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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2008 Nov;19(11):2098–2107. doi: 10.1681/ASN.2007111261

CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy

Tri Q Nguyen *, Peggy Roestenberg *, Frans A van Nieuwenhoven *, Niels Bovenschen *, Zeke Li †, Leon Xu †, Noelynn Oliver †, Jan Aten ‡, Jaap A Joles §, Cecilia Vial ‖, Enrique Brandan ‖, Karen M Lyons , Roel Goldschmeding *
PMCID: PMC2573007  PMID: 18632843

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 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 of diabetic CTGF+/+ and CTGF+/− mice, phosphorylation of the BMP signal transduction protein 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 = −0.86) and MMP activity (R = 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.

Connective tissue growth factor (CTGF) is considered an important factor in the development of diabetic nephropathy. The renal expression of CTGF mRNA and protein is upregulated in human and experimental diabetic nephropathy.1–3 In patients with diabetic nephropathy, both plasma CTGF levels and urinary CTGF excretion are increased and correlate with clinical markers of renal disease.4–7 Recently, it was demonstrated that specific downregulation of CTGF by antisense oligonucleotide treatment attenuated albuminuria and mesangial matrix expansion in experimental type 1 and type 2 diabetic nephropathy.8 These observations suggest that CTGF is a critical determinant of structural and functional damage in diabetic nephropathy.

Several mechanisms have been proposed for a pathogenic role of CTGF in diabetic nephropathy. Studies with renal cells demonstrated that CTGF is involved in diabetes-associated changes such as extracellular matrix synthesis, cell migration, cellular hypertrophy, and epithelial-to-mesenchymal transition.2,9–11 CTGF might exert these effects in diabetes by modulating the activity of other growth factors. For example, CTGF is known to enhance profibrotic activity of TGF-β1 and IGF-1, which involves physical interaction of CTGF with these growth factors.12,13 In contrast, binding to CTGF potently antagonizes the signaling activity of bone morphogenetic protein 4 (BMP-4) in osteogenesis assays and in embryonic patterning.12 In the kidney, the importance of several BMP, including BMP-4, has been demonstrated mainly in developmental studies14–16; however, thus far, only BMP-7 has also been studied for its contribution as an antifibrotic and proregenerative factor in response to injury of the adult kidney.17,18 Renal expression of BMP-7 is progressively decreased during the course of human and experimental diabetic nephropathy and in podocytes cultured in high glucose medium,19–21 whereas restoration of BMP-7 availability has resulted in prevention or even reversal of functional and structural changes of diabetic nephropathy.22–24 Although it has been hypothesized that CTGF might inhibit BMP-7,25,26 this has not been addressed experimentally; therefore, we set out to investigate the impact of CTGF on BMP-7 signal transduction and target gene expression in experimental diabetic nephropathy and in cultured renal cells.

RESULTS

BMP-7 Deficiency in Diabetic Nephropathy Is Accompanied by Decrease of pSmad1/5 and BMP-Target Gene Expression

Induction of diabetes in C57BL6/J mice by intraperitoneal injection of streptozotocin resulted in characteristic features of diabetic nephropathy, including persistent hyperglycemia; increased glycosylated hemoglobin levels; proteinuria; structural changes of the kidney; and increased CTGF levels in kidney, urine, and plasma.3 In diabetic mice, renal cortical expression of BMP-7 mRNA was decreased 2.6-fold. This was accompanied by reduced levels of pSmad1/5 protein, and also BMP-7 downstream target Id1 was decreased (Figure 1).

Figure 1.

Figure 1.

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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 ± SD. *P < 0.05.

CTGF+/− Mice Have Lower Levels of CTGF mRNA and Protein

After 17 wk of diabetes, CTGF mRNA and protein expression in renal cortex and CTGF levels in plasma and urine were increased in diabetic CTGF+/+ mice as compared with nondiabetic CTGF+/+ mice. In diabetic CTGF+/− mice, expression of CTGF mRNA and protein in renal cortex and CTGF levels in plasma were not significantly different from nondiabetic mice. Although urinary CTGF excretion in diabetic CTGF+/− mice seemed to be lower than in diabetic CTGF+/+ mice, this difference did not reach statistical significance. In nondiabetic CTGF+/− mice, CTGF expression seemed slightly lower than in nondiabetic CTGF+/+, but this difference was NS (Figure 2, A through D).

Figure 2.

Figure 2.

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Levels of CTGF mRNA and protein are decreased in CTGF+/− mice. Diabetes was induced in CTGF+/+ and CTGF+/− mice. (A through D) CTGF mRNA and protein in renal cortex and CTGF levels in plasma were two-fold increased in diabetic CTGF+/+ mice, as compared with diabetic CTGF+/− mice. Also urinary CTGF excretion tended to be lower in diabetic CTGF+/− mice than in diabetic CTGF+/+ mice (P = 0.29). (E) CTGF immunohistochemistry showed more prominent glomerular CTGF staining in diabetic CTGF+/+ mice than in diabetic CTGF+/− mice and nondiabetic mice. Data are means ± SD. *P < 0.05 versus control CTGF+/+ mice; **P < 0.05 versus diabetic CTGF+/+ mice.

CTGF immunohistochemistry showed more prominent glomerular CTGF staining in diabetic CTGF+/+ mice than in diabetic CTGF+/− mice and in nondiabetic mice. No prominent CTGF staining was observed in tubuli (Figure 2E).

Diabetic Nephropathy Is Attenuated in CTGF+/− Mice

Albuminuria was increased in diabetic CTGF+/+ mice as compared with nondiabetic mice. In diabetic CTGF+/− mice, albuminuria was significantly less pronounced than in diabetic CTGF+/+ mice (111 ± 39 versus 176 ± 26 mg/g creatinine; P = 0.024; Figure 3A).

Figure 3.

Figure 3.

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Diabetic nephropathy is attenuated in CTGF+/− mice. Diabetic nephropathy in CTGF+/+ and CTGF+/− mice was assessed by albuminuria, GBM thickening, and MMP activity. (A) In diabetic CTGF+/− mice, albuminuria was significantly less pronounced than in diabetic CTGF+/+ mice. (B) GBM thickening in diabetic CTGF+/+ mice, as determined by electron microscopy, was absent in diabetic CTGF+/− mice. (C and D) In situ zymography on renal sections (green, MMP activity; red, nuclear counterstain; C) and colorimetric detection in renal lysates (D) showed that MMP activity was decreased in diabetic CTGF+/+ mice. In contrast, MMP activity was preserved in diabetic CTGF+/− mice. Data are means ± SD. *P < 0.05 versus control CTGF+/+ mice; **P < 0.05 versus diabetic CTGF+/+ mice.

Examination of ultrathin sections by electron microscopy showed that the thickness of the glomerular basement membrane (GBM) in diabetic CTGF+/+ mice was increased compared with control CTGF+/+ mice; however, no increase in GBM thickness was observed in diabetic CTGF+/− mice as compared with diabetic CTGF+/+ mice (154 ± 3.0 versus 174 ± 2.3 nm; P < 0.01; Figure 3B).

In situ zymography showed that gelatinase activity, representing activity of matrix metalloproteinase 2 (MMP-2) and MMP-9, was localized mainly in glomeruli. Activity of gelatinase was decreased in diabetic CTGF+/+ mice but was preserved in diabetic CTGF+/− mice (Figure 3C). This was confirmed by quantification of gelatinolytic activity in renal lysates, showing a reduction by approximately 40% in diabetic CTGF+/+ mice (P = 0.034) but not in diabetic CTGF+/− mice (Figure 3D).

Preserved BMP Signaling Activity in Diabetic CTGF+/− Mice: Correlation of CTGF Level with Albuminuria and MMP Activity

Renal cortical expression of BMP-7 mRNA was similar in diabetic CTGF+/+ and CTGF+/− mice. Diabetic CTGF+/− mice had relatively preserved pSmad1/5 protein levels and Id1 mRNA expression as compared with diabetic CTGF+/+ mice, whereas total Smad5 protein was not different (Figure 4, A through C). Furthermore, Id1 mRNA correlated with albuminuria (R = −0.86, P = 0.011) and with MMP activity in renal lysates (R = 0.76, P = 0.037; Figure 4, D and E).

Figure 4.

Figure 4.

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CTGF inhibits BMP signaling activity in diabetic nephropathy. Renal cortex of diabetic CTGF+/+ and CTGF+/− mice was harvested 17 wk after induction of diabetes. (A) Renal cortical expression of BMP-7 mRNA was similar in diabetic CTGF+/+ and CTGF+/− mice. (B and C) In diabetic CTGF+/− mice, pSmad1/5 protein and Id1 mRNA were higher than in diabetic CTGF+/+ mice, whereas total Smad5 was not different. (D and E) Id1 mRNA correlated with albuminuria and MMP activity. Data are means ± SD. *P < 0.05.

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 = 0.0083; Figure 5B). Injection with vehicle did not alter renal pSmad1/5 (Figure 5C).

Figure 5.

Figure 5.

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Injection of CTGF impairs renal cortical BMP-7 activity in nondiabetic mice. Recombinant CTGF or vehicle only was injected intraperitoneally into nondiabetic BALBc mice. (A) CTGF levels in plasma were increased at 10 min after injection with rhCTGF. (B) Renal cortical pSmad1/5 protein levels were significantly decreased 4 h after injection with rhCTGF. (C) Renal cortical pSmad1/5 levels were not different 4 h after vehicle injection. Data are means ± SD. *P < 0.05.

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. Addition 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 significant, although less pronounced, increase of Id1 mRNA expression, which was reduced by addition of CTGF (Figure 6E).

Figure 6.

Figure 6.

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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 epithelial cells (HK-2), and renal interstitial fibroblasts (TK173). (A and B) Treatment of rat mesangial cells with BMP-7 resulted in an increase of Smad1/5 phosphorylation and Id1 mRNA expression, both of which were inhibited by co-stimulation with CTGF. (C and D) Treatment of HK-2 cells with BMP-7 resulted in an increase of pSmad1/5 protein and Id1 mRNA, both of which were inhibited by co-stimulation with CTGF. (E) Treatment of podocytes with BMP-7 resulted in increase of Id1 mRNA, which was inhibited by co-stimulation with CTGF. (F) TK173 cells were transfected with the BRE-luciferase construct. Treatment with BMP-7 of cells co-transfected with control vector pCAGGS-lacZ resulted in increase of BRE-luciferase activity, whereas co-transfection with pCAGGS-CTGF inhibited BMP-7–induced luciferase activity. Data are means ± SD. *P < 0.05 versus only 50 ng/ml BMP-7; #P < 0.05 versus p-lacZ with 10 ng/ml rhBMP-7; †P < 0.05 versus p-lacZ with 50 ng/ml BMP-7.

CTGF also inhibited activation of a BMP-responsive element (BRE)-luciferase construct, which specifically reports Smad1/5-mediated gene transcription.27 When TK173 cells were co-transfected with BRE-luciferase and pCAGGS-mCTGF, BMP-7–induced luciferase activity was significantly lower than in cells co-transfected with pCAGGS-lacZ control vector (Figure 6F).

CTGF Binds BMP-7 with High Affinity

Co-immunoprecipitation experiments showed that captured BMP-7 on anti–BMP-7 mAb-coated agarose beads was able to bind CTGF. In the absence of BMP-7, only a weak CTGF band was detected, which is due to known nonspecific binding of CTGF to agarose beads. Incubation with captured HGF on anti-HGF mAb-coated beads and with BMP-7–and IgG1-coated beads also did not result in significant pull-down of CTGF (Figure 7A). Similarly, solid-phase binding assay showed direct physical interaction between BMP-7 and CTGF but not between HGF and CTGF or BSA and CTGF. Increased binding between was observed as the concentration of either BMP-7 or CTGF was increased (Figure 7B).

Figure 7.

Figure 7.

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CTGF binds directly to BMP-7. Physical interaction between CTGF and BMP-7 was demonstrated by co-immunoprecipitation, solid-phase binding assay, and surface plasmon resonance. (A) Anti–BMP-7 mAb–coated agarose beads were incubated with rhBMP-7 and/or rxCTGF-flag. As controls, beads were coated with anti-HGF and incubated 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 CTGF was run as control. (B) Increasing concentrations of rhCTGF were added to microtiter plates coated with rhBMP-7, rhHGF, or 1% BSA. Bound proteins were detected with an AP-conjugated antibody against rhCTGF. (C) Purified CTGF protein was run over BMP-7 sensor chips. Association and dissociation were monitored by a change in the resonance units.

The binding affinity of CTGF for BMP-7 was determined by surface plasmon resonance analysis. CTGF displayed time-dependent association with immobilized BMP-7 followed by dissociation, which was dose-dependent because higher response was observed at higher CTGF concentrations (Figure 7C) and at higher BMP-7 density (data not shown). The data showed complex binding behavior in which multiple components were involved; therefore, a heterogeneous two-site binding model was required to describe accurately the binding behavior. This resulted in Kd values describing a high- and low-affinity component of 14 ± 6 and 316 ± 190 nM for the interaction between CTGF and BMP-7 [kon1 = 2.4 ± 0.9 × 105 M/s, koff1 = 3.3 ± 0.8 × 10−3, kon2 = 1.3 ± 0.5 × 105, koff2 = 4.0 ± 1.8 × 10−2].

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 CTGF+/+ mice with those in diabetic CTGF+/− mice. The latter mice have lower CTGF expression in kidney, plasma, and urine as compared with diabetic CTGF+/+ mice, but they have equal TGF-β1 expression (data not shown). Thus, diabetic CTGF+/− mice constitute a unique model to assess the impact of CTGF level on downstream BMP-7 activity in diabetic nephropathy. BMP-7 mRNA expression in renal cortex was similar in diabetic CTGF+/+ and CTGF+/− mice, indicating that CTGF level does not influence BMP-7 expression; however, pSmad1/5 protein and Id1 mRNA levels were lower in diabetic CTGF+/+ mice, as compared with diabetic CTGF+/− mice. This indicates that CTGF might be an important determinant of the diabetes-induced reduction in signaling activity of residual BMP-7. CTGF is known also to modulate BMP-4 and TGF-β1 directly.12 In addition, the level of BMP signaling activity in the kidney is subject to the influence of other members of the TGF-β superfamily, BMP receptors, and of BMP modulators such as gremlin, noggin, kielin/chordin-like protein, or uterine sensitization-associated gene 128; therefore, the relative contribution of BMP-7 inhibition by CTGF remains to be established.

Albuminuria, thickening of the GBM, and decreased activity of MMP all are hallmarks of human and experimental diabetic nephropathy.29–31 Recently, it was shown that renal CTGF protein correlates with GBM thickness and prospective albuminuria in a nonhuman primate model of diabetes.32 We observed that these alterations and also decreased MMP activity were attenuated or absent in diabetic CTGF+/− mice. This indicates that CTGF plays a pathogenic role in at least these characteristic manifestations of diabetic nephropathy. Similarly, decrease of albuminuria in experimental models of type 1 and type 2 diabetic nephropathy has been observed in mice treated with an anti-CTGF antibody and with a CTGF antisense oligonucleotide, respectively.8,33 In the latter study, inhibition of CTGF also resulted in reduction of serum creatinine and inhibition of mesangial matrix expansion, which were not observed in our diabetic CTGF+/− mouse model (data not shown). A possible explanation for this discrepancy is that CTGF level derived from a single functional allele might suffice to mediate diabetes-induced increase in serum creatinine and matrix accumulation. Treatment with antisense oligonucleotides might have lowered CTGF availability to levels below those in our diabetic CTGF+/− mice, resulting in more complete protection; however, we cannot directly compare actual reduction of CTGF levels in the different studies, and BMP signaling activity was not addressed after anti-CTGF antibody or CTGF-antisense oligonucleotide treatment.

The strong correlation of Id1 with both albuminuria and MMP activity suggests that CTGF-dependent suppression of this target gene of BMP signaling might be involved directly in the pathogenesis of diabetic nephropathy. Accordingly, diabetic transgenic mice overexpressing BMP-7 in podocytes and proximal tubular epithelial cells had higher Id1:PAI mRNA ratio and preserved MMP activity and developed less albuminuria, as compared with diabetic wild-type mice.23

As in overexpression of endogenous CTGF in diabetic mice, injection of recombinant human CTGF into nondiabetic 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 sustained 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 exemplified by inhibition of BMP-7–induced pSmad1/5, Id1, and BRE-luciferase activity. The inhibitory effect of CTGF was robust, in the sense that it was observed with exogenously added human and Xenopus CTGF, as well as with transfected mouse CTGF.

Direct physical interaction between CTGF and BMP-7 was evidenced by co-immunoprecipitation and in a solid-phase binding assay. Furthermore, surface plasmon resonance analysis demonstrated that the interaction between CTGF and BMP-7 was complex and consisted of a high- and a low-affinity component (Kd values of 14 and 316 nM, respectively). This not only may result from inherent biologic properties of both CTGF and BMP-7 but also may be due to partial blocking of CTGF-interactive sites during the immobilization of BMP-7. The high binding affinity of CTGF and BMP-7 was comparable to that described for CTGF and BMP-4, which had a Kd value of 5 nM.12

In conclusion, overexpression of CTGF inhibits BMP-7 signal transduction in the diabetic kidney and contributes significantly to altered gene transcription, as well as to reduced MMP activity, and to GBM thickening and albuminuria, which all are hallmarks of diabetic nephropathy.

CONCISE METHODS

Animal Experiments

Signaling activity of BMP-7 was studied in diabetic mice.3 Briefly, diabetes was induced in nine 12-wk-old female C57Bl/6J mice by a single intraperitoneal injection of 200 mg/kg streptozotocin (Sigma, St. Louis, MO) in sodium citrate buffer. Six control animals were administered an injection of vehicle only. Hyperglycemia was determined 3 d after injection by measurement of blood glucose levels. Slow-release insulin pellets (Linshin; Scarborough, Ontario, Canada) were implanted to stabilize the condition of the diabetic animals. Mice were killed 9 wk after injection. Renal cortex was harvested by dissecting small caps of the upper and the lower poles. Before homogenization, absence of medulla was checked in frozen sections of the cut surface.

Effects of CTGF level on BMP-7 signaling activity were studied in diabetic CTGF+/+ and CTGF+/− mice. Outbred male BALBc/129Sv CTGF+/− mice, in which exon 1 of one CTGF allele has been replaced by a neomycin resistance gene,34 were mated with female C57Bl/6J mice. From their first offspring, female CTGF+/− mice and female CTGF+/+ littermates were used for this study. Diabetes was induced in five 16-wk-old CTGF+/− mice and four CTGF+/+ mice by injection of streptozotocin. Nine control mice were administered an injection of vehicle only. After 9 wk, unilateral nephrectomy was performed on all animals to aggravate the diabetic nephropathy model. Mice were killed 17 wk after induction of diabetes. Albumin levels were determined by sandwich ELISA using a goat–anti-mouse albumin antibody (Bethyl Laboratories, Montgomery, TX). Urinary creatinine excretion was determined by enzymatic assays (J2L Elitech, Labarthe Inard, France).

Effects of CTGF on BMP-7 signaling activity in renal cortex of normoglycemic mice were studied in 24 BALBc mice, which were administered an intraperitoneal injection of rhCTGF (FibroGen, South San Francisco, CA) at a dosage of 20 μg/kg diluted in 50 mM Tris-HCl buffer containing 800 mM NaCl. Mice were killed 0, 5, 10, 15, 30, 60, 120, and 240 min after injection (three mice for each time point). Six control mice were administered an injection of vehicle only and killed 0 and 240 min after injection.

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 pellet laboratory diet and had free access to water. The experiments were performed with the approval of the Experimental Animal Ethics Committee of the University of Utrecht.

CTGF ELISA

CTGF levels in plasma, urine, and renal lysates were determined by sandwich ELISA using two distinct specific antibodies (FibroGen), both directed against CTGF.3 The assay detects full-length and N-terminal fragments of CTGF. CTGF levels are expressed as pmol/L.

CTGF Immunohistochemistry

CTGF immunohistochemistry was performed as described previously.3 Briefly, antigen retrieval was performed by predigestion with Protease XXIV (Sigma). Sections were incubated with a CTGF-specific human mAb (FibroGen), followed by incubation with rabbit–anti-human IgG (Dako, Glostrup, Denmark) and goat–anti-rabbit Powervision-PO (Klinipath, Duiven, Netherlands). Bound antibody was visualized with Nova RED (Vector Laboratories, Burlingame, 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 dehydrated 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 uranyl 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-sections of GBM per glomerulus at a magnification of ×5000 and analyzed by computer image analysis (ImageJ; National Institutes of Health, www.rsb.info.nih.gov/ij/).

In Situ Zymography

Glomerular MMP activity was visualized by in situ zymography and confocal laser-scanning microscopy. Frozen tissue sections were incubated with DQTM gelatin from pig skin (Invitrogen, Carlsbad, CA), diluted 1:20 in 50 mM Tris-HCl buffer containing 10 mol/L CaCl2, 0.05% Brij 35, and 5 mmol/L PMSF (pH 7.4). Slides were incubated in a dark humidified chamber at 37°C for 19 h. An MMP inhibitor, 1,10-phenanthroline monohydrate (2 μg/ml), was used to verify that the obtained gelatinase activity specifically represented MMP activity. Nuclei were counterstained in red with propidium iodide.

Gelatinase Activity in Tissue Lysates

From frozen kidneys, 10 sections of 20 μm were cut and dissolved in lysis buffer (50 mM Tris, 150 mM NaCl, and 1% Triton X-100 [pH 7.4]). Gelatinase activity was measured with an EnzChek Gelatinase assay kit (Invitrogen). Collagenase was used as standard, and specificity of the gelatinase activity was verified using 1,10-phenanthroline monohydrate. Protein concentrations of the lysates were determined colorimetrically and used for normalization of collagenase activity per lysate.

Cell Culture

The immortalized human proximal tubular epithelial cell line HK-2,35 the SV40-transformed human renal fibroblast cell line TK173 (gift of F. Strutz, Göttingen, Germany),36 rat mesangial cells,9 and conditionally immortalized mouse podocytes were used to study the effects of CTGF on BMP-7 signaling activity. HK-2, TK-173, and rat mesangial cells were maintained in DMEM with 10% FBS, penicillin, and streptomycin (Invitrogen). Podocyte cultures were maintained at 33°C in RPMI with 10% FBS in the presence of IFN-γ (R&D Systems, Abingdon, UK). For differentiation, podocytes were plated in collagen I–coated wells at 37°C and cultured for another 2 wk in RPMI with 10% FBS in the absence of IFN-γ. HK-2, rat mesangial cells, and podocytes were seeded at a density of 2 × 105 cells per well in six-well plates. Cells were serum-starved for 24 h, followed by stimulation with and without 50 ng/ml rhBMP-7 (R&D Systems) in the presence of 0, 1, 50, or 100 ng/ml rhCTGF (FibroGen) or rxCTGF from Xenopus laevis.12 Cells were harvested after 1 h for Western blot analysis of pSmad1/5, and after 2 h for quantitative PCR.

For transfection experiments, TK173 cells were seeded at 1 × 105 cells per well in six-well plates. After overnight culture, cells were washed and transfected with the BRE-luciferase reporter construct (provided by P. ten Dijke, Leiden, Netherlands). Cells were co-transfected with pCAGGS-CTGF or pCAGGS-lacZ. The plasmid pCAGGS-mCTGF was constructed by insertion of mouse CTGF cDNA into an EcoRI cloning site of the pCAGGS expression vector (provided by J. Miyazaki, Osaka, Japan).37 Transfection was carried out using Lipofectamine 2000 (Invitrogen) with 2.0 μg of reporter construct, 2.0 μg of pCAGGS plasmid, and 40 ng of pRL-TK-Renilla (Promega, Madison, WI) as a control to normalize transfection efficiency. After 24 h, cells were washed, serum-starved for 12 h, and exposed to 10 or 50 ng/ml rhBMP-7 for 24 h. Luciferase activity was quantified using the Dual-Luciferase Reporter 1000 Assay System (Promega).

Quantitative PCR

Total RNA was extracted from 30 mg of frozen renal cortex or from cultured cells using RNeasy columns (Qiagen, Venlo, Netherlands). After cDNA synthesis, expression of BMP-7, Id1, and CTGF mRNA was assessed by quantitative real-time PCR using TaqMan Gene Expression Assays with predesigned probe and primers (Applied Biosystems, Foster City, CA). TATA-box binding protein and β-actin were used as internal reference.

Western Blot Analysis

Cells or sections of renal cortex were homogenized in lysis buffer (20 mM Tris at pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 0.5% sodium deoxycholate, 50 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 and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated with polyclonal antibody specifically directed against pSmad1/5 or total Smad5 (Cell Signaling Technology, Beverly, MA) overnight, washed, and incubated with horseradish peroxidase–conjugated secondary antibody. For detection, membranes were incubated with SuperSignal West Dura Chemiluminescent Substrate (Pierce). Actin antibody (Sigma) was used on the same blot for loading control. Phosphorylated Smad1/5 and total Smad5 staining were performed on the same blots (with in between stripping) to control for possible regulation of total Smad5 expression.

Co-immunoprecipitation

Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were coated with monoclonal anti–BMP-7 (R&D Systems) and blocked with 1% BSA. Beads were subsequently preincubated with rhBMP-7 (R&D Systems) at 4°C. As controls, beads coated with monoclonal anti-HGF and preincubated with rhHGF (both R&D Systems) or beads coated with IgG1 and preincubated with rhBMP-7 were used. The next day, beads were washed and rxCTGF-flag was added for 4 h at 37°C. Beads were washed and resuspended in 20 μl of PBS, and bound proteins were eluted and denatured in SDS sample buffer and separated under reducing conditions by SDS-PAGE. Membranes were blocked and incubated with monoclonal anti-flag (Sigma). After incubation with horseradish peroxidase–conjugated rat–anti-mouse antibody, detection was performed as described already.

Solid-Phase Binding Assay

Microtiter plates were coated overnight at 4°C with 0, 25, 50, and 100 ng/ml rhBMP-7 or with 150 ng/ml rhHGF. Wells were rinsed and blocked with 1% BSA for 2 h. After washing, a range of 0 to 500 ng/ml of rhCTGF (FibroGen) was added, followed by alkaline phosphatase–conjugated mAb against human CTGF (FibroGen). After incubation for 2.5 h at 37°C, plates were washed and substrate solution containing p-nitrophenyl phosphate was added. Absorbance was read at 405 nm.

Surface Plasmon Resonance Analysis

Real-time binding experiments were performed on the Biacore 2000 (GE Healthcare, Uppsala, Sweden). Carrier-free recombinant BMP-7 (R&D Systems) was immobilized on the CM5 sensor-chip surface at 66 and 122 fmol/mm2. One control flow channel was routinely activated and blocked in the absence of protein. Association of rhCTGF (5 to 300 nM) was assessed in triplicate in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20 for 2 min, at a flow rate of 20 μl/min at 25°C. Dissociation was allowed for 2 min in 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 μl/min. Data were corrected for both refractive index changes, and association and dissociation rate constants were determined by nonlinear regression analysis using the BIAevaluation Software 3.1 (GE Healthcare).

Statistical Analysis

Data are presented as means ± SD. Differences between groups were analyzed by t test or ANOVA with Bonferroni correction for multiple comparisons. Correlations were assessed by linear regression. For all comparisons, P < 0.05 was considered to be significant (two-tailed).

DISCLOSURES

NO., Z.L., and L.X. are employees of FibroGen, supplier of rhCTGF and anti-CTGF antibodies; R.G. has received research support grants and consultancy fees from FibroGen.

Acknowledgments

This work was supported by the Netherlands Organization for Scientific Research (Mozaïek grant 017.003.037) and by the Dutch Kidney Foundation (C05.2144).

We thank C. Vink, R. Verheul, A. Rietdijk, J.W. Leeuwis, and N. Veldhuijzen for technical assistance; J. Miyazaki for providing the pCAGGS plasmid; F. Strutz for providing the TK173 cell line; and P. ten Dijke for providing the BRE-luciferase construct.

Published online ahead of print. Publication date available at www.jasn.org.

P.R.'s current affilation is Department of Biochemistry, Radboud University Nijmegen Medical Center, Nijmegan, Netherlands; F.A.v.N.'s current affiliation is Department of Physiology, CARIM, Maastricht University, Maastricht, Netherlands.

REFERENCES

  • 1.Ito Y, Aten J, Bende RJ, Oemar BS, Rabelink TJ, Weening JJ, Goldschmeding R: Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 53: 853–861, 1998 [DOI] [PubMed] [Google Scholar]
  • 2.Riser BL, deNichilo M, Cortes P, Baker C, Grondin JM, Yee J, Narins RG: Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 11: 25–38, 2000 [DOI] [PubMed] [Google Scholar]
  • 3.Roestenberg P, van Nieuwenhoven FA, Joles JA, Trischberger C, Martens PP, Oliver N, Aten J, Hoppener JW, Goldschmeding R: 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 Renal Physiol 290: F1344–F1354, 2006 [DOI] [PubMed] [Google Scholar]
  • 4.Roestenberg P, van Nieuwenhoven FA, Wieten L, Boer P, Diekman T, Tiller AM, Wiersinga WM, Oliver N, Usinger W, Weitz S, Schlingemann RO, Goldschmeding R: Connective tissue growth factor is increased in plasma of type 1 diabetic patients with nephropathy. Diabetes Care 27: 1164–1170, 2004 [DOI] [PubMed] [Google Scholar]
  • 5.Riser BL, Cortes P, deNichilo M, Deshmukh PV, Chahal PS, Mohammed AK, Yee J, Kahkonen D: Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: Preliminary report. Kidney Int 64: 451–458, 2003 [DOI] [PubMed] [Google Scholar]
  • 6.Nguyen TQ, Tarnow L, Andersen S, Hovind P, Parving HH, Goldschmeding R, van Nieuwenhoven FA: Urinary connective tissue growth factor excretion correlates with clinical markers of renal disease in a large population of type 1 diabetic patients with diabetic nephropathy. Diabetes Care 29: 83–88, 2006 [DOI] [PubMed] [Google Scholar]
  • 7.Gilbert RE, Akdeniz A, Weitz S, Usinger WR, Molineaux C, Jones SE, Langham RG, Jerums G: Urinary connective tissue growth factor excretion in patients with type 1 diabetes and nephropathy. Diabetes Care 26: 2632–2636, 2003 [DOI] [PubMed] [Google Scholar]
  • 8.Guha M, Xu ZG, Tung D, Lanting L, Natarajan R: Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes. FASEB J 21: 3355–3368, 2007 [DOI] [PubMed] [Google Scholar]
  • 9.Blom IE, van Dijk AJ, Wieten L, Duran K, Ito Y, Kleij L, deNichilo M, Rabelink TJ, Weening JJ, Aten J, Goldschmeding R: In vitro evidence for differential involvement of CTGF, TGFbeta, and PDGF-BB in mesangial response to injury. Nephrol Dial Transplant 16: 1139–1148, 2001 [DOI] [PubMed] [Google Scholar]
  • 10.Abdel-Wahab N, Weston BS, Roberts T, Mason RM: Connective tissue growth factor and regulation of the mesangial cell cycle: Role in cellular hypertrophy. J Am Soc Nephrol 13: 2437–2445, 2002 [DOI] [PubMed] [Google Scholar]
  • 11.Burns WC, Twigg SM, Forbes JM, Pete J, Tikellis C, Thallas-Bonke V, Thomas MC, Cooper ME, Kantharidis P: Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: Implications for diabetic renal disease. J Am Soc Nephrol 17: 2484–2494, 2006 [DOI] [PubMed] [Google Scholar]
  • 12.Abreu JG, Ketpura NI, Reversade B, De Robertis EM: Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4: 599–604, 2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lam S, van der Geest RN, Verhagen NA, van Nieuwenhoven FA, Blom 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 [DOI] [PubMed] [Google Scholar]
  • 14.Raatikainen-Ahokas A, Hytonen M, Tenhunen A, Sainio K, Sariola H: BMP-4 affects the differentiation of metanephric mesenchyme and reveals an early anterior-posterior axis of the embryonic kidney. Dev Dyn 217: 146–158, 2000 [DOI] [PubMed] [Google Scholar]
  • 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 [DOI] [PubMed] [Google Scholar]
  • 16.Godin RE, Robertson EJ, Dudley AT: Role of BMP family members during kidney development. Int J Dev Biol 43: 405–411, 1999 [PubMed] [Google Scholar]
  • 17.Vukicevic S, Basic V, Rogic D, Basic N, Shih MS, Shepard A, Jin D, Dattatreyamurty B, Jones W, Dorai H, Ryan S, Griffiths D, Maliakal J, Jelic M, Pastorcic M, Stavljenic A, Sampath TK: Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat. J Clin Invest 102: 202–214, 1998 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, Kalluri R: BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9: 964–968, 2003 [DOI] [PubMed] [Google Scholar]
  • 19.Wang SN, Lapage J, Hirschberg R: Loss of tubular bone morphogenetic protein-7 in diabetic nephropathy. J Am Soc Nephrol 12: 2392–2399, 2001 [DOI] [PubMed] [Google Scholar]
  • 20.De Petris L, Hruska KA, Chiechio S, Liapis H: Bone morphogenetic protein-7 delays podocyte injury due to high glucose. Nephrol Dial Transplant 22: 3442–3450, 2007 [DOI] [PubMed] [Google Scholar]
  • 21.Mitu GM, Wang S, Hirschberg R: BMP7 is a podocyte survival factor and rescues podocytes from diabetic injury. Am J Physiol Renal Physiol 293: F1641–F1648, 2007 [DOI] [PubMed] [Google Scholar]
  • 22.Wang S, Chen Q, Simon TC, Strebeck F, Chaudhary L, Morrissey J, Liapis H, Klahr S, Hruska KA: Bone morphogenic protein-7 (BMP-7), a novel therapy for diabetic nephropathy. Kidney Int 63: 2037–2049, 2003 [DOI] [PubMed] [Google Scholar]
  • 23.Wang S, de CM, Kopp J, Mitu G, Lapage J, Hirschberg R: Renal bone morphogenetic protein-7 protects against diabetic nephropathy. J Am Soc Nephrol 17: 2504–2512, 2006 [DOI] [PubMed] [Google Scholar]
  • 24.Sugimoto H, Grahovac G, Zeisberg M, Kalluri R: Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced glycation end product inhibitors. Diabetes 56: 1825–1833, 2007 [DOI] [PubMed] [Google Scholar]
  • 25.Neilson EG: Setting a trap for tissue fibrosis. Nat Med 11: 373–374, 2005 [DOI] [PubMed] [Google Scholar]
  • 26.Wahab NA, Mason RM: A critical look at growth factors and epithelial-to-mesenchymal transition in the adult kidney: Interrelationships between growth factors that regulate EMT in the adult kidney. Nephron Exp Nephrol 104: e129–e134, 2006 [DOI] [PubMed] [Google Scholar]
  • 27.Korchynskyi O, Ten DP: Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem 277: 4883–4891, 2002 [DOI] [PubMed] [Google Scholar]
  • 28.Zeisberg M: Bone morphogenic protein-7 and the kidney: Current concepts and open questions. Nephrol Dial Transplant 21: 568–573, 2006 [DOI] [PubMed] [Google Scholar]
  • 29.Wolf G, Ziyadeh FN: Cellular and molecular mechanisms of proteinuria in diabetic nephropathy. Nephron Physiol 106: 26–31, 2007 [DOI] [PubMed] [Google Scholar]
  • 30.MCLennan SV, Kelly DJ, Cox AJ, Cao Z, Lyons JG, Yue DK, Gilbert RE: Decreased matrix degradation in diabetic nephropathy: Effects of ACE inhibition on the expression and activities of matrix metalloproteinases. Diabetologia 45: 268–275, 2002 [DOI] [PubMed] [Google Scholar]
  • 31.Del PD, Anglani F, Forino M, Ceol M, Fioretto P, Nosadini R, Baggio B, Gambaro G: Down-regulation of glomerular matrix metalloproteinase-2 gene in human NIDDM. Diabetologia 40: 1449–1454, 1997 [DOI] [PubMed] [Google Scholar]
  • 32.Thomson SE, McLennan SV, Kirwan PD, Heffernan SJ, Hennessy A, Yue DK, Twigg SM: Renal connective tissue growth factor correlates with glomerular basement membrane thickness and prospective albuminuria in a non-human primate model of diabetes: Possible predictive marker for incipient diabetic nephropathy. J Diabetes Complications April 15, 2008 [epub ahead of print] [DOI] [PubMed]
  • 33.Flyvbjerg A, Khatir D, Jensen LJN, Lomongsod E, Liu DY, Rasch R, Usinger WR: Long-term renal effects of a neutralizing connective tissue growth factor (CTGF)-antibody in obese type 2 diabetic mice [Abstract]. J Am Soc Nephrol 15: 261A, 2004. 14747372 [Google Scholar]
  • 34.Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM: Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130: 2779–2791, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Ryan MJ, Johnson G, Kirk J, Fuerstenberg SM, Zager RA, Torok-Storb B: HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int 45: 48–57, 1994 [DOI] [PubMed] [Google Scholar]
  • 36.Muller GA, Frank J, Rodemann HP, Engler-Blum G: Human renal fibroblast cell lines (tFKIF and tNKF) are new tools to investigate pathophysiologic mechanisms of renal interstitial fibrosis. Exp Nephrol 3: 127–133, 1995 [PubMed] [Google Scholar]
  • 37.Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193–199, 1991 [DOI] [PubMed] [Google Scholar]

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