Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan
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ABSTRACT |
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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.
transforming growth factor-; in situ hybridization; obstructive
nephropathy; antisense oligonucleotide; reporter assay
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INTRODUCTION |
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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 impairment 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 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.
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METHODS |
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Unilateral ureteral obstruction. All animal experiments were conducted in accordance with our institutional guidelines for animal research. Male Wistar rats weighing 200- 250 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 µg 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-micrometer-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 µg/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 µg/ml) were added to hybridization solution containing 50% deionized formamide, 10% dextran sulfate, 1× Denhardt's solution, 10 mM Tris · HCl, 600 mM NaCl, 1 mM EDTA, 0.25% SDS, 250 µg/ml denatured salmon testis DNA, and 250 µg/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 µg/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-micrometer-thick sections were stained with Masson's trichrome.
For immunohistochemical analysis ofCell 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 µg/ml streptomycin. For
TGF- stimulation, cells at ~90% confluence were made quiescent in
serum-free DMEM supplemented with 10 µg/ml insulin, 10 µg/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 µg 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 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 µM 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 µg 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 · 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 µg/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).
Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using analysis of variance followed by Scheffé's test. P < 0.05 was considered statistically significant.
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RESULTS |
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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 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 (4.1- and 6.9-fold of control, respectively).
This increase was 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).
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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).
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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 macrophages
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).
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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 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.
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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
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.
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DISCUSSION |
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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 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,
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), 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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: muko{at}kuhp.kyoto-u.ac.jp).
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.
10.1152/ajprenal.00122.2001
Received 17 April 2001; accepted in final form 1 November 2001.
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