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 andchronic 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 nephropa-thy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, acollagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagensynthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 inthe kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renalexpression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, wehave performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of α-smooth muscleactin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomeru-lar epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III andtype IV collagens, α-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins inparaffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in theexpression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal depositionof types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of α-smooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) wasdetected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys ofchronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiationand progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly supporta pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which isassociated with glomerulosclerosis and tubulointerstitial fibrosis.

Introduction

Heat shock protein (HSP) 47 or colligin is a collagen-specificchaperone that was originally identified by Kurkinen et al.(Kurkinen et al. 1984) from murine parietal endoderm cells.Subsequently, HSP47 from human, rabbit, rat, mouse andchicken cDNA have been cloned and revealed a high degree ofhomology in their sequences (Wang & Gudas 1990, Hirayoshiet al. 1991, Clarke & Sanwal 1992, Takechi et al. 1992, Hartet al. 2000). HSP47 binds specifically to various types of col-lagens including types I–V in vitro (Natsume et al. 1994). Ithas been shown to be present in the endoplasmic reticulumand numerous studies have shown its expression in collagen-producing cells and tissues. The biochemical properties,intracellular localization and tissue distribution of HSP47,suggest its role in post-transcriptional regulation of procol-lagens (Nagata 1998). The crucial role of HSP47 in collagenbiosynthesis is well-documented, and HSP47 gene disrup-tion resulted in embryonic lethality in mice. The HSP47 null

mice died by 11.5 days post-coitus and showed a molecularabnormality in procollagen (Nagai et al. 2000).

Recent studies have convincingly shown a pathogenic roleof HSP47 in the development and progression of varioushuman and experimental fibrotic diseases (Masuda et al.1994, Razzaque & Taguchi 1997, 2002 Razzaque et al.1998a, Razzaque et al. 2001). For instance, the expres-sion of HSP47 augmented CCl4-induced liver cirrhosis,bleomycin-induced pulmonary fibrosis and anti-thymocyteserum-induced glomerulosclerosis in rats (Masuda et al.1994, Razzaque & Taguchi 1997, Razzaque et al. 1998a).However, the expression and significance of HSP47 inkidneys of acute and chronic diabetic rats and its role in dia-betic nephrosclerosis are not yet clear. Streptozotocin (STZ)-induced diabetic rats are widely used as a model to studyrenal changes (Osterby et al. 1967). STZ treatment results ininsulin deficiency in rats and the morphological changes in thekidney closely resemble those of human disease (Wilson &Letter 1990). In this preliminary study, we have studied the

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expression of HSP47 and collagens (types III and IV) in thekidneys obtained from STZ-induced diabetic rats, in bothshort- and long-term diabetes.

Materials and methods

Animal study

Six-week-old male Sprague–Dawley rats (n = 36) were usedfor the study. 18 rats were injected with STZ (50 mg/kg bodyweight) via a tail vein. The rats remained untreated for thedeveloping diabetes. A second set of 18 age-matched normalrats served as a control. All rats had unlimited access to waterand chow. The body weight and blood glucose levels of eachrat were measured periodically. Three rats from each groupwere sacrificed on days 1, 3 and 14 and weeks 4, 12 and 24of the experiment.

Tissue collection

Rats were sacrificed by exsanguinations under ether anes-thesia. Both kidneys were removed via midline incision andfixed immediately in Carnoy’s solution for 2 h (for immuno-histochemistry) and fixed in 10% formalin for 24 h (for bothimmunohistochemistry and histological examination).

Histological studies

Tissues were routinely processed and embedded in paraffinwax, cut into 4 µm sections and stained with haematoxylinand eosin (HE), periodic acid-Schiff (PAS), periodic acid-Schiff-methenamine silver (PAM) or Masson’s trichome.The extent of glomerular and tubulointerstitial damageswere determined in these stained slides by light microscopeexamination.

Immunohistochemistry

Paraffin sections (4 µm-thick) of kidneys were cut, andmounted on slides coated with aminoalkylsaline. Conven-tional immunoperoxidase staining was performed using theHistofine SAB kit (Nichirei, Tokyo) [for details of theprocedure, 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) inthe paraffin embedded renal sections. Briefly, paraffin sec-tions were deparaffinized with xylene, and rehydrated inethanol (from 100% to 70%), and incubated with hydro-gen peroxide. The sections were blocked with either 10%goat serum or 10% rabbit serum for 1 h, and then incubatedovernight at 4 ˚C with the above-listed primary antibodies.After washing with phosphate-buffered saline (PBS), sec-tions were processed further using the Nichirei Histostainkit (Nichirei, Tokyo, Japan). The reaction products were

developed with a mixture of 3,3′-diaminobenzine 4 HCl(DAB) and H2O2.

Double staining

Dual staining was performed to localize HSP47 withα-smooth muscle actin, vimentin or desmin as describedearlier (Razzaque et al. 1998c, Razzaque & Taguchi1999a). Paraffin sections were deparaffinized and rehy-drated routinely, and then HSP47-expressing cells identifiedon the sections by the streptavidin-alkaline phosphatasemethod. The sections were developed with 5-bromo-4-chloro-3-indolylphosphate (BCIP)-nitroblue tetrazolium(NBT), which produced dark purple coloured staining. TheHSP47-stained sections were counterstained with α-smoothmuscle actin, vimentin or desmin by the streptavidin-biotinperoxidase method, and developed with aminoethylcarbazole(ACE)-hydrogen peroxide, producing a red-coloured stain.

Control experiments

The following control experiments were performed to ver-ify the specificity of the immunostaining of HSP47. (i) Insome sections, primary antibodies were replaced with either0.01 m PBS or mouse IgG diluted with PBS (similar con-centration to that of the primary antibody). (ii) In someadjacent sections primary antibodies were replaced with asolution containing a 10-fold excess of recombinant HSP47(Stressgen Biotechnologies Corp, Canada) in addition to anti-HSP47 antibody [the antibody preabsorbed for HSP47 withthe recombinant HSP47 overnight at 4 ˚C before use]. Thesetreatments abolished the antigen-specific staining.

Semiquantitation of immunoperoxidase staining

Immunoperoxidase staining of kidney sections was anal-ysed and graded semiquantitatively according to the propor-tion of a particular structure in the kidney on the followingscale: (−) = no staining, (±) = staining involved <5%,(+) = staining involved 5–25%, (++) = staining involved25–75%, (+ + +) = staining involved >75%. This analysiswas performed in a blind and randomized way.

Results

Morphological analysis

Through out the study period, no significant fibrotic changescompared to control rat kidneys (Figure 1A) were noted in thekidneys of acute [days 1, 3 and 14] diabetic rats (Figure 1B,C).In contrast, gradual thickening of the glomerular basementmembrane, glomerulosclerosis, tubular damage, and intersti-tial fibrosis were noted in the kidneys of chronic [weeks 4, 12and 24] diabetic rats (Figure 1D–F). The extent of renal dam-age increased gradually with the time course and was mostsevere at week 24 (Figure 1F).

HSP47 in experimental diabetic nephropathy 623

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 kidneysof 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 andis most severe in week 24 (F). [PAS stain]

Immunohistochemical localization of type IIIand type IV collagens

Compared to control rat kidneys (Figure 2A and 3A), nosignificant changes in the expression of type III collagen(Figure 2B,C) and type IV collagen (Figure 3B,C) wasnoted in the kidneys obtained from acute diabetic rats. Inthe kidneys of control rats (Figure 2A) and acute diabeticrats, immunostaining of type III collagen (Figure 2B,C)was detected mainly in the interstitium. As for the type IVcollagen, the mesangium, glomerular basement membraneand tubular basement membrane were positively stainedin the kidneys of control rats (Figure 3A) and acute dia-betic rats (Figure 3B,C). Compared to kidneys of acutediabetic rats, an increased deposition of type III collagen(Figure 2D) and type IV collagen (Figure 3D) was detectedin kidneys of chronic diabetic rats, although the stainingpattern and distribution was different. Type III collagen(Figure 2D) was located predominantly in the widen inter-stitium, while type IV collagen (Figure 3D) was mainlylocated in the glomerulosclerosis and along the thickenedtubular basement membrane. The grading of semiquantitation

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

Immunohistochemical localization of HSP47

The expression of HSP47 was weakly detected in the intra-glomerular cells (mesangial and epithelial cells) and occa-sionally in the interstitial cells of the kidneys obtained fromcontrol rats (Figure 4A) and acute diabetic rats (Figure 4B,C).We did not find any significant changes in the expression ofHSP47, at least at the protein level, in the kidneys of acute dia-betic rats. In contrast, a markedly increased immunostainingfor HSP47 was noted in the glomerular cells (mesangial andepithelial cells), interstitial cells, and tubular epithelial cells inthe kidneys obtained from chronic diabetic rats (Figure 4D).When monoclonal antibody for HSP47 was replaced with a10-fold excess of recombinant HSP47, in addition to anti-HSP47 antibody, no specific immunostaining was detectedin the kidney (data not shown). The semiquantitative data ofthe expression of HSP47 in the kidneys of acute and chronicphases of diabetes are shown in Table 1.

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Figure 2. Immunoperoxidase staining (brown) for type III collagen insections 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 dia-betic rats, there is an increased interstitial deposition of type III collagen(arrow) in the kidney section obtained from a chronic diabetic rat (D).

Immunohistochemical localization ofα-smooth muscle actin, vimentin and desmin

No immunostaining for α-smooth muscle actin was detectedin the glomeruli of kidneys of control rats (Figure 5A) andacute diabetic rats (Figure 5B,C), while it was present inthe mesangial cells of the kidneys obtained from chronicdiabetic rats (Figure 5D), revealing a phenotypic alterationof mesangial cells in chronic diabetic rats. Vimentin waspresent in the glomeruli, but absent in the tubular epithe-lial cells in the kidneys of control rats (Figure 6A) and acutediabetic rats (Figure 6B,C). In contrast to kidneys of acute dia-betic rats, tubular epithelial cells and interstitial cells in kid-neys of rats in the chronic phase of diabetes showed markedimmunostaining for vimentin (Figure 6D), suggesting phe-notypic changes of tubular epithelial cells in chronic dia-betic rats. For desmin, weak immunostaining was noted inthe glomeruli in the kidneys of control rats (Figure 7A) andacute diabetic rats (Figure 7B,C), while strong immunos-taining for desmin was detected in the glomerular epithe-lial cells in the kidneys of chronic diabetic rats (Figure 7D),

Figure 3. Immunostaining for type IV collagen in sections of the kidneysobtained 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 ofcontrol (A) and acute diabetic rats (B, C), an increased glomerular depo-sition of type IV collagen is noted in the kidney section obtained from achronic diabetic rat (D). An increased deposition of type IV collagen isalso noted in the thickened tubular basement membrane (arrow).

revealing a phenotypic modulation of glomerular epithelialcells. Although the pattern of immunostaining was mostlysimilar in the kidneys of rats in the chronic phase of diabetes, itwas most marked in the kidneys of STZ-treated rats sacrificedon week 24. Semiquantitative data of the immunostaining ofα-smooth muscle actin, vimentin and desmin are shown inTable 1.

Double immunostaining HSP47 andα-smooth muscle actin, vimentin or desmin

Double immunostaining was performed to identify the cellsexpressing HSP47 in the kidneys of chronic diabetic rats.As expected, most of these phenotypically-altered α-smoothmuscle actin-positive mesangial cells (Figure 8A,B), desmin-positive glomerular epithelial cells (Figure 8C,D), vimentin-positive tubular epithelial cells (Figure 8E,F), and α-smoothmuscle actin-positive interstitial cells, are found to be HSP47-expressing cells (Razzaque et al. 1998d, Razzaque & Taguchi1999a).

HSP47 in experimental diabetic nephropathy 625

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

Antibody for: Glomeruli Tubules Interstitium

Control Acute∗ Chronic∗ Control Acute∗ Chronic∗ Control Acute∗ Chronic∗

(day 3) (week 24) (day 3) (week 24) (day 3) (week 24)

Collagen III − − ++ − − − ++ ++ +++Collagen IV + + +++ + + ++ ± ± +HSP47 + + +++ ± ± +++ + + +++α-SMA∗ − ± + − − − − ± ++Desmin + + +++ − − − ± ± +Vimentin ++ ++ ++ − − +++ ± ± +∗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 sectionsof a control rat (A), after 3 days of hyperglycaemia (B), after 14 daysof hyperglycaemia (C), and after 24 weeks of hyperglycaemia (D) indiabetic rats. Note that compared to the kidneys of control rat (A) andacute diabetic rats (B, C), a markedly increased expression of HSP47is 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).

Discussion

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

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

an important role in the correct folding of polypeptide chains,and thus has an important part in the post-translational mod-ification of collagens. Hence, the level of HSP47 appears tobe a marker for the rate at which collagen is being produced,and laid down in the matrix. We believe that the renal level ofHSP47 is an adequate indication and marker of when the kid-neys of diabetic rats initiate fibrosis. Immunohistochemistry

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

on kidney sections of acute and chronic STZ-induced diabeticrats has allowed us to examine the rates of types III and IVcollagen accumulation during the disease process and to cor-relate this with the expression of HSP47. Our results showedan obvious increase in the deposition of types III and IV col-lagens in kidneys of rats sacrificed on week 24 after STZinjection (the chronic phase of diabetes) rather than in theacute phase of diabetes, when morphologically the kidneysshowed no significant fibrotic changes.

HSP47, a collagen-binding protein, was examined in thisstudy by immunocytochemistry, to determine its cellularlocalization within kidneys of STZ-induced diabetic rats,in both short- and long-term diabetes. The semiquantitativescoring method we used allowed the immunoreactivity ofcollagens and HSP47 in the kidney to be determined. A par-allel increase in the expression of collagens and HSP47 wasnoted in the kidneys of chronic diabetic rats, while no sig-nificant changes were seen in the kidneys of acute diabeticrats. No major changes in the expression of HSP47 in thekidneys of STZ-treated rats, in the acute stage of diabetes,

Figure 7. Immunostaining for desmin in sections of kidneys from normalcontrol rat (A), acute [day 3 and 14] diabetic rats (B, C) and a chronicdiabetic rat (D). In contrast to the immunostaining in kidneys of con-trol rats (A) and acute diabetic rats (B, C), a strong immunostaining fordesmin 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 ofthe diseases is not just an acute toxic effect of the drug STZ,but a consequence of the disease process. This result is ofclinical interest because it raises the possibility of delayingand/or blocking the progression of the renal scarring evenin the chronic stages of diabetes, by therapeutic manipula-tion of the expression of HSP47. Preliminary studies haveshown that in vivo blocking or modulation of the biologicalactivities of HSP47 resulted in decreased production of colla-gens, and thereby less fibrotic changes in kidneys (Sunamotoet al 1998, Razzaque & Taguchi 1999b). We have shownthat an increased expression of HSP47 in human diabeticnephropathy is associated with glomerulosclerosis and tubu-lointerstitial fibrosis (Razzaque et al. 1998b). However, wewere not able to determine the sequential expression patternof HSP47 in kidneys using human renal biopsies. In thisstudy, using a STZ-induced diabetic model, we showed atime-course expression of HSP47 in acute and chronic dia-betic nephropathy. We have demonstrated that, in contrast tothe acute phase of diabetes, the levels of expression of HSP47were significantly increased in glomeruli and tubules of the

HSP47 in experimental diabetic nephropathy 627

Figure 8. Double staining for HSP47 (dark purple) with α-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 colocalizationof HSP47 with α-smooth muscle actin (arrows) and desmin-positive (arrows) glomerular cells, suggesting that mesangial cells (A, B) and glomerularepithelial cells (C, D) are HSP47-expressing cells. Some of the HSP47-expressing interstitial cells (arrowheads) are α-smooth muscle actin-positivemyofibroblasts (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-treatedrats; the increased expression of HSP47 corresponded withthat of collagen deposition in the kidneys.

In this study, we also identified the phenotypically-alteredand/or damaged cells in kidneys of STZ-treated diabetic rats.Phenotypically-altered glomerular mesangial cells, glomeru-lar epithelial cells, and tubular epithelial cells were detected

in the kidneys of chronic diabetic rats. Previously it wasshown that all these phenotypically-altered renal cells areHSP47-producing cells (Razzaque et al. 1998b, Razzaque &Taguchi 1999b). Consistent with these earlier studies, wehave shown here that phenotypically-altered renal cells are themain source of HSP47 in the kidneys of chronic STZ-treatedrats. Interestingly, in the acute phase of the diabetes, we could

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not detect many phenotypically-altered cells in the kidney,which may be one of the reasons why HSP47 expression isnot as high as it is in the chronic phase of the disease, whenphenotypically-altered cells are widespread in the kidney.

In summary, the main conclusion made from this prelim-inary study is that the increased deposition of collagens inthe scarring kidney in the chronic phase of diabetes corre-sponds well with increased levels of HSP47. Importantly,HSP47 up-regulation coincided with the initiation and pro-gression of fibrogenesis, as determined by the expression anddeposition of collagens. These results suggest that the HSP47could be a key factor in the initiation and progression of fibro-sis that occurs in kidneys of chronic diabetic rats. From thecollagen synthesizing abilities of the HSP47, it is suggestedthat HSP47 may play a key regulatory role in the excessiveassembly and synthesis of collagen during renal scarring indiabetic nephropathy. It also appears from this study thatHSP47 is mostly involved in the chronic phase of the dis-ease, and therefore could be a potential target for innovativetherapies designed to limit fibrosis, even in the chronic phaseof diabetes.

References

Clarke EP, Sanwal BD (1992) Cloning of a human collagen-bindingprotein, and its homology with rat gp46, chick hsp47 and mouse J6proteins. 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 rabbitconnective 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 surface-associated proteins which bind native type IV collagen or gelatin. J BiolChem 259: 5915–5922.

Masuda H, Fukumoto M, Hirayoshi K, Nagata K (1994) Coexpression ofthe collagen-binding stress protein HSP47 gene and the alpha 1(I) andalpha 1(III) collagen genes in carbon tetrachloride induced rat liverfibrosis. 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 hsp47knockout 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: acollagen-specific molecular chaperone in the endoplasmic reticulum.Matrix Biol 16: 379–386.

Natsume T, Koide T, Yokota S, Hirayoshi K, Nagata K (1994) Interactionsbetween collagen-binding stress protein HSP47 and collagen. Analysis

of kinetic parameters by surface plasmon resonance biosensor. J BiolChem 269: 31224–31228.

Osterby R, Lundbaek K, Olsen TS, Orskov H (1967) Kidney lesions inrats with severe long term alloxan diabetes. Lab Invest 17: 675–692.

Razzaque MS, Foster CS, Ahmed AR (2001) Tissue and molecular eventsin human conjunctival scarring in ocular cicatricial pemphigoid. HistolHistopathol 16: 1203–1212

Razzaque MS, Hossain MA, Kohno S, Taguchi T (1998a) Bleomycin-induced pulmonary fibrosis in rat is associated with increased expres-sion of collagen-binding heat shock protein (HSP) 47. Virchows Arch432: 455–460.

Razzaque MS, Koji T, Harada T, Taguchi T (1999) Localization in situof type VI collagen protein and its mRNA in mesangial proliferativeglomerulonephritis using renal biopsy sections. Histochem Cell Biol111: 1–6.

Razzaque MS, Kumatori A, Harada T, Taguchi T (1998b) Coexpres-sion of collagens and collagen-binding heat shock protein 47 inhuman diabetic nephropathy and IgA nephropathy. Nephron 80:434–443.

Razzaque MS, Nazneen A, Taguchi T (1998c) Immunolocalization ofcollagen and collagen-binding heat shock protein 47 in fibrotic lungdiseases. 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 over-expression 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 expres-sion of collagens and collagen-binding heat shock protein 47 in agedFischer 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 glomeru-lonephritis. J Pathol 183: 24–29.

Razzaque MS, Taguchi T (1999a) Localization of HSP47 in cisplatin-treated rat kidney: possible role in tubulointerstitial damage. Clin ExpNephrol 3: 222–228.

Razzaque MS, Taguchi T (1999b) The possible role of colligin/HSP47,a collagen-binding protein, in the pathogenesis of human and experi-mental fibrotic diseases. Histol Histopathol 14: 1199–1212.

Razzaque MS, Taguchi T (2002) Cellular and molecular events lead-ing 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 pro-tein HSP47 suppress collagen accumulation in experimental glomeru-lonephritis. 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 thedifferentiation of F9 teratocarcinoma cells. Eur J Biochem 206:323–329.

Wang SY, Gudas LJ (1990) A retinoic acid-inducible mRNA from F9teratocarcinoma cells encodes a novel protease inhibitor homologue.J Biol Chem 265: 15818–15822.

Wilson GL, Letter EH (1990) Streptozotocin interactions with pancreaticbeta cells and the induction of insulin-dependent diabetes. Curr TopMicrobiol Immunol 156: 27–54.