Role of connective tissue growth factor in fibronectin expression and ...

Am J Physiol Renal Physiol 282: F933–F942, 2002; 10.1152/ajprenal.00122.2001.

Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis HIDEKI YOKOI, MASASHI MUKOYAMA, AKIRA SUGAWARA, KIYOSHI MORI, TETSUYA NAGAE, HISASHI MAKINO, TAKAYOSHI SUGANAMI, KENSEI YAHATA, YURIKO FUJINAGA, ISSEI TANAKA, AND KAZUWA NAKAO Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan Received 17 April 2001; accepted in final form 1 November 2001

Yokoi, Hideki, Masashi Mukoyama, Akira Sugawara, Kiyoshi Mori, Tetsuya Nagae, Hisashi Makino, Takayoshi Suganami, Kensei Yahata, Yuriko Fujinaga, Issei Tanaka, and Kazuwa Nakao. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol 282: F933–F942, 2002; 10.1152/ajprenal.00122.2001.—Connective tissue growth factor (CTGF) is one of the candidate factors mediating downstream events of transforming growth factor-␤ (TGF-␤), but its role in fibrogenic properties of TGF-␤ and in tubulointerstitial fibrosis has not yet been clarified. Using unilateral ureteral obstruction (UUO) in rats, we analyzed gene expression of TGF-␤1, CTGF, and fibronectin. We further investigated the effect of blockade of endogenous CTGF on TGF-␤-induced fibronectin expression in cultured rat renal fibroblasts by antisense oligodeoxynucleotide (ODN) treatment. After UUO, CTGF mRNA expression in the obstructed kidney was significantly upregulated subsequent to TGF-␤1, followed by marked induction of fibronectin mRNA. By in situ hybridization, CTGF mRNA was detected mainly in the interstitial fibrotic areas and tubular epithelial cells as well as in parietal glomerular epithelial cells in the obstructed kidney. The interstitial cells expressing CTGF mRNA were also positive for ␣-smooth muscle actin. CTGF antisense ODN transfected into cultured renal fibroblasts significantly attenuated TGF␤-stimulated upregulation of fibronectin mRNA and protein compared with control ODN transfection, together with inhibited synthesis of type I collagen. With the use of a reporter assay, rat fibronectin promoter activity was increased by 2.5-fold with stimulation by TGF-␤1, and this increase was abolished with antisense CTGF treatment. Thus CTGF plays a crucial role in fibronectin synthesis induced by TGF-␤, suggesting that CTGF blockade could be a possible therapeutic target against tubulointerstitial fibrosis.

TUBULOINTERSTITIAL FIBROSIS is a common feature of progressive renal diseases regardless of the initiating insult (4, 41). It has been shown in a number of clinical as well as experimental studies that tubulointerstitial injury is a more consistent predictor of functional im-

pairment than glomerular damage (41, 44). Mechanisms by which the interstitial fibrosis progresses are not well understood, but various cytokines are thought to be involved in fibrogenic and inflammatory processes by stimulating fibroblast proliferation, macrophage infiltration, and extracellular matrix (ECM) accumulation (18). Among them, multiple lines of evidence have indicated transforming growth factor-␤ (TGF-␤) as a key cytokine underlying the development of tissue fibrosis, including tubulointerstitial fibrosis as well as glomerulosclerosis (5, 18). TGF-␤ enhances the synthesis of ECM proteins such as collagen types I, III, and IV, fibronectin, and laminin (5, 18, 46). TGF-␤ also promotes ECM accumulation by increasing the production of protease inhibitors such as plasminogen activator inhibitor-1 and by decreasing the activity of proteases such as matrix metalloproteinases (5, 18, 46). Furthermore, TGF-␤ stimulates fibroblast migration and proliferation (5) and also is chemotactic for monocytes and macrophages (5, 18). Transgenic mice overexpressing TGF-␤1 in the liver with high plasma levels of active TGF-␤1 develop marked tubulointerstitial fibrosis with severe glomerulonephritis (48). In accordance with these observations, elevated renal expression of TGF-␤ mRNA or protein has been reported in nearly every experimental model of renal failure characterized by fibrosis (3). In an obstructive nephropathy model, augmented expression of TGF-␤ in fibrotic tissue, produced mainly in the interstitial fibroblasts and macrophages, greatly paralleled the increased interstitial expression of fibronectin and collagen types I, III, and IV (4, 14, 18, 33, 34). On the basis of these findings, it has been suggested that blocking of TGF-␤ or its downstream pathway becomes a potential antifibrotic strategy for chronic renal diseases (6). However, the molecular mechanisms for profibrotic effects of TGF-␤ have not yet been fully elucidated. Connective tissue growth factor (CTGF), originally isolated from conditioned media of human umbilical vein endothelial cells (10), belongs to a new family of

Address for reprint requests and other correspondence: M. Mukoyama, Dept. of Medicine and Clinical Science, Kyoto Univ. Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

transforming growth factor-␤; in situ hybridization; obstructive nephropathy; antisense oligonucleotide; reporter assay

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0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society

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ROLE OF CTGF IN RENAL FIBROSIS

cysteine-rich growth factors (the CCN family) that consists of CTGF/fisp-12, cef10/cyr61, and nov (8, 47). In cultured fibroblasts, CTGF gene expression is strongly induced by TGF-␤ but not by other growth factors, such as epidermal growth factor, platelet-derived growth factor, or basic fibroblast growth factor (26). Addition of CTGF, in turn, potently stimulates fibroblast proliferation and ECM protein synthesis (19). In human diseases, CTGF gene expression was detected in fibroblasts of sclerotic lesions from patients with systemic sclerosis (25) and in fibrotic areas of atherosclerotic plaques (42). Recently, it has been shown that CTGF expression is upregulated in proliferative and fibrotic glomerular lesions of various human renal diseases, including glomerulonephritis and diabetic nephropathy (29). Subsequent reports using in vitro and animal models revealed that CTGF mRNA and protein are increased in cultured mesangial cells as well as in the renal cortex in a diabetic milieu, suggesting the involvement of CTGF in the pathogenesis of diabetic glomerulosclerosis (39, 45). Although these observations have led to the hypothesis that CTGF is a candidate factor mediating fibrogenic properties of TGF-␤ (22), the role of CTGF in tubulointerstitial fibrosis still remains unclarified. In the present study, to explore the implication of CTGF in tubulointerstitial fibrosis, we investigated the time course of TGF-␤, CTGF, and fibronectin gene expression in an obstructive nephropathy model in rats by Northern blot analysis. Localization of CTGF mRNA expression was also investigated by in situ hybridization. Furthermore, to evaluate the contribution of CTGF to fibronectin and collagen expression induced by TGF-␤, we inhibited endogenous CTGF by antisense oligodeoxynucleotide (ODN) in cultured rat renal fibroblasts and analyzed the effect of its blockade by Northern blot analysis, immunoblotting, and luciferase reporter assay. METHODS

Unilateral ureteral obstruction. All animal experiments were conducted in accordance with our institutional guidelines for animal research. Male Wistar rats weighing 200250 g were subjected to either unilateral ureteral obstruction (UUO) or sham operation (14, 33, 40). In UUO rats under pentobarbital anesthesia, the right ureter was ligated with 4–0 silk at two points through a midline abdominal incision and cut between the ligatures to prevent retrograde infection. Rats were killed at 12 h and 3, 6, or 14 days after UUO or sham operation (n ⫽ 4 at each time point), and both the obstructed kidney and the contralateral kidney were harvested. Northern blot analysis was performed using 40 ␮g of total RNA prepared from each kidney. In situ hybridization. In situ hybridization was performed as described, with some modifications (11, 38). A cDNA fragment of rat CTGF (nucleotides 1221–1803) (57) subcloned into pGEM-T-Easy vector (Promega, Madison, WI) was used to produce antisense and sense riboprobes. After digestion with a restriction enzyme SpeI or Eco47III, antisense and sense cRNA riboprobes were transcribed in vitro from the linearized plasmids using digoxigenin (DIG)-labeled UTP and an RNA labeling kit (DIG RNA Labeling Kit SP6/T7; Roche Diagnostics, Mannheim, Germany). Seven-micromeAJP-Renal Physiol • VOL

ter-thick sections of paraffin-embedded renal tissues were placed on silanized slides (DAKO Japan, Kyoto, Japan). Sections were deparaffinized, treated with 0.2 M HCl for 20 min, digested with 10 ␮g/ml proteinase K for 10 min at 37°C, fixed with 4% paraformaldehyde for 5 min, and treated with 2 mg/ml glycine for 30 min. The specimens were incubated with prehybridization buffer [50% deionized formamide/5⫻ standard saline citrate (SSC)] for 30 min in a humidified chamber at 45°C. Then, DIG-labeled riboprobes (final concentration, 1 ␮g/ml) were added to hybridization solution containing 50% deionized formamide, 10% dextran sulfate, 1⫻ Denhardt’s solution, 10 mM Tris 䡠 HCl, 600 mM NaCl, 1 mM EDTA, 0.25% SDS, 250 ␮g/ml denatured salmon testis DNA, and 250 ␮g/ml yeast tRNA. Hybridization was performed in a humidified chamber for 16 h at 45°C. Thereafter, the slides were washed once with 5⫻ SSC and once with 2⫻ SSC containing 50% formamide at 45°C. Then, they were treated with 20 ␮g/ml RNase A for 30 min at 37°C. Washing was continued once with 2⫻ SSC and twice with 0.1⫻ SSC. The sections were blocked with 1% blocking reagent (Roche), washed with 100 mM maleic acid buffer (pH 7.5) containing 150 mM NaCl, and then incubated with alkaline phosphatase-conjugated Fab fragments of sheep anti-DIG antibody (Roche) at a dilution of 1:500 for 30 min at room temperature. They were visualized on reaction with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate for 16 h at room temperature according to the DIG Nucleic Acid Detection Kit protocol (Roche). The slides were counterstained with hematoxylin. Histology and immunohistochemistry. For histological analysis, sagittal kidney sections were fixed with 4% buffered formaldehyde and embedded in paraffin. Two-micrometerthick sections were stained with Masson’s trichrome. For immunohistochemical analysis of ␣-smooth muscle actin (␣-SMA), serial paraformaldehyde-fixed paraffin sections were deparaffinized, washed with PBS, and treated with 3% H2O2 in methanol for 10 min to quench endogenous peroxidase activity. The specimens were incubated with murine anti-␣-SMA antibody (DAKO) for 1 h at room temperature, processed using LSAB⫹ kit (DAKO), and developed with 3,3⬘-diaminobenzidine tetrahydrochloride. Cell culture. Normal rat kidney fibroblasts (NRK-49F cells) and normal rat kidney epithelial cells (NRK-52E cells) were obtained from American Type Culture Collection (Rockville, MD) and maintained in DMEM (GIBCO BRL, Rockville, MD) containing 10% fetal calf serum (FCS; Sanko Junyaku, Tokyo, Japan), 100 U/ml penicillin, and 100 ␮g/ml streptomycin. For TGF-␤ stimulation, cells at ⬃90% confluence were made quiescent in serum-free DMEM supplemented with 10 ␮g/ml insulin, 10 ␮g/ml transferrin, and 10 ng/ml selenium (ITS; Sigma, St. Louis, MO). After 24 h of serum starvation, cells were stimulated with 1–10 ng/ml of recombinant human TGF-␤1 (R&D Systems, Minneapolis, MN) and further incubated for 1–48 h. Northern blot analysis was performed using 20 ␮g of total RNA prepared from each culture. Northern blot analysis. Total RNA from the whole kidney or cultured cells was extracted by the acid guanidinium thiocyanate-phenol-chloroform method. Northern blot analysis was performed as described previously (38, 40). Briefly, total RNA was electrophoresed on a 1.4% agarose gel and transferred to a nylon membrane filter (Biodyne; Pall BioSupport, East Hills, NY). Hybridization was performed at 42°C overnight with 32P-labeled cDNA probes for rat CTGF (nucleotides 1221–1803) (57), TGF-␤1 (1142–1546) (52), and fibronectin (619–1082) (50), which were prepared by standard reverse-transcription PCR method. The membranes

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were washed at 55°C in 1⫻ SSC/0.1% SDS, and autoradiography was performed for 12 h with the BAS-2500 system (Fuji Photo Film, Tokyo, Japan). The amount of RNA loaded in each lane was normalized with 28S or 18S rRNA. CTGF antisense oligonucleotide transfection. Transfection of antisense ODN into cultured cells was performed as described (31) with some modifications. The sequences of phosphorothioate oligonucleotides (Kurabo, Osaka, Japan) for rat CTGF used in this study 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 (57) around the translation initiation codon (underlined in sequences). Transfection into NRK-49F cells was carried out by cationic lipofection with TransFast Reagent (Promega) according to the manufacturer’s instructions. Cells (1 ⫻ 106/dish) were plated into 10-cm dishes and serum-starved in DMEM with ITS for 24 h. Oligonucleotide and the reagent in a charge ratio of 1:1 were allowed to aggregate for 10 min at room temperature, and cells were transfected with 0.5 ␮M of ODN in serum-free DMEM. After 2 h of incubation, the cells were overlaid with growth medium containing FCS to achieve the final concentration of 5% and then stimulated with 3 ng/ml TGF-␤1 for 12–48 h. Northern blot analysis was performed using 15 ␮g of total RNA prepared from each culture. Western blot analysis. Western blot analysis was performed as described previously (55). Cells were lysed on ice in a solution containing 1 M Tris 䡠 HCl (pH 7.5), 12 mM ␤-glycerophosphate, 0.1 M EGTA, 1 mM pyrophosphate, 5 mM NaF, 10 mg/ml aprotinin, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. Cell lysates were centrifuged at 15,000 rpm for 20 min at 4°C, and the supernatants were treated with Laemmli’s sample buffer. Equal amounts of samples (40 ␮g/lane) were separated by 12.5% SDS-PAGE and electrophoretically transferred onto Immobilon polyvinylidine difluoride filters (Millipore, Bedford, MA) in 25 mM Tris, 192 mM glycine, and 5% methanol at 100 V for 1 h. Filters were incubated with antibodies against fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA) or type I collagen (Calbiochem, San Diego, CA) overnight at 4°C. Immunoblots were then developed by an enhanced chemiluminescence protocol using horseradish peroxidase-linked donkey anti-rabbit IgG (Bio-Rad Laboratories, Richmond, CA) and a chemiluminescence kit (Amersham, Arlington Heights, IL). ␣-Tubulin (antibody from Sigma) was used as an internal control. Plasmid construction and luciferase reporter assay. The promoter region (nucleotide ⫺531/⫹1) of the rat fibronectin gene (13) was PCR amplified from Wistar rat genomic DNA with the following primers: forward, 5⬘-GGA CAA GGT AGT GGC CAC TTA ACG-3⬘ and antisense, 5⬘-GCG GCT GAG CCC CAA GAG CAG AGG-3⬘. The PCR product of the expected size was subcloned into pGEM-T-Easy vector (Promega) to construct pGEM-T-rFN531. pGL3-Basic vector (Promega) was digested with HindIII, then blunted with Klenow fragment, and ligated with EcoRI-Linker (Takara, Tokyo, Japan). The rat fibronectin promoter region was cleaved with EcoRI from pGEM-T-rFN531 and inserted into the EcoRI sites of pGL3-Basic vector (rFN531-Luc). The sequence and direction of the inserted fragment were confirmed by the dideoxy chain-termination method using a Dye Terminator cycle sequencing kit FS and 373B DNA sequencer (Applied Biosystems, Foster City, CA). A luciferase reporter assay was performed as described elsewhere (17). In brief, NRK-49F cells were plated into six-well plates at 5 ⫻ 104/well and serum-starved in DMEM with ITS. After 24 h of incubation, cells were transfected in AJP-Renal Physiol • VOL

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serum-free DMEM by cationic lipofection with 2 ␮g/well of rFN531-Luc and 50 nM of phosphorothioate ODN (antisense or control ODN for rat CTGF), along with 0.1 ␮g/well of pRL-SV40 (Promega) as an internal control for transfection efficiency. After 6 h of incubation, the medium was changed to DMEM with ITS, and the cells were stimulated with 3 ng/ml TGF-␤1 for an additional 24 h. Cells were then lysed, and the luciferase activity was determined using a Dual Luciferase Reporter Assay Kit (Toyo Ink, Tokyo, Japan). Statistical analysis. Data are expressed as means ⫾ SE. Statistical analysis was performed using analysis of variance followed by Scheffe´ ’s test. P ⬍ 0.05 was considered statistically significant. RESULTS

CTGF expression in obstructive nephropathy. To investigate the involvement of CTGF in tubulointerstitial fibrosis, we examined changes in CTGF gene expression together with those in TGF-␤1 and fibronectin messages in rat kidney after obstructive nephropathy. Figure 1 illustrates the expression of TGF-␤1, CTGF, and fibronectin mRNA at 12 h and 3, 6, and 14 days after ureteral obstruction. The staining intensity of 28S rRNA verified equivalent loading of RNA samples in each lane (Fig. 1A). TGF-␤1 mRNA expression was increased in the right obstructed kidneys as early as

Fig. 1. Expression of transforming growth factor (TGF)-␤1, connective tissue growth factor (CTGF), and fibronectin mRNA in the kidney after unilateral ureteral obstruction (UUO) in rats. Forty micrograms of total RNA from the whole kidney in each lane were electrophoresed and hybridized with rat TGF-␤1, CTGF, and fibronectin cDNA probes. A: representative Northern blots. FN, fibronectin; S, sham-operated rat; U, UUO rat; 0.5, 12 h; 3, day 3; 6, day 6; 14, day 14; R, right obstructed kidney; L, left contralateral kidney. B: the relative mRNA levels of TGF-␤1, CTGF, and fibronectin in the obstructed (filled bars) and contralateral (open bars) kidneys at 12 h and 3, 6, and 14 days after UUO. mRNA levels are normalized with 28S rRNA. The mean level of the right kidney from sham-operated rats is regarded as 1.0 arbitrary unit; n ⫽ 4. *P ⬍ 0.05, **P ⬍ 0.01 vs. sham operation. #P ⬍ 0.05, ##P ⬍ 0.01 vs. contralateral kidney.

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12 h compared with control sham-operated or contralateral kidneys and remained high at day 14 (Fig. 1B), as reported in previous studies (14, 33, 34, 40). CTGF mRNA expression was significantly upregulated

subsequent to TGF-␤1 in the obstructed kidneys at day 3 (1.7-fold of control) (Fig. 1, A and B). The upregulation became more pronounced at days 6 and 14 (4.1and 6.9-fold of control, respectively). This increase was

Fig. 2. In situ hybridization for CTGF mRNA in the kidney after UUO in rats. Representative results from control sham-operated rats (A and B; ⫻400) and UUO rats at day 14 (C and D; ⫻400) after surgery are shown. In the control kidney [cortex (A) and medulla (B)], CTGF mRNA expression was confined only in the glomerular tuft, presumably in podocytes (arrows). At day 14 after UUO [cortex (C) and medulla (D)], CTGF mRNA was upregulated in glomerular epithelial cells (arrows) and in mesangial areas (asterisk) as well as in the interstitial fibrotic areas (arrowheads) and tubular epithelial cells (double arrow). The contralateral kidney at day 14 [cortex (E) and medulla (F)] showed increased CTGF mRNA expression in the glomerulus (asterisk) but not in the tubulointerstitial areas. No significant signal was detected from the same sample as in C with the sense probe (G; ⫻400). Masson’s trichromestained sections from the sham-operated kidney showed normal appearance (H; ⫻400); after UUO, the obstructed kidney at day 14 exhibited increased extracellular matrix deposition with markedly atrophic tubules (I; ⫻400).

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followed by marked augmentation of fibronectin mRNA induction in the obstructed kidney, which appeared progressively throughout the time course and was more prominent than that of CTGF (17-fold of control at day 14) (Fig. 1B). These findings indicate that the upregulation of TGF-␤1 and CTGF gene expression precedes that of fibronectin expression after obstructive nephropathy, suggesting that CTGF may mediate the fibronectin upregulation induced by TGF-␤1 during the course of tubulointerstitial fibrosis. The contralateral kidney showed a slight but significant increase in CTGF and fibronectin gene expression at day 14, without a significant change in TGF-␤1 expression (Fig. 1B). In situ hybridization and histology. In situ hybridization for CTGF mRNA gave signals only in the glomerular tuft and no apparent signal in the tubulointerstitial area in the sham-operated kidney (Fig. 2, A and B). In the obstructed kidney, on the other hand, CTGF mRNA expression was increased in fibrotic areas and tubular epithelial cells as well as parietal glomerular epithelial and mesangial cells at day 14 after UUO (Fig. 2, C and D). The contralateral kidney showed a slight increase in CTGF expression in the glomerulus, presumably in the mesangial area, but signals were not apparent in the tubulointerstitial area (Fig. 2, E and F). No significant signal was detected when serial sections from the same sample were hybridized with the sense probe (Fig. 2G). Histologically, the sham-operated kidney had a normal appearance (Fig. 2H). In the obstructed kidney at day 14, collecting ducts and distal tubules displayed tubular atrophy and epithelial flattening with marked tubulointerstitial fibrosis (Fig. 2I). By immunohistochemistry, interstitial expression of ␣-SMA, a marker for myofibroblasts (15), was markedly increased after UUO (Fig. 3B), as reported previously (27). The interstitial cells expressing CTGF mRNA were also positive for ␣-SMA staining (Fig. 3, A and B, arrows). TGF-␤-stimulated expression of CTGF and fibronectin mRNA in cultured cells. In models of tubulointerstitial fibrosis, interstitial fibroblasts are thought to be the major cell type of TGF-␤ production and action to stimulate ECM accumulation (4, 18), whereas macro-

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phages may be another important source of TGF-␤ production (14, 18). In situ hybridization has revealed that CTGF is induced in the tubulointerstitium mainly in the interstitial fibroblasts and tubular epithelial cells. Although TGF-␤ is already shown to stimulate the expression of both CTGF and fibronectin in cultured fibroblasts (22, 26), their relationship has not been evaluated. To investigate this, we first examined the dose- and time-dependent induction by TGF-␤ of CTGF and fibronectin gene expression using cultured rat renal fibroblasts and renal epithelial cells (Fig. 4). After confirming that TGF-␤1 showed a well-paralleled CTGF and fibronectin gene upregulation in NRK-49F cells in a dose-dependent manner (Fig. 4A), we examined the time course of CTGF and fibronectin mRNA induction by using 3 ng/ml of TGF-␤1 (Fig. 4B). CTGF gene expression was significantly upregulated by TGF-␤1 stimulation in 3 h, showed a peak ⬃12 h after exposure (3.9-fold above baseline), and then declined at 48 h. In contrast, fibronectin mRNA expression was gradually increased from 6 h and showed a significant upregulation at 24 and 48 h after stimulation. Thus, as observed in the obstructive nephropathy model, the upregulation of fibronectin expression by TGF-␤1 was rather delayed compared with that of CTGF. TGF-␤1 also stimulated CTGF and fibronectin expression in NRK-52E cells, but the extent of induction was less remarkable than in NRK-49F cells (Fig. 4C). Effects of CTGF antisense transfection on fibronectin and type I collagen synthesis. The findings that TGF-␤ induces CTGF expression in an earlier time course than fibronectin and that TGF-␤ and CTGF share a number of effects including fibronectin induction have led to the hypothesis that CTGF may serve as a downstream mediator of TGF-␤ action (22). To explore the role of CTGF in TGF-␤-induced fibronectin expression, we examined the effect of CTGF antisense ODN transiently transfected into NRK-49F cells by the cationic lipofection method. As shown in Fig. 5, CTGF antisense ODN markedly inhibited TGF-␤1-induced CTGF mRNA expression at 12 and 48 h after stimulation compared with control reverse ODN, indicating efficient transfection of ODN into the cells. Under this condition, TGF-␤1-induced fibronectin mRNA expression was significantly (by ⬃70%) attenuated at 48 h

Fig. 3. Interstitial fibrotic area in the obstructed kidney from UUO rats. A: in situ hybridization of CTGF mRNA reveals increased expression in the interstitial cells (arrows). B: immunohistochemical staining for ␣-smooth muscle actin (␣-SMA) of the same section shows that CTGF-positive cells are also positive for ␣-SMA.


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Fig. 5. Inhibitory effects of CTGF antisense transfection on TGF-␤induced expression of fibronectin mRNA. NRK-49F cells were transiently transfected with 0.5 ␮M of reverse or antisense oligodeoxynucleotide (ODN) for CTGF by cationic lipofection. After 2 h of transfection, cells were stimulated with 3 ng/ml of TGF-␤1. After 12 or 48 h of stimulation, cells were harvested for RNA extraction, and 15 ␮g of total RNA in each lane were subjected to Northern blot analysis. A: representative Northern blots. Veh, vehicle without TGF-␤1 stimulation; R, reverse ODN transfection with TGF-␤1 stimulation; AS, antisense ODN transfection with TGF-␤1 stimulation. B: the relative mRNA levels of CTGF and fibronectin at 12 and 48 h after stimulation, which are normalized with 18S rRNA levels; n ⫽ 6. *P ⬍ 0.05, **P ⬍ 0.01.

Fig. 4. Northern blot analysis for TGF-␤-induced expression of CTGF and fibronectin mRNA in NRK-49F and NRK-52E cells. Twenty micrograms of total RNA from NRK-49F cells (A and B) and NRK-52E cells (C) in each lane were electrophoresed and hybridized with rat CTGF and fibronectin cDNA probes. The relative mRNA levels of CTGF and fibronectin normalized with 28S ribosomal RNA are shown. A: NRK-49F cells were stimulated with various concentrations of TGF-␤1 for 24 h; n ⫽ 6. *P ⬍ 0.05, **P ⬍ 0.01 vs. 0 ng/ml. #P ⬍ 0.05, ##P ⬍ 0.01. B: NRK-49F cells were stimulated with 3 ng/ml of TGF-␤1 for 1–48 h; n ⫽ 6. **P ⬍ 0.01 vs. 0 h; ##P ⬍ 0.01. C: NRK-52E cells were stimulated with various concentrations of TGF-␤1 for 24 h; n ⫽ 6. *P ⬍ 0.05 vs. 0 ng/ml. AJP-Renal Physiol • VOL

after transfection in antisense ODN-treated fibroblasts compared with control ODN-treated cells (⫹19 vs. ⫹60% of vehicle-treated cells, P ⬍ 0.05) (Fig. 5B). Western blot analysis confirmed the inhibited synthesis of fibronectin protein by CTGF antisense treatment (Fig. 6). Furthermore, CTGF antisense ODN also inhibited TGF-␤1-stimulated production of type I collagen at 48 h after transfection (Fig. 6). These findings strongly suggest that the increased production of fibronectin and type I collagen by TGF-␤1 is mediated by, for the most part, the induction of CTGF expression in cultured renal fibroblasts. Effects of CTGF antisense transfection on fibronectin promoter activity. We next examined the effect of the blockade of CTGF expression on fibronectin promoter activity. For this purpose, we constructed a luciferase reporter plasmid carrying the promoter region (⫺531 to ⫹1) of the rat fibronectin gene (13). This fibronectin promoter construct rFN531-Luc showed a 2.5-fold increase in luciferase activity on stimulation by 3 ng/ml TGF-␤1 (Fig. 7). Treatment with control ODN had no significant effect on luciferase activity, showing that a nonspecific effect by ODN transfection was negligible. In this condition, treatment with CTGF antisense ODN almost completely abolished TGF-␤1-stimulated

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Fig. 6. Inhibitory effects of CTGF antisense transfection on TGF-␤induced upregulation of fibronectin and type I collagen protein. NRK-49F cells were transiently transfected with 0.5 ␮M of reverse or antisense ODN for CTGF by cationic lipofection. After 2 h of transfection, cells were stimulated with 3 ng/ml of TGF-␤1. After 48 h of stimulation, cells were harvested for immunoblotting, and 40 ␮g of cell lysates in each lane were analyzed. Representative Western blots of 3 independent experiments are shown. Lane 1, vehicle without TGF-␤1 stimulation; lane 2, reverse ODN transfection with TGF-␤1 stimulation; lane 3, antisense ODN transfection with TGF-␤1 stimulation. Mean levels normalized with ␣-tubulin are 1.0, 1.5, and 0.72 of fibronectin staining and 1.0, 1.3, and 1.0 of type I collagen staining for lanes 1, 2, and 3, respectively.

induction of fibronectin promoter activity (Fig. 7). These results indicate that CTGF plays a critical role in TGF-␤1-induced transcriptional activation of the fibronectin gene in cultured renal fibroblasts. DISCUSSION

Previous studies have shown the close relationship between the increased expression of TGF-␤ and the progression of glomerulosclerosis and tubulointerstitial fibrosis, suggesting a role of this cytokine in the pathogenesis of fibrotic renal diseases (3, 5, 18, 46). Indeed, blocking of TGF-␤1 with neutralizing antiserum or antisense oligonucleotide effectively suppresses matrix protein accumulation and mesangial expansion in experimental glomerulonephritis (1, 7). Similarly, the beneficial effects on renal histology and function by treatment with anti-TGF-␤ antibody have been reported in experimental diabetic nephropathy models (53, 58). Upregulation of TGF-␤ is consistently documented in experimental obstructive nephropathy (3, 14, 33, 34), but the pathogenic role of TGF-␤ in this particular fibrosis model is less defined. Studies so far have indicated the importance of the activated renin-angiotensin system in stimulating TGF-␤ after UUO, and the inhibition of angiotensin II generation or its receptor signaling has been shown to successfully prevent the progression of fibrosis concomitantly with reduced TGF-␤ expression (3, 28, 34, 43). Recently, blocking of TGF-␤ in obstructive nephropathy using TGF-␤1 antisense ODN (27) or anti-TGF-␤ antibody (37) has been reported, resulting in significant amelioration of tubulointerstitial fibrosis. These studies have provided plausible evidence for TGF-␤ as a potential therapeutic target against renal fibrosis (6). An important caveat for this strategy, however, is that long-term suppression of TGF-␤, a multifunctional cytokine, might be detrimental. TGF-␤ has a modulatory role in the immune system, mainly suppressing the inflammatory AJP-Renal Physiol • VOL

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response (9). In fact, TGF-␤1-null mice exhibit excessive inflammation with tissue necrosis in specific organs, leading finally to organ failure and death (49). Moreover, TGF-␤1 may also function as an endogenous antiangiogenic and antitumor factor for certain malignancies (21), thus rendering this methodology less feasible in humans. Therefore, to design antifibrotic strategies, it is important to elucidate the mechanisms and downstream pathways specific to the profibrotic action of TGF-␤. In the present study, we reveal that CTGF is a likely mediator of TGF-␤-stimulated fibronectin induction in a rat model of interstitial fibrosis and in cultured renal interstitial fibroblasts on the basis of the following findings. CTGF expression was markedly upregulated subsequent to TGF-␤ from an early stage of tubulointerstitial fibrosis after ureteral obstruction, followed by a marked increase in fibronectin mRNA (Fig. 1). This observation is compatible with different time courses for TGF-␤ activation between CTGF and fibronectin noted in renal fibroblasts in culture (Fig. 4B). Interstitial fibroblasts have been shown to be the major site of TGF-␤ upregulation in obstructive nephropathy (27). Using in situ hybridization, we for the first time demonstrated the upregulation of CTGF mRNA in the cells of interstitial fibrotic areas as well as in tubular epithelial cells in the obstructive nephropathy model (Fig. 2). In the sham-operated kidney, we detected CTGF mRNA expression only in cells of the glomerular tuft (Fig. 2A), presumably podocytes (29, 30). It has been reported that myofibroblasts and fibroblasts may be the main source of CTGF expression in tubulointerstitial fibrosis in a human renal biopsy specimen (29). Consistent with this observation, the immunohistochemical staining for ␣-SMA, a myofibroblast marker,

Fig. 7. Effects of CTGF antisense transfection on rat fibronectin promoter activity. NRK-49F cells were transiently transfected by cationic lipofection with 50 nM of ODN (reverse or antisense ODN for CTGF) and 2 ␮g/well of the reporter plasmid (rFN531-Luc) harboring the rat fibronectin promoter region, together with 0.1 ␮g/well of pRL-SV40 as an internal control for transfection efficiency. After 6 h of incubation, the cells were stimulated with 3 ng/ml TGF-␤1. After 24 h of stimulation, the cells were harvested for dual luciferase assay as described in METHODS. R, reverse ODN transfection; AS, antisense ODN transfection; n ⫽ 4. *P ⬍ 0.05 vs. vehicle. #P ⬍ 0.01.

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revealed that most of the CTGF-positive cells were also positive for ␣-SMA in the obstructed kidney (Fig. 3). Together with the potent profibrotic property of CTGF (19, 22), these findings suggest CTGF mediation of TGF-␤-dependent fibronectin induction. One interesting finding in the UUO model is a significant upregulation of CTGF mRNA in the glomeruli of the contralateral kidney without a change of TGF-␤1 expression (Figs. 1B and 2E), suggesting a possible TGF-␤-independent stimulation. A previous report showed a similar increase in collagen IV mRNA in the contralateral kidney but along with TGF-␤1 upregulation, which was abolished by angiotensin-converting enzyme inhibition (28). Although TGF-␤ dependence is not clear at present, this might reflect mechanical or humoral signals to the contralateral kidney for increased matrix production in this model. To clarify CTGF dependence of TGF-␤-stimulated fibronectin induction, we employed antisense strategy in cultured rat renal fibroblasts. Northern blot analysis indicated that the introduction of CTGF antisense ODN abolished TGF-␤-induced CTGF expression at 12 h and thereafter significantly attenuated fibronectin mRNA and protein synthesis (Figs. 5 and 6). Furthermore, the antisense CTGF treatment almost completely abolished the increased fibronectin promoter activity stimulated by TGF-␤ (Fig. 7). These results strongly suggest that TGF-␤-induced fibronectin expression is mostly dependent on CTGF in cultured renal fibroblasts. The difference in inhibition magnitude among these experiments may be due to the difference in transfection efficiency. CTGF antisense ODN also inhibited TGF-␤-stimulated type I collagen synthesis (Fig. 6). Recently, CTGF has been reported to mediate TGF-␤-induced collagen synthesis in NRK fibroblasts using cells treated with anti-CTGF antibodies or those stably transfected with an antisense CTGF gene (16). Such CTGF dependence in this cell line has also been demonstrated in anchorage-independent growth induced by TGF-␤ (35). Taken together, these findings indicate that CTGF plays a crucial role in mediating various important actions of TGF-␤. Increased CTGF expression has been shown in a variety of human and experimental diseases characterized by fibrosis, including studies in the kidney (29, 30, 39, 45), skin (19, 25, 26), blood vessels (42), lung (36), and liver (22). Whether CTGF plays a role in vivo in fibrosis progression of these disease states still remains to be elucidated and obviously requires further clarification. Whether CTGF upregulation in those conditions is TGF-␤ dependent is another issue to be clarified. Of note, it has been reported that dexamethasone potently induces CTGF while suppressing TGF-␤ (12), suggesting the presence of a TGF-␤-independent pathway of CTGF activation and also the possible involvement of CTGF in profibrotic actions of glucocorticoids. Besides stimulating fibrogenesis, CTGF exerts various biological actions, including endothelial cell migration and proliferation (2, 54), angiogenesis (2, 54), and vascular smooth muscle cell apoptosis (23), AJP-Renal Physiol • VOL

thereby potentially participating in tissue remodeling in various disease states. Fibronectin is a major ECM protein serving as a scaffold for the deposition of other proteins; it also functions as a fibroblast chemoattractant (20). Furthermore, fibronectin promotes differentiation of fibroblasts to myofibroblasts (51), which may be a crucial phenomenon in the pathogenesis of tubulointerstitial fibrosis (15, 32). We demonstrate that CTGF plays a critical role in fibronectin gene induction activated by TGF-␤, but the signaling mechanisms of this cascade activation are yet to be determined. TGF-␤-induced fibronectin expression has been shown to be dependent on the activation of c-Jun N-terminal kinase in human fibrosarcoma cells (24). It has also been shown that the activator protein-1 element in the rat fibronectin gene is important in angiotensin II-stimulated fibronectin induction in vascular smooth muscle cells (56). TGF-␤ and CTGF share multiple biological actions including fibrogenesis but, at the same time, have other actions that may not overlap one another (22). Therefore, it is important to elucidate the signaling pathway and mechanisms by which CTGF induces fibronectin expression. In summary, the present study demonstrates that CTGF expression is upregulated in obstructive nephropathy, followed by a marked induction of fibronectin expression. CTGF blockade resulted in a marked inhibition of TGF-␤-induced fibronectin expression and its promoter activity in cultured renal fibroblasts, suggesting that CTGF is crucial in mediating fibronectin induction in the TGF-␤-stimulated pathway. Our study opens the possibility that blockade of CTGF should provide a novel therapeutic strategy for treating various renal diseases leading to fibrosis. We gratefully acknowledge Drs. K. Ebihara, H. Chusho, and T. Miyazawa for technical advice, J. Nakamura and A. Wada for technical assistance, and S. Doi and A. Sonoda for secretarial assistance. This work was supported in part by research grants from the Japanese Ministry of Education, Science, Sports, and Culture, the Japanese Ministry of Health and Welfare, “Research for the Future (RFTF)” of the Japan Society for the Promotion of Science, the Smoking Research Foundation, and the Salt Science Research Foundation. REFERENCES 1. Akagi Y, Isaka Y, Arai M, Kaneko T, Takenaka M, Moriyama T, Kaneda Y, Ando A, Orita Y, Kamada T, Ueda N, and Imai E. Inhibition of TGF-␤1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 50: 148–155, 1996. 2. Babic AM, Chen CC, and Lau LF. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin ␣v␤3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19: 2958– 2966, 1999. 3. Basile DP. The transforming growth factor beta system in kidney disease and repair: recent progress and future directions. Curr Opin Nephrol Hypertens 8: 21–30, 1999. 4. Becker GJ and Hewitson TD. The role of tubulointerstitial injury in chronic renal failure. Curr Opin Nephrol Hypertens 9: 133–138, 2000. 5. Border WA and Noble NA. Transforming growth factor-␤ in tissue fibrosis. N Engl J Med 331: 1286–1292, 1994.

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ROLE OF CTGF IN RENAL FIBROSIS 6. Border WA and Noble NA. TGF-␤ in kidney fibrosis: a target for gene therapy. Kidney Int 51: 1388–1396, 1997. 7. Border WA, Okuda S, Languino LR, Sporn MB, and Ruoslahti E. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor ␤1. Nature 346: 371–374, 1990. 8. Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 327: 125–130, 1993. 9. Bottinger EP, Letterio JJ, and Roberts AB. Biology of TGF-␤ in knockout and transgenic mouse models. Kidney Int 51: 1355–1360, 1997. 10. Bradham DM, Igarashi A, Potter RL, and Grotendorst GR. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 114: 1285–1294, 1991. 11. Chusho H, Tamura N, Ogawa Y, Yasoda A, Suda M, Miyazawa T, Nakamura K, Nakao K, Kurihara T, Komatsu Y, Itoh H, Tanaka K, Saito Y, Katsuki M, and Nakao K. Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc Natl Acad Sci USA 98: 4016–4021, 2001. 12. Dammeier J, Beer HD, Brauchle M, and Werner S. Dexamethasone is a novel potent inducer of connective tissue growth factor expression: implications for glucocorticoid therapy. J Biol Chem 273: 18185–18190, 1998. 13. Dean DC, Blakeley MS, Newby RF, Ghazal P, Hennighausen L, and Bourgeois S. Forskolin inducibility and tissue-specific expression of the fibronectin promoter. Mol Cell Biol 9: 1498–1506, 1989. 14. Diamond JR, Kees-Folts D, Ding G, Frye JE, and Restrepo NC. Macrophages, monocyte chemoattractant peptide-1, and TGF-␤1 in experimental hydronephrosis. Am J Physiol Renal Fluid Electrolyte Physiol 266: F926–F933, 1994. 15. Diamond JR, van Goor H, Ding G, and Engelmyer E. Myofibroblasts in experimental hydronephrosis. Am J Pathol 146: 121–129, 1995. 16. Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, and Grotendorst GR. Connective tissue growth factor mediates transforming growth factor ␤-induced collagen synthesis: down-regulation by cAMP. FASEB J 13: 1774–1786, 1999. 17. Ebihara K, Ogawa Y, Isse N, Mori K, Tamura N, Masuzaki H, Kohno K, Yura S, Hosoda K, Sagawa N, and Nakao K. Identification of the human leptin 5⬘-flanking sequences involved in the trophoblast-specific transcription. Biochem Biophys Res Commun 241: 658–663, 1997. 18. Eddy AA. Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 7: 2495–2508, 1996. 19. Frazier K, Williams S, Kothapalli D, Klapper H, and Grotendorst GR. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107: 404–411, 1996. 20. Gharaee-Kermani M, Wiggins R, Wolber F, Goyal M, and Phan SH. Fibronectin is the major fibroblast chemoattractant in rabbit anti-glomerular basement membrane disease. Am J Pathol 148: 961–967, 1996. 21. Gohongi T, Fukumura D, Boucher Y, Yun CO, Soff GA, Compton C, Todoroki T, and Jain RK. Tumor-host interactions in the gallbladder suppress distal angiogenesis and tumor growth: involvement of transforming growth factor ␤1. Nat Med 5: 1203–1208, 1999. 22. Gupta S, Clarkson MR, Duggan J, and Brady HR. Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney Int 58: 1389–1399, 2000. 23. Hishikawa K, Oemar BS, Tanner FC, Nakaki T, Fujii T, and Luscher TF. Overexpression of connective tissue growth factor gene induces apoptosis in human aortic smooth muscle cells. Circulation 100: 2108–2112, 1999. 24. Hocevar BA, Brown TL, and Howe PH. TGF-␤ induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J 18: 1345–1356, 1999. AJP-Renal Physiol • VOL

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25. Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Grotendorst GR, and Takehara K. Significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. J Invest Dermatol 105: 280–284, 1995. 26. Igarashi A, Okochi H, Bradham DM, and Grotendorst GR. Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4: 637–645, 1993. 27. Isaka Y, Tsujie M, Ando Y, Nakamura H, Kaneda Y, Imai E, and Hori M. Transforming growth factor-␤1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int 58:1885–1892, 2000. 28. Ishidoya S, Morrissey J, McCracken R, and Klahr S. Delayed treatment with enalapril halts tubulointerstitial fibrosis in rats with obstructive nephropathy. Kidney Int 49: 1110–1119, 1996. 29. Ito Y, Aten J, Bende RJ, Oemar BS, Rabelink TJ, Weening JJ, and Goldschmeding R. Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 53: 853–861, 1998. 30. Ito Y, Goldschmeding R, Bende RJ, Claessen N, Chand MA, Kleij L, Rabelink TJ, Weening JJ, and Aten J. Kinetics of connective tissue growth factor expression during experimental proliferative glomerulonephritis. J Am Soc Nephrol 12: 472– 484, 2001. 31. Itoh H, Mukoyama M, Pratt RE, Gibbons GH, and Dzau VJ. Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II. J Clin Invest 91: 2268–2274, 1993. 32. Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl P, Floege J, and Schwartz SM. Renal injury from angiotensin II-mediated hypertension. Hypertension 19: 464–474, 1992. 33. Kaneto H, Morrissey J, and Klahr S. Increased expression of TGF-␤1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 44: 313–321, 1993. 34. Kaneto H, Morrissey J, McCracken R, Reyes A, and Klahr S. Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed rat kidney. Kidney Int 45: 1637–1647, 1994. 35. Kothapalli D, Frazier KS, Welply A, Segarini PR, and Grotendorst GR. Transforming growth factor ␤ induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway. Cell Growth Differ 8: 61–68, 1997. 36. Lasky JA, Ortiz LA, Tonthat B, Hoyle GW, Corti M, Athas G, Lungarella G, Brody A, and Friedman M. Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol 275: L365–L371, 1998. 37. Miyajima A, Chen J, Lawrence C, Ledbetter S, Soslow RA, Stern J, Jha S, Pigato J, Lemer ML, Poppas DP, Vaughan ED, and Felsen D. Antibody to transforming growth factor-␤ ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int 58: 2301–2313, 2000. 38. Mori K, Ogawa Y, Ebihara K, Tamura N, Tashiro K, Kuwahara T, Mukoyama M, Sugawara A, Ozaki S, Tanaka I, and Nakao K. Isolation and characterization of CA XIV, a novel membrane-bound carbonic anhydrase from mouse kidney. J Biol Chem 274: 15701–15705, 1999. 39. Murphy M, Godson C, Cannon S, Kato S, Mackenzie HS, Martin F, and Brady HR. Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 274: 5830–5834, 1999. 40. Nagae T, Mukoyama M, Sugawara A, Mori K, Yahata K, Kasahara M, Suganami T, Makino H, Fujinaga Y, Yoshioka T, Tanaka I, and Nakao K. Rat receptor-activitymodifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and upregulation in obstructive nephropathy. Biochem Biophys Res Commun 270: 89–93, 2000. 41. Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis 20: 1–17, 1992.

282 • MAY 2002 •

www.ajprenal.org

F942


42. Oemar BS, Werner A, Garnier JM, Do DD, Godoy N, Nauck M, Marz W, Rupp J, Pech M, and Luscher TF. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation 95: 831–839, 1997. 43. Pimentel JL Jr, Sundell CL, Wang S, Kopp JB, Montero A, and Martinez-Maldonado M. Role of angiotensin II in the expression and regulation of transforming growth factor-␤ in obstructive nephropathy. Kidney Int 48: 1233–1246, 1995. 44. Risdon RA, Sloper JC, and de Wardener HE. Relationship between renal function and histological changes found in renalbiopsy specimens from patients with persistent glomerular nephritis. Lancet 2: 363–366, 1968. 45. Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, and 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. 46. Roberts AB, McCune BK, and Sporn MB. TGF-␤: regulation of extracellular matrix. Kidney Int 41: 557–559, 1992. 47. Ryseck RP, Macdonald-Bravo H, Mattei MG, and Bravo R. Structure, mapping, and expression of fisp-12, a growth factorinducible gene encoding a secreted cysteine-rich protein. Cell Growth Differ 2: 225–233, 1991. 48. Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, Roberts AB, Sporn MB, and Thorgeirsson SS. Hepatic expression of mature transforming growth factor ␤1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA 92: 2572–2576, 1995. 49. Schull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, and Doetschman T. Targeted disruption of the mouse transforming growth factor-␤1 gene results in multifocal inflammatory disease. Nature 359: 693–699, 1992. 50. Schwarzbauer JE, Patel RS, Fonda D, and Hynes RO. Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J 6: 2573–2580, 1987. 51. Seini G, Bochaton-Piallat ML, Ropraz P, Geinoz A, Borsi L, Zardi L, and Gabbiani G. The fibronectin domain ED-A is


52. 53.

54.

55.

56.

57.

58.

crucial for myofibroblastic phenotype induction by transforming growth factor-␤1. J Cell Biol 142: 873–881, 1998. Shankland SJ, Scholey JW, Ly H, and Thai K. Expression of transforming growth factor-␤1 during diabetic renal hypertrophy. Kidney Int 46: 430–442, 1994. Sharma K, Jin Y, Guo J, and Ziyadeh FN. Neutralization of TGF-␤ by anti-TGF-␤ antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZinduced diabetic mice. Diabetes 45: 522–530, 1996. Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tazuka K, Takemura M, Matsumura T, and Takigawa M. Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126: 137–145, 1999. Suganami T, Tanaka I, Mukoyama M, Kotani M, Muro S, Mori K, Goto M, Ishibashi R, Kasahara M, Yahata K, Makino H, Sugawara A, and Nakao K. Altered growth response to prostaglandin E2 and its receptor signaling in mesangial cells from stroke-prone spontaneously hypertensive rats. J Hypertens 19: 1095–1103, 2001. Tamura K, Nyui N, Tamura N, Fujita T, Kihara M, Toya Y, Takasaki I, Takagi N, Ishii M, Oda K, Horiuchi M, and Umemura S. Mechanism of angiotensin II-mediated regulation of fibronectin gene in rat vascular smooth muscle cells. J Biol Chem 273: 26487–26496, 1998. Xu J, Smock SL, Safadi FF, Rosenzweig AB, Odgren PR, Marks SC Jr, Owen TA, and Popoff SN. Cloning the fulllength cDNA for rat connective tissue growth factor: implications for skeletal development. J Cell Biochem 77: 103–115, 2000. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-de la Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, and Sharma K. Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-␤ antibody in db/db diabetic mice. Proc Natl Acad Sci USA 97: 8015–8020, 2000.

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