Angiotensin II Increases Connective Tissue Growth Factor in the Kidney
2003; Elsevier BV; Volume: 163; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63552-3
ISSN1525-2191
AutoresMónica Rupérez, Marta Ruíz-Ortega, Vanesa Esteban, Óscar Lorenzo, Sergio Mezzano, Juan José González Plaza, Jesús Egido,
Tópico(s)Systemic Sclerosis and Related Diseases
ResumoConnective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and is induced by different factors involved in renal injury. Angiotensin II (AngII) participates in the pathogenesis of kidney damage, contributing to fibrosis; however, whether AngII regulates CTGF in the kidney has not been explored. Systemic infusion of AngII into normal rats for 3 days increased renal CTGF mRNA and protein levels. At day 7, AngII-infused rats presented overexpression of CTGF in glomeruli, tubuli, and renal arteries, as well as tubular injury and elevated fibronectin deposition. Only treatment with an AT1 receptor antagonist, but not an AT2, diminished CTGF and fibronectin overexpression and ameliorated tubular damage. In rats with immune complex nephritis, renal overexpression of CTGF was diminished by the ACE inhibitor quinapril, correlated with a diminution in fibrosis. In cultured renal cells (mesangial and tubular epithelial cells) AngII, via AT1, increased CTGF mRNA and protein production, and a CTGF antisense oligonucleotide decreased AngII-induced fibronectin synthesis. Our data show that AngII regulates CTGF in the kidney and cultured in mesangial and tubular cells. This novel finding suggests that CTGF could be a mediator of the profibrogenic effects of AngII in the kidney. Connective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and is induced by different factors involved in renal injury. Angiotensin II (AngII) participates in the pathogenesis of kidney damage, contributing to fibrosis; however, whether AngII regulates CTGF in the kidney has not been explored. Systemic infusion of AngII into normal rats for 3 days increased renal CTGF mRNA and protein levels. At day 7, AngII-infused rats presented overexpression of CTGF in glomeruli, tubuli, and renal arteries, as well as tubular injury and elevated fibronectin deposition. Only treatment with an AT1 receptor antagonist, but not an AT2, diminished CTGF and fibronectin overexpression and ameliorated tubular damage. In rats with immune complex nephritis, renal overexpression of CTGF was diminished by the ACE inhibitor quinapril, correlated with a diminution in fibrosis. In cultured renal cells (mesangial and tubular epithelial cells) AngII, via AT1, increased CTGF mRNA and protein production, and a CTGF antisense oligonucleotide decreased AngII-induced fibronectin synthesis. Our data show that AngII regulates CTGF in the kidney and cultured in mesangial and tubular cells. This novel finding suggests that CTGF could be a mediator of the profibrogenic effects of AngII in the kidney. The connective tissue growth factor (CTGF), a member of the CCN family of early response genes, has been recently described as a new fibrotic mediator.1Lau LF Lam SCT The CCN family of angiogenic regulators: the integrin connection.Exp Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar CTGF has been detected in many human tissues and biological fluids, being most abundant in the kidney.2Gupta S Clarkson MR Duggan J Brady HR Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Crossref PubMed Scopus (268) Google Scholar, 3Ito Y Aten J Bende RJ Oemar BS Rabelink TJ Weening JJ Goldschmeding R Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Crossref PubMed Scopus (512) Google Scholar This growth factor is overexpressed in a variety of fibrotic disorders, such as skin, vascular, and lung diseases.1Lau LF Lam SCT The CCN family of angiogenic regulators: the integrin connection.Exp Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar, 2Gupta S Clarkson MR Duggan J Brady HR Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Crossref PubMed Scopus (268) Google Scholar, 3Ito Y Aten J Bende RJ Oemar BS Rabelink TJ Weening JJ Goldschmeding R Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Crossref PubMed Scopus (512) Google Scholar, 4Oemar BS Luscher TF Connective tissue growth factor: friend or foe?.Arterioscler Thromb Vasc Biol. 1997; 17: 1483-1489Crossref PubMed Scopus (148) Google Scholar Fibrosis is a common final pathway of renal diseases of diverse etiology, including inflammation, hemodynamics, and metabolic injury. In several human kidney diseases, CTGF was strongly up-regulated in the glomeruli and tubulointerstitium in association with scarring and sclerosis.2Gupta S Clarkson MR Duggan J Brady HR Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Crossref PubMed Scopus (268) Google Scholar, 3Ito Y Aten J Bende RJ Oemar BS Rabelink TJ Weening JJ Goldschmeding R Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Crossref PubMed Scopus (512) Google Scholar In experimental models of renal wound repair and scarring, elevated renal CTGF expression was found correlated with cellular proliferation and extracellular matrix (ECM) accumulation.2Gupta S Clarkson MR Duggan J Brady HR Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Crossref PubMed Scopus (268) Google Scholar, 5Murphy M Godson C Cannon S Kato S Mackenzie HS Martin F 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. 1999; 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37: 328-335Crossref PubMed Scopus (65) Google Scholar and mediates many of the profibrotic actions of TGF-β.1Lau LF Lam SCT The CCN family of angiogenic regulators: the integrin connection.Exp Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar, 5Murphy M Godson C Cannon S Kato S Mackenzie HS Martin F 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. 1999; 26: 5830-5834Crossref Scopus (355) Google Scholar, 7Blom IE van Dijk AJ Wieten L Duran K Ito Y Kleij L deNichilo M Rabelink TJ Weening JJ Aten J Goldschmeding R In vitro evidence for differential involvement of CTGF, TGF-β, and PDGF-BB in mesangial response to injury.Nephrol Dial Transplant. 2001; 16: 1139-1148Crossref PubMed Scopus (111) Google Scholar, 10Yokoi H Sugawara A Mukoyama M Mori K Makino H Suganami T Nagae T Yahata K Fujinaga Y Tanaka I Nakao K Role of connective tissue growth factor in profibrotic action of transforming growth factor-β: a potential target for preventing renal fibrosis.Am J Kidney Dis. 2001; 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13: 1774-1778Google Scholar Depending on the cell types, CTGF has diverse bioactivities, including induction of mitogenesis, chemotaxis, cellular adhesion, ECM production, regulation of proliferation/apoptosis, and angiogenesis.1Lau LF Lam SCT The CCN family of angiogenic regulators: the integrin connection.Exp Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar, 2Gupta S Clarkson MR Duggan J Brady HR Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Crossref PubMed Scopus (268) Google Scholar, 3Ito Y Aten J Bende RJ Oemar BS Rabelink TJ Weening JJ Goldschmeding R Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Crossref PubMed Scopus (512) Google Scholar, 4Oemar BS Luscher TF Connective tissue growth factor: friend or foe?.Arterioscler Thromb Vasc Biol. 1997; 17: 1483-1489Crossref PubMed Scopus (148) Google Scholar, 18Hisikawa K Nakaki T Fujii T Transforming growth factor-β1 induces apoptosis via connective tissue growth factor in human aortic smooth muscle cells.Eur J Pharmacol. 1999; 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83: 1182-1191Crossref PubMed Scopus (427) Google Scholar 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.20Egido J Vasoactive hormones and renal sclerosis.Kidney Int. 1996; 49: 578-597Crossref PubMed Scopus (207) Google Scholar, 21Matsubara H Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases.Circ Res. 1998; 83: 1182-1191Crossref PubMed Scopus (427) Google Scholar, 22Wolf G Neilson EG Angiotensin II as a renal growth factor.J Am Soc Nephrol. 1993; 3: 1531-1540PubMed Google Scholar, 23Ruiz-Ortega M Egido J Angiotensin II modulates cell growth-related events and synthesis of matrix proteins in renal interstitial fibroblasts.Kidney Int. 1997; 52: 1497-1510Crossref PubMed Scopus (183) Google Scholar, 24Mezzano S Ruiz-Ortega M Egido J Angiotensin II and renal fibrosis.Hypertension. 2001; 38: 635-638Crossref PubMed Scopus (414) Google Scholar A close relation between AngII and TGF-β has already been established. Systemic infusion of AngII into normal rats increases glomerular TGF-β.25Border WA Noble NA Interactions of transforming growth factor-β and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar In renal cells AngII increases TGF-β mRNA expression and synthesis as well as TGF-β conversion to its active form. Moreover, neutralizing antibodies to TGF-β remarkably reduce AngII-induced ECM production.20Egido J Vasoactive hormones and renal sclerosis.Kidney Int. 1996; 49: 578-597Crossref PubMed Scopus (207) Google Scholar, 22Wolf G Neilson EG Angiotensin II as a renal growth factor.J Am Soc Nephrol. 1993; 3: 1531-1540PubMed Google Scholar, 23Ruiz-Ortega M Egido J Angiotensin II modulates cell growth-related events and synthesis of matrix proteins in renal interstitial fibroblasts.Kidney Int. 1997; 52: 1497-1510Crossref PubMed Scopus (183) Google Scholar, 24Mezzano S Ruiz-Ortega M Egido J Angiotensin II and renal fibrosis.Hypertension. 2001; 38: 635-638Crossref PubMed Scopus (414) Google Scholar, 25Border WA Noble NA Interactions of transforming growth factor-β and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar In experimental models of kidney damage, renal RAS activation and renal TGF-β 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-β and fibrosis.25Border WA Noble NA Interactions of transforming growth factor-β and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar, 26Ruiz-Ortega M González S Serón D Condom E Bustos C Largo R González E Ortiz A Egido J ACE inhibition reduces proteinuria, glomerular lesions and extracellular matrix production in a normotensive rat model of immune complex nephritis.Kidney Int. 1995; 48: 1778-1791Crossref PubMed Scopus (120) Google Scholar, 27Wu LL Cox A Roe CJ Dziadek M Cooper ME Gilbert RE Transforming growth factor-β1 and renal injury following subtotal nephrectomy in the rat: role of the renin-angiotensin system.Kidney Int. 1997; 51: 1555-1567Crossref Scopus (202) Google Scholar 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-β profibrotic activities,1Lau LF Lam SCT The CCN family of angiogenic regulators: the integrin connection.Exp Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (575) Google Scholar, 2Gupta S Clarkson MR Duggan J Brady HR Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis.Kidney Int. 2000; 58: 1389-1399Crossref PubMed Scopus (268) Google Scholar, 3Ito Y Aten J Bende RJ Oemar BS Rabelink TJ Weening JJ Goldschmeding R Expression of connective tissue growth factor in human renal fibrosis.Kidney Int. 1998; 53: 853-861Crossref PubMed Scopus (512) Google Scholar, 4Oemar BS Luscher TF Connective tissue growth factor: friend or foe?.Arterioscler Thromb Vasc Biol. 1997; 17: 1483-1489Crossref PubMed Scopus (148) Google Scholar, 16Grotendorst GR Connective tissue growth factor: a mediator of TGF-β action on fibroblasts.Cytokine Growth Factor Rev. 1997; 8: 171-179Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 17Duncan MR Frazier KS Abramson S Williams S Klapper H Huang X Grotendorst GR Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: down-regulation by cAMP.EMBO J. 1999; 13: 1774-1778Google Scholar the potential link between renal RAS and CTGF has not yet been investigated. 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.28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Systemic Infusion of angiotensin II into normal rats activates nuclear factor κB and AP-1 in the kidney: role of AT1 and AT2 receptors.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar 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.26Ruiz-Ortega M González S Serón D Condom E Bustos C Largo R González E Ortiz A Egido J ACE inhibition reduces proteinuria, glomerular lesions and extracellular matrix production in a normotensive rat model of immune complex nephritis.Kidney Int. 1995; 48: 1778-1791Crossref PubMed Scopus (120) Google Scholar 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. 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,28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Systemic Infusion of angiotensin II into normal rats activates nuclear factor κB and AP-1 in the kidney: role of AT1 and AT2 receptors.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar 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.28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Systemic Infusion of angiotensin II into normal rats activates nuclear factor κB and AP-1 in the kidney: role of AT1 and AT2 receptors.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar 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.28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Systemic Infusion of angiotensin II into normal rats activates nuclear factor κB and AP-1 in the kidney: role of AT1 and AT2 receptors.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar 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 (×40). 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. Mesangial cells were cultured from isolated rat glomeruli by sequential sieving and differential centrifugation.29Lorenzo O Ruiz-Ortega M Suzuki Y Rupérez M Esteban V Sugaya T Egido J Angiotensin III activates nuclear transcription factor κB in cultured mesangial cells mainly via AT2 receptors: studies with AT1 receptor-knockout mice.J Am Soc Nephrol. 2002; 13: 1162-1171Crossref PubMed Scopus (37) Google Scholar 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.29Lorenzo O Ruiz-Ortega M Suzuki Y Rupérez M Esteban V Sugaya T Egido J Angiotensin III activates nuclear transcription factor κB in cultured mesangial cells mainly via AT2 receptors: studies with AT1 receptor-knockout mice.J Am Soc Nephrol. 2002; 13: 1162-1171Crossref PubMed Scopus (37) Google Scholar 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.28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Systemic Infusion of angiotensin II into normal rats activates nuclear factor κB and AP-1 in the kidney: role of AT1 and AT2 receptors.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 29Lorenzo O Ruiz-Ortega M Suzuki Y Rupérez M Esteban V Sugaya T Egido J Angiotensin III activates nuclear transcription factor κB in cultured mesangial cells mainly via AT2 receptors: studies with AT1 receptor-knockout mice.J Am Soc Nephrol. 2002; 13: 1162-1171Crossref PubMed Scopus (37) Google Scholar 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. 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.6Riser BL Denichillo M Cortes P Baker C Grodin JM Yee J 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. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar Northern blot analysis was carried out as described.23Ruiz-Ortega M Egido J Angiotensin II modulates cell growth-related events and synthesis of matrix proteins in renal interstitial fibroblasts.Kidney In
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