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From the Renal and Hypertension Unit,*Fundación Jimenez Diaz, Universidad Autónoma de Madrid, Madrid, Spain, and the Division of Nephrology,School of Medicine, Universidad Austral, Valdivia, Chile
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Abstract |
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Activation of renin angiotensin system (RAS) has been described in human and experimental kidney diseases.20 Angiotensin II (AngII), the main peptide of RAS, is a renal growth factor that acts through its binding to the specific receptors AT1 and AT2.20,21 This vasoactive peptide activates mesangial and tubular cells, and interstitial fibroblasts inducing hyperplasia/hypertrophy, depending on the cell type, and increasing the expression and synthesis of ECM. Both AT1 and AT2 receptors are involved in the regulation of cell growth and ECM.20-24 A close relation between AngII and TGF-ß has already been established. Systemic infusion of AngII into normal rats increases glomerular TGF-ß.25 In renal cells AngII increases TGF-ß mRNA expression and synthesis as well as TGF-ß conversion to its active form. Moreover, neutralizing antibodies to TGF-ß remarkably reduce AngII-induced ECM production.20,22-25 In experimental models of kidney damage, renal RAS activation and renal TGF-ß overexpression correlated with increased ECM mRNA expression and deposition have been described. In some of these models, the blockade of AngII actions by angiotensin converting enzyme (ACE) inhibitors and AT1 antagonists reduced TGF-ß and fibrosis.25-27 CTGF appears to play a role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis. Although emerging evidence suggests that the novel profibrogenic cytokine CTGF may be an important downstream mediator of TGF-ß profibrotic activities,1-4,16,17 the potential link between renal RAS and CTGF has not yet been investigated.
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Materials and Methods |
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The in vivo effect of AngII was evaluated by systemic infusion of AngII (dissolved in saline) into female Wistar rats (subcutaneously by osmotic minipumps, Alza Corp., Palo Alto, CA), at the dose of 50 ng/kg/min. Animals were sacrificed at 3 and 7 days (n = 8 rats in each group). Then, tissue samples were immediately removed and further processed for histological studies and RNA. To determine the role of AngII receptors, a group of rats was treated with the AT1 antagonist Losartan (10 mg/kg/day in the drinking water, n = 8 rats each group) or the AT2 antagonist PD123319 (30 mg/kg/day, subcutaneously by osmotic minipumps, n = 4 rats) from 24 hours before AngII infusion. Losartan was kindly provided by Merck Sharp and Dome (Madrid, Spain), and PD123319 was from Sigma (St. Louis, MO). The doses of Losartan and PD123319 have previously demonstrated to cause an effective blockade of AT1 and AT2, respectively.28 Control groups of animals of the same age, untreated or treated (AT antagonists and saline-infused), were also studied. Systolic arterial blood pressure was measured in conscious, restrained rats by the tail-cuff sphygmomanometer (NARCO Biosystems, Houston, TX). The blood pressure value for each rat was calculated as the average of three separate measurements at each session.
Immune complex nephritis was induced according to a previously described protocol.26 Briefly, rats received an initial subcutaneous injection of ovalbumin in complete Freund’s adjuvant, followed by reimmunization 3 weeks later, and 1 week later, daily administration of ovalbumin for 9 weeks was started. When proteinuria appeared, animals were randomly distributed into two groups: untreated and quinapril-treated (ACE inhibitor; 100 mg/L in the drinking water) and studied 3 weeks later.
Renal Histopathological Studies
Kidney samples were studied by staining with hematoxylin and eosin and Masson, and examined by light microscopy. The presence of CTGF in renal tissue was determined by immunohistochemistry with a purified rabbit anti-CTGF antibody, which reacts with mouse and rat CTGF (Torrey Pines Biolabs, San Diego, CA), and a mouse anti-fibronectin monoclonal antibody (Chemicon, Temecula, CA) was used. Paraffin-embedded renal tissue sections (4 µm) were mounted on poly-L-lysine-coated slides. The slides were deparaffinized with xylene and graded concentrations of ethanol and then rehydrated. The endogenous peroxidase was blocked by incubation in 3% H2O2/methanol (1:1) at 25°C for 30 minutes. The slides were subsequently incubated in PBS with 4% bovine serum albumin (BSA) and 6% horse or sheep serum, respectively, for 1 hour at 37°C to reduce nonspecific background staining, and then incubated overnight at 4°C with anti-CTGF or anti-fibronectin antibodies in PBS containing 4% BSA and 1% serum. After being washed with PBS, the sections were incubated with a secondary anti-IgG HRP-conjugated antibody diluted 1:100 in 4% BSA/PBS for 1 hour, and after washing, they were stained with 3,3'-diaminobenzidine (Dako Diagnositcs, Barcelona, Spain) in 0.3% H202 for 10 minutes. The sections were counterstained with Mayer’s hematoxylin and mounted in Pertex (Medite, Burgdorf, Germany). In each experiment, negative controls without the primary antibody, or using an unrelated antibody, were included to check nonspecific staining (not shown).
Morphology was scored by semiquantitative determination as previously reported,28 and graded as follows: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, marked staining. Identification of different cell types was based on topographical criteria as published.28 The mean number of positive cells per glomerular cross-section was determined by evaluating 10 to 15 glomeruli. The whole interstitium was examined from each animal, separately evaluating proximal, distal, and collecting ducts. Tubular damage was defined as flattening of epithelium, lumen increase, vacuolization, desquamations, necrosis, and loss of brush border in proximal tubules, and glomerular damage such as increase in mesangial matrix expansion, as described.28 Immunohistochemical quantification was evaluated by image analysis using a KZ 300 imaging system 3.0 (Zeiss, Munchen-Hallbergmoos, Germany). Briefly, the percentage of the stained area was calculated as the ratio of suitable binary thresholded image and the total field area. For each sample, the mean staining area was obtained by analysis of 20 different fields (x40). The staining score is expressed as density per mm2. The immunohistochemistry experiments were performed from two to four kidney sections from each experimental animal, which were stained and analyzed to obtain a mean score for each of them. In all cases, evaluations were performed by two independent observers in a blinded fashion, and the mean score value was then calculated for each rat.
Cell Cultures
Mesangial cells were cultured from isolated rat glomeruli by sequential sieving and differential centrifugation.29 Cells were grown in RPMI 1640 medium, pH 7.4, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/L glutamine, and 10% fetal calf serum (FCS), at 37°C in 5% CO2. These cells were characterized by phase-contrast microscopy, positive staining for desmin and vimentin, and negative staining for keratin and factor VIII antigen, excluding epithelial and endothelial contamination, respectively.29 The murine proximal tubuloepithelial cells (MCT line), kindly supplied by Dr. E. G. Neilson (University of Pennsylvania, Philadelphia, PA), were grown in RPMI. Mesangial and tubular epithelial cells have AT1 and AT2 receptors.28,29 After confluence, cells were growth-arrested by incubation with 0.5% FCS for 48 hours before the experiments. The experiments were done with different preparations of cultured mesangial cells with 0 or 1 passage.
RNA Studies
Total RNA was isolated using a standard method with Trizol reagent (Gibco-BRL, Grand Island, NY) and CTGF mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and/or Northern blot.6
Northern blot analysis was carried out as described.23
Briefly, 40 µg of total RNA were denatured and electrophoresed on 1% agarose-formaldehyde gels, and then transferred to nylon membranes (Genescreen; New England Nuclear). Membranes were hybridized with the 
[32P]-labeled DNA probes, and were exposed to autoradiographic films. G3PDH was used as control of RNA loading. For RT-PCR specific primers used were: CTGF (sense: 5'-GAGTGGGTGTGTGACGAGCCCAAGG-3', antisense: 5'-ATGTCTCCGTACATCTTCCTGTAGT-3') and G3PDH (sense: 5'-AATGCATCCTGCACCACCAA-3', antisense: 5'-GTAGCCATATTCATTGTCATA-3'),which yield a 376 bp and 515 bp product, and PCR (1 minute at 94°C, 1 minute at 63°/54°C and 2 minutes at 68°C; 25 cycles), respectively. In control experiments we have determined the CTGF mRNA levels of control and AngII-treated samples, using different amount of mRNA and analyzing several PCR cycles (from 20 to 40, in 5-cycle interval), and then 25 cycles were chosen to compare groups because they provide submaximal cDNA amplifications. Other additional controls were done: RT-PCR with RNA samples but without AMV reverse transcriptase. In parallel experiments, CTGF PCR products were purified, sequences were confirmed and then used as DNA probes for Northern blot. The PCR products were analyzed on 6% polyacrylamide gels, which were dried and then exposed to X-OMAT AS films (Eastman Kodak, Rochester, NY). The autoradiographs were scanned using the GS-800 Calibrated Densitometer (Quantity One, Bio-Rad, Spain), obtaining densitometric arbitrary units. Data were normalized against those of the corresponding G3PDH data. To compare different groups, ratio CTGF/G3PDH in control rats is arbitrary equal to 1. In addition, all samples were analyzed in duplicate to obtain each sample mRNA value. Then, results were calculated as n-fold increase over control and expressed as mean ± SEM of n animals of each group. In another set of control experiments using RNA samples from cells, Northern blot, and RT-PCR analysis were compared. We have obtained similar results with both methods (data not shown), confirming the validation of RT-PCR for analysis of CTGF gene levels in renal samples, where the total amount of RNA obtained is not enough to perform Northern blot.
Western Blot
Cells were homogenized in lysis buffer (170 mmol/L TrisHCl, 22% glycerol, 2,2% sodium dodecyl sulfate (SDS), 0,1 mmol/L phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail) and then separated by 12% SDS-polyacrylamide gel electrophoresis under reducing conditions. After electrophoresis, samples were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked in PBS containing 0.1% Tween-20, and 5% dry skimmed milk for 1 hour at 37°C, and then incubated in the same buffer with specific CTGF or fibronectin antibodies for 18 hours at 4°C. After washing, detection was made by incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham). In all experiments protein content was determined by BCA method. In addition, Red Ponceau staining was used to show quality of proteins and the efficacy of protein transfer to the membrane (not shown). In experiments of cell-associated proteins -tubuline was used as a loading control.
To neutralize TGF-ß bioactivity we have used a neutralizing antibody against active TGF-ß (R&D Systems, UK). The neutralization dose (10 µg/ml) was selected following the manufacturer instructions. To block CTGF actions, we used a CTGF antisense oligonucleotide, constructed with a 16 mer derived from the starting translation site, which contained the initial ATG whose sequence is 5'-TACTGGCGGCGGTCAT-3'.18 The CTGF antisense oligonucleotide (20 µg/ml) was added directly to the serum-free medium without any transfection compounds. CTGF sense oligonucleotide was used as a control.
Statistical Analysis
Results are expressed as n-fold increase over control in densitometric arbitrary units, normalized by internal controls, and expressed as mean ± SEM of the experiments done. Significance was established by the GraphPAD Instat using Student’s t-test (GraphPAD Software), Mann-Whitney test (non-parametric t-test) and analysis of variance nonparametric (Kruskal-Wallis test), and differences were considered significant if the P value was less than 0.05.
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Results |
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CTGF transcripts have been detected in the kidney by RT-PCR and in situ hybridization, showing that CTGF expression transcripts were low in the glomeruli of control mice.6
We have investigated the direct in vivo effect of AngII by systemic infusion into normal rats evaluating CTGF levels by RT-PCR. AngII-infused rats for 3 days presented elevated renal CTGF mRNA levels (5.9-fold versus control, P < 0.05, n = 8, RT-PCR; Figure 1
). We evaluated CTGF production in the kidney of these animals using immunohistochemistry (Figure 2)
. Control animals did not present staining for CTGF. After 3 days of AngII infusion, CTGF immunostaining was observed in glomeruli (located mainly in mesangial area), tubuli (mainly in distal tubular epithelial cells), and renal arteries (mainly in -smooth muscle actin-positive cells). In AngII-infused rats for 7 days, CTGF overexpression remained elevated in the same renal structures (Figure 2C)
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. At day 7, animals presented mild mesangial matrix expansion and focal areas of tubular injury (Table 1)
, as described.28,30
To further study the implication of AngII on matrix accumulation, we determined fibronectin deposition by immunohistochemistry. In the normal kidney, fibronectin is present in the glomeruli, interstitium, and tubules along the basement membrane. During renal injury, fibronectin deposition is frequently increased.31
Infusion of AngII for 3 days almost did not modify fibronectin deposition (not shown), but after 7 days fibronectin was markedly increased in glomerular and tubulointerstitial areas (Figure 3)
. These data show that up-regulation of CTGF production occurs earlier than fibronectin deposition, suggesting that CTGF could be a mediator of AngII-induced renal fibrosis.
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, suggesting that AngII-induced CTGF overexpression was mediated by the AT1 receptor. In AngII-infused rats for 7 days, the AT1 antagonist normalized blood pressure (101 ± 3 vs. 150 ± 12 mmHg in AngII-infused alone, n = 8, P < 0.05), decreased renal CTGF (Figure 2)
and fibronectin protein deposition (Figure 3)
and ameliorated glomerular sclerosis and tubular damage (Table 1)
. All these results show that AngII via AT1 regulates CTGF, ECM proteins overexpression and fibrosis. RAS Blockade Diminished CTGF Overexpression in an Experimental Model of Normotensive Immune Complex Nephritis
In human and experimental nephritis, renal CTGF overexpression has been described.2,3
Our second aim was to determine whether RAS blockade could modulate CTGF during kidney injury. In a normotensive model of immune complex nephritis in rats, characterized by elevated renal AngII production and mRNA overexpression of TGF-ß and ECM proteins, associated with fibrosis,26
we have investigated whether RAS blockade did modulate renal CTGF expression. In nephritic animals, CTGF mRNA was increased compared to controls (Figure 4
; 8.2-fold RT-PCR, n = 8, P < 0.05 vs. control). The ACE inhibitor quinapril diminished CTGF overexpression (Figure 4)
, as well as the gene expression of TGF-ß and ECM proteins, and ameliorated proteinuria and fibrosis.26
These data show that during renal injury, blockade of renal AngII generation, without modification of blood pressure, diminished CTGF up-regulation and fibrosis.
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To investigate whether AngII could regulate CTGF in renal tissue, independently of hemodynamic changes, we studied the direct effect of AngII on CTGF expression in two renal cell types, in which we have observed up-regulation of CTGF after AngII infusion in vivo: mesangial cells (MC) and tubuloepithelial cells (MCT).
By Northern blot we have observed that growth-arrested mesangial cells expressed low CTGF mRNA levels, as shown as a band of 2.4 kb, that increased in AngII-treated cells (Figure 5A)
. This effect was seen within 1 hour, and CTGF mRNA levels remained elevated after 24 hours. The PKC activator PMA was used as positive control. Similar results were seen by RT-PCR (not shown).
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. CTGF gene was also increased after at 24 hours, showing a similar behavior to that of mesangial cells. This up-regulation was accompanied by increased CTGF protein synthesis after 18 hours, prolonged for at least 48 hours (Figure 7)
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. These results suggest that AngII-induced CTGF up-regulation was mediated by AT1 receptors and confirm the in vivo data.
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TGF-ß is the most important regulator of ECM, since it increases matrix production, inhibits matrix degradation, and modulates integrin receptors.25
Previous studies have demonstrated that TGF-ß is a direct stimulus for CTGF gene transcription.8,11
In human fibroblasts, TGF-ß presents a rapid and maintained CTGF gene up-regulation,11,15,33
similar to that observed in mesangial cells treated with AngII. In contrast, platelet-derived growth factor (PDGF), epidermal growth factor, fibroblast growth factor, serotonin, and lysophosphatidic acid provoked only a transient and weak response.33-35
We, and others, have previously demonstrated that the blockade of TGF-ß, with antibodies that neutralize active TGF-ß, blocked AngII-induced ECM production in renal cells.23,25
For this reason, we evaluated whether TGF-ß could be involved in CTGF regulation in response to AngII. The presence of a TGF-ß neutralizing antibody diminished AngII-induced CTGF mRNA induction after 24 hours and protein production after 48 hours (Figure 9)
, suggesting that CTGF production caused by AngII is, at least in part, mediated by endogenous TGF-ß synthesis.
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We investigated whether CTGF was involved in AngII-induced fibrosis, evaluating its effect on soluble fibronectin production by Western blot. We analyzed the effects of endogenous CTGF, blocking its production using an antisense CTGF oligodeoxynucleotide. In cultured mesangial cells, AngII significantly increased fibronectin release into the medium after 48 and 72 hours of incubation. Coincubation with the CTGF antisense oligonucleotide diminished AngII-induced fibronectin production at both times (Figure 10)
, suggesting that CTGF has a crucial role in the profibrotic action of AngII in renal cells.
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Discussion |
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In human normal kidney, CTGF mRNA was mainly expressed in epithelial cells and some interstitial cells,8 as observed in rats.9 In human and experimental models of renal injury, such as crescentic glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, lupus nephritis, chronic transplant rejection, focal glomerulosclerosis, membranoproliferative glomerulonephritis, 5/6 nephrectomy and diabetic nephropathy, CTGF was strongly up-regulated.3,6,8-10 In contrast, CTGF expression did not appear to be increased in minimal change nephrotic syndrome and membranous nephropathy, glomerular diseases characterized by non-inflammatory lesions and proteinuria.3 We have observed a marked renal CTGF overexpression in two different models of renal injury, AngII-induced renal damage and immune complex nephritis. These findings clearly support the hypothesis that CTGF is a common growth factor involved in renal damage.
In several models of kidney damage, as unilateral ureteral obstruction, anti-Thy-1.1 nephritis and experimental diabetic glomerulosclerosis, TGF-ß1 and CTGF were coordinately up-regulated from the early stage of interstitial fibrosis followed by marked induction of ECM proteins.8-10 In diabetic nephropathy, CTGF overexpression was observed when mesangial expansion was mild, and interstitial disease and proteinuria were absent.6,8 In AngII-induced renal damage, CTGF up-regulation was observed after 3 days, preceding elevated fibronectin deposition, which was found after 7 days, when tubular damage was also observed. In rats with immune complex nephritis, renal CTGF mRNA expression was strongly up-regulated coincidentally with overexpression of TGF-ß and ECM proteins. In this phase of the disease, animals also present renal damage, fibrosis, and severe proteinuria.26 In a model of skin fibrosis, CTGF mRNA levels remained elevated in areas of persistent fibrosis,36 as we observed after 7 days of AngII infusion. In addition, injection of CTGF into the skin induces the formation of fibrous tissue.12 These data strongly suggest that CTGF could be a mediator of AngII-induced fibrosis in the kidney.
Pharmacological blockade of the RAS has demonstrated beneficial effects in several kidney diseases. In experimental models of renal damage, ACE inhibitors and AT1 antagonists reduced gene up-regulation of TGF-ß and ECM proteins as well as fibrosis.25-27 We have observed that an ACE inhibitor decreased CTGF overexpression in rats with immune nephritic and an AT1 antagonist diminished AngII-induced CTGF up-regulation, as well as renal fibrosis. These data show that the beneficial effects of RAS blockers could be due to the modulation of growth factors such as CTGF. Our data demonstrate that drugs controlling AngII are probably some of the best ways of avoiding fibrosis in progressive renal diseases.
Hypertension causes structural changes in the kidney, including cellular hypertrophy and ECM accumulation, which have been attributed to hemodynamic changes associated to mechanical stress and to factors like AngII and TGF-ß. Mechanical strains, such as stretch, shear stress, and static pressure, are possible regulatory elements in CTGF expression.6,37 In mesangial cells, high static pressure and cyclic mechanical stretching up-regulated CTGF and ECM mRNA.37-39 These effects were reversed by a CTGF antisense oligonucleotide.39 In contrast, Riser et al6,8 did not find any increase in CTGF protein after 4 to 72 hours of cyclin stress either under serum or serum-depleted conditions. Systemic infusion of AngII into normal rats caused a slight blood pressure elevation; however, several data suggest that the cellular actions of AngII are responsible for CTGF up-regulation. In a rat model of cyclosporine A and high sodium diet-induced hypertension, an AT1 antagonist normalized increased cardiac CTGF expression, while it only partially diminished blood pressure elevation.40 In rats with immune complex nephritis, renal CTGF mRNA expression was strongly up-regulated in the absence of systemic hypertension, and blockade of renal AngII generation by ACE inhibition diminished CTGF mRNA expression. In AngII-infused rats, the AT1 antagonist Losartan diminished renal CTGF up-regulation. Moreover, in cultured mesangial cells the AT1 antagonist also decreased CTGF gene expression and synthesis. All these in vivo evidence, together with our in vitro data, suggest that CTGF is up-regulated by a direct, non-pressure-mediated, AngII-effect.
Mesangial cells are the main cells involved in the development of glomerulosclerosis. In these cells, CTGF significantly increased fibronectin and collagen type I and IV production, induced its own expression, regulated mesangial cell migration, but did not increase cell proliferation.6-8
CTGF is up-regulated by high extracellular glucose, TGF-ß, and, as we have observed, AngII, but not by PDGF.6-8
Other factors, including TNF-, cAMP, and NO, suppress CTGF.17,41,42
AngII, via AT1, regulates mesangial cell behavior, inducing cell proliferation or hypertrophy and increasing ECM accumulation.20
These processes seem to be mediated by the endogenous production of several factors, including TGF-ß20,23,24
and CTGF. In this regard, we have observed that an antisense CTGF oligonucleotide diminished AngII-induced fibronectin production. In mesangial cells, CTGF is a downstream mediator of profibrogenic actions of TGF-ß.7,8
Using a neutralizing TGF-ß antibody, CTGF induction caused by AngII was diminished, showing that TGF-ß mediates, at least in part, CTGF production. In AngII-infused rats, CTGF immunostaining was elevated in the mesangial area, and preceded mesangial matrix expansion observed after 7 days. These data suggest that CTGF is a mediator of AngII-induced mesangial matrix accumulation.
The chronic accumulation of ECM in glomeruli and tubulointerstitial regions is critical in the progressive loss of function in renal diseases.24 Tubulointerstitial fibrosis is considered to be a predictor of poor outcome in renal diseases. In human nephritis, the increase in the number of cells expressing CTGF mRNA was observed at sites of chronic tubulointerstitial damage, which correlated with the degree of damage.3 We have found that AngII increases CTGF in tubular cells in vivo and in vitro. Moreover, AngII infusion for 7 days caused tubular damage. In murine proximal tubuloepithelial cells AngII via AT1 induces hypertrophy, production of TGF-ß and ECM accumulation22 and, as observed by us, CTGF. In these cells, other factors also up-regulate CTGF including TGF-ß, calcium oxalate, and mechanical wounding, but not high glucose,43-45 and CTGF increased fibronectin but not collagen type I and type III expression.43 These data suggest that CTGF could also be involved in AngII-induced fibrotic responses in tubular cells.
Cell-derived CTGF may act on renal cells by an autocrine mode, contributing to the increased expansion of ECM proteins. The primary target cells for CTGF in the renal interstitium are myofibroblasts, which are responsible for providing ECM proteins in interstitial fibrosis. In this sense, in normal rat kidney (NRK) fibroblasts, CTGF increases ECM proteins.14-17 In these cells AngII also increases cell proliferation, TGF-ß, and ECM production.23 CTGF is also involved in myofibroblast differentiation.46 In the rat remnant kidney model, CTGF is highly expressed in interstitial fibroblasts, coinciding with damage, regeneration, and fibrosis. The description of CTGF mRNA expression in the proximity of regenerative epithelial cells and those transdifferentiating to myofibroblasts suggests that CTGF may modulate renal tubular epithelial differentiation.15 These results indicate that CTGF has a crucial role in the profibrotic action of TGF-ß and AngII in renal cells, providing a potential therapeutic target against tubulointerstitial fibrosis.
On the whole, we have presented information showing that AngII via CTGF is implicated in the process of renal fibrosis. Our results suggest that CTGF up-regulation is an important factor in the pathogenesis of AngII-induced renal fibrosis, regulating mesangial matrix accumulation, progressive glomerulosclerosis, and tubular damage, acting downstream of AngII. TGF-ß is considered the main factor involved in matrix regulation. The blockade of TGF-ß actions with neutralizing antibodies and decorin has demonstrated reduction of fibrosis in experimental models; however, the TGF-ß knockout mice is lethal, presenting a hyperinflammatory phenotype,47 suggesting that other more specific targets for antifibrotic therapy are necessary. Since there are no data available on the involvement of CTGF in modulation of inflammation and immune reactions, and this growth factor is a downstream mediator of TGF-ß and, as shown here, of AngII-induced fibrosis, CTGF maybe a useful target for novel antifibrotic therapies.
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Footnotes |
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Supported by grants from Fondo de Investigación Sanitaria (01/3130, PI020513, and PI020822), Comunidad Autónoma de Madrid (08.4/0017/2000 and 08.4/0018/2001), Sociedad Española de Nefrología, Fundación Renal Iñigo Alvarez de Toledo, European grant of shared-cost Research and Technological Development actions (QLRT-200101215), Fondecyt 1000433, and Fondo Europeo de Desarrollo Regional.
M. R., O. L., and V. E. are fellows of Fondo de Investigación Sanitaria.
Accepted for publication July 29, 2003.
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References |
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B and AP-1 in the kidney: role of AT1 and AT2 receptors. Am J Pathol 2001, 158:1743-1756B in cultured mesangial cells mainly via AT2 receptors: studies with AT1 receptor-knockout mice. J Am Soc Nephrol 2002, 13:1162-1171 suppresses the induction of connective tissue growth factor by transforming growth factor-ß in normal and scleroderma fibroblasts. J Biol Chem 2000, 275:15220-15225This article has been cited by other articles:
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 B. F. Schrijvers, A. S. De Vriese, and A. Flyvbjerg From Hyperglycemia to Diabetic Kidney Disease: The Role of Metabolic, Hemodynamic, Intracellular Factors and Growth Factors/Cytokines Endocr. Rev., December 1, 2004; 25(6): 971 - 1010. [Abstract] [Full Text] [PDF]
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 G. Zhou, C. Li, and L. Cai Advanced Glycation End-Products Induce Connective Tissue Growth Factor-Mediated Renal Fibrosis Predominantly through Transforming Growth Factor {beta}-Independent Pathway Am. J. Pathol., December 1, 2004; 165(6): 2033 - 2043. [Abstract] [Full Text] [PDF]
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38 to 42 kd, corresponding to the predicted CTGF size, was detected. A: A representative Western blot of CTGF and 
