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Expression of connective tissue growth factor in human renal fibrosis

1998; Elsevier BV; Volume: 53; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1755.1998.00820.x

1523-1755

Yasuhiko Ito, Jan Aten, Richard J. Bende, Barry S. Oemar, Ton J. Rabelink, Jan J. Weening, Roel Goldschmeding,

Systemic Sclerosis and Related Diseases

Expression of connective tissue growth factor in human renal fibrosis. Chronic renal failure may occur in etiologically diverse renal diseases and can be caused by hemodynamic, immunologic and metabolic factors. Initial damage may evoke irreversible scarring, which involves production of a number of proinflammatory and fibrogenic cytokines, including platelet-derived growth factor (PDGF) and transforming growth factor β (TGF-β). Connective tissue growth factor (CTGF), a cytokine of the family of growth regulators comprising cef10, cyr61, CTGF and nov, has recently been described in association with scleroderma and other scarring conditions. We investigated CTGF mRNA expression in 65 human renal biopsy specimens of various renal diseases by in situ hybridization. In control human kidney CTGF mRNA was mainly expressed in visceral epithelial cells, parietal epithelial cells, and some interstitial cells. Connective tissue growth factor was strongly up-regulated in the extracapillary and severe mesangial proliferative lesions of crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis and diabetic nephropathy. An increase in the number of cells expressing CTGF mRNA was observed at sites of chronic tubulointerstitial damage, which correlated with the degree of damage. In the tubulointerstitial area the majority of the CTGF mRNA positive cells coexpressed α-smooth muscle actin, and were negative for macrophage markers. Our results indicate that CTGF may be a common growth factor involved in renal fibrosis. Expression of connective tissue growth factor in human renal fibrosis. Chronic renal failure may occur in etiologically diverse renal diseases and can be caused by hemodynamic, immunologic and metabolic factors. Initial damage may evoke irreversible scarring, which involves production of a number of proinflammatory and fibrogenic cytokines, including platelet-derived growth factor (PDGF) and transforming growth factor β (TGF-β). Connective tissue growth factor (CTGF), a cytokine of the family of growth regulators comprising cef10, cyr61, CTGF and nov, has recently been described in association with scleroderma and other scarring conditions. We investigated CTGF mRNA expression in 65 human renal biopsy specimens of various renal diseases by in situ hybridization. In control human kidney CTGF mRNA was mainly expressed in visceral epithelial cells, parietal epithelial cells, and some interstitial cells. Connective tissue growth factor was strongly up-regulated in the extracapillary and severe mesangial proliferative lesions of crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis and diabetic nephropathy. An increase in the number of cells expressing CTGF mRNA was observed at sites of chronic tubulointerstitial damage, which correlated with the degree of damage. In the tubulointerstitial area the majority of the CTGF mRNA positive cells coexpressed α-smooth muscle actin, and were negative for macrophage markers. Our results indicate that CTGF may be a common growth factor involved in renal fibrosis. α-smooth muscle actin anti-neutrophil cytoplasmic antibody connective tissue growth factor diethyl pyrocarbonate digoxigenin-labeled uridine-triphosphate diabetes mellitus focal glomerulosclerosis glomerular visceral epithelial cells hematoxylin and eoson stain human umbilical vein endothelial cells IgA nephropathy lupus nephritis minimal change nephrotic syndrome idiopathic membranous nephropathy nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate nucleotide-triphosphate periodic acid-Schiff stain phosphate buffered saline platelet-derived growth factor transforming growth factor-β Fibrosis is the final common pathway for almost all forms of renal disease that progress to end-stage renal failure including immunologically mediated glomerulonephritis and tubulointerstitial nephritis, hemodynamic disorders, metabolic diseases, and hereditary diseases1.Kelly C.J. Neilson E.G. Tubulointerstitial diseases,.The Kidney. 1996; vol 2 (edited by BRENNER BM, Philadelphia, WB Saunders Company): 1655-1679Google Scholar. Histologically, such scarring consists of glomerulosclerosis, tubulointerstitial fibrosis, as well as vascular hyalinosis and sclerosis. Whatever the primary disturbance, further mediators are required to cause renal scarring characterized by cell proliferation and accumulation of matrix constituents2.Eddy A.A. Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar. An important role in this process was shown for the cytokines platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), as indicated by in vivo modulation of the activity of these cytokines3.Border W.A. Noble N.A. Transforming growth factor β in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (3007) Google Scholar. Blocking of the activity of PDGF or TGF-β was shown to prevent extracellular matrix expansion and glomerulosclerosis4.Johnson R.J. Raines E.W. Floege J. Yoshimura A. Pritzl P. Alpers C. Ross R. Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor.J Exp Med. 1992; 175: 1413-1416https://doi.org/10.1084/jem.175.5.1413Crossref PubMed Scopus (352) Google Scholar, 5.Border W.A. Okuda S. Languino L.R. Sporn M.B. Ruoslahti E. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor β1.Nature. 1990; 346: 371-374Crossref PubMed Scopus (933) Google Scholar, 6.Border W.A. Noble N.A. Yamamoto T. Harper J.R. Yamaguchi Y. Pierschbacher M.D. Ruoslahti E. Natural inhibitor of transforming growth factor-β protects against scarring in experimental kidney disease.Nature. 1992; 360: 361-364Crossref PubMed Scopus (926) Google Scholar, 7.Isaka Y. Brees D.K. Ikegaya K. Kaneda Y. Imai E. Nobel N.A. Border W.A. Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney.Nature Med. 1996; 2: 418-423https://doi.org/10.1038/nm0496-418Crossref PubMed Scopus (460) Google Scholar. Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators, which are comprised of cef 10, cyr 61, CTGF and nov8.Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. Connective tissue growth factor: A cystein-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10.J Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (809) Google Scholar, 9.Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor.FEBS Lett. 1993; 327: 125-130Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 10.Simmons D.L. Levy D.B. Yannoni Y. Erikson R.L. Identification of a phorbol ester-repressible v-src-inducible gene.Proc Natl Acad Sci USA. 1989; 86: 1178-1182Crossref PubMed Scopus (296) Google Scholar, 11.O'BRIEN T.P. Yang G.P. Sanders L. Lau L.F. Expression of cry61, a growth factor-inducible immediate-early gene.Mol Cell Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (271) Google Scholar, 12.Joliot V. Marinerie C. Dambrine G. Plassiart G. Brisac M. Crochet J. Perbal B. Proviral rearrangements and overexpression of a new celluar gene (nov) in myeloblastosis-associated virus type-1 induced nephroblastomas.Mol Cell Biol. 1992; 12: 10-21Crossref PubMed Scopus (278) Google Scholar. Connective tissue growth factor was originally cloned from human umbilical vein endothelial cells (HUVEC)8.Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. Connective tissue growth factor: A cystein-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10.J Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (809) Google Scholar. Subsequently, skin fibroblasts in scleroderma lesions were found to overexpress CTGF13.Kikuchi K. Kadono T. Ihn H. Sato S. Igarashi A. Nakagawa H. Tamaki K. Takehara K. Growth regulation in scleroderma fibroblasts: Increased response to transforming growth factor-β1.J Invest Dermatol. 1995; 105: 128-132https://doi.org/10.1111/1523-1747.ep12313452Abstract Full Text PDF PubMed Scopus (93) Google Scholar. By using a differential cloning technique, a cDNA clone identical to CTGF was isolated from a human atherosclerotic aorta cDNA library14.Oemar B.S. Werner A. Garnier J.M. Do D.D. Godoy N. Nauck M. MÄRZ W. Rupp J. Pech M. LÜSCHER T.F. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions.Circulation. 1997; 95: 831-839Crossref PubMed Scopus (290) Google Scholar, in which expression was found to be 50- to 100-fold increased as compared to normal arteries14.Oemar B.S. Werner A. Garnier J.M. Do D.D. Godoy N. Nauck M. MÄRZ W. Rupp J. Pech M. LÜSCHER T.F. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions.Circulation. 1997; 95: 831-839Crossref PubMed Scopus (290) Google Scholar. Connective tissue growth factor was shown to induce kidney fibroblast proliferation and extracellular matrix synthesis15.Frazier K. Williams S. Kothapalli D. Klapper H. Grotendorst G.R. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor.J Invest Dermatol. 1996; 107: 404-411https://doi.org/10.1111/1523-1747.ep12363389Abstract Full Text PDF PubMed Scopus (673) Google Scholar. Effects of TGF-β on fibroblasts were found to be partially mediated by CTGF16.Kothapalli D. Frazier K.S. Welply A. Segarini P.R. Grotendorst G.R. Transforming growth factor β induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway.Cell Growth Differ. 1997; 8: 61-68PubMed Google Scholar. The present study proposes the possible involvement of CTGF in renal fibrosis. As a first step in the elucidation of its possible role in renal sclerosis, we investigated the expression of human CTGF mRNA in human biopsy specimens of various renal diseases by in situ hybridization. Sixty-five specimens from control and diseased human kidneys were studied. Kidney samples were obtained from patients undergoing diagnostic evaluation at the Academic Medical Center of the University of Amsterdam. Control human kidney specimens (N = 5) were taken from normal portions of nephrectomy specimens of patients who underwent surgery because of localized renal tumors. Specimens from diseased kidneys were obtained by percutaneous renal biopsy from the patients with various renal diseases. The histologic diagnoses are listed in Table 1. These included minimal change nephrotic syndrome (MCNS), IgA nephropathy (IgAN), idiopathic membranous nephropathy (MN), focal glomerulosclerosis (FGS), crescentic anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, lupus nephritis (LN), diabetic glomerulosclerosis (DM), membranoproliferative glomerulonephritis (MPGN), chronic rejection of transplanted kidneys, acute postinfectious glomerulonephritis, and nephrosclerosis. The classification of LN was based on the WHO histomorphological classification17.Churg J. Sobin L.H. Lupus nephritis,.Renal Disease: Classification and Atlas of Glomerular Diseases. Igaku-shoin, Tokyo1982: 127Google Scholar. For light microscopy, kidney tissues were fixed in 4% buffered formalin overnight, dehydrated and embedded in paraffin by conventional techniques. Sections were stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and silver methenamine (Jones).Table 1Table 1. Renal biopsy cases evaluated for connective tissue growth factor (CTGF) mRNA expression The 1.5-kb EcoRI/KpnI fragment of CL59 cDNA of human CTGF was used to produce antisense and sense probes14.Oemar B.S. Werner A. Garnier J.M. Do D.D. Godoy N. Nauck M. MÄRZ W. Rupp J. Pech M. LÜSCHER T.F. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions.Circulation. 1997; 95: 831-839Crossref PubMed Scopus (290) Google Scholar. Antisense and sense cRNA riboprobes were generated after digestion with restriction enzymes EcoRI and XbaI. In vitro transcription was performed using an RNA Labeling Kit (Boehringer-Mannheim, Germany). One microgram of linear template DNA was used in each labeling reaction. The labeling reaction components including transcription buffer, nucleotide-triphosphate (NTP) substrate, and T3 or T7 polymerase were mixed with the linearized DNA. The mixture was incubated at 37°C for two hours. During the transcription, digoxigenin-labeled uridine-triphosphate (DIG-UTP) was incorporated into the transcription products. The transcribed riboprobe was precipitated by 70% ethanol, dissolved in 100 μl diethyl pyrocarbonate (DEPC; Sigma, St Louis, MO, USA) treated water. The amount of DIG-labeled RNA was measured with DIG quantification test strips and control test strips (Boehringer-Mannheim). Four-micrometer serial sections of formalin-fixed paraffin-embedded renal tissues were placed on 3-aminopropyltriethoxysilane (Sigma) coated slides. Sections were deparaffinized, treated with 0.2 M HCl for 20 minutes, digested with 10 μg/ml proteinase K for 20 minutes at 37°C, treated with 2 mg/ml glycine for two minutes and post-fixed with 4% paraformaldehyde in PBS for 20 minutes. After treatment with 10 mM EDTA the sections were subjected to hybridization. Digoxigenin (DIG)-labeled riboprobes (final concentration 0.3 μg/ml) were added to hybridization solution containing 50% deionized formamide, 10% dextran sulfate, 2 × SSC, 2 × Denhardt's solution, 0.1% Triton X, 200 μg/ml Herring sperm DNA (Boehringer-Mannheim), 100 μg/ml yeast transfer RNA (Boehringer-Mannheim). Hybridizations were performed in a humidified chamber for 18 hours at 52°C. The slides were washed twice with 1 × SSC containing 50% formamide at 52°C. After washing with 1 × SSC they were treated with 20 μg/ml RNAse A for 30 minutes at 37°C. Washing was then continued with 1 × SSC twice and with 0.1 × SSC once. After post-hybridization washing, they were incubated with alkaline phosphatase-conjugated F(ab) fragments of sheep anti-DIG antibody and then visualized with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt (NBT/BCIP) according to the DIG nucleic acid detection kit protocol (Boehringer-Mannheim). The sections were counterstained with hematoxylin or PAS without hematoxylin. Slides were rinsed in distilled water and covered with DAKO glycergel mounting medium (DAKO, Glostrup, Denmark). Serial formalin-fixed paraffin sections were deparaffinized with xylene, rehydrated, and washed with phosphate-buffered saline (PBS). After blocking endogenous peroxidase activity with 0.3% hydrogen peroxide and free protein binding sites with normal goat serum, the tissue was incubated with primary murine monoclonal antibodies, for α-smooth muscle actin (α-SMA, 1A4; DAKO) or for CD68 (PG-M1; DAKO) during two hours at room temperature. Immunoreactivity for CD68 was enhanced by microwave oven heating of sections in 10 mM sodium citrate, pH 6.0, for 10 minutes. Immobilized mouse antibodies were detected by the immunoalkaline phosphatase anti-alkaline phosphatase method with rabbit anti-mouse immunoglobulin (DAKO) and APAAP complex (DAKO), or a streptavidin-biotin-immunoperoxidase technique using the strept ABC complex/HRP kit (DAKO). The specificity was checked by omission of primary antibodies and use of non-immune mouse IgG as negative control. In situ hybridization in combination with immunohistochemistry was performed on the same section to simultaneously detect CD68 or α-SMA and CTGF mRNA. Sections were first hybridized with DIG-RNA probe and transcripts were detected. After washing with PBS, endogenous biotin present in renal tissue was blocked with 1 mg/ml streptavidin (Zymed, San Francisco, CA, USA) and 0.1 mg/ml d-biotin (Sigma) in two successive steps. Then tissues were stained with α-smooth muscle actin or CD68 monoclonal antibody. Microwave pretreatment enhanced the reactivity for CD68. Presence of CTGF mRNA was visualized with NBT/BCIP (purple-black) and α-SMA or CD68 were stained with diaminobenzidine (brown). The results of the double staining experiments showed staining patterns and staining intensities similar to those obtained in simultaneously performed single staining experiments. The correlation between interstitial CTGF expression and chronic tubulointerstitial injury was assessed in 45 cases. Cases of acute renal injury (6 cases of LN, and 7 cases of crescentic ANCA-associated glomerulonephritis) were excluded. Another seven cases were excluded because these lacked adequate tissue for quantification of the interstitium. Tubulointerstitial expression of the CTGF gene was determined by counting the CTGF mRNA positive cells within the whole cortical area of each biopsy. At least twenty fields of 0.044 mm2 were assessed and the average number of positive cells in 0.1 mm2 was calculated. Tubulointerstitial injury was classified into four groups, according to the extent of cortical interstitial fibrosis and of tubular atrophy and degeneration: (1) normal; (2) involvement up to 25% of the cortex; (3) involvement of 26 to 50% of cortex; and (4) extensive damage involving more than 50% of the cortex. This is comparable to the chronic change score in the Banff classification of kidney transplant pathology18.Solez K. Axelsen R.A. Benediktsson H. Burdick J.F. Cohen A.H. Colvin R.B. Croker B.P. Droz D. Dunnill M.S. Halloran P.F. HÄYRY P. Jennette J.C. Keown P.A. Marcussen N. Mihatsch M. Morozumi K. Myers B.D. Nast C.C. Olsen S. Racusen L.C. Ramos E.L. Rosen S. Sachs D.H. Salomon D.R. Sanfilippo F. Verani R. VON WILLEBRAND E. Yamaguchi Y. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology.Kidney Int. 1993; 44: 411-422Abstract Full Text PDF PubMed Scopus (1289) Google Scholar. For this study an established cell-line of glomerular visceral epithelial cells (GVEC) was used, derived from Sprague-Dawley rat glomeruli as has been described elsewhere19.Coers W. Brouwer E. Vos J.T.W.M. Chand A. Huitema S. Heeringa P. Kallenberg C.G.M. Weening J.J. Podocyte expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1) in experimental pauci-immune crescentic glomerulonephritis.Clin Exp Immunol. 1994; 98: 279-286Crossref PubMed Scopus (49) Google Scholar, 20.Quigg R.J. Cybulsky A.V. Jacobs J.B. Salant D.J. Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.Kidney Int. 1988; 34: 43-52Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 21.O'MEARA Y.M. Natori Y. Minto A.W. Goldstein D.J. Manning E.C. Salant D.J. Nephrotoxic antiserum identifies a β1-integrin on rat glomerular epithelial cells.Am J Physiol. 1992; 262: F1083-F1091PubMed Google Scholar. This cell line was found to express a ganglioside reported to be specific for visceral and not parietal epithelial cells of the glomerulus22.Reivinen J. HOLTHÖFER H. Miettinen A. A cell-type specific ganglioside of glomerular podocytes in rat kidney: An O-acetylated GD3.Kidney Int. 1992; 42: 624-631Abstract Full Text PDF PubMed Scopus (44) Google Scholar. The cell line was cultured and maintained in K1 medium containing hormone mix (all ingredients from Sigma), 100 U/ml penicillin (Gibco, Gaithersburg, MD, USA), 100 μg/ml streptomycin (Gibco) and 5% NuSerum (Collaborative Research Inc., Bedford, MA, USA) on a collagen gel (Vitrogen 100; Collagen Corp, CA, USA) in a humidified 5% CO2—95% air incubator, as described19.Coers W. Brouwer E. Vos J.T.W.M. Chand A. Huitema S. Heeringa P. Kallenberg C.G.M. Weening J.J. Podocyte expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1) in experimental pauci-immune crescentic glomerulonephritis.Clin Exp Immunol. 1994; 98: 279-286Crossref PubMed Scopus (49) Google Scholar. Experiments were performed with GVEC at passage numbers 25 to 30. Total RNA was isolated from renal cortical tissues of nephrectomy specimens and from cultured GVEC by the TRIzol method (Life Technologies, MD, USA). Fifteen micrograms of total RNA were size separated by electrophoresis on a 0.22 M formaldehyde-1% agarose gel, transferred to a nylon membrane, (Boehringer-Mannheim), and U.V. cross-linked. Before transfer to a nylon membrane, ethidium-stained gels were visualized by ultraviolet illumination to determine the position of 28S and 18S ribosomal RNA and to assess the integrity of the RNA. Prehybridization and hybridization was performed in DIG Easy Hyb (Boehringer-Mannheim), containing yeast RNA or sheared heat-denatured herring sperm DNA. Membranes were hybridized with a DIG-labeled CTGF cRNA probe or with a 18S rRNA oligonucleotide probe23.Mendez R.E. Pfeffer J.M. Ortola F.V. Bloch K.D. Anderson S. Seidman J.G. Brenner B.M. Atrial natriuretic peptide transcription, storage, and release in rats with myocardial infarction.Am J Physiol. 1987; 253: H1449-H1455PubMed Google Scholar, respectively. The latter was used as a positive control and was labeled using a DIG 3′-End Labeling Kit (Boehringer-Mannheim). Following hybridization, the blots were washed twice with 2 × SSC containing 0.1% SDS at room temperature. Subsequently, the blots were washed twice with 0.5 × SSC, 0.1% SDS at 62°C for the CTGF probe and at 42°C for the 18S rRNA oligonucleotide probe. Hybridized probes were detected using alkaline phosphatase-conjugated F(ab) fragments of sheep anti-DIG antibody and the chemiluminescent substrate CSPD (Boehringer- Mannheim), according to the manufacturer's instructions. All values are expressed as mean ±SD. Statistical analysis was performed with Statview IV software (Abacus Concepts, Berkeley, CA, USA). Statistical significance was evaluated by one way analysis of variance with the modified t-test using Bonferroni correction. The Spearman rank correlation coefficient was used for analysis of correlation. In the glomeruli of control human kidney, CTGF mRNA was expressed mainly by visceral epithelial cells. CTGF transcripts were also detected in some parietal epithelial cells Figure 1a. Some interstitial cells in the peritubular and periglomerular areas also showed CTGF expression Figure 1b. To determine the cellular origin of the CTGF mRNA positive cells in the tubulointerstitial area, tissue sections were double-labeled for α-SMA and CTGF mRNA and also for CD68 and CTGF mRNA. The majority of the CTGF mRNA positive cells coexpressed α-SMA and did not express CD68 antigen. CD68 positive cells also did not coexpress CTGF mRNA Figure 1c, d. These cells resemble interstitial fibroblasts, but we could not completely exclude the possibility that CTGF mRNA might be expressed in endothelial cells of peritubular capillaries. All sections hybridized with a sense probe were negative and binding of the antisense probes to these cells was prevented by preincubation with RNAse. Connective tissue growth factor expression was found to be normal as compared to control or only marginally increased in glomerular diseases characterized by non-inflammatory lesions and proteinuria, such as MCNS and MN, and in acute postinfectious exsudative glomerulonephritis. In contrast, CTGF expression was found to be markedly increased in inflammatory glomerular and tubulointerstital lesions, associated with cellular proliferation and matrix accumulation. These lesions include IgA nephropathy, chronic transplant rejection, crescentic glomerulonephritis, FGS, lupus nephritis (WHO class IV) and MPGN. In three out of five MCNS and three out of seven MN without glomerulosclerosis no up-regulation was observed as compared to that in control renal tissue samples. In the other two cases of MCNS and in the remaining four cases of MN without glomerulosclerosis, CTGF mRNA expression was slightly increased in visceral epithelial cells Figure 2. In disorders with extracapillary proliferative lesions, a significant increase of CTGF mRNA was noted in the glomeruli. In the cellular crescents of crescentic glomerulonephritis, IgA nephropathy, and diffuse lupus nephritis CTGF expression was strongly increased Figure 3a, b. According to immunostaining for CD68 and α-SMA, macrophages and myofibroblasts are not the main components of the crescents Figure 3 C, D). This finding suggests that in these crescents CTGF is mainly expressed by proliferating epithelial cells. In the few remaining cells in fibrocellular crescents, CTGF mRNA expression was still high. Connective tissue growth factor expression was also increased in other extracapillary lesions, including segmental sclerosis with adhesion to Bowman's capsule observed in FGS Figure 4 and MN with fibrosis. Although CTGF expressing cells were mainly epithelial cells, severe mesangial proliferative lesions in IgAN, diabetic diffuse lesions, and diffuse lupus nephritis also expressed CTGF Figure 5a, b . In contrast, CTGF was not up-regulated in postinfectious endocapillary proliferative nephritis in spite of the numerous infiltrating neutrophils Figure 5c, d.Figure 3Renal biopsy from a patient with crescentic glomerulonephritis. PAS staining (A), connective tissue growth factor (CTGF) mRNA (B), CD68 (C), and α-smooth muscle actin (D). In the cellular crescent, CTGF mRNA is mainly expressed by proliferated epithelial cells (×200).View Large Image Figure ViewerDownload (PPT)Figure 4Renal biopsy from a patient with focal glomerularsclerosis (FGS). PAS staining (A), and connective tissue growth factor (CTGF) mRNA (B). CTGF expression is increased in segmental sclerotic lesions with adhesion to Bowman's capsule (×125).View Large Image Figure ViewerDownload (PPT)Figure 5In situ hybridization for connective tissue growth factor (CTGF) mRNA reveals CTGF upregulation in severe mesangial proliferative lesions of diabetes mellitus (DM) nephropathy diffuse lesion (A) and IgA nephropathy (B). In contrast, CTGF is not up-regulated in acute postinfectious glomerulonephritis (C, PAS stain; D, CTGF; ×200).View Large Image Figure ViewerDownload (PPT) Uninjured portions of renal cortical interstitial tissues were generally indistinguishable from the interstitial patterns of sparse CTGF mRNA expression described above in control kidneys. In the case of MCNS, FGS and MN without interstitial fibrosis, CTGF mRNA positive cells were rare in the tubulointerstitial area Figures 2 and 4. In contrast, increased numbers of CTGF mRNA positive cells were identified within the tubulointerstitial fibrotic areas in chronic transplant rejection, and in the chronic interstitial damage in the context of glomerulonephritides. Within these lesions, the distribution of CTGF mRNA positive cells was similar to that of α-SMA staining Figure 6. Also in periglomerular fibrosis, CTGF mRNA was strongly up-regulated in α-SMA positive cells Figure 7. A statistically highly significant correlation between the extent of tubulointerstitial fibrosis and the number of CTGF mRNA positive cells per surface area was demonstrated Figure 8 using the Spearman rank correlation coefficient (r = 0.849; P < 0.001).Figure 7Connective tissue growth factor (CTGF) expression in periglomerular fibrosis in a case of focal glomerulosclerosis (FGS) with hypertension (A, PAS stain; B, CTGF mRNA; and C, α-smooth muscle actin stain on serial sections). CTGF expression is increased in the periglomerular lesion and in lesions of proliferated visceral and parietal epithelial cells. CTGF is expressed by α-smooth muscle actin positive cells in the periglomerular interstitial fibrosis (×200).View Large Image Figure ViewerDownload (PPT)Figure 8Relationship between the number of interstitial connective tissue growth factor (CTGF)-expressing cells and the extent of tubulointerstitial lesions. Data are expressed as the mean ±SD.*P < 0.0001, as compared to the group without tubulointerstitial lesions, scored as 0%.View Large Image Figure ViewerDownload (PPT) In atherosclerotic arteries not only smooth muscle cells, but also endothelial cells showed CTGF gene expression (not shown). By Northern analysis, a 2.4 Kb CTGF transcript was detected in RNA extracted from control human kidney Figure 9a and from cultured podocytes Figure 9b. A widely accepted paradigm in concepts of development of renal fibrosis is that TGF-β up-regulation is a main factor responsible for fibrotic changes and scarring in response to renal injury3.Border W.A. Noble N.A. Transforming growth factor β in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (3007) Google Scholar. Its importance in glomerulosclerosis has been established by in vivo studies and by analysis of glomerular cells in culture24.Yamamoto T. Nobel N.A. Cohen A.H. Nast C.C. Hishida A. Gold L.I. Border W.A. Expression of transforming growth factor-β isoforms in human glomerular diseases.Kidney Int. 1996; 49: 461-469Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 25.Yamamoto T. Nakamura T. Nobel N.A. Ruoslahti E. Border W.A. Expression of transforming growth factor-β is elevated in human and experimental diabetic nephropathy.Proc Natl Acad Sci USA. 1993; 90: 1814-1818Crossref PubMed Scopus (813) Google Scholar, 26.Yoshioka K. Takemura T. Murakami K. Okada M. Hino S. Miyamoto H. Maki S. Transforming growth factor-β protein and mRNA in glomeruli in normal and diseased human kidneys.Lab Invest. 1993;