Cellular Stress Responses in Renal Diseases

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 mrazzaque@hms.harvard.edu E-Mail taguchi@net.nagasaki-u.ac.jp
and
Department of Pathology
Nagasaki University Graduate
School of Biomedical Sciences
1–12–4, Sakamoto, Nagasaki 852–8523 (Japan)
E-Mail razzaque@net.nagasaki-u.ac.jp
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
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© 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 informa-
tion 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 devel-
opment 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 restor-
ing the biological activities of abnormal proteins. Cellular stress responses include a tran-
sient 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 con-
stitutively expressed and actively involved in maintaining cellular homeostasis,
by acting as molecular chaperones [5–7]. The HSPs regulate folding and assem-
bly of nascent and unfolded peptides, help in transporting proteins to a partic-
ular 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 surface-
exposed hydrophobic portions of proteins, and ultimately enhance their fold-
ing. Certain HSPs exert anti-inflammatory effects by modulating the
transcriptional activation of proinflammatory cytokines and adhesion mole-
cules [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 transcription-

ally 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 eukary-
otes [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 trimeriza-
tion, 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
HSP
HSF P
HSP
HSF HSE
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 dis-
eases, 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 impor-

tant roles of several stress proteins in various renal pathophysiological condi-
tions, 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 protec-
tive role of HSP27 in glomerular epithelial cells injury, and postulate that the
manipulation of HSP27 expression could be potentially beneficial by modulat-
ing 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 aggra-
vating factors. In view of the fact that some of the immunoinflammatory fea-
tures 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.
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ence in trimer cooperativity. Mol Cell Biol 1994;14:7592–7603.
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21 Harrison CJ, Bohm AA, Nelson HCM: Crystal structure of the DNA binding domain of the heat
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2005;148:21–34.
25 Bijian K, Cybulsky AV: Stress Proteins in glomerular epithelial cell injury. Contrib Nephrol
2005;148:8–20.
26 Kelly KJ: Heat shock (stress response) proteins and renal ischemia/reperfusion injury. Contrib
Nephrol 2005;148:86–106.
27 Akagi R, Takahashi T, Sassa S: Cytoprotective effects of heme oxygenase in acute renal failure.
Contrib Nephrol 2005;148:70–85.
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2005;148:35–56.
29 Nagata K: HSP47 as a collagen-specific molecular chaperone: Function and expression in normal
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30 Razzaque MS, Foster CS, Ahmed AR: Role of collagen-binding heat shock protein 47 and trans-
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thymocyte serum (ATS)-induced glomerulonephritis. J Pathol 1997;183:24–29.
32 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.
33 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:
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34 Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T: Age-related nephropathy in the
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protein 47. Cell Tissue Res 1998;293:471–478.
35 Razzaque MS, Taguchi T: Heat shock protein 47 and renal fibrogenesis. Contrib Nephrol
2005;148:57–69.
36 Pockley MR, Muthana M: Heat shock proteins and allograft rejection. Contrib Nephrol 2004;148:
2005;148:122–134.
37 Cochrane AL, Ricardo SD: Oxidant stress and regulation of chemokines in the development of
renal interstitial fibrosis. Contrib Nephrol 2003;139:102–119.
38 Abid MR, Razzaque MS, Taguchi T: Oxidant stress in renal pathophysiology. Contrib Nephrol
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logical 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 mrazzaque@hms.harvard.edu

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, spe-
cialized 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 phospholi-
pase 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.
Introduction
Stress proteins are involved in diverse cellular processes, including assem-

bly 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 con-
stitutively, 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, includ-
ing 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 accumu-
lation 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 condi-
tions. Induction of the UPR may allow cells to recover from ER stress; how-
ever, 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 poly-
merization and stress fiber formation [11]. This process may allow for remod-
eling of the actin cytoskeleton following stress [12]. Small Hsps, including
Hsp27 in their multimeric forms may act as molecular chaperones, e.g., pre-
venting protein aggregation, and facilitating protein refolding after stressful
stimuli [13].
Hsp47
Hsp47 is an ER resident glycoprotein that is heat-inducible, and may func-

tion as a molecular chaperone for collagens. Hsp47 has been shown to interact
with collagens I–V and procollagens, and may participate in the proper pro-
cessing 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 assem-

bly 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 assem-
bles 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 necro-
sis. 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 permselec-
tivity defect of the glomerular capillary wall in PHN. On the other hand, com-
plement 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

a neo cPLA2
HIS, 24h
HIS, 24h
NS, 24h
NS, 24h
HIS, 4h
HIS, 6h
HIS, 4h
HIS, 6h
NS, 4 h
NS, 6 h
NS, 4 h
NS, 6 h
Tunic
Tunic
Untr
Untr
bip
grp94
b
2.6 2.2
2.4 bip grp94
2.0
2.2
Fold increase
2.0 1.8
1.8 1.6
1.6
1.4
1.4
1.2 1.2
1.0 1.0
4h
6h
24h
Tunic
4h
6h
24h
Tunic
4h
6h
24h
Tunic
4h
6h
24h
Tunic
neo cPLA2 neo cPLA2
c d 1.8 bip
C8DS ⫹ C8
NS ⫹ MAFP
1.6
1.8 1.4
Fold increase
Fold increase
C8DS
1.2
HIS
NS
1.4 1.0
1.8
bip bip grp94
1.0 1.6
bip
grp94
1.4
grp94
grp94 1.2
1.0
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 immunoblot-
ted with antibodies to bip or grp94. a Representative immunoblot. b Densitometric quantifi-
cation 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 phos-
pholipid hydrolysis (and presumably membrane injury), and were not due to
products of phospholipid hydrolysis (e.g., arachidonic acid, lysophosphatidyl-
choline) or their metabolites (i.e., prostanoids).
To determine if C5b-9-mediated induction of ER stress proteins is func-
tionally 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 induc-
tion 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 flu-
orophosphonate (MAFP; 25 ␮M), or heat-inactivated serum in controls, for 24 h. A repre-
sentative 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].

40
LDH release (%) 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 cyto-
toxicity was determined by measuring release of lactate dehydrogenase (LDH) into cell super-
natants. 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 cytotoxic-
ity 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 aminonu-
cleoside increased expression of bip. When these treated GEC were subse-
quently 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 complement-
mediated lysis. To model ischemia-reperfusion injury in vitro, GEC were incu-
bated 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 grp94 bip grp94 bip
1.0
ⴙ
**
(arbitrary units)
*
Densitometry
0.8
0.6
0.4
Ctrl
PHN
Ctrl
PHN
Ctrl
ADR
Ctrl
ADR
Ctrl
Tun
Ctrl
Tun
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 C5b-
9-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 lev-
els 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

PHN-ADR
PHN-Tun
1,500
PHN
Proteinuria (mg/24h)
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 protein-
uria 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, prein-
duction 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 patho-
genesis of several sclerotic/fibrotic diseases. Previous studies have demon-
strated 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 infiltra-
tion 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 signifi-
cant 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 hypercholes-
terolemic rats, as compared with controls. Double immunostaining for Hsp47
and desmin (a marker of phenotypically altered GEC), ED-1 (marker of infil-
trating monocytes/macrophages), or ␣-smooth muscle actin (marker of pheno-
typically altered mesangial cells) confirmed that increased Hsp47 was present
specifically in GEC.
Another study addressed the renal expression of Hsp47 and collagens dur-
ing the acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) phases of
streptozotocin-induced diabetic nephropathy in rats [29]. Morphological analy-
sis 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 thicken-
ing of the glomerular basement membrane, glomerulosclerosis, tubular dam-
age, and interstitial fibrosis. Immunohistochemical analysis of these kidney
sections revealed increased deposition of collagen III and IV only in the sam-
ples obtained from chronic diabetic rats, consistent with the aforementioned
morphological changes. Furthermore, immunocytochemical studies demon-
strated 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.

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 syn-
drome 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 glomeru-
losclerosis, which features GEC injury and heavy proteinuria. Using combined
immunofluorescence and electron microscopic studies, the authors showed signif-
icant 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 rearrange-
ment 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 signifi-
cant decreases in cell survival, polymerized actin content and cell area after treat-
ment, whereas sense mRNA-expressing cells had increased cell survival and cell
area. Protection against puromycin aminonucleoside-induced microfilament dis-
ruption 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 phosphoryla-
tion, 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. Further-
more, 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 activa-
tion. 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.
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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 andrey.cybulsky@mcgill.ca.
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 mecha-
nisms 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.
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 num-
ber of adverse consequences such as DNA damage, growth arrest and inhibi-
tion of protein synthesis [1, 2], are most prominent in cells of the renal medulla.
As a result of the renal countercurrent system, extracellular NaCl concentra-
tions 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 mecha-
nism 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 exonucle-
ase complex, which normally resides in the nucleus and is part of this DNA
repair system, is excluded from the nucleus following NaCl-mediated hyper-
tonic 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 concentra-
tions of both betaine and myo-inositol increase rather than decrease protein syn-
thesis, 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 func-
tion. 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 distribu-
tion pattern is observed for sorbitol, which, however, usually is below detection
limit in the cortex. The betaine concentration gradient between the cortico-
medullary 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 trimethy-
lamine 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

1,400
Organic osmolytes, mmol/kg protein 1,200

6
1,000
5
HSP70, ng/µg protein

800
4
600
3
400 2
200 1
0 0
ul r
ul l
x
ul r
ul r
pi of
pi of
ed ota
ed te
te
ed te
ed nne
te
u
la
or
la
pa ip
pa e
m Ou
la
la
lla
lla
or
rm T
O
s
T
I
C
Ba
C
a b
m
m
ne
in
GPC Inositol
Betaine Amino acids
Sorbitol
Fig. 1. a Intrarenal distribution of organic osmolytes in hydropenic rats (urine osmo-

lality 3,364 mosm/kg H2O) [96]. Amino acids analyzed include: asparate, glutamate, gluta-
mine, 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 accom-

plished 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 phospholi-
pase activity [16, 17]. After prolonged exposure to high NaCl (but not urea)
concentrations, the activity of GPC:choline phosphodiesterase, i.e., GPC degra-
dation, 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 long-
term 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 prefer-
entially 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 oxi-
dation 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 sub-
stantially 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 regula-
tion 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 sor-
bitol [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 con-
trast, 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

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 cotrans-
porter activity [24, 26]. SMIT mRNA abundance, which, in antidiuresis is high-
est 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 sul-
fonic ␤-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 kid-
ney [54]. Compared with trimethylamines or polyols, however, the quantitative
contribution of free amino acids to the adaptation of medullary cells to long-
term 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 var-

ious 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 cellu-
lar protein [58, 60]. Experiments involving forced overexpression or down-
regulation 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 sim-
ilarities, osmotic stress protein 94 and HSP110 [66–68]. Since these chaper-
ones 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

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 immediate-
early 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 mam-
malian 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 upregu-
lation 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 expres-
sion 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 trans-
porters, 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 essen-
tial 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 appar-
ently 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 trans-
duction pathway by which cells sense changes in ambient tonicity are not fully
Neuhofer/Beck 28
Hypertonicity
TonEBP
AR, BGT1, SMIT HSP70 UT-A
Compatible
Urea
osmolyte
accumulation
accumulation
Protection from Urea-induced

hypertonicity 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 ⫽ vasopressin-
regulated urea transporters.
understood so far. However, recent studies have demonstrated that the transcrip-
tional 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 mecha-
nism is stimulated and medullary cells have to adapt to increased solute concen-
trations. 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].

In this context, the observation that long-term use of nonsteroidal anti-
inflammatory drugs is associated occasionally with papillary injury and deteri-
oration 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 inter-
feres 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.
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Franz-X. Beck, MD
Physiologisches Institut der Universität
Pettenkoferstrasse 12, DE–80336 München (Germany)
Tel. ⫹49 0 89218075534, Fax ⫹49 0 89218075512
E-Mail FX.Beck@physiol.med.uni-muenchen.de
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,
b
Bayer Health Care AG, PH-R&D-EU Enabling Technologies, Wuppertal,
c
Institute of Medical Immunology, Martin Luther University, Halle, Germany
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 tar-
get for immunotherapies. So far, only a few RCC-associated antigens have been character-
ized. 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 reg-
ulation 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.
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 metanephric adenoma

RCA, papillary type tubulo-papillary adenoma
RCA, oncocytic type oncocytic adenoma
Renal cell carcinoma (RCC) RCC, clear cell type clear cell carcinoma
RCC, papillary type papillary carcinoma
RCC, chromophobic type chromophobic carcinoma
RCC, collecting duct type collecting duct carcinoma
RCC, neuroendocrine type
RCC, unclassified 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 correla-
tion 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
Phenotypic
Initiation association Promotion Adenoma Progression Carcinoma
VHl-Mutation ⫺8p, ⫺9p, ⫺11p,

⫹5q, ⫺17p, ⫺14p,
⫺3p Clear cell type
⫹12, ⫹20
Clear cell type
(Proximal)
tubule Metanephric t(Xp11.2)
system ⫺2p13 ⫹7,⫹17
c-Met-Mutation ⫺3p21
Papillary Papillary type
⫹7, ⫹17, ⫺Y ⫺9p, ⫺11q, ⫺14q, ⫹20
? ⫺X
(Distal) t (5;11) Oncocytic
tubule (q35;q13) ⫺10, ⫺13, ⫺21, ⫺6 Chromophobic
system Hybrid/ ⫺2, ⫺17, ⫺3, ⫺9 type
Collecting ⫺1, ⫺Y chromophobic
duct system
⫺1, ⫺4, ⫺6, ⫺8,
Tubular ?
⫺9, ⫺13, ⫺14,
mucinous
⫺15, ⫺22
tumor
⫺18, ⫺1q, ⫺6p, ⫺8p, Collecting duct
?
⫺13q, ⫺21q type
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 serolog-
ical 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 immuno-
genicity [11–13]. Furthermore, HSPs have been described as molecular chaper-
ones 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 pre-
sent in both prokaryotic and eukaryotic cells. Due to their high level of conser-
vation, 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 situa-
tions, 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 mis-
folded 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
Table 2. Characteristics of human heat shock protein families
HSP family Cellular localization Proposed function
HSP27 (sHSP) cytosol, nucleus microfilament stabilization,

anti-apoptotic activity, suppression
of aggregation and heat
native hot
HSP60 mitochondria refolds proteins and prevents
aggregation of denatured proteins,
pro-apoptotic
HSP70 family: cytosol, mitochondria, anti-apoptotic effects
nucleus
HSP72 (Hsp70) cytosol, nucleus protein folding, cytoprotection
HSP73 (Hsc70) cytosol, nucleus molecular chaperones
HSP78/BIP (GRP78) ER cytoprotection, molecular
chaperones
HSP90 cytosol role in signal transduction, cell
proliferation, protein translocation
GRP94, gp96 ER molecular chaperone, antigen
processing
HSP110/104 cytosol 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 trans-

duction, 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 partic-
ipating 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 refold-
ing and prevention of aggregation of denatured proteins in vitro and may fur-
ther 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

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 constitu-
tive 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 trans-
activating 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).
Table 3. HSP expression in RCC analyzed by various methods
Method Number of Number of Frequency of Literature

RCC lesions/ normal kidney upregulation (%)
cell lines lesions/cell lines
IHC 15 n.d. Takashi et al. [29]

(1998)
31 24 Seliger et al. [9]
(2003, unpubl.)
Western blot 3 1 n.d. Lichtenfels et al.
[32] (2002)
Proteomics 3 1 n.d.* Lichtenfels et al.
[32] (2002)
PROTEOMEX 3 3 n.d. Lichtenfels et al.
[32] (2002)
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 cytoplas-
mic 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 avail-
able 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 signif-
icantly 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 con-
text, it is noteworthy that HSP72 expression was increased following IFN-␥ treat-
ment for 48 h. Thus, HSP72 may represent a favorable prognostic factor
independent of tumor staging and grading which is upregulated by IFN-␥ [30].

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 experi-
mental strategies. These include immunohistochemistry, Western blot analysis,
proteomics and PROTEOMEX. Immunohistochemical staining with an anti-
HSP27-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 unal-
tered 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 con-
ventional proteomics consisting of 2D gel electrophoresis followed by mass
spectrometry of the differentially expressed protein spots, different HSP pro-
teins 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 prog-
nostic 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].
Expression of HSP27 in RCC
of clear cell subtype
5%
33% 19% Strong positive
Intermediate
Weak
Negative
43%
a
b
Expression of HSP27 in RCC
of chromophobic subtype
Strong positive
Intermediate
Weak
0% 10% Negative
50%
40%
c
d
Fig. 2. Expression of HSP27 in normal kidney epithelium and RCCs of different sub-
types. 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 inter-
mediate positive staining for HSP27, whereas 40% of these tumors reveal weak positive
staining, whereas 50% stain negative for this antigen.

Table 4. Heat shock protein recognition by PROTEOMEX
Immunoreactivity of patient sera (n ⫽ 3) with Immunoreactivity of control sera (n ⫽ 3) with

Atkins/Lichtenfels/Seliger
HSP family
member
RCC line 1 RCC line 2 RCC line 3 NN* line 3 RCC line 1 RCC line 2 RCC line 3 NN* line 3
HSP27 none 2 out of 3 none 2 out of 3 none none 2 out of 3 none

variant A
HSP27 none 2 out of 3 none none 1 out of 3 1 out of 3 1 out of 3 none
variant B
HSP27 none 1 out of 3 none 1 out of 3 2 out of 3 none 2 out of 3 none
variant C
HSP60 all all all all 1 out of 3 all 2 out of 3 1 out of 3
HSP70 all 2 out of 3 all 2 out of 3 1 out of 3 2 out of 3 all 2 out of 3
HSP75 all 2 out of 3 1 out of 3 all 1 out of 3 1 out of 3 1 out of 3 2 out of 3
GRP78 2 out of 3 1 out of 3 2 out of 3 none 2 out of 3 2 out of 3 none none
HSP90 2 out of 3 2 out of 3 2 out of 3 2 out of 3 none 2 out of 3 1 out of 3 2 out of 3
GRP94 none none 1 out of 3 none none none none 1 out of 3
*NN ⫽ Cell line derived from normal renal tissue. Differences in the recognition pattern are indicated in bold.
44
Mitochondria Membrane
receptor
Cytochrome C
Apaf-1/
HSP70 Caspase 9
HSP27
Caspase 8
Caspase 3 Pro-caspase 3
Apoptosis
Fig. 3. Inhibition of apoptotic cell death by HSP70 and HSP27. HSPs interact with dif-
ferent 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 chro-
mophobic 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

further investigation, because its precise underlying mechanism is as yet not
understood.
Furthermore, HSP70 has been shown to interact with mutant, but not wild-
type 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 substrate-
specific 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 autophos-
phorylation 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.].
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 HSP90-
Raf-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 vascu-
larization 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 con-
sist 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 chap-
erones 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 ana-
lyzed, but appears to be independent of tumor staging, grading and metastatic
spread [9, 56].

It has been postulated that HSPs are involved in the augmentation and
priming of HLA class I-restricted CTL [57–59]. Besides targeting ubiquiti-
nated 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 immunopro-
teasome [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 depen-
dent. 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 post-
proteasomal carrier, chaperoning antigenic peptides to the TAP system, thereby
preventing further degeneration by cytosolic peptidases [60, 63, 64]. In conse-
quence, 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 pro-
cessing 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 immunothera-
peutic 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
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 stress-
induced 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].

Among the HSPs demonstrated to display prophylactic as well as thera-
peutic 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 sup-
press 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 immuno-
logical and clinical activity of HSP preparations in cancer patients, multiple ran-
domized 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 treat-
ment 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 pathophysio-
logical 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 fur-
ther 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.
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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 Barbara.Seliger@medizin.uni-halle.de
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 collagen-
specific 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 profi-
brotic 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.
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 mecha-
nisms 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 encod-
ing 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 process-
ing 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 modifica-
tions 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, triple-
helix 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 HSP47

formation
Dissociation
Secretion
Propeptide
cleavage
Fibril formation
Fig. 1. Simplified schematic diagram showing involvement of HSP47 during biosyn-

thesis 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
HSP47 and increased deposition of collagens in various human and experi-
mental 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

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, phenotypi-
cally 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
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

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
expression of HSP47 with accumulation of collagen was detected in bleomycin-
induced 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 responsi-
ble for enhanced accumulation of interstitial collagens in the scarring tissues.
In a separate study, the correlation of HSP47 expression and collagen accumu-
lation 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 fibro-
blasts, 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

for modulating renal fibroproliferative diseases. However, because of complex
molecular interactions, despite identifying crucial molecules involved in fibroge-
nesis, 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 thera-
peutic 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 oligodeoxynu-
cleotides 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.
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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 mrazzaque@hms.harvard.edu

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 acceler-
ate 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-reperfusion-
mediated 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 patho-
physiology is summarized, with a particular emphasis on its protective role in the ischemic
acute renal failure.
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
Oxidative stress heat shock, inflammation, UV
heavy metals, NO, heme
ROS Lipid peroxidation
Protein modification
ER
Hemeproteins
(CYP)
V M
M V
HO
N N H H H
Fe2+ O N N N N O
N N
M M NADPH NADP V M M P P M V M
P P O2 CO Biliverdin IX␣
Heme NADPH/NADH
Iron
BVR
NADP/NAD
Ferritin
H H H H
O N N N N O
M; -CH3
P; -CH2-CH2-COOH V M M P P M V M
V; -CH2=CH2
Bilirubin IX␣ 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 oxygen-
ase (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 reduc-
tase 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 pro-
oxidant 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 reduc-
tase (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 num-
ber 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, anti-
inflammatory, 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 activa-
tion, 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 sur-

vival since they must rely on the use of molecular oxygen for various enzy-
matic 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 mod-
erate 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, regula-
tory 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 NO-
mediated 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 devel-
oped 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 pro-
tein 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.

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 hyperlipid-
emia, increased haptoglobin levels, and extremely high concentration of heme
(490 ␮M), associated with undetectable levels of hemopexin. The patient suf-
fered 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 pro-
teinuria 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 compen-
satory 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 accom-
panies 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 fol-
lowed 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 irre-
versible 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
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 reperfu-
sion [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, pre-
sumably 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 aggra-
vation 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/reperfu-
sion 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

4
1.4 Control level
Serum creatinine (mg/dl)

IARF
Heme concentration
1.2 3
SnCl2⫹IARF
(nmol/mg prot)
1.0 SnMP ⫹IARF
0.8 2
0.6
0.4
SnMP⫹IARF
SnCl2⫹IARF
1 HO-1 immunostaining
Control
0.2
IARF
0 0
0 6 12 18 24 (h)
0
(pmol bilirubin/60 min/mg prot)
IARF
50
100
HO activity
150
200 SnCl2 ⫹IARF
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.
Table 1. HO expression in experimental acute renal failure
Procedure Animal HO induction/inhibition Reference
Ischemia/reperfusion Rats, Sprague-Dawley – [49]

Rats, Sprague-Dawley – [50]
Rats, Sprague-Dawley Induction by endotoxin [63]
Rats, Sprague-Dawley Induction by nephrotoxic serum [83]
Rats, Sprague-Dawley Inhibition by Sn-MP [51]
Mice, HO-1 knockout – [40]
Rats, Sprague-Dawley Induction by SnCl2 [60]
Rats, Sprague-Dawley Induction by SnCl2 [84]
Inhibition by Sn-MP
Rats, Wistar Induction by BSO [85]
Rats, Wistar Induction by hemolysate [86]
Glycerol-induced Rats, Inbred strains Induction by hemoglobin [52]
Inhibition by Zn-PP
Rats, Sprague-Dawley Induction by endotoxin [63]
Rats, Sprague-Dawley Induction by nephrotoxic serum [83]
Rats, Wistar – [87]
Mice, Inbred C57BL Induction by hemoglobin [88]
Mice, Mutant Strains
Rats, Sprague-Dawley Induction by bile duct ligation [89]
Cisplatin-induced Rats, Sprague-Dawley Inhibition by Sn-PP [53]
Hemoglobin-induced Rats, Sprague-Dawley Induction by endotoxin [63]
Mice, Inbred C57BL Induction by hemoglobin [88]
Mice, Mutant strains
Gentamicin-induced Mice, Inbred C57BL Induction by hemoglobin [88]
Mice, Mutant strains
Cyclosporine A-, Mice, Inbred C57BL Induction by hemoglobin [88]
Gold sodium Mice, Mutant strains
thiomalate-induced
FeSO4–, Ca2⫹ Rats, Sprague-Dawley Induction by glycerol [90]
ionophore-,
cytochalasin D-,
PLA2-induced
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.

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 apop-
tosis [66]. Furthermore, CO produced from heme by HO can suppress apoptosis
of endothelial cells via the activation of p38 MAPK [16]. Thus, all these metabo-
lites 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 impor-
tant 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 sub-

strate, 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
1,000 b
MARE
5' 3'
CdRE AP-1
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5
⫺4,000 b
HSE E-box
5' Exon 1 3'
⫺400 b IL- 6 IL- 6
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 activ-
ity 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 pre-
vent 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 acute-
phase 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 recog-
nition 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

involve the small Maf proteins, either with Nrf or Bach 1. The small Maf pro-
teins 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 com-
petitive interplay between the Bach 1-containing repressor dimers and Nrf-con-
taining 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 DNA-
binding activity of Bach 1 through a direct interaction [80, 81], mediated by an
evolutionarily conserved multiple heme regulatory motif including the cysteine-
proline 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 acti-
vation 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 inhibi-
tion of HO activity, and suppression of ho-1 gene expression, lead to aggrava-
tion 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 treat-
ment 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.
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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 akagirei@fhw.oka-pu.ac.jp

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 mole-
cular chaperones that facilitate the folding of enzymes and other proteins into functional
conformations. After injury, HSPs are believed to facilitate the restoration of normal func-
tion 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 func-
tional 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.
Acute renal ischemia injury is common in hospitalized patients. The mor-

tality remains ⬎50% in many series and has not improved significantly in sev-
eral 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 proce-

dure 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 ani-
mals after daily ischemia for 6 but not 3 days. Islam et al. [12] found no pro-
tection 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 cre-
atinine. 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, precondition-
ing 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 hyper-
thermia, 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 acti-
vate 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
Denatured protein DNA
RNA
Stress Polypeptide
Stress
HSPs
HSPs
Aggregated
HSPs
Irreparably Correctly protein
damaged folded,
protein functional Stress
protein
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 intra-
cellular 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 poten-
tial 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 poten-
tially 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

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 seg-
ments 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 local-
ized 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 eukary-
otic 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 metabo-
lism. 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 Postulated significance

(Prokaryotic homolog)
Small HSPs Form large multimeric structures;

restricted to specific tissues
HSP32 Cytosol Antioxidant
HSP25/27 Cytosol/nuclear Inhibits actin polymerization;
may limit apoptosis
HSP22 Cytosol Binds denatured proteins
HSP10 (GroES) Mitochondria Regulates HSP60
P20 Cytosol Interacts with HSP25/27
MKBD Cytosol Maintains myofibril integrity;
increased in myotonic dystrophy
HSP60 (GroEL; Large oligomers
chaperonins)
HSP 60 Mitochondria Molecular chaperone; limits protein
aggregation and facilitates folding
TCP-1 Cytosol Facilitates folding of cytoskeletal
proteins
HPS70 (DnaK)
HSP72/HSP70 Cytosol/nuclear with Stress-inducible chaperone; limits
stress protein aggregation; inhibits apoptosis
HSP73/HSC70 (Ssa) Cytosol/peroxisome Constitutive/CFTR binding
GRP75 Mitochondria Molecular chaperone
GRP78/BiP (Kar2) Endoplasmic reticulum Molecular chaperone
HSP90 (HtpG)
HSP90␣ (HSP86) Cytosol Part of steroid hormone receptor
complex; molecular chaperone
HSP90␤ (HSP84) Cytosol
GRP94 Endoplasmic reticulum Calcium-binding chaperone
HSP 110
(C1p/SSE families)
HSP110 Cytosol/nucleus Proteolysis
HSP105␣/␤ Cytosol Solubilizes aggregated proteins
OSP94/APG-1 Cytosol (renal medulla) Induced by hyperosmotic stress
Other
GRP170
HSP56 Cytosol Part of steroid receptor complex;
binds FK 506
Calnexin Endoplasmic reticulum Glycoprotein folding
Calreticulin Endoplasmic reticulum Glycoprotein folding
Ubiquitin Cytosol/nuclear/membrane Protein degradation
(only eukaryotes)

Table 2 (continued)
HSP family Subcellular localization Postulated significance

(Prokaryotic homolog)
HSP47 Endoplasmic reticulum Processing of procollagen

HSP40 (DnaJ) Cytosol Regulates HSP70; minimizes
protein aggregation
HSP10 (GroES) Mitochondria 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 aggre-
gation [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 cytoskele-
ton 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 ischemia-
resistant and tolerance-acquired neurons in the gerbil brain [75].
HSP90, with other chaperones, assists in the maturation of steroid hor-
mone 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 colla-
gen 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].
HSPs and Renal Ischemia
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

50
Urea nitrogen (mg/dl)

40
30
6h
20 12h
48h
Sham
10
0 2 4 6
Time (days)
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] sug-
gesting that this protein is important in organogenesis and thus may be import-
ant 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 func-
tion 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 induc-
tion of several different HSPs results in decreased apoptosis [105–108] and
necrosis [33]. Apoptosis has been demonstrated in human ARF [109] and limit-
ing 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

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) expres-
sion 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 pul-
monary 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 neu-
trophil infiltration and mucosal injury after intestinal ischemia [129]. Induction
of HSP72 via hyperthermia or exposure to sodium arsenite results in attenu-
ation of neutrophil-mediated necrosis of endothelial cells in vitro [130]. Ischemia-
induced 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 hyperther-
mia 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 extra-
cellular, 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 prolif-
erate in response to HSP72 [137]. In human acute and chronic allograft rejec-
tion, a marked increase in the stress response proteins HSP60 and HDJ2 (a
member of the HSP40 family of chaperones) has been found [138]. Anti-
HSP60 antibodies are found in patients with ischemic heart disease and correl-
ate 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 epithe-
lia 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 pre-
conditioning 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

size and better survival [144, 145]. In both the liver and heart, ischemic pre-
conditioning 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-pyridinecarboximidoil-
chloride 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 bimo-
clomol 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 ele-
vations 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, herbi-
mycin 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 expres-
sion 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 con-
siderations in designing potential therapies, but one consideration is early diag-
nosis. 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 bio-
marker 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.
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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 kajkelly@iupui.edu
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 microenviron-
mental changes following cisplatin treatment is a complex process and could be broadly cat-
egorized 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 var-
ious phases of cisplatin-induced renal injuries.
Introduction
Cis-dichlorodiaminoplatinum (II), cisplatin, is one of the most widely used

antineoplastic drugs. Cisplatin is an inorganic complex formed by an atom of plat-
inum surrounded by chlorine and ammonia atoms in the cis position of a horizon-
tal 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 dam-
age, induces cell cycle arrest in the G2 phase, reduces ATPase activity, alters cel-
lular transport system, eventually leading to apoptotic and/or necrotic cell death.
Cisplatin is the single most active antitumor agent against testicular, blad-
der, ovarian, lung, head and neck tumors (table 1). The use of cisplatin in com-
bination 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 potas-
sium [8] could be partly restored by supplementation [9, 10]. In addition, both
human and experimental studies have shown that the use of diuretics and hydra-
tion 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 var-
ious 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 infil-
tration 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, inflam-
matory 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 dis-
rupts 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 oxida-
tive 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 mito-
chondrial 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 relation-
ship 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 Fas-
mediated cisplatin-induced apoptosis has been reported in neuroblastoma [36],
leukemia [35] and hepatoma cells [34] and thymocytes [33]; in contrast a Fas-
independent cisplatin-induced apoptosis has also been reported in various tumors
cell lines [38, 39] including lung cancer cells. It appears likely that cisplatin-
induced apoptosis does not always take a uniform pathway, and there might be a
cell-specific mode of apoptosis. Early cytotoxic events following cisplatin treat-
ment are usually associated with inflammatory changes in the kidneys.
Inflammatory Events
Detailed inflammatory phenotypes of infiltrating cells in kidney of cisplatin-
treated 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 umbili-
cal vein [43]. Increased renal expression of tumor necrosis factor-␣, transform-
ing 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

a
Fig. 1. Infiltration of ED-1-positive macrophages (arrows) in control (a) and cisplatin-

treated 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 cisplatin-
treated rats, an increased expression of TGF-␤1 has been detected in tubular
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 signifi-
cant 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 stud-
ies 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 num-
ber of these above-mentioned molecules were detected in the kidneys of
cisplatin-treated rats [62].

HSP47, a collagen-specific molecular chaperone, is involved in the biosyn-
thesis 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 kid-
neys 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 synthe-
sis of collagens, whereas resorption of the extracellular matrix is mediated pre-
dominantly 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 patholog-
ical 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 nephropa-
thy, while the expression decreased in later stages (on day 14). Decreased renal
expression of MMP-1 has been shown to be associated with increased intersti-
tial 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 cisplatin-
related side effects. Numerous human and experimental studies have been per-
formed 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 inten-
sive 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
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 dys-
function 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 cyto-
protective 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 com-
plex 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, atten-
uated 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 apo-
ptotic death, implicating that hepatocyte growth factor may ameliorate cis-
platin-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 cisplatin-
treated rats, taurine-treated rats showed relatively less renal damage, as deter-
mined by histo-morphometric analysis. Taurine-treatment resulted in less
macrophage accumulation and delayed interstitial fibrotic changes in cisplatin-
treated 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 apop-
tosis [80]. Use of a novel free radical scavenger, 3-methyl-1-phenyl-pyrazolin-
5-one (MCI-186; edaravone) has also been shown to protect the kidneys from

Cisplatin
Oxidative injury Chemokines
Cytotoxicity Macrophages
Cytochrome c
caspase-3, -9
Fas/FasL
Phenotypic alteration
HSP47 of tubulointerstitial TGF-␤1
cells
Apoptosis
Matrix
PDGF
remodeling
Tubulointerstitial
injury
Fig. 3. Schematic diagram showing involvement of various molecules involved in ini-

tiation 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 pre-
vent 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. Cisplatin-
induced 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
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 char-
acterizing 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 mem-
bers 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.
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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 taguchi@net.nagasaki-u.ac.jp

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 reactiv-
ity drive transplant rejection responses. However, although these proteins can activate innate
immune cells such as monocytes, macrophages and dendritic cells and can promote the devel-
opment 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 pro-
longs 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.
Introduction
The first study to link heat shock protein (Hsp) expression and organ trans-
plantation was published by Currie et al. in 1987 [1]. This investigated protein
synthesis in heterotopically transplanted rat hearts and other tissues, and demon-
strated 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 oxy-
genation 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/reper-
fusion injury with which organ procurement, preservation, storage and trans-
plantation are inevitably associated. This early phase appears to lead to the
induction of the stress proteins Hsp60, Hsp70 and the endoplasmic reticulum-
associated 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 car-
diac 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 mole-
cular 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; how-
ever, 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 tar-
geted 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 proin-
flammatory 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 lipopolysac-
charide (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 pre-
sent in the extracellular environment.
In the mammalian system, Hsps have typically been regarded as being
exclusively intracellular proteins, and their presence in the extracellular envi-
ronment 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, extra-
cellular 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 inter-
cellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 expres-
sion 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) matura-
tion 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

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 demon-
strate 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 Hsp-
derived 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 trans-
planted 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 find-
ings 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 indi-
viduals [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 mole-
cules 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 implica-
tions 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 physio-
logical 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 dis-
ease 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 autolo-
gous Hsps [68, 101–104]. Self-Hsp reactive T cells recognizing self-Hsp
epitopes in stressed tissues via low-affinity interactions might lead to the

generation of Th2 (IL-4-producing), Th3 (TGF␤-producing) or Tr1 (IL-10-
producing) 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 popu-
lations 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 trans-
plantation 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 potenti-
ating 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 identi-
fied as an autoantigen in rheumatoid arthritis and shown to stimulate the pro-
liferation 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 gener-
ation 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.
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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 g.pockley@sheffield.ac.uk
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 cen-
tral 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.
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, con-
tribute to the development of renal fibroproliferative lesions. Although ongoing
studies have significantly increased our understanding of molecular mecha-
nisms 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-cell-
recruiting 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 pre-
cede 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, uncon-
trolled regulation of proinflammatory and profibrogenic molecules, including
platelet-derived growth factor (PDGF), interleukin-1, insulin-like growth fac-
tor, epidermal growth factor, tumor necrosis factor-, basic fibroblast growth
factor, and transforming growth factor-1 (TGF-1), continue to induce exces-
sive synthesis of extracellular matrix proteins, and in some instances inhibit
degradation of matrix proteins, eventually leading to the development of fibro-
proliferative 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 con-
tinuously 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 mecha-
nisms 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 nonphago-
cytic cells [32–44]. Agonist-stimulated ROS have been shown to activate down-
stream 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 sur-
vival/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 cel-
lular 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
Receptor tyrosine
gp91 p22 kinase
Rac
p47
p67 NAD(P) H PI3K
NAD(P)H e p PKC
MAPK
O2
•O2 Protein tyrosine
phosphatase
MnSOD
Cu ZnSOD H2O2
Cell survival
Proliferation
Migration
•O2 Catalase
H2O2
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 tyro-
sine 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, pro-
liferation, migration, and other phenotypic changes. NADPH Nicotinamide adenine dinu-
cleotide phosphate; PI3K phosphatidylinositol-3 kinase; MAPK mitogen-activated
protein kinase; SOD superoxide dismutase.
process that involves translocation of the cytosolic components to the mem-

brane-bound flavoenzyme cytochrome b558 and generation of millimolar quan-
tities of O2 within seconds. Although first described in phagocytic cells,
various components of the leukocyte NADPH oxidase complex have been iden-
tified in nonphagocytic cells, including endothelial cells, vascular smooth mus-
cle 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 non-
phagocytic NAPDH oxidases posses few functional differences from their
leukocyte counterparts. The nonphagocytic NADPH oxidases show: (1) a basal
or ambient level activity in different cells; (2) a slower and less potent activa-
tion 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 kid-
ney, 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 sig-
naling 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

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 PDGF-
mediated 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. ROS-
mediated 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 signal-
ing 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 compart-
ment or subcellular location may be communicated to other signaling path-
ways, 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
apoptosis through Jun-N-terminal kinase and mitochondria. However, no gen-
eralized 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 fibro-
proliferative 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 exper-
imental 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) pro-
duced tubulointerstitial injuries, reduced glomerular filtration rate, and protein-
uria. 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 oxida-
tive 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 pre-
treatment of a synthetic antioxidant (probucol) could improve renal structural
damages in obstructed kidneys [145]. A pathological role of hyperglycemia-
induced 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

AGE AT II
High glucose ROS TGF-1
Epithelial–
Increased ECM Decreased ECM
mesenchymal
production degradation
transition
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 remod-
eling 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 sys-
tem. 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 metal-
loproteinase; 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-mesenchy-
mal 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.
One important question that requires further study is how ROS is gener-
ated 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 oxi-
dase, with resultant ROS generation. In a separate study, it has been demon-
strated 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 collagen-
producing 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 antioxi-
dant 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 activ-
ities [164]. Another group showed that MnSOD activity returned to normal lev-
els 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

DM Nephritis Drug Hypertension
Renal injury
Chemokines
Accumulation of ROS
EMT
inflammatory cells
TGF-1 Apoptosis
CTGF
IL-4 bFGF
IL-13 ET-1
TGF-1 AT-II
HSP47
Induction of Glomerulosclerosis
ECM 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, TGF-
1 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 epithelial-
mesenchymal 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 characteriza-
tion 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.
Acknowledgment
This work was supported, in part, by the National Scientist Development Grant from
the American Heart Association to MRA.
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Benoit G: Antioxidant enzymatic activities and renal warm ischemia: Correlation with the dura-
tion 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 hyperten-
sive 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.
168 Kashihara N, Sugiyama H, Makino H: Implication of apoptosis in progression of renal diseases.
169 Razzaque MS, Taguchi T: Cellular and molecular events leading to renal tubulointerstitial fibro-
sis. 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.
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 taguchi@net.nagasaki-u.ac.jp

Author Index
Abid, M.R. 107, 135 Le, V.T. 57 Razzaque, M.S. VII, 1, 57,
Akagi, R. 70 Lichtenfels, R. 35 107, 135
Atkins, D. 35
Muthana, M. 122 Sassa, S. 70
Beck, F.-X. 21 Seliger, B. 35
Bijian, K. 8
Neuhofer, W. 21
Cybulsky, A.V. 8 Nazneen, A. 107 Taguchi, T. VII, 1, 57,
107, 135
Kelly, K.J. 86 Pockley, A.G. 122 Takahashi, T. 70
154
Subject Index
Acute renal failure (ARF) initial cytotoxic effects 109, 110

heme oxygenase probenecid enhancement 107, 108
deficiency and disease induction prophylaxis 114–117
73, 74 side effects 108, 109
induction in experimental models transport 107
74, 75 Collagen
inhibition and induction effects in HSP47 role in synthesis 59–61
ischemic ARF 75, 77 processing 58, 59
metabolites of reaction and structure 58
cytoprotection 78 Complement, see Glomerular visceral
ischemia, see Ischemia/reperfusion epithelial cell injury
injury Cyclooxygenase-2 (COX-2), inhibitors
Allograft, see Organ transplantation and renal risks in dehydration 30
Amifostine, cisplatin cytoprotection 115
Amino acids, osmotic stress adaptation Diabetic nephropathy
of medullary cells 26 HSP47 expression in glomerular visceral
Apoptosis epithelial cells 17, 18
cisplatin modulation 110, 115 inflammatory cells and oxidative stress
heat shock protein modulation after 136, 141–143
ischemia 95, 96
stress protein modulation in renal cell Ebselen, cisplatin cytoprotection 115
carcinoma 45, 46 Edaravone, cisplatin cytoprotection 115,
116
Betaine, osmotic stress adaptation of ErbB receptors, signaling modulation by
medullary cells 24, 25 stress proteins in renal cell carcinoma
Bip, see grp78 46, 47
Cisplatin Fibrosis
indications 108 cisplatin effects 111–114
nephrotoxicity common pathways in disease 57, 58
fibroproliferative effects 111–114 HSP47
inflammatory events 111 collagen synthesis role 59–61
155
Fibrosis (continued) function 90, 92
HSP47 (continued) ischemia response 94
expression HSP25 function 92
human non-scarring renal diseases HSP27
64, 65 expression in glomerulopathies and
human scarring renal diseases 63, 64 glomerular visceral epithelial cell
nephritis experimental models actin cytoskeleton effects 18, 19
61–63 function 10
regulation 65 renal cell carcinoma expression 42
tubulointerstitial fibrosis 63 HSP32 function 92
therapeutic targeting 65, 66 HSP47
renal fibroproliferative diseases 61 cisplatin induction and fibrosis 114
collagen synthesis role 59–61
Glomerular visceral epithelial cell (GEC) expression
injury human non-scarring renal diseases
complement-mediated injury and stress 64, 65
protein response 11, 13–16 human scarring renal diseases 63, 64
HSP27 expression in glomerulopathies hyperlipidemia and diabetic
and actin cytoskeleton effects 18, 19 nephropathy 17, 78
HSP47 expression in hyperlipidemia and nephritis experimental models 61–63
diabetic nephropathy 17, 18 regulation 65
Glycerophosphorylcholine (GPC), osmotic tubulointerstitial fibrosis 63
stress adaptation of medullary cells 23, 24 function 10
grp78 therapeutic targeting in fibrosis 65, 66
glomerular visceral epithelial cell trafficking 59
complement-mediated injury and HSP60 function 92, 93
stress protein response 11, 13–16 HSP70
protein synthesis role 9, 93 ischemia response 95, 96, 98
renal cell carcinoma expression 42 medullary cell adaptation to high urea
grp94 concentrations 27
glomerular visceral epithelial cell renal cell carcinoma expression 42, 46
complement-mediated injury and HSP72
stress protein response 11, 13–16 ischemia response 95–97, 99
protein synthesis role 9, 93 renal cell carcinoma expression 41
HSP73 function 93
Heat shock factor (HSF), heat shock human leukocyte antigen class I
protein expression regulation 2, 3, 40 processing modulation 47, 48
Heat shock proteins (HSPs) ischemic preconditioning 87
allograft expression, see Organ overview in renal disease 4, 5
transplantation postischemic inflammation studies 96, 97
classification 38–40, 90–92 protein folding chaperone 3
DNA binding 2 trimer formation 2
domains 2, 3 vaccination as renal cell carcinoma
expression regulation 2, 3, 40 immunotherapy 49, 50
functional overview 38, 39 Heme oxygenase (HO)
history of study 1 activation in oxidative tissue injuries 72,
HSP22 73
Subject Index 156

acute renal failure and heme oxygenase cytoprotective effects 124, 125
deficiency and disease induction 73, 74 HSP70 expression in experimental
induction in experimental models 74, heart transplantation 122, 123
75 inflammatory response regulation
inhibition and induction effects in 127, 128
ischemic ARF 75, 77 innate immunity activation 125,
metabolites of reaction and 126
cytoprotection 78 therapeutic modulation 128, 129
heme degradation pathway 71, 72 phases of stress protein expression 123
ho-1 gene expression regulation in rat Osmotic stress, medullary cell response
78–80 adaptation
oxidative stress response 70–72 high salt concentrations
Hyperlipidemia, HSP47 expression in long-term adaptation 23–26
glomerular visceral epithelial cells 17, 18 short-term adaptation 22, 23
high urea concentrations 26, 27
Inflammation overview 21, 22
allograft response regulation by heat pathophysiology 29, 30
shock proteins 127, 128 solute signaling through tonicity-
oxidative stress, see Oxidative stress responsive enhancer-binding protein
myo-inositol, osmotic stress adaptation of 27–29
medullary cells 26 Oxidative stress
Ionomycin, endoplasmic reticulum stress antioxidants
induction 10 protective effects in renal disease
Ischemia/reperfusion injury 141
ischemic preconditioning 87 systems 137
oxidative stress 143 diabetic nephropathy role 139, 141–143
stress protein response and functions heme oxygenase response 70–72
apoptosis modulation after ischemia inflammatory cells and renal disease
95, 96 progression 136
HSP22 94 ischemia/reperfusion injury 143
HSP70 95, 96, 98 NADPH-oxidase complex 137–139
HSP72 95–97, 99 reactive oxygen species
negative effects 97 signal transduction 139–141
overview 88–90, 92, 93 types 137
postischemic inflammation and heat
shock proteins 96, 97 Passive Heymann nephritis (PHN),
therapeutic induction 97–99 complement-mediated injury and stress
protein response 11, 13–16
Medullary cells, see Osmotic stress, Peroxynitrite, renal injury 136
medullary cell response Probenecid, enhancement of cisplatin
nephrotoxicity 107, 108
NADPH oxidase, oxidative stress 137–139
Reactive oxygen species, see Oxidative
Organ transplantation stress
heat shock protein expression Receptor tyrosine kinases, reactive
adaptive immunity activation 126, 127 oxygen species signal transduction 139,
clinical studies 124 140
Subject Index 157

Renal cell carcinoma (RCC) Proteomex approach 42, 44, 49
classification 36 treatment 37, 38
epidemiology 35, 36
markers 37, 38 Scarring, see Fibrosis
stress protein expression Sorbitol, osmotic stress adaptation of
apoptosis modulation 45, 46 medullary cells 25, 26
assays 40, 41
growth factor receptor signaling
Taurine, cisplatin cytoprotection 115
modulation 46, 47
Tonicity-responsive enhancer-binding
GRP75 42
protein (TonEBP), solute signaling in
GRP78 42
medullary cells 27–29
heat shock proteins
Transforming growth factor-␤ (TGF-␤),
HSP27 42
cisplatin induction 111–113
HSP70 42, 46
Transplantation, see Organ transplantation
HSP72 41
Tunicamycin, endoplasmic reticulum stress
mutants as tumor-associated antigens
induction 10, 14, 16
48
vaccination as immunotherapy 49, 50
human leukocyte antigen class I Unfolded protein response (UPR), grp
processing modulation 47, 48 protein roles 9, 10
Subject Index 158

Cellular Stress Responses in Renal Diseases

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Cellular Stress Responses in Renal Diseases

Claudio Ronco Vicenza

Mohammed S. Razzaque Boston, Mass.

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

Basel · Freiburg · Paris · London · New York ·

Mohammed S. Razzaque Takashi Taguchi

Library of Congress Cataloging-in-Publication Data

Cellular stress responses in renal diseases / volume editors, Mohammed S.

© Copyright 2005 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)

1 Involvement of Stress Proteins in Renal Diseases

8 Stress Proteins in Glomerular Epithelial Cell Injury

21 Response of Renal Medullary Cells to Osmotic Stress

35 Heat Shock Proteins in Renal Cell Carcinomas

57 Heat Shock Protein 47 and Renal Fibrogenesis

70 Cytoprotective Effects of Heme Oxygenase in Acute Renal Failure

86 Heat Shock (Stress Response) Proteins and Renal Ischemia/

107 Cisplatin-Associated Nephrotoxicity and Pathological Events

135 Oxidant Stress in Renal Pathophysiology

154 Author Index

155 Subject Index

Studies on heat shock proteins (HSPs) and stress responses following an

Mohammed S. Razzaque, Boston, Mass.

Involvement of Stress Proteins

The heat shock response, first observed by Ritossa in Drosophila in 1962

The stress responses in mammalian cells are thought to be transcription-

HSP HSF HSP

Fig. 1. Simplified schematic diagram showing the transcriptional regulation of heat

budding yeasts, HSFs also have a carboxyl-terminal hydrophobic repeat, which

Stress Proteins in Renal Diseases 3

In the accompanying chapters, the authors have elaborated on the impor-

Extensive research work in the last couple of years has significantly

Stress Proteins in Renal Diseases 5

Mohammed S. Razzaque, MBBS, PhD

Stress Proteins in Renal Diseases 7

Stress Proteins in Glomerular

Stress proteins are involved in diverse cellular processes, including assem-

The ER serves as a site for folding, assembly and degradation of proteins

Stress Proteins in Glomerular Epithelial Cell Injury 9

Although Hsps were originally shown to be induced in cells exposed to

Hsp47 is an ER resident glycoprotein that is heat-inducible, and may func-

Activation of the complement cascade near a cell surface leads to assem-

Stress Proteins in Glomerular Epithelial Cell Injury 11

Fig. 1. Effect of complement and cPLA2 on expression of ER stress proteins in GEC.

expression of ER stress proteins. Antibody-sensitized GEC that overexpress cPLA2 were

Stress Proteins in Glomerular Epithelial Cell Injury 13

Fig. 2. Complement-mediated cytotoxicity in GEC that express bip antisense mRNA.

complement-dependent injury. Thus, induction of ER stress proteins is an

Stress Proteins in Glomerular Epithelial Cell Injury 15

Fig. 4. Pretreatment of rats with a subnephritogenic dose of adriamycin or tunicamycin

tunicamycin also enhanced glomerular expression of bip and grp94 without

The ability of Hsp47 to bind to newly synthesized procollagen in the ER

Stress Proteins in Glomerular Epithelial Cell Injury 17

Hsp27 and the GEC Actin Cytoskeleton in Glomerulopathies

Hsp27 has been implicated in the actin polymerization/depolymerization

Stress Proteins in Glomerular Epithelial Cell Injury 19

Response of Renal Medullary

Adaptation to High NaCl Concentrations

Osmoadaptation in the Renal Medulla 23

Organic osmolytes, mmol/kg protein 1,200

HSP70, ng/µg protein

Fig. 1. a Intrarenal distribution of organic osmolytes in hydropenic rats (urine osmo-

[15]. The degradation of GPC to choline and glycerol-3-phosphate is accom-

Osmoadaptation in the Renal Medulla 25

Adaptation to High Urea Concentrations