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Reduction in Connective Tissue Growth Factor by Antisense Treatment Ameliorates Renal Tubulointerstitial Fibrosis

Hideki Yokoi^*, Masashi Mukoyama^*, Tetsuya Nagae^*, Kiyoshi Mori^*, Takayoshi Suganami^*, Kazutomo Sawai^*, Tetsuro Yoshioka^*, Masao Koshikawa^*, Takashi Nishida, Masaharu Takigawa, Akira Sugawara^* and Kazuwa Nakao^*

*Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; and Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Correspondence to Dr. Masashi Mukoyama, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: +81-75-751-4285; Fax: +81-75-771-9452; E-mail: muko{at}kuhp.kyoto-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
ABSTRACT. Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-–induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and 1(I) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and 1(I) collagen genes, whereas TGF- gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of -smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tubulointerstitial fibrosis is a common feature of progressive renal injury in almost all forms of renal diseases. It has been shown that tubulointerstitial injury is a more consistent predictor of functional impairment than glomerular damage (1,2). Among proposed mechanisms by which the renal injury progresses, TGF- is postulated to play a central role in the development of tubulointerstitial fibrosis as well as glomerulosclerosis (3–5). TGF- promotes the accumulation of extracellular matrix (ECM), through the enhanced synthesis of ECM proteins and inhibition of their degradation (3–5). Elevated renal expression of TGF- has been shown in nearly every experimental model of renal injury characterized by fibrosis (5); transgenic overproduction of TGF-1 into the circulation in mice resulted in marked tubulointerstitial fibrosis with severe glomerulonephritis (6). These studies have provided plausible evidence for TGF- and its downstream pathway as a potential therapeutic target against renal fibrosis (5,6). Long-term suppression of TGF-, however, is still controversial because of its antiproliferative and anti-inflammatory effects (6). These notions have therefore prompted us to investigate the mechanisms specific to the profibrotic action of TGF-.

Connective tissue growth factor (CTGF/CCN2) (7) is thought to be an important factor that mediates some of downstream events of fibrogenic properties of TGF- (8–11). In cultured fibroblasts, CTGF gene expression is strongly induced by TGF- (10–12), in a smad 3/smad 4-dependent manner (13). Addition of CTGF in turn potently stimulates fibroblast proliferation and ECM protein synthesis (14). In the kidney, enhanced expression of CTGF has been shown in proliferative and fibrotic lesions of various human and experimental renal diseases, including glomerulonephritis and diabetic nephropathy (15–18). We have reported that CTGF mRNA is markedly upregulated, subsequent to TGF-1, in the interstitial fibrotic areas and tubular epithelial cells as well as in parietal glomerular epithelial cells in a rat model of tubulointerstitial fibrosis (19). These observations led to the hypothesis that CTGF could serve as a downstream effector of TGF- in the kidney. Indeed, we reveal that the blockade of CTGF using antisense oligonucleotide (ODN) efficiently inhibits TGF-–induced fibronectin production in cultured renal interstitial fibroblasts (11,19). Moreover, it has already been shown that TGF-–induced collagen synthesis is CTGF dependent, using the cells treated with neutralizing CTGF antibody or those stably transfected with an antisense CTGF gene (20). Although these observations strongly suggest that CTGF is a key mediator of ECM production, the role of CTGF in renal fibrogenesis in vivo still remains to be elucidated.

Previous reports have shown that proliferation of both tubular and interstitial cells was observed after unilateral ureteral obstruction (UUO) (21,22). Inhibition of the renin-angiotensin system attenuates cell proliferation in the obstructed kidney (21). However, the blockade of TGF-, which has an antiproliferative action, increases tubular and interstitial cell proliferation (22). CTGF stimulates both cell proliferation and ECM accumulation in cultured fibroblasts (10), but its action in interstitial fibroblasts in vivo remains unclear. In the present study, we investigated the effects of CTGF antisense ODN, with a kidney-targeted gene transfer technique using hydrodynamic pressure (23), on ECM production and interstitial fibrosis during the progression of obstructive nephropathy. Furthermore, we evaluated the proliferation of tubular and interstitial cells in obstructive nephropathy with CTGF antisense treatment.

	Materials and Methods

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In Vivo Gene Transfer Using Hydrodynamics-Based Technique
All animal experiments were conducted in accordance with our institutional guidelines for animal research. Naked plasmid or ODN was introduced into rat kidney by means of a recently developed hydrodynamics-based gene transfer technique (23). In brief, male Wistar rats that weighed 200 g were anesthetized with pentobarbital, and the left kidney was exposed by midline incision. To first examine functional expression of the transgene introduced, 1 mg of pCAGGS-LacZ (a gift from Dr. J. Miyazaki, Osaka University) diluted in 750 µl of Ringer solution was injected into the left kidney via the renal vein using a 24-gauge SURFLO intravenous catheter (Terumo, Tokyo, Japan), after clamping the left renal vein. Blood flow was reestablished immediately after injection. Two days after injection, frozen kidney sections (4 µm thick) embedded in OCT compound were fixed in 1.5% glutaraldehyde, washed with PBS, and incubated with X-gal staining solution containing 1 mg/ml X-gal, 2 mM MgCl₂, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, and 0.5% Nonidet P-40 in PBS at 37°C for 3 h.

For assessing the transfection efficiency and site of ODN introduced, FITC-labeled ODN (200 µg) in 750 µl of Ringer solution was injected into the left kidney as described. The sequence of phosphorothioate ODN (Bex, Tokyo, Japan) for rat CTGF used was as follows: 5'-GAC GGA GGC GAG CAT GGT-3'. Ten minutes after injection, the kidneys were perfused with PBS. For locating FITC-labeled ODN, 4-µm-thick cryostat sections in OCT compound fixed in cold acetone were incubated with rabbit anti-collagen IV (Chemicon International, Temecula, CA) for 1 h, followed by incubation with rhodamine-conjugated anti-rabbit IgG for 1 h. The specimens were also stained with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The slides were developed with confocal laser scanning microscopy (LSM 5 Pascal; Carl Zeiss Vision, Munich, Germany).

UUO with CTGF Antisense ODN Transfer
After rats were anesthetized, the left ureter was ligated with 4-0 silk at two points (19). Antisense or control ODN (200 µg) in 750 µl of Ringer solution was then injected into the left kidney via the renal vein. The sequences of phosphorothioate ODN (Bex) for rat CTGF used were as follows: antisense ODN, 5'-GAC GGA GGC GAG CAT GGT-3'; and control reverse ODN, 5'-TGG TAC GAG CGG AGG CAG-3'. The antisense sequence is complementary to rat CTGF cDNA around the translation initiation codon (underlined in sequences); the sequence was effective in blocking CTGF expression in vitro (11,19). For sham operation, vehicle was injected into the kidney without ureteral ligation. Rats were killed 7 d after UUO or sham operation (n = 7 each), and the kidneys were harvested.

Northern Blot Analysis
Total RNA from the whole kidney was extracted by the acid guanidinium thiocyanate-phenol-chloroform method. Northern blot analysis was performed using 30 µg of total RNA as described (19). Briefly, hybridization was performed at 42°C overnight with [³²P]-labeled probes for rat CTGF, TGF-1, fibronectin (19), fibronectin extra domain (ED)-A (nucleotides 5370 to 5632) (24), or human pro-1(I) collagen cDNA (11). The membranes were washed at 55°C in 1x SSC/0.1% SDS, and autoradiography was performed for 24 h with BAS-2500 system (Fuji Photo Film, Tokyo, Japan). The amount of RNA loaded was normalized with human glyceraldehyde-3-phosphate dehydrogenase (Clontech, Palo Alto, CA).

Histology and Immunohistochemistry
For histologic analysis, sagittal kidney sections fixed with 4% buffered paraformaldehyde were embedded in paraffin, and 2-µm-thick sections were stained with Masson’s trichrome (19). The fibrotic area was measured quantitatively using a computer-aided manipulator (KS400, Carl Zeiss Vision) with slight modifications (25,26). Thirty randomly selected fields were examined by two investigators without knowledge of the origin of the slides. Then the results were expressed as the percentage area of interstitial fibrosis (27).

For immunohistochemical analyses of CTGF, type I collagen, -smooth muscle actin (-SMA), proliferating cell nuclear antigen (PCNA), and Ki-67, the sections were deparaffinized, washed with PBS, and treated with 3% H₂O₂ in methanol for 10 min (19). The samples were incubated with rabbit polyclonal anti-CTGF (Abcam, Cambridge, UK), goat polyclonal anti-type I collagen (Southern Biotechnology, Birmingham, AL), mouse monoclonal anti–-SMA (DAKO Japan, Kyoto, Japan), mouse monoclonal anti-PCNA (DAKO), or mouse monoclonal anti–Ki-67 (DAKO) antibody for 1 h. After incubation with biotin-conjugated secondary antibodies, the specimens were processed using LSAB+ kit (DAKO) and developed with 3,3'-diaminobenzidine tetrahydrochloride. PCNA-positive cells of renal tubules and the interstitium were separately counted in 20 high-power fields (21). For immunofluorescence study of fibronectin ED-A, cryostat sections (4 µm thick) fixed in cold acetone were incubated with mouse anti-fibronectin ED-A (Abcam) for 1 h, followed by incubation with FITC-labeled anti-mouse IgG (Biosource International, Camarillo, CA). The slides were developed by confocal laser scanning microscopy. Ratio of positively stained areas was computer analyzed using NIH Image and expressed as percentages (27).

Western Blot Analysis
Western blot analysis was performed as described (19) with some modifications. The whole kidneys were homogenized in ice-cold solution that contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 4 mM EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 10 mM Na₄P₂O₇, 10 mM NaF, 10 mg/ml aprotinin, 2 mM dithiothreitol, 2 mM sodium orthovanadate, and 1 mM PMSF. The homogenates were centrifuged at 15,000 rpm for 15 min at 4°C, and the supernatants were treated with Laemmli’s sample buffer. Samples (30 µg protein/lane) were separated by 12.5% SDS-PAGE and electrophoretically transferred onto Immobilon polyvinylidine difluoride filter (Millipore, Bedford, MA). Filters were incubated with anti–-SMA or anti-PCNA for 1 h, and immunoblots were then developed using horseradish peroxidase–linked donkey anti-mouse Ig (BioRad, Richmond, CA) and a chemiluminescence kit (Amersham, Arlington Heights, IL). -Actin (antibody from Sigma) was used as an internal control.

Statistical Analyses
Data are expressed as the mean ± SEM. Statistical analysis was performed using one-way ANOVA or the Kruskal-Wallis test as appropriate. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Localization of FITC-Labeled CTGF Antisense ODN
For investigating the validity of the gene transfer method, pCAGGS-LacZ plasmid was injected into the kidney. Positive X-gal staining was observed most prominently in the interstitial cells 2 d after transfection (Figure 1A). Similar staining was observed at day 7 (not shown). Then we transferred FITC-labeled ODN into the kidney. Green fluorescence was seen in the interstitial areas throughout the left kidney (Figure 1B) but not in the contralateral kidney (not shown). Using double staining with DAPI, a substantial portion of FITC-labeled ODN was introduced into the nuclei (Figure 1C). When the specimens were stained with anti-collagen type IV, ODN was observed outside the basement membrane of the tubular epithelium (Figure 1D). Thus, most of the transgene introduced was present in the interstitial areas and active in the interstitial cells, consistent with a previous report (23).

View larger version (72K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Gene transfer into rat kidney by the hydrodynamics-based gene transfer method. (A) A kidney section 2 d after transfection with pCAGGS-LacZ (1 mg) stained with X-gal. Arrows denote a glomerulus. (B) Transfected FITC-labeled oligonucleotide (ODN; 200 µg) is observed in the interstitial areas of the kidney. (C) FITC-labeled ODN (green) was introduced into the nuclei (blue) after transfection. (D) FITC-ODN (green) is detected outside the basement membrane of tubular cells by staining with anti-collagen type IV antibody (red). Magnifications: x200 in A and B, x1000 in C and x400 in D.

Effect of CTGF Antisense ODN on CTGF and TGF-1 Gene Expression

View larger version (50K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Northern blot analyses for connective tissue growth factor (CTGF), TGF-1, fibronectin (FN), fibronectin ED-A, and 1(I) collagen (COL1A1) mRNA expression in the obstructed kidney on day 7 after introduction of CTGF antisense ODN in rats. Thirty micrograms of total RNA from the whole kidney in each lane were electrophoresed. (A) Representative Northern blots. Rat kidney was injected with CTGF antisense ODN (AS) or control reverse ODN (R), after the left ureter was ligated or sham-operated. Seven days after introduction, mRNA expression was analyzed. Glyceraldehyde-3-phosphate dehydrogenase is used as an internal control. (B) The relative mRNA levels of CTGF, TGF-1, fibronectin, fibronectin ED-A, and 1(I) collagen. The mean level of the whole kidney from sham-operated rats is regarded as 1.0 arbitrary unit. S, sham operation; U, unilateral ureteral obstruction (UUO) with vehicle injection; R, UUO with CTGF reverse ODN (200 µg) introduction; AS, UUO with CTGF antisense ODN (200 µg) introduction. Mean ± SE; n = 7; *P < 0.05; **P < 0.01; #P < 0.01 between AS and S; unless marked, not significantly different between AS and S.

View larger version (185K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Immunohistochemical study for CTGF in the kidneys 7 d after UUO with the treatment of ODN. S, sham operation; U, UUO with vehicle injection; R, UUO with CTGF reverse ODN (200 µg) introduction; AS, UUO with CTGF antisense ODN (200 µg) introduction. In control kidneys (S), CTGF was positive in the arteriole (white arrow) and glomerular epithelial cells. Seven days after UUO (U), CTGF staining was positive in the arteriole (white arrow) and was markedly increased in the interstitial cells (black arrowheads) and tubular cells (black arrows). With the CTGF antisense ODN treatment (AS), CTGF staining was attenuated in the interstitium (white arrowhead) but not in the arteriole (white arrow), as compared with control ODN (R).

Effect of CTGF Antisense ODN on ECM Accumulation and Interstitial Fibrosis
To evaluate the role of CTGF in promoting ECM accumulation, we examined the expression of fibronectin, fibronectin ED-A, and 1(I) collagen genes after CTGF antisense ODN treatment by Northern blotting (Figure 2). Fibronectin mRNA expression was upregulated in the obstructed kidneys (2.0-fold of sham operation) and significantly attenuated (by 55 ± 8%; P < 0.05, n = 7) with the transfer of CTGF antisense ODN. Expression of fibronectin ED-A and 1(I) collagen genes was markedly upregulated after UUO, and such induction was also significantly suppressed in CTGF antisense ODN treatment (by 45 ± 8% and 66 ± 10% of vehicle, respectively; Figure 2B).

Immunohistochemical analyses revealed that the expression of fibronectin ED-A was markedly increased after UUO (Figure 4A). Fibronectin ED-A was deposited around the Bowman’s capsules (arrows) and interstitial fibrotic areas (arrowheads). Treatment with CTGF antisense ODN reduced the amount of fibronectin ED-A deposition in the interstitial areas but not around the Bowman’s capsules (Figure 4A). Similar results were observed for the expression of type I collagen (Figure 4B). Type I collagen deposition was markedly enhanced in the interstitial fibrotic areas of the untreated group and significantly reduced with the introduction of CTGF antisense ODN (Figure 4B).

View larger version (71K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. (A) Immunofluorescence study for fibronectin (FN) ED-A in the kidneys 7 d after UUO with the treatment of ODN. S, sham operation; U, UUO with vehicle injection; R, UUO with CTGF reverse ODN (200 µg) introduction; AS, UUO with CTGF antisense ODN (200 µg) introduction. In control kidneys (S), FN ED-A was slightly deposited in the mesangial area. Seven days after UUO (U), FN ED-A was mostly deposited around the Bowman’s capsules (arrows) and interstitial fibrotic areas (arrowheads). FN ED-A staining in the fibrotic areas was attenuated with the CTGF antisense ODN treatment (AS) as compared with control ODN (R). Bar graph shows the ratio of the positively stained area to the total cortical field. Mean ± SE; n = 7; *P < 0.05; **P < 0.01. (B) Immunohistochemical study for type I collagen in the kidneys 7 d after UUO with introduction of ODN. Type I collagen staining was also weaker in the CTGF antisense ODN-treated kidney (AS). Bar graph shows the ratio of the positively stained area to the total cortical field. Mean ± SE; n = 7; *P < 0.05; **P < 0.01. Magnification, x200.

View larger version (89K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Treatment with CTGF antisense ODN suppresses interstitial fibrosis. (A) Representative Masson’s trichrome–stained sections. S, sham operation; U, UUO with vehicle injection; R, UUO with CTGF reverse ODN (200 µg) introduction; AS, UUO with CTGF antisense ODN (200 µg) introduction. (B) Semiquantitative score of tubulointerstitial fibrosis in the cortex from the kidneys. Mean ± SE; n = 7; *P < 0.05; **P < 0.01; #P < 0.01 between AS and S. Magnification, x200.

Effect of CTGF Antisense ODN on -SMA Expression

View larger version (99K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. -Smooth muscle actin (-SMA) expression in the kidney after UUO with introduction of ODN. (A) Representative Western blots of -SMA 7 d after UUO. S, sham operation; U, UUO with vehicle injection; R, UUO with CTGF reverse ODN (200 µg) introduction; AS, UUO with CTGF antisense ODN (200 µg) introduction. -Actin is used as an internal control. (B) Quantification of relative -SMA levels analyzed with densitometer. The -SMA expression level from the obstructed kidney (U) was significantly higher than that from the sham-operated one (S). The extent of -SMA expression in the obstructed kidney with antisense ODN treatment (AS) was significantly decreased as compared with reverse ODN treatment (R). Mean ± SE; n = 6; *P < 0.05; **P < 0.01. (C) Immunohistochemical examination for -SMA revealed increased staining in the interstitial fibrotic areas of the reverse ODN-treated kidney (R), whereas it was significantly attenuated in the CTGF antisense ODN-treated kidney (AS). Magnification, x200.

View larger version (88K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Cell proliferation in the kidney after UUO with introduction of ODN. (A) Representative Western blots of PCNA 7 d after UUO. S, sham operation; U, UUO with vehicle injection; R, UUO with CTGF reverse ODN (200 µg) introduction; AS, UUO with CTGF antisense ODN (200 µg) introduction. -Actin is used as an internal control. (B) Quantification of relative proliferating cell nuclear antigen (PCNA) levels analyzed with densitometer. The PCNA expression level from the CTGF antisense ODN-treated obstructed kidney (AS) was unchanged compared with the vehicle-injected (U) or reverse ODN-treated (R) ones. (C) Immunohistochemical examination revealed that PCNA-positive cells were renal tubular cells (arrows) and interstitial cells (arrowheads). No significant differences in PCNA staining between the CTGF reverse (R)- or antisense (AS)-treated kidneys. (D) No significant differences in the ratio of PCNA-positive renal tubular or interstitial cells between the CTGF reverse (R)- or antisense (AS)-treated kidneys. (E) Ki-67–positive cells were renal tubular cells (arrows) and interstitial cells (arrowheads). Ki-67 staining also showed no difference between the CTGF reverse (R)- or antisense (AS)-treated kidneys. (F) The ratio of Ki-67–positive renal tubular or interstitial cells was not different between the CTGF reverse (R)- or antisense (AS)-treated kidneys. Mean ± SE; n = 7; *P < 0.05; **P < 0.01; #P < 0.01 between AS and S. Magnification, x400.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We demonstrate in this study that the reduction in CTGF effectively attenuates the upregulation of ECM proteins in a rat model of obstructive nephropathy. Northern blot analysis exhibited that the transfer of CTGF antisense ODN greatly reduces fibronectin, fibronectin ED-A, and 1(I) collagen mRNA expression (Figure 2). Fibronectin ED-A, a splice variant of fibronectin, is strongly induced by TGF- and is crucial for the alteration to the myofibroblastic phenotype (24). In UUO rats, fibronectin ED-A is mostly deposited around the Bowman’s capsules and fibrotic areas (34). Immunohistochemical analysis revealed that the treatment with CTGF antisense ODN reduced the amount of fibronectin ED-A deposition in the interstitial fibrotic areas but not around the Bowman’s capsules (Figure 4A). CTGF antisense ODN also significantly reduced type I collagen deposition in the interstitial areas, leading to the amelioration of tubulointerstitial fibrosis (Figure 5). It has been reported that, by this hydrodynamics-based gene transfer technique, the transgene is introduced mainly into renal interstitial fibroblasts (23,27). Together with the present data, these observations suggest that this strategy could provide specific knockdown of CTGF in the interstitial areas of the targeted kidney.

Interestingly, our results show that CTGF antisense treatment does not affect TGF-1 mRNA expression in the kidney (Figure 2). A number of reports so far have shown that the treatment of renal fibrosis in obstructive nephropathy is generally associated with the inhibited expression of TGF-, such as upon blocking of the renin-angiotensin system (21), in PAI-1–deficient mice (35), and by directly inhibiting TGF- with antisense ODN (36) or neutralizing antibodies (22). Hepatocyte growth factor also ameliorates renal fibrosis with the inhibition of TGF- (37). These studies have suggested that TGF- is a final common pathway leading to renal fibrosis. However, a recent report has shown that the treatment of hepatocyte growth factor ameliorates renal fibrosis through the attenuation of CTGF induction in 5/6 nephrectomized TGF-1 transgenic mice, without reducing TGF-1, 2, or 3 (38). It has been shown that the addition of recombinant CTGF does not stimulate TGF-1 mRNA expression in renal interstitial fibroblasts (14) or in mesangial cells (17). Thus, a decrease in CTGF may lessen renal fibrosis without affecting TGF- expression. In the present study, >80% reduction of CTGF mRNA resulted in 50 to 60%, but not complete, inhibition of ECM deposition after UUO (Figures 2 through 5). This may be due to the incomplete efficacy of this type of treatment or to the presence of other major pathways to interstitial fibrosis. Nevertheless, these findings strongly suggest that CTGF should serve as one of the bone fide downstream mediators of TGF-’s profibrotic action in vivo and could become a more specific target against renal fibrosis.

Fibronectin ED-A is thought to be a key player in the differentiation into myofibroblasts, which are characterized by -SMA expression (28,29). Anti-fibronectin ED-A antibody specifically blocks TGF-–stimulated -SMA induction (24). Our results reveal that -SMA expression is potently inhibited by blocking CTGF expression in the obstructed kidneys. Therefore, CTGF may function to induce myofibroblast differentiation in vivo, by activating fibronectin ED-A or by acting synergistically with it.

Proliferation of both tubular and interstitial cells is documented during obstructive nephropathy (21,22). This process might involve some repair mechanisms, because blocking of TGF- in the obstructed kidney results in significantly increased tubular proliferation along with the protection against interstitial fibrosis (22). CTGF stimulates cell proliferation in some cells (10,30–32), and CTGF-null mice exhibit decreased PCNA-positive cells in the chondrocytes (33). CTGF therefore may be related to cell proliferation after UUO. It is interesting however, that the proliferation of the tubular and interstitial cells in the obstructed kidney was not inhibited by the treatment with CTGF antisense ODN (Figure 7). These findings suggest that CTGF does not play a crucial role in proliferation in the obstructed kidney but is an important mediator specific to fibrogenesis. The reason that the blockade of TGF- and CTGF causes different effects on proliferation remains to be elucidated and requires further clarification.

CTGF is potently activated by TGF- but may also be induced through TGF-–independent pathways, e.g., by glucocorticoids (39), angiotensin II/calcineurin (40), and advanced glycation end products (41). This notion further supports that CTGF can become a promising therapeutic target as a final common pathway of renal fibrosis. Recently, CTGF has been shown to bind to TGF-1 to activate phosphorylation of smad 2 (42). These findings suggest that CTGF may transduce its signals in part through binding to TGF-1.

Downstream pathways in the CTGF signaling still remain unclear. CTGF binds directly to integrin v3 and IIb3, which are candidate receptors for CTGF to promote cell adhesion, migration, and proliferation (30,43). CTGF also binds to LDL receptor-related protein (44), which serves as a functional receptor for MMP-9 (45). We and others have already shown that Cyr61, another member of the CCN family, may exert distinct actions from CTGF in terms of ECM production and mesangial activation, while potentially sharing the same receptors (8,9,46). Further clarifications therefore are essential to elucidate the signaling pathways and mechanisms by which CTGF induces ECM production in vitro and renal fibrosis in vivo.

In summary, the present study reveals that the treatment with CTGF antisense ODN by a kidney-specific gene transfer reduces ECM production in experimental obstructive nephropathy, ameliorating the development of interstitial fibrosis. Our study demonstrates the potential role of CTGF in renal fibrosis in vivo and opens up the possibility of a novel therapeutic approach for a variety of renal diseases leading to tubulointerstitial fibrosis.

	Acknowledgments

 
This work was supported in part by research grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare; Research for the Future (RFTF) of Japan Society for the Promotion of Science; Smoking Research Foundation; and the Salt Science Research Foundation.

We gratefully acknowledge Dr. J. Miyazaki (Osaka University) for providing plasmid, Dr. H. Maruyama (Niigata University) and Drs. M. Nagata and M. Kanemoto (Tsukuba University) for valuable discussion, Ms. J. Nakamura and Ms. S. Saito for technical assistance, and Ms. S. Doi and Ms. A. Sonoda for secretarial assistance.

	References

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