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CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy

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CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy BASIC RESEARCH www.jasn.org CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy Tri Q. Nguyen,* Peggy Roestenberg,* Frans A. van Nieuwenhoven,* Niels Bovenschen,* ʈ Zeke Li,† Leon Xu,† Noelynn Oliver,† Jan Aten,‡ Jaap A. Joles,§ Cecilia Vial, ʈ Enrique Brandan, Karen M. Lyons,¶ and Roel Goldschmeding* Departments of *Pathology and §Nephrology, University Medical Center Utrecht, Utrecht, and ‡Department of Pathology, Academic Medical Center, Amsterdam, Netherlands; †FibroGen, Inc., South San Francisco, and ¶Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California; and ʈ Department of Cell and Molecular Biology, Catholic University of Chile, Santiago, Chile ABSTRACT In diabetic nephropathy, connective tissue growth factor (CTGF) is upregulated and bone morpho- genetic protein 7 (BMP-7) is downregulated. CTGF is known to inhibit BMP-4, but similar cross-talk between BMP-7 and CTGF has not been studied. In this study, it was hypothesized that CTGF acts as an inhibitor of BMP-7 signaling activity in diabetic nephropathy. Compared with diabetic wild-type ϩ ϩ ϩ Ϫ CTGF / mice, diabetic CTGF / mice had approximately 50% lower CTGF mRNA and protein, less severe albuminuria, no thickening of the glomerular basement membrane, and preserved matrix metalloproteinase (MMP) activity. Although the amount of BMP-7 mRNA was similar in the kidneys ϩ ϩ ϩ Ϫ of diabetic CTGF / and CTGF / mice, phosphorylation of the BMP signal transduction protein ϩ ϩ Smad1/5 and expression of the BMP target gene Id1 were lower in diabetic CTGF / mice. Moreover, renal Id1 mRNA expression correlated with albuminuria (R ϭϪ0.86) and MMP activity (R ϭ 0.76). In normoglycemic mice, intraperitoneal injection of CTGF led to a decrease of pSmad1/5 in the renal cortex. In cultured renal glomerular and tubulointerstitial cells, CTGF diminished BMP-7 signaling activity, evidenced by lower levels of pSmad1/5, Id1 mRNA, and BMP-responsive element– luciferase activity. Co-immunoprecipitation, solid-phase binding assay, and surface plasmon reso- nance analysis showed that CTGF binds BMP-7 with high affinity (Kd approximately 14 nM). In conclusion, upregulation of CTGF inhibits BMP-7 signal transduction in the diabetic kidney and contributes to altered gene transcription, reduced MMP activity, glomerular basement membrane thickening, and albuminuria, all of which are hallmarks of diabetic nephropathy. J Am Soc Nephrol 19: 2098–2107, 2008. doi: 10.1681/ASN.2007111261 Connective tissue growth factor (CTGF) is con- critical determinant of structural and functional sidered an important factor in the development damage in diabetic nephropathy. of diabetic nephropathy. The renal expression of CTGF mRNA and protein is upregulated in hu- Received November 29, 2007. Accepted May 21, 2008. 1–3 man and experimental diabetic nephropathy. Published online ahead of print. Publication date available at In patients with diabetic nephropathy, both www.jasn.org. plasma CTGF levels and urinary CTGF excretion P.R.’s current affilation is Department of Biochemistry, Radboud are increased and correlate with clinical markers University Nijmegen Medical Center, Nijmegan, Netherlands; 4–7 F.A.v.N.’s current affiliation is Department of Physiology, CARIM, of renal disease. Recently, it was demonstrated Maastricht University, Maastricht, Netherlands. that specific downregulation of CTGF by anti- Correspondence: Dr. Roel Goldschmeding, University Medical Cen- sense oligonucleotide treatment attenuated ter Utrecht, Department of Pathology, H04.312, Heidelberglaan albuminuria and mesangial matrix expansion in 100, 3584 CX, Utrecht, Netherlands. Phone: ϩ31-88-755-6559; Fax: experimental type 1 and type 2 diabetic nephrop- ϩ31-30-254-4990; E-mail: [email protected] athy.8 These observations suggest that CTGF is a Copyright ᮊ 2008 by the American Society of Nephrology 2098 ISSN : 1046-6673/1911-2098 J Am Soc Nephrol 19: 2098–2107, 2008 www.jasn.org BASIC RESEARCH Figure 1. Decrease of BMP-7 signal transduction and target gene expression in diabetic nephropathy. Diabetes was induced in C57BL6/J mice by injection of streptozotocin. Renal cortex was harvested 9 wk after injection. Gene expression of BMP-7 and Id1 was evaluated by quantitative PCR, and pSmad1/5 protein level was analyzed by Western blotting. (A through C) In diabetic mice, expression of BMP-7 was decreased 2.6-fold. This was accompanied by reduced levels of pSmad1/5 protein and Id1 mRNA. Data are means Ϯ SD. *P Ͻ 0.05. Several mechanisms have been proposed for a patho- urine, and plasma.3 In diabetic mice, renal cortical expression genic role of CTGF in diabetic nephropathy. Studies with of BMP-7 mRNA was decreased 2.6-fold. This was accompa- renal cells demonstrated that CTGF is involved in diabetes- nied by reduced levels of pSmad1/5 protein, and also BMP-7 associated changes such as extracellular matrix synthesis, downstream target Id1 was decreased (Figure 1). cell migration, cellular hypertrophy, and epithelial-to-mes- enchymal transition.2,9–11 CTGF might exert these effects in CTGF؉/؊ Mice Have Lower Levels of CTGF mRNA and diabetes by modulating the activity of other growth factors. Protein For example, CTGF is known to enhance profibrotic activity After 17 wk of diabetes, CTGF mRNA and protein expression of TGF-␤1 and IGF-1, which involves physical interaction in renal cortex and CTGF levels in plasma and urine were in- ϩ ϩ of CTGF with these growth factors.12,13 In contrast, binding creased in diabetic CTGF / mice as compared with nondia- to CTGF potently antagonizes the signaling activity of bone betic CTGFϩ/ϩ mice. In diabetic CTGFϩ/Ϫ mice, expression of morphogenetic protein 4 (BMP-4) in osteogenesis assays CTGF mRNA and protein in renal cortex and CTGF levels in and in embryonic patterning.12 In the kidney, the impor- plasma were not significantly different from nondiabetic mice. tance of several BMP, including BMP-4, has been demon- Although urinary CTGF excretion in diabetic CTGFϩ/Ϫ mice ϩ ϩ strated mainly in developmental studies14–16; however, thus seemed to be lower than in diabetic CTGF / mice, this dif- far, only BMP-7 has also been studied for its contribution as ference did not reach statistical significance. In nondiabetic an antifibrotic and proregenerative factor in response to CTGFϩ/Ϫ mice, CTGF expression seemed slightly lower than ϩ ϩ injury of the adult kidney.17,18 Renal expression of BMP-7 is in nondiabetic CTGF / , but this difference was NS (Figure 2, progressively decreased during the course of human and A through D). experimental diabetic nephropathy and in podocytes cul- CTGF immunohistochemistry showed more prominent ϩ ϩ tured in high glucose medium,19–21 whereas restoration of glomerular CTGF staining in diabetic CTGF / mice than in BMP-7 availability has resulted in prevention or even rever- diabetic CTGFϩ/Ϫ mice and in nondiabetic mice. No promi- sal of functional and structural changes of diabetic ne- nent CTGF staining was observed in tubuli (Figure 2E). phropathy.22–24 Although it has been hypothesized that CTGF might inhibit BMP-7,25,26 this has not been addressed Diabetic Nephropathy Is Attenuated in CTGF؉/؊ Mice experimentally; therefore, we set out to investigate the im- Albuminuria was increased in diabetic CTGFϩ/ϩ mice as com- pact of CTGF on BMP-7 signal transduction and target gene pared with nondiabetic mice. In diabetic CTGFϩ/Ϫ mice, al- expression in experimental diabetic nephropathy and in buminuria was significantly less pronounced than in diabetic cultured renal cells. CTGFϩ/ϩ mice (111 Ϯ 39 versus 176 Ϯ 26 mg/g creatinine; P ϭ 0.024; Figure 3A). Examination of ultrathin sections by electron microscopy RESULTS showed that the thickness of the glomerular basement mem- brane (GBM) in diabetic CTGFϩ/ϩ mice was increased com- ϩ ϩ BMP-7 Deficiency in Diabetic Nephropathy Is pared with control CTGF / mice; however, no increase in ϩ Ϫ Accompanied by Decrease of pSmad1/5 and BMP- GBM thickness was observed in diabetic CTGF / mice as ϩ ϩ Target Gene Expression compared with diabetic CTGF / mice (154 Ϯ 3.0 versus Induction of diabetes in C57BL6/J mice by intraperitoneal in- 174 Ϯ 2.3 nm; P Ͻ 0.01; Figure 3B). jection of streptozotocin resulted in characteristic features of In situ zymography showed that gelatinase activity, repre- diabetic nephropathy, including persistent hyperglycemia; in- senting activity of matrix metalloproteinase 2 (MMP-2) and creased glycosylated hemoglobin levels; proteinuria; structural MMP-9, was localized mainly in glomeruli. Activity of gelatinase changes of the kidney; and increased CTGF levels in kidney, was decreased in diabetic CTGFϩ/ϩ mice but was preserved in J Am Soc Nephrol 19: 2098–2107, 2008 CTGF Inhibits BMP-7 Activity 2099 BASIC RESEARCH www.jasn.org whereas total Smad5 protein was not dif- ferent (Figure 4, A through C). Further- more, Id1 mRNA correlated with albu- minuria (R ϭϪ0.86, P ϭ 0.011) and with MMP activity in renal lysates (R ϭ 0.76, P ϭ 0.037; Figure 4, D and E). Injection of Recombinant CTGF Impairs Renal Cortical BMP-7 Activity in Nondiabetic Mice Intraperitoneal injection of recombinant CTGF in nondiabetic mice resulted in transient elevation of plasma CTGF lev- els (Figure 5A). Although renal cortical expression of BMP-7 and Id1 mRNA was not different at the various time points (data not shown), pSmad1/5 protein lev- els were significantly decreased 4 h after injection (P ϭ 0.0083; Figure 5B). In- jection with vehicle did not alter renal pSmad1/5 (Figure 5C). CTGF Inhibits BMP-7 Signal Transduction and Target Gene Expression in Renal Cells Treatment of rat mesangial cells and HK-2 cells with BMP-7 resulted in in- creased phosphorylation of Smad1/5 protein and Id1 mRNA expression. Ad- dition of CTGF partially inhibited phos- phorylation of Smad1/5 and reduced ex- pression of Id1 mRNA (Figure 6, A through D).
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