Overexpression of connective tissue growth factor in podocytes worsens diabetic nephropathy in mice
2007; Elsevier BV; Volume: 73; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5002722
ISSN1523-1755
AutoresHideki Yokoi, Masashi Mukoyama, Kiyoshi Mori, Masato Kasahara, Takayoshi Suganami, Kenji Sawai, Takeshi Yoshioka, Yoko Saito, Yoshihiro Ogawa, Takashige Kuwabara, Akira Sugawara, Kazuwa Nakao,
Tópico(s)Lymphatic System and Diseases
ResumoConnective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes. Connective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes. Diabetic nephropathy is a major complication in diabetes and the leading cause of end-stage renal disease worldwide.1.Ritz E. Orth S.R. Nephropathy in patients with type 2 diabetes mellitus.N Engl J Med. 1999; 341: 1127-1133Crossref PubMed Scopus (657) Google Scholar Diabetic nephropathy is characterized by microalbuminuria, renal and glomerular hypertrophy, mesangial expansion with increased thickness of the glomerular basement membrane (GBM), arteriolar hyalinosis, and global glomerular sclerosis, which ultimately cause the progression of proteinuria and renal failure.2.Mauer S.M. Steffes M.W. Ellis E.N. et al.Structural-functional relationships in diabetic nephropathy.J Clin Invest. 1984; 74: 1143-1155Crossref PubMed Scopus (1062) Google Scholar Accumulation of the extracellular matrix (ECM) components in the mesangium, GBM, and tubulointerstitium plays an important role in its pathology.3.Ziyadeh F.N. The extracellular matrix in diabetic nephropathy.Am J Kidney Dis. 1993; 22: 736-744Abstract Full Text PDF PubMed Scopus (231) Google Scholar,4.Park I.S. Kiyomoto H. Abboud S.L. et al.Expression of transforming growth factor-β and type IV collagen in early streptozotocin-induced diabetes.Diabetes. 1997; 46: 473-480Crossref PubMed Google Scholar Evidence has shown that such renal lesions are driven, in part, by transforming growth factor-β (TGF-β) in humans and experimental models.4.Park I.S. Kiyomoto H. Abboud S.L. et al.Expression of transforming growth factor-β and type IV collagen in early streptozotocin-induced diabetes.Diabetes. 1997; 46: 473-480Crossref PubMed Google Scholar, 5.Yamamoto T. Nakamura T. Noble N.A. et al.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, 6.Ziyadeh F.N. Hoffman B.B. Han D.C. et al.Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-β antibody in db/db diabetic mice.Proc Natl Acad Sci USA. 2000; 97: 8015-8020Crossref PubMed Scopus (805) Google Scholar Recently, functional and structural abnormalities in glomerular podocytes have become highlighted as one of the earliest events in the development of diabetic glomerular injury.7.Wolf G. Chen S. Ziyadeh F.N. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy.Diabetes. 2005; 54: 1626-1634Crossref PubMed Scopus (506) Google Scholar Podocyte loss and injury are found at very early stages in patients with diabetic nephropathy, being associated with the acceleration of glomerular structural abnormalities.8.Pagtalunan M.E. Miller P.L. Jumping-Eagle S. et al.Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest. 1997; 99: 342-348Crossref PubMed Scopus (901) Google Scholar Causes and consequences of podocyte injury during early diabetic nephropathy, however, remain poorly understood. Connective tissue growth factor (CTGF, also known as CCN2) belongs to a family of cysteine-rich growth factors, the CCN family, that consists of cysteine-rich protein 61 (Cyr61/CCN1), CTGF/CCN2, nephroblastoma overexpressed (Nov/CCN3), and Wnt-induced secreted proteins (WISP)-1, 2, and 3 (CCN4, 5, and 6, respectively).9.Bradham D.M. Igarashi A. Potter R.L. et al.Connective tissue growth factor: a cysteine-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, 10.Brigstock D.R. Goldschmeding R. Katsube I. et al.Proposal for a unified CCN nomenclature.Mol Pathol. 2003; 56: 127-128Crossref PubMed Scopus (205) Google Scholar, 11.Perbal B. CCN proteins: multifunctional signalling regulators.Lancet. 2004; 363: 62-64Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar Accumulating evidence has demonstrated that CTGF is crucially involved in the fibrogenic properties of TGF-β.12.Gupta S. Clarkson M.R. Duggan J. et al.Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 13.Yokoi H. Mukoyama M. Sugawara A. et al.Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.Am J Physiol. 2002; 282: F933-F942Crossref PubMed Scopus (167) Google Scholar, 14.Yokoi H. Sugawara A. Mukoyama M. et al.Role of connective tissue growth factor in profibrotic action of transforming growth factor-β: a potential target for preventing renal fibrosis.Am J Kidney Dis. 2001; 38: S134-S138Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar CTGF gene expression is strongly induced by TGF-β, and recombinant CTGF potently stimulates fibroblast proliferation and ECM protein synthesis.12.Gupta S. Clarkson M.R. Duggan J. et al.Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 13.Yokoi H. Mukoyama M. Sugawara A. et al.Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.Am J Physiol. 2002; 282: F933-F942Crossref PubMed Scopus (167) Google Scholar, 14.Yokoi H. Sugawara A. Mukoyama M. et al.Role of connective tissue growth factor in profibrotic action of transforming growth factor-β: a potential target for preventing renal fibrosis.Am J Kidney Dis. 2001; 38: S134-S138Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15.Holmes A. Abraham J.D. Sa S. et al.CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling.J Biol Chem. 2001; 276: 10594-10601Crossref PubMed Scopus (375) Google Scholar, 16.Frazier K. Williams S. Kothapalli D. et al.Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor.J Invest Dermatol. 1996; 107: 404-411Abstract Full Text PDF PubMed Scopus (673) Google Scholar TGF-β-induced collagen synthesis in vitro is shown to be CTGF dependent, which is shown by a neutralizing antibody or the antisense gene targeting CTGF.17.Duncan M.R. Frazier K.S. Abramson S. et al.Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: down-regulation by cAMP.FASEB J. 1999; 13: 1774-1786Crossref PubMed Scopus (578) Google Scholar Furthermore, we have demonstrated in vivo that knockdown of CTGF gene expression with antisense gene transfer into rat kidney ameliorates tubulointerstitial fibrosis in obstructive nephropathy.18.Yokoi H. Mukoyama M. Nagae T. et al.Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis.J Am Soc Nephrol. 2004; 15: 1430-1440Crossref PubMed Scopus (224) Google Scholar CTGF has been proposed to be a crucial mediator for the development of diabetic glomerulosclerosis.19.Riser B.L. Denichilo M. Cortes P. et al.Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar, 20.Roestenberg P. van Nieuwenhoven F.A. Joles J.A. et al.Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice.Am J Physiol. 2006; 290: F1344-F1354Crossref PubMed Scopus (73) Google Scholar, 21.Makino H. Mukoyama M. Sugawara A. et al.Roles of connective tissue growth factor and prostanoids in early streptozotocin-induced diabetic rat kidney: the effect of aspirin treatment.Clin Exp Nephrol. 2003; 7: 33-40Crossref PubMed Scopus (33) Google Scholar, 22.Umezono T. Suzuki D. Toyoda M. et al.Localization of connective tissue growth factor mRNA in human diabetic nephropathy by in situ hybridization.Clin Exp Nephrol. 2002; 6: 13-20Crossref Scopus (8) Google Scholar, 23.Wahab N.A. Yevdokimova N. Weston B.S. et al.Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy.Biochem J. 2001; 359: 77-87Crossref PubMed Scopus (146) Google Scholar, 24.Ito Y. Aten J. Bende R.J. et al.Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar CTGF mRNA is mainly expressed at podocytes and detected in some parietal epithelial cells of glomeruli under normal conditions in humans24.Ito Y. Aten J. Bende R.J. et al.Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar and rats.13.Yokoi H. Mukoyama M. Sugawara A. et al.Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.Am J Physiol. 2002; 282: F933-F942Crossref PubMed Scopus (167) Google Scholar Under the diabetic milieu, CTGF expression is markedly upregulated in podocytes, mesangial cells, and tubulointerstitial cells, being associated with fibrotic lesions and epithelial-to-mesenchymal transformation.19.Riser B.L. Denichilo M. Cortes P. et al.Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar, 20.Roestenberg P. van Nieuwenhoven F.A. Joles J.A. et al.Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice.Am J Physiol. 2006; 290: F1344-F1354Crossref PubMed Scopus (73) Google Scholar, 21.Makino H. Mukoyama M. Sugawara A. et al.Roles of connective tissue growth factor and prostanoids in early streptozotocin-induced diabetic rat kidney: the effect of aspirin treatment.Clin Exp Nephrol. 2003; 7: 33-40Crossref PubMed Scopus (33) Google Scholar, 22.Umezono T. Suzuki D. Toyoda M. et al.Localization of connective tissue growth factor mRNA in human diabetic nephropathy by in situ hybridization.Clin Exp Nephrol. 2002; 6: 13-20Crossref Scopus (8) Google Scholar, 23.Wahab N.A. Yevdokimova N. Weston B.S. et al.Role of connective tissue growth factor in the pathogenesis of diabetic nephropathy.Biochem J. 2001; 359: 77-87Crossref PubMed Scopus (146) Google Scholar, 24.Ito Y. Aten J. Bende R.J. et al.Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar CTGF may also enhance ECM accumulation through inhibition of matrix degradation.25.McLennan S.V. Wang X.Y. Moreno V. et al.Connective tissue growth factor mediates high glucose effects on matrix degradation through tissue inhibitor of matrix metalloproteinase type 1: implications for diabetic nephropathy.Endocrinology. 2004; 145: 5646-5655Crossref PubMed Scopus (100) Google Scholar Although these observations are consistent with CTGF as a key mediator of the progression of diabetic nephropathy, the precise role of CTGF in diabetic glomeruli, especially in podocytes, still remains elusive. To explore the roles of CTGF in podocytes and in diabetic nephropathy, we generated a transgenic mouse model harboring the podocyte-specific expression of the CTGF gene under the control of the nephrin promoter. We demonstrated acceleration of diabetic nephropathy in CTGF-overexpressing mice, indicating that dysregulated expression of CTGF in podocytes may play a role in mesangial expansion and podocyte loss in diabetic nephropathy. We constructed a transgene using a fragment of the human nephrin gene (Nphs1),26.Wong M.A. Cui S. Quaggin S.E. Identification and characterization of a glomerular-specific promoter from the human nephrin gene.Am J Physiol. 2000; 279: F1027-F1032PubMed Google Scholar rabbit β-globin intron, mouse CTGF cDNA, and polyA signal (Figure 1a). Transgenic founder lines carrying the human Nphs1 promoter-mouse CTGF transgene were identified by Southern blot analysis. We obtained five founder CTGF-transgenic (CTGF-Tg) mice, and semiquantitative analysis showed 10 (line 30), 10 (line 68), 15 (line 56), 20 (line 52), and 5 (line 12) transgene copies in these mice (Figure 1b). Northern blot analyses for CTGF revealed that the transgene was expressed abundantly in the isolated glomeruli of CTGF-Tg mice, but not in other tissues, including aorta, brain, heart, lung, liver, spleen, stomach, and pancreas (Figure 1c). The size of transgene-derived CTGF mRNA (1.7 kb) was smaller than the endogenous one, because we used only CTGF coding region (Figure 1c). The glomerular CTGF protein level in CTGF-Tg mice (line 12) was five times higher than that in wild-type mice (Figure 1d). All CTGF-Tg mice were fertile, grew normally, and showed normal gross appearance including kidney size and renal histology, as represented in line 12 (Figure 1f). Immunofluorescent analysis revealed that CTGF expression in podocytes was upregulated compared with their non-transgenic (non-Tg) littermates (Figure 1g and h). Blood pressure, blood glucose, serum creatinine, urea nitrogen, and urinary albumin excretion in CTGF-Tg mice line 12 were not different from those in control non-Tg littermates (Table 1). Another CTGF-Tg line (line 52) showed essentially similar results (data not shown).Table 1Baseline characteristics of non-Tg and CTGF-Tg miceNon-Tg miceCTGF-Tg mice line 12Body weight (g)20.8±1.620.0±0.4Systolic blood pressure (mm Hg)108.5±3.4108.0±2.5Blood glucose (mg per 100 ml)162±11151±11Serum creatinine (mg per 100 ml)0.12±0.010.13±0.01Serum urea nitrogen (mg per 100 ml)30.7±2.630.7±3.5Urine volume (ml per day)1.0±0.21.3±0.2Urinary albumin excretion (μg per mg Cr)41.3±4.242.8±5.4CTGF, connective tissue growth factor; Tg, transgenic.Values are expressed as the mean±s.e. for non-Tg mice (n=13) and CTGF-Tg mice line 12 (n=9). Open table in a new tab CTGF, connective tissue growth factor; Tg, transgenic. Values are expressed as the mean±s.e. for non-Tg mice (n=13) and CTGF-Tg mice line 12 (n=9). Next, to examine the role of CTGF expressed at podocytes in diabetic nephropathy, we induced diabetes in these mice by intraperitoneal injection of streptozotocin. We used CTGF-Tg mice line 12 and line 52, both with high transgene expression, in this experiment. Blood glucose, HbA1c, and serum creatinine levels as well as body weights in diabetic CTGF-Tg mice were not different from those in diabetic non-Tg mice at 12 weeks after disease induction (Table 2). Diabetic CTGF-Tg mice exhibited a tendency of renal hypertrophy compared with diabetic non-Tg mice, as shown by the increased kidney weight to body weight ratio. Systolic blood pressure and urine volume were not significantly different among diabetic groups. All diabetic groups showed increased albuminuria by 4 weeks after induction. Interestingly, diabetic CTGF-Tg mice revealed more pronounced elevation of urinary albumin excretion than diabetic non-Tg mice at 4 weeks, and became more aggravated during the experimental period (Figure 2). At 12 weeks, diabetic non-Tg mice exhibited 3.6-fold higher albumin excretion than non-diabetic control, and both lines of diabetic CTGF-Tg mice revealed significantly enhanced albuminuria by 2.8-fold compared with diabetic non-Tg mice (Figure 2). These results indicate that overexpression of CTGF in podocytes enhances proteinuria in diabetic nephropathy.Table 2Characteristics of diabetic mice at 12 weeks after induction of diabetesControlDiabetesNon-TgTg line 12Tg line 52Non-TgTg line 12Tg line 52Blood glucose (mg per 100 ml)144±14123±20106±16732±134*P<0.01 vs control non-Tg mice.718±48*P<0.01 vs control non-Tg mice.804±156*P<0.01 vs control non-Tg mice.HbA1c (%)3.3±0.23.1±0.22.9±0.211.4±0.7*P<0.01 vs control non-Tg mice.10.0±0.5*P<0.01 vs control non-Tg mice.9.3±1.8*P<0.01 vs control non-Tg mice.Serum creatinine (mg per 100 ml)0.16±0.020.14±0.010.17±0.020.21±0.03#P<0.05 vs control non-Tg mice.0.25±0.03#P<0.05 vs control non-Tg mice.0.24±0.06#P<0.05 vs control non-Tg mice.Serum urea nitrogen (mg per 100 ml)25.7±1.824.3±2.329.6±3.049.7±2.5*P<0.01 vs control non-Tg mice.44.0±3.6*P<0.01 vs control non-Tg mice.50.6±±10.4*P<0.01 vs control non-Tg mice.Body weight (g)24.6±1.825.7±0.623.1±1.023.1±0.522.1±2.222.2±0.4Kidney weight (g)0.14±0.020.13±0.010.12±0.020.22±0.02*P<0.01 vs control non-Tg mice.0.22±0.01*P<0.01 vs control non-Tg mice.0.24±0.05*P<0.01 vs control non-Tg mice.Kidney weight/body weight ratio (g per 100 g BW)0.56±0.020.52±0.020.47±0.020.96±0.04*P<0.01 vs control non-Tg mice.0.99±0.09*P<0.01 vs control non-Tg mice.1.04±0.04*P<0.01 vs control non-Tg mice.Systolic blood pressure (mm Hg)105.4±1.1107.2±0.3110.3±0.3108.2±0.3108.6±1.0110.8±1.6Urine volume (ml per day)1.1±0.31.2±0.40.5±0.532.1±0.9*P<0.01 vs control non-Tg mice.26.6±7.4*P<0.01 vs control non-Tg mice.25.8±5.3*P<0.01 vs control non-Tg mice.BW, body weight; Hb, hemoglobin; Tg, transgenic.Values are expressed as the mean±s.e. for control non-Tg mice (n=4), control CTGF-Tg mice line 12 (n=5), control CTGF-Tg mice line 52 (n=5), diabetic non-Tg mice (n=10), diabetic CTGF-Tg mice line 12 (n=8), and diabetic CTGF-Tg mice line 52 (n=7).* P<0.01 vs control non-Tg mice.# P<0.05 vs control non-Tg mice. Open table in a new tab BW, body weight; Hb, hemoglobin; Tg, transgenic. Values are expressed as the mean±s.e. for control non-Tg mice (n=4), control CTGF-Tg mice line 12 (n=5), control CTGF-Tg mice line 52 (n=5), diabetic non-Tg mice (n=10), diabetic CTGF-Tg mice line 12 (n=8), and diabetic CTGF-Tg mice line 52 (n=7). Microscopic examination showed that diabetic mice at 12 weeks after induction of diabetes exhibited marked mesangial expansion with glomerular hypertrophy (Figure 3c and d). Morphometric analysis revealed a significant increase in the mesangial area of diabetic CTGF-Tg mice line 12 compared with diabetic non-Tg mice (Figure 3f). In electron microscopic analysis, podocytes of both lines of diabetic CTGF-Tg mice revealed diffuse vacuolation (Figure 4b and c), which was rarely observed in diabetic non-Tg mice (Figure 4a) or non-diabetic CTGF-Tg mice. The thickness of the GBM was similar among diabetic groups (Figure 4d–f). Thus, CTGF overexpressed at podocytes causes enhanced mesangial matrix expansion and podocyte structural changes under diabetic conditions. Since we found enhanced ECM accumulation in diabetic CTGF-Tg mice compared with diabetic non-Tg mice, we investigated glomerular expression of total CTGF, endogenous CTGF, and TGF-β1, which are key inducers of ECM, as well as fibronectin, α1(IV) collagen (COL4A1), and α3(IV) collagen (COL4A3), which are representative ECM components. Total CTGF indicates the sum of transgene-derived and endogenous CTGF. Total CTGF expression in glomeruli from non-diabetic CTGF-Tg mice was upregulated by 2.5-fold compared with control non-Tg mice (Figure 5a). In diabetic CTGF-Tg mice, total CTGF mRNA expression was significantly upregulated by 4.0-fold compared with diabetic non-Tg mice (Figure 5a). Endogenous CTGF expression in diabetic CTGF-Tg mice was 3.0 times higher than that in diabetic non-Tg mice, indicating that CTGF upregulation in diabetic CTGF-Tg mice was due to the increase of endogenous CTGF. Fibronectin mRNA expression in glomeruli was increased in diabetic mice, but the levels were not significantly different between diabetic CTGF-Tg and diabetic non-Tg mice (Figure 5a). TGF-β1 and COL4A1 mRNA expressions in glomeruli also increased or tended to increase in diabetic conditions, but there was no significant difference among diabetic groups. There was no significant alteration in COL4A3 mRNA expression between CTGF-Tg and non-Tg mice in diabetic or non-diabetic conditions (Figure 5a). Next, we investigated glomerular expression of ECM degradation enzymes. Matrix metalloproteinase (MMP)-2 mRNA expression in glomeruli was significantly increased in diabetic condition (Figure 5a). In diabetic CTGF-Tg mice, MMP-2 mRNA expression was reduced compared with diabetic non-Tg mice. Expressions of tissue inhibitor of metalloproteinase (TIMP)-1 and -2, inhibitors of MMPs, were also upregulated in diabetic conditions, but there was no significant difference among diabetic groups. We next examined glomerular MMP-2 activity in diabetic CTGF-Tg mice, using gelatin zymography. The gelatinase activity of pro- and active- MMP-2 was significantly elevated in diabetic non-Tg mice (Figure 5b). In diabetic CTGF-Tg mice, the gelatinase activity was significantly decreased compared with diabetic non-Tg mice (Figure 5b). These findings suggest that CTGF causes ECM accumulation in mesangium by suppression of degradation rather than by enhancement of ECM expression itself. The number of podocytes was evaluated by immunohistochemical study for the Wilms' tumor gene (WT1). The number of WT1-positive cells was significantly (by 29%) low in a glomerular cross-section of diabetic CTGF-Tg mice line 12 (Figure 6d) than in diabetic non-Tg mice (Figure 6c). CTGF-Tg line 52 showed virtually similar results (not shown). Immunofluorescent study revealed that in diabetic non-Tg mice CTGF expression was upregulated in podocytes and mesangial cells (Figure 7c). Diabetic CTGF-Tg mice showed further increased CTGF protein expression in podocytes and mesangial cells compared with diabetic non-Tg mice (Figure 7c and d). Immunostaining of podocin became downregulated in diabetic CTGF-Tg mice as compared with diabetic non-Tg mice (Figure 7g and h). Double immunostaining for CTGF and podocin showed that CTGF protein in non-diabetic CTGF-Tg mice was located mainly in podocytes (yellow) (Figure 7j) and that CTGF was increased in the mesangial area in diabetic CTGF-Tg mice (green) (Figure 7l). These findings suggest that CTGF overexpression at podocytes resulted in loss of and functional impairment of podocytes and augmented induction of CTGF mainly at the mesangial area in streptozotocin-induced diabetic nephropathy. In this study, to clarify the role of CTGF in diabetic nephropathy, we have generated podocyte-specific CTGF-Tg mice under the control of the human nephrin promoter.26.Wong M.A. Cui S. Quaggin S.E. Identification and characterization of a glomerular-specific promoter from the human nephrin gene.Am J Physiol. 2000; 279: F1027-F1032PubMed Google Scholar Glomerular expressions of CTGF mRNA and protein of CTGF-Tg mice were increased 2.5 and 5 times, respectively, compared with non-Tg mice. Immunofluorescent analysis revealed that CTGF expression was upregulated at podocytes in CTGF-Tg mice. Both lines of CTGF-Tg mice exhibited normal blood pressure, serum urea nitrogen, and urinary albumin excretion levels as well as normal renal histology, suggesting that increased CTGF expression at podocytes alone is not sufficient to evoke renal abnormality. After induction of diabetes, CTGF-Tg mice exhibited enhanced CTGF mRNA expression by 4.0-fold as compared with diabetic non-Tg mice. In contrast, non-diabetic CTGF-Tg mice showed only 2.5-fold elevated CTGF mRNA compared with control, non-Tg mice. The origin of CTGF upregulation in diabetic CTGF-Tg mice was mainly endogenous CTGF in glomeruli determined by real-time reverse transcription-PCR (RT-PCR). Immunofluorescent staining for CTGF also revealed that diabetic CTGF-Tg mice exhibited enhanced CTGF expression mainly in the mesangial area. Such upregulation of CTGF expression associated with diabetes suggests a ‘vicious cycle’ whereby transgene-derived CTGF induces endogenous CTGF gene expression. It has already been shown that CTGF administration stimulates CTGF expression in cultured mesangial cells,19.Riser B.L. Denichilo M. Cortes P. et al.Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar supporting this hypothesis. Diabetic CTGF-Tg mice showed significantly increased mesangial expansion. Several lines of evidence indicate that humoral factors produced by podocytes have effects on mesangial and endothelial cells. Quaggin et al.27.Eremina V. Sood M. Haigh J. et al.Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases.J Clin Invest. 2003; 111: 707-716Crossref PubMed Scopus (1081) Google Scholar have reported that heterozygous disruption of vascular endothelial growth factor allele in podocytes results in endothelial cell damages and mesangial expansion. Another report showed that podocyte-specific injury in adult mice leads to mesangial expansion and glomerulosclerosis.28.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (213) Google Scholar Taken together, it is well conceivable that CTGF produced by podocytes should exert an effect on mesangial cells to stimulate CTGF expression and ECM accumulation in diabetic conditions. CTGF has been shown to inhibit ECM degradation in mesangial cells and fibroblasts.12.Gupta S. Clarkson M.R. Duggan J. et al.Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 19.Riser B.L. Denichilo M. Cortes P. et al.Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.J Am Soc Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar, 25.McLennan S.V. Wang X.Y. Moreno V. et al.Connective tissue growth factor mediates high glucose effects on matrix degradation through tissue inhibitor of matrix metalloproteinase type 1: implications for diabetic nephropathy.Endocrinology. 2004; 145: 5646-5655Crossref PubMed Scopus (100) Google Scholar This study showed that glomerular COL4A3 or fibronectin mRNA expression was not changed among diabetic groups. Of note, glomerular MMP-2 mRNA expression in diabetic CTGF-Tg mice was significantly decreased compared with diabetic non-Tg mice. Glomerular MMP-2 activity in diabetic CTGF-Tg mice was also reduced compared with diabetic non-Tg mice. MMP-2 mRNA expression has been shown to be located in mesangial cells, as well as in visceral epithelial, glomerular epithelial, and endothelial cells in the kidney from patients of type I diabetes with diabetic nephropathy.29.Suzuki D. Yagame M. Kim Y. et al.Renal in situ hybridization studies of extracellular matrix related molecules in type I diabetes mellitus.Nephron. 2002; 92: 564-572Crossref PubMed Scopus (19) Google Scholar Our results, that the reduced activity of MMP-2 in glomeruli is associated with mesangial expansion in diabetic CTGF-Tg mice, may be consistent with this finding. Although the mechanism for the enhanced mesangial expansion in CTGF-Tg mice should await further clarification, our study suggests that the enhanced mesangial expansion in diabetic CTGF-Tg mice may be due to inhibition of ECM degradation through decreased expression and activity of MMP-2. Increased CTGF expression has already been demonstrated in various cell types of human and rodent diabetic kidneys,19
Referência(s)