Renin-stimulated TGF-β1 expression is regulated by a mitogen-activated protein kinase in mesangial cells
2007; Elsevier BV; Volume: 72; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5002243
ISSN1523-1755
AutoresYü Huang, Nancy A. Noble, J. Zhang, Chuanming Xu, Wayne A. Border,
Tópico(s)Hormonal Regulation and Hypertension
ResumoRecent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-β1 (TGF-β1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-β1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin. Recent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-β1 (TGF-β1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-β1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin. Renin is an aspartic protease known to cleave angiotensinogen to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin–angiotensin–aldosterone system (RAAS), Ang II.1Griendling 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 Recent studies suggest that renin, in addition to its role to convert angiotensinogen to Ang I, might have direct, receptor-mediated actions.2Nguyen 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,3Nguyen 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 Renin was reported to bind to human mesangial cells in culture inducing hypertrophy and increased plasminogen activator inhibitor-1 (PAI-1) protein levels. Receptor-bound renin was neither internalized nor degraded.2Nguyen 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 receptor has been cloned and is a 350-amino-acid membrane-associated polypeptide.3Nguyen 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 It is also known that renin binding to mesangial cells in the presence of losartan leads to intracellular signaling by activating the pathway of the mitogen-activated protein kinase (MAPK) -1 and -2 (extracellular-signal regulated kinase 1 and 2 (ERK1 and ERK2)).3Nguyen 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 We have recently shown that human and rat recombinant renin, at physiologically relevant concentrations, induces marked dose- and time-dependent increases in transforming growth factor-β1 (TGF-β1) in human and rat mesangial cells that were not altered by addition of losartan or enalapril.4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar The actions of rat renin were blocked completely by silencing the renin receptor with small-inhibiting RNA (siRNA). Taken together, these studies suggest that renin has novel receptor-mediated actions, independent of renin's role in Ang II generation that could play a role in renal fibrosis. The great potential clinical relevance of these findings rests in the possibility that the high plasma and local tissue renin levels induced by therapeutic Ang II blockade may contribute to renal fibrosis and thereby limit the effectiveness of this therapy. The underlying cellular mechanism for this new renin action is unclear. It is known that the MAPK signaling pathways play a pivotal role in induction of cell growth and differentiation in response to extracellular stimulation.5Ohren J.F. Chen H. Pavlovsky A. et al.Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition.Nat Struct Mol Biol. 2004; 11: 1192-1197Crossref PubMed Scopus (488) Google Scholar The MAPK signaling pathway is a complex linear kinase cascade of serine/threonine kinases in which MAPK kinase kinase (or Raf) phosphorylates and activates MAPK kinase proteins, which in turn phosphorylate and activate MAPK proteins. At least seven MAPK kinase homologs and four MAPK families (ERK1/2, also called as p42/p44 MAPKs, c-Jun NH2 terminal kinase, P38 and ERK5) have been identified. Among them, extracellular signal-regulated protein kinase (ERK) is the most extensively studied. Although its function is typically associated with cell proliferative events, it is now clear that MAPKs function in a more pleiotropic manner to regulate a variety of cell functions.6Cobb M.H. Goldsmith E.J. How MAP kinases are regulated.J Biol Chem. 1995; 270: 14843-14846Crossref PubMed Scopus (1640) Google Scholar Nguyen et al.3Nguyen 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 has shown that human renin induces activation of ERK1/2 MAPK signaling independent of Ang II. Here we investigate whether renin activates this signaling pathway in rat mesangial cells, whether this activation is receptor mediated, how renin activation compares with Ang II activation in this system and whether ERK1/2 MAPK signaling mediates the action of renin to increase TGF-β and PAI-1, both important profibrotic molecules. To investigate involvement of MAPK in renin-mediated TGF-β1 induction, we first examined the activation kinetics of ERK1/2 in rat mesangial cells by Western blot analysis using phospho-specific antibodies. The results, shown in Figure 1, indicate that addition of 10-8 M recombinant rat renin resulted in a rapid and significant activation of ERK1/2. Phosphorylation of ERK (p-ERK) was significantly increased at 2.5 min, peaked at 5 min with a 12-fold increase over control and then remained sixfold elevated until the end of the 30 min study (Figure 1). The effect of increasing doses of renin on ERK1/2 phosphorylation at 2.5 min is shown in Figure 2. ERK1/2 signal was significantly phosphorylated by renin at concentrations of 10-12 M or higher and was greatest at the highest concentration used (10-7 M) showing a 4.5-fold increase over control (Figure 2). Blots for total ERK1/2 signals confirmed that gels were equally loaded. The ratio of active to total ERK1/2 was determined using the Bio-Rad GS 700 imaging densitometer. These results indicate that renin activates the ERK pathway in mesangial cells in a time- and dose-dependent manner.Figure 2Effect of renin dose on ERK1/2 phosphorylation in mesangial cells. (a) Quiescent rat mesangial cells were incubated for 2.5 min in the presence of various concentrations of renin, and Western blotting was performed. (b) Graph shows the relative levels of intracellular phosphorylated ERK1/2. Values are expressed relative to the no-additive, control, which was set at unity. *P<0.05 compared to the control.View Large Image Figure ViewerDownload (PPT) Even though reports suggest that mesangial cells have all the components necessary to synthesize Ang II,7Vidotti 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,8Andrade 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 we have demonstrated previously that the limited amount of angiotensinogen mRNA present in mesangial cells fails to produce significant Ang II either in untreated or renin-treated mesangial cells.4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar To further determine renin-induced ERK1/2 phosphorylation independent of Ang II, cells were pre-incubated with the angiotensin-converting enzyme inhibitor enalaprilate (the active form of enalapril) or the angiotensin II type 1 receptor antagonist losartan 30 min at 10-5 M and then treated with 10-8 M renin for 15 min. The results seen in Figure 3a and b revealed that enalaprilate or losartan treatment had no effect on renin-induced activation of ERK1/2 signaling. Since ERK signaling is typically associated with cell proliferative events, levels of mesangial cell proliferation were evaluated. During our initial studies, we observed that primary cultures of mesangial cells in RPMI 1640 with 10% fetal bovine serum (FBS) grew quickly, reaching ∼90% confluence by 24 h. Cell number increased to a greater extent after 24 h (Figure 4a). Addition of recombinant rat renin in serum-free RPMI 1640 increased cell proliferation rates in a dose-dependent manner, compared with cells cultured without renin treatment (Figure 4a). This effect was not altered by addition of enalaprilate or losartan either, but it was significantly inhibited by a specific MAPK/ERK kinase inhibitor, 1-,4-diamino-2,3-dicyano-1,4bis[2-aminophenylthio] butadiene, U0126 (Figure 4b). As we have previously shown,4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar and as shown in Figure 5a and b, silencing the rat renin receptor by transfecting mesangial cells for 72 h with 500 pmol Stealth™ renin receptor siRNA effectively reduced the renin receptor to undetectable levels. This treatment also significantly reduced renin-induced induction of phospho-ERK1/2 (Figure 5a and c). Therefore, the mesangial cell renin receptor mediates renin-induced activation of ERK1/2 signaling and cell proliferation. Because enalapril and losartan had no effect on renin-induced induction of phospho-ERK1/2 (Figure 3), this effect is independent of Ang II generation or action. Since it was previously2Nguyen 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,4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar shown that renin binding to the mesangial cell receptor induces PAI-1, TGF-β1, and matrix proteins fibronectin and collagen I, and we have shown here that renin-induced phosphorylation of ERK is also receptor-mediated, we next asked whether activation of the ERK1/2 pathway mediates TGF-β upregulation by renin. To this end, the specific MAPK/ERK kinase inhibitor U0126 was used. The efficacy of this inhibitor in our in vitro system is shown in Figure 6. Addition of 0.1–50 μM U0126 for 10 min substantially reduced baseline ERK1/2 phosphorylation without renin (Figure 6a). As shown in Figure 6b, the increase in ERK1/2 phosphorylation seen with renin was decreased below control values with increasing concentrations of U0126. In terms of ERK phosphorylation mediating renin-induced increases in TGF-β1 mRNA, Northern blotting revealed that co-incubation of renin with 50 μM U0126 for 2 h completely prevented the renin-induced increases in TGF-β1 mRNA expression by mesangial cells (Figure 7).Figure 7Renin-induced TGF-β1 mRNA expression is ERK dependent. Quiescent rat mesangial cells were co-incubated with 10-8 M renin and the ERK inhibitor U0126 (50 μM) for 2 h. TGF-β1 mRNA levels were determined by Northern blot analysis and standardized for densitometric analysis to GAPDH mRNA levels. Densitometric values are expressed relative to the no-additive control, which was set at unity. *P<0.05 compared to the untreated control. #P<0.05 vs renin-alone-treated cells.View Large Image Figure ViewerDownload (PPT) We have previously shown that the renin-induced increases in mesangial cell PAI-1 originally reported by Nguyen et al.,2Nguyen 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 were in part mediated by renin-induced increases in TGF-β1 as indicated by the fact that PAI-1 induction by renin was blocked by 68% with excess TGF-β antibody.4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar We now ask whether activation of ERK mediates renin-induced increases in PAI-1 expression. Mesangial cells were exposed to 10-8 M renin and 50 μM U0126 for 2 h. As shown in Figure 8, the ERK inhibitor U0126 substantially inhibited both basal and renin-induced increases in PAI-1 mRNA expression. Addition of the TGF-β-neutralizing antibody, 1D11 (5 μg/ml, R&D Systems Inc., Minneapolis, MN, USA), and the ERK inhibitor U0126 for 2 h completely inhibited renin-induced increases in PAI-1 expression (Figure 9).Figure 9ERK inhibitor U0126 blocks renin-mediated TGF-β 1action on PAI-1 mRNA expression. Quiescent rat mesangial cells were co-incubated with 10-8 M renin, the neutralizing antibody, 1D11, and/or the ERK inhibitor U0126 (50 μM) for 2 h. PAI-1mRNA levels were determined by Northern blot and standardized for densitometric analysis to GAPDH mRNA levels. Densitometric values are expressed relative to the U0126 alone treated control, which was set at unity. *P<0.05 compared to the U0126-alone-treated control. #P<0.05 vs renin-alone-treated cells.View Large Image Figure ViewerDownload (PPT) This study shows that rat renin induces rapid activation of cellular MAPKs ERK1 and ERK2 in rat mesangial cells, findings consistent with a previous study using human renin on human mesangial cells.3Nguyen 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 In addition, we show here that knockdown of the renin receptor with siRNA blocked renin induction of ERK1/2 phosphorylation indicating that this effect is renin receptor mediated. Because Ang II blockers enalapril and losartan had no effect, renin activation of ERK1/2 is independent of Ang II generation or action. This is the first direct demonstration that renin triggers intracellular signaling by binding to renin receptor in addition to its role in Ang II generation. It is apparent from previous2Nguyen 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,4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar and present studies that renin binds to a cell-member receptor on glomerular mesangial cells. Through this binding a number of molecules involved in renal fibrosis are upregulated. This renin receptor is thought to be a functional renin receptor, different from the other renin-binding proteins that have been reported.9Schmitz C. Gotthardt M. Hinderlich S. et al.Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor.J Biol Chem. 2000; 275: 15357-15362Crossref PubMed Scopus (48) Google Scholar, 10Saris J.J. Derkx F.H. De Bruin R.J. et al.High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin.Am J Physiol Heart Circ Physiol. 2001; 280: H1706-H1715PubMed Google Scholar, 11Sealey 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 However, similar functional studies have not been done with the renin-binding proteins. Further studies are needed to determine whether there is a cross-talk among these receptors and binding proteins. The ability of renin to induce ERK1/2 phosphorylation raised the question of the subsequent pathophysiological role of the signaling pathways following activation. ERK1/2 is one of the cascades of serine/threonine kinases that transduce signals from the cell surface to the nucleus in response to growth factors and cellular stress.6Cobb M.H. Goldsmith E.J. How MAP kinases are regulated.J Biol Chem. 1995; 270: 14843-14846Crossref PubMed Scopus (1640) Google Scholar,12Lewis T.S. Shapiro P.S. Ahn N.G. Signal transduction through MAP kinase cascades.Adv Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar ERK pathway activation is sufficient to drive mitogenesis in many cell types. Recent data demonstrate that this signaling cascade, traditionally recognized for its role in growth, also plays a critical role in other cell functions.6Cobb M.H. Goldsmith E.J. How MAP kinases are regulated.J Biol Chem. 1995; 270: 14843-14846Crossref PubMed Scopus (1640) Google Scholar Renin has been known to induce cell hypertrophy and proliferation and to upregulate TGF-β1, PAI-1, and matrix protein production through a receptor-mediated, Ang II-independent pathway.2Nguyen 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,4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar The present study further documented that rat renin promotes mesangial cell proliferation. The renin-induced hyperproliferative phenotype in rat mesangial cells was mediated by ERK activation through Ang II-independent pathway since the increased mitotic activity was reversed by the ERK1/2 kinase inhibitor, U0126, and not by Ang II blockade, enalaprilate, or losartan. The present study also adds data indicating that renin induction of TGF-β1 production in rat mesangial cells is dependent on signaling through ERK1/2, providing an important link between renin and its potentially important profibrotic, angiotensin II-independent action. Renin binds to its receptor to activate the ERK MAPK and subsequent signaling that leads to cell proliferation and TGF-β induction. Further studies are required to determine whether renin activates other members of the MAPK family such as p38 MAPK and c-Jun NH2 terminal kinase. Very recently, it was first observed by Saris et al. that human prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated Hsp-27 in neonatal rat cardiomyocytes, independent of Ang II generation and M6P receptor.13Saris J.J. t Hoen P.A. Garrelds I.M. et al.Prorenin induces intracellular signaling in cardiomyocytes independently of angiotensin II.Hypertension. 2006; 48: 564-571Crossref PubMed Scopus (205) Google Scholar Through its regulation of actin filament dynamics, Hsp-27 is believed to be involved in maintaining the integrity of cell architecture, growth, motility, survival, and death.14Burridge K. Wennerberg K. Rho and Rac take center stage.Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar Protesome differential display experiments supported the downstream effects of Hsp-27 on actin cytoskeleton, suggesting the potential role of prorenin-induced signaling in the severe cardiac hypertrophy. This action of prorenin may be consistent with the previous report in rats with hepatic prorenin overexpression.15Veniant M. Menard J. Bruneval P. et al.Vascular damage without hypertension in transgenic rats expressing prorenin exclusively in the liver.J Clin Invest. 1996; 98: 1966-1970Crossref PubMed Scopus (153) Google Scholar In contrast, human prorenin application to cardiomyocytes did not result in detectable ERK1/2 MAPK activation or PAI-1 release as renin did in mesangial cells. Increased PAI-1 only occurred when both human angiotensinogen and prorenin were added. Eprosartan, but not PD123319 (antagonist of AT2-subtype receptor) or mannose 6-phosphate (M6P), blocked this effect.13Saris J.J. t Hoen P.A. Garrelds I.M. et al.Prorenin induces intracellular signaling in cardiomyocytes independently of angiotensin II.Hypertension. 2006; 48: 564-571Crossref PubMed Scopus (205) Google Scholar It has been shown that the specific renin receptor binds prorenin as effectively as it binds renin. Prorenin bound to this receptor cleaves angiotensinogen with kinetics similar to that of fully active renin in solution.16Jan Danser A.H. Saris J.J. Prorenin uptake in the heart: a prerequisite for local angiotensin generation?.J Mol Cell Cardiol. 2002; 34: 1463-1472Abstract Full Text PDF PubMed Scopus (24) Google Scholar Therefore, these data from Saris et al. indicate that increased PAI-1 secretion in neonatant rat cardiomyocytes stimulated by human prorenin is entirely dependent on Ang II generation and subsequent AT1 receptor activation. Although it is not known whether there is the species specificity of the receptor-prorenin binding, renin and prorenin seem to trigger different cellular signaling. The clarified receptor-mediated action of prorenin and its cellular action remain to be further determined. TGF-β and PAI-1 are both key mediators of tissue fibrosis.17Border 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 TGF-β1 enhances both extracellular matrix synthesis and extracellular matrix deposition.18Border W.A. Noble N.A. Transforming growth factor beta in tissue fibrosis.New Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2902) Google Scholar,19Noble N.A. Border W.A. Angiotensin II in renal fibrosis: should TGF-b rather than blood pressure be the therapeutic target?.Semin Nephrol. 1997; 17: 455-466PubMed Google Scholar TGF-β1 also increases PAI-1 production whereby plasmin generation is decreased, protease-dependent fibrinolytic activity is decreased and matrix turnover is decreased.20Manchanda N. Schwartz B.S. Single chain urokinase.J Biol Chem. 1991; 266: 14580-14584Abstract Full Text PDF PubMed Google Scholar,21Huang Y. Noble N. An unexpected role of plasminogen activator inhibitor-type 1 (PAI-1) in renal fibrosis.Kidney Int. 2005; 67: 2502-2503Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Renin treatment increases both TGF-β1 and PAI-1 production2Nguyen 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,4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar and we have previously shown that renin increases PAI-1 through both TGF-β1-dependent and -independent pathways because addition of the TGF-β neutralizing antibody, 1D11,4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar only partially blocks the renin-induced increase in PAI-1. In the present study, the renin-induced increase in PAI-1 expression is completely inhibited by treatment with the ERK inhibitor, U0126. This suggests that both the TGF-β-dependent and TGF-β1-independent pathways by which renin increases PAI-1 are mediated by ERK phosphorylation. Also suggesting that TGF-β1 induces PAI-1 expression through the ERK-MAPK pathway are data showing that TGF-β stimulation of rat mesangial cell PAI-1 mRNA is significantly inhibited by the specific MAPK kinase inhibitor, PD98059 or U0126.22Guo B. Inoki K. Isono M. et al.MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-beta in rat mesangial cells.Kidney Int. 2005; 68: 972-984Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,23Kutz S.M. Hordines J. McKeown-Longo P.J. Higgins P.J. TGF-beta1-induced PAI-1 gene expression requires MEK activity and cell-to-substrate adhesion.J Cell Sci. 2001; 114: 3905-3914Crossref PubMed Google Scholar Therefore, both renin and TGF-β1 appear to share common elements in the downstream signaling cascades involved in PAI-1 upregulation. Taken together, our findings suggest that the receptor-mediated activation of the ERK1/2 signaling pathway is an important molecular mechanism by which renin can increase expression of molecules implicated in renal fibrosis. Of interest is work where a human renin receptor transgenic rat was developed to study the mechanisms underlying the pathogenic effects of (pro)renin receptor activation in diabetic nephropathy.24Kaneshiro Y. Ichihara A. Takemitsu T. et al.Increased expression of cyclooxygenase-2 in the renal cortex of human prorenin receptor gene-transgenic rats.Kidney Int. 2006; 70: 641-646Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Because cell culture data showed increased binding of renin and prorenin at the cell surface after transfection of human or rat renin receptor cDNA,3Nguyen 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,25Burckle C.A. Jan Danser A.H. Muller D.N. et al.Elevated blood pressure and heart rate in human renin receptor transgenic rats.Hypertension. 2006; 47: 552-556Crossref PubMed Scopus (186) Google Scholar the human receptor transgenic animals would be expected to bind rat prorenin/renin in vivo and activate the human receptor. These animals were found to have higher levels of cyclooxygenase 2 and increased ERK1/2 activation in renal cortex.24Kaneshiro Y. Ichihara A. Takemitsu T. et al.Increased expression of cyclooxygenase-2 in the renal cortex of human prorenin receptor gene-transgenic rats.Kidney Int. 2006; 70: 641-646Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Although receptor activation was not proven directly, and renin and prorenin effects were not distinguished, the data indicate that human (pro)renin receptor directly or indirectly contributes to cellular ERK signaling in vivo, as it does in vitro in the present study, and this signaling may have profibrotic consequences in kidney disease. Many molecules activate the MAPK pathway, particularly in the RAAS. Ang II, as a critical effecter of the RAAS, stimulates the activation of ERK in cultured mesangial cells.26Gorin Y. Ricono J.M. Wagner B. et al.Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells.Biochem J. 2004; 381: 231-239Crossref PubMed Scopus (106) Google Scholar Ang II-induced increases in protein synthesis and hypertrophy in mesangial cells have been shown to involve ERK activation.26Gorin Y. Ricono J.M. Wagner B. et al.Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells.Biochem J. 2004; 381: 231-239Crossref PubMed Scopus (106) Google Scholar Furthermore, Ang II-mediated increases in TGF-β1 and PAI-1 mRNAs were inhibited by U0126 or PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase (MEK).27Naito T. Masaki T. Nikolic-Paterson D.J. et al.Angiotensin II induces thrombospondin-1 production in human mesangial cells via p38 MAPK and JNK: a mechanism for activation of latent TGF-beta1.Am J Physiol Renal Physiol. 2004; 286: F278-F287Crossref PubMed Scopus (122) Google Scholar,28Perlman A. Lawsin L.M. Kolachana P. et al.Angiotensin II regulation of TGF-beta in murine mesangial cells involves both PI3 kinase and MAP kinase.Ann Clin Lab Sci. 2004; 34: 277-286PubMed Google Scholar These data indicate that activation of MAPK pathway is one of important events involved in a variety of responses induced by Ang II to stimulate extracellular matrix accumulation in mesangial cells. Similarly, aldosterone, a potent effector molecule in the RAAS, was also demonstrated recently to stimulate proliferation of mesangial cells by activating ERK1/2.29Terada Y. Kobayashi T. Kuwana H. et al.Aldosterone stimulates proliferation of mesangial cells by activating mitogen-activated protein kinase 1/2, cyclin D1, and cyclin A.J Am Soc Nephrol. 2005; 16: 2296-2305Crossref PubMed Scopus (71) Google Scholar,30Nishiyama A. Yao L. Fan Y. et al.Involvement of aldosterone and mineralocorticoid receptors in rat mesangial cell proliferation and deformability.Hypertension. 2005; 45: 710-716Crossref PubMed Scopus (116) Google Scholar Although there are no data available linking aldosterone-induced MAPK1/2 signaling to matrix protein expression by mesangial cells, it has been shown that aldosterone stimulates collagen gene expression and synthesis via activation of ERK1/2 in rat renal fibroblasts.31Nagai Y. Miyata K. Sun G.P. et al.Aldosterone stimulates collagen gene expression and synthesis via activation of ERK1/2 in rat renal fibroblasts.Hypertension. 2005; 46: 1039-1045Crossref PubMed Scopus (132) Google Scholar Also, chronic aldosterone/salt treatment of non-nephrectomized rats induces severe glomerular mesangial injury and tubulointerstitial fibrosis beyond aldosterone effects on blood pressure and hemodynamic changes. This damage is associated with the activation of MAPKs including ERK1/2.32Nishiyama A. Abe Y. Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effects of aldosterone blockade.J Pharmacol Sci. 2006; 100: 9-16Crossref PubMed Scopus (50) Google Scholar These data indicate that MAPKs are important signaling molecules mediating aldosterone-induced renal injury and possibly renal fibrosis. The results of the present study, as well as previously published data, suggest that the three primary effector molecules of the RAAS may promote growth and renal damage through a common pathway involving ERK stimulation that is independent of the effects of Ang II as a vasoconstrictor, independent of renin's role as an aspartyl protease, and independent of the effects of aldosterone as a regulator of sodium and volume. In addition, there is some evidence that RAAS components act synergistically. Min et al.33Min L.J. Mogi M. Li J.M. et al.Aldosterone and angiotensin II synergistically induce mitogenic response in vascular smooth muscle cells.Circ Res. 2005; 97: 434-442Crossref PubMed Scopus (141) Google Scholar reported that Ang II and aldosterone synergistically activate ERK signaling and thereby synergistically induce cellular mitogenic responses. It will be of interest to determine whether renin and Ang II or renin and aldosterone have synergistic effects on ERK signaling and induction of profibrotic proteins or whether they simply back up one another in the fibrotic milieu. Such studies will expand our understanding of the roles of all RAAS components in tissue fibrosis. In conclusion, this study demonstrates that rat renin rapidly and dramatically activates intracellular ERK MAPK signaling by mesangial cells through a receptor-mediated mechanism, independent of Ang II generation or action. Renin-induced phosphorylation of ERK1/2 in turn triggers cell proliferation and cellular TGF-β1 and PAI-1 gene overexpression. The data presented indicate that the renin-induced ERK pathway could play a role in renal fibrosis. Considering that both Ang II and aldosterone, the two other major components of RAAS, also exert their profibrotic effects via the MAPK pathway, and considering that Ang II and aldosterone synergistically induce hypertrophy through activated ERK signaling, the demonstration here that MAPK signaling mediates renin's profibrotic actions raises the intriguing possibility that blockade of this common signaling pathway may result in more effective therapy. Rat recombinant renin was made as described previously.4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar A specific MAPK/ERK kinase inhibitor, U0126 was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Unless otherwise indicated, other reagents were purchased from Sigma (St Louis, MO, USA). Rat mesangial cells were derived from intact glomeruli of four- to six-week-old Wistar rats and characterized according to published methods.34Okuda S. Languino L.R. Ruoslahti E. Border W.A. Elevated expression of transforming growth factor-β and proteoglycan production in experimental glomerulonephritis.J Clin Invest. 1990; 86: 453-462Crossref PubMed Scopus (517) Google Scholar,35Border W.A. Okuda S. Languino L.R. Ruoslahti E. Transforming growth factor-beta regulates production of proteoglycans by mesangial cells.Kidney Int. 1990; 37: 689-695Abstract Full Text PDF PubMed Scopus (325) Google Scholar Cells were used between passages 4 and 7 and were maintained in RPMI 1640 medium supplemented with 20% FBS (Hyclone Laboratory, Logan, UT), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.66 U/ml insulin, 25 mmol N-2-hydroxyethylppiperazine-N′-2-ethanesulfonic acid (HEPES) buffer at 37°C in a 5% CO2 incubator. Subconfluent mesangial cells seeded on six-well plates were made quiescent by serum-free medium for 48 h before experimental studies. Mesangial cell mitotic activity was evaluated using an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) cell proliferation assay according to the manufacturer's instruction. The MTT assay is colorimetric method for determining the total number of viable cells. The yellow tetrazolium MTT is reduced by dehydrogenase in mitochondria of live cells and lead to the resulting intracellular purple formazan, which can be solubilized and quantified by an enzyme-linked immunosorbent assay plate reader. The proliferative viability of cells corresponds to the amount of MTT reduced. Our preliminary study confirmed a significant correlation between counted cell numbers and optical density values at 520 nm. Each experiment was typically performed with n=8 separate wells of mesangial cells in 96-well plates under identical conditions. The administration of 10% FBS was used as the positive control. Cellular ERK activation was measured by Western blotting as levels of phosphorylation of ERK using mouse monoclonal anti-phospho-pERK1/2 MAPK(Tyr204) E4 monoclonal antibody and rabbit anti-total ERK1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunostaining was visualized by enhanced chemiluminescense (ECL)™ Western blotting detection reagents (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). Quantitation of the bands on autoradiograms was performed using a Bio-Rad GS 700 imaging densitometer. Changes in phosphate ERK1/2 were determined by correcting the densitometric intensity of its total EKR1/2 for each sample. For comparison, this ratio was set at unity for normal control samples and other lanes on the same gel were expressed as fold-increase over this value. Before transfection, rat mesangial cells of 50% confluence were maintained in 2.5% fetal calf serum medium and then transfected with lipofectamine™ 2000 (Invitrogen Life Technologies Inc., Gaithersburg, MD, USA) and rat renin receptor Stealth™ siRNA as described previously.4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar Briefly, cells were transfected with 500 pmol Stealth™ siRNA complexed with lipofectamine™ 2000 (This condition was determined previously to induce a 75% reduction in receptor mRNA expression at 72 h incubation4Huang Y. Wongamorntham S. Kasting J. et al.Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.Kidney Int. 2006; 69: 105-113Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar) in 500 μl Opti-minimum essential medium (MEM) (Invitrogen Life Technologies Inc.) at 37°C in a CO2 incubator, following the siRNA transfection protocols provided by Invitrogen Life Technologies Inc. Opti-MEM medium with and without lipofectamine™ 2000 were added to cells as controls. After a 48 h transfection, medium was replaced with serum-free and antibiotic-free RPMI 1640. Cells were then made quiescent for 24 h. After a total transfection of 72 h, cells were treated with 10-8 M renin for 10 min for analysis of ERK1/2 signaling after renin receptor depletion. Rat renin receptor mRNA levels were determined using Northern blot assay. Total RNA was isolated from rat mesangial cells using Trizol™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Northern blotting was performed using Ambion's NorthernMax Gly System (Austin, TX, USA) as described previously. Probes were labeled with 32P using the Strip-EZ labeling kit (Ambion) and hybridized overnight in Ultrahyb hybridization buffer (Ambion). After washing, blots were exposed to films that were scanned and analyzed by densitometry. Changes in mRNA levels were determined by correcting the densitometric intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample to correct for loading variations. For comparison, this ratio was set at unity for normal control samples, and other lanes on the same gel were expressed as fold-increase over this value. Probes used included cDNAs for TGF-β1, PAI-1, rat renin receptor cDNA, and GAPDH and were used as previously described.36Huang Y. Haraguchi M. Lawrence D.A. et al.A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis.J Clin Invest. 2003; 112: 379-388Crossref PubMed Scopus (125) Google Scholar Results are presented as mean±s.d. A value of P<0.05 was considered statistically significant (Student's t-test). Duplicate wells were analyzed for each experiment, and each experiment was performed independently at least three times. We thank Ms Linda Hoge for her excellent technical assistance. National Institutes of Health Grants DK 43609 (WAB) and DK60508 (NAN) supported this work. Correction to "Renin-stimulated TGFβ1 expression is regulated by mitogen-activated protein kinases in mesangial cells"Kidney InternationalVol. 75Issue 1PreviewCorrection to: Kidney International (2007) 72, 45–52; doi: 10.1038/sj.ki.5002243 Full-Text PDF Open Archive
Referência(s)