CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy

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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 morphogenetic 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 resonance 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

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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-mesenchymal 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 membrane (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

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whereas total Smad5 protein was not different (Figure 4, A through C). Further- more, Id1 mRNA correlated with albuminuria (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 levels (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 levels 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 increased phosphorylation of Smad1/5 protein and Id1 mRNA expression. Ad- dition of CTGF partially inhibited phosphorylation of Smad1/5 and reduced expression of Id1 mRNA (Figure 6, A through D). Also, in mouse podocytes, BMP-7 stimulation resulted in a signifi-

ϩ Ϫ cant, although less pronounced, increase Figure 2. Levels of CTGF mRNA and protein are decreased in CTGF / mice. Diabetes ϩ ϩ ϩ Ϫ of Id1 mRNA expression, which was re- was induced in CTGF / and CTGF / mice. (A through D) CTGF mRNA and protein in ϩ ϩ duced by addition of CTGF (Figure 6E). renal cortex and CTGF levels in plasma were two-fold increased in diabetic CTGF / ϩ Ϫ mice, as compared with diabetic CTGF / mice. Also urinary CTGF excretion tended to CTGF also inhibited activation of a ϩ Ϫ ϩ ϩ be lower in diabetic CTGF / mice than in diabetic CTGF / mice (P ϭ 0.29). (E) CTGF BMP-responsive element (BRE)-lucif- immunohistochemistry showed more prominent glomerular CTGF staining in diabetic erase construct, which specifically re- ϩ ϩ ϩ Ϫ CTGF / mice than in diabetic CTGF / mice and nondiabetic mice. Data are means Ϯ ports Smad1/5-mediated gene transcrip- ϩ ϩ ϩ ϩ SD. *P Ͻ 0.05 versus control CTGF / mice; **P Ͻ 0.05 versus diabetic CTGF / mice. tion.27 When TK173 cells were co- transfected with BRE-luciferase and diabetic CTGFϩ/Ϫ mice (Figure 3C). This was confirmed by pCAGGS-mCTGF, BMP-7–induced luciferase activity was sig- quantification of gelatinolytic activity in renal lysates, showing nificantly lower than in cells co-transfected with pCAGGS- a reduction by approximately 40% in diabetic CTGFϩ/ϩ mice lacZ control vector (Figure 6F). (P ϭ 0.034) but not in diabetic CTGFϩ/Ϫ mice (Figure 3D). CTGF Binds BMP-7 with High Affinity Preserved BMP Signaling Activity in Diabetic CTGF؉/؊ Co-immunoprecipitation experiments showed that captured Mice: Correlation of CTGF Level with Albuminuria and BMP-7 on anti–BMP-7 mAb-coated agarose beads was able to MMP Activity bind CTGF. In the absence of BMP-7, only a weak CTGF band Renal cortical expression of BMP-7 mRNA was similar in dia- was detected, which is due to known nonspecific binding of betic CTGFϩ/ϩ and CTGFϩ/Ϫ mice. Diabetic CTGFϩ/Ϫ mice CTGF to agarose beads. Incubation with captured HGF on had relatively preserved pSmad1/5 protein levels and Id1 anti-HGF mAb-coated beads and with BMP-7– and IgG1- mRNA expression as compared with diabetic CTGFϩ/ϩ mice, coated beads also did not result in significant pull-down of

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were involved; therefore, a heteroge- neous two-site binding model was re- quired to describe accurately the binding behavior. This resulted in Kd values de- scribing a high- and low-affinity component of 14 Ϯ 6 and 316 Ϯ 190 nM for the interaction between CTGF and BMP-7 ϭ Ϯ ϫ 5 ϭ [kon1 2.4 0.9 10 M/s, koff1 Ϯ ϫ Ϫ3 ϭ Ϯ ϫ 5 3.3 0.8 10 , kon2 1.3 0.5 10 , ϭ Ϯ ϫ Ϫ2 koff2 4.0 1.8 10 ].

DISCUSSION

The results of this study reveal how diabetes-induced increase of CTGF expression contributes to impairment of renal BMP signaling activity and that this is associated with severity of structural and functional hallmarks of diabetic nephropathy. To determine how CTGF expression level might relate to reduced BMP-7 signaling in diabetic nephropathy, we compared pSmad1/5 and Id1 levels in diabetic CTGFϩ/ϩ mice with those in diabetic CTGFϩ/Ϫ mice. The latter mice have lower CTGF expression in kidney, plasma, and urine as compared with diabetic CTGFϩ/ϩ mice, but they have equal TGF-␤1 expression (data not shown). Thus, diabetic CTGFϩ/Ϫ mice constitute a unique model to assess the impact of CTGF level on downstream ϩ Ϫ Figure 3. Diabetic nephropathy is attenuated in CTGF / mice. Diabetic nephropathy BMP-7 activity in diabetic nephropathy. ϩ ϩ ϩ Ϫ in CTGF / and CTGF / mice was assessed by albuminuria, GBM thickening, and MMP ϩ Ϫ BMP-7 mRNA expression in renal cortex activity. (A) In diabetic CTGF / mice, albuminuria was significantly less pronounced than ϩ/ϩ ϩ/ϩ ϩ/ϩ was similar in diabetic CTGF and in diabetic CTGF mice. (B) GBM thickening in diabetic CTGF mice, as determined ϩ/Ϫ ϩ Ϫ CTGF mice, indicating that CTGF by electron microscopy, was absent in diabetic CTGF / mice. (C and D) In situ zymog- level does not influence BMP-7 expres- raphy on renal sections (green, MMP activity; red, nuclear counterstain; C) and colori- metric detection in renal lysates (D) showed that MMP activity was decreased in diabetic sion; however, pSmad1/5 protein and ϩ ϩ ϩ Ϫ CTGF / mice. In contrast, MMP activity was preserved in diabetic CTGF / mice. Data Id1 mRNA levels were lower in diabetic ϩ ϩ ϩ/ϩ are means Ϯ SD. *P Ͻ 0.05 versus control CTGF / mice; **P Ͻ 0.05 versus diabetic CTGF mice, as compared with dia- ϩ ϩ ϩ/Ϫ CTGF / mice. betic CTGF mice. This indicates that CTGF might be an important determi- CTGF (Figure 7A). Similarly, solid-phase binding assay nant of the diabetes-induced reduction in signaling activity of showed direct physical interaction between BMP-7 and CTGF residual BMP-7. CTGF is known also to modulate BMP-4 and but not between HGF and CTGF or BSA and CTGF. Increased TGF-␤1 directly.12 In addition, the level of BMP signaling activity binding between was observed as the concentration of either in the kidney is subject to the influence of other members of the BMP-7 or CTGF was increased (Figure 7B). TGF-␤ superfamily, BMP receptors, and of BMP modulators The binding affinity of CTGF for BMP-7 was determined by such as gremlin, noggin, kielin/chordin-like protein, or uterine surface plasmon resonance analysis. CTGF displayed time-de- sensitization-associated gene 128; therefore, the relative contribu- pendent association with immobilized BMP-7 followed by dis- tion of BMP-7 inhibition by CTGF remains to be established. sociation, which was dose-dependent because higher response Albuminuria, thickening of the GBM, and decreased activ- was observed at higher CTGF concentrations (Figure 7C) and ity of MMP all are hallmarks of human and experimental dia- at higher BMP-7 density (data not shown). The data showed betic nephropathy.29–31 Recently, it was shown that renal complex binding behavior in which multiple components CTGF protein correlates with GBM thickness and prospective

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betic mice also resulted in decrease of pSmad1/5; however, this did not affect levels of Id1 mRNA. Thus, transient two- fold increase of plasma CTGF in a nondiabetic environment seemed not to be sufficient for inhibition of Id1 mRNA, which might require higher or more sus- tained elevation of CTGF or additional diabetes-induced changes. As for the nature of CTGF–BMP-7 interaction in the kidney, exogenous CTGF protein as well as CTGF transfection inhibited BMP-7 signaling activity in cultured mesangial cells, podocytes, proximal tubular epithelial cells, and renal interstitial fibroblasts. This was exempli- fied by inhibition of BMP-7–induced pS- Figure 4. CTGF inhibits BMP signaling activity in diabetic nephropathy. Renal cortex of ϩ ϩ ϩ Ϫ mad1/5, Id1, and BRE-luciferase activity. diabetic CTGF / and CTGF / mice was harvested 17 wk after induction of diabetes. ϩ ϩ (A) Renal cortical expression of BMP-7 mRNA was similar in diabetic CTGF / and The inhibitory effect of CTGF was ro- ϩ Ϫ ϩ Ϫ CTGF / mice. (B and C) In diabetic CTGF / mice, pSmad1/5 protein and Id1 mRNA were bust, in the sense that it was observed ϩ ϩ higher than in diabetic CTGF / mice, whereas total Smad5 was not different. (D and E) Id1 with exogenously added human and Xe- mRNA correlated with albuminuria and MMP activity. Data are means Ϯ SD. *P Ͻ 0.05. nopus CTGF, as well as with transfected mouse CTGF. albuminuria in a nonhuman primate model of diabetes.32 We Direct physical interaction between CTGF and BMP-7 was observed that these alterations and also decreased MMP activ- evidenced by co-immunoprecipitation and in a solid-phase ity were attenuated or absent in diabetic CTGFϩ/Ϫ mice. This binding assay. Furthermore, surface plasmon resonance anal- indicates that CTGF plays a pathogenic role in at least these ysis demonstrated that the interaction between CTGF and characteristic manifestations of diabetic nephropathy. Simi- BMP-7 was complex and consisted of a high- and a low-affinity larly, decrease of albuminuria in experimental models of type 1 component (Kd values of 14 and 316 nM, respectively). This and type 2 diabetic nephropathy has been observed in mice not only may result from inherent biologic properties of both treated with an anti-CTGF antibody and with a CTGF anti- CTGF and BMP-7 but also may be due to partial blocking of sense oligonucleotide, respectively.8,33 In the latter study, inhi- CTGF-interactive sites during the immobilization of BMP-7. bition of CTGF also resulted in reduction of serum creatinine The high binding affinity of CTGF and BMP-7 was comparable and inhibition of mesangial matrix expansion, which were not to that described for CTGF and BMP-4, which had a Kd value ϩ Ϫ observed in our diabetic CTGF / mouse model (data not of5nM.12 shown). A possible explanation for this discrepancy is that In conclusion, overexpression of CTGF inhibits BMP-7 sig- CTGF level derived from a single functional allele might suffice nal transduction in the diabetic kidney and contributes signif- to mediate diabetes-induced increase in serum creatinine and icantly to altered gene transcription, as well as to reduced matrix accumulation. Treatment with antisense oligonucleo- MMP activity, and to GBM thickening and albuminuria, tides might have lowered CTGF availability to levels below which all are hallmarks of diabetic nephropathy. those in our diabetic CTGFϩ/Ϫ mice, resulting in more com- plete protection; however, we cannot directly compare actual reduction of CTGF levels in the different studies, and BMP CONCISE METHODS signaling activity was not addressed after anti-CTGF antibody or CTGF-antisense oligonucleotide treatment. Animal Experiments The strong correlation of Id1 with both albuminuria and Signaling activity of BMP-7 was studied in diabetic mice.3 Briefly, MMP activity suggests that CTGF-dependent suppression of diabetes was induced in nine 12-wk-old female C57Bl/6J mice by a this target gene of BMP signaling might be involved directly in single intraperitoneal injection of 200 mg/kg streptozotocin (Sigma, the pathogenesis of diabetic nephropathy. Accordingly, dia- St. Louis, MO) in sodium citrate buffer. Six control animals were betic transgenic mice overexpressing BMP-7 in podocytes and administered an injection of vehicle only. Hyperglycemia was deter- proximal tubular epithelial cells had higher Id1:PAI mRNA mined 3 d after injection by measurement of blood glucose levels. ratio and preserved MMP activity and developed less albumin- Slow-release insulin pellets (Linshin; Scarborough, Ontario, Canada) uria, as compared with diabetic wild-type mice.23 were implanted to stabilize the condition of the diabetic animals. Mice As in overexpression of endogenous CTGF in diabetic were killed 9 wk after injection. Renal cortex was harvested by dissect- mice, injection of recombinant human CTGF into nondia- ing small caps of the upper and the lower poles. Before homogeniza-

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Figure 6. CTGF inhibits BMP-7 signal transduction and target gene expression in renal cells. Effects of CTGF on BMP-7 were studied in rat mesangial cells, mouse podocytes, proximal tubular Figure 5. Injection of CTGF impairs renal cortical BMP-7 activity epithelial cells (HK-2), and renal interstitial fibroblasts (TK173). (A in nondiabetic mice. Recombinant CTGF or vehicle only was and B) Treatment of rat mesangial cells with BMP-7 resulted in an injected intraperitoneally into nondiabetic BALBc mice. (A) CTGF increase of Smad1/5 phosphorylation and Id1 mRNA expression, levels in plasma were increased at 10 min after injection with both of which were inhibited by co-stimulation with CTGF. (C and rhCTGF. (B) Renal cortical pSmad1/5 protein levels were signifi- D) Treatment of HK-2 cells with BMP-7 resulted in an increase of cantly decreased 4 h after injection with rhCTGF. (C) Renal cortical pSmad1/5 protein and Id1 mRNA, both of which were inhibited pSmad1/5 levels were not different 4 h after vehicle injection. by co-stimulation with CTGF. (E) Treatment of podocytes with Ϯ Ͻ Data are means SD. *P 0.05. BMP-7 resulted in increase of Id1 mRNA, which was inhibited by co-stimulation with CTGF. (F) TK173 cells were transfected with tion, absence of medulla was checked in frozen sections of the cut the BRE-luciferase construct. Treatment with BMP-7 of cells co- transfected with control vector pCAGGS-lacZ resulted in increase surface. of BRE-luciferase activity, whereas co-transfection with pCAGGS- Effects of CTGF level on BMP-7 signaling activity were studied in ϩ/ϩ ϩ/Ϫ CTGF inhibited BMP-7–induced luciferase activity. Data are diabetic CTGF and CTGF mice. Outbred male BALBc/129Sv means Ϯ SD. *P Ͻ 0.05 versus only 50 ng/ml BMP-7; #P Ͻ 0.05 ϩ/Ϫ CTGF mice, in which exon 1 of one CTGF allele has been replaced versus p-lacZ with 10 ng/ml rhBMP-7; †P Ͻ 0.05 versus p-lacZ with by a neomycin resistance gene,34 were mated with female C57Bl/6J 50 ng/ml BMP-7. mice. From their first offspring, female CTGFϩ/Ϫ mice and female CTGFϩ/ϩ littermates were used for this study. Diabetes was induced were determined by sandwich ELISA using a goat–anti-mouse albu- in five 16-wk-old CTGFϩ/Ϫ mice and four CTGFϩ/ϩ mice by injec- min antibody (Bethyl Laboratories, Montgomery, TX). Urinary cretion of streptozotocin. Nine control mice were administered an injec- atinine excretion was determined by enzymatic assays (J2L Elitech, tion of vehicle only. After 9 wk, unilateral nephrectomy was per- Labarthe Inard, France). formed on all animals to aggravate the diabetic nephropathy model. Effects of CTGF on BMP-7 signaling activity in renal cortex of Mice were killed 17 wk after induction of diabetes. Albumin levels normoglycemic mice were studied in 24 BALBc mice, which were

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ervision-PO (Klinipath, Duiven, Nether- lands). Bound antibody was visualized with Nova RED (Vector Laboratories, Burlin- game, CA).

Electron Microscopy Tissue samples were fixed in Karnovsky solution. Upon embedding, samples were rinsed with 0.1 M Na-cacodylate buffer, followed by fixation with 1% osmiumtetroxide, and de- hydrated with acetone and embedded in Epon. Ultrathin sections of 95 nm were cut and mounted on copper one-hole specimen support grids. Sections were stained with ura- nyl acetate and lead citrate to provide contrast. Ultrathin sections were photographed using a transmission electron microscope (JEM-1200 EX; JEOL, Peabody, MA). GBM thickness was measured in five random glomeruli per mouse at 10 perpendicular cross- Figure 7. CTGF binds directly to BMP-7. Physical interaction between CTGF and BMP-7 sections of GBM per glomerulus at a magni- was demonstrated by co-immunoprecipitation, solid-phase binding assay, and surface fication of ϫ5000 and analyzed by computer plasmon resonance. (A) Anti–BMP-7 mAb–coated agarose beads were incubated with image analysis (ImageJ; National Institutes of rhBMP-7 and/or rxCTGF-flag. As controls, beads were coated with anti-HGF and incu- Health, www.rsb.info.nih.gov/ij/). bated with rhHGF or coated with IgG1 and incubated with rhBMP-7 and/or rxCTGF-flag. Bound proteins were separated by SDS-PAGE and probed with anti-flag. Recombinant In Situ Zymography CTGF was run as control. (B) Increasing concentrations of rhCTGF were added to Glomerular MMP activity was visualized by microtiter plates coated with rhBMP-7, rhHGF, or 1% BSA. Bound proteins were detected in situ zymography and confocal laser-scan- with an AP-conjugated antibody against rhCTGF. (C) Purified CTGF protein was run over ning microscopy. Frozen tissue sections were BMP-7 sensor chips. Association and dissociation were monitored by a change in the incubated with DQTM gelatin from pig skin resonance units. (Invitrogen, Carlsbad, CA), diluted 1:20 in 50 mM Tris-HCl buffer containing 10 mol/L

administered an intraperitoneal injection of rhCTGF (FibroGen, CaCl2, 0.05% Brij 35, and 5 mmol/L PMSF (pH 7.4). Slides were South San Francisco, CA) at a dosage of 20 ␮g/kg diluted in 50 mM incubated in a dark humidified chamber at 37°C for 19 h. An MMP Tris-HCl buffer containing 800 mM NaCl. Mice were killed 0, 5, 10, inhibitor, 1,10-phenanthroline monohydrate (2 ␮g/ml), was used to 15, 30, 60, 120, and 240 min after injection (three mice for each time verify that the obtained gelatinase activity specifically represented point). Six control mice were administered an injection of vehicle MMP activity. Nuclei were counterstained in red with propidium only and killed 0 and 240 min after injection. iodide. All mice were housed in standard cages in a room with constant temperature and a 12-h light-dark cycle. Mice were fed a standard Gelatinase Activity in Tissue Lysates ␮ pellet laboratory diet and had free access to water. The experiments From frozen kidneys, 10 sections of 20 m were cut and dissolved in were performed with the approval of the Experimental Animal Ethics lysis buffer (50 mM Tris, 150 mM NaCl, and 1% Triton X-100 [pH Committee of the University of Utrecht. 7.4]). Gelatinase activity was measured with an EnzChek Gelatinase assay kit (Invitrogen). Collagenase was used as standard, and specific- ity of the gelatinase activity was verified using 1,10-phenanthroline CTGF ELISA monohydrate. Protein concentrations of the lysates were determined CTGF levels in plasma, urine, and renal lysates were determined by colorimetrically and used for normalization of collagenase activity per sandwich ELISA using two distinct specific antibodies (FibroGen), lysate. both directed against CTGF.3 The assay detects full-length and N- terminal fragments of CTGF. CTGF levels are expressed as pmol/L. Cell Culture CTGF Immunohistochemistry The immortalized human proximal tubular epithelial cell line HK- CTGF immunohistochemistry was performed as described previous- 2,35 the SV40-transformed human renal fibroblast cell line TK173 ly.3 Briefly, antigen retrieval was performed by predigestion with Pro- (gift of F. Strutz, Go¨ttingen, Germany),36 rat mesangial cells,9 and tease XXIV (Sigma). Sections were incubated with a CTGF-specific conditionally immortalized mouse podocytes were used to study the human mAb (FibroGen), followed by incubation with rabbit–anti- effects of CTGF on BMP-7 signaling activity. HK-2, TK-173, and rat human IgG (Dako, Glostrup, Denmark) and goat–anti-rabbit Pow- mesangial cells were maintained in DMEM with 10% FBS, penicillin,

2104 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 2098–2107, 2008 www.jasn.org BASIC RESEARCH and streptomycin (Invitrogen). Podocyte cultures were maintained at Co-immunoprecipitation 33°C in RPMI with 10% FBS in the presence of IFN-␥ (R&D Systems, Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Santa Abingdon, UK). For differentiation, podocytes were plated in colla- Cruz, CA) were coated with monoclonal anti–BMP-7 (R&D Systems) gen I–coated wells at 37°C and cultured for another 2 wk in RPMI and blocked with 1% BSA. Beads were subsequently preincubated with 10% FBS in the absence of IFN-␥. HK-2, rat mesangial cells, and with rhBMP-7 (R&D Systems) at 4°C. As controls, beads coated with podocytes were seeded at a density of 2 ϫ 105 cells per well in six-well monoclonal anti-HGF and preincubated with rhHGF (both R&D plates. Cells were serum-starved for 24 h, followed by stimulation Systems) or beads coated with IgG1 and preincubated with rhBMP-7 with and without 50 ng/ml rhBMP-7 (R&D Systems) in the presence were used. The next day, beads were washed and rxCTGF-flag was of 0, 1, 50, or 100 ng/ml rhCTGF (FibroGen) or rxCTGF from Xeno- added for4hat37°C. Beads were washed and resuspended in 20 ␮lof pus laevis.12 Cells were harvested after 1 h for Western blot analysis of PBS, and bound proteins were eluted and denatured in SDS sample pSmad1/5, and after 2 h for quantitative PCR. buffer and separated under reducing conditions by SDS-PAGE. For transfection experiments, TK173 cells were seeded at 1 ϫ 105 Membranes were blocked and incubated with monoclonal anti-flag cells per well in six-well plates. After overnight culture, cells were (Sigma). After incubation with horseradish peroxidase–conjugated washed and transfected with the BRE-luciferase reporter construct rat–anti-mouse antibody, detection was performed as described al- (provided by P. ten Dijke, Leiden, Netherlands). Cells were co-trans- ready. fected with pCAGGS-CTGF or pCAGGS-lacZ. The plasmid pCAGGS-mCTGF was constructed by insertion of mouse CTGF Solid-Phase Binding Assay cDNA into an EcoRI cloning site of the pCAGGS expression vector Microtiter plates were coated overnight at 4°C with 0, 25, 50, and 100 (provided by J. Miyazaki, Osaka, Japan).37 Transfection was carried ng/ml rhBMP-7 or with 150 ng/ml rhHGF. Wells were rinsed and out using Lipofectamine 2000 (Invitrogen) with 2.0 ␮g of reporter blocked with 1% BSA for 2 h. After washing, a range of 0 to 500 ng/ml construct, 2.0 ␮g of pCAGGS plasmid, and 40 ng of pRL-TK-Renilla of rhCTGF (FibroGen) was added, followed by alkaline phosphatase– (Promega, Madison, WI) as a control to normalize transfection effi- conjugated mAb against human CTGF (FibroGen). After incubation ciency. After 24 h, cells were washed, serum-starved for 12 h, and for 2.5 h at 37°C, plates were washed and substrate solution contain- exposed to 10 or 50 ng/ml rhBMP-7 for 24 h. Luciferase activity was ing p-nitrophenyl phosphate was added. Absorbance was read at 405 quantified using the Dual-Luciferase Reporter 1000 Assay System nm. (Promega). Surface Plasmon Resonance Analysis Real-time binding experiments were performed on the Biacore 2000 Quantitative PCR (GE Healthcare, Uppsala, Sweden). Carrier-free recombinant BMP-7 Total RNA was extracted from 30 mg of frozen renal cortex or from (R&D Systems) was immobilized on the CM5 sensor-chip surface at cultured cells using RNeasy columns (Qiagen, Venlo, Netherlands). 66 and 122 fmol/mm2. One control flow channel was routinely acti- After cDNA synthesis, expression of BMP-7, Id1, and CTGF mRNA vated and blocked in the absence of protein. Association of rhCTGF (5 was assessed by quantitative real-time PCR using TaqMan Gene Ex- to 300 nM) was assessed in triplicate in 10 mM HEPES (pH 7.4), 150 pression Assays with predesigned probe and primers (Applied Biosys- mM NaCl, 3 mM EDTA, and 0.005% surfactant P20 for 2 min, at a tems, Foster City, CA). TATA-box binding protein and ␤-actin were flow rate of 20 ␮l/min at 25°C. Dissociation was allowed for 2 min in used as internal reference. the same buffer flow. Sensor chips were regenerated using several pulses of 20 mM HEPES (pH 7.4) and 1 M NaCl at a flow rate of 20 Western Blot Analysis ␮l/min. Data were corrected for both refractive index changes, and Cells or sections of renal cortex were homogenized in lysis buffer (20 association and dissociation rate constants were determined by non- mM Tris at pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 10% linear regression analysis using the BIAevaluation Software 3.1 (GE glycerol, 1 mM EDTA, 1 mM EGTA, 0.5% sodium deoxycholate, 50 Healthcare). mM NaF, and 2 mM Na3VO4) containing 5% Protease Inhibitor Cocktail (Sigma). Protein quantity was determined by BCA protein assay kit (Pierce, Rockford, IL). Samples were run on 8% SDS-PAGE Statistical Analysis and transferred onto polyvinylidene difluoride membranes. After Data are presented as means Ϯ SD. Differences between groups were blocking, membranes were incubated with polyclonal antibody spe- analyzed by t test or ANOVA with Bonferroni correction for multiple cifically directed against pSmad1/5 or total Smad5 (Cell Signaling comparisons. Correlations were assessed by linear regression. For all Technology, Beverly, MA) overnight, washed, and incubated with comparisons, P Ͻ 0.05 was considered to be significant (two-tailed). horseradish peroxidase–conjugated secondary antibody. For detection, membranes were incubated with SuperSignal West Dura Chemiluminescent Substrate (Pierce). Actin antibody (Sigma) was ACKNOWLEDGMENTS used on the same blot for loading control. Phosphorylated Smad1/5 and total Smad5 staining were performed on the same blots (with in This work was supported by the Netherlands Organization for Scien- between stripping) to control for possible regulation of total Smad5 tific Research (Mozaı¨ek grant 017.003.037) and by the Dutch Kidney expression. Foundation (C05.2144).

J Am Soc Nephrol 19: 2098–2107, 2008 CTGF Inhibits BMP-7 Activity 2105 BASIC RESEARCH www.jasn.org

We thank C. Vink, R. Verheul, A. Rietdijk, J.W. Leeuwis, and N. 12. Abreu JG, Ketpura NI, Reversade B, De Robertis EM: Connective- Veldhuijzen for technical assistance; J. Miyazaki for providing the tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4: 599–604, 2002 pCAGGS plasmid; F. Strutz for providing the TK173 cell line; and P. 13. Lam S, van der Geest RN, Verhagen NA, van Nieuwenhoven FA, Blom ten Dijke for providing the BRE-luciferase construct. IE, Aten J, Goldschmeding R, Daha MR, van Kooten C: Connective tissue growth factor and IGF-I are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose. Diabetes 52: 2975–2983, 2003 DISCLOSURES 14. Raatikainen-Ahokas A, Hytonen M, Tenhunen A, Sainio K, Sariola H: NO., Z.L., and L.X. are employees of FibroGen, supplier of rhCTGF and BMP-4 affects the differentiation of metanephric mesenchyme and anti-CTGF antibodies; R.G. has received research support grants and consul- reveals an early anterior-posterior axis of the embryonic kidney. Dev tancy fees from FibroGen. Dyn 217: 146–158, 2000 15. Dudley AT, Lyons KM, Robertson EJ: A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev 9: 2795–2807, 1995 REFERENCES 16. Godin RE, Robertson EJ, Dudley AT: Role of BMP family members during kidney development. Int J Dev Biol 43: 405–411, 1999 1. Ito Y, Aten J, Bende RJ, Oemar BS, Rabelink TJ, Weening JJ, Gold- 17. Vukicevic S, Basic V, Rogic D, Basic N, Shih MS, Shepard A, Jin D, schmeding R: Expression of connective tissue growth factor in human Dattatreyamurty B, Jones W, Dorai H, Ryan S, Griffiths D, Maliakal renal fibrosis. Kidney Int 53: 853–861, 1998 J, Jelic M, Pastorcic M, Stavljenic A, Sampath TK: Osteogenic 2. Riser BL, deNichilo M, Cortes P, Baker C, Grondin JM, Yee J, Narins protein-1 (bone morphogenetic protein-7) reduces severity of in- RG: Regulation of connective tissue growth factor activity in cultured jury after ischemic acute renal failure in rat. J Clin Invest 102: rat mesangial cells and its expression in experimental diabetic glo- 202–214, 1998 merulosclerosis. J Am Soc Nephrol 11: 25–38, 2000 18. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, 3. Roestenberg P, van Nieuwenhoven FA, Joles JA, Trischberger C, Kalluri R: BMP-7 counteracts TGF-beta1-induced epithelial-to-mesen- Martens PP, Oliver N, Aten J, Hoppener JW, Goldschmeding R: chymal transition and reverses chronic renal injury. Nat Med 9: 964– Temporal expression profile and distribution pattern indicate a role 968, 2003 of connective tissue growth factor (CTGF/CCN-2) in diabetic ne- 19. Wang SN, Lapage J, Hirschberg R: Loss of tubular bone morphoge- phropathy in mice. Am J Physiol Renal Physiol 290: F1344–F1354, netic protein-7 in diabetic nephropathy. J Am Soc Nephrol 12: 2392– 2006 2399, 2001 4. Roestenberg P, van Nieuwenhoven FA, Wieten L, Boer P, Diekman T, 20. De Petris L, Hruska KA, Chiechio S, Liapis H: Bone morphogenetic Tiller AM, Wiersinga WM, Oliver N, Usinger W, Weitz S, Schlingemann protein-7 delays podocyte injury due to high glucose. Nephrol Dial RO, Goldschmeding R: Connective tissue growth factor is increased in Transplant 22: 3442–3450, 2007 plasma of type 1 diabetic patients with nephropathy. Diabetes Care 21. Mitu GM, Wang S, Hirschberg R: BMP7 is a podocyte survival factor 27: 1164–1170, 2004 and rescues podocytes from diabetic injury. Am J Physiol Renal 5. Riser BL, Cortes P, deNichilo M, Deshmukh PV, Chahal PS, Moham- Physiol 293: F1641–F1648, 2007 med AK, Yee J, Kahkonen D: Urinary CCN2 (CTGF) as a possible 22. Wang S, Chen Q, Simon TC, Strebeck F, Chaudhary L, Morrissey J, predictor of diabetic nephropathy: Preliminary report. Kidney Int 64: Liapis H, Klahr S, Hruska KA: Bone morphogenic protein-7 (BMP-7), a 451–458, 2003 novel therapy for diabetic nephropathy. Kidney Int 63: 2037–2049, 6. Nguyen TQ, Tarnow L, Andersen S, Hovind P, Parving HH, Gold- 2003 schmeding R, van Nieuwenhoven FA: Urinary connective tissue 23. Wang S, de CM, Kopp J, Mitu G, Lapage J, Hirschberg R: Renal bone growth factor excretion correlates with clinical markers of renal dis- morphogenetic protein-7 protects against diabetic nephropathy. ease in a large population of type 1 diabetic patients with diabetic J Am Soc Nephrol 17: 2504–2512, 2006 nephropathy. Diabetes Care 29: 83–88, 2006 24. Sugimoto H, Grahovac G, Zeisberg M, Kalluri R: Renal fibrosis and 7. Gilbert RE, Akdeniz A, Weitz S, Usinger WR, Molineaux C, Jones SE, glomerulosclerosis in a new mouse model of diabetic nephropathy Langham RG, Jerums G: Urinary connective tissue growth factor ex- and its regression by bone morphogenic protein-7 and advanced cretion in patients with type 1 diabetes and nephropathy. Diabetes glycation end product inhibitors. Diabetes 56: 1825–1833, 2007 Care 26: 2632–2636, 2003 25. Neilson EG: Setting a trap for tissue fibrosis. Nat Med 11: 373–374, 8. Guha M, Xu ZG, Tung D, Lanting L, Natarajan R: Specific down- 2005 regulation of connective tissue growth factor attenuates progression 26. Wahab NA, Mason RM: A critical look at growth factors and epithelial- of nephropathy in mouse models of type 1 and type 2 diabetes. to-mesenchymal transition in the adult kidney: Interrelationships be- FASEB J 21: 3355–3368, 2007 tween growth factors that regulate EMT in the adult kidney. Nephron 9. Blom IE, van Dijk AJ, Wieten L, Duran K, Ito Y, Kleij L, deNichilo M, Exp Nephrol 104: e129–e134, 2006 Rabelink TJ, Weening JJ, Aten J, Goldschmeding R: In vitro evidence 27. Korchynskyi O, Ten DP: Identification and functional characterization for differential involvement of CTGF, TGFbeta, and PDGF-BB in mes- of distinct critically important bone morphogenetic protein-specific angial response to injury. Nephrol Dial Transplant 16: 1139–1148, response elements in the Id1 promoter. J Biol Chem 277: 4883–4891, 2001 2002 10. Abdel-Wahab N, Weston BS, Roberts T, Mason RM: Connective tissue 28. Zeisberg M: Bone morphogenic protein-7 and the kidney: Current growth factor and regulation of the mesangial cell cycle: Role in concepts and open questions. Nephrol Dial Transplant 21: 568–573, cellular hypertrophy. J Am Soc Nephrol 13: 2437–2445, 2002 2006 11. Burns WC, Twigg SM, Forbes JM, Pete J, Tikellis C, Thallas-Bonke 29. Wolf G, Ziyadeh FN: Cellular and molecular mechanisms of proteinuria V, Thomas MC, Cooper ME, Kantharidis P: Connective tissue in diabetic nephropathy. Nephron Physiol 106: 26–31, 2007 growth factor plays an important role in advanced glycation end 30. MCLennan SV, Kelly DJ, Cox AJ, Cao Z, Lyons JG, Yue DK, Gilbert RE: product-induced tubular epithelial-to-mesenchymal transition: Im- Decreased matrix degradation in diabetic nephropathy: Effects of plications for diabetic renal disease. J Am Soc Nephrol 17: 2484– ACE inhibition on the expression and activities of matrix metallopro- 2494, 2006 teinases. Diabetologia 45: 268–275, 2002

2106 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 2098–2107, 2008 www.jasn.org BASIC RESEARCH

31. Del PD, Anglani F, Forino M, Ceol M, Fioretto P, Nosadini R, 34. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Baggio B, Gambaro G: Down-regulation of glomerular matrix met- Daluiski A, Lyons KM: Connective tissue growth factor coordinates alloproteinase-2 gene in human NIDDM. Diabetologia 40: 1449– chondrogenesis and angiogenesis during skeletal development. De- 1454, 1997 velopment 130: 2779–2791, 2003 32. Thomson SE, McLennan SV, Kirwan PD, Heffernan SJ, Hennessy A, 35. Ryan MJ, Johnson G, Kirk J, Fuerstenberg SM, Zager RA, Torok-Storb Yue DK, Twigg SM: Renal connective tissue growth factor correlates B: HK-2: An immortalized proximal tubule epithelial cell line from with glomerular basement membrane thickness and prospective albu- normal adult human kidney. Kidney Int 45: 48–57, 1994 minuria in a non-human primate model of diabetes: Possible predic- 36. Muller GA, Frank J, Rodemann HP, Engler-Blum G: Human renal tive marker for incipient diabetic nephropathy. J Diabetes Complica- fibroblast cell lines (tFKIF and tNKF) are new tools to investigate tions April 15, 2008 [epub ahead of print] pathophysiologic mechanisms of renal interstitial fibrosis. Exp Nephrol 33. Flyvbjerg A, Khatir D, Jensen LJN, Lomongsod E, Liu DY, Rasch R, 3: 127–133, 1995 Usinger WR: Long-term renal effects of a neutralizing connective 37. Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expres- tissue growth factor (CTGF)-antibody in obese type 2 diabetic mice sion transfectants with a novel eukaryotic vector. Gene 108: 193–199, [Abstract]. J Am Soc Nephrol 15: 261A, 2004 1991

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