Connective tissue growth factor (CTGF) is a 38-kD cysteine-rich peptide that belongs to the emerging CCN (CTGF, cyr 61/cef 10, nov) family of multifunctional growth factors (1–4). Murine CTGF, which is also called fisp-12, promotes chemotaxis, migration, adhesion, proliferation, and differentiation or formation of the extracellular matrix (ECM), depending on whether the target cell is a fibroblast, chondrocyte, or vascular endothelial cell (2). CTGF is induced exclusively by TGF-β and is thought to mediate the latter’s profibrotic effects by modulating fibroblast cell growth and ECM protein synthesis (1,4). CTGF expression has been shown to increase in a variety of human diseases and experimental disease models that are characterized by fibrosis, including those that affect the kidney, skin, blood vessels, lung, and liver (1,2). In the case of the kidney, CTGF mRNA was shown to be expressed primarily in glomerular mesangial, epithelial, and endothelial cells in IgA nephropathy, focal and segmental glomerulosclerosis, and diabetic nephropathy (5,6). Moreover, CTGF mRNA overexpression was found in tubular epithelial cells and interstitial cells at sites of chronic interstitial damage (5,6). In the remnant kidney of the subtotal nephrectomy (SNx) model and in the kidneys with ureteral obstruction, tubular CTGF was shown to be expressed in response to renal interstitial fibrosis (7,8). These findings strongly suggest that renal interstitial fibrogenesis is mediated by CTGF that is expressed in the tubular epithelial cells. However, whether CTGF plays a direct role in vivo in renal interstitial fibrogenesis remains to be elucidated. In the present study, we demonstrated, using neutralization protocols, that tubular CTGF directly and significantly contributed to TGF-β1-dependent renal interstitial fibrogenesis.
Materials and Methods
CTGF Antisense Oligodeoxynucleotides
Phosphorothioate-capped oligodeoxynucleotides (ODN) were synthesized by an automated synthesizer. After deprotection, ODN were dissolved in water, extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and redissolved in water. The CTGF sense ODN sequence was 5′-ATGCTCGCCTCCGTCGCA-3′, the antisense ODN sequence was 5′-TGCGACGGAGGCGAGCAT-3′, and the mutated-antisense ODN sequence was 5′-TGCAGTGGAAATGAGTGC-3′; these sequences were chosen, after analyzing the murine CTGF (fisp12) gene (9), as likely to disrupt ribosomal docking because of their proximity to the initiation site.
Cultured murine proximal tubular epithelial cells (mProx24) and tubulointerstitial fibroblasts (TFB) were maintained in DMEM that contained 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin (growth medium) (7). Pilot experiments revealed that recombinant human TGF-β1 (rhTGF-β1; approximately 0.1 to 30 ng/ml; R&D Systems, Minneapolis, MN) did not induce expression of the α1(I) procollagen (α1COLI) gene in mProx24 or the CTGF gene in TFB (7,10). The latter finding, which was also reported in a previous study (11), was possibly due to the spontaneous transformation of the TFB in culture.
TFB were seeded in six-well plates (5 × 104 cells/well) and incubated overnight in growth medium. The medium was then changed to DMEM that contained 0.5% FCS to render the cells quiescent. After 48 h, 20 ng/ml recombinant human CTGF (rhCTGF) (7) was added to the cultures, and cells were harvested for mRNA extraction as described below 2 or 12 h later.
TFB were seeded (1.5 × 104 cells/well) in six-well plates, and mProx24 were seeded (3 × 104 cells/well) in cell culture inserts (Falcon Cell Culture Inserts, 0.4-μm pore size; Becton Dickinson, Franklin Lakes, NJ). On day 1, the medium was changed to K-1 medium (50:50 Ham’s F-12/DMEM with 5 μg/ml transferrin, 5 μg/ml insulin, and 5 × 10−8 M hydrocortisone) to induce cell quiescence. At that time, the inserts that contained the mProx24 were placed into the wells that contained the TFB. On day 4, the medium was changed to fresh K-1 medium that contained 1.0 ng/ml rhTGF-β1 alone or in combination with either neutralizing anti-CTGF antibody (10 μg/ml) (7) or anti-PDGF antibody (20 μg/ml; R&D Systems). The doses of neutralizing antibodies were determined in the pilot, dose-response experiments. The anti-CTGF antibody was raised in rabbits by immunizing them with a synthetic CTGF peptide that was composed of 20 amino acids, the sequence of which was the same as that of Fisp12. We confirmed that this antibody was able to neutralize the proliferative effects of rhCTGF on bovine aortic endothelial cells in vitro (12). Cells that were cultured in K-1 medium alone served as the negative control. After 24 h of stimulation, total RNA was extracted from the TFB.
mProx24 cells (5 × 106) were washed in serum-free medium and then exposed to dithiothreitol-activated streptolysin-O (SL-O; Sigma, St. Louis, MO) at 150 U/ml and 30 μM ODN in 200 μl of permeabilization buffer (137 mM NaCl and 100 mM PIPES [pH 7.4] that contained 5.6 mM glucose, 2.7 mM KCl, 2.7 mM EGTA, 1 mM Na-ATP, and 0.1% BSA). After an incubation of 20 min at 37°C, 1 ml of growth medium was used to reseal the cells. Twenty minutes later, the cells were transferred to culture dishes that contained 10 ml of growth medium and were incubated for 4 h later. The cells were treated with 1.0 ng/ml rhTGF-β1 for another 3 h, after which the cells were harvested for mRNA extraction. Untreated cells were permeabilized with SL-O without ODN treatment and stimulated with rhTGF-β1; control cells were also permeabilized with SL-O without ODN treatment but were not stimulated with rhTGF-β1.
Five-week-old TGF-β1 transgenic mice that had undergone SNx were used in this study and served as a model of renal fibrosis (7,13). These mice reproducibly develop significant interstitial fibrosis at approximately 8 to 10 wk after surgery. TGF-β1 transgenic mice that had been anesthestized and received wound suture but were not nephrectomized were used as negative controls. We confirmed that the control, transgenic mice did not show any significant renal fibrosis up to 17 wk of age, although we have reported that some older animals do develop renal fibrosis (7,13). Our laboratory and others reported that when a given antisense ODN was injected intravenously into rodents, it was absorbed into the proximal tubular epithelium, where it was retained for nearly 48 h and during which time it could block transcription of a target gene (14–16). Animals received an intravenous injection of the CTGF antisense and mutated antisense ODN at a concentration of 1.0 mg/kg every 2 d from day 29 to day 56 (SNx mice that received an injection of antisense ODN, n = 10; mice that received an injection of mutated-antisense ODN, n = 10; mice that were not treated with any ODN, n = 10; negative control mice, n = 8). Two mice from each group were killed 48 h later to assess the effect of the first injection (i.e., day 31) on renal CTGF expression. After the mice were killed on day 31 or 56, half of each remnant kidney was fixed in 4% paraformaldehyde in PBS overnight and processed into paraffin blocks for histopathologic analysis. A sampling of fixed tissues was serially rinsed in different concentrations of sucrose and then snap-frozen for in situ hybridization. The other half of each remnant kidney was used for RNA extraction as described below.
Morphologic Examination and Immunohistochemistry
Four-micrometer sections were cut from the paraffin blocks and processed for hematoxylin-eosin and Masson’s trichrome (MT) staining. Interstitial fibrosis in MT-stained sections was assessed quantitatively at ×200 magnification using a Mac SCOPE (Version 2.5; Mitani Corp., Fukui, Japan). All glomeruli and vessels were subtracted from a given field, yielding a target area of tubulointerstitium. Collagenous, fibrotic areas in blue then were quantified and expressed as a mean percentage area per field of 10 randomly chosen cortical fields (17). For indirect immunoperoxidase staining, sections were deparaffinized, rehydrated, and treated with proteinase K. These sections then were boiled in citrate buffer in a microwave to unmask antigenic sites. Endogenous biotin was blocked by using a Biotin Blocking System (X0590; DAKO Corp., Carpinteria, CA), after which sections were immersed in 3% H2O2 in methanol to inactivate endogenous peroxidase and treated with 1% nonfat milk in PBS to block nonspecific binding. Rabbit polyclonal anti-CTGF (1:200), anti–TGF-β1 (1:500) (18), anti-COLI (1:400) (18), anti-fibronectin (anti-FN) (1:400; Life Technologies BRL, Grand Island, NY), anti–plasminogen activator inhibitor-1 (anti–PAI-1; 1:200; American Diagnostica, Greenwich, CT), anti–FSP-1 (1:500) (13), goat polyclonal anti–tissue inhibitor of metalloproteinase-1 (anti–TIMP-1; 1:200; G-T Research Product, Minneapolis, MN), and rat monoclonal anti-Mac1 (1:100; PharMingen, San Diego, CA) antibodies were applied to particular sections as the primary antibodies. Subsequently, sections were incubated with appropriate biotin-conjugated, secondary antibodies. A Catalyzed Signal Amplification System Peroxidase (K1500; DAKO) was used to visualize the antibody reactions according to the manufacturer’s instructions. The secondary antibody was isolated by immunoaffinity chromatography. Negative control sections were treated as described above, but the primary antibody was omitted. The stained areas were assessed quantitatively as described above.
In Situ Hybridization
In situ hybridization was performed as described previously using cRNA probes generated from a cDNA clone encoding mouse CTGF (fisp-12) (7), with a small modification in the washing procedure. This cDNA fragment of CTGF was prepared in our laboratory by reverse transcriptase-PCR (RT-PCR) according to the GenBank data (accession no. M70642). In this procedure, the final wash with 0.2× SSC was performed twice at 50°C for 15 min.
Total RNA was extracted from renal tissues and cultured cells by homogenization in TRIzol (Life Technologies BRL) according to the manufacturer’s instructions. All of the RNA samples were treated with the RNase-free DNase I (Qiagen, Basel, Switzerland) before the RT-PCR. Real-time quantitative one-step RT-PCR assay was performed to quantify mRNA using QuantiTect SYBR Green RT-PCR (Qiagen) and an ABI PRISM 7700 sequence detection system (Applied Biosystems, Tokyo, Japan). The following mRNA were quantified: The profibrotic growth factors CTGF and TGF-β1, the ECM proteins α1COLI and FN EIIIA isoform (FN-EIIIA), the proteinase inhibitors PAI-1 and TIMP-1, and glyceraldehyde-3-phosphate dehydrogenase. The primers used for real-time RT-PCR were as follows: CTGF primer, forward 5′-CTTCTGCGATTTCGGCTCC-3′, reverse 5′-TACACCGACCCACCGAAGA-3′; TGF-β1 primer, forward 5′-CAGTGGCTGAACCAAGGAGAC-3′, reverse 5′-ATCCCGTTGATTTCCACGTG-3′; α1COLI primer, forward 5′-TGTAAACTCCCTCCACCCCA-3′, reverse 5′-TCGTCTGTTTCCAGGGTTGG-3′; FN-EIIIA primer, forward 5′-ATCCGGGAGCTTTTCCCTG-3′, reverse 5′-TGCAAGGCAACCACACTGAC-3′; PAI-1 primer, forward 5′-TCGTGGAACTGCCCTACCAG-3′, reverse 5′-ATGTTGGTGAGGGCGGAGAG-3′; TIMP-1 primer, forward 5′-TGTGGGAAATGCCGCAGATA-3′, reverse 5′-TTCACTGCGGTTCTGGGACT-3′; and glyceraldehyde-3-phosphate dehydrogenase primer, forward 5′-TGCAGTGGCAAAGTGGAGATT-3′, reverse 5′-TTGAATTTGCCGTGAGTGGA-3′. All of these ODN were designed by using Primer Express software (Perkin Elmer, Foster City, CA). Preliminary RT-PCR experiments with these primer sets yielded single products of the expected sizes.
All values were expressed as the mean ± SD. An ANOVA followed by Bonferroni/Dunnett test was used to determine the significance of differences between groups after multiple comparisons were carried out. P < 0.05 was considered to be statistically significant.
Recombinant CTGF and CTGF Derived from Tubular Epithelial Cells (mProx24) after TGF-β1 Treatment Increased the mRNA Levels of Profibrotic Molecules in TFB
In the first instance, the increase in mRNA levels of profibrotic molecules by CTGF was tested. rhCTGF (20 ng/ml) significantly increased mRNA levels of the ECM proteins α1COLI and FN-EIIIA and the proteinase inhibitors PAI-1 and TIMP-1 in TFB in the monolayer culture in variable manners (Figure 1A). For examining the profibrotic effects of CTGF derived from rhTGF-β1–stimulated tubular epithelial cells, an in vitro culture model of the renal subepithelial mesenchyme was set up by using TFB and mProx24. TFB in the monolayer culture that were treated only with 1.0 ng/ml rhTGF-β1 showed a small but statistically insignificant increase in their mRNA levels of α1COLI and FN-EIIIA (Figure 1, B and C). Conversely, rhTGF-β1–treated TFB that were co-cultured with mProx24 showed a significant increase in their mRNA levels of α1COLI and FN-EIIIA; these elevations were significantly inhibited by treatment with neutralizing anti-CTGF antibody but not with neutralizing anti-PDGF antibody, supporting the notion that these effects were mediated by CTGF (Figure 1, B and C). Because the TFB had lost their capacity to produce CTGF, the CTGF in these cultures must have been derived solely from the mProx24. Other profibrotic actions of CTGF, such as enhancement of mRNA levels of PAI-1 and TIMP-1 in TFB, also were tested. In these experiments, rhTGF-β1-stimulated TFB also showed the enhanced mRNA levels of these molecules, which was similarly blocked by neutralizing anti-CTGF but not anti-PDGF antibodies (Figure 1, D and E).
CTGF Antisense ODN Reduced the Level of CTGF mRNA in Tubular Epithelial Cells In Vitro and In Vivo
CTGF antisense ODN significantly blocked an increase in CTGF mRNA in mProx24 that were treated with TGF-β1, whereas sense and mutated antisense ODN did not (Figure 2A). These effects were not due to a nonspecific ODN toxicity because all of the cells that were stimulated with rhTGF-β1 increased their mRNA levels of TGF-β1 regardless of ODN treatment (Figure 2A). Administration of the antisense ODN intravenously significantly reduced the level of CTGF mRNA in the tubular epithelium in the remnant kidney of SNx mice, whereas neither the mutated-antisense ODN nor the sense ODN had such an effect (Figure 2, B through D).
Reduction in CTGF Expression in the Tubular Epithelium Significantly Attenuated Renal Interstitial Fibrogenesis and Functional Impairment
In the following in vivo experiments, sense ODN was not used because mutated antisense ODN acted similarly in vitro and in vivo as described above. A reduction in the level of CTGF mRNA in the tubular epithelium by antisense ODN (Figure 3) significantly decreased the number of interstitial cells such as FSP-1+ fibroblasts and Mac1+ monocytes (data not shown) and attenuated interstitial fibrosis in the remnant kidney of SNx mice (Figure 4). Tubular expression of CTGF protein was significantly reduced in the day 56 remnant kidney after multiple injections of antisense ODN (Figure 3, B and C). In contrast, because glomerular CTGF expression was not significantly affected by antisense ODN treatment (Figure 3C), glomerulosclerosis developed in the day 56 remnant kidney of animals that received such treatment, despite their attenuated interstitial fibrosis (Figure 4C). Although TGF-β1 expression was not significantly affected by CTGF antisense ODN treatment (Figure 3, D through F), expression of the ECM protein α1COLI and FN-EIIIA (Figure 5) and the proteinase inhibitors PAI-1 and TIMP-1 (Figure 6) fell in parallel with decreased expression of CTGF. Immunohistochemistry revealed that the protein expression of COLI, FN, PAI-1, and TIMP-1 was significantly reduced in the CTGF antisense ODN-treated day 56 remnant kidneys (Figures 5, A through C, and 6, A and B, and data not shown); these findings were consistent with the mRNA levels of these factors. Mutated antisense ODN treatment was without significant effects. Although CTGF antisense ODN treatment did not markedly affect urinary protein excretion, it significantly attenuated renal functional impairment in day 56 mice that had remnant kidneys (Table 1).
Our results showed, for the first time, that CTGF expression in tubular epithelial cells plays a pivotal role in TGF-β1-dependent interstitial fibrogenesis in the remnant kidney of the SNx model. The close association between increased TGF-β expression and renal fibrogenesis has been observed in a number of renal diseases (19,20), and it has been shown that blockade of TGF-β activity using a neutralizing antibody, antisense ODN, or the natural inhibitor decorin significantly suppressed renal fibrogenesis (21–23). These data point to an important role for TGF-β in promoting renal fibrosis. Several downstream mediators of the profibrotic action of TGF-β have been proposed (4,24). One such mediator is CTGF, which was detected at the mRNA level not only in glomerular cells but also in tubular epithelial cells and interstitial cells in a variety of chronic renal diseases (5,6). We previously reported that a high dose of hepatocyte growth factor (HGF) attenuated the induction of CTGF by TGF-β1 in tubular epithelial cells and that this was at least partially responsible for the significant suppression of renal interstitial fibrosis seen in the remnant kidney of SNx model under HGF treatment (7,10). These findings suggested that tubular CTGF played an important role in renal fibrogenesis.
In support of this notion are our current results that showed that treatment with rhCTGF increased the mRNA levels of the profibrotic molecules α1COLI, FN-EIIIA, PAI-1, and TIMP-1 in TFB. Although others have reported similar findings such as COLI and FN in rat renal fibroblasts (8) and FN and PAI-1 in human mesangial cells (25), to our knowledge, this is the first report to show an increase in TIMP-1 mRNA levels in murine renal fibroblasts in response to CTGF treatment. rhTGF-β1 (1.0 ng/ml) increased the mRNA levels of α1COLI and FN-EIIIA in TFB that were co-cultured with mProx24 to a significantly greater degree than in TFB that were cultured alone, suggesting the involvement of profibrotic humoral factors secreted by the mProx24. To confirm that tubular CTGF acted as a mediator of the profibrotic effects of TGF-β1 in TGF-β-dependent renal interstitial fibrogenesis, we neutralized CTGF that was secreted into the medium of cultures that contained TFB, mProx24, and rhTGF-β1 and found that such treatment abolished these effects.
In addition to CTGF, PDGF and fibroblast growth factor-2 (FGF-2) are potential mediators of the profibrotic effects of TGF-β (26,27). However, anti-PDGF and anti–FGF-2 (R&D Systems) antibodies did not demonstrate any neutralizing effects in our study (the latter data not shown), whereas neutralizing anti-CTGF antibody significantly attenuated the expression of α1COLI and FN-EIIIA in TFB, suggesting that neither PDGF nor FGF-2 had profibrotic effects in our co-culture system. It was interesting that the neutralization of CTGF significantly reduced TGF-β1’s increases in mRNA levels of α1COLI, PAI-1, and TIMP-1 in our study. Because the expression of these genes is controlled by Smad3 (28), TGF-β is thought to induce their transcription directly. CTGF was shown recently to downregulate the expression of Smad7, which is an inhibitor of the Smad signaling pathway. CTGF likely promotes increased TGF-β signaling through this pathway by decreasing the availability of Smad7 (29). CTGF may accelerate binding of TGF-β to its receptor and thereby promote TGF-β signaling (30). All of these effects could account for the results that we obtained when CTGF was neutralized in vitro. They could also help to explain why CTGF derived from mProx24 so significantly enhanced TGF-β’s effects on the mRNA levels of these genes in TFB in our co-culture system.
When CTGF gene expression was blocked in the tubular epithelial cells in the remnant kidney of TGF-β1 transgenic mice, intravenously administered CTGF antisense ODN reduced the CTGF mRNA level in the tubular epithelial cells, in addition to the mRNA levels of other ECM proteins and proteinase inhibitors, and suppressed interstitial fibrogenesis despite the sustained levels of TGF-β1 mRNA. These in vivo findings support the notion that tubular CTGF acts as the primary mediator of the profibrotic effects of TGF-β1 in TGF-β-dependent renal interstitial fibrogenesis. It is likely that HGF attenuates renal fibrogenesis by altering tubular CTGF expression (7,10). In this study, reduced CTGF mRNA levels in the tubular epithelial cells were also associated with a decrease in the number of infiltrating monocytes. We demonstrated that direct contact between fibroblasts and monocytes enhanced monocyte chemoattractant protein-1 synthesis in fibroblasts, facilitating monocyte infiltration (31). Oda et al. (32) reported that interstitial monocyte infiltration was attenuated in the ureteral obstruction model in the PAI-1 gene knockout mice. Treatment with CTGF antisense ODN decreased the number of interstitial fibroblasts and the PAI-1 mRNA levels in the remnant kidney, which therefore, at least partially, accounted for its anti-inflammatory effects. Because it is very difficult to transduce ODN into renal interstitial cells via the systemic circulation, it was unlikely that CTGF expression in interstitial fibroblasts was suppressed by injected CTGF antisense ODN. As mentioned above, the actions of TGF-β are significantly accelerated by coexisting CTGF, and we suppose that early, essential supply of CTGF from tubular epithelial cells was blocked by CTGF antisense ODN and resulted in significant attenuation of profibrotic effects of TGF-β, e.g., induction of CTGF and ECM molecules, in the tubulointerstitial area.
In addition to tubular epithelial cells, glomerular visceral epithelial cells were reported to synthesize CTGF (5). In contrast to mesenchymal cells such as mesangial cells, fibroblasts, endothelial cells, and chondrocytes, epithelial cells have not been well investigated as a potential source of CTGF production (2,3). As in mesenchymal cells, TGF-β is an important inducer of CTGF in renal tubular epithelial cells (7,10,33). Both basal control element-1 (formerly called TGF-β response element) and the Smad pathway (via the Smad binding element in the CTGF promoter) were shown to be involved in basal and TGF-β-induced CTGF expression in these cells (3). Moreover, Ras/MEK/Erk, protein kinase C, and tyrosine kinase activity was reported to contribute to the basal- and TGF-β-induced CTGF expression in mesangial cells in a Smad-independent manner (34). Although these molecular mechanisms are also likely to be involved in the expression of CTGF in tubular epithelial cells, this remain to be determined.
Recently, Yokoi et al. (35) used antisense ODN technology to demonstrate that CTGF expression in interstitial cells played an important role in interstitial fibrogenesis in the unilateral ureteral obstruction model. Their findings, coupled with ours, support the notion that CTGF acts as an important profibrotic mediator of TGF-β’s effects in the renal tubulointerstitium while not eliminating the possibility that it plays a similar role in the glomerulus. In fact, a number of studies have shown that mesangial CTGF expression plays a role in the pathogenesis of glomerulosclerosis, especially in diabetic nephropathy (5,6,11,25,36). Because intravenously injected antisense ODN is not appreciably absorbed by glomerular cells, it cannot significantly block their expression of the target genes (14,15). Thus, in this study, it was unlikely that glomerular CTGF expression was blocked by treatment of mice with antisense ODN. The degree of interstitial pathologic changes is known to correlate with renal function better than does glomerular pathology. Therefore, suppression of interstitial fibrosis, as opposed to glomerular damage, by CTGF antisense ODN treatment would be expected to improve renal function but not correct urinary protein excretion.
In conclusion, we found that tubular CTGF induces renal interstitial fibrogenesis in the remnant kidney of the SNx model. Because TGF-β has a host of effects in different cell types, blockage of this growth factor is not a realistic therapeutic option for the treatment of this condition (37). Conversely, in light of the above data, CTGF may be a promising target for antifibrotic therapy in TGF-β-dependent renal diseases such as diabetic nephropathy; however, further studies are needed to provide support for future intervention strategies in renal fibrogenesis.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
A portion of this work was presented at the 35th Annual Meeting of the American Society of Nephrology; November 13–16, 2003; San Diego, CA.
We are grateful to M. Otobe for technical assistance.
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