Renin increases mesangial cell transforming growth factor-β1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms
2005; Elsevier BV; Volume: 69; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5000011
ISSN1523-1755
AutoresYufeng Huang, S. Wongamorntham, J. Kasting, David J. McQuillan, R. T. Owens, Ling Yu, Nancy A. Noble, Wayne A. Border,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoRecent evidence suggesting a strong interplay between components of the renin–angiotensin system and key mediators of fibrosis led us to hypothesize that renin, independent of its enzymatic action to enhance angiotensin (Ang) II synthesis, directly increases production of the fibrogenic cytokine transforming growth factor (TGF)-β. Human or rat mesangial cells (MCs) were treated with human recombinant renin (HrRenin) or rat recombinant renin (RrRenin) and the effects on TGF-β1, plasminogen activator inhibitor-type 1 (PAI-1), fibronectin (FN) and collagen 1 mRNA and protein were investigated. Blockade of the rat MC renin receptor was achieved using siRNA. HrRenin or RrRenin, at doses shown to be physiologically relevant, induced marked dose- and time-dependent increases in TGF-β1. These effects were not altered by adding an inhibitor of renin's enzymatic action (RO 42-5892), the Ang II receptor antagonist losartan or the Ang-converting enzyme inhibitor enalapril. RrRenin also induced PAI-1, FN and collagen 1 mRNA and PAI-1 and FN protein in a dose-dependent manner. Neutralizing antibodies to TGF-β partially blocked these effects. Supernatant and cell lysate Ang I and Ang II levels were extremely low. MC angiotensinogen mRNA was undetectable both with and without added renin. Targeting of the rat renin receptor mRNA with siRNA blocked induction of TGF-β1. We conclude that renin upregulates MC TGF-β1 through a receptor-mediated mechanism, independent of Ang II generation or action. Renin-induced increases in TGF-β1 in turn stimulate increases in PAI-1, FN and collagen I. Thus, renin may contribute to renal fibrotic disease, particularly when therapeutic Ang II blockade elevates plasma renin. Recent evidence suggesting a strong interplay between components of the renin–angiotensin system and key mediators of fibrosis led us to hypothesize that renin, independent of its enzymatic action to enhance angiotensin (Ang) II synthesis, directly increases production of the fibrogenic cytokine transforming growth factor (TGF)-β. Human or rat mesangial cells (MCs) were treated with human recombinant renin (HrRenin) or rat recombinant renin (RrRenin) and the effects on TGF-β1, plasminogen activator inhibitor-type 1 (PAI-1), fibronectin (FN) and collagen 1 mRNA and protein were investigated. Blockade of the rat MC renin receptor was achieved using siRNA. HrRenin or RrRenin, at doses shown to be physiologically relevant, induced marked dose- and time-dependent increases in TGF-β1. These effects were not altered by adding an inhibitor of renin's enzymatic action (RO 42-5892), the Ang II receptor antagonist losartan or the Ang-converting enzyme inhibitor enalapril. RrRenin also induced PAI-1, FN and collagen 1 mRNA and PAI-1 and FN protein in a dose-dependent manner. Neutralizing antibodies to TGF-β partially blocked these effects. Supernatant and cell lysate Ang I and Ang II levels were extremely low. MC angiotensinogen mRNA was undetectable both with and without added renin. Targeting of the rat renin receptor mRNA with siRNA blocked induction of TGF-β1. We conclude that renin upregulates MC TGF-β1 through a receptor-mediated mechanism, independent of Ang II generation or action. Renin-induced increases in TGF-β1 in turn stimulate increases in PAI-1, FN and collagen I. Thus, renin may contribute to renal fibrotic disease, particularly when therapeutic Ang II blockade elevates plasma renin. In the classical renin–angiotensin system, renin has been considered to have no biological activity except to enzymatically cleave angiotensinogen (ANG) to angiotensin (Ang) I.1.Griendling K.K. Murphy T.J. Alexander R.W. Molecular biology of the renin–angiotensin system.Circulation. 1993; 87: 1816-1828Crossref PubMed Scopus (325) Google Scholar Although several cell membrane proteins have been shown to bind renin, no functional effects of such binding have been reported.2.van Kesteren C.A. Danser A.H. Derkx F.H. et al.Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells.Hypertension. 1997; 30: 1389-1396Crossref PubMed Scopus (125) Google Scholar, 3.Admiraal P.J. van Kesteren C.A. Danser A.H. et al.Uptake and proteolytic activation of prorenin by cultured human endothelial cells.J Hypertens. 1999; 17: 621-629Crossref PubMed Scopus (64) Google Scholar, 4.Sealey J.E. Catanzaro D.F. Lavin T.N. et al.Specific prorenin/renin binding (ProBP) – identification and characterization of a novel membrane site.Am J Hypertens. 1996; 9: 491-502Crossref PubMed Scopus (82) Google Scholar Recently, a renin receptor was identified on human mesangial cells (MCs).5.Nguyen G. Delarue F. Berrou J. et al.Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen.Kidney Int. 1996; 50: 1897-1903Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 6.Nguyen G. Delarue F. Burckle C. et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1137) Google Scholar Binding of renin to this receptor induced a hypertrophic effect and increased the synthesis of plasminogen activator inhibitor-type 1 (PAI-1).5.Nguyen G. Delarue F. Berrou J. et al.Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen.Kidney Int. 1996; 50: 1897-1903Abstract Full Text PDF PubMed Scopus (212) Google Scholar This effect is not likely due to renin proteolytic activity leading to Ang II generation, as an inhibitor of renin's enzymatic action had no effect. This receptor has been cloned and is a 350-amino-acid protein with a single transmembrane domain and no homology with any known membrane protein.6.Nguyen G. Delarue F. Burckle C. et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1137) Google Scholar Binding to this protein is specific for renin and prorenin. Very recently, a closely related renin receptor was cloned from rats.7.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the ‘handle’ region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar Although there are no data on cellular effects of the rat renin receptor, human receptor-bound renin induces phosphorylation of serine and tyrosine residues, associated with activation of mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1 and ERK2. This action was not altered in the presence of either an Ang-converting enzyme inhibitor (ACEi) or an Ang II receptor antagonist (ARB), further confirming that the receptor-mediated events are independent of Ang II generation or action. In addition, mice that lack Ang II or Ang II receptor binding because of genetic deficiencies in ANG, ACE or Ang II receptors have high renin levels and develop vascular and glomerular sclerosis.8.Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (310) Google Scholar, 9.Tsuchida S. Matsusaka T. Chen X. et al.Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes.J Clin Invest. 1998; 101: 755-760Crossref PubMed Scopus (278) Google Scholar, 10.Esther Jr, C.R. Howard T.E. Marino E.M. et al.Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 74: 953-965PubMed Google Scholar Taken together, these data suggest that renin may have specific, receptor-mediated actions in addition to its role as an aspartyl protease. In the hypertrophic juxtaglomerular apparatus (JGA) of a hypertensive human, renin has been shown to colocalize with the profibrotic cytokine transforming growth factor (TGF)-β.11.Ray P.E. McCune B. Gomez R.A. et al.Expression of transforming growth factor b3 in hypertrophic juxtaglomerular apparatus.N Engl J Med. 1994; 330: 68-69Crossref PubMed Scopus (10) Google Scholar Treatment of rats with ACEi further increases renin and TGF-β in the JGA,12.Ray P.E. McCune B.K. Gomez R.A. et al.Renal vascular induction of TGF-β2 and renin by potassium depletion.Kidney Int. 1993; 44: 1006-1013Abstract Full Text PDF PubMed Scopus (29) Google Scholar suggesting that the production of renin and TGF-β is coregulated there and that Ang II is not mediating increases in TGF-β.12.Ray P.E. McCune B.K. Gomez R.A. et al.Renal vascular induction of TGF-β2 and renin by potassium depletion.Kidney Int. 1993; 44: 1006-1013Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 13.Border W.A. Noble N.A. Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar Knowledge that renin and TGF-β may be coregulated in the JGA, that renin upregulates PAI-1 synthesis through a receptor on MCs, that TGF-β is a potent stimulator of PAI-1 synthesis14.Border W.A. Noble N.A. Transforming growth factor beta in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2902) Google Scholar, 15.Noble N.A. Border W.A. Angiotensin II in renal fibrosis: should TGF-β rather than blood pressure be the therapeutic target?.Semin Nephrol. 1997; 17: 455-466PubMed Google Scholar, 16.Tomooka S. Border W.A. Marshall B.C. Noble N.A. Glomerular matrix accumulation is linked to inhibition of the plasmin protease system.Kidney Int. 1992; 42: 1462-1469Abstract Full Text PDF PubMed Scopus (194) Google Scholar and that animals deficient in Ang II have high renin levels and develop glomerulosclerosis8.Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (310) Google Scholar led us to hypothesize that renin may act through receptor-mediated, Ang II-independent mechanisms to upregulate synthesis of MC TGF-β and thereby contribute directly to renal fibrosis. Investigation of this hypothesis is of great clinical relevance because the most effective antifibrotic therapies currently available are ACEis and ARBs and these agents markedly elevate plasma renin levels. Human and animal data suggest that, even at high doses, these drugs only slow down progression to end-stage organ failure. The limited effectiveness of these drugs may be due, in part, to the elevated renin levels they induce and the Ang II-independent, profibrotic actions of renin. Human recombinant renin (HrRenin)-treated human MCs showed progressive increases in TGF-β1 levels from the first time point of 4 h up to 72 h, both in control and in renin-treated cell culture supernatants (Figure 1a). Because the difference in TGF-β production between renin-treated and control cells became significant at 24 h (P<0.02), 24 h was used for further experiments. A dose-dependent increase in TGF-β1 was seen, with a four-fold increase at the highest dose of 10-6 M HrRenin (P<0.01; Figure 1b). Treatment of cells with RO 42-5892, which blocks human renin's enzymatic conversion of ANG to Ang I, the ACEi enalapril or the ARB losartan had no effect on renin-induced increases in TGF-β1 production (Figure 1c). The recombinant prorenin elution profile revealed three peaks containing prorenin (Figure 2). When prorenin (∼43 kDa) was digested by trypsin, it was cleaved to renin (∼34 kDa) (Figure 3a). The enzymatic activity of renin increased more than 1000-fold after trypsin digestion (Figure 3b). There was no measurable renin activity in the control buffer.Figure 3(a) Processing of rat recombinant prorenin by immobilized TPCK trypsin. Active renin was generated by incubation of purified (His)6-prorenin with agarose-immobilized TPCK trypsin (Pierce, Rockford, IL, USA) for 6 h at 37°C. Trypsin was removed by centrifugation and the (His)6-propeptide was removed from the supernatant by Ni2+ affinity chromatography. Concentrated prorenin and renin were applied to SDS-PAGE gels, stained and photographed. (b) Renin activity of rat recombinant prorenin and renin determined by an Ang I generation assay. Samples were collected from prorenin, before and after trypsin digestion. The same volume of phosphate-buffered saline was trypsin digested and used to control for an effect of trypsin on renin activity. Renin activity was determined as the ability of a sample to generate Ang I from nephrectomized rat serum substrate (containing ANG).View Large Image Figure ViewerDownload (PPT) Plasma renin activity and glomerular renin content did not differ between control and nephritic rats (Table 1). Using the assumption that plasma and glomerular renin have the same specific activity (U/mol) as rat recombinant renin (RrRenin), the physiological plasma renin concentration in control and diseased rats was estimated to be 1.0 × 10-11 M (Table 1). Glomerular renin concentration was about 100-fold greater at 10-9 M. Enalapril treatment of nephritic rats increased glomerular renin mRNA expression 13-fold (Figure 4). Plasma and glomerular renin activities were increased 5.6- and 8.9-fold, respectively, bringing the estimated glomerular renin concentration in enalapril-treated, nephritic rats into the 10-8 M range (Table 1). Therefore, renin concentrations around 10-8 M were used in further experiments.Table 1Renin activity and concentration in normal and nephritic rats and in RrReninRenin activityRenin content (M)Plasma (ng Ang I/ml/h)Glomeruli (ng Ang I/ml/ 104 glom/h)PlasmaGlomeruliNormal control (NC; N=6)10.84±1.851145±1431.00 × 10-111.06 × 10-9Disease control (DC; N=6)10.25±0.991160±2120.95 × 10-111.07 × 10-9DC+enalapril (N=6)60.31±2.5510 276±3285.58 × 10-110.95 × 10-8RrRenin2.7 × 1062.5 × 10-6 Open table in a new tab TGF-β1 mRNA expression was elevated significantly by almost two-fold (P<0.05) at 1/2 h after renin treatment and remained more than three-fold increased from 2 h to at least 8 h (Figure 5). Dose-dependent increases in TGF-β1 mRNA were seen after 4 h (P<0.01; Figure 6a and b). That mRNA increases resulted in protein increases is shown in Figure 6c where TGF-β1 protein was increased 165% after 48 h.Figure 6Effect of RrRenin dose on TGF-β1 mRNA (a, b) and protein production (c). TGF-β1 mRNA (b) and protein (c) values are expressed relative to the no-additive control, which was set at unity. *P<0.05 vs control. C: control.View Large Image Figure ViewerDownload (PPT) Renin stimulated PAI-1 mRNA 9-fold at 1 h and 13-fold at 2 h (P<0.05). PAI-1 mRNA then declined to control levels by 6 h (Figure 7). PAI-1 mRNA increased in a dose-dependent manner between 10-10 and 10-8 M RrRenin, resulting in a 215% increase in protein after 48 h (Figure 8).Figure 8Effect of RrRenin dose on PAI-1 mRNA (a, b) and protein production (c, d). PAI-1 mRNA (b) and protein (d) values are expressed relative to the no-additive control, which was set at unity. *P<0.05 vs control.View Large Image Figure ViewerDownload (PPT) Treatment of cultures with the TGF-β-neutralizing antibody (Ab), 1D11, or control IgG for 2 or 4 h significantly, but not completely, blocked the stimulation of PAI-1 mRNA synthesis. 1D11 reduced RrRenin-induced increases in PAI-1 mRNA by 12% at 2 h and by 68% at 4 h (Figure 9). Significant increases in fibronectin (FN) and type 1 collagen mRNA were seen at two renin concentrations at 4 h (P<0.05; Figure 10a–c). RrRenin significantly increased FN protein production after 24 h of incubation, in a dose-dependent manner (Figure 10d). Ab to TGF-β, but not control Ab, significantly reduced production of FN by 39% (Figure 10e).Figure 10(a–c) Effect of RrRenin dose on FN-EDA+ and collagen I mRNA expression at 4 h. Densitometric values are expressed relative to the no-additive control, which was set at unity. *P vs control <0.05. **P<0.05 vs 10-10 M renin. (d) Dose response of FN protein production by RrRenin after 24 h incubation. FN protein values are expressed relative to the no-additive control in (d) and to the control+IgG value in (e), both of which were set at unity. *P<0.05 vs control. (e) Effect of neutralizing TGF-β Ab (5 μg/ml, 1D11) on renin-induced increases in FN production. *P<0.05 vs control+IgG, #P<0.05 vs RrRenin+IgG.View Large Image Figure ViewerDownload (PPT) Reports that MCs contain ANG mRNA at very low levels, and renin, ACE and Ang II receptors suggest that these cells have all the components necessary to synthesize Ang II.17.Vidotti D.B. Casarini D.E. Cristovam P.C. et al.High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells.Am J Physiol Renal Physiol. 2004; 286: F1039-F1045Crossref PubMed Scopus (172) Google Scholar, 18.Andrade A.Q. Casarini D.E. Schor N. Boim M.A. Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells.Braz J Med Biol Res. 2002; 35: 17-24PubMed Google Scholar Thus, it is important to rule out the possibility that renin effects on TGF-β in vitro are mediated by increased Ang II production. Two previous publications suggest that renin receptor signaling and upregulation of PAI-1 are independent of Ang II generation or action.5.Nguyen G. Delarue F. Berrou J. et al.Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen.Kidney Int. 1996; 50: 1897-1903Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 7.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the ‘handle’ region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar We provide here seven additional lines of evidence that renin's effects on TGF-β are Ang II independent. (1) As previously published for PAI-1,5.Nguyen G. Delarue F. Berrou J. et al.Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen.Kidney Int. 1996; 50: 1897-1903Abstract Full Text PDF PubMed Scopus (212) Google Scholar data presented in Figure 1c indicate that an inhibitor of renin's enzymatic action to convert ANG to Ang I had no effect on renin-induced upregulation of TGF-β. This suggests that renin does not act through increasing Ang II generation. (2) ANG mRNA was undetectable in cultured rat MCs by reverse transcriptase-polymerase chain reaction (RT-PCR) even when 1000-fold the recommended amount of RNA (1 μg) was used (Figure 11a, left). A 3 μg portion of RNA produced a very weak band after 40 cycles of PCR (data not shown). In contrast, there is abundant ANG mRNA in rat liver tissue (Figure 11a). That gels were loaded with equal amounts of RNA is shown by the gel on the right of Figure 11a, where β-actin and ANG primers were used. (3) There was no visible ANG mRNA signal in MCs treated with 0–10-7 M RrRenin or 0–10-6 M Ang II for 4 h (data not shown). Additional amplification of the first RT-PCR products did not alter the results, further confirming that MC ANG mRNA is very low. Thus, renin does not induce MC production of ANG, which might be converted to Ang II. (4) Consistent with ANG mRNA levels, Ang I levels were below the detectable range of pg/ml in both cultured supernatant and cell lysate, indicating that a reservoir of Ang I is not available to convert to Ang II (data not shown). (5) Ang II levels in culture supernatants were in the 10-11 M range, much lower than physiological levels,19.Seikaly M.G. Arant Jr, B.S. Seney Jr, F.D. Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat.J Clin Invest. 1990; 86: 1352-1357Crossref PubMed Scopus (385) Google Scholar below the level of significant induction of TGF-β reported at 10-8 M Ang II20.Kagami S. Border W.A. Miller D.E. Noble N.A. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.J Clin Invest. 1994; 93: 2431-2437Crossref PubMed Scopus (965) Google Scholar and were not increased by renin treatment (data not shown). (6) Ang II was undetectable in cell lysates and was not increased by renin treatment (data not shown). (7) Treatment of cells with enalaprilate or losartan had no effect on TGF-β1 mRNA levels either in the absence or presence of RrRenin, indicating that blocking the action of any Ang II present does not alter renin's effect (Figure 11b–e). These results indicate that the limited amount of ANG mRNA present in MCs fails to produce significant Ang II either in untreated or renin-treated MCs. As a first step to determining whether renin upregulation of TGF-β1 is renin receptor-mediated, we determined by RT-PCR that renin/prorenin receptor mRNA is abundant in rat MCs (data not shown). Then, transfection of a Stealth™ siRNA molecule that targets the renin receptor was shown to induce a dose-dependent decrease in receptor mRNA after 48 h (Figure 12a) and a time-dependent, 75% reduction in receptor mRNA at 72 h (Figure 12b). Treatment of cells with Lipofectamine™ 2000 or Opti-MEM medium alone did not affect renin receptor mRNA expression. Rat MCs transfected for 72 h with 500 pmol Stealth™ siRNA molecule had undetectable renin receptor mRNA and showed significantly reduced induction of TGF-β1 mRNA with RrRenin treatment (Figure 13), indicating that the MC renin receptor mediates renin-induced increases in TGF-β1 expression.Figure 13Effect of Stealth™ siRNA on renin-induced TGF-β1 mRNA expression and rat renin receptor mRNA expression. Renin receptor and TGF-β1 mRNA levels were determined by Northern blot and standardized for densitometric analysis to GAPDH mRNA levels. The rat renin receptor cDNA was synthesized by RT-PCR and purified using the NucleoSpin® Extraction kit (Clontech Laboratories Inc.). Densitometric values are expressed relative to the Lipofectamine™ 2000 (Lipo)-transfected, no renin added control, which was set at unity. *P<0.05 vs control, #P<0.05 vs Stealth™ siRNA-transfected cells plus renin.View Large Image Figure ViewerDownload (PPT) We have shown here that HrRenin induces a dose-dependent and marked increase in TGF-β1. To more thoroughly explore this finding, it was necessary to produce RrRenin. The assessment of human renin function in rats is impossible because the species specificity of the renin–ANG reaction makes the reaction rate of human renin with rat ANG extremely slow21.Ganten D. Wagner J. Zeh K. et al.Species specificity of renin kinetics in transgenic rats harboring the human renin and angiotensinogen genes.Proc Natl Acad Sci USA. 1992; 89: 7806-7810Crossref PubMed Scopus (197) Google Scholar and because human ANG is cleaved to Ang I by human but not by rat renin.22.Bohlender J. Menard J. Wagner J. et al.Human renin-dependent hypertension in rats transgenic for human angiotensinogen.Hypertension. 1996; 27: 535-540Crossref PubMed Google Scholar We then showed that RrRenin increases TGF-β1 mRNA and protein at concentrations shown to be physiologically relevant by in vivo measurement of renin levels in enalapril-treated, nephritic rats. These effects extend to increased synthesis of PAI-1 and fibrotic extracellular matrix (ECM) components FN and collagen I, and are partially mediated by TGF-β. That the renin-induced increases in PAI-1 mRNA and FN protein were only blocked by 68 and 39%, respectively, with excess TGF-β Ab indicates that TGF-β-independent pathways are responsible for part of the profibrotic action of renin. siRNA targeting of the rat renin receptor completely blocked the renin-induced increase in TGF-β1 expression, indicating that this action is clearly renin receptor dependent. Considerable evidence presented here supports the idea that these renin effects are independent of Ang II generation or action. In addition, it is important to note that in vivo data consistent with an Ang II-independent profibrotic effect of renin exist in the literature. Both ANG and double Ang II receptor Type 1A and 1B knockout mice show increased renal renin staining, which is associated with increased TGF-β staining and sclerosis.8.Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (310) Google Scholar, 9.Tsuchida S. Matsusaka T. Chen X. et al.Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes.J Clin Invest. 1998; 101: 755-760Crossref PubMed Scopus (278) Google Scholar The data presented here provide a potential mechanism for the fibrosis observed in these animals. Perhaps the most important and clinically relevant finding in this study is that renin acts through Ang II-independent mechanisms to promote fibrosis by inducing overexpression of TGF-β1. Ang II, the major effector molecule of the renin–angiotensin system, has been strongly implicated in renal fibrosis.15.Noble N.A. Border W.A. Angiotensin II in renal fibrosis: should TGF-β rather than blood pressure be the therapeutic target?.Semin Nephrol. 1997; 17: 455-466PubMed Google Scholar, 23.Gaedeke J. Peters H. Noble N.A. Border W.A. Angiotensin II, TGF-β and renal fibrosis.Contrib Nephrol. 2001; 135: 153-160Crossref PubMed Google Scholar Numerous experimental studies, using pharmacological or genetic means to manipulate Ang II action, as well as clinical studies, have shown that Ang II blockade reduces renal injury and fibrosis in part by reducing TGF-β.24.Sharma K. Eltayeb B.O. McGowan T.A. et al.Captopril-induced reduction of serum levels of transforming growth factor-beta1 correlates with long-term renoprotection in insulin-dependent diabetic patients.Am J Kidney Dis. 1999; 34: 818-823Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 25.Laviades C. Varo N. Diez J. Transforming growth factor beta in hypertensives with cardiorenal damage.Hypertension. 2000; 36: 517-522Crossref PubMed Scopus (113) Google Scholar Ang II blockade is the greatest therapeutic breakthrough in renal fibrosis in the past two decades. However, Ang II blockade does not normalize TGF-β levels, and disease progression is slowed, but not halted. Indeed, studies from our laboratory using superpharmacological doses of ACEi or ARB alone, or in combination, suggest that the maximal reduction in disease markers with Ang II blockade is about 50% in experimental nephritis.13.Border W.A. Noble N.A. Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar, 26.Peters H. Border W.A. Noble N.A. Targeting TGF-beta overexpression in renal disease: maximizing the antifibrotic action of angiotensin II blockade.Kidney Int. 1998; 54: 1570-1580Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 27.Border W.A. Noble N.A. Maximizing hemodynamic-independent effects of angiotensin II antagonists in fibrotic diseases.Semin Nephrol. 2001; 21: 563-572Abstract Full Text PDF PubMed Scopus (34) Google Scholar These findings are entirely consistent with the limitations of Ang II blockade to halt renal fibrosis in human clinical medicine. The therapeutic limitations of Ang II blockade strongly suggest that other molecules contribute to TGF-β overexpression. Ang II inhibits both renin secretion and renin synthesis through the Ang II type 1 receptor in the renal JGA.28.Kurtz A. Cellular control of renin secretion.Rev Physiol Biochem Pharmacol. 1989; 113: 1-40Crossref PubMed Google Scholar, 29.Johns W.D. Peach M.J. Gomez R.A. et al.Angiotensin II regulates renin gene expression.Am J Physiol. 1990; 259: F882-F887PubMed Google Scholar, 30.Kurtz A. Wagner C. Regulation of renin secretion by angiotensin II-AT1 receptors.J Am Soc Nephrol. 1999; 10: S162-S168PubMed Google Scholar Conversely, blockade of Ang II with either ACEi or ARB increases plasma and renal renin expression by 5- to 10-fold in both human patients and in experimental models,29.Johns W.D. Peach M.J. Gomez R.A. et al.Angiotensin II regulates renin gene expression.Am J Physiol. 1990; 259: F882-F887PubMed Google Scholar, 31.Azizi M. Bissery A. Lamarre-Cliche M. Menard J. Integrating drug pharmacokinetics for phenotyping individual renin response to angiotensin II blockade in humans.Hypertension. 2004; 43: 785-790Crossref PubMed Scopus (35) Google Scholar, 32.Gomez R.A. Chevalier R.L. Everett A.D. et al.Recruitment of renin gene-expressing cells in adult rat kidneys.Am J Physiol. 1990; 259: F660-F665PubMed Google Scholar, 33.Cheng H.F. Wang S.W. Zhang M.Z. et al.Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1.Am J Physiol Regul Integr Comp Physiol. 2002; 283: R638-R646Crossref PubMed Scopus (37) Google Scholar, 34.Wagner C. Kurtz A. Effects of candesartan on the renin system in conscious rats.J Am Soc Nephrol. 1999; 10: S169-S171PubMed Google Scholar, 35.Kramer B.K. Ritthaler T. Schweda F. et al.Effects of the angiotensin II type-1 receptor antagonist ZD7155 on angiotensin ll-mediated regulation of renin secretion and renal renin gene expression, renal vasoconstriction, and blood pressure in rats.J Cardiovasc Pharmacol. 1998; 31: 700-705Crossref PubMed Scopus (12) Google Scholar data that are confirmed in Table 1 and Figure 4. Our results raise the important possibility that the elevated renin seen with Ang II blockade may, independent of Ang II, contribute to overproduction of TGF-β1, PAI-1 and fibrotic matrix components and thereby contribute to disease progression. The present study demonstrates that human and rat renin rapidly and dramatically upregulate TGF-β1 production by MCs through a receptor-mediated mechanism, independent of Ang II generation. Renin-induced increases in TGF-β1 in turn stimulate increases in PAI-1, FN and collagen I. While further in vivo work is needed to determine whether the in vitro renin effects seen here play a role in fibrotic renal disease, the data presented raise the intriguing possibility that the high plasma renin levels induced by therapeutic Ang II blockade may, in fact, contribute to the limited effectiveness of this therapy.
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