased interstitial expression of ␣-smooth muscle actin (arrowheads) is noted in cisplatin-treated rat kidney (b), suggesting phenotypically altered myofibroblast proliferation following cisplatin treatment. No significant expression of ␣-smooth muscle actin was detected in the glomeruli (denoted as G) in both control and kidneys of cisplatin-treated rat. For vimentin, only intraglomerular staining (arrows) is noted in the control rat kidney (c). Note no staining for vimentin in the tubular epithelial cells in the control rat kidney. Strong positive staining for vimentin is noted in the tubular epithelial cells (arrowheads) and interstitial cells in cisplatin-treated rat kidney (d), suggesting phenotypically altered tubular epithelial cells following cisplatin treatment.

epithelial cells and interstitial cells, by in situ hybridization [58]. Further studies are needed to determine the effects of increased expression of TGF-␤1 in cisplatin nephritis, and the role of TGF-␤1-induced molecules, including connective tissue growth factor, in such fibroproliferative lesions [59–61]. In addition, c-myc, ets-1, platelet-derived growth factor, ILs, interferon-␥, tumor necrosis factor, epidermal growth factor, insulin-like growth factor and its binding proteins, angiotensin II and tissue transglutaminase, have shown to play roles in the development of fibroproliferative lesions in various human and experimental renal diseases. Interestingly, by microarray analysis, a number of these above-mentioned molecules were detected in the kidneys of cisplatin-treated rats [62].


113

HSP47, a collagen-specific molecular chaperone, is involved in the biosynthesis and secretion of procollagens [63]. HSP47 has shown to play important role in the development of fibroproliferative changes by post-transcriptionally regulating increased production of collagens. For instance, upregulation in the expression of HSP47 with increased interstitial accumulation of collagens (types I and III) has been reported in various human and experimental fibrotic renal diseases [53, 64, 65]. Similar upregulation of HSP47, in association with increased accumulation of type I and III collagens, was also detected in kidneys of cisplatin-treated rats [47]. Phenotypically altered tubular epithelial cells, interstitial fibroblasts and myofibroblasts were HSP47-expressing cells in kidneys of cisplatin-treated rats [47]. Although further studies are warranted, at this stage, HSP47 appears to play a role in the development of fibroproliferative lesions in the kidneys following cisplatin treatment. In addition to HSP47, induction of several other HSPs (HSP-70, -90) has been reported during early stages of cisplatin nephropathy [66]. Production of extracellular matrix is mainly achieved through the synthesis of collagens, whereas resorption of the extracellular matrix is mediated predominantly by the matrix metalloproteinases (MMPs). A delicate balance between matrix synthesis and its degrading enzymes (MMPs) is essential for maintaining normal structural stability and integrity of tissues and organs. An imbalance in the production and utilization of matrix proteins lead to pathological matrix remodeling. In the kidneys of cisplatin-treated rats, the expression of MMP-1 has shown to increase in early stages (on day 3) of cisplatin nephropathy, while the expression decreased in later stages (on day 14). Decreased renal expression of MMP-1 has been shown to be associated with increased interstitial accumulation of type III collagen in kidneys of cisplatin-treated rats [67], suggesting a pathological role of MMPs in cisplatin-nephropathy.

Modulation of Cisplatin-Induced Nephrotoxicity

The beneficial antineoplastic use of cisplatin is often limited because of its significant side effects, including nephrotoxicity. Following standard-dose regimens, one third of patients usually develop varying degrees of cisplatinrelated side effects. Numerous human and experimental studies have been performed to understand the mechanism of cisplatin-associated nephrotoxicity, and thereby to minimize its side effects. Several strategies have been explored to reduce the side effects of cisplatin therapy, including the use of less intensive treatment, replacement of the nephro- and neurotoxic cisplatin by its less toxic analog carboplatin. Carboplatin generates a reactive species much more slowly than with cisplatin. Therefore its pharmacokinetic and toxicological


114

characteristics are different. Moreover, plasma half-life of carboplatin is several-fold longer than that of cisplatin. Needless to mention that carboplatin also exerts unwarranted side effects that include fatigue, bone marrow dysfunction and loss of fertility. Aggressive hydration with saline, often with the addition of mannitol, has been used to reduce cisplatin-induced nephrotoxicity. Two liters of 5% dextrose in 0.5 N saline over 12–24 h before treatment and at least 24 h of intravenous fluid afterward is helpful in minimizing the kidney damage after cisplatin treatment. Amifostine (Ethyol) is an organic thiophosphate compound with a cytoprotective potential. The active free thiol metabolite can reduce the toxic effects of cisplatin on the kidney, possibly by binding to free radicals generated in the tissues. Patients treated with amifostine prior to cisplatin therapy were reported to have less renal damage compared with patients treated with cisplatin alone [68–70]. In experimental models, preadministration of a zinc-histidine complex has been reported to reduce cisplatin-induced renal damage, possibly by preventing peroxidative damage [71]. Recently heme oxygenase-1 (HO-1), a 32-kDa microsomal enzyme, has been shown to attenuate cisplatin-induced apoptosis and necrosis. It has been shown that compared to wild-type mice (HO-1⫹/⫹), cisplatin-treatment intensified renal injury in homozygous mice with a targeted deletion of the HO-1 gene (HO-1⫺/⫺) [72]. Studies have also shown that the upregulation of p21, a cyclin-dependent kinase inhibitor, attenuated cisplatin-induced renal dysfunction, apoptotic cell death and tubular damage [73]. A protective role of p21 has also been shown in p21 knockout mice treated with cisplatin [74]. In vitro treatment of renal epithelial cells (mIMCD-3) with cisplatin could induce apoptosis, while constitutive expression of hepatocyte growth factor by transfection in mIMCD-3 cells developed resistance to cisplatin-induced apoptotic death, implicating that hepatocyte growth factor may ameliorate cisplatin-associated renal injury, by protecting renal epithelial cells from undergoing apoptosis [75]. Cisplatin-associated nephrotoxicity has been reported to be modified by taurine treatment in rats. Compared to cisplatintreated rats, taurine-treated rats showed relatively less renal damage, as determined by histo-morphometric analysis. Taurine-treatment resulted in less macrophage accumulation and delayed interstitial fibrotic changes in cisplatintreated rat kidneys [76, 77]. Recently, ebselen has shown to be nephroprotective in cisplatin-treated rats, possibly exerting its beneficial effects by modulating the antioxidant system [78, 79]. Similarly, treatment of myeloma cells with N-acetylcysteine completely blocked cisplatin-associated intracellular GSH oxidation, ROS generation, poly(ADP-ribose) polymerase cleavage and apoptosis [80]. Use of a novel free radical scavenger, 3-methyl-1-phenyl-pyrazolin5-one (MCI-186; edaravone) has also been shown to protect the kidneys from


115

Cisplatin

Oxidative injury

Chemokines

Cytotoxicity

Macrophages

Cytochrome c caspase-3, -9 Fas/FasL HSP47

Phenotypic alteration of tubulointerstitial cells

TGF-␤1

Apoptosis Matrix remodeling

PDGF

Tubulointerstitial injury

Fig. 3. Schematic diagram showing involvement of various molecules involved in initiation and progression of cisplatin-induced nephrotoxicity. TGF-␤1 ⫽ Transforming growth factor; PDGF ⫽ platelet-derived growth factor; HSP47 ⫽ heat shock protein 47.

developing acute renal failure following cisplatin treatment [81]; edaravone, a lipophilic compound, has been shown to trap both hydroxyl radicals and prevent iron-induced peroxidative injuries [82]. These studies suggest a beneficial role in the use of a free radical scavenger in modulating cisplatin-associated nephrotoxicity.

Conclusion

Despite prophylactic intensive hydration and forced diuresis, irreversible renal damage occurs in about one third of cisplatin-treated patients. Cisplatininduced renal damage is usually associated with acute stress-related injuries, focal necrosis and apoptosis of the tubular epithelial cells and dilatation of tubules with cast formation. Inflammatory events initiated due to cytotoxic


116

stress responses of cisplatin facilitate activation of resident cells to release of profibrogenic factors, which induces excessive production of matrix proteins, resulting in irreversible tubulointerstitial injuries (fig. 3). Further studies characterizing the molecules involved in acute stress responses following cisplatin treatment, and determining their molecular interactions in various stages of nephrotoxicity, would help in developing strategies to make a focused approach to minimize cisplatin-associated nephrotoxicity, without reducing or interfering with its antitumor effects. At this stage, modulating oxidative stress following cisplatin treatment appears to be a promising option to reduce its side effects, including nephrotoxicity. Acknowledgments We deeply appreciate the kind cooperation and the technical assistance of staff members of the Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences. Our apology goes to all authors whose work could not be cited due to space limitations.

References 1 2

3

4 5

6 7 8 9 10 11 12

Dresser MJ, Leabman MK, Giacomini KM: Transporters involved in the elimination of drugs in the kidney: Organic anion transporters and organic cation transporters. J Pharm Sci 2001;90: 397–421. Chao CC, Shieh TC, Huang H: Use of a monoclonal antibody to detect DNA damage caused by the anticancer drug cis-diamminedichloroplatinum (II) in vivo and in vitro. FEBS Lett 1994;354: 103–109. Burstyn JN, Heiger-Bernays WJ, Cohen SM, Lippard SJ: Formation of cis-diamminedichloroplatinum(II) 1,2-intrastrand cross-links on DNA is flanking-sequence independent. Nucleic Acids Res 2000;28:4237–4243. Moul JW, Robertson JE, George SL, Paulson DF, Walther PJ: Complications of therapy for testicular cancer. J Urol 1989;142:1491–1496. Daugaard G, Rossing N, Rorth M: Effects of cisplatin on different measures of glomerular function in the human kidney with special emphasis on high-dose. Cancer Chemother Pharmacol 1988;21:163–167. Safirstein R, Winston J, Goldstein M, Moel D, Dikman S, Guttenplan J: Cisplatin nephrotoxicity. Am J Kidney Dis 1986;8:356–367. van de Loosdrecht AA, Gietema JA, van der Graaf WT: Seizures in a patient with disseminated testicular cancer due to cisplatin-induced hypomagnesaemia. Acta Oncol 2000;39:239–240. Buckley JE, Clark VL, Meyer TJ, Pearlman NW: Hypomagnesemia after cisplatin combination chemotherapy. Arch Intern Med 1984;144:2347–2348. Whang R, Whang DD, Ryan MP: Refractory potassium repletion. A consequence of magnesium deficiency. Arch Intern Med 1992;152:40–45. Rodriguez M, Solanki DL, Whang R: Refractory potassium repletion due to cisplatin-induced magnesium depletion. Arch Intern Med 1989;149:2592–2594. Roth BJ, Einhorn LH, Greist A: Long-term complications of cisplatin-based chemotherapy for testis cancer. Semin Oncol 1988;15:345–350. Cornelison TL, Reed E: Nephrotoxicity and hydration management for cisplatin, carboplatin, and ormaplatin. Gynecol Oncol 1993;50:147–158.


117

13 14 15 16 17 18 19 20 21

22

23

24

25

26 27

28

29

30 31

32

33

34

Groth S, Nielsen H, Sorensen JB, Christensen AB, Pedersen AG, Rorth M: Acute and long-term nephrotoxicity of cis-platinum in man. Cancer Chemother Pharmacol 1986;17:191–196. Lau AH: Apoptosis induced by cisplatin nephrotoxic injury. Kidney Int 1999;56:1295–1298. Razzaque MS, Ahsan N, Taguchi T: Role of apoptosis in fibrogenesis. Nephron 2002;90: 365–372. Lieberthal W, Menza SA, Levine JS: Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells. Am J Physiol 1998;274(2 pt 2):F315–F327. Schaaf GJ, Maas RF, de Groene EM, Fink-Gremmels J: Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells. Free Radic Res 2002;36:835–843. De Martinis BS, Bianchi MD: Effect of vitamin C supplementation against cisplatin-induced toxicity and oxidative DNA damage in rats. Pharmacol Res 2001;44:317–320. Tedeschi M, De Cesare A, Oriana S, Perego P, Silva A, Venturino P, Zunino F: The role of glutathione in combination with cisplatin in the treatment of ovarian cancer. Cancer Treat Rev 1991;18:253–259. Plaxe S, Freddo J, Kim S, Kirmani S, McClay E, Christen R, Braly P, Howell S: Phase I trial of cisplatin in combination with glutathione. Gynecol Oncol 1994;55:82–86. Fontanelli R, Spatti G, Raspagliesi F, Zunino F, Di Re F: A preoperative single course of highdose cisplatin and bleomycin with glutathione protection in bulky stage IB/II carcinoma of the cervix. Ann Oncol 1992;3:117–121. Di Re F, Bohm S, Oriana S, Spatti GB, Zunino F: Efficacy and safety of high-dose cisplatin and cyclophosphamide with glutathione protection in the treatment of bulky advanced epithelial ovarian cancer. Cancer Chemother Pharmacol 1990;25:355–360. Smyth JF, Bowman A, Perren T, Wilkinson P, Prescott RJ, Quinn KJ, Tedeschi M: Glutathione reduces the toxicity and improves quality of life of women diagnosed with ovarian cancer treated with cisplatin: Results of a double-blind, randomised trial. Ann Oncol 1997;8:569–573. Colombo N, Bini S, Miceli D, Bogliun G, Marzorati L, Cavaletti G, Parmigiani F, Venturino P, Tedeschi M, Frattola L, Buratti C, Mangioni C: Weekly cisplatin ⫹/⫺ glutathione in relapsed ovarian carcinoma. Int J Gynecol Cancer 1995;5:81–86. Cascinu S, Cordella L, Del Ferro E, Fronzoni M, Catalano G: Neuroprotective effect of reduced glutathione on cisplatin-based chemotherapy in advanced gastric cancer: A randomized doubleblind placebo-controlled trial. J Clin Oncol 1995;13:26–32. Bauman JW, Liu J, Liu YP, Klaassen CD: Increase in metallothionein produced by chemicals that induce oxidative stress. Toxicol Appl Pharmacol 1991;110:347–354. Komatsuda A, Wakui H, Oyama Y, Imai H, Miura AB, Itoh H, Tashima Y: Overexpression of the human 72 kDa heat shock protein in renal tubular cells confers resistance against oxidative injury and cisplatin toxicity. Nephrol Dial Transplant 1999;14:1385–1390. Tudor G, Aguilera A, Halverson DO, Laing ND, Sausville EA: Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels. Cell Death Differ 2000;7:574–586. Henkels KM, Turchi JJ: Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines. Cancer Res 1999;59:3077–3083. Zhan Y, van de Water B, Wang Y, Stevens JL: The roles of caspase-3 and bcl-2 in chemicallyinduced apoptosis but not necrosis of renal epithelial cells. Oncogene 1999;18:6505–6512. Kruidering M, van de Water B, Zhan Y, Baelde JJ, Heer E, Mulder GJ, Stevens JL, Nagelkerke JF: Cisplatin effects on F-actin and matrix proteins precede renal tubular cell detachment and apoptosis in vitro. Cell Death Differ 1998;5:601–614. van de Water B, Nagelkerke JF, Stevens JL: Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J Biol Chem 1999;274:13328–13337. Eichhorst ST, Muerkoster S, Weigand MA, Krammer PH: The chemotherapeutic drug 5-fluorouracil induces apoptosis in mouse thymocytes in vivo via activation of the CD95(APO-1/Fas) system. Cancer Res 2001;61:243–248. Muller M, Strand S, Hug H, Heinemann EM, Walczak H, Hofmann WJ, Stremmel W, Krammer PH, Galle PR: Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. J Clin Invest 1997;99: 403–413.


118

35 36 37

38

39

40

41

42

43

44 45 46

47 48

49 50

51

52 53

54 55

Friesen C, Herr I, Krammer PH, Debatin KM: Involvement of the CD95 (APO-1/FAS) receptor/ ligand system in drug-induced apoptosis in leukemia cells. Nat Med 1996;2:574–577. Fulda S, Sieverts H, Friesen C, Herr I, Debatin KM: The CD95 (APO-1/Fas) system mediates drug-induced apoptosis in neuroblastoma cells. Cancer Res 1997;57:3823–3829. Razzaque MS, Koji T, Kumatori A, Taguchi T: Cisplatin-induced apoptosis in human proximal tubular epithelial cells is associated with the activation of the Fas/Fas ligand system. Histochem Cell Biol 1999;111:359–365. Ferreira CG, Tolis C, Span SW, Peters GJ, van Lopik T, Kummer AJ, Pinedo HM, Giaccone G: Drug-induced apoptosis in lung cancer cells is not mediated by the Fas/FasL (CD95/APO1) signaling pathway. Clin Cancer Res 2000;6:203–212. Eischen CM, Kottke TJ, Martins LM, Basi GS, Tung JS, Earnshaw WC, Leibson PJ, Kaufmann SH: Comparison of apoptosis in wild-type and Fas-resistant cells: Chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. Blood 1997;90:935–943. Yamate J, Tatsumi M, Nakatsuji S, Kuwamura M, Kotani T, Sakuma S: Immunohistochemical observations on the kinetics of macrophages and myofibroblasts in rat renal interstitial fibrosis induced by cis-diamminedichloroplatinum. J Comp Pathol 1995;112:27–39. Yamate J, Ishida A, Tsujino K, Tatsumi M, Nakatsuji S, Kuwamura M, Kotani T, Sakuma S: Immunohistochemical study of rat renal interstitial fibrosis induced by repeated injection of cisplatin, with special reference to the kinetics of macrophages and myofibroblasts. Toxicol Pathol 1996;24:199–206. Yamate J, Sato K, Ide M, Nakanishi M, Kuwamura M, Sakuma S, Nakatsuji S: Participation of different macrophage populations and myofibroblastic cells in chronically developed renal interstitial fibrosis after cisplatin-induced renal injury in rats. Vet Pathol 2002;39:322–333. Shi Y, Inoue S, Shinozaki R, Fukue K, Kougo T: Release of cytokines from human umbilical vein endothelial cells treated with platinum compounds in vitro. Jpn J Cancer Res 1998;89: 757–767. Ramesh G, Reeves WB: TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity. J Clin Invest. 2002;110:835–842. Ramesh G, Reeves WB: Salicylate reduces cisplatin nephrotoxicity by inhibition of tumor necrosis factor-alpha. Kidney Int 2004;65:490–499. Iguchi S, Nishi S, Ikegame M, Hoshi K, Yoshizawa T, Kawashima H, Arakawa M, Ozawa H, Gejyo F: Expression of osteopontin in cisplatin-induced tubular injury. Nephron Exp Nephrol 2004;97:e96–e105. Razzaque MS, Taguchi T: Localisation of HSP47 in cisplatin-treated kidney: Possible role in tubulointerstitial damage. Clin Exp Nephrol 1999;3:123–132. Razzaque MS, Shimokawa I, Koji T, Higami Y, Taguchi T: Life-long caloric restriction suppresses age-associated Fas expression in the Fischer 344 rat kidney. Mol Cell Biol Res Commun 1999;1: 82–85. Cheng M, Razzaque MS, Nazneen A, Taguchi T: Expression of the heat shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 1998;79:125–132. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T: Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47. Cell Tissue Res 1998:293:471–478. Razzaque MS, Shimokawa I, Nazneen A, Liu D, Naito T, Higami Y, Taguchi T: Life-long dietary restriction modulates the expression of collagens and collagen-binding heat shock protein 47 in aged Fischer 344 rat kidney. Histochem J 1999;31:123–132. Razzaque MS, Taguchi T: Collagen-binding heat shock protein (HSP) 47 expression in antithymocyte serum (ATS)-induced glomerulonephritis. J Pathol 1997;183:24–29. Razzaque MS, Kumatori A, Harada T, Taguchi T: Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 1998;80: 434–443. Tamaki K, Okuda S: Role of TGF-beta in the progression of renal fibrosis. Contrib Nephrol 2003;139:44–65. Moriyama T, Imai E: Role of myofibroblasts in progressive renal diseases. Contrib Nephrol 2003;139:120–140.


119

56 57 58 59

60 61 62

63 64 65 66

67 68 69

70 71

72

73 74 75 76 77

78

79

Basile DP: Transforming growth factor-beta as a target for treatment in diabetic nephropathy. Am J Kidney Dis 2001;38:887–892. Frishberg Y, Kelly CJ: TGF-beta and regulation of interstitial nephritis. Miner Electrolyte Metab 1998;24:181–189. Razzaque MS, Cheng M, Horita Y, Taguchi T: Cellular localization of TGF-b1 in cisplatin-induced tubulointerstitial damage in rat kidney. Jpn J Nephrol 1996;38:41. Razzaque MS, Foster CS, Ahmed AR: Role of connective tissue growth factor in the pathogenesis of conjunctival scarring in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44: 1998–2003. Oh KH, Margetts PJ: Cytokines and growth factors involved in peritoneal fibrosis of peritoneal dialysis patients. Int J Artif Organs 2005;28:129–134. Leask A, Abraham DJ: TGF-beta signaling and the fibrotic response. FASEB J 2004;18:816–827. Huang Q, Dunn RT 2nd, Jayadev S, DiSorbo O, Pack FD, Farr SB, Stoll RE, Blanchard KT: Assessment of cisplatin-induced nephrotoxicity by microarray technology. Toxicol Sci 2001;63: 196–207. Nagata K: HSP47 as a collagen-specific molecular chaperone: Function and expression in normal mouse development. Semin Cell Dev Biol 2003;14:275–282. Razzaque MS, Nazneen A, Taguchi T: Immunolocalization of collagen and collagen-binding heat shock protein 47 in fibrotic lung diseases. Mod Pathol 1998;11:1183–1188. Razzaque MS, Taguchi T: The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999;14:1199–1212. Satoh K, Wakui H, Komatsuda A, Nakamoto Y, Miura AB, Itoh H, Tashima Y: Induction and altered localization of 90-kDa heat-shock protein in rat kidneys with cisplatin-induced acute renal failure. Ren Fail 1994;16: 313–323. Nazneen A, Razzaque MS, Liu D, Taguchi T: Possible role of Ets-1 and MMP-1 in matrix remodeling in experimental cisplatin nephropathy. Med Electron Microsc 2002;35:242–247. Hayek M, Srinivasan A: Amifostine protects against cisplatin-induced nephrotoxicity in a child with medulloblastoma. Pediatr Hematol Oncol 2003;20: 253–256. Hartmann JT, Knop S, Fels LM, van Vangerow A, Stolte H, Kanz L, Bokemeyer C: The use of reduced doses of amifostine to ameliorate nephrotoxicity of cisplatin/ifosfamide-based chemotherapy in patients with solid tumors. Anticancer Drugs 2000;11:1–6. Capizzi RL: Amifostine reduces the incidence of cumulative nephrotoxicity from cisplatin: Laboratory and clinical aspects. Semin Oncol 1999;26(suppl7):72–81. Srivastava RC, Farookh A, Ahmad N, Misra M, Hasan SK, Husain MM: Reduction of cis-platinum induced nephrotoxicity by zinc histidine complex: The possible implication of nitric oxide. Biochem Mol Biol Int 1995;36:855–862. Shiraishi F, Curtis LM, Truong L, Poss K, Visner GA, Madsen K, Nick HS, Agarwal A: Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am J Physiol Renal Physiol 2000;278:F726–F736. Miyaji T, Kato A, Yasuda H, Fujigaki Y, Hishida A: Role of the increase in p21 in cisplatininduced acute renal failure in rats. J Am Soc Nephrol 2001;12:900–908. Megyesi J, Safirstein RL, Price PM: Induction of p21WAF1/CIP1/SDI1 in kidney tubule cells affects the course of cisplatin-induced acute renal failure. J Clin Invest 1998;101:777–782. Liu Y, Sun AM, Dworkin LD: Hepatocyte growth factor protects renal epithelial cells from apoptotic cell death. Biochem Biophys Res Commun 1998;246:821–826. Saad SY, Al-Rikabi AC: Protection effects of Taurine supplementation against cisplatin-induced nephrotoxicity in rats. Chemotherapy 2002;48:42–48. Sato S, Yamate J, Saito T, Hosokawa T, Saito S, Kurasaki M: Protective effect of taurine against renal interstitial fibrosis of rats induced by cisplatin. Naunyn Schmiedebergs Arch Pharmakol 2002;365:277–283. Yoshida M, Iizuka K, Terada A, Hara M, Nishijima H, Akinori, Shimada, Nakada K, Satoh Y, Akama Y: Prevention of nephrotoxicity of cisplatin by repeated oral administration of ebselen in rats. Tohoku J Exp Med 2000;191:209–220. Husain K, Morris C, Whitworth C, Trammell GL, Rybak LP, Somani SM: Protection by ebselen against cisplatin-induced nephrotoxicity: Antioxidant system. Mol Cell Biochem 1998;178: 127–133.


120

80

81

82

Godbout JP, Pesavento J, Hartman ME, Manson SR, Freund GG: Methylglyoxal enhances cisplatin-induced cytotoxicity by activating protein kinase Cdelta. J Biol Chem 2002;277: 2554–2561. Satoh M, Kashihara N, Fujimoto S, Horike H, Tokura T, Namikoshi T, Sasaki T, Makino H: A novel free radical scavenger, edarabone, protects against cisplatin-induced acute renal damage in vitro and in vivo. J Pharmacol Exp Ther 2003;305:1183–1190. Murota S, Morita I, Suda N: The control of vascular endothelial cell injury. Ann NY Acad Sci 1990;598:182–187.

Takashi Taguchi MD, PhD Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences 1–12–4, Sakamoto machi Nagasaki 852–8523 (Japan) Tel. ⫹81 958 497 053, Fax ⫹81 958 497 056, E-Mail [email protected]


121


Heat Shock Proteins and Allograft Rejection Alan Graham Pockley, Munitta Muthana Immunobiology Research Unit, University of Sheffield, Sheffield, UK

Abstract Heat shock proteins (Hsps) are ubiquitously expressed and highly conserved families of molecules, immune reactivity to which has been implicated in the pathogenesis of inflammatory conditions such as autoimmune and cardiovascular disease. The observations that Hsp expression is induced by ischemia-reperfusion injury and is elevated in transplanted organs, and that rejecting allografts are infiltrated by Hsp-specific lymphocyte populations have prompted the suggestions that Hsps and the development of anti-Hsp immune reactivity drive transplant rejection responses. However, although these proteins can activate innate immune cells such as monocytes, macrophages and dendritic cells and can promote the development of proinflammatory immune responses, they are also cytoprotective and have been shown to improve organ viability and function after ischemia-reperfusion injury in a number of experimental models. In addition, the induction of immunity to Hsp60, Hsp70 and Grp78 attenuates experimental autoimmune disease and the induction of immunity to Hsp60 prolongs murine skin allograft survival. It would, therefore, appear that the expression of Hsps and the presence of Hsp-specific lymphocyte populations are not necessarily indicative of a deleterious response; indeed they might reflect an anti-inflammatory, protective response. This chapter reviews current knowledge in the area of Hsps, anti-Hsp reactivity and allograft rejection. Copyright © 2005 S. Karger AG, Basel

Introduction

The first study to link heat shock protein (Hsp) expression and organ transplantation was published by Currie et al. in 1987 [1]. This investigated protein synthesis in heterotopically transplanted rat hearts and other tissues, and demonstrated an upregulation in the expression of a stress-induced (heat shock) protein having a molecular mass of 71 kDa (designated as SP71, but now termed Hsp70)

in rejecting donor hearts. Data suggested that other factors such as the surgical procedure and organ storage might also contribute to the induction of stress protein expression [1] and work published by the same author in the same year reported Hsp70 expression to be induced after prolonged storage of rat hearts at a range of temperatures (4, 20 and 30⬚C) [2]. To some degree at least, this induction was related to hypoxia as it could be reduced by continuous oxygenation of the storage buffer [2]. It has since been shown that the induction of Hsp or stress protein expression after organ transplantation can be attributed to three defined stages in the procedure [3]. As indicated by Currie et al. [1, 2], the initial stage involves the physiological stress induced by the surgical procedure and the ischemia/reperfusion injury with which organ procurement, preservation, storage and transplantation are inevitably associated. This early phase appears to lead to the induction of the stress proteins Hsp60, Hsp70 and the endoplasmic reticulumassociated stress protein Grp78 (otherwise known as immunoglobulin heavy chain-binding protein, BiP). Lymphocyte infiltration of the transplanted graft induces the expression of Grp78 and Grp94 (otherwise known as Gp96) and a range of Hsps are induced by the inflammatory response, which is induced by the induction and progression of acute rejection.

Stress/Hsp Expression after Transplantation and during Allograft Rejection

Experimental Animal Studies In rats, Hsp70 gene and protein expression progressively increase as cardiac allograft rejection develops [4–6]. Hsp70 expression appears to correlate with the evolution of allograft rejection, as it has been shown to be attenuated when animals are treated with, and rejection is prevented by the administration of the immunosuppressant cyclosporine A [6]. In rat cardiac allografts, Hsp70 appears to be preferentially expressed in cardiomyocytes [4]. The experimental heterotopic cardiac transplant model allows gene and protein expression in the transplanted and native hearts to be compared; however, the data in this area are equivocal, with one study reporting no elevation of Hsp70 expression in the native heart [4] and another reporting increases in expression [5]. Studies using a rat heterotopic intestinal transplantation model have reported a selective induction of Hsp expression during allograft rejection, in that villus epithelial expression of Hsp60 increases, whereas the expression of Hsp70 does not [7]. In contrast, the expression of both Hsps is induced in the lamina propria. Interestingly, a systemic stress response also occurs in this model as Hsp expression was also induced in the native intestine [7].

Heat Shock Proteins and Allograft Rejection

123

Clinical Studies

Despite the evidence from experimental cardiac transplantation models, an induction of Hsp70 expression in rejecting human cardiac allografts has not been reported, although an increase in Hsp27 expression has [8]. The synthesis of Hsp40 and Hsp60 are significantly elevated in kidney biopsies from individuals undergoing acute and chronic rejection [9]. The expression of the collagen-specific stress protein Hsp47, which acts as a molecular chaperone during the processing and/or secretion of procollagen [10] and is closely related to collagen synthesis in an experimental model of interstitial renal fibrosis [11–14] is increased in the interstitium of fibrotic regions of transplanted kidneys [15]. Elevated levels of Hsp60, Hsp70 and the constitutively expressed 70-kDa stress protein Hsc70 have been identified in rejecting human renal transplants on the basis of semi-quantitative immunohistochemical scoring criteria; however, quantitative differences were only observed in the case of Hsp70 [16]. In the same study, a de novo expression of Hsp60 in the vascular compartment of rejecting allografts was observed [16], and this might explain the presence of Hsp-reactive T cells in rejecting human renal and cardiac allografts [17, 18].

Hsp Expression after Transplantation – Friend or Foe?

During steady state conditions Hsps fulfill a range of functions, including the intracellular assembly, folding and translocation of oligomeric proteins [19]. Under conditions of cellular stress they act as cytoprotective agents by binding to misfolded proteins and protecting them from denaturation [20]. Given their cytoprotective capacity, the induction of Hsps in the peri- and immediate posttransplantation periods is likely to be a protective response targeted toward the maintenance of cell and tissue integrity. This proposition is supported by evidence that the induction of Hsps using appropriate stressors or via gene therapy approaches attenuates preservation and ischemia-reperfusion injury and has been shown to improve organ viability and function in a number of experimental models [2, 21–35]. Hsps have also been shown to protect endothelial cells from neutrophil-mediated necrosis [36] and a variety of cell types from oxidative injury [37–39]. In the clinical setting, lower levels of Hsp70 in biopsies prior to liver transplantation and organ perfusates have been associated with early graft loss [40]. In addition to their direct cytoprotective effects, intracellular Hsps also appear to be anti-inflammatory, in that the induction of Hsp70 represses proinflammatory gene transcription by inhibiting nuclear factor-␬B activation

Pockley/Muthana

124

[41–43]. The induction of intracellular Hsp expression has also been shown to inhibit the adhesion of leukocytes to vascular endothelium during lipopolysaccharide (LPS)-induced inflammatory responses in vivo [44]. Such an inhibition might greatly influence the establishment and progression of inflammatory events, including those induced by organ preservation, ischemia-reperfusion injury and developing rejection responses. However, the cytoprotective and anti-inflammatory effects of intracellular Hsps might be offset by their ability to influence the activity of the innate and adaptive immune systems when present in the extracellular environment. In the mammalian system, Hsps have typically been regarded as being exclusively intracellular proteins, and their presence in the extracellular environment has been taken to reflect tissue damage and cellular necrosis. The release of Hsps would provide a ‘danger’ signal, which would activate innate and adaptive immune responses [45, 46]. Hsps as Activators of Innate Immunity after Transplantation? In contrast to the protective and anti-inflammatory effects of Hsps, extracellular forms of proteins such as Hsp60, Hsp70 and Gp96, all of which are induced in the post-transplant period [3], have been shown to stimulate the innate arm of the immune system. Bacterial and mycobacterial Hsps induce proinflammatory cytokine expression [47–49] and bacterial Hsps induce intercellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression on human vascular endothelial cells [47]. Chlamydial and human Hsp60 activate human vascular endothelial cells to express E-selectin, intercellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 and activate vascular endothelial cells, smooth muscle cells and monocytes/macrophages to secrete inflammatory cytokines such as IL-1␤, IL-6 and TNF␣ [50, 51]. With kinetics that are in some instances similar to those induced by LPS, mammalian Hsp60 induces the production of TNF␣, IL-12, IL-15 and nitric oxide by macrophages [52, 53]. Hsp60 also induces human dendritic cell (DC) maturation and their secretion of proinflammatory cytokines such as TNF␣, IL-12, and IL-1␤ [54, 55]. Hsp70 matures DCs to a lesser extent, but induces the release of proinflammatory cytokines from human monocytes and immature DCs as efficiently as Hsp60 [54]. A number of studies have also shown Gp96 to be capable of activating inflammatory innate immune responses [56–59], although others have reported Gp96 to have no such effect [54, 60]. As a caveat, despite the measurement and reporting of LPS levels in the published works, some concern has been expressed that the in vitro responses induced by recombinant Hsp70 produced in E. coli might result from the effects of LPS or other proteins present as either a contaminant of the preparation or chaperoned by the Hsp under investigation [61, 62]. Endotoxin contamination


125

has been reported to be responsible for the human Hsp70 preparation-induced induction of TNF␣ release from murine macrophages [63], and its ability to induce the activation of human DCs [64]. Similar findings have been reported for Hsp60 [65] and at least some of the capacity of Gp96 to stimulate macrophages, might be related to bound/chaperoned endotoxin [60]. Arguing against LPS being responsible for such activities are experiments which demonstrate that the activity is lost on boiling the Hsp under investigation, but retained when LPS is removed using polymyxin B. In addition, synthesized Hsp-derived peptides are capable of eliciting biological effects [66, 67]. No doubt, this controversy will continue for some time to come. Hsps as Inducers of Adaptive Immunity after Transplantation? Hsps are immunodominant molecules and a significant element of the immune response to pathogenic microorganisms is directed towards Hspderived peptides [68, 69]. The conserved nature of Hsp combined with their inherent immunogenicity has prompted the suggestion that they might act as autoantigens that are capable of initiating and driving adaptive inflammatory events [68], and that immune recognition of cross-reactive Hsp epitopes provides a link between infection and autoimmunity [70]. It might, therefore, be that an inappropriate or prolonged expression of Hsps in postischemic transplanted tissue promotes the expansion of autoreactive, Hsp-specific T cell populations and their infiltration into Hsp-expressing tissue. Intragraft expression of Hsps and the generation of anti-Hsp immune reactivity might, therefore, fuel the development of acute and chronic allograft rejection [21, 71, 72]. Supporting such a proposition have been the observations that T cells from rejected human renal allografts respond to Hsp70 [17], that cells infiltrating rat cardiac allografts proliferate in response to mycobacterial Hsp65 and Hsp70 [73] and that the mycobacterial Hsp65-induced growth of graft infiltrating lymphocytes from human endomyocardial biopsies correlates with cardiac graft rejection [18]. However (and crucially), the phenotype (proinflammatory vs. regulatory) of the T cells responding to Hsps after transplantation has not been identified. Allograft outcome might also be influenced by the development of humoral immunity to Hsps, given that Hsp antibodies can be pathogenic and mediate the cytotoxicity of stressed endothelial cells [74, 75]. In this area findings are equivocal, with one group reporting that higher levels of anti-Hsp60 and anti-Hsp70 antibodies are associated with a poorer prognosis after human cardiac transplantation [76, 77], whereas another has reported there to be no relationship between anti-Hsp60 and anti-Hsp70 antibody titers and clinical course after human renal transplantation [78]. It should be noted that the

Pockley/Muthana

126

presence of anti-Hsp60 and anti-Hsp70 antibodies does not necessarily reflect pathogenic processes, as circulating Hsp antibodies are present in normal individuals [79–82].

Hsps as Regulators of Inflammatory Responses

Although for many years the perception has been that Hsps are intracellular molecules that are only released from nonviable (necrotic) cells, these molecules can be released from a variety of viable (non-necrotic) mammalian cell types, including cultured rat embryo cells [83], human islet cells [84], rat glial cells and a human neuroblastoma cell line [85], and cultured vascular smooth muscle cells exposed to oxidative stress [86]. These observations have implications for the perceived role of these proteins as exclusively proinflammatory intercellular signaling molecules and ‘danger’ signals. This is especially so given that Hsp60 and Hsp70 are also present in the peripheral circulation of normal individuals [79–81, 87–90]. Indeed, data from a number of studies provide a basis for the proposition that self-Hsp immune reactivity is a physiological mechanism, which is a capable of downregulating rather than promoting inflammatory disease processes [91]. The induction of T cell reactivity to Hsp60 and Hsp70 downregulates disease in a number of experimental arthritis models, by a mechanism that appears to involve the induction of self-Hsp-specific Th2-type CD4⫹ T cells producing the regulatory cytokines IL-4 and IL-10 [92–99]. In the clinical setting, T cells from the synovial fluid of patients with rheumatoid arthritis respond to self (human)-Hsp60 by predominantly producing regulatory Th2-type cytokine responses, whereas cells stimulated with bacterial Hsp60 produce higher levels of IFN␥, which is consistent with a proinflammatory Th1-type response [100]. In addition, T cell lines generated from the synovial fluid of patients with rheumatoid arthritis in response to self-Hsp60 suppress the production of the proinflammatory cytokine TNF␣ by peripheral blood mononuclear cells, whereas cells generated using mycobacterial Hsp65 have no such regulatory effect [100]. It might, therefore, be that appropriately expressed Hsps serve to control inflammatory events that are associated with the induction and progression of allograft rejection. The precise mechanisms by which self-Hsp reactivity might regulate inflammatory disease have yet to be fully elucidated; however, a number of possibilities exist [91]. As has been shown for many other self-peptides, the normal T cell repertoire includes low affinity T cells reactive against autologous Hsps [68, 101–104]. Self-Hsp reactive T cells recognizing self-Hsp epitopes in stressed tissues via low-affinity interactions might lead to the


127

generation of Th2 (IL-4-producing), Th3 (TGF␤-producing) or Tr1 (IL-10producing) regulatory T cells responses. It is clear from the studies described above that immune responses to Hsps can downregulate as well as induce and exacerbate inflammatory events. The induction of Hsp expression and the presence of Hsp-specific T cell populations might, therefore, not necessarily drive allograft rejection responses. Data providing a mechanistic insight into the influence of Hsps on allograft rejection are limited and it appears that only one published study has addressed this issue directly [66]. Evidence from this study, which used a murine skin transplant model, suggests that Hsp60 can both potentiate and attenuate the graft rejection process [66]. A direct involvement of Hsp60 in the rejection process has been suggested by the observations that skin from transgenic mice overexpressing Hsp60 transplanted into allogeneic recipients rejects more rapidly than skin transplanted from wild-type donors. In addition, the transplantation of skin from wild-type donors into Hsp60 transgenic mice, in which spontaneous autoimmunity to Hsp60 is reduced, rejects more slowly than skin transplanted into wild-type recipients [66]. In contrast to the apparent potentiating effects of Hsp60 immunoreactivity on allograft rejection, in the same model, rejection could be delayed by immunizing animals with self-Hsp60 or Hsp60-derived peptides that have the capacity to shift Hsp60-immunoreactivity from a Th1 (proinflammatory) to a Th2 (regulatory) phenotype [66]. Although attention has to date been focused on Hsp60 and Hsp70, other Hsps might also influence allograft survival. A notable candidate is Grp78 (BiP) which is a member of the Hsp70 family of molecules [105] and has been shown to be expressed in transplanted and rejecting tissue [3]. Although its influence on allograft survival has not been evaluated, Grp78 has been identified as an autoantigen in rheumatoid arthritis and shown to stimulate the proliferation of synovial T cells from patients with rheumatoid arthritis [106]. It has also been shown to prevent the induction of arthritis in experimental models of the disease by a mechanism which might, in part at least, involve the generation of IL-10-secreting regulatory T cells [106, 107].

Conclusions

Hsps that are expressed in tissue following ischemia-reperfusion injury and at different phases in the post-transplantation period have been shown to influence a variety of immunological events. Hsp60, Hsp70 and gp96 have all been reported to activate and induce the secretion of proinflammatory cytokines from monocytes, macrophages and DCs and their expression in transplanted tissue might, therefore, promote inflammation, tissue damage and the induction

Pockley/Muthana

128

of allograft rejection. However, intracellular Hsps are cytoprotective molecules and the induction of immunity to extracellular Hsp60, Hsp70 and grp78 has been shown to temper inflammatory events and to induce an immunoregulatory response which is capable of controlling autoimmune disease and, in one instance at least, allograft rejection. The precise roles that Hsps have in the sequelae that follow transplantation are unclear. However, the varied attributes of Hsps suggest that they might be valuable as immunotherapeutic agents for the control of inflammatory conditions and further work aimed at exploring these aspects of these ubiquitously expressed molecules in the field of organ transplantation is clearly warranted.

Acknowledgments Heat shock protein-related studies in the author’s laboratory are supported by funding from the National Heart, Lung and Blood Institute, USA (grant HL 69726) and the Association for International Cancer Research.

References 1 2 3

4

5

6

7 8

9 10 11

Currie RW, Sharma VK, Stepkowski SM, Payce RF: Protein synthesis in heterotopically transplanted rat hearts. Exp Cell Biol 1987;55:46–56. Currie RW: Effects of ischemia and perfusion temperature on the synthesis of stress-induced (heat shock) proteins in isolated and perfused rat hearts. J Mol Cell Cardiol 1987;19:795–808. Qian J, Moliterno R, Donovan-Peluso MA, Liu K, Suzow J, Valdivia L, Pan F, Duquesnoy RJ: Expression of stress proteins and lymphocyte reactivity in heterotopic cardiac allografts undergoing cellular rejection. Transplant Immunol 1995;3:114–123. Baba HA, Schmid C, Wilhelm MJ, Blasius S, Scheld HH, Bocker W, Dockhorn-Dworniczak B: Inducible heat shock protein 70 in rat cardiac allograft and its immunohistochemical localization in cardiac myocytes. Transplantation 1997;64:1035–1040. Mehta NK, Carroll M, Sykes DE, Tan Z, Bergsland J, Canty JJ, Bhayana JN, Hoover EL, Salerno TA: Heat shock protein 70 expression in native and heterotopically transplanted rat hearts. J Surg Res 1997;70:151–155. Davis EA, Wang BH, Stagg CA, Baldwin WM 3rd, Baumgartner WA, Sanfilippo F, Udelsman R: Induction of heat shock protein in cardiac allograft rejection – A cyclosporine-suppressible response. Transplantation 1996;61:279–284. Ogita K, Hopkinson K, Nakao M, Wood RFM, Pockley AG: Stress responses in graft and native intestine after rat heterotopic small bowel transplantation. Transplantation 2000;69:2273–2277. Schimke I, Lutsch G, Schernes U, Kruse I, Dubel HP, Pregla R, Hummel M, Meyer R, Stahl J: Increased level of HSP27 but not of HSP72 in human heart allografts in relation to acute rejection. Transplantation 2000;70:1694–1697. Alevy YG, Brennan D, Durriya S, Howard T, Mohanakumar T: Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidney. Transplantation 1996;61:963–967. Nakai A, Satoh M, Hirayoshi K, Nagata K: Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J Cell Biol 1992;117:903–914. Nagata K, Yamada KM: Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts. J Biol Chem 1986;261:7531–7536.


129

12

13 14 15

16 17 18

19 20 21 22

23

24

25 26 27

28

29

30

31

32

33

Masuda H, Fukumoto M, Hirayoshi K, Nagata K: Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachlorideinduced rat liver fibrosis. J Clin Invest 1994;94:2481–2488. Cheng M, Razzaque MS, Nazneen A, Taguchi T: Expression of the heat shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 1998;79:125–132. Moriyama T, Kawada N, Ando A, Yamauchi A, Horio M, Nagata K, Imai E, Hori M: Up-regulation of HSP47 in the mouse kidneys with unilateral ureteral obstruction. Kidney Int 1998;54:110–119. Abe K, Ozono Y, Miyazaki M, Koji T, Shioshita K, Furusu A, Tsukasaki S, Matsuya F, Hosokawa N, Harada T, Taguchi T, Nagata K, Kohno S: Interstitial expression of heat shock protein 47 and alpha-smooth muscle actin in renal allograft failure. Nephrol Dial Transplant 2000;15:529–535. Trieb K, Dirnhofer S, Krumbock N, Blahovec H, Sgonc R, Margreiter R, Feichtinger H: Heat shock protein expression in the transplanted human kidney. Transpl Int 2001;14:281–286. Trieb K, Grubeck-Loebenstein B, Eberl T, Margreiter R: T cells from rejected human kidney allografts respond to heat shock protein 72. Transplant Immunol 1996;4:43–45. Moliterno R, Woan M, Bentlejewski C, Zeevi A, Pham S, Griffith BP, Duquesnoy RJ: Heat shock protein-induced T lymphocyte propagation from endomyocardial biopsies in heart transplantation. J Heart Lung Transplant 1995;14:329–337. Hightower LE: Heat shock, stress proteins, chaperones and proteotoxicity. Cell 1991;66:191–197. Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996;381:571–579. Perdrizet GA: The heat shock response and organ transplantation. Transplant Rev 1996;10:78–98. Chen G, Kelly C, Stokes K, Wang JH, Leahy A, Bouchier-Hayes D: Induction of heat shock protein 72 kDa expression is associated with attenuation of ischaemia-reperfusion induced microvascular injury. J Surg Res 1997;69:435–439. Suzuki K, Sawa Y, Kaneda Y, Ichikawa H, Shirakura R, Matsuda H: In vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion injury in rat. J Clin Invest 1997;99:1645–1650. Hiratsuka M, Yano M, Mora BN, Nagahiro I, Cooper JD, Patterson GA: Heat shock pretreatment protects pulmonary isografts from subsequent ischemia-reperfusion injury. J Heart Lung Transplant 1998;17:1238–1246. Hiratsuka M, Mora BN, Yano M, Mohanakumar T, Patterson GA: Gene transfer of heat shock protein 70 protects lung grafts from ischemia-reperfusion injury. Ann Thorac Surg 1999;67:1421–1427. Squiers EC, Bruch D, Buelow R, Tice DG: Pretreatment of small bowel isograft donors with cobalt-protoporphyrin decreases preservation injury. Transplant Proc 1999;31:585–586. Suzuki K, Smolenski RT, Jayakumar J, Murtuza B, Brand NJ, Yacoub MH: Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart. Circulation 2000;102 (suppl 3):III216–III221. Jayakumar J, Suzuki K, Khan M, Smolenski RT, Farrell A, Latif N, Raisky O, Abunasra H, Sammut IA, Murtuza B, Amrani M, Yacoub MH: Gene therapy for myocardial protection: Transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury. Circulation 2000;102(suppl 3):III302–III306. Jayakumar J, Suzuki K, Sammut IA, Smolenski RT, Khan M, Latif N, Abunasra H, Murtuza B, Amrani M, Yacoub MH: Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury. Circulation 2001;104(suppl 1):I303–I307. Redaelli CA, Wagner M, Kulli C, Tian YH, Kubulus D, Mazzucchelli L, Wagner AC, Schilling MK: Hyperthermia-induced HSP expression correlates with improved rat renal isograft viability and survival in kidneys harvested from non-heart-beating donors. Transpl Int 2001;14:351–360. Redaelli CA, Tien YH, Kubulus D, Mazzucchelli L, Schilling MK, Wagner AC: Hyperthermia preconditioning induces renal heat shock protein expression, improves cold ischemia tolerance, kidney graft function and survival in rats. Nephron 2002;90:489–497. Kevelaitis E, Patel AP, Oubenaissa A, Peynet J, Mouas C, Yellon DM, Menasche P: Backtable heat-enhanced preconditioning: A simple and effective means of improving function of heart transplants. Ann Thorac Surg 2001;72:107–112. Fudaba Y, Ohdan H, Tashiro H, Ito H, Fukuda Y, Dohi K, Asahara T: Geranylgeranylacetone, a heat shock protein inducer, prevents primary graft nonfunction in rat liver transplantation. Transplantation 2001;72:184–189.

Pockley/Muthana

130

34

35

36

37

38

39 40

41 42 43

44

45 46 47

48

49

50

51

52 53 54

Katori M, Buelow R, Ke B, Ma J, Coito AJ, Iyer S, Southard D, Busuttil RW, Kupiec-Weglinski JW: Heme oxygenase-1 over-expression protects rat hearts from cold ischemia/reperfusion injury via an anti-apoptotic pathway. Transplantation 2002;73:287–292. Wagner M, Cadetg P, Ruf R, Mazzucchelli L, Ferrari P, Redaelli CA: Heme oxygenase-1 attenuates ischemia/reperfusion-induced apoptosis and improves survival in rat renal allografts. Kidney Int 2003;63:1564–1573. Wang JH, Redmond HP, Watson RWG, Condron C, Bouchier-Hayes D: Induction of heat shock protein 72 prevents neutrophil-mediated human endothelial necrosis. Arch Surg 1995;130: 1260–1265. Gill RR, Gbur CJJ, Fisher BJ, Hess ML, Fowler AA 3rd, Kukreja RC, Sholley MM: Heat shock provides delayed protection against oxidative injury in cultured human umbilical vein endothelial cells. J Mol Cell Cardiol 1998;30:2739–2749. Amrani M, Latif N, Morrison K, Gray CC, Jayakumar J, Corbett J, Goodwin AT, Dunn MJ, Yacoub MH: Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: Implications for myocardial protection. J Thorac Cardiovasc Surg 1998;115:200–209. Martin JL, Mestril R, Hilal-Dandan R, Brunton LL, Dillman WH: Small heat shock proteins and protection against ischemic injury in cardiac myocytes. Circulation 1997;96:4138–4140. Flohé S, Speidel N, Flach R, Lange R, Erhard J, Ulrich Schade F: Expression of Hsp70 as a potential prognostic marker for acute rejection in human liver transplantation. Transplant Int 1998; 11:89–94. Cahill CM, Lin HS, Price BD, Bruce JL, Calderwood SK: Potential role of heat shock transcription factor in the expression of inflammatory cytokines. Adv Exp Med Biol 1997;400B:625–630. Cahill CM, Waterman WR, Xie Y, Auron PE, Calderwood SK: Transcriptional repression of the prointerleukin 1␤ gene by heat shock factor 1. J Biol Chem 1996;271:24874–24879. Housby JN, Cahill CM, Chu B, Prevelige R, Bickford K, Stevenson MA, Calderwood SK: Nonsteroidal anti-inflammatory drugs inhibit the expression of cytokines and induce HSP70 in human monocytes. Cytokine 1999;11:347–358. House SD, Guidon PT Jr, Perdrizet GA, Rewinski M, Kyriakos R, Bockman RS, Mistry T, Gallagher PA, Hightower LE: Effects of heat shock, stannous chloride, and gallium nitrate on the rat inflammatory response. Cell Stress Chaperones 2001;6:164–171. Matzinger P: Tolerance, danger, and the extended family. Ann Rev Immunol 1994;12:991–1045. Srivastava PK, Ménoret A, Basu S, Binder RJ, McQuade KL: Heat shock proteins come of age: Primitive functions acquire new roles in an adaptive world. Immunity 1998;8:657–665. Galdiero M, de l’Ero GC, Marcatili A: Cytokine and adhesion molecule expression in human monocytes and endothelial cells stimulated with bacterial heat shock proteins. Infect Immun 1997;65:699–707. Retzlaff C, Yamamoto Y, Hoffman PS, Friedman H, Klein TW: Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures. Infect Immun 1994;62:5689–5693. Peetermans WE, Raats CJ, Langermans JA, van Furth R: Mycobacterial heat shock protein 65 induces proinflammatory cytokines but does not activate human mononuclear phagocytes. Scand J Immunol 1994;39:613–617. Kol A, Bourcier T, Lichtman A, Libby P: Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J Clin Invest 1999; 103:571–577. Asea A, Kraeft S-K, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK: Hsp70 stimulates cytokine production through a CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nature Med 2000;6:435–442. Chen W, Syldath U, Bellmann K, Burkart V, Kold H: Human 60-kDa heat-shock protein: A danger signal to the innate immune system. J Immunol 1999;162:3212–3219. Habich C, Kempe K, van der Zee R, Burkart V, Kolb H: Different heat shock protein 60 species share pro-inflammatory activity but not binding sites on macrophages. FEBS Lett 2003;533:105–109. Bethke K, Staib F, Distler M, Schmitt U, Jonuleit H, Enk AH, Galle PR, Heike M: Different efficiency of heat shock proteins to activate human monocytes and dendritic cells: Superiority of HSP60. J Immunol 2002;169:6141–6148.


131

55

56 57

58 59 60

61 62 63 64

65 66

67

68 69 70

71 72 73 74 75

76 77

Flohé SB, Bruggemann J, Lendemans S, Nikulina M, Meierhoff G, Flohé S, Kolb H: Human heat shock protein 60 induces maturation of dendritic cells versus a Th1-promoting phenotype. J Immunol 2003;170:2340–2348. Panjwani NN, Popova L, Srivastava PK: Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs. J Immunol 2002;168:2997–3003. Vabulas RM, Braedel S, Hilf N, Singh-Jasuja H, Herter S, Ahmad-Nejad P, Kirschning CJ, Da Costa C, Rammensee HG, Wagner H, Schild H: The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J Biol Chem 2002;277:20847–20853. Hilf N, Singh-Jasuja H, Schild H: The heat shock protein Gp96 links innate and specific immunity. Int J Hyperthermia 2002;18:521–533. Zheng H, Dai J, Stoilova D, Li Z: Cell surface targetting of heat shock protein gp96 induces dendritic cell maturation and anti-tumour immunity. J Immunol 2001;167:6734–6735. Reed RC, Berwin B, Baker JP, Nicchitta CV: GRP94/gp96 elicits ERK activation in murine macrophages. A role for endotoxin contamination in NF-kappa B activation and nitric oxide production. J Biol Chem 2003;278:31853–31860. Wallin RPA, Lundqvist A, Moré SH, von Bonin A, Kiessling R, Ljunggren H-G: Heat-shock proteins as activators of the innate immune system. Trends Immunol 2002;23:130–135. Gaston JSH: Heat shock proteins and innate immunity. Clin Exp Immunol 2002;127:1–3. Gao B, Tsan MF: Endotoxin contamination in recombinant human Hsp70 preparation is responsible for the induction of TNF␣ release by murine macrophages. J Biol Chem 2003;278:174–179. Bausinger H, Lipsker D, Ziylan U, Manie S, Briand JP, Cazenave JP, Muller S, Haeuw JF, Ravanat C, de la Salle H, Hanau D: Endotoxin-free heat-shock protein 70 fails to induce APC activation. Eur J Immunol 2002;32:3708–3713. Gao B, Tsan MF: Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor alpha from murine macrophages. J Biol Chem 2003;278:22523–22529. Birk OS, Gur SL, Elias D, Margalit R, Mor F, Carmi P, Bockova J, Altmann DM, Cohen IR: The 60-kDa heat shock protein modulates allograft rejection. Proc Natl Acad Sci USA 1999;96: 5159–5163. Wang Y, Kelly CG, Singh M, McGowan EG, Carrara AS, Bergmeier LA, Lehner T: Stimulation of Th1-polarizing cytokines, C-C chemokines, maturation of dendritic cells, and adjuvant function by the peptide binding fragment of heat shock protein 70. J Immunol 2002;169:2422–2429. Kaufmann SHE: Heat shock proteins and the immune response. Immunol Today 1990;11: 129–136. Young RA: Stress proteins and immunology. Ann Rev Immunol 1990;8:401–420. Lamb JR, Bal V, Mendez-Samperio A, Mehlert A, So J, Rothbard JB, Jindal S, Young RA, Young DB: Stress proteins may provide a link between the immune response to infection and autoimmunity. Int Immunol 1989;1:191–196. Duquesnoy RJ, Moliterno R: Role of heat shock protein immunity in transplantation; in van Eden W, Young DB (eds): Stress Proteins in Medicine. New York, Marcel Dekker, 1995, pp 327–343. Duquesnoy RJ, Liu K, Fu X-F, Murase N, Ye Q, Demetris AJ: Evidence for heat shock protein immunity in a rat cardiac allograft model of chronic rejection. Transplantation 1999;67:156–164. Moliterno R, Valdivia L, Pan F, Duquesnoy RJ: Heat shock protein reactivity of lymphocytes isolated from heterotopic rat cardiac allografts. Transplantation 1995;59:598–604. Schett G, Xu Q, Amberger A, van der Zee R, Reiches H, Willeit J, Wick G: Autoantibodies against heat shock protein mediate endothelial cytotoxicity. J Clin Invest 1995;96:2569–2577. Mayr M, Metzler B, Kiechl S, Willeit J, Scett G, Xu Q, Wick G: Endothelial cytotoxicity mediated by serum antibodies to heat shock proteins of Escherichia coli and Chlamydia pneumonia. Immune reactions to heat shock proteins as a possible link between infection and atherosclerosis. Circulation 1999;99:1560–1566. Latif N, Rose ML, Yacoub MH, Dunn MJ: Association of pretransplantation antiheart antibodies with clinical course after heart transplantation. J Heart Lung Transplant 1995;14:119–126. Latif N, Yacoub MH, Dunn MJ: Association of pre-transplant anti-heart antibodies against human heat shock protein 60 with clinical course following cardiac transplantation. Transplant Proc 1997;29:1039–1040.

Pockley/Muthana

132

78 79 80

81 82 83 84 85

86

87

88 89 90

91

92

93 94

95 96

97

98

99

Trieb K, Gerth R, Berger P, Margreiter R: Serum antibodies to heat shock proteins are of no diagnostic value for human kidney allograft rejection. Transpl Int 2000;13:46–48. Pockley AG, Shepherd J, Corton J: Detection of heat shock protein 70 (Hsp70) and anti-Hsp70 antibodies in the serum of normal individuals. Immunol Invest 1998;27:367–377. Pockley AG, Bulmer J, Hanks BM, Wright BH: Identification of human heat shock protein 60 (Hsp60) and anti-Hsp60 antibodies in the peripheral circulation of normal individuals. Cell Stress Chaperones 1999;4:29–35. Pockley AG, Wu R, Lemne C, Kiessling R, de Faire U, Frostegård J: Circulating heat shock protein 60 is associated with early cardiovascular disease. Hypertension 2000;36:303–307. Buhlin K, Gustafsson A, Pockley A, Frostegård J, Klinge B: Risk factors for cardiovascular disease in patients with periodontitis. Eur Heart J 2003;24:2099–2107. Hightower LE, Guidon PT: Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. J Cell Physiol 1989;138:257–266. Child DF, Williams CP, Jones RP, Hudson PR, Jones M, Smith CJ: Heat shock protein studies in Type 1 and Type 2 diabetes and human islet cell culture. Diabet Med 1995;12:595–599. Bassan M, Zamostiano R, Giladi E, Davidson A, Wollman Y, Pitman J, Hauser J, Brenneman DE, Gozes I: The identification of secreted heat shock 60-like protein from rat glial cells and a human neuroblastoma cell line. Neurosci Lett 1998;250:37–40. Liao D-F, Jin Z-G, Baas AS, Daum G, Gygi SP, Aebersold R, Berk BC: Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells. J Biol Chem 2000;275:189–196. Xu Q, Schett G, Perschinka H, Mayr M, Egger G, Oberhollenzer F, Willeit J, Kiechl S, Wick G: Serum soluble heat shock protein 60 is elevated in subjects with atherosclerosis in a general population. Circulation 2000;102:14–20. Rea IM, McNerlan S, Pockley AG: Serum heat shock protein and anti-heat shock protein antibody levels in aging. Exp Gerontol 2001;36:341–352. Lewthwaite J, Owen N, Coates A, Henderson B, Steptoe A: Circulating human heat shock protein 60 in the plasma of British civil servants. Circulation 2002;106:196–201. Pockley AG, de Faire U, Kiessling R, Lemne C, Thulin T, Frostegård J: Circulating heat shock protein and heat shock protein antibody levels in established hypertension. J Hypertens 2002;20: 1815–1820. van Eden W, van der Zee R, Paul AGA, Prakken BJ, Wendling U, Anderton SM, Wauben MHM: Do heat shock proteins control the balance of T-cell regulation in inflammatory diseases? Immunol Today 1998;19:303–307. van den Broek MF, Hogervorst EJM, van Bruggen MCJ, van Eden W, van der Zee R, van den Berg W: Protection against streptococcal cell wall induced arthritis by pretreatment with the 65 kD heat shock protein. J Exp Med 1989;170:449–466. Thompson SJ, Rook GAW, Brealey RJ, van der Zee R, Elson CJ: Autoimmune reactions to heat shock proteins in pristane induced arthritis. Eur J Immunol 1990;20:2479–2484. Anderton SM, van der Zee R, Prakken B, Noordzij A, van Eden W: Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis. J Exp Med 1995;181:943–952. Anderton SM, van Eden W: T lymphocyte recognition of hsp60 in experimental arthritis; in van Eden W, Young D (eds): Stress Proteins in Medicine. New York, Marcel Dekker, 1996, pp 73–91. Paul AGA, van Kooten PJS, van Eden W, van der Zee R: Highly autoproliferative T cells specific for 60-kDa heat shock protein produce IL-4/IL-10 and IFN-␥ and are protective in adjuvant arthritis. J Immunol 2000;165:7270–7277. Kingston AE, Hicks CA, Colston MJ, Billingham MEJ: A 71-kD heat shock protein (hsp) from Mycobacterium tuberculosis has modulatory effects on experimental rat arthritis. Clin Exp Immunol 1996;103:77–82. Tanaka S, Kimura Y, Mitani A, Yamamoto G, Nishimura H, Spallek R, Singh M, Noguchi T, Yoshikai Y: Activation of T cells recognizing an epitope of heat-shock protein 70 can protect against rat adjuvant arthritis. J Immunol 1999;163:5560–5565. Wendling U, Paul L, van der Zee R, Prakken B, Singh M, van Eden W: A conserved mycobacterial heat shock protein (hsp) 70 sequence prevents adjuvant arthritis upon nasal administration and


133

100

101

102 103

104

105 106

107

induces IL-10-producing T cells that cross-react with the mammalian self-hsp70 homologue. J Immunol 2000;164:2711–2717. van Roon JAG, van Eden W, van Roy JLAM, Lafeber FJPG, Bijlsma JWJ: Stimulation of suppressive T cell responses by human but not bacterial 60-kD heat shock protein in synovial fluid of patients with rheumatoid arthritis. J Clin Invest 1997;100:459–463. Munk ME, Schoel B, Modrow S, Karr RW, Young RA, Kaufmann SHE: T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein. J Immunol 1989;143:2844–2849. Cohen IR: Heat shock protein 60 and the regulation of autoimmunity; in van Eden W, Young DB (eds): Stress Proteins in Medicine. New York, Marcel Dekker, 1996, pp 93–102. Ramage JM, Young JL, Goodall JC, Hill Gaston JS: T cell responses to heat shock protein 60: Differential responses by CD4⫹ T cell subsets according to their expression of CD45 isotypes. J Immunol 1999;162:704–710. Macht LM, Elson CJ, Kirwan JR, Gaston JSH, Lamont AG, Thompson JM, Thompson SJ: Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60. Immunology 2000;99:208–214. Munro S, Pelham HR: An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 1986;46:291–300. Corrigall VM, Bodman-Smith MD, Fife MS, Canas B, Myers LK, Wooley P, Soh C, Staines NA, Pappin DJ, Berlo SE, van Eden W, van der Zee R, Lanchbury JS, Panayi GS: The human endoplasmic reticulum molecular chaperone BiP is an autoantigen for rheumatoid arthritis and prevents the induction of experimental arthritis. J Immunol 2001;166:1492–1498. Bodman-Smith MD, Corrigall VM, Kemeny DM, Panayi GS: BiP, a putative autoantigen in rheumatoid arthritis, stimulates IL-10-producing CD8⫹ T cells from normal individuals. Rheumatology (Oxford) 2003;42:637–644.

A. Graham Pockley, PhD Immunobiology Research Unit, Clinical Sciences Centre Northern General Hospital, Herries Road, Sheffield, S5 7AU, UK Tel. ⫹44 114 271 4450, Fax ⫹44 114 271 4450 E-Mail [email protected]

Pockley/Muthana

134


Oxidant Stress in Renal Pathophysiology M. Ruhul Abida, Mohammed S. Razzaqueb,c, Takashi Taguchic a Division of Molecular and Vascular Medicine, Department of Medicine, and Vascular Biology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass., USA; bDepartment of Oral and Developmental Biology, Harvard School of Dental Medicine, Boston, Mass., USA; cDepartment of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Abstract Despite recent progress in identifying a number of important factors that may play central roles in various renal diseases, the precise molecular basis of renal injuries remains unclear. Recent studies have documented an important role for oxidant stress in several renal diseases. Oxidant stress by overproduction of reactive oxygen species, generation of reactive nitrogen species, and/or modulation of cellular antioxidant enzyme activities, resulting in the activation of certain transcription factors, and synthesis and/or release of inflammatory cytokines, chemokines, growth factors, and extracellular matrix proteins. These changes alter the balance in the microenvironment of the kidney, and may activate signaling cascades that induce and propagate renal injury. Complex molecular interactions and cross-talk between the activated signaling pathways, in turn, define the nature and clinical course of the disease process. In this article, we will briefly present the relevance of the oxidant stress in the pathogenesis of various renal diseases. Copyright © 2005 S. Karger AG, Basel

Introduction

The recruitment of inflammatory cells is an important pathological event that eventually determine the clinical course of many renal diseases. Infiltrating inflammatory cells through the generation of reactive oxygen species (ROS), and by influencing production of inflammatory and fibrogenic molecules, contribute to the development of renal fibroproliferative lesions. Although ongoing studies have significantly increased our understanding of molecular mechanisms of progressive renal diseases, molecular interactions among various

immunoinflammatory pathways that eventually lead to chronic renal diseases remain to be elucidated. Recent studies have shed light on the pathological role of oxidant stress in renal injury [1–8]. An increased production of ROS, reactive nitrogen species, and modulation of cellular antioxidant enzymes are thought to be involved in early phase of renal injury [9–15]. For instance, inducible nitric oxide (NO) synthase-mediated superoxide (O2) and peroxynitrite (ONOO) have been shown to mediate macrophage-associated renal injuries [16]. It has also been shown that ONOO, an oxidant formed by the interaction of (O2) and NO, may regulate inflammatory phase of various renal diseases, by facilitating chemotactic activities of monocytes [17, 18]. Similarly, ROS and other free radical intermediates are involved in the activation of inflammatory-cellrecruiting molecules, including monocyte chemoattractant protein 1, that are involved in determining the inflammatory phenotypes following renal injury [19]. In this review, we will briefly present recent molecular understanding of oxidant stress, and its role in various renal diseases.

Inflammatory Cells and Progression of Renal Diseases

Renal accumulation of various acute and chronic inflammatory cells, including macrophages play major roles in the severity of the disease process, and eventual renal failure [20]. Studies have convincingly demonstrated that therapeutic manipulation of macrophage infiltration resulted in relatively less severe renal injuries [21, 22], emphasizing an important role of macrophages in the pathophysiology of various renal diseases. In diabetic nephropathy, macrophage infiltration has been shown to precede the development of tubulointerstitial injury [23]. Infiltrating macrophages are important source of proinflammatory cytokines and cytotoxic mediators, which induce a cascade of events to initiate repair process. However, uncontrolled regulation of proinflammatory and profibrogenic molecules, including platelet-derived growth factor (PDGF), interleukin-1, insulin-like growth factor, epidermal growth factor, tumor necrosis factor-, basic fibroblast growth factor, and transforming growth factor-1 (TGF-1), continue to induce excessive synthesis of extracellular matrix proteins, and in some instances inhibit degradation of matrix proteins, eventually leading to the development of fibroproliferative lesions in various organs, including kidney [24–27]. In addition to releasing proinflammatory molecules, macrophages are one of the major sources of ROS that mediate renal injury, possibly by exerting the cytotoxic effects on resident renal cells.

Abid/Razzaque/Taguchi

136

Reactive Oxygen Species ROS such as hydrogen peroxide (H2O2) or hypochloride (HOCl), and free radicals such as O2, hydroxyl radical (OH), and nitric oxide (NO), are continuously generated in vivo. Thus, detection of ROS per se could not be defined as oxidative stress; nevertheless, attenuated balance between formation of ROS and antioxidative defense systems results in generation of oxidative stress. This delicate balance between formation of ROS and antioxidative defense mechanisms mostly depends on enzymatic activities, such as superoxide dismutases (SODs), catalase, NO synthase, and glutathione peroxidase (GPx). ROS include molecules that have unpaired electrons, such as O2, OH, and NO, or that have oxidizing ability but do not possess free electrons, such as H2O2, HOCl, and ONOO. Although first described in phagocytes as part of a protective mechanism in the immune system [28–31], where they have been mostly viewed as cytotoxic molecules, ROS are now recognized to play critical roles in signal transduction and transcriptional regulation in many nonphagocytic cells [32–44]. Agonist-stimulated ROS have been shown to activate downstream signaling targets to induce the expression of redox-sensitive genes and mediate cell proliferation and migration [41, 45–51]. Eukaryotic cells have evolutionarily evolved a well co-ordinated system to maintain redox balance within and around the cells, which is critical for proper maintenance of cell survival/apoptosis, growth, and proliferation. There are many potential systems that modulate the redox state of cells. Pro-oxidants include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, cycloxygenase, lipoxygenases, mitochondrial respiration, phosphatidic acid, and cytochrome P450. Antioxidant systems include GPx, SOD family that includes cytosolic copper-zinc-containing SOD (Cu-ZnSOD), mitochondrial manganese-containing SOD (MnSOD) and extracellular Cu-ZnSOD, catalase, thioredoxin, and heme oxygenase. In physiological state, O2 is converted to H2O2 by SOD. H2O2 is then reduced to water and molecular oxygen by catalase and extracellular GPx. An imbalance between the activities of the pro- and antioxidant enzymes may result in an increase in the net concentration of ROS, which is known as oxidative or oxidant stress [52–61]. Therefore, oxidant stress may result from an overproduction of ROS and/or downregulation or reduced activity of the cellular antioxidant enzymes. The membranous pro-oxidant, NADPH oxidase complex, was first described in phagocytes, where it is a potent source of O2 and plays a major role in phagocytosis. NADPH oxidase consists of a membrane component cytochrome b558, comprising two subunits, gp91phox (flavin-containing subunit) and p22phox, and several cytosolic components including p40phox, p47 phox, p67 phox and the small guanosine triphosphatase Rac (Rac1 or Rac2) (fig. 1). NADPH oxidase in resting neutrophils is dormant but is transiently stimulated in a

Oxidant Stress and Renal Injuries

137

Growth factors hormones gp91

Receptor tyrosine kinase

p22

Rac p47 NAD(P) H

p67

p

e

NAD(P)H

•O2

MnSOD

O2

Cu ZnSOD

•O2

Protein tyrosine phosphatase

H2O2

Catalase H2O2

PI3K PKC MAPK

Cell survival Proliferation Migration

H2O O2

Fig. 1. NADPH-oxidase-derived ROS increases the intensity and/or duration of the activation of receptor tyrosine kinase (RTK) by transiently inactivating tyrosine phosphatases. Agonist-stimulated activation of RTK generates NADPH-oxidase-derived superoxide that is catalyzed to hydrogen peroxide (H2O2) by cytosolic Cu-ZnSOD. H2O2 reversibly inhibits tyrosine phosphatase by transiently oxidizing cysteine residues (SH groups to S-S) present in the catalytic site of the phosphatase. This transient and reversible conversion of the phosphatase to sulfenic acid helps propagate signaling downstream to RTK, resulting in cell survival, proliferation, migration, and other phenotypic changes. NADPH Nicotinamide adenine dinucleotide phosphate; PI3K phosphatidylinositol-3 kinase; MAPK mitogen-activated protein kinase; SOD superoxide dismutase.

process that involves translocation of the cytosolic components to the membrane-bound flavoenzyme cytochrome b558 and generation of millimolar quantities of O2 within seconds. Although first described in phagocytic cells, various components of the leukocyte NADPH oxidase complex have been identified in nonphagocytic cells, including endothelial cells, vascular smooth muscle cells, renal mesangial, and tubular epithelial cells, fibroblasts, and chondrocytes [62–76]. In these cells, NADPH oxidase has been shown to be the major source of agonist-induced ROS. Although structurally related, the nonphagocytic NAPDH oxidases posses few functional differences from their leukocyte counterparts. The nonphagocytic NADPH oxidases show: (1) a basal


138

or ambient level activity in different cells; (2) a slower and less potent activation upon agonist stimulation, and (3) unlike phagocytic NADPH oxidases, an intracellular influx of ROS [32, 54, 59, 60, 64, 77–88]. Several different stimuli may cause activation of NADPH oxidase. In endothelial cells, activation of NADPH oxidase has been shown to occur in response to steady-state and oscillatory shear stress, ischemia, cyclical strain, high concentrations of potassium, high glucose and their advanced glycation end products, growth factors, cytokines, and hyperlipidemia [59, 70, 89–104]. In vascular smooth muscle cells, NADPH oxidase activity has been reported to increase by PDGF, thrombin, angiotensin II, tumor necrosis factor-, lipophilic substrates, membrane permeant oxidants (e.g. H2O2) [42, 105–111]. In the kidney, O2-generating NADPH oxidase has been shown to be expressed in the vasculature, interstitium, juxtaglomerular apparatus, and the distal nephron [60, 76, 112–117]. Signal Transduction by ROS Very little is known about the precise mechanism of signal transduction by which ROS bring about phenotypic changes in cell. Most of the ROS are short lived and act in the immediate vicinity of the site of their production, e.g. O2 and singlet oxygen (1O2), which are also unable to penetrate cellular membrane components. As mentioned above, it is well established that ROS may play a wide variety of roles within the cell ranging from gene transcription, cell survival/growth to apoptosis. This raises an important question – how do the short-lived and mostly locally acting ROS initiate a milieu of cellular responses that result in far-reaching changes in the phenotype? The following are the major pathways currently believed to be used by ROS to transduce signal within the cells: (1) a lightning-fast direct oxidation of a signaling molecule(s) in the immediate vicinity of their production; (2) an enzymatic reaction resulting in the conversion of the short-lived ROS (e.g. conversion of O2 by SOD to H2O2) into a more stable and diffusible one that can react with cellular signaling molecules, and (3) by changing the redox state of the cell, which may also affect pH, and thereby modulating the kinetics of cellular metabolic pathways [118, 119]. O2 usually acts by the first pathway of direct oxidation of the signaling molecule and/or local lipid or membrane components. However, H2O2 is relatively stable but unreactive with most cellular molecules and can travel freely within the cellular compartments. The longevity and abundance of H2O2 are dependent on their enzymatic conversion by catalase into CO2 and H2O. Recent studies have suggested that H2O2 exert its effect through the reversible oxidation and inactivation of protein tyrosine phosphatases (PTP), including inositol phosphatase (phosphatase and tensin homolog) and dual specificity phosphatases [120–123]. PTPs are cellular inhibitors of receptor tyrosine


139

kinases (RTK; e.g. PDGF receptor, insulin/insulin-like growth factor receptor, vascular endothelial growth factor receptor). Therefore, transient inhibition of phosphatases by ROS that reversibly oxidize cysteine residues (to sulfenic acid) present at the active sites of the PTPs would allow RTKs and other non-RTKs to propagate signals to downstream molecules. The development of an elegant in vivo alkylation method using iodoacetic acid [124, 125] has remarkably advanced the ROS signal transduction studies. This indirect assay is based on the premise that oxidized (inactive) phosphatase is protected from alkylation by a disulfide bond between Cys residues in intact cells, and therefore retains phosphatase activity when reduced by dithiothreitol in in vitro assays (and vice versa). For example, using this assay, Meng et al. [120] showed that PDGFmediated activation of PDGF receptor and downstream mitogen-activated protein kinase depends on ROS-mediated oxidation (and inactivation) of the Cys residues at the active site of the receptor-bound PTP, SHP-2. ROSmediated inactivation of phosphatase and tensin homolog via the oxidation of the active site Cys residue [125, 126] and ROS-mediated activation of an upstream phosphatidylinositol-3 kinase-regulatory kinase (e.g. non-RTK src [127], focal adhesion kinase [123, 128]) have also been reported. Angiotensin II-mediated Akt phosphorylation has also been shown to be dependent on NADPH-oxidase-derived ROS in vascular cells [129]. However, ROS may act on any of the above signaling molecules at a particular point of the temporal and/or spatial axis to modulate downstream and/or lateral signal transduction from that specific point, depending on the site and type of ROS generated, and the agonist and cell type. ROS may also act by selectively modulating a signaling component of a specific pathway at an upstream location and thereby affecting the entire downstream pathway. Another mechanism of regulation of signal transduction by ROS may occur through a shift in the oxidation/redox state of a localized, subcellular space or component of the cell. Changes in the ROS content of one compartment or subcellular location may be communicated to other signaling pathways, for example, the O2 generated by oxidative phosphorylation within the mitochondria can diffuse into the cytosol only after their (O2) conversion into H2O2 by mitochondrial MnSOD [38] (fig. 1). Cells usually maintain a reduced state within to protect themselves from the injury of the oxidative products that are produced as a result of metabolism [119]. The Cys residues of glutathione, the major component of the cellular redox balance, form disulfide bonds when oxidized. The ratio of glutathione to glutathione disulfide is critical and works as a sensor for the global redox state of the cell. A decrease in this ratio causes an increase in the amount of oxidized (thiolated) thioredoxin. Oxidized thioredoxin has been shown to release and activate apoptosis signal-regulating kinase 1 [130, 131], which then carries out


140

apoptosis through Jun-N-terminal kinase and mitochondria. However, no generalized conclusion or global model for ROS-mediated signal transduction can be drawn at this point due to variability of cell types, their redox components, and specificity of the ligand/receptor signaling. Oxidative Stress and Renal Diseases Although ROS, including the production of reactive nitrogen species (ONOO, nitrogen dioxide) derived from NO are involved in maintaining normal physiological functions, including host defense, studies have documented that oxidative stress plays an important role in initiating and progressing various renal diseases. As mentioned earlier, oxidant stress may result from an overproduction of ROS and/or downregulation or reduced capacity of the cellular antioxidant enzymes, including SODs, catalase, and GPx. Oxidative stress is now believed to be not only involved in renal cell injury, but also associated with activation of various immunoinflammatory and fibrogenic molecules, leading to renal fibroproliferative diseases [132]. To determine the effects of modulation of oxidant levels, -tocopherol (vitamin E) – a known antioxidant, has been used in various human diseases and experimental models [133–136]. The protective effects of the antioxidants vitamin E and -lipoic acid in renal diseases, including nephritis [132], diabetic nephropathy [135, 136], cyclosporin nephrotoxicity [137], and ischemic acute renal failure [138], have been documented in a number of experimental and clinical studies. In contrast, Nath et al. [139] have shown that rats on diet containing low amounts of antioxidants (less selenium and vitamin E) produced tubulointerstitial injuries, reduced glomerular filtration rate, and proteinuria. Recent studies have found an association between uremia and enhanced oxidative stress [140, 141], and hemodialysis or peritoneal dialysis could execrate such stress [142, 143], possibly by membrane-induced activation of macrophages; loss of antioxidant activity, e.g. vitamin E deficiency, could also aggravate oxidative stress in uremic patients. In obstructed kidneys, an increased generation of ROS and impaired antioxidant defense systems, as determined by downregulated expression of Cu-ZnSOD and catalase has been documented [144], and that pretreatment of a synthetic antioxidant (probucol) could improve renal structural damages in obstructed kidneys [145]. A pathological role of hyperglycemiainduced ROS has been suggested in diabetic nephropathy. For instance, relatively less renal Cu-ZnSOD and catalase activity was detected in experimental models of streptozotocin-induced diabetic nephropathy, while prolonged administration of antioxidants could delay the onset of early diabetic retinopathy in rats [146]. In accord with the experimental studies, a similar decrease in renal Cu-ZnSOD and GPx activities were associated with tubular injuries in patients with type 1 diabetes mellitus [147]. High glucose, advanced glycation end products, angiotensin II, and TGF-1 have shown to induce intracellular generation of


141

AGE

AT II

High glucose

ROS

Increased ECM production

Epithelial– mesenchymal transition

TGF-1

Decreased ECM degradation

Matrix remodeling

Diabetic nephropathy

Fig. 2. Schematic diagram showing involvement of ROS in the development of diabetic nephropathy. ROS, induced by AGE, AT-II, TGF-1, and high glucose, regulate matrix remodeling by inducing synthesis of various collagens (mainly types I, III and IV) and modulating rate of matrix degradation by influencing both PA/plasmin/PAI system and MMP/TIMP system. ROS Reactive oxygen species; TGF-1; transforming growth factor 1; AT-II angiotensin II; AGE advanced glycation end products; ECM extracellular matrix; PA plasminogen activator; PAI plasminogen activator inhibitor; MMP matrix metalloproteinase; TIMP tissue inhibitors of matrix metalloproteinase.

ROS in renal cells, and contribute to the development and progression of diabetic renal injury [148–151] (fig. 2). ROS also play a role in high glucose- and TGF-1-induced plasminogen activator inhibitor-1 expression and decreased plasmin activity in rat mesangial cells, and antioxidants, such as NAC, catalase, and trolox, could effectively reverse such effects on plasminogen system [152]. Moreover, exogenous H2O2 as well as TGF-1 could induce epithelial-mesenchymal transition in tubular epithelial cells and antioxidants could effectively inhibit TGF-1-induced epithelial-mesenchymal transition [152]. These studies suggest an important role for ROS in epithelial-mesenchymal transition and matrix remodelling, and eventual tubulointerstitial fibrosis in diabetic kidney.


142

One important question that requires further study is how ROS is generated in diabetic kidney. A recent study has shown that adenoviral-mediated overexpression of protein kinase C (PKC)- in cultured mesangial cells could enhance generation of ROS, by modulating the membranous translocation of p47phox and p67phox [153], suggesting that oxidative stress is primarily enhanced in the diabetic glomeruli due to a PKC--dependent activation of NADPH oxidase, with resultant ROS generation. In a separate study, it has been demonstrated that long-term in vivo inhibition of PKC activation, by using a PKC- inhibitor (LY333531) could ameliorate glomerular pathologies of db/db mice, a model for type 2 diabetes, implicating that PKC- inhibition might be useful in the treatment of diabetic nephropathy [154]. Similar beneficial effects of LY333531 in ameliorating vascular dysfunctions have also been shown in rat models of diabetes [155]. In addition to the activation of proinflammatory molecules, including monocyte chemoattractant protein 1, intercellular adhesion molecule, and other cell adhesion molecules, and exerting cytotoxic effects on various intrinsic renal cells, ROS play a role in the development of renal fibroproliferative lesions by directly inducing synthesis of collagens [156–158]. Furthermore, connective tissue growth factor, a potent inducer of collagen synthesis has shown to be induced by H2O2, possibly via JAK-2/-3 activation [159]. ROS is also involved in the phenotypic transformation of fibroblasts to collagenproducing myofibroblasts, and thereby intensifying fibroproliferative lesions. Oxidant stress appears to play a significant role in renal ischemia and reperfusion injury, a major cause of acute renal failure. As many as 5% of all hospitalized patients are affected by acute renal failure [160, 161], and has a high rate of mortality [162]. Renal ischemia and reperfusion not only increases O2, and its two reaction products OH and ONOO, but also deplete antioxidant enzymes, including SOD, GPx, and catalase [163]. In a rat model of ischemia, MnSOD and catalase levels were significantly reduced as early as 15 min after ischemia, followed by a reduction in GPx by 30 min; after 45 min of ischemia, there was also a significant drop in the antioxidant enzyme activities [164]. Another group showed that MnSOD activity returned to normal levels during reperfusion [165]. All these studies have compellingly demonstrated that oxidant stress is an important contributory factor in acute and chronic renal diseases.

Conclusion

Elucidation of molecular mechanisms of renal injuries in acute and chronic progressive renal diseases is essential for designing any effective therapeutic


143

DM

Nephritis

Drug

Hypertension

Renal injury Chemokines

Accumulation of inflammatory cells

ROS

EMT

TGF-1 CTGF bFGF ET-1 AT-II HSP47

IL-4 IL-13 TGF-1

Induction of ECM

Apoptosis

Glomerulosclerosis Tubular atrophy Capillary obliteration MMP/TIMP Renal fibrosis

Fig. 3. Schematic diagram showing involvement of ROS in various events of multistep and multifactorial disease processes that eventually lead to end-stage renal fibrosis. In order to keep the diagram simple, we have only included key molecules that are known to be involved in the complex process of fibrogenesis. ROS Reactive oxygen species; IL-4 interleukin-4; IL-13 interleukin-13; ET-1 endothelin 1; AT-II angiotensin II, TGF1 transforming growth factor 1; CTGF connective tissue growth factor, bFGF basic fibroblast growth factor; HSP47 heat shock protein 47; MMP matrix metalloproteinase; TIMP tissue inhibitor of metalloproteinase; EMT epithelialmesenchymal transition; ECM extracellular matrix; DM diabetes mellitus.

modalities. Recent studies have convincingly demonstrated pathophysiological roles for ROS in various renal diseases. A detailed understanding of antioxidant defense system that is important for eliminating ROS, and further characterization of signaling cascade following oxidant stress in various renal diseases would open new avenues for selective, yet effective, early interventions. In this brief review, we have discussed the relevance of oxidant stress in pathogenesis of different renal diseases. We realize that renal diseases are multistep and -factorial phenomena [166–171] (fig. 3). To that end, we have presented basic information on the generation and regulation of oxidant stress in the various renal diseases that could be significant, and play important role in our understanding of initiation and progression of renal diseases.


144

Acknowledgment This work was supported, in part, by the National Scientist Development Grant from the American Heart Association to MRA.

References 1

2

3 4

5 6 7 8

9 10 11 12 13 14 15

16 17

18

19

Al-Ghamdi SS, Chatterjee PK, Raftery MJ, Thiemermann C, Yaqoob MM: Role of cytochrome P4502E1 activation in proximal tubular cell injury induced by hydrogen peroxide. Ren Fail 2004;26:103–110. Drager LF, Andrade L, Barros de Toledo JF, Laurindo FR, Machado Cesar LA, Seguro AC: Renal effects of N-acetylcysteine in patients at risk for contrast nephropathy: Decrease in oxidant stressmediated renal tubular injury. Nephrol Dial Transplant 2004;19:1803–1807. Goligorsky MS, Brodsky SV, Noiri E: NO bioavailability, endothelial dysfunction, and acute renal failure: New insights into pathophysiology. Semin Nephrol 2004;24:316–323. Kaushal GP, Liu L, Kaushal V, Hong X, Melnyk O, Seth R, Safirstein R, Shah SV: Regulation of caspase-3 and -9 activation in oxidant stress to RTE by forkhead transcription factors, Bcl-2 proteins, and MAP kinases. Am J Physiol Renal Physiol 2004;287:F1258–F1268. Zhang JJ, Bledsoe G, Kato K, Chao L, Chao J: Tissue kallikrein attenuates salt-induced renal fibrosis by inhibition of oxidative stress. Kidney Int 2004;66:722–732. Touyz RM: Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: What is the clinical significance? Hypertension 2004;44:248–252. Modlinger PS, Wilcox CS, Aslam S: Nitric oxide, oxidative stress, and progression of chronic renal failure. Semin Nephrol 2004;24:354–365. Lu C, He JC, Cai W, Liu H, Zhu L, Vlassara H: Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to AGE in mesangial cells. Proc Natl Acad Sci USA. 2004;101:11767–11772. Ha H, Lee HB: Reactive oxygen species and matrix remodeling in diabetic kidney. J Am Soc Nephrol 2003;14:S246–S249. Li JM, Shah AM: ROS generation by nonphagocytic NADPH oxidase: Potential relevance in diabetic nephropathy. J Am Soc Nephrol 2003;14:S221–S226. Thevenod F: Nephrotoxicity and the proximal tubule. Insights from cadmium. Nephron Physiol 2003;93:p87–p93. Hammes HP: Pathophysiological mechanisms of diabetic angiopathy. J Diabetes Complications 2003;17:16–19. Rodrigo R, Rivera G: Renal damage mediated by oxidative stress: A hypothesis of protective effects of red wine. Free Radic Biol Med 2002;33:409–422. Kovacic P, Sacman A, Wu-Weis M: Nephrotoxins: Widespread role of oxidative stress and electron transfer. Curr Med Chem 2002;9:823–847. Davila-Esqueda ME, Vertiz-Hernandez AA, Martinez-Morales F: Comparative analysis of the renoprotective effects of pentoxifylline and vitamin E on streptozotocin-induced diabetes mellitus. Ren Fail 2005;27:115–122. Xia Y, Zweier JL: Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA 1997;94:6954–6958. Noiri E, Nakao A, Uchida K, Tsukahara H, Ohno M, Fujita T, Brodsky S, Goligorsky MS: Oxidative and nitrosative stress in acute renal ischemia. Am J Physiol Renal Physiol 2001;281: F948–F957. Heigold S, Bauer G: RAW 264.7 macrophages induce apoptosis selectively in transformed fibroblasts: Intercellular signaling based on reactive oxygen and nitrogen species. J Leukoc Biol 2002;72:554–563. Schlondorff D: The role of chemokines in the initiation and progression of renal disease. Kidney Int Suppl 1995;49:S44–S47.


145

20 21

22

23 24

25 26 27 28

29

30

31 32 33 34

35 36

37 38

39 40

41

Nikolic-Paterson DJ, Lan HY, Hill PA, Atkins RC: Macrophages in renal injury. Kidney Int Suppl 1994;45:S79–S82. Yokoyama Y, Kuebler JF, Matsutani T, Schwacha MG, Bland KI, Chaudry IH. Mechanism of the salutary effects of 17beta-estradiol following trauma-hemorrhage: Direct downregulation of Kupffer cell proinflammatory cytokine production. Cytokine 2003;21:91–97. Feng L, Chen S, Garcia GE, Xia Y, Siani MA, Botti P, Wilson CB, Harrison JK, Bacon KB: Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication. Kidney Int 1999;56:612–620. Young BA, Johnson RJ, Alpers CE, Eng E, Gordon K, Floege J, Couser WG, Seidel K: Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int 1995;47:935–944. Razzaque MS, Foster CS, Ahmed AR: Role of collagen-binding heat shock protein 47 and transforming growth factor-beta1 in conjunctival scarring in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44:1616–1621. Razzaque MS, Ahmed BS, Foster CS, Ahmed AR: Effects of IL-4 on conjunctival fibroblasts: Possible role in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci 2003;44:3417–3423. Razzaque MS, Kumari S, Foster CS, Ahmed AR: Expression profiles of collagens, HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita. Cytokine 2003;21:207–213. Tamaki K, Okuda S: Role of TGF-beta in the progression of renal fibrosis. Contrib Nephrol 2003;139:44–65. Nauseef WM, Volpp BD, Clark RA: Immunochemical and electrophoretic analyses of phosphorylated native and recombinant neutrophil oxidase component p47-phox. Blood 1990;76: 2622–2629. Okamura N, Babior BM, Mayo LA, Peveri P, Smith RM, Curnutte JT: The p67-phox cytosolic peptide of the respiratory burst oxidase from human neutrophils. Functional aspects. J Clin Invest 1990;85:1583–1587. Cassatella MA, Bazzoni F, Flynn RM, Dusi S, Trinchieri G, Rossi F: Molecular basis of interferongamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components. J Biol Chem 1990;265: 20241–20246. Heyworth PG, Cross AR, Curnutte JT: Chronic granulomatous disease. Curr Opin Immunol 2003;15:578–584. Finkel T: Signal transduction by reactive oxygen species in non-phagocytic cells. J Leukoc Biol 1999;65:337–340. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD: Cell transformation by the superoxide-generating oxidase Mox1. Nature 1999;401:79–82. Nauseef WM, Volpp BD, McCormick S, Leidal KG, Clark RA: Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components. J Biol Chem 1991;266:5911–5917. Liu X, Brutlag DL, Liu JS: BioProspector: Discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. Pac Symp Biocomput 2001:127–138. Gorlach A, Brandes RP, Bassus S, Kronemann N, Kirchmaier CM, Busse R, Schini-Kerth VB: Oxidative stress and expression of p22phox are involved in the up-regulation of tissue factor in vascular smooth muscle cells in response to activated platelets. FASEB J 2000;14:1518–1528. Wang S, Leonard SS, Ye J, Ding M, Shi X: The role of hydroxyl radical as a messenger in Cr(VI)induced p53 activation. Am J Physiol Cell Physiol 2000;279:C868–C875. Abid MR, Tsai JC, Spokes KC, Deshpande SS, Irani K, Aird WC: Vascular endothelial growth factor induces manganese-superoxide dismutase expression in endothelial cells by a Rac1-regulated NADPH oxidase-dependent mechanism. FASEB J 2001;15:2548–2550. Sauer H, Wartenberg M, Hescheler J: Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001;11:173–186. Bao J, Sato K, Li M, Gao Y, Abid R, Aird W, Simons M, Post MJ: PR-39 and PR-11 peptides inhibit ischemia-reperfusion injury by blocking proteasome-mediated IkappaBalpha degradation. Am J Physiol Heart Circ Physiol 2001;281:H2612–H2618. Brar SS, Kennedy TP, Sturrock AB, Huecksteadt TP, Quinn MT, Whorton AR, Hoidal JR: An NAD(P)H oxidase regulates growth and transcription in melanoma cells. Am J Physiol Cell Physiol 2002;282:C1212–C1224.


146

42

43

44

45

46

47

48

49

50

51 52 53 54 55 56 57 58 59

60 61 62

63

Brandes RP, Miller FJ, Beer S, Haendeler J, Hoffmann J, Ha T, Holland SM, Gorlach A, Busse R: The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression. Free Radic Biol Med 2002;32:1116–1122. Barbieri SS, Eligini S, Brambilla M, Tremoli E, Colli S: Reactive oxygen species mediate cyclooxygenase-2 induction during monocyte to macrophage differentiation: Critical role of NADPH oxidase. Cardiovasc Res 2003;60:187–197. Wang T, Qin L, Liu B, Liu Y, Wilson B, Eling TE, Langenbach R, Taniura S, Hong JS: Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia. J Neurochem 2004;88:939–947. Heinloth A, Heermeier K, Raff U, Wanner C, Galle J: Stimulation of NADPH oxidase by oxidized low-density lipoprotein induces proliferation of human vascular endothelial cells. J Am Soc Nephrol 2000;11:1819–1825. Teshima S, Kutsumi H, Kawahara T, Kishi K, Rokutan K: Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1. Am J Physiol Gastrointest Liver Physiol 2000;279:G1169–G1176. Brar SS, Kennedy TP, Sturrock AB, Huecksteadt TP, Quinn MT, Murphy TM, Chitano P, Hoidal JR: NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2002;282:L782–L795. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M: Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 2000;20:2175–2183. Gao XP, Standiford TJ, Rahman A, Newstead M, Holland SM, Dinauer MC, Liu QH, Malik AB: Role of NADPH oxidase in the mechanism of lung neutrophil sequestration and microvessel injury induced by Gram-negative sepsis: Studies in p47phox/ and gp91phox/ mice. J Immunol 2002;168:3974–3982. Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fujimoto M, Quinn MT, Pagano PJ, Johnson C, Alexander RW: Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res 2002;91:1160–1167. Abid MR, Kachra Z, Spokes KC, Aird WC: NADPH oxidase activity is required for endothelial cell proliferation and migration. FEBS Lett 2000;486:252–256. Sorescu D, Szocs K, Griendling KK: NAD(P)H oxidases and their relevance to atherosclerosis. Trends Cardiovasc Med 2001;11:124–131. Zalba G, San Jose G, Moreno MU, Fortuno MA, Fortuno A, Beaumont FJ, Diez J: Oxidative stress in arterial hypertension: Role of NAD(P)H oxidase. Hypertension 2001;38:1395–1399. Wilcox CS: Reactive oxygen species: Roles in blood pressure and kidney function. Curr Hypertens Rep 2002;4:160–166. Morena M, Cristol JP, Senecal L, Leray-Moragues H, Krieter D, Canaud B: Oxidative stress in hemodialysis patients: Is NADPH oxidase complex the culprit? Kidney Int Suppl 2002:109–114. Terada LS: Oxidative stress and endothelial activation. Crit Care Med 2002;30:S186–S191. Liu Y, Gutterman DD: The coronary circulation in diabetes: Influence of reactive oxygen species on K channel-mediated vasodilation. Vascul Pharmacol 2002;38:43–49. Byrne JA, Grieve DJ, Cave AC, Shah AM: Oxidative stress and heart failure. Arch Mal Coeur Vaiss 2003;96:214–221. Potente M, Fisslthaler B, Busse R, Fleming I: 11,12-Epoxyeicosatrienoic acid-induced inhibition of FOXO factors promotes endothelial proliferation by down-regulating p27Kip1. J Biol Chem 2003;278:29619–29625. Wilcox CS: Redox regulation of the afferent arteriole and tubuloglomerular feedback. Acta Physiol Scand 2003;179:217–223. Yamawaki H, Haendeler J, Berk BC: Thioredoxin: A key regulator of cardiovascular homeostasis. Circ Res 2003;93:1029–1033. Jones SA, Wood JD, Coffey MJ, Jones OT: The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts. FEBS Lett 1994;355:178–182. Fukui T, Lassegue B, Kai H, Alexander RW, Griendling KK: Cytochrome b-558 alpha-subunit cloning and expression in rat aortic smooth muscle cells. Biochim Biophys Acta 1995;1231:215–219.


147

64

65

66 67

68 69

70

71

72 73

74

75 76

77 78 79

80 81 82 83

84 85

Bayraktutan U, Draper N, Lang D, Shah AM: Expression of functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells. Cardiovasc Res 1998;38: 256–262. Lee YS, Kang YS, Lee SH, Kim JA: Role of NAD(P)H oxidase in the tamoxifen-induced generation of reactive oxygen species and apoptosis in HepG2 human hepatoblastoma cells. Cell Death Differ 2000;7:925–932. Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD: Homologs of gp91phox: Cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 2001;269:131–140. Krijnen PA, Meischl C, Hack CE, Meijer CJ, Visser CA, Roos D, Niessen HW: Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction. J Clin Pathol 2003;56:194–199. Bokoch GM, Knaus UG: NADPH oxidases: Not just for leukocytes anymore! Trends Biochem Sci 2003;28:502–508. Teshima S, Rokutan K, Nikawa T, Kishi K: Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system. Gastroenterology 1998;115: 1186–1196. Al-Mehdi AB, Zhao G, Dodia C, Tozawa K, Costa K, Muzykantov V, Ross C, Blecha F, Dinauer M, Fisher AB: Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K. Circ Res 1998;83:730–737. Hannken T, Schroeder R, Stahl RA, Wolf G. Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals. Kidney Int 1998;54:1923–1933. Geiszt M, Kopp JB, Varnai P, Leto TL: Identification of renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci USA 2000;97:8010–8014. Yang ZZ, Zhang AY, Yi FX, Li PL, Zou AP: Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells. Am J Physiol Renal Physiol 2003;284: F1207–F1215. Jones SA, Hancock JT, Jones OT, Neubauer A, Topley N: The expression of NADPH oxidase components in human glomerular mesangial cells: Detection of protein and mRNA for p47phox, p67phox, and p22phox. J Am Soc Nephrol 1995;5:1483–1491. Greiber S, Munzel T, Kastner S, Muller B, Schollmeyer P, Pavenstadt H: NAD(P)H oxidase activity in cultured human podocytes: Effects of adenosine triphosphate. Kidney Int 1998;53:654–663. Etoh T, Inoguchi T, Kakimoto M, Sonoda N, Kobayashi K, Kuroda J, Sumimoto H, Nawata H: Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment. Diabetologia 2003;46:1428–1437. Cai H, Griendling KK, Harrison DG: The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 2003;24:471–478. Li JM, Shah AM: Differential NADPH-versus NADH-dependent superoxide production by phagocyte-type endothelial cell NADPH oxidase. Cardiovasc Res 2001;52:477–486. Li JM, Mullen AM, Yun S, Wientjes F, Brouns GY, Thrasher AJ, Shah AM: Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ Res 2002;90:143–150. Li JM, Shah AM: Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells. J Biol Chem 2002;277:19952–19960. Li JM, Shah AM: Mechanism of endothelial cell NADPH oxidase activation by angiotensin II. Role of the p47phox subunit. J Biol Chem 2003;278:12094–12100. Griendling KK, Sorescu D, Ushio-Fukai M: NAD(P)H oxidase: Role in cardiovascular biology and disease. Circ Res 2000;86:494–501. Irani K: Oxidant signaling in vascular cell growth, death, and survival: A review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res 2000;87:179–183. Finkel T: Neutrophils with a license to kill: Permeabilized, not stirred. Dev Cell 2003;4:146–148. Zou AP, Cowley AW Jr: Reactive oxygen species and molecular regulation of renal oxygenation. Acta Physiol Scand 2003;179:233–241.


148

86 Li P, Lee H, Guo S, Unterman TG, Jenster G, Bai W: AKT-independent protection of prostate cancer cells from apoptosis mediated through complex formation between the androgen receptor and FKHR. Mol Cell Biol 2003;23:104–118. 87 Meier B: Superoxide generation of phagocytes and nonphagocytic cells. Protoplasma 2001;217: 117–124. 88 Rey FE, Pagano PJ: The reactive adventitia: Fibroblast oxidase in vascular function. Arterioscler Thromb Vasc Biol 2002;22:1962–1971. 89 De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK: Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: Role of a superoxide-producing NADH oxidase. Circ Res 1998;82:1094–1101. 90 Li A, Prasad A, Mincemoyer R, Satorius C, Epstein N, Finkel T, Quyyumi AA: Relationship of the C242T p22phox gene polymorphism to angiographic coronary artery disease and endothelial function. Am J Med Genet 1999;86:57–61. 91 Hu Q, Ziegelstein RC: Hypoxia/reoxygenation stimulates intracellular calcium oscillations in human aortic endothelial cells. Circulation 2000;102:2541–2547. 92 Rueckschloss U, Galle J, Holtz J, Zerkowski HR, Morawietz H: Induction of NAD(P)H oxidase by oxidized low-density lipoprotein in human endothelial cells: Antioxidative potential of hydroxymethylglutaryl coenzyme A reductase inhibitor therapy. Circulation 2001;104:1767–1772. 93 Yeh LH, Kinsey AM, Chatterjee S, Alevriadou BR: Lactosylceramide mediates shear-induced endothelial superoxide production and intercellular adhesion molecule-1 expression. J Vasc Res 2001;38:551–559. 94 Heitzer T, Wenzel U, Hink U, Krollner D, Skatchkov M, Stahl RA, MacHarzina R, Brasen JH, Meinertz T, Munzel T: Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C. Kidney Int 1999;55: 252–260. 95 Fleming I, Michaelis UR, Bredenkotter D, Fisslthaler B, Dehghani F, Brandes RP, Busse R: Endothelium-derived hyperpolarizing factor synthase (Cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res 2001;88:44–51. 96 Sohn HY, Krotz F, Gloe T, Keller M, Theisen K, Klauss V, Pohl U: Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine. Cardiovasc Res 2003;58:638–646. 97 Poppa V, Miyashiro JK, Corson MA, Berk BC: Endothelial NO synthase is increased in regenerating endothelium after denuding injury of the rat aorta. Arterioscler Thromb Vasc Biol 1998;18:1312–1321. 98 Gorlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, Kietzmann T, Busse R: Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase. Circ Res 2001;89:47–54. 99 Schuppe-Koistinen I, Gerdes R, Moldeus P, Cotgreave IA: Studies on the reversibility of protein S-thiolation in human endothelial cells. Arch Biochem Biophys 1994;315:226–234. 100 Kashiwagi A, Asahina T, Nishio Y, Ikebuchi M, Tanaka Y, Kikkawa R, Shigeta Y: Glycation, oxidative stress, and scavenger activity: Glucose metabolism and radical scavenger dysfunction in endothelial cells. Diabetes 1996;45(suppl 3):S84–S86. 101 Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H: High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 2000;49:1939–1945. 102 Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T: Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 2001;88:E14–E22. 103 Ido Y, Carling D, Ruderman N: Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: Inhibition by the AMP-activated protein kinase activation. Diabetes 2002;51:159–167. 104 Christ M, Bauersachs J, Liebetrau C, Heck M, Gunther A, Wehling M: Glucose increases endothelialdependent superoxide formation in coronary arteries by NAD(P)H oxidase activation: Attenuation


149

105

106

107

108

109 110

111

112 113

114 115

116 117

118 119 120 121 122

123

124

by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin. Diabetes 2002;51:2648–2652. Marumo T, Schini-Kerth VB, Brandes RP, Busse R: Glucocorticoids inhibit superoxide anion production and p22 phox mRNA expression in human aortic smooth muscle cells. Hypertension 1998;32:1083–1088. Sorescu D, Somers MJ, Lassegue B, Grant S, Harrison DG, Griendling KK: Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells. Free Radic Biol Med 2001;30:603–612. Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, Griendling KK: Novel gp91(phox) homologues in vascular smooth muscle cells: Nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res 2001;88: 888–894. De Keulenaer GW, Alexander RW, Ushio-Fukai M, Ishizaka N, Griendling KK: Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochem J 1998;329(Pt 3):653–657. Berk BC: Redox signals that regulate the vascular response to injury. Thromb Haemost 1999;82:810–817. Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, Runge MS: Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo. J Biol Chem 1999;274:19814–19822. Li WG, Miller FJ Jr., Zhang HJ, Spitz DR, Oberley LW, Weintraub NL: H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury. J Biol Chem 2001;276:29251–29256. Zou AP, Li N, Cowley AW Jr: Production and actions of superoxide in the renal medulla. Hypertension 2001;37:547–553. Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, Wilcox CS: Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension 2002;39:269–274. Mori T, Cowley AW Jr: Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla. Hypertension 2003;42:588–593. Klechikov VZ, Dobrodeev GV: [Interrelation of changes in the epithelium of kidney tubules and interstitium in nephrosclerosis based on morphometric and histochemical data]. Arkh Patol 1987;49:54–60. Agarwal R, Campbell RC, Warnock DG: Oxidative stress in hypertension and chronic kidney disease: Role of angiotensin II. Semin Nephrol 2004;24:101–114. Kerschbaum HH, Huang S, Xie M, Hermann A: NADPH-diaphorase activity and nitric oxide synthase activity in the kidney of the clawed frog, Xenopus laevis. Cell Tissue Res 2000;301: 405–411. Rhee SG, Bae YS, Lee SR, Kwon J: Hydrogen peroxide: A key messenger that modulates protein phosphorylation through cysteine oxidation. Sci STKE 2000;2000:PE1. Kochevar IE: Singlet oxygen signaling: From intimate to global. Sci STKE 2004;2004:PE7. Meng TC, Fukada T, Tonks NK: Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell 2002;9:387–399. Lee SR, Kwon KS, Kim SR, Rhee SG: Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J Biol Chem 1998;273:15366–15372. Denu JM, Tanner KG: Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: Evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 1998;37:5633–5642. Chiarugi P, Pani G, Giannoni E, Taddei L, Colavitti R, Raugei G, Symons M, Borrello S, Galeotti T, Ramponi G: Reactive oxygen species as essential mediators of cell adhesion: The oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion. J Cell Biol 2003;161: 933–944. Federici M, Menghini R, Mauriello A, Hribal ML, Ferrelli F, Lauro D, Sbraccia P, Spagnoli LG, Sesti G, Lauro R: Insulin-dependent activation of endothelial nitric oxide synthase is impaired by


150

125 126 127

128 129

130

131 132 133

134 135

136

137

138 139 140 141

142

143

144

145

O-linked glycosylation modification of signaling proteins in human coronary endothelial cells. Circulation 2002;106:466–472. Leslie NR, Bennett D, Lindsay YE, Stewart H, Gray A, Downes CP: Redox regulation of PI 3-kinase signalling via inactivation of PTEN. EMBO J 2003;22:5501–5510. Lee SR, Yang KS, Kwon J, Lee C, Jeong W, Rhee SG: Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem 2002;277:20336–20342. Esposito F, Chirico G, Montesano Gesualdi N, Posadas I, Ammendola R, Russo T, Cirino G, Cimino F: Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity. J Biol Chem 2003;278:20828–20834. Ben Mahdi MH, Andrieu V, Pasquier C: Focal adhesion kinase regulation by oxidative stress in different cell types. IUBMB Life 2000;50:291–299. Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, Griendling KK: Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 1999;274:22699–22704. Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, Minowa O, Miyazono K, Noda T, Ichijo H: ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2001;2:222–228. Tobiume K, Saitoh M, Ichijo H: Activation of apoptosis signal-regulating kinase 1 by the stressinduced activating phosphorylation of pre-formed oligomer. J Cell Physiol 2002;191:95–104. Cochrane AL, Ricardo SD: Oxidant stress and regulation of chemokines in the development of renal interstitial fibrosis. Contrib Nephrol 2003;139:102–119. Trachtman H, Chan JC, Chan W, Valderrama E, Brandt R, Wakely P, Futterweit S, Maesaka J, Ma C: Vitamin E ameliorates renal injury in an experimental model of immunoglobulin A nephropathy. Pediatr Res 1996;40:620–626. Scheinman JI, Trachtman H, Lin CY, Langman CB, Chan JC: IgA nephropathy: To treat or not to treat? Nephron 1997;75:251–258. Hahn S, Kuemmerle NB, Chan W, Hisano S, Saborio P, Krieg RJ Jr., Chan JC: Glomerulosclerosis in the remnant kidney rat is modulated by dietary alpha-tocopherol. J Am Soc Nephrol 1998;9:2089–2095. Hahn S, Krieg RJ Jr., Hisano S, Chan W, Kuemmerle NB, Saborio P, Chan JC: Vitamin E suppresses oxidative stress and glomerulosclerosis in rat remnant kidney. Pediatr Nephrol 1999;13: 195–198. Satyanarayana PS, Singh D, Chopra K: Quercetin, a bioflavonoid, protects against oxidative stress-related renal dysfunction by cyclosporine in rats. Methods Find Exp Clin Pharmacol 2001;23:175–181. Takaoka M, Ohkita M, Kobayashi Y, Yuba M, Matsumura Y: Protective effect of alpha-lipoic acid against ischaemic acute renal failure in rats. Clin Exp Pharmacol Physiol 2002;29:189–194. Nath KA, Grande J, Croatt A, Haugen J, Kim Y, Rosenberg ME: Redox regulation of renal DNA synthesis, transforming growth factor-beta1 and collagen gene expression. Kidney Int 1998;53:367–381. Vaziri ND: Oxidative stress in uremia: Nature, mechanisms, and potential consequences. Semin Nephrol 2004;24:469–473. Toutain HJ, Sarsat JP, Bouant A, Hoet D, Leroy D, Moronvalle-Halley V: Precision-cut dog renal cortical slices in dynamic organ culture for the study of cisplatin nephrotoxicity. Cell Biol Toxicol 1996;12:289–298. Toborek M, Wasik T, Drozdz M, Klin M, Magner-Wrobel K, Kopieczna-Grzebieniak E: Effect of hemodialysis on lipid peroxidation and antioxidant system in patients with chronic renal failure. Metabolism 1992;41:1229–1232. Epperlein MM, Nourooz-Zadeh J, Jayasena SD, Hothersall JS, Noronha-Dutra A, Neild GH: Nature and biological significance of free radicals generated during bicarbonate hemodialysis. J Am Soc Nephrol 1998;9:457–463. Ricardo SD, Ding G, Eufemio M, Diamond JR: Antioxidant expression in experimental hydronephrosis: Role of mechanical stretch and growth factors. Am J Physiol 1997;272: F789–F798. Modi KS, Morrissey J, Shah SV, Schreiner GF, Klahr S: Effects of probucol on renal function in rats with bilateral ureteral obstruction. Kidney Int 1990;38:843–850.


151

146 Kowluru RA, Tang J, Kern TS: Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes 2001;50:1938–1942. 147 Yaqoob M, McClelland P, Patrick AW, Stevenson A, Mason H, White MC, Bell GM: Evidence of oxidant injury and tubular damage in early diabetic nephropathy. Qjm 1994;87:601–607. 148 Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB: Role of high glucose-induced nuclear factorkappaB activation in monocyte chemoattractant protein-1 expression by mesangial cells. J Am Soc Nephrol 2002;13:894–902. 149 Scivittaro V, Ganz MB, Weiss MF: AGEs induce oxidative stress and activate protein kinase Cbeta(II) in neonatal mesangial cells. Am J Physiol Renal Physiol 2000;278:F676–F683. 150 Yamagishi S, Inagaki Y, Okamoto T, Amano S, Koga K, Takeuchi M: Advanced glycation end products inhibit de novo protein synthesis and induce TGF-beta overexpression in proximal tubular cells. Kidney Int 2003;63:464–473. 151 Jaimes EA, Galceran JM, Raij L: Angiotensin II induces superoxide anion production by mesangial cells. Kidney Int 1998;54:775–784. 152 Lee HB, Yu MR, Yang Y, Jiang Z, Ha H: Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol 2003;14:S241–S245. 153 Kitada M, Koya D, Sugimoto T, Isono M, Araki S, Kashiwagi A, Haneda M: Translocation of glomerular p47phox and p67phox by protein kinase C-beta activation is required for oxidative stress in diabetic nephropathy. Diabetes 2003;52:2603–2614. 154 Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, Kikkawa R: Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J 2000;14:439–447. 155 Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 1996;272:728–731. 156 Sorescu D, Griendling KK: Reactive oxygen species, mitochondria, and NAD(P)H oxidases in the development and progression of heart failure. Congest Heart Fail 2002;8:132–140. 157 Zent R, Ailenberg M, Downey GP, Silverman M: ROS stimulate reorganization of mesangial cellcollagen gels by tyrosine kinase signaling. Am J Physiol 1999;276:F278–F287. 158 Kobayashi T, Uehara S, Ikeda T, Itadani H, Kotani H: Vitamin D3 up-regulated protein-1 regulates collagen expression in mesangial cells. Kidney Int 2003;64:1632–1642. 159 Park SK, Kim J, Seomun Y, Choi J, Kim DH, Han IO, Lee EH, Chung SK, Joo CK: Hydrogen peroxide is a novel inducer of connective tissue growth factor. Biochem Biophys Res Commun 2001;284:966–971. 160 DuBose TD Jr., Warnock DG, Mehta RL, Bonventre JV, Hammerman MR, Molitoris BA, Paller MS, Siegel NJ, Scherbenske J, Striker GE: Acute renal failure in the 21st century: Recommendations for management and outcomes assessment. Am J Kidney Dis 1997;29:793–799. 161 Rahman TM, Treacher D: Management of acute renal failure on the intensive care unit. Clin Med 2002;2:108–113. 162 Smithies MN, Cameron JS: Can we predict outcome in acute renal failure? Nephron 1989;51:297–300. 163 Davies SJ, Reichardt-Pascal SY, Vaughan D, Russell GI: Differential effect of ischaemia-reperfusion injury on anti-oxidant enzyme activity in the rat kidney. Exp Nephrol 1995;3:348–354. 164 Conti M, Eschwege P, Ahmed M, Paradis V, Droupy S, Loric S, Bedossa P, Charpentier B, Legrand A, Benoit G: Antioxidant enzymatic activities and renal warm ischemia: Correlation with the duration of ischemia. Transplant Proc 2000;32:2740–2741. 165 Dobashi K, Ghosh B, Orak JK, Singh I, Singh AK: Kidney ischemia-reperfusion: Modulation of antioxidant defenses. Mol Cell Biochem 2000;205:1–11. 166 Razzaque MS, Azouz A, Shinagawa T, Taguchi T: Factors regulating the progression of hypertensive nephrosclerosis. Contrib Nephrol 2003;139:173–186. 167 Matsuo S, Morita Y, Maruyama S, Manchang L, Yuzawa Y: Proteinuria and tubulointerstitial injury: The causative factors for the progression of renal diseases. Contrib Nephrol 2003;139: 20–31.


152

168 Kashihara N, Sugiyama H, Makino H: Implication of apoptosis in progression of renal diseases. Contrib Nephrol 2003;139:156–172. 169 Razzaque MS, Taguchi T: Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc 2002;35:68–80. 170 Razzaque MS, Le VT, Taguchi T: Heat shock protein 47 and renal fibrogenesis. Contrib Nephrol 2005;148:57–69. 171 Razzaque MS, Taguchi T: Factors that influence and contribute to the regulation of fibrosis. Contrib Nephrol 2003;139:1–11.

Takashi Taguchi MD, PhD Department of Pathology Nagasaki University Graduate School of Biomedical Sciences 1–12–4, Sakamoto machi, Nagasaki 852–8523 (Japan) Tel. 81 958 497 053, Fax 81 958 497 056, E-Mail [email protected]


153

Author Index

Abid, M.R. 107, 135 Akagi, R. 70 Atkins, D. 35 Beck, F.-X. 21 Bijian, K. 8

Le, V.T. 57 Lichtenfels, R. 35

Razzaque, M.S. VII, 1, 57, 107, 135

Muthana, M. 122

Sassa, S. 70 Seliger, B. 35

Cybulsky, A.V. 8

Neuhofer, W. 21 Nazneen, A. 107

Kelly, K.J. 86

Pockley, A.G. 122

Taguchi, T. VII, 1, 57, 107, 135 Takahashi, T. 70

154

Subject Index

Acute renal failure (ARF) heme oxygenase deficiency and disease induction 73, 74 induction in experimental models 74, 75 inhibition and induction effects in ischemic ARF 75, 77 metabolites of reaction and cytoprotection 78 ischemia, see Ischemia/reperfusion injury Allograft, see Organ transplantation Amifostine, cisplatin cytoprotection 115 Amino acids, osmotic stress adaptation of medullary cells 26 Apoptosis cisplatin modulation 110, 115 heat shock protein modulation after ischemia 95, 96 stress protein modulation in renal cell carcinoma 45, 46

initial cytotoxic effects 109, 110 probenecid enhancement 107, 108 prophylaxis 114–117 side effects 108, 109 transport 107 Collagen HSP47 role in synthesis 59–61 processing 58, 59 structure 58 Complement, see Glomerular visceral epithelial cell injury Cyclooxygenase-2 (COX-2), inhibitors and renal risks in dehydration 30 Diabetic nephropathy HSP47 expression in glomerular visceral epithelial cells 17, 18 inflammatory cells and oxidative stress 136, 141–143

Betaine, osmotic stress adaptation of medullary cells 24, 25 Bip, see grp78

Ebselen, cisplatin cytoprotection 115 Edaravone, cisplatin cytoprotection 115, 116 ErbB receptors, signaling modulation by stress proteins in renal cell carcinoma 46, 47

Cisplatin indications 108 nephrotoxicity fibroproliferative effects 111–114 inflammatory events 111

Fibrosis cisplatin effects 111–114 common pathways in disease 57, 58 HSP47 collagen synthesis role 59–61

155

Fibrosis (continued) HSP47 (continued) expression human non-scarring renal diseases 64, 65 human scarring renal diseases 63, 64 nephritis experimental models 61–63 regulation 65 tubulointerstitial fibrosis 63 therapeutic targeting 65, 66 renal fibroproliferative diseases 61 Glomerular visceral epithelial cell (GEC) injury complement-mediated injury and stress protein response 11, 13–16 HSP27 expression in glomerulopathies and actin cytoskeleton effects 18, 19 HSP47 expression in hyperlipidemia and diabetic nephropathy 17, 18 Glycerophosphorylcholine (GPC), osmotic stress adaptation of medullary cells 23, 24 grp78 glomerular visceral epithelial cell complement-mediated injury and stress protein response 11, 13–16 protein synthesis role 9, 93 renal cell carcinoma expression 42 grp94 glomerular visceral epithelial cell complement-mediated injury and stress protein response 11, 13–16 protein synthesis role 9, 93 Heat shock factor (HSF), heat shock protein expression regulation 2, 3, 40 Heat shock proteins (HSPs) allograft expression, see Organ transplantation classification 38–40, 90–92 DNA binding 2 domains 2, 3 expression regulation 2, 3, 40 functional overview 38, 39 history of study 1 HSP22

Subject Index

function 90, 92 ischemia response 94 HSP25 function 92 HSP27 expression in glomerulopathies and glomerular visceral epithelial cell actin cytoskeleton effects 18, 19 function 10 renal cell carcinoma expression 42 HSP32 function 92 HSP47 cisplatin induction and fibrosis 114 collagen synthesis role 59–61 expression human non-scarring renal diseases 64, 65 human scarring renal diseases 63, 64 hyperlipidemia and diabetic nephropathy 17, 78 nephritis experimental models 61–63 regulation 65 tubulointerstitial fibrosis 63 function 10 therapeutic targeting in fibrosis 65, 66 trafficking 59 HSP60 function 92, 93 HSP70 ischemia response 95, 96, 98 medullary cell adaptation to high urea concentrations 27 renal cell carcinoma expression 42, 46 HSP72 ischemia response 95–97, 99 renal cell carcinoma expression 41 HSP73 function 93 human leukocyte antigen class I processing modulation 47, 48 ischemic preconditioning 87 overview in renal disease 4, 5 postischemic inflammation studies 96, 97 protein folding chaperone 3 trimer formation 2 vaccination as renal cell carcinoma immunotherapy 49, 50 Heme oxygenase (HO) activation in oxidative tissue injuries 72, 73

156

acute renal failure and heme oxygenase deficiency and disease induction 73, 74 induction in experimental models 74, 75 inhibition and induction effects in ischemic ARF 75, 77 metabolites of reaction and cytoprotection 78 heme degradation pathway 71, 72 ho-1 gene expression regulation in rat 78–80 oxidative stress response 70–72 Hyperlipidemia, HSP47 expression in glomerular visceral epithelial cells 17, 18 Inflammation allograft response regulation by heat shock proteins 127, 128 oxidative stress, see Oxidative stress myo-inositol, osmotic stress adaptation of medullary cells 26 Ionomycin, endoplasmic reticulum stress induction 10 Ischemia/reperfusion injury ischemic preconditioning 87 oxidative stress 143 stress protein response and functions apoptosis modulation after ischemia 95, 96 HSP22 94 HSP70 95, 96, 98 HSP72 95–97, 99 negative effects 97 overview 88–90, 92, 93 postischemic inflammation and heat shock proteins 96, 97 therapeutic induction 97–99 Medullary cells, see Osmotic stress, medullary cell response

cytoprotective effects 124, 125 HSP70 expression in experimental heart transplantation 122, 123 inflammatory response regulation 127, 128 innate immunity activation 125, 126 therapeutic modulation 128, 129 phases of stress protein expression 123 Osmotic stress, medullary cell response adaptation high salt concentrations long-term adaptation 23–26 short-term adaptation 22, 23 high urea concentrations 26, 27 overview 21, 22 pathophysiology 29, 30 solute signaling through tonicityresponsive enhancer-binding protein 27–29 Oxidative stress antioxidants protective effects in renal disease 141 systems 137 diabetic nephropathy role 139, 141–143 heme oxygenase response 70–72 inflammatory cells and renal disease progression 136 ischemia/reperfusion injury 143 NADPH-oxidase complex 137–139 reactive oxygen species signal transduction 139–141 types 137 Passive Heymann nephritis (PHN), complement-mediated injury and stress protein response 11, 13–16 Peroxynitrite, renal injury 136 Probenecid, enhancement of cisplatin nephrotoxicity 107, 108

NADPH oxidase, oxidative stress 137–139 Organ transplantation heat shock protein expression adaptive immunity activation 126, 127 clinical studies 124

Subject Index

Reactive oxygen species, see Oxidative stress Receptor tyrosine kinases, reactive oxygen species signal transduction 139, 140

157

Renal cell carcinoma (RCC) classification 36 epidemiology 35, 36 markers 37, 38 stress protein expression apoptosis modulation 45, 46 assays 40, 41 growth factor receptor signaling modulation 46, 47 GRP75 42 GRP78 42 heat shock proteins HSP27 42 HSP70 42, 46 HSP72 41 mutants as tumor-associated antigens 48 vaccination as immunotherapy 49, 50 human leukocyte antigen class I processing modulation 47, 48

Subject Index

Proteomex approach 42, 44, 49 treatment 37, 38 Scarring, see Fibrosis Sorbitol, osmotic stress adaptation of medullary cells 25, 26 Taurine, cisplatin cytoprotection 115 Tonicity-responsive enhancer-binding protein (TonEBP), solute signaling in medullary cells 27–29 Transforming growth factor-␤ (TGF-␤), cisplatin induction 111–113 Transplantation, see Organ transplantation Tunicamycin, endoplasmic reticulum stress induction 10, 14, 16 Unfolded protein response (UPR), grp protein roles 9, 10

158

Cellular Stress Responses In Renal Diseases (Contributions to Nephrology)

Renal Fibrosis (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Diseases Renal Parenchyma

Acute Kidney Injury (Contributions to Nephrology, 156)

Obesity And the Kidney (Contributions to Nephrology)

Yeast Stress Responses

Cardiorenal Syndromes in Critical Care (Contributions to Nephrology, Vol. 165)

Kidney Transplantation: Strategies to Prevent Organ Rejection (Contributions to Nephrology)

Advances in Genetics Volume 28 Genomic Responses to Environmental Stress

Plant Responses to Abiotic Stress (Topics in Current Genetics)

Environmental Stress and Cellular Response in Arthropods

Lipids and Cellular Membranes in Amyloid Diseases

Nutrition and Kidney Disease: A New Era (Contributions to Nephrology)

Acute Blood Purification (Contributions to Nephrology, Vol. 166)

Hyperuricemic Syndromes: Pathophysiology and Therapy (Contributions to Nephrology)

Oxidative stress in aging: From model systems to human diseases

Peritoneal Dialysis: A Clinical Update (Contributions to Nephrology)

Fluid Overload: Diagnosis and Management (Contributions to Nephrology, Vol. 164)

Hemodialysis Vascular Access and Peritoneal Dialysis Access (Contributions to Nephrology)

Gene Therapy for Renal Diseases and Transplantation

CNS Injuries: Cellular Responses and Pharmacological Strategies

Stress Responses of Lactic Acid Bacteria

Intensive Care in Nephrology

Ion Channels and Plant Stress Responses

A Practical Manual of Renal Medicine: Nephrology, Dialysis and Transplantation

Redox Proteomics: From Protein Modifications to Cellular Dysfunction and Diseases

Abiotic Stress Responses in Plants: Metabolism, Productivity and Sustainability

Vital Nephrology

Cellular Stress Responses In Renal Diseases (Contributions to Nephrology)

Renal Fibrosis (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Proteomics in Nephrology: Towards Clinical Applications (Contributions to Nephrology)

Diseases Renal Parenchyma

Acute Kidney Injury (Contributions to Nephrology, 156)

Obesity And the Kidney (Contributions to Nephrology)

Yeast Stress Responses

Cardiorenal Syndromes in Critical Care (Contributions to Nephrology, Vol. 165)

Kidney Transplantation: Strategies to Prevent Organ Rejection (Contributions to Nephrology)

Advances in Genetics Volume 28 Genomic Responses to Environmental Stress

Plant Responses to Abiotic Stress (Topics in Current Genetics)

Environmental Stress and Cellular Response in Arthropods

Lipids and Cellular Membranes in Amyloid Diseases

Nutrition and Kidney Disease: A New Era (Contributions to Nephrology)

Acute Blood Purification (Contributions to Nephrology, Vol. 166)

Hyperuricemic Syndromes: Pathophysiology and Therapy (Contributions to Nephrology)

Oxidative stress in aging: From model systems to human diseases

Peritoneal Dialysis: A Clinical Update (Contributions to Nephrology)

Fluid Overload: Diagnosis and Management (Contributions to Nephrology, Vol. 164)

Hemodialysis Vascular Access and Peritoneal Dialysis Access (Contributions to Nephrology)

Gene Therapy for Renal Diseases and Transplantation

CNS Injuries: Cellular Responses and Pharmacological Strategies

Stress Responses of Lactic Acid Bacteria

Intensive Care in Nephrology

Ion Channels and Plant Stress Responses

A Practical Manual of Renal Medicine: Nephrology, Dialysis and Transplantation

Redox Proteomics: From Protein Modifications to Cellular Dysfunction and Diseases

Abiotic Stress Responses in Plants: Metabolism, Productivity and Sustainability

Vital Nephrology

Recommend Documents