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  , . 183: 24–29 (1997)

COLLAGEN-BINDING HEAT SHOCK PROTEIN (HSP) 47 EXPRESSION IN ANTI-THYMOCYTE SERUM (ATS)-INDUCED GLOMERULONEPHRITIS   *    Second Department of Pathology, Nagasaki University School of Medicine, Nagasaki, Japan

SUMMARY An increased accumulation of extracellular matrix (ECM), predominantly collagens, is the main component of the expanded mesangial matrix in anti-thymocyte serum (ATS)-induced glomerulonephritis (GN). Heat shock protein (HSP) 47 is a collagen-binding stress protein and has been shown to have a specific role in the intracellular processing of procollagen molecules. It is a collagen-specific molecular chaperone in various organs, but its role in the kidney in relation to matrix expansion is not yet known. This study was designed to assess whether increased ECM accumulation in ATS-induced GN is associated with HSP47. The expression of type I, type III and type IV collagens, with their molecular chaperone HSP47, was investigated in ATS-induced GN rat kidneys. Fifteen male Wistar rats were divided into two groups: ATS-induced GN rats (group I) and age-matched controls (group II). GN was induced by injecting a single dose of ATS (0·8 ml/100 g body weight). All the rats were killed on the third and tenth day of the experiment. In group I, 3 days after ATS injection, histological examination revealed a reduction in glomerular cell number with mesangiolysis. However, 10 days after ATS injection, histologically severe mesangial cell proliferation with expansion of the mesangial matrix was noted in group I rats. By semiquantitative analysis, compared with controls, increased type I, type III, and type IV collagen immunostaining was observed in the expanded mesangial matrix in ATS-induced GN (group I) rats on day 10. Immunoreactive HSP47 expression was weak in the intraglomerular cells and was occasionally seen in the interstitial cells in control kidneys. In contrast, strong immunostaining for HSP47 was noted in the glomeruli of the ATS-treated rat kidneys on day 10. In this study, there was a parallel increase of various collagens and their molecular chaperone HSP47 in the ATS-treated rat kidneys. Compared with controls, no significant difference in HSP47 expression was found in the ATS-treated rat kidneys without mesangial matrix expansion (3 days after ATS injection). It is concluded that overexpression of HSP47 might play a significant role in the excessive assembly of collagens and could subsequently contribute to the expansion of mesangial matrix found in ATS-treated rat kidneys. ? 1997 by John Wiley & Sons, Ltd. J. Pathol. 183: 24–29, 1997. No. of Figures 4. No. of Tables 0. KEY WORDS—HSP47;

No. of References 21.

collagen; anti-thymocyte serum; glomerulonephritis

INTRODUCTION Mesangial matrix expansion as a result of increased deposition of various extracellular matrix (ECM) components is found not only in various form of human glomerulonephritis (GN), but also in other nonneoplastic human renal diseases,1 including diabetic nephropathy2 and hypertensive nephrosclerosis.3 The exact mechanism of the increased deposition of ECM is not fully understood. Heat shock protein (HSP) 47, a 47 kD heat shock protein with unique collagen-binding ability,4,5 resides in the endoplasmic reticulum (ER). It is thought that HSP47 binds procollagen immediately after it cotranslationally enters the ER, and it dissociates from the procollagen before its secretion. In the case of conformationally abnormal procollagen, HSP47 binds for a much longer time in the ER, suggesting a specific role of HSP47 in the intracellular processing of procollagen molecules as a collagen-specific molecular chaperone.6 *Correspondence to: M.S. Razzaque, MBBS, PhD, Second Department of Pathology, Nagasaki University School of Medicine, 1-12-4, Sakamoto, Nagasaki 852, Japan. E-mail: razzaque@net.nagasaki-u. ac.jp

CCC 0022–3417/97/090024–06 $17.50 ? 1997 by John Wiley & Sons, Ltd.

HSP47 is assumed to be a molecular chaperone for collagen synthesis in various organs and its significance is reported elsewhere.7–9 It is possible that HSP47 is involved in the increased deposition of various collagens in glomerulonephritis. Rat GN induced by the injection of anti-thymocyte serum (ATS) is a useful model for investigating the mechanisms involved in the development of mesangial matrix expansion.10 In this model, early mesangiolytic changes are followed by mesangial cell proliferation and expansion of matrix components.11 To the best of our knowledge, it has yet to be determined whether mesangial matrix expansion is related to the collagenbinding HSP47. This preliminary study was undertaken to evaluate the expression and distribution of HSP47 in ATS-induced GN. MATERIALS AND METHODS Animals Fifteen male Wistar rats aged 6 weeks were used for the study. All the rats were given free access to normal rat chow and tap water throughout the experimental period. Received 20 August 1996 Revised 10 December 1996 Accepted 4 March 1997


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Experimental design The rats were divided into two experimental groups. Group I (n=9) were injected with a single dose of ATS (0·8 ml/100 g body weight). Group II (n=6) were agematched control rats. Three rats from each group were killed on the third day after ATS injection and the remaining rats were killed on the tenth day. Tissue collection Rats were killed by cardiac puncture under ether anaesthesia. Both kidneys were then removed via a midline incision and a portion of the kidney was immediately stored at "80)C for further use. The remaining portion of the kidney was fixed immediately either in Carnoy’s solution for 2 h, or in 10 per cent formalin for 24 h. Histological studies Tissues were processed to paraffin and 4 ìm sections were stained with haematoxylin and eosin (H & E), periodic acid Schiff (PAS), and periodic acid Schiff methenamine silver (PAM). The degree of glomerular injury was assessed from the state of the mesangial cells and matrix. Immunohistochemistry This was performed to analyse the localization of type I, type III, and type IV collagens, along with their molecular chaperone HSP47, in control and ATStreated kidneys. Briefly, paraffin wax sections (4 ìm) were dewaxed in xylene and rehydrated in a graded ethanol series. The slides were preincubated with 0·3 per cent hydrogen peroxide in methanol (20 min) to block endogenous peroxidase activity. The sections were incubated with either 10 per cent goat or 10 per cent rabbit serum (30 min) to block non-specific binding. As a negative control, the primary antibodies were replaced by either 0·01  PBS or mouse IgG or rabbit IgG in a concentration similar to that of the primary antibodies. Polyclonal antibodies against type I collagen (Cosmo Bio, Japan), type III collagen (Chemicon, U.S.A.), type IV collagen (Cosmo Bio), and monoclonal antibody against HSP47 (Biotechnologies Corp., Canada) were diluted 1 in 100 with 0·01  PBS (pH 7·2) except for HSP47, which was diluted 1 in 200 with 0·01  PBS. The sections were allowed to react with the primary antibodies for 1 h and were processed further by the streptavidin–biotin method with a Histofine SAB kit (Nichirei, Tokyo), used as directed by the manufacturer. The sections were developed with 3,3*diaminobenzidine/H2O2. The staining intensity of the collagens and HSP47 was assessed visually and graded as follows: 0, no staining; +, mild staining; ++, moderate staining; and +++, strong staining. Double immunostaining This was performed to reveal two distinct antigens in the same renal section, using a Histostain-DS kit ? 1997 by John Wiley & Sons, Ltd.

Fig. 1—Histological features of a control (A) and ATS-treated glomeruli (B, C). (A) Glomerulus of an age-matched control rat, killed on the tenth day of the experiment, showing no significant histological changes. (B) Glomerulus of an ATS-injected rat, killed on the third day of the experiment, revealing mesangiolytic changes. (C) Glomerulus of an ATS-injected rat, killed on the tenth day of the experiment, showing mesangial cell proliferation and expansion of the matrix components. (PAS stain)

(Zymed Laboratories Inc., U.S.A.) as directed by the manufacturer. Briefly, paraffin sections (4 ìm) were deparaffinized, denatured with 0·3 per cent hydrogen peroxide in methanol (30 min), reacted with 10 per cent non-immune goat serum (10 min), and incubated with the monoclonal antibody against HSP47 for 1 h. The sections were treated further with the biotinylated second antibody (10 min) and streptavidin–alkaline phosphatase (10 min) successively and developed with   , . 183: 24–29 (1997)


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Fig. 2—Immunohistochemistry for type III collagen (A) and type IV collagen (C) in age-matched control renal sections. Immunostaining for type III collagens is noted mainly in the interstitium of the control kidney. The mesangium, GBM, and TBM are positively stained for type IV collagen in the control kidney. In contrast, immunostaining for type III collagen (B) and type IV collagen (D) is strongly present in ATS-treated kidneys on day 10, although the pattern of distribution is different; type III collagen is particularly present in the pericapsular fibrosis and type IV collagen is mainly located in expanded mesangial matrix. (Bars=50 ìm)

BCIP/NBT, which produces a dark purple stain. Then the sections were reacted with double-staining enhancer reagent (provided in the DS kit) for 30 min, blocked with 10 per cent non-immune goat serum (10 min), and incubated with polyclonal antibodies against type I, type III, and type IV collagens (1 h). The sections were further treated with the biotinylated second antibody (10 min) and streptavidin–peroxidase (HRP) (10 min) successively. The antigen–antibody complex was visualized using aminoethyl carbazole (AEC)/H2O2, which produces an intense red stain. As the immunohistochemical control, primary antibodies were replaced with either 0·01  PBS or mouse/rabbit IgG diluted with PBS at a concentration similar to that of the primary antibody. RESULTS Histological changes after ATS treatment Throughout the experimental period, age-matched control rats showed no significant histological changes ? 1997 by John Wiley & Sons, Ltd.

(Fig. 1A). In contrast, ATS-treated rats showed marked mesangiolysis and aneurysm of the capillary loops, with a decrease in the number of cells in the glomeruli on day 3 (Fig. 1B). Subsequently, severe mesangial cell proliferation with expansion of the mesangial matrix, periglomerular fibrosis, and inflammatory cell infiltration were noted on day 10 (Fig. 1C). Immunohistochemical localization of type I, type III, and type IV collagens Immunostaining for type I (data not shown) and type III (Fig. 2A) collagens was similar and found mainly in the interstitium (+), while type IV collagen was weakly positive (+) in the mesangium and glomerular basement membrane (GBM) of the control glomeruli (Fig. 2C). Compared with the control rats (group II), increased immunostaining (+ + +) for type I collagen (data not shown), type III collagen (Fig. 2B), and type IV collagen (Fig. 2D) was observed in the expanded mesangial matrix and in the pericapsular fibrosis in group I rats killed 10 days after ATS treatment, suggesting increased   , . 183: 24–29 (1997)


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HSP47 IN ATS-INDUCED GLOMERULONEPHRITIS

Fig. 3—Immunohistochemistry for HSP47 in an age-matched control renal section, showing positive staining for HSP47 in the intraglomerular cells and interstitial cells (A). In ATS-treated rat kidneys on day 10, markedly increased HSP47 immunostaining is noted in the fibroblasts in the pericapsular fibrosis (B) and in the expanded mesangial areas (C). (Bars=50 ìm)

accumulation of type I, type III, and type IV collagens in ATS-treated rat glomeruli. The expression of collagens was not increased in group I rats killed 3 days after ATS injection (data not shown). Immunohistochemical localization of HSP47 Immunoreactive HSP47 expression was detected mainly in the intraglomerular cells including mesangial (+) and epithelial cells (+), interstitial cells (+), and occasionally in the tubular epithelial cells (+) in control kidney (Fig. 3A). However, markedly increased HSP47 immunostaining was noted mainly in the mesangial proliferative lesions and interstitial fibroblasts in and around the pericapsular fibrosis in the kidneys of group I rats killed 10 days after ATS injection (Figs 3B and 3C). Increased expression of HSP47 was always noted in all the glomeruli with expanded mesangial matrix and pericapsular fibrosis. In contrast, no evidence of increased expression of HSP47 was found in the kidneys of group I rats killed 3 days after ATS injection (data not shown). When monoclonal HSP47 was replaced ? 1997 by John Wiley & Sons, Ltd.

with a similar concentration of mouse IgG, no specific staining was noted (data not shown). Co-localization of HSP47 and collagens To determine whether the increased expression of HSP47 in ATS-induced GN is associated with increased collagen accumulation, double staining for HSP47 and collagens was performed on the same renal section. Co-expression of HSP47 and type I (data not shown), type III (Figs 4A and 4B), and type IV collagens (Figs 4C and 4D) was always noted in all the glomeruli with expanded mesangial matrix and pericapsular fibrosis in the kidneys of group I rats killed 10 days after ATS treatment. DISCUSSION The mechanism by which glomerulosclerosis occurs in various renal diseases remains one of several important unanswered questions. Although several reports have   , . 183: 24–29 (1997)


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Fig. 4—Double staining on a paraffin section of ATS-induced GN using antibodies against HSP47 and type III collagen (A, B) and HSP47 and type IV collagen (C, D). HSP47 is stained dark purple and collagens are stained an intense red. Note that the immunostaining for HSP47 is associated with increased collagen deposition in and around the glomeruli in ATS-induced GN on day 10. (Bars=50 ìm)

documented increased synthesis of ECM, including various collagens, by the intrarenal cells at the mRNA and protein level during the development of glomerulosclerosis in experimental and human renal diseases,1–3,12–14 very little is known about the intracellular processing of the collagen molecules, and even less information is available about the association between glomerulosclerosis and collagen-binding HSP47. HSP47 is one of the heat shock proteins that exert important biological effects on collagen synthesis and thus could actively be involved in the mesangial matrix expansion found in ATS-treated rats. The histological changes in ATS-treated rats include early mesangiolytic changes followed by mesangial cell proliferation and expansion of matrix components. Increased deposition of ECM, including various collagens, is mainly responsible for these structural alterations in ATS-treated rat kidneys. We used this model to evaluate the expression and distribution of HSP47 in relation to mesangial matrix expansion. In the present study, increased expression of various collagens correlated with the increased expression of their molecular chaperone HSP47 in the ATS-treated rat ? 1997 by John Wiley & Sons, Ltd.

kidney. By immunohistochemistry, we were able to demonstrate the precise localization of HSP47 in control and ATS-treated rat kidneys. The expression of HSP47 was substantially increased in the glomeruli in parallel with the increased expression of type I, type III, and type IV collagens in the kidneys of group I rats killed 10 days after ATS treatment. However, no significant difference in HSP47 expression was found in control rats and in group I rats with no significant expansion of the mesangial matrix (3 days after ATS injection), indicating that an elevated level of HSP47 protein was not necessarily due to the direct effect of ATS as a stressor, but could be related to the progression of glomerulosclerosis. Our results are in accord with earlier reports in which the expression of HSP47 mRNA was markedly induced during the progression of fibrosis, in parallel with alpha 1(I) and alpha 1(III) collagen mRNAs, in carbon tetrachloride-induced liver fibrosis in rats.15 Localization of HSP47 was also found only in collagensecreting cells, such as chondrocytes in cartilage, smooth muscle cells in the gastrointestinal tract and blood vessels, vitamin A storage cells in sinusoidal areas of   , . 183: 24–29 (1997)


HSP47 IN ATS-INDUCED GLOMERULONEPHRITIS

liver, endothelial cells in blood vessels, epithelial cells of renal glomeruli and tubules, and basal cells of epidermis.16 In developing long bone, HSP47 expression was related to type I collagen expression.17 In this preliminary study, immunostaining for HSP47 was strongly positive in the glomerular mesangial and epithelial cells in ATS-treated rat kidneys, along with increased expression of various collagens. The exact nature of the association has yet to be defined, but in view of the fact that HSP47 plays an important part in the synthesis, processing, and assembly of various collagens,15,18–20 elevated levels of HSP47 may play a significant role in the subsequent manifestation of mesangial matrix expansion. Our preliminary studies revealed that the expression of HSP47 was also elevated at both protein and mRNA levels in cisplatin-induced tubulointerstitial nephritis in parallel with increased expression of various collagens.21 These pieces of evidence seem to indicate that HSP47 is not only associated with glomerulosclerosis, but might also contribute significantly to the tubulointerstitial fibrotic process. Further in vivo and in vitro studies are required to clarify the factors regulating the increased synthesis of HSP47 and to determine whether therapeutic intervention of HSP47 could partially block or slow down the sclerotic process in the kidney. In fact, experimental studies have shown that phosphorothioate antisense oligodeoxynucleotides to HSP47 both inhibited HSP47 production and consequently diminished the production of á(1) chains of type I procollagen.18 In conclusion, these data show that ATS-induced mesangial matrix expansion is associated with an increased expression of HSP47 in the glomeruli, in parallel with the increased expression of various collagens. We conclude that increased HSP47 in ATStreated rat kidneys might contribute significantly to the expansion of mesangial matrix and pericapsular fibrosis in this model.

2. 3.

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

16. 17. 18.

ACKNOWLEDGEMENTS

19.

We thank Ms R. Togawa and Mrs Y. Yamashita for preparing paraffin sections during this study. Special thanks are due to Drs M. Cheng and A. Nazneen for their technical help.

20.

REFERENCES 1. Petern EP, Striker LJ, Carome MA, Elliot SJ, Yang CW, Striker GE. The contribution of increased collagen synthesis to human glomerulosclerosis: a

? 1997 by John Wiley & Sons, Ltd.

21.

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quantitative analysis of a 2 IV collagen mRNA expression by competitive polymerase chain reaction. J Exp Med 1992; 176: 1571–1576. Razzaque MS, Koji T, Taguchi T, Harada T, Nakane PK. In situ localization of type III and type IV collagen expressing cells in human diabetic nephropathy. J Pathol 1994; 174: 131–138. Razzaque MS, Koji T, Kawano H, Harada T, Nakane PK, Taguchi T. Glomerular expression of type III and type IV collagens in benign nephrosclerosis: immunohistochemical and in situ hybridization study. Pathol Res Pract 1994; 190: 493–499. Nagata K, Yamada KM. Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts. J Biol Chem 1986; 261: 7531–7536. Nagata K, Saga S, Yamada KM. A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein. J Cell Biol 1986; 103: 223–229. Nakai A, Satoh M, Hirayoshi K, Nagata K. Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J Cell Biol 1992; 117: 903–914. Cates GA, Nandan D, Brickenden AM, Sanwal BD. Differentiation defective mutants of skeletal myoblasts altered in a gelatin-binding glycoprotein. Biochem Cell Biol 1987; 65: 767–775. Cates GA, Brickenden AM, Sanwal BD. Possible involvement of a cell surface glycoprotein in the differentiation of skeletal myoblasts. J Biol Chem 1984; 259: 2646–2650. Saga S, Nagata K, Chen WT, Yamada KM. pH-dependent function, purification, and intracellular location of a major collagen-binding glycoprotein. J Cell Biol 1987; 105: 517–527. Yamamoto T, Wilson CB. Complement dependence of antibody-induced mesangial cell injury in the rat. J Immunol 1987; 138: 3758–3765. Yamamoto T, Wilson CB. Quantitative and qualitative studies of antibodyinduced mesangial cell damage in the rat. Kidney Int 1987; 32: 514–525. Border WA, Okuda S, Nakamura T. Extracellular matrix and glomerular disease. Semin Nephrol 1989; 9: 307–317. Jones CL, Buch S, Post M, McCullock L, Liu E, Eddy AA. Renal extracellular matrix accumulation in acute puromycin aminonucleoside nephrosis in rats. Am J Pathol 1992; 141: 1381–1396. Razzaque MS, Taguchi T. Expression of type III collagen mRNA in renal biopsy specimens of patients with idiopathic membranous glomerulonephritis. J Clin Pathol (Mol Pathol) 1996; 49: M40–M42. Masuda H, Fukumoto M, Hirayoshi K, Nagata K. Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. J Clin Invest 1994; 94: 2481–2488. Miyaishi O, Sakata K, Matsuyama M, Saga S. Distribution of the collagen binding heat-shock protein in chicken tissues. J Histochem Cytochem 1992; 40: 1021–1029. Shroff B, Smith T, Norris K, Pileggi R, Sauk JJ. Hsp47 is localized to regions of type I collagen production in developing murine femurs and molars. Connective Tissue Res 1993; 29: 273–286. Sauk JJ, Smith T, Norris K, Ferreira L. Hsp47 and the translation– translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen. J Biol Chem 1994; 269: 3941–3946. Kambe K, Yamamoto A, Yoshimori T, Hirayoshi K, Ogawa R, Tashiro Y. Preferential localization of heat shock protein 47 in dilated endoplasmic reticulum of chicken chondrocytes. J Histochem Cytochem 1994; 42: 833–841. Ferreira LR, Norris K, Smith T, Hebert C, Sauk JJ. Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones. J Cell Biochem 1994; 56: 518–526. Razzaque MS, Cheng M, Horita Y, Taguchi T, Harada T. Expression of collagen binding heat shock protein 47 (HSP47) in cisplatin-induced tubulointerstitial nephritis in rats. J Am Soc Nephrol 1996; 7: 1845 (Abstract).

  , . 183: 24–29 (1997)


The Histochemical Journal 33: 621–628, 2001. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats Diange Liu, Mohammed S. Razzaque, Ming Cheng & Takashi Taguchi∗ Second Department of Pathology, Nagasaki University School of Medicine, Nagasaki, Japan ∗

Author for correspondence

Received 22 October 2001 and in revised form 31 January 2002

Summary Glomerulosclerosis and tubulointerstitial fibrosis are the main structural changes found in the later stages of diabetic nephropathy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, a collagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagen synthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47 in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 in the kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renal expression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, we have performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of α-smooth muscle actin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomerular epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III and type IV collagens, α-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins in paraffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in the expression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47 was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal deposition of types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of αsmooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) was detected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys of chronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiation and progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly support a pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which is associated with glomerulosclerosis and tubulointerstitial fibrosis. Introduction Heat shock protein (HSP) 47 or colligin is a collagen-specific chaperone that was originally identified by Kurkinen et al. (Kurkinen et al. 1984) from murine parietal endoderm cells. Subsequently, HSP47 from human, rabbit, rat, mouse and chicken cDNA have been cloned and revealed a high degree of homology in their sequences (Wang & Gudas 1990, Hirayoshi et al. 1991, Clarke & Sanwal 1992, Takechi et al. 1992, Hart et al. 2000). HSP47 binds specifically to various types of collagens including types I–V in vitro (Natsume et al. 1994). It has been shown to be present in the endoplasmic reticulum and numerous studies have shown its expression in collagenproducing cells and tissues. The biochemical properties, intracellular localization and tissue distribution of HSP47, suggest its role in post-transcriptional regulation of procollagens (Nagata 1998). The crucial role of HSP47 in collagen biosynthesis is well-documented, and HSP47 gene disruption resulted in embryonic lethality in mice. The HSP47 null

mice died by 11.5 days post-coitus and showed a molecular abnormality in procollagen (Nagai et al. 2000). Recent studies have convincingly shown a pathogenic role of HSP47 in the development and progression of various human and experimental fibrotic diseases (Masuda et al. 1994, Razzaque & Taguchi 1997, 2002 Razzaque et al. 1998a, Razzaque et al. 2001). For instance, the expression of HSP47 augmented CCl4 -induced liver cirrhosis, bleomycin-induced pulmonary fibrosis and anti-thymocyte serum-induced glomerulosclerosis in rats (Masuda et al. 1994, Razzaque & Taguchi 1997, Razzaque et al. 1998a). However, the expression and significance of HSP47 in kidneys of acute and chronic diabetic rats and its role in diabetic nephrosclerosis are not yet clear. Streptozotocin (STZ)induced diabetic rats are widely used as a model to study renal changes (Osterby et al. 1967). STZ treatment results in insulin deficiency in rats and the morphological changes in the kidney closely resemble those of human disease (Wilson & Letter 1990). In this preliminary study, we have studied the


622 expression of HSP47 and collagens (types III and IV) in the kidneys obtained from STZ-induced diabetic rats, in both short- and long-term diabetes.

D. Liu et al. developed with a mixture of 3,3 -diaminobenzine 4 HCl (DAB) and H2 O2 . Double staining

Materials and methods Animal study Six-week-old male Sprague–Dawley rats (n = 36) were used for the study. 18 rats were injected with STZ (50 mg/kg body weight) via a tail vein. The rats remained untreated for the developing diabetes. A second set of 18 age-matched normal rats served as a control. All rats had unlimited access to water and chow. The body weight and blood glucose levels of each rat were measured periodically. Three rats from each group were sacrificed on days 1, 3 and 14 and weeks 4, 12 and 24 of the experiment. Tissue collection Rats were sacrificed by exsanguinations under ether anesthesia. Both kidneys were removed via midline incision and fixed immediately in Carnoy’s solution for 2 h (for immunohistochemistry) and fixed in 10% formalin for 24 h (for both immunohistochemistry and histological examination). Histological studies Tissues were routinely processed and embedded in paraffin wax, cut into 4 µm sections and stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS), periodic acidSchiff-methenamine silver (PAM) or Masson’s trichome. The extent of glomerular and tubulointerstitial damages were determined in these stained slides by light microscope examination. Immunohistochemistry Paraffin sections (4 µm-thick) of kidneys were cut, and mounted on slides coated with aminoalkylsaline. Conventional immunoperoxidase staining was performed using the Histofine SAB kit (Nichirei, Tokyo) [for details of the procedure, see Razzaque et al. 1998c, Razzaque et al. 1999] to localize HSP47 (Stressgen Biotechnologies Corp, Canada), α-smooth muscle actin (Dako Corp, Denmark), vimentin (Dako Corp), desmin (Dako Corp), type III collagen (Chemicon, USA), and type IV collagen (Chemicon) in the paraffin embedded renal sections. Briefly, paraffin sections were deparaffinized with xylene, and rehydrated in ethanol (from 100% to 70%), and incubated with hydrogen peroxide. The sections were blocked with either 10% goat serum or 10% rabbit serum for 1 h, and then incubated overnight at 4 ˚C with the above-listed primary antibodies. After washing with phosphate-buffered saline (PBS), sections were processed further using the Nichirei Histostain kit (Nichirei, Tokyo, Japan). The reaction products were

Dual staining was performed to localize HSP47 with α-smooth muscle actin, vimentin or desmin as described earlier (Razzaque et al. 1998c, Razzaque & Taguchi 1999a). Paraffin sections were deparaffinized and rehydrated routinely, and then HSP47-expressing cells identified on the sections by the streptavidin-alkaline phosphatase method. The sections were developed with 5-bromo4-chloro-3-indolylphosphate (BCIP)-nitroblue tetrazolium (NBT), which produced dark purple coloured staining. The HSP47-stained sections were counterstained with α-smooth muscle actin, vimentin or desmin by the streptavidin-biotin peroxidase method, and developed with aminoethylcarbazole (ACE)-hydrogen peroxide, producing a red-coloured stain. Control experiments The following control experiments were performed to verify the specificity of the immunostaining of HSP47. (i) In some sections, primary antibodies were replaced with either 0.01 m PBS or mouse IgG diluted with PBS (similar concentration to that of the primary antibody). (ii) In some adjacent sections primary antibodies were replaced with a solution containing a 10-fold excess of recombinant HSP47 (Stressgen Biotechnologies Corp, Canada) in addition to antiHSP47 antibody [the antibody preabsorbed for HSP47 with the recombinant HSP47 overnight at 4 ˚C before use]. These treatments abolished the antigen-specific staining. Semiquantitation of immunoperoxidase staining Immunoperoxidase staining of kidney sections was analysed and graded semiquantitatively according to the proportion of a particular structure in the kidney on the following scale: (−) = no staining, (±) = staining involved <5%, (+) = staining involved 5–25%, (++) = staining involved 25–75%, (+ + +) = staining involved >75%. This analysis was performed in a blind and randomized way. Results Morphological analysis Through out the study period, no significant fibrotic changes compared to control rat kidneys (Figure 1A) were noted in the kidneys of acute [days 1, 3 and 14] diabetic rats (Figure 1B,C). In contrast, gradual thickening of the glomerular basement membrane, glomerulosclerosis, tubular damage, and interstitial fibrosis were noted in the kidneys of chronic [weeks 4, 12 and 24] diabetic rats (Figure 1D–F). The extent of renal damage increased gradually with the time course and was most severe at week 24 (Figure 1F).


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Figure 1. Histological features of kidneys of contol rat (A), and STZ-treated rat after 3 days (B), after 14 days (C), after 4 weeks (D), after 12 weeks (E) and after 24 weeks (F). Compared to control rat kidneys (A), no significant changes are seen in the kidneys obtained from acute [day 3] (B), and [day 14] (C) diabetic rats. In contrast gradual thickening of GBM, glomerulosclerosis, tubular damage, and interstitial fibrosis are noted in the kidneys of chronic [week 4] (D), [week 12] (E), and [week 24] (F) diabetic rats. The extent of renal damage was gradually increased with the time course and is most severe in week 24 (F). [PAS stain]

Immunohistochemical localization of type III and type IV collagens Compared to control rat kidneys (Figure 2A and 3A), no significant changes in the expression of type III collagen (Figure 2B,C) and type IV collagen (Figure 3B,C) was noted in the kidneys obtained from acute diabetic rats. In the kidneys of control rats (Figure 2A) and acute diabetic rats, immunostaining of type III collagen (Figure 2B,C) was detected mainly in the interstitium. As for the type IV collagen, the mesangium, glomerular basement membrane and tubular basement membrane were positively stained in the kidneys of control rats (Figure 3A) and acute diabetic rats (Figure 3B,C). Compared to kidneys of acute diabetic rats, an increased deposition of type III collagen (Figure 2D) and type IV collagen (Figure 3D) was detected in kidneys of chronic diabetic rats, although the staining pattern and distribution was different. Type III collagen (Figure 2D) was located predominantly in the widen interstitium, while type IV collagen (Figure 3D) was mainly located in the glomerulosclerosis and along the thickened tubular basement membrane. The grading of semiquantitation

of immunostaining of collagens in kidneys of different stages of diabetes is shown in Table 1.

Immunohistochemical localization of HSP47 The expression of HSP47 was weakly detected in the intraglomerular cells (mesangial and epithelial cells) and occasionally in the interstitial cells of the kidneys obtained from control rats (Figure 4A) and acute diabetic rats (Figure 4B,C). We did not find any significant changes in the expression of HSP47, at least at the protein level, in the kidneys of acute diabetic rats. In contrast, a markedly increased immunostaining for HSP47 was noted in the glomerular cells (mesangial and epithelial cells), interstitial cells, and tubular epithelial cells in the kidneys obtained from chronic diabetic rats (Figure 4D). When monoclonal antibody for HSP47 was replaced with a 10-fold excess of recombinant HSP47, in addition to antiHSP47 antibody, no specific immunostaining was detected in the kidney (data not shown). The semiquantitative data of the expression of HSP47 in the kidneys of acute and chronic phases of diabetes are shown in Table 1.


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Figure 2. Immunoperoxidase staining (brown) for type III collagen in sections of the kidneys obtained from a normal control rat (A), acute [days 3 and 14] diabetic rats (B, C) and a chronic [week 24] diabetic rat (D). Note that compared to the kidney sections of control and acute diabetic rats, there is an increased interstitial deposition of type III collagen (arrow) in the kidney section obtained from a chronic diabetic rat (D).

Figure 3. Immunostaining for type IV collagen in sections of the kidneys obtained from a normal control rat (A), acute [day 3 and 14] diabetic rats (B, C) and a chronic diabetic rat (D). Compared to the kidney sections of control (A) and acute diabetic rats (B, C), an increased glomerular deposition of type IV collagen is noted in the kidney section obtained from a chronic diabetic rat (D). An increased deposition of type IV collagen is also noted in the thickened tubular basement membrane (arrow).

Immunohistochemical localization of α-smooth muscle actin, vimentin and desmin

revealing a phenotypic modulation of glomerular epithelial cells. Although the pattern of immunostaining was mostly similar in the kidneys of rats in the chronic phase of diabetes, it was most marked in the kidneys of STZ-treated rats sacrificed on week 24. Semiquantitative data of the immunostaining of α-smooth muscle actin, vimentin and desmin are shown in Table 1.

No immunostaining for α-smooth muscle actin was detected in the glomeruli of kidneys of control rats (Figure 5A) and acute diabetic rats (Figure 5B,C), while it was present in the mesangial cells of the kidneys obtained from chronic diabetic rats (Figure 5D), revealing a phenotypic alteration of mesangial cells in chronic diabetic rats. Vimentin was present in the glomeruli, but absent in the tubular epithelial cells in the kidneys of control rats (Figure 6A) and acute diabetic rats (Figure 6B,C). In contrast to kidneys of acute diabetic rats, tubular epithelial cells and interstitial cells in kidneys of rats in the chronic phase of diabetes showed marked immunostaining for vimentin (Figure 6D), suggesting phenotypic changes of tubular epithelial cells in chronic diabetic rats. For desmin, weak immunostaining was noted in the glomeruli in the kidneys of control rats (Figure 7A) and acute diabetic rats (Figure 7B,C), while strong immunostaining for desmin was detected in the glomerular epithelial cells in the kidneys of chronic diabetic rats (Figure 7D),

Double immunostaining HSP47 and α-smooth muscle actin, vimentin or desmin Double immunostaining was performed to identify the cells expressing HSP47 in the kidneys of chronic diabetic rats. As expected, most of these phenotypically-altered α-smooth muscle actin-positive mesangial cells (Figure 8A,B), desminpositive glomerular epithelial cells (Figure 8C,D), vimentinpositive tubular epithelial cells (Figure 8E,F), and α-smooth muscle actin-positive interstitial cells, are found to be HSP47expressing cells (Razzaque et al. 1998d, Razzaque & Taguchi 1999a).


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Table 1. Semiquantitative analysis of immunostaining in kidney sections of control, acute and chronic diabetic rats. Antibody for:

Collagen III Collagen IV HSP47 α-SMA∗ Desmin Vimentin ∗

Glomeruli

Tubules

Interstitium

Control

Acute∗ (day 3)

Chronic∗ (week 24)

Control

Acute∗ (day 3)

Chronic∗ (week 24)

Control

Acute∗ (day 3)

Chronic∗ (week 24)

− + + − + ++

− + + ± + ++

++ +++ +++ + +++ ++

− + ± − − −

− + ± − − −

− ++ +++ − − +++

++ ± + − ± ±

++ ± + ± ± ±

+++ + +++ ++ + +

α-SMA: α smooth muscle actin, Acute: Acute diabetes on day 3, Chronic: Chronic diabetes in week 24.

Figure 4. Immunoperoxidase staining for HSP47 in the kidney sections of a control rat (A), after 3 days of hyperglycaemia (B), after 14 days of hyperglycaemia (C), and after 24 weeks of hyperglycaemia (D) in diabetic rats. Note that compared to the kidneys of control rat (A) and acute diabetic rats (B, C), a markedly increased expression of HSP47 is present in the glomerular mesangial cells, glomerular epithelial cells (solid arrows), interstitial cells (open arrows), tubular epithelial cells (arrowheads) in the kidneys of chronic diabetic rats (D).

Figure 5. Immunoperoxidase staining for α-smooth muscle actin in kidneys of a control rat (A), after 3 days of hyperglycaemia (B), after 14 days of hyperglycaemia (C), and after 24 weeks of hyperglycaemia (D) in diabetic rats. Mainly blood vessels show immnostaining for α-smooth muscle actin, while immunoreactivity is mostly absent in the glomeruli of control rat kidney (A) and acute diabetic rat kidneys (B, C). In contrast, an increased expression of α-smooth muscle actin is present in the glomeruli of the chronic diabetic rat kidneys (D).

Discussion Procollagen assembly is a multi-step process within the endoplasmic reticulum (ER), where the collagen polypeptide α-chains require correct folding to form the collagen protein. The N- and C-terminal propeptides are removed by amino and carboxyl procollagen peptidases, and this occurs before collagen is laid down in the extracellular matrix. HSP47 plays

an important role in the correct folding of polypeptide chains, and thus has an important part in the post-translational modification of collagens. Hence, the level of HSP47 appears to be a marker for the rate at which collagen is being produced, and laid down in the matrix. We believe that the renal level of HSP47 is an adequate indication and marker of when the kidneys of diabetic rats initiate fibrosis. Immunohistochemistry


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Figure 6. Immunostaining for vimentin in sections of kidneys of a normal control rat (A), acute [day 3 and 14] diabetic rats (B, C) and a chronic diabetic rat (D). Vimentin is immunopositive in glomeruli, but mostly absent in tubular epithelial cells in the kidneys of normal control rats (A) and acute diabetic rats (B, C). In chronic diabetic rat kidney, strongly positive immunostaining for vimentin is noted in the tubular epithelial cells (arrows) (D).

on kidney sections of acute and chronic STZ-induced diabetic rats has allowed us to examine the rates of types III and IV collagen accumulation during the disease process and to correlate this with the expression of HSP47. Our results showed an obvious increase in the deposition of types III and IV collagens in kidneys of rats sacrificed on week 24 after STZ injection (the chronic phase of diabetes) rather than in the acute phase of diabetes, when morphologically the kidneys showed no significant fibrotic changes. HSP47, a collagen-binding protein, was examined in this study by immunocytochemistry, to determine its cellular localization within kidneys of STZ-induced diabetic rats, in both short- and long-term diabetes. The semiquantitative scoring method we used allowed the immunoreactivity of collagens and HSP47 in the kidney to be determined. A parallel increase in the expression of collagens and HSP47 was noted in the kidneys of chronic diabetic rats, while no significant changes were seen in the kidneys of acute diabetic rats. No major changes in the expression of HSP47 in the kidneys of STZ-treated rats, in the acute stage of diabetes,

D. Liu et al.

Figure 7. Immunostaining for desmin in sections of kidneys from normal control rat (A), acute [day 3 and 14] diabetic rats (B, C) and a chronic diabetic rat (D). In contrast to the immunostaining in kidneys of control rats (A) and acute diabetic rats (B, C), a strong immunostaining for desmin is evident in the glomeruli of the chronic diabetic rat kidney (D) with most marked staining in the glomerular epithelial cells (arrows).

reveals that up-regulation of HSP47 in the chronic phase of the diseases is not just an acute toxic effect of the drug STZ, but a consequence of the disease process. This result is of clinical interest because it raises the possibility of delaying and/or blocking the progression of the renal scarring even in the chronic stages of diabetes, by therapeutic manipulation of the expression of HSP47. Preliminary studies have shown that in vivo blocking or modulation of the biological activities of HSP47 resulted in decreased production of collagens, and thereby less fibrotic changes in kidneys (Sunamoto et al 1998, Razzaque & Taguchi 1999b). We have shown that an increased expression of HSP47 in human diabetic nephropathy is associated with glomerulosclerosis and tubulointerstitial fibrosis (Razzaque et al. 1998b). However, we were not able to determine the sequential expression pattern of HSP47 in kidneys using human renal biopsies. In this study, using a STZ-induced diabetic model, we showed a time-course expression of HSP47 in acute and chronic diabetic nephropathy. We have demonstrated that, in contrast to the acute phase of diabetes, the levels of expression of HSP47 were significantly increased in glomeruli and tubules of the


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Figure 8. Double staining for HSP47 (dark purple) with Îą-smooth muscle actin (intense red) (A, B), HSP47 (dark purple) with desmin (intense red) (C, D), and HSP47 (dark purple) with vimentin (intense red) (E, F) in sections of the kidneys obtained from chronic diabetic rats. Note colocalization of HSP47 with Îą-smooth muscle actin (arrows) and desmin-positive (arrows) glomerular cells, suggesting that mesangial cells (A, B) and glomerular epithelial cells (C, D) are HSP47-expressing cells. Some of the HSP47-expressing interstitial cells (arrowheads) are Îą-smooth muscle actin-positive myofibroblasts (A, B). Vimentin-positive tubular epithelial cells (arrows) are co-expressing HSP47 in the chronic diabetic rat kidneys (E, F).

kidney during the chronic phase of the disease in STZ-treated rats; the increased expression of HSP47 corresponded with that of collagen deposition in the kidneys. In this study, we also identified the phenotypically-altered and/or damaged cells in kidneys of STZ-treated diabetic rats. Phenotypically-altered glomerular mesangial cells, glomerular epithelial cells, and tubular epithelial cells were detected

in the kidneys of chronic diabetic rats. Previously it was shown that all these phenotypically-altered renal cells are HSP47-producing cells (Razzaque et al. 1998b, Razzaque & Taguchi 1999b). Consistent with these earlier studies, we have shown here that phenotypically-altered renal cells are the main source of HSP47 in the kidneys of chronic STZ-treated rats. Interestingly, in the acute phase of the diabetes, we could


628 not detect many phenotypically-altered cells in the kidney, which may be one of the reasons why HSP47 expression is not as high as it is in the chronic phase of the disease, when phenotypically-altered cells are widespread in the kidney. In summary, the main conclusion made from this preliminary study is that the increased deposition of collagens in the scarring kidney in the chronic phase of diabetes corresponds well with increased levels of HSP47. Importantly, HSP47 up-regulation coincided with the initiation and progression of fibrogenesis, as determined by the expression and deposition of collagens. These results suggest that the HSP47 could be a key factor in the initiation and progression of fibrosis that occurs in kidneys of chronic diabetic rats. From the collagen synthesizing abilities of the HSP47, it is suggested that HSP47 may play a key regulatory role in the excessive assembly and synthesis of collagen during renal scarring in diabetic nephropathy. It also appears from this study that HSP47 is mostly involved in the chronic phase of the disease, and therefore could be a potential target for innovative therapies designed to limit fibrosis, even in the chronic phase of diabetes.

References Clarke EP, Sanwal BD (1992) Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins. Biochim Biophys Acta 1129: 246–248. Hart DA, Reno C, Hellio Le Graverand MP, Hoffman L, Kulyk W (2000) Expression of heat shock protein 47 (Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy. Biochem Cell Biol 78: 511–518. Hirayoshi K, Kudo H, Takechi H, Nakai A, Iwamatsu A, Yamada KM, Nagata K (1991) HSP47: a tissue-specific, transformation-sensitive, collagen binding heat shock protein of chicken embryo fibroblasts. Mol Cell Biol 11: 4036–4044. Kurkinen M, Taylor A, Garrels JI, Hogan BL (1984) Cell surfaceassociated proteins which bind native type IV collagen or gelatin. J Biol Chem 259: 5915–5922. Masuda H, Fukumoto M, Hirayoshi K, Nagata K (1994) Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride induced rat liver fibrosis. J Clin Invest 94: 2481–2488. Nagai N, Hosokawa M, Itohara S, Adachi E, Matsushita T, Hosokawa N, Nagata K (2000) Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol 150: 1499–1506. Nagata K (1998) Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 16: 379–386. Natsume T, Koide T, Yokota S, Hirayoshi K, Nagata K (1994) Interactions between collagen-binding stress protein HSP47 and collagen. Analysis

D. Liu et al. of kinetic parameters by surface plasmon resonance biosensor. J Biol Chem 269: 31224–31228. Osterby R, Lundbaek K, Olsen TS, Orskov H (1967) Kidney lesions in rats with severe long term alloxan diabetes. Lab Invest 17: 675–692. Razzaque MS, Foster CS, Ahmed AR (2001) Tissue and molecular events in human conjunctival scarring in ocular cicatricial pemphigoid. Histol Histopathol 16: 1203–1212 Razzaque MS, Hossain MA, Kohno S, Taguchi T (1998a) Bleomycininduced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat shock protein (HSP) 47. Virchows Arch 432: 455–460. Razzaque MS, Koji T, Harada T, Taguchi T (1999) Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections. Histochem Cell Biol 111: 1–6. Razzaque MS, Kumatori A, Harada T, Taguchi T (1998b) Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 80: 434–443. Razzaque MS, Nazneen A, Taguchi T (1998c) Immunolocalization of collagen and collagen-binding heat shock protein 47 in fibrotic lung diseases. Modern Pathol 11: 1183–1188. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T (1998d) Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47. Cell Tissue Res 293: 471–478. Razzaque MS, Shimokawa I, Nazneen A, Liu D, Naito T, Higami Y, Taguchi T (1999b) Life-long dietary restriction modulates the expression of collagens and collagen-binding heat shock protein 47 in aged Fischer 344 rat kidney. Histochem J 31: 123–132. Razzaque MS, Taguchi T (1997) Collagen-binding heat shock protein (HSP) 47 expression in anti-thymocyte serum (ATS)-induced glomerulonephritis. J Pathol 183: 24–29. Razzaque MS, Taguchi T (1999a) Localization of HSP47 in cisplatintreated rat kidney: possible role in tubulointerstitial damage. Clin Exp Nephrol 3: 222–228. Razzaque MS, Taguchi T (1999b) The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 14: 1199–1212. Razzaque MS, Taguchi T (2002) Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc 35: (in press) Sunamoto M, Kuze K, Tsuji H, Ohishi N, Yagi K, Nagata K, Kita T, Doi T (1998) Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis. Lab Invest 78: 967–972. Takechi H, Hirayoshi K, Nakai A, Kudo H, Saga S, Nagata K (1992) Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells. Eur J Biochem 206: 323–329. Wang SY, Gudas LJ (1990) A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue. J Biol Chem 265: 15818–15822. Wilson GL, Letter EH (1990) Streptozotocin interactions with pancreatic beta cells and the induction of insulin-dependent diabetes. Curr Top Microbiol Immunol 156: 27–54.


Role of Collagen-Binding Heat Shock Protein 47 and Transforming Growth Factor-␤1 in Conjunctival Scarring in Ocular Cicatricial Pemphigoid Mohammed S. Razzaque,1,2 C. Stephen Foster,3 and A. Razzaque Ahmed1,2 PURPOSE. Submucosal fibrosis due to excessive accumulation of collagens is an important histologic feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). Heat shock protein 47 (HSP47), a collagen-binding protein, plays an important role in the biosynthesis of procollagens. In the present study, we examined the role of HSP47 in conjunctival scarring in patients with OCP. METHODS. Biopsy specimens of the conjunctiva of 15 patients with OCP and 5 normal subjects were studied for the expression of HSP47, transforming growth factor (TGF)-␤1, type I collagen, and type III collagen. The role of TGF-␤1 on the induction of HSP47 and type I collagen by conjunctival fibroblasts was studied by immunostaining, Western blot analysis, and quantitative real-time PCR. RESULTS. Compared with the control, increased accumulations of type I and type III collagens were detected by immunohistochemistry in fibrotic conjunctiva of patients with OCP. Weak and sparse expression of HSP47 was detected in the epithelial cells and stromal fibroblasts in control conjunctival tissues. In contrast to the control, the expression of HSP47 was markedly increased in the stromal fibroblasts in conjunctival tissues obtained from patients with OCP, as detected by immunohistochemistry. By quantitative real-time PCR, compared with control conjunctival tissues, a 3.4-fold increase in the expression of HSP47 was noted in the conjunctival tissues obtained from patients with OCP. Similar to conjunctival tissues, fibroblasts isolated from conjunctiva of patients with OCP exhibited 4.8fold increase in the expression of HSP47, compared with control fibroblasts. When conjunctival fibroblasts were treated with various concentration of TGF-␤1, upregulation in the expression of HSP47 and type I collagen was detected. CONCLUSIONS. This study demonstrated increased expression of HSP47 and TGF-␤1 by conjunctival fibroblasts in biopsy specimens obtained from patients with OCP. TGF-␤1 induced the expression of HSP47 and type I collagen by conjunctival fibroblasts. Increased levels of TGF-␤1 and HSP47 may regulate increased synthesis, assembly, and production of collagens and

From the 1Department of Dermatology, Harvard Medical School, Boston, Massachusetts; the 2Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts; the 3Department of Ophthalmology, Immunology and Uveitis Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts. Supported by National Eye Institute Grant EY08379. Submitted for publication July 1, 2002; revised September 30, 2002; accepted October 29, 2002. Disclosure: M.S. Razzaque, None; C.S. Foster, None; A.R. Ahmed, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: A. Razzaque Ahmed, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115; razzaque_ahmed@hms.harvard.edu.

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thereby could significantly contribute to the process of conjunctival scarring in patients with OCP. (Invest Ophthalmol Vis Sci. 2003;44:1616 –1621) DOI:10.1167/iovs.02-0644

R

ecent research has greatly increased our knowledge concerning the molecular mechanisms of the synthesis and processing of collagen.1 Procollagen assembly is a complex process within the endoplasmic reticulum (ER), in which the C-propeptide domains of three polypeptide ␣-chains fold individually and subsequently interact and trimerize to form a triple helix. Heat shock protein (HSP) 47 is a 47-kDa stress protein that resides in the ER of collagen-producing cells.1 It binds specifically to native collagens and acts as a collagenspecific molecular chaperone during the biosynthesis and intracellular processing of newly formed procollagen polypeptides and assists in the formation of triple helices within the ER and cis-Golgi vesicles.1–3 The crucial role of HSP47 in the collagen biosynthesis is well documented, and HSP47 gene– disrupted mice demonstrate molecular abnormalities in procollagen and embryonic death.4 A close association between the increased expression of HSP47 and increased accumulation of collagens has been demonstrated in various fibrotic diseases of the lung, liver, and kidney.5–9. In these disease processes, HSP47 is thought to exert an important biological effect on procollagen synthesis and subsequent fibrosis. Moreover, in a rat model of renal fibrosis, in vivo treatment with HSP47-specific antisense oligonucleotides has been shown to decrease the accumulation of collagen.10 Although several studies have elucidated the role of HSP47 in human and experimental fibrotic diseases, its role in conjunctival scarring in response to injury or in pathologic states such as ocular cicatricial pemphigoid (OCP) is not yet known. Mucous membrane pemphigoid is a rare autoimmune vesiculobullous disease. When ocular involvement is present, it is referred to as OCP. It is usually characterized by recurrent episodes of inflammation in the conjunctiva with progressive subepithelial fibrosis, resulting in shortening of the fornix. In a number of patients, despite immunosuppressive therapy, it progresses and eventually leads to blindness.11 HSP47 is closely involved in the folding, assembly, and/or posttranslational modification of procollagen.1 Consequently, it has a potential role in conjunctival fibrosis. In the present study, we investigated the role of HSP47 and transforming growth factor (TGF)-␤1 in conjunctival scarring in patients with OCP.

MATERIALS

AND

METHODS

Conjunctival Specimens Samples of the conjunctiva were obtained from 15 patients with OCP. The diagnosis of OCP was based on clinical presentation, histology, and direct immunofluorescence of the conjunctiva demonstrating IgG and C3 at the basement membrane zone. Biopsy specimens from conjunctiva of normal individuals, to be used as the control, were obtained from five patients who underwent routine cataract surgery. Conjunctival biopsy specimens were also obtained from three subjects Investigative Ophthalmology & Visual Science, April 2003, Vol. 44, No. 4 Copyright © Association for Research in Vision and Ophthalmology


IOVS, April 2003, Vol. 44, No. 4 with atopic diseases of the conjunctiva during episodes of inflammation. The study adhered to the guidelines of the Declaration of Helsinki for research involving human subjects.

Immunohistochemistry Immunohistochemistry was performed on paraffin and frozen sections as described previously.12,13 Briefly, biopsy sections of the conjunctiva were blocked with either 10% goat serum or 10% rabbit serum for 1 hour, and then incubated overnight at 4°C with the following primary antibodies: HSP47 (StressGen Biotechnologies Corp., Victoria, British Columbia, Canada), type I collagen (Sigma Chemical Co., St. Louis, MO), type III collagen (Fuji Chemical Industries, Tokyo, Japan), and TGF-␤1 (R&D Systems, Minneapolis, MN). After a wash with PBS, sections were processed further using a kit (Histostain; Nichirei, Tokyo, Japan), and reaction products were developed with a mixture of 3,3⬘-diaminobenzine-4 HCl (DAB) and H2O2. Preabsorption of the primary antibody with excess recombinant HSP47 peptide (StressGen Biotechnologies) and normal mouse or goat IgG was used as the negative control. The staining pattern was graded semiquantitatively according to the intensity and distribution of the staining, as described in our earlier reports.5

Isolation of Fibroblasts from Conjunctiva Fibroblasts from conjunctiva of normal control subjects and patients with OCP were isolated as described earlier.14 Briefly, conjunctival tissue was cut into explants of approximately 2 ⫻ 2 mm2, placed into tissue culture dishes, and covered with DMEM containing FCS, gentamicin, and amphotericin B and incubated overnight, at 37°C with 95% humidity and 5% CO2. Medium was changed three times weekly thereafter for 2 weeks. The isolated fibroblasts were subcultured with 0.1% trypsin and 0.02% EDTA in Ca⫹-free minimum essential medium (MEM) at 80% to 90% confluence. Fibroblasts isolated from conjunctiva of normal control subjects and patients with OCP were grown on the glass slides, fixed with methanol, and used for immunostaining for HSP47 and TGF-␤1, as just described. In addition RNA and proteins extracted from fibroblasts isolated from conjunctiva of normal control subjects and patients with OCP were used for real-time PCR and Western blot analysis. Cells at passages 3 to 7 were used in this study.

Effects of TGF-␤1 on Expression of HSP47 in Conjunctival Fibroblasts Conjunctival fibroblasts were treated with various concentration (1, 10, and 100 ng/mL) of recombinant TGF-␤1 (R&D Systems), for 24 hours, in an incubator at 37°C with 95% humidity and 5% CO2. The total RNA and proteins were extracted from conjunctival fibroblasts and were used for quantitative real-time PCR and Western blot analysis. Conjunctival fibroblasts, grown on the glass slides and treated with various concentrations of TGF-␤1 for 24 hours and then fixed with cold methanol, were used for the detection of HSP47 by immunohistochemistry.

Blocking the Effects of TGF-␤1 on the Expression of HSP47 in Conjunctival Fibroblasts Conjunctival fibroblasts were incubated with various concentrations (2 and 20 ng/mL) of TGF-␤ type II receptor–neutralizing antibody (R&D Systems) for 12 hours at 37°C with 95% humidity and 5% CO2. The fibroblasts were then treated with 10 ng/mL recombinant TGF-␤1 for 24 hours, and the extracted total RNA and proteins were analyzed by quantitative real-time PCR and Western blot analysis.

Western Blot Analysis of Protein Extracted from Conjunctival Fibroblasts Protein extracted from TGF␤1–treated and nontreated conjunctival fibroblasts were electrophoresed on 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane, as described earlier.15 The membrane was blocked with 20 mg/mL bovine serum

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TABLE 1. The Oligonucleotide Sequences for the Primers and Probe Used in Quantitative Real-Time PCR HSP47 Forward: CGC CAT GTT CTT CAA GCC A Reverse: CAT GAA GCC ACG GTT GTC C Probe: FAM-CTG GGA TGA GAA ATT CCA CCA CAA GAT GGTAMRA TGF-␤1 Forward: CGA GAA GCG GTA CCT GAA C Reverse: TGA GGT ATC GCC AGG AAT TGT Probe: FAM-CAG CAC GTG GAG CTG TAC CAG AAA TAC AGCTAMRA Type I collagen Forward: CCT CAA GGG CTC CAA CGA G Reverse: TCA ATC ACT GTC TTG CCC CA Probe: FAM-ATG GCT GCA CGA GTC ACA CCG GA-TAMRA

albumin dissolved in PBS solution for 2 hours at room temperature. The expression of HSP47 was detected with a mouse monoclonal antibody to HSP47 (overnight at 4°C). Blots were then washed and incubated with horseradish peroxidase (HRP)– conjugated antibody against mouse IgG for 1 hour. Immunoreactive protein was visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, UK). Blots were also reacted with anti-␤-actin antibody (Sigma Chemical Co.), to assess the variations in protein loading between various samples. As a positive control, recombinant HSP47 was loaded along with samples, electrophoresed, transferred to the membrane, and reacted with anti-mouse antibody to HSP47. The negative control consisted of incubating the blots with mouse IgG, instead of primary antibody.

Quantitative Real-Time PCR The total RNA isolated from conjunctival tissues and conjunctival fibroblasts of control subjects and patients with OCP was used to detect relative expression of HSP47, TGF-␤1, and type I collagen mRNA. The principle of quantitative real-time PCR has been described elsewhere.16,17 The quantification of transcription of real-time PCR takes advantage of the 5⬘ nuclease activity of Taq DNA polymerase. Total RNA was extracted from conjunctival tissues and conjunctival fibroblasts using a RNA isolation kit (Qiagen, Valencia, CA). The primers and probe used for detection of HSP47, TGF-␤1, and type I collagen are listed in Table 1. Each PCR reaction contained equivalent amounts of total RNA. Real-time PCR was always performed in duplicate with a PCR reagent kit (TaqMan; Applied Biosystems, Foster City, CA). All the reactions were controlled by standards (nontemplate control and standard positive control). The quantity of mRNA was calculated by normalizing the cycle threshold (CT) of HSP47, TGF-␤1, or type I collagen to the CT of the housekeeping gene 18S or GAPDH of the same RNA sample, according to the following formula: the average 18S or GAPDH CT (each multiplex PCR was performed in duplicate) was subtracted from the average HSP47, TGF-␤1, or type I collagen CT. This result represents the ⌬CT, which is specific and can be compared with the ⌬CT of a calibration sample (for example control conjunctival tissues or control conjunctival fibroblasts). The subtraction of control ⌬CT from the ⌬CT of OCP samples or fibroblasts of OCP samples was referred as ⌬⌬CT. The relative expression of HSP47, TGF-␤1, or type I collagen (in comparison to the control) in tissues and fibroblasts obtained from conjunctiva of patients with OCP was determined by 2⫺⌬⌬CT. For all the probes the quencher dye was 6-carboxy-tetramethyrhodamine (TAMRA), the reporter dye was 6-carboxy fluorescein (FAM) for HSP47, TGF-␤1, and type I collagen, and VIC for 18S or GAPDH.

RESULTS Expression of HSP47 in Conjunctival Tissue The expression of HSP47 was weakly and sparsely detected in the conjunctival epithelium and stromal cells in sections from


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FIGURE 1. Immunohistochemistry for HSP47 in conjunctival sections obtained from a control subject (A) and an a patient with OCP (B). Note increased submucosal HSP47-expressing cells (arrows) in conjunctival section of the patient (B). When mouse serum was used instead of antibody for HSP47, no specific staining was noted (C).

normal control conjunctiva (Fig. 1A). In contrast, in sections of conjunctiva from patients with OCP, both intensity and number of stromal cells expressing HSP47 were increased (Fig. 1B). Moderately increased expression of HSP47 was also noted in the epithelial cells, in comparison to control conjunctival sections. Moreover, compared with control conjunctival tissue, a 3.4-fold increase in the expression of HSP47 was detected by quantitative real-time PCR in the conjunctival tissue obtained from patients with OCP. (Fig. 2). However, no such increased expression of HSP47 was noted by quantitative real-time PCR. in conjunctival tissues obtained from patients with atopic conjunctivitis.

Expression of Type I and Type III Collagens in Conjunctival Tissue Interstitial type I and type III collagens was weakly expressed in the submucosal stroma and around the blood vessels in the conjunctival sections of normal control subjects (Figs. 3A, 3C). Compared with the control, an increased deposition of type I and type III collagens was detected in the fibrotic interstitium in conjunctiva of patients with OCP (Figs. 3B, 3D).

Expression of HSP47 in Conjunctival Fibroblasts

fibroblasts isolated from conjunctiva of patients with OCP (Fig. 4B). Similarly, a 4.8-fold increase in the expression of HSP47 mRNA was detected in the fibroblasts isolated from conjunctiva of patients with OCP (Fig. 2).

Expression of Type I Collagen and TGF-␤1 in Conjunctival Fibroblasts The expression levels of type I collagen and TGF-␤1 in conjunctival fibroblasts was examined with quantitative real-time PCR and immunohistochemistry. Compared with control conjunctival fibroblasts, increased expression of type I collagen and TGF-␤1 was detected by real-time PCR in the fibroblasts isolated from conjunctiva of patients with OCP. Compared with control conjunctival fibroblasts, increased expression of TGF-␤1 was detected by immunohistochemistry in fibroblasts isolated from conjunctiva of patients with OCP (data not shown).

Induction of HSP47 and Type I Collagen by TGF-␤1 in Cultured Conjunctival Fibroblasts Conjunctival fibroblasts were treated with various concentrations of TGF-␤1 (1, 10, and 100 ng/mL) for 24 hours to eluci-

Fibroblasts isolated from conjunctiva of normal control subjects and patients with OCP were studied by immunostaining and real-time PCR, to examine the expression of HSP47. Compared with control conjunctival fibroblasts (Fig. 4A), increased cytoplasmic immunostaining for HSP47 was observed in the

FIGURE 2. Quantitative real-time PCR analysis of HSP47 in conjunctival tissues and fibroblasts of patients with OCP. Compared with control conjunctival tissue, a 3.4-fold increase in the expression of HSP47 was detected by quantitative real-time PCR in the conjunctival tissue obtained from patients with OCP. Real-time PCR was always performed in duplicate, and the data are the mean relative expression of HSP47 in comparison with the control. Similarly, compared with control conjunctival fibroblasts, a 4.8-fold increase in the expression of HSP47 mRNA was detected in the fibroblasts isolated from conjunctiva of patients with OCP.

FIGURE 3. Immunohistochemistry for type I (A, B) and type III (C, D) collagens in conjunctival sections obtained from control subjects (A, C) and patients with OCP (B, D). Compared with control conjunctival sections (A, C) there was an increased submucosal deposition of type I (B) and type III (D) collagens in conjunctival sections of patients with OCP.


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FIGURE 5. Conjunctival fibroblasts were treated with various concentrations of TGF-␤1 (1, 10, and 100 ng/mL) for 24 hours, and the induction of HSP47 was determined at the mRNA level by quantitative real-time PCR. Real-time PCR was always performed in duplicate, and the data are the mean relative expression of HSP47 in TGF-␤1–treated fibroblasts in comparison with the control. There was increased expression of HSP47 mRNA in the TGF-␤1–treated fibroblasts.

body (for 12 hours) and then treated with 10 ng/mL TGF-␤1 (for 24 hours). Compared with the TGF-␤1–treated conjunctival fibroblasts, TGF-␤ type II receptor–neutralizing antibody– treated fibroblasts demonstrated relatively less expression of HSP47 by quantitative real-time PCR (data not shown).

DISCUSSION

FIGURE 4. Immunoexpression of HSP47 in fibroblasts isolated from conjunctiva of a control subject (A) and a patient with OCP (B), revealing increased cytoplasmic expression of HSP47 in fibroblast obtained from conjunctiva of the patient (B). Increased cytoplasmic staining for HSP47 was noted in the TGF-␤1–treated fibroblasts isolated from conjunctiva of a normal subject (C). Preabsorption of the antiHSP47 antibody with recombinant HSP47 resulted in no specific immunostaining (D).

In this study, we have shown a possible role of HSP47 in the pathogenesis of conjunctival scarring in patients with OCP. Our results indicate that the expression of HSP47 was increased, at both the mRNA and protein levels, in conjunctiva obtained from patients with OCP. Increased expression of HSP47 was accompanied with an increased deposition of type I and type III collagens in the fibrotic conjunctiva of patients with OCP. The staining patterns and distribution of both type I and type III collagens was essentially similar.18 HSP47 is a collagen-specific chaperone that was originally identified by Kurkinen et al.19 from murine parietal endoderm

date the effect of TGF-␤1 on the expression of HSP47. Compared with the nontreated fibroblasts (Fig. 4A), increased cytoplasmic staining for HSP47 was detected in the TGF-␤1– treated conjunctival fibroblasts (Fig. 4C). When the conjunctival fibroblasts were incubated with anti-HSP47 antibody, preabsorbed with recombinant HSP47, no specific staining was detected (Fig. 4D). This elimination of staining demonstrated the specificity of the HSP47 staining. In addition, compared with nontreated fibroblasts, TGF-␤1–treated fibroblasts showed upregulated expression of HSP47, at both the mRNA level, detected by quantitative real-time PCR (Fig. 5) and the protein level, detected by Western blot analysis (Fig. 6). The increased expression of HSP47 in TGF-␤1–treated conjunctival fibroblasts was correlated with the increased expression of type I collagen, as detected by quantitative real-time PCR (Fig. 7).

Inhibition of TGF-␤1–Induced HSP47 and Collagen Expression in Cultured Conjunctival Fibroblasts by TGF-␤ Type II Receptor–Neutralizing Antibody Conjunctiva fibroblasts were treated with two concentrations (2 and 20 ng/mL) of TGF-␤ type II receptor–neutralizing anti-

FIGURE 6. Expression of HSP47 in control conjunctival fibroblasts (lane 1) and fibroblasts isolated from a patient with OCP (lane 2), showing increased expression of HSP47 by Western blot analysis. When conjunctival fibroblasts were treated with various concentration of TGF-␤1 (1, 10, and 100 ng/mL; lanes 3– 5) for 24 hours, an induction of HSP47 was noted, by Western blot analysis.


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FIGURE 7. Conjunctival fibroblasts were treated with various concentrations of TGF-␤1 (1, 10, and 100 ng/mL) for 24 hours, and the induction of type I collagen was determined at the mRNA level by quantitative real-time PCR. Data show the relative expression of type I collagen in TGF-␤1–treated fibroblasts in comparison with control fibroblasts. There was increased expression of type I collagen in the TGF-␤1–treated fibroblasts.

cells. Subsequently, HSP47 from human, rabbit, rat, mouse, and chicken cDNA have been cloned and show a high degree of homology in their sequences.20 –24 Numerous studies have shown expression of HSP47 in collagen-producing cells and tissues. Its role in embryogenesis and fibrogenesis has been reported.5–9,25–27 Studies have demonstrated that HSP47 specifically binds to various types of collagens (type I–V collagens), and has been shown to be present in the ER of collagensecreting cells.28 The biochemical properties, intracellular localization, and tissue distribution of HSP47, implicate its role in posttranscriptional regulation of procollagens.1 The important observation of this study is that, the expression of HSP47 was increased in conjunctiva obtained from patients with OCP. This upregulated expression of HSP47 correlated with the increased expression and accumulation of interstitial collagens. Furthermore, enhanced expression of HSP47 was observed in conjunctival fibroblasts isolated from patients with OCP, at both the mRNA and protein levels. Because multiple sequential studies were not performed (in the same patient), a direct causative relationship cannot be shown at this time. Indeed, such studies are limited by the fact that repetitive conjunctival biopsy specimens can aggravate or advance the existing disease process of OCP. Nevertheless, based on the role of HSP47 in the biosynthesis of procollagens, it appears very likely that it plays an important role in excessive biosynthesis of collagens and subsequent conjunctival scarring in patients with OCP. There are several studies that have documented the upregulation in the expression of HSP47 and increased accumulation of collagens in both in human and experimental models of fibrosis.5–9,29 –31 A coordinated expression of HSP47 with synthesis and deposition of collagen has been reported in CCl4induced liver cirrhosis, bleomycin-induced pulmonary fibrosis, and 5–9,anti-thymocyte serum-induced glomerulosclerosis in 29 –31 rats. Various profibrotic factors, such as interleukin (IL)-1, -4, and -6; TGF-␤1; and connective tissue growth factor (CTGF) have the potential to mediate fibrogenesis in human and experimental animals. Among these, TGF-␤1 is an extensively studied molecule during fibrogenesis.32 It is expressed at high levels during tissue remodeling and affects the formation of connective tissue, by stimulating the transcription of genes encoding for extracellular matrix proteins.33,34 Both in vitro and in vivo studies have convincingly shown that modulation of TGF-␤1 suppresses collagen production and subsequently modulates the fibrotic process.35,36 Recently, a fibrogenic role for all three

IOVS, April 2003, Vol. 44, No. 4 isoforms of TGF-␤1 has been reported during the development of mouse conjunctival fibrosis,37 and mitomycin-C has been shown to reverse this fibrosis in mice.38 Moreover, increased expression of TGF-␤1 and -␤3 has been reported in conjunctiva of patients with OCP.39 Epithelial cells and fibroblasts have been shown to produce increased levels of TGF-␤ in conjunctiva of patients with OCP, as detected by in situ hybridization.39 In our present study, compared with the control conjunctival fibroblasts, increased expression of TGF-␤1 was detected in conjunctival fibroblasts isolated from patients with OCP. Furthermore, when conjunctival fibroblasts were treated with recombinant TGF-␤1, upregulation in the expression of both HSP47 and type I collagen was noted. Hence, the observation of this study suggests that TGF-␤1 is one of the essential molecules that may regulate the expression of HSP47 and type I collagen in conjunctiva of patients with OCP. Our results are in accord with earlier studies that have shown activation and/or induction of HSP47 by TGF-␤1.40,41 Both in vitro and in vivo studies have shown that suppression of the expression of HSP47 modulates collagen production.10,42 Using in vitro studies, Sauk et al.42 demonstrated that phosphorothioate antisense oligodeoxynucleotides to HSP47 inhibits the production of HSP47 and consequently diminishes the production of type I procollagen. In a rat nephritis model, the inhibition of HSP47 overexpression by administration of HSP47 antisense oligodeoxynucleotides resulted in the suppression of collagen production and the attenuation of glomerulosclerosis.10 Similarly, modulation of the expression of HSP47 by calorie restriction has been shown to delay ageassociated renal scarring in Fischer 344 rats.43 Hence, the multistep, multifactorial scarring process44,45 could be molecularly modulated, to reduce, inhibit, and possibly reverse the conjunctival scarring process, which may ultimately prevent blindness in some patients with OCP. In conclusion, the present study demonstrates increased expression of HSP47 with excessive accumulation of collagens in the conjunctiva of patients with OCP. This upregulation of HSP47 and collagens appears to be induced by TGF-␤1. We realize that it is possible that other molecules may also be involved in the process of this upregulation,44 and, if that is the case, then TGF-␤1 may be working synergistically with them. A detailed study of the sequential events and factors that facilitate or enhance matrix remodeling in the conjunctiva, could be important in understanding the molecular mechanism of OCP and could provide specific sites for molecular intervention for treatment or arrest of the progression of disease.

Acknowledgments The authors thank Takashi Taguchi, MD, PhD, Nagasaki University Graduate School of Medical Sciences, for kindly providing antibodies and immunostaining kits, and for critical advice and Suman Kumari, PhD, and Victoria Patkova, PhD, for technical advice.

References 1. Nagata K. Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol. 1998;16:379 –386. 2. Nagata K, Yamada KM. Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts. J Biol Chem. 1986;261:7531–7536. 3. Nagata K. Hsp47: a collagen-specific molecular chaperone. Trends Biochem Sci. 1996;21:22–26. 4. Nagai N, Hosokawa M, Itohara S, et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol. 2000;150:1499 –1506.


IOVS, April 2003, Vol. 44, No. 4 5. 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. 6. Razzaque MS, Hossain MA, Kohno S, Taguchi T. Bleomycin-induced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat shock protein (HSP) 47. Virchows Arch. 1998;432:455– 460. 7. Masuda H, Fukumoto M, Hirayoshi K, Nagata K. Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride induced rat liver fibrosis. J Clin Invest. 1994;94:2481–2488. 8. Razzaque MS, Kumatori A, Harada T, Taguchi T. Coexpression of collagens and collagen-binding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 1998;80: 434 – 443. 9. Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T. Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47. Cell Tissue Res. 1998;293:471– 478. 10. Sunamoto M, Kuze K, Tsuji H, et al. Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis. Lab Invest. 1998;78:967–972. 11. Foster CS. Cicatricial pemphigoid. Trans Am Ophthalmol Soc. 1986;84:527– 663. 12. Razzaque MS, Koji T, Harada T, Taguchi T. Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections. Histochem Cell Biol. 1999;111:1– 6. 13. Razzaque MS, Taguchi T. Localization of HSP47 in cisplatin-treated rat kidney: possible role in tubulointerstitial damage. Clin Exp Nephrol. 1999;3:222–228. 14. Razzaque MS, Foster CS, Ahmed AR. Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid. Invest Ophthalmol Vis Sci. 2002;43:2977–2983. 15. Naito T, Razzaque MS, Nazneen A, Liu D, Nihei H, Koji T, Taguchi T. Renal expression of the Ets-1 proto-oncogene during progression of rat crescentic glomerulonephritis. J Am Soc Nephrol. 2000; 11:2243–2255. 16. Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res. 1996;6:995–1001. 17. Razzaque MS, Ahmed AR. Collagens, collagen-binding heat shock protein 47 and transforming growth factor ␤1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis. Cytokine. 2002;17:311–316. 18. Razzaque MS, Foster CS, Ahmed AR. Tissue and molecular events in human conjunctival scarring in ocular cicatricial pemphigoid. Histol Histopathol. 2001;16:1203–1212. 19. Kurkinen M, Taylor A, Garrels JI, Hogan BL. Cell surface-associated proteins which bind native type IV collagen or gelatin. J Biol Chem. 1984;259:5915–5922. 20. Clarke EP, Sanwal BD. Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins. Biochim Biophys Acta. 1992;1129:246 –248. 21. Hirayoshi K, Kudo H, Takechi H, et al. HSP47: a tissue-specific, transformation-sensitive, collagen binding heat shock protein of chicken embryo fibroblasts. Mol Cell Biol. 1991;11:4036 – 4044. 22. Takechi H, Hirayoshi K, Nakai A, Kudo H, Saga S, Nagata K. Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells. Eur J Biochem. 1992; 206:323–329. 23. Hart DA, Reno C, Hellio Le Graverand MP, Hoffman L, Kulyk W. Expression of heat shock protein 47(Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy. Biochem Cell Biol. 2000;78:511–518. 24. Wang SY, Gudas LJ. A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue. J Biol Chem. 1990;265:15818 –15822.

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25. Razzaque MS, Taguchi T. The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol. 1999;14:1199 – 1212. 26. Tanaka Y, Kobayashi K, Kita M, et al. Expression of 47 kDa heat shock protein (HSP47) during development of mouse cornea. Exp Eye Res. 1996;63:383–393. 27. Liu D, Razzaque MS, Cheng M, Taguchi T. The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats. Histochem J. 2001;33:623– 630. 28. Natsume T, Koide T, Yokota S, Hirayoshi K, Nagata K. Interactions between collagen-binding stress protein HSP47 and collagen: analysis of kinetic parameters by surface plasmon resonance biosensor. J Biol Chem. 1994;269:31224 –31228. 29. Razzaque MS, Taguchi T. Collagen-binding heat shock protein (HSP) 47 expression in anti-thymocyte serum (ATS)-induced glomerulonephritis. J Pathol. 1997;183:24 –29. 30. Liu D, Razzaque MS, Nazneen A, Naito T, Taguchi T. Role of heat shock protein 47 on tubulointerstitium in experimental radiation nephropathy. Pathol Int. 2002;52:340 –347. 31. Razzaque MS, Taguchi T. Possible role of glomerular epithelial cell-derived HSP47 in experimental lipid nephropathy. Kidney Int. 1999;56:S256 –S259. 32. Wells RG. Fibrogenesis. V. TGF-beta signaling pathways. Am J Physiol. 2000;279:G845–G850. 33. McGowan SE. Extracellular matrix and the regulation of lung development and repair. FASEB J. 1992;6:2895–2904. 34. McWhirter A, Colosetti P, Rubin K, Miyazono K, Black C. Collagen type I is not under autocrine control by transforming growth factor-beta 1 in normal and scleroderma fibroblasts. Lab Invest. 1994;71:885– 894. 35. Hori Y, Katoh T, Hirakata M, et al. Anti-latent TGF-beta binding protein-1 antibody or synthetic oligopeptides inhibit extracellular matrix expression induced by stretch in cultured rat mesangial cells. Kidney Int. 1998;53:1616 –1625. 36. McCormick LL, Zhang Y, Tootell E, Gilliam AC. Anti-TGF-beta treatment prevents skin and lung fibrosis in murine sclerodermatous graft-versus-host disease: a model for human scleroderma. J Immunol. 1999;163:5693–5699. 37. Cordeiro MF, Gay JA, Khaw PT. Human anti-transforming growth factor-beta2 antibody: a new glaucoma anti-scarring agent. Invest Ophthalmol Vis Sci. 1999;40:2225–2234. 38. Cordeiro MF, Reichel MB, Gay JA, D’Esposita F, Alexander RA, Khaw PT. Transforming growth factor-beta1, -beta2, and -beta3 in vivo: effects on normal and mitomycin C-modulated conjunctival scarring. Invest Ophthalmol Vis Sci 1999;40:1975–1982. 39. Elder MJ, Dart JK, Lightman S. Conjunctival fibrosis in ocular cicatricial pemphigoid-the role of cytokines. Exp Eye Res. 1997; 65:165–176. 40. Yamamura I, Hirata H, Hosokawa N, Nagata K. Transcriptional activation of the mouse HSP47 gene in mouse osteoblast MC3T3–E1 cells by TGF-beta 1. Biochem Biophys Res Commun. 1998;244:68 –74. 41. Sasaki H, Sato T, Yamauchi N, et al. Induction of heat shock protein 47 synthesis by TGF-beta and IL-1 beta via enhancement of the heat shock element binding activity of heat shock transcription factor 1. J Immunol. 2002;168:5178 –5183. 42. Sauk JJ, Smith T, Norris K, Ferreira L. Hsp47 and the translationtranslocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen. J Biol Chem. 1994;269:3941–3946. 43. Razzaque MS, Shimokawa I, Nazneen A, et al. Life-long dietary restriction modulates the expression of collagens and collagenbinding heat shock protein 47 in aged Fischer 344 rat kidney. Histochem. J. 1999;31:123–132. 44. Razzaque MS, Taguchi T. Cellular and molecular events leading to renal tubulointerstitial fibrosis. Med Electron Microsc. 2002;35: 68 – 80. 45. Razzaque MS, Ahsan N, Taguchi T. Role of apoptosis in fibrogenesis. Nephron 2002;90:365–372.


Hypothesis Accepted: January 7, 2000

Nephron 2000;86:339–341

Heat Shock Protein 47 in Renal Scarring M.S. Razzaque a, b N. Ahsan b T. Taguchi a a 2nd

Department of Pathology, Nagasaki University School of Medicine, Nagasaki, Japan; of Nephrology, Pennsylvania State University College of Medicine, Hershey, Pa., USA

b Division

Key Words Heat shock protein 47 W Collagen W Renal scarring

Abstract Heat shock protein 47 (HSP47) is a collagen-binding protein, thought to play an essential mechanistic role in the assembly and processing of procollagens. HSP47 is increasingly being implicated in the pathogenesis of several human and experimental fibrotic diseases. HSP47 could mediate increased accumulation of collagens in the fibrotic mass, possibly by regulating increased assembly of procollagens. Therefore, modulation of HSP47 might be a valuable tool for manipulation of some fibrotic diseases, including renal scarring Copyright © 2000 S. Karger AG, Basel

Introduction

Increased deposition of various types of collagen is a typical feature in the development of renal fibrosis. Clarification of the molecular mechanisms underlying fibrotic disorders and the development of effective therapy are both of clinical importance. It has been shown that heat shock protein 47 (HSP47) is an important collagen-specific stress protein and possibly involved in the assembly

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and secretion of procollagens and that, as a consequence, it may play a pivotal role in the fibrotic changes that follow tissue damage in many vital organs, including liver, lung, and kidney.

Heat Shock Protein 47

Environmental, chemical, or physiological stress induces the expression of a group of highly conserved proteins called heat shock proteins (HSPs). The cellular response to stress was discovered by Ritossa [1, 2], and so far, at least two general roles of HSPs have been suggested for helping cells cope with stress-induced protein damage. Some HSPs can promote degradation of abnormal proteins, whereas others can reactivate stress-damaged proteins by preventing the aggregation or by promoting the proper refolding of denatured proteins [3]. HSP47, a 47-kD stress protein, is localized on the endoplasmic reticulum of collagen-producing cells and is thought to participate in procollagen processing as a collagen-specific chaperone. Kurkinen et al. [4] first identified a collagen-binding protein from murine parietal endodermal cells and found it to bind specifically to collagens. Thereafter, species-specific collagen-binding proteins were characterized in human and rat as gp46 [5, 6], in the chick as HSP47 [7], and in the mouse as J6 [8].

M.S. Razzaque, MBBS, PhD Division of Nephrology, Pennsylvania State University College of Medicine Hershey Medical Center, PO Box 850 H040 Hershey, PA 17033 (USA) E-Mail razzaque@net.nagasaki-u.ac.jp


The formation of a triple-helical structure is one of the important posttranslational events in the collagen synthesis. In the endoplasmic reticulum, HSP47 binds to the alpha polypeptide chains, possibly to assist in the alignment and folding of the triple helix. Afterwards, HSP47 dissociates from the procollagen molecule once it enters the Golgi apparatus [9, 10]. Substantial in vitro evidence now points to HSP47 as a collagen-specific chaperone that assists in the intracellular processing, alignment, folding, and/or assembly of procollagens. Here we will discuss the possible in vivo role of HSP47 in human and experimental renal fibrotic diseases.

HSP47 and the Kidney

HSP47 and Experimental Glomerular Scarring Early inflammatory reactions cause proliferation and activation of glomerular cells, which, without intervention, ultimately lead to progressive glomerular scarring. In experimental nephritis induced by antithymocyte serum, an increased glomerular expression of HSP47 was associated with increased deposition of collagens in the scleroproliferative glomeruli [11]. Furthermore, within the scleroproliferative glomeruli, colocalization study revealed that phenotypically altered collagen-producing glomerular myofibroblasts (alpha-smooth muscle actin positive) were the main source of HSP47. Also, in the rat antiGBM (glomerular basement membrane) autoimmune glomerulonephritis model, marked accumulation of HSP47 was seen in developing and sclerosing crescents. Since HSP47 is a molecular chaperone that is intimately involved in synthesizing procollagen, we suggest that increased levels of glomerular HSP47 may regulate synthesis and assembly of the various procollagens and thus contribute to the glomerular sclerotic process. HSP47 and Experimental Tubulointerstitial Scarring The progression of tubulointerstitial fibrosis is characterized by the appearance of interstitial myofibroblasts that express alpha-smooth muscle actin [12], and tubular epithelial cells expressing vimentin [13] with interstitial inflammatory cell infiltration. Earlier studies have convincingly shown that increased synthesis of collagens by interstitial myofibroblasts and tubular epithelial cells results in tubulointerstitial fibrosis. In rat models, an increased expression of HSP47 was consistently observed in the interstitial myofibroblasts and tubular epithelial cells during the process of experimental and spontaneous tubulointerstitial fibrosis [14â&#x20AC;&#x201C;16]. Increased expression of

340

Nephron 2000;86:339â&#x20AC;&#x201C;341

HSP47 was seen in and around areas of interstitial fibrosis, and it colocalizes with collagens (types I and III). In these areas, infiltrating monocytes/macrophages (ED-1 positive) in the interstitium did not express HSP47. HSP47 and Human Sclerotic/Fibrotic Diseases Few studies deal with the expression of HSP47 in the normal human adult kidney [17]. However, in adult human kidneys HSP47 has been detected at very low levels in intraglomerular cells, tubular epithelial cells, and interstitial cells. HSP47 was strongly expressed in proliferating mesangial cells in human IgA nephropathy, diabetic kidneys, and during crescentic glomerulonephritis. In all types of nephropathy studied, HSP47 was strongly expressed in the tubular epithelial cells and in alphasmooth muscle actin positive myofibroblastic cells associated with areas of interstitial scarring [17, 18]. Available data suggest that irrespective of primary disease, upregualtion of HSP47 was seen during collagenization of glomeruli and tubulointerstitium [17].

Modulating HSP47 for Therapeutic Purposes

Calorie restriction has been known not only to suppress spontaneous neoplasms [19], but also to reduce the spontaneous age-associated renal scarring [20, 21]. The increased expression of HSP47 was closely associated with age-associated renal scarring in Fischer 344 rats. Furthermore, calorie restriction suppressed HSP47 expression and was associated with improvement of renal scarring [21]. In experimental nephritis models, the inhibition of HSP47 overexpression by administration of HSP47 antisense oligodeoxynucleotides resulted in the suppression of collagen production. Furthermore, this treatment attenuated the histological manifestations of glomerulosclerosis [22]. The results of both these in vivo studies [21, 22] raise the possibility of beneficial effects of therapeutic intervention interfering with HSP47 in renal fibrotic diseases. Blocking the relatively early factors for cell proliferation (i.e, platelet-derived growth factor) and matrix synthesis (i.e., transforming growth factor beta) has been shown to have some beneficial effects on the renal fibrotic process. However, HSP47 is playing a role in the later stage of collagen biosynthesis. Thus, targeting HSP47 could have a greater chance to reverse the fibrotic course, even in the later stages of the process.

Razzaque/Ahsan/Taguchi


Conclusions

Renal fibrosis accounts for considerable chronic morbidity in various renal diseases and could be a target of therapy. Since HSP47 plays a role in the fibrotic process [11, 14–17, 23–26] it will be of interest to determine whether monitoring HSP47 expression might have a predictive value in defining patients at risk of developing renal fibrotic complications and in assessing the response to conventional therapy. In addition, devising therapeutic

strategies directed at interfering with the HSP47 could be beneficial. As preliminary studies [21, 22] have shown, suppressing the expression of HSP47 can modulate renal scarring.

Acknowledgment We thank Dr. Jacqueline M. Crisman for critical reading and insightful comments on the manuscript.

References 1 Ritossa F: A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962;18:571–573. 2 Ritossa F: Discovery of the heat shock response. Cell Stress Chaperones 1996;1:97–98. 3 Parsell DA, Lindquist S: The function of heatshock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu Rev Genet 1993;27:437–496. 4 Kurkinen M, Taylor A, Garrels JI, Hogan BL: Cell surface-associated proteins which bind native type IV collagen or gelatin. J Biol Chem 1984;259:5915–5922. 5 Clarke EP, Sanwal BD: Cloning of a human collagen-binding protein and its homology with rat gp46, chick hsp47 and mouse J6 proteins. Biochim Biophys Acta 1992;1129:246–248. 6 Cates GA, Brickenden AM, Sanwal BD: Possible involvement of a cell surface glycoprotein in the differentiation of skeletal myoblasts. J Biol Chem 1984;259:2646–2650. 7 Hirayoshi K, Kudo H, Takechi H, Nakai A, Iwamatsu A, Yamada KM, Nagata K: HSP47: A tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts. Mol Cell Biol 1991;11: 4036–4044. 8 Wang SY, Gudas LJ: A retinoic acid-inducible mRNA from F9 teratocarcinoma cells encodes a novel protease inhibitor homologue. J Biol Chem 1990;265:15818–15822. 9 Satoh M, Hirayoshi K, Yokota S, Hosokawa N, Nagata K: Intracellular interaction of collagenspecific stress protein HSP47 with newly synthesized procollagen. J Cell Biol 1996;133: 469–483. 10 Nagata K: Expression and function of heat shock protein 47: A collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 1998;16:379–386.

Heat Shock Protein 47 in Renal Scarring

11 Razzaque MS, Taguchi T: Collagen-binding heat shock protein (HSP) 47 expression in antithymocyte serum (ATS)-induced glomerulonephritis. J Pathol 1997;183:24–29. 12 Muchaneta-Kubara EC, el Nahas AM: Myofibroblast phenotype expression in experimental renal scarring. Nephrol Dial Transplant 1997; 12:904–915. 13 Nakatsuji S, Yamate J, Sakuma S: Relationship between vimentin expressing renal tubules and interstitial fibrosis in chronic progressive nephropathy in aged rats. Virchows Arch 1998; 433:359–367. 14 Razzaque MS, Taguchi T: Localization of HSP47 in cisplatin-treated rat kidney: A possible role in tubulointerstitial damage. Clin Exp Nephrol 1999;3:222–228. 15 Razzaque MS, Shimokawa I, Nazneen A, Higami Y, Taguchi T: Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47. Cell tissue Res 1998;293: 471–478. 16 Cheng M, Razzaque MS, Nazneen A, Taguchi T: Expression of the heat shock protein 47 in gentamicin-treated rat kidneys. Int J Exp Pathol 1998;79:125–132. 17 Razzaque MS, Kumatori A, Harada T, Taguchi T: Coexpression of collagens and collagenbinding heat shock protein 47 in human diabetic nephropathy and IgA nephropathy. Nephron 1998;80:434–443. 18 Nakahama H, Nakamura H, Kitada O, Sugita M: Chronic drug-induced tubulointerstitial nephritis with renal failure associated with propylthiouracil therapy. Nephrol Dial Transplant 1999;14:1263–1265.

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19 Hursting SD, Perkins SN, Brown CC, Haines DC, Phang JM: Calorie restriction induces a p53-independent delay of spontaneous carcinogenesis in p53-deficient and wild-type mice. Cancer Res 1997;57:2843–2846. 20 Maeda H, Gleiser CA, Masoro EJ, Murata I, McMahan CA, Yu BP: Nutritional influences on aging of Fischer 344 rats. II. Pathology. J Gerontol 1985;40:671–688. 21 Razzaque MS, Shimokawa I, Nazneen A, Liu D, Naito T, Higami Y, Taguchi T: Life-long dietary restriction modulates the expression of collagens and collagen-binding heat shock protein 47 in aged Fisher 344 rat kidney. Histochem J 1999;31:123–132. 22 Sunamoto M, Kuze K, Tsuji H, Ohishi N, Yagi K, Nagata K, Kita T, Doi T: Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis. Lab Invest 1998;78:967–972. 23 Razzaque MS, Nazneen A, Taguchi T: Immunolocalization of collagen and collagen-binding heat shock protein 47 in fibrotic lung diseases. Modern Pathol 1998;11:1183–1188. 24 Kawada N, Kuroki T, Kobayashi K, Inoue M, Nakatani K, Kaneda K, Nagata K: Expression of heat-shock protein 47 in mouse liver. Cell Tissue Res 1996;284:341–346. 25 Razzaque MS, Taguchi T: The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999;14: 1199–1212. 26 Razzaque MS, Hossain MA, Kohno S, Taguchi T: Bleomycin-induced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat shock protein (HSP) 47. Virchows Arch 1998;432:455–460.

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@hsp47=JP+DN+IOVS+NEPHRON  

INTRODUCTION *Correspondence to: M.S. Razzaque, MBBS, PhD, Second Depart- ment of Pathology, Nagasaki University School of Medicine, 1-12-4,...

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