Effects And Mechanisms Of AT₂R Activation On The Regulation Of TGF-βRⅡ In Human Renal Proximal Cells

Background and objectiveRenal tubulointerstitial fibrosis is often regarded as the final outcome of a wide range of progressive chronic kidney disease and is a final common pathway to end-stage chronic kidney disease whose severity correlates with renal prognosis. Proximal tubular epithelial cells play a pivotal role in renal tubulointerstitial fibrosis. Emerging evidence suggests that a critical step in the pathogenesis of tubulointerstitial fibrosis is epithelial-mesenchymal transition（EMT）, a pathological process characterized by a phenotypic conversion from epithelial cells to fibroblast-like morphology. During EMT, tubular epithelial cells lose their epithelial phenotype and acquire a mesenchymal phenotype. This phenotypic conversion involves the de novo synthesis of mesenchymal markers such as α-smooth muscle actin（α-SMA）, and a downregulation of epithelial markers such as E-cadherin that is essential for the structural integrity of renal epithelium.It is generally accepted that of a variety of cytokines and growth factors that trigger EMT, transforming growth factor-β（TGF-β） is the major profibrotic cytokine that contributes to tubulointerstitial damage and renal fibrosis via numerous intracellular signal transduction pathways. Active TGF-β initiates cell signaling by binding to its transmembrane serine/threonine kinase receptor type I（TGF-βRI） and type Ⅱ（TGF-βRⅡ）. Binding of TGF-β to receptor type Ⅱ leads to the recruitment and phosphorylation of receptor type I, which further activates its downstream signaling via the Smad-dependent or-independent pathways and directly leads to the initiation of EMT. Moreover, many other cytokines such as interleukin-1 and angiotensin Ⅱ（Ang Ⅱ） also have effects on EMT indirectly via the induction of TGF-β. In addition, the effects of other cytokines such as tumor necrosis factor-alpha（TNF-α） may be synergistic with that of TGF-β. Since the subtypes of receptors are primarily engaged in the initial binding of TGF-β, the potent effect of TGF-β on the induction of EMT is dependent on its receptors. So how to suppress its profibrotic receptors activation-induced EMT in renal tubular epithelial cells is an important issue to prevent renal tubulointerstitial fibrosis.Ang Ⅱ, considered as the primary mediator of classic renin-angiotensin system（RAS）, exerts its action by binding totwo major receptor subtypes, namely type 1（AT1R） and type 2（AT₂R）. AT1 R mediates the major actions of Ang Ⅱ, including vasoconstriction, renal tubule sodium reabsorption, inflammation, and aldosterone secretion. However, AT₂ R is generally considered to be a functional antagonist of AT1 R and is thought to exert beneficial effects, including promoting natriuresis, preventing fibrogenesis, lowering blood pressure, and modulating inflammation. In recent years, studies have paid more attention to the interaction between Ang Ⅱ receptors and TGF-β receptors in the cardiovascular system and kidney. Activation of AT1 R enhances the expression of TGF-βRI; but transfection of the AT₂ R gene suppresses the expression of TGF-βRI in vascular smooth muscle cells（VSMCs）. In proximal tubular cells, stimulation of AT1 R increases TGF-βRⅡ expression; however, TGF-β1 stimulation decreases AT1 R level in VSMCs. Because both AT₂ R and TGF-β receptors are well-expressed in renal proximal tubular, we hypothesize that AT₂ R may also regulate TGF-β receptors expression and function in kidney.Based on above, we observed the effect of AT₂ R on the expression of TGF-βRⅡ and its-mediated EMT in human proximal tubule cells（HK-2）; studied the direct interaction between AT₂ R and TGF-βRⅡ using immunofluorescence confocal microscopy and co-immunoprecipitation, explored the regulatory molecular signal mechanism of AT₂ R on the expression of TGF-βRⅡ. These will definite the effect of AT₂ R on the expression and function of TGF-βRⅡ in the kidney,which may rovide a new strategy for the prevention and treatment of renal tubulointerstitial fibrosis.Methods1. Human proximal tubule cells（HK-2） were used in this experiment. Effects of AT₂ R on the expression of TGF-β receptors was observed by immunoblotting. The difference between TGF-βRI and TGF-βRⅡ regulated by AT₂ R was determined. Whether or not the effect is tissue-specific is also evaluated.2. The morphological changes of HK-2 cells were observed after treatment with TGF-β1 and AT₂ R agonist CGP42112 A. After treatment with TGF-β1 and AT₂ R agonist CGP42112 A, the expression of α-SMA, a mesenchymal marker and E-cadherin, a epithelial marker, were determined using immunofluorescence staining and immunoblotting.3. The TGF-β1 expression in HK-2 cells was determined using immunoblotting; the level of TGF-β1 in culture medium was also measured by enzyme-linked immuno sorbent assay（ELISA）.4. After treatment with AT₂ R agonist CGP42112 A or not, the co-localization of AT₂ R and TGF-βRⅡ in HK-2 cells was evaluated using immunofluorescence confocal microscopy.5. The direct interaction between AT₂ R and TGF-βRⅡ was determined by co-immunoprecipitation after treatment with AT₂ R agonist CGP42112 A or not.6. The degradation of TGF-βRⅡ protein in HK-2 cells after stimulation with or without AT₂ R agonist CGP42112 A was measured.7. After treatment with AT₂ R agonist CGP42112 A and the nitric oxide synthase inhibitor L-NAME,the expression of TGF-βRⅡ was determined using immunoblotting.The expression of α-SMA,a mesenchymal marker and E-cadherin,a epithelial marker,were also determined.Results1. Treatment with the AT₂ R agonist CGP42112 A decreased TGF-βRⅡ expression in a concentration- and time-dependent manner in HK-2 cells. The AT₂ R antagonists PD123319 or PD123177 had no effect on TGF-βRⅡ expression by themselves, but reversed the inhibitory effect of CGP42112 A on TGF-βRⅡ expression. The inhibitory effect of the AT₂ R on TGF-βRⅡ expression was receptor-specific because stimulation of the AT₂ R had no effect on TGF-βRI expression in HK-2 cells. Moreover, in immortalized cardiomyocytes, stimulation of the AT₂ R also had no effect on TGF-βRⅡ expression.2. TGF-β1 treatment increased EMT in HK-2 cells in a time-dependent manner, which resulted in the gradual increase in expression of α-SMA, an important marker of myofibroblast, and decrease in expression of E-cadherin, a typical phenotypic marker of epithelial cell.3. HK2 cells treated with TGF-β1 underwent phenotypic conversion from epithelial cells to myofibroblast-like cells. The AT₂ R agonist CGP42112 A had no effect on the phenotypic conversion by itself, but reversed the effect of TGF-β1 on the phenotypic conversion of HK-2 cells. TGF-β1 increased the expression of α-SMA, but decreased that of E-cadherin. Pretreatment with CGP42112 A dramatically abrogated TGF-β1-induced α-SMA expression and restored E-cadherin expression in a dose-dependent manner. However, CGP42112 A per se had no effect on the expression of α-SMA and E-cadherin. Furthermore, the inhibitory effect of AT₂ R on the TGF-β1-induced EMT in HK-2 cells was also confirmed with evaluation of the expression of α-SMA and E-cadherin via immunofluorescence method.4. Treatment with CGP42112 A did not change the amount of TGF-β1 secreted by HK-2 cells. Furthermore, we also evaluated the expression of TGF-β1 in cell lysates via immunoblotting, and observed that CGP42112 A treatment did not change the protein expression of TGF-β1 in HK-2 cells.5. Immunofluorescence laser confocal microscopy showed that AT₂ R and TGF-βRⅡ colocalized in HK-2 cells, which is enhanced by the stimulation of AT₂ R. A direct physical interaction between AT₂ R and TGF-βRⅡ was confirmed by coimmunoprecipitation in the basal state, which was also increased following activation of the AT₂ receptor with CGP42112 A. The AT₂ receptor antagonist, PD123319, by itself, had no effect, but reversed the stimulatory effect of CGP42112 A on the coimmunoprecipitation of AT₂ R and TGF-βRⅡ.6. Either vehicle or CGP42112 A had no regulatory effect on TGF-βRⅡ protein expression for up to 3 h without cycloheximide treatment; however, in the presence of cycloheximide, stimulation of AT₂ R with CGP42112 A accelerated the degradation of TGF-βRⅡ protein in HK-2 cells, compared with the cells treated with vehicle.7. The nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester（L-NAME）, by itself, had no effect on TGF-βRⅡ expression; however, inhibition of nitric oxide production blocked the inhibitory effect of AT₂ R on TGF-βRⅡ expression. Furthermore, inhibition of nitric oxide production via L-NAME also blocked the degradation of TGF-βRⅡ induced by AT₂ activation. In addition, L-NAME inhibited the suppressive effect of the AT₂ R on the EMT induced by TGF-β1.Conclusions1. Stimulation of AT₂ R with CGP42112 A decreases TGF-βRⅡ expression in human renal tubular epithelial cells. This effect is clearly exerted at the AT₂ R because an AT₂ R antagonist, either PD123319 or PD123177, completely blocks the effect of CGP42112 A. The inhibitory effect of the AT₂ R on TGF-βRⅡ expression is both receptor-specific and tissue-specific.2. The interaction of AT₂ R and TGF-βRⅡ has physiological significance in HK-2 cells since pre-treatment with CGP42112 A reversed the induction effect of TGF-β1 on the EMT.3. Stimulation of AT₂ R with CGP42112 A increases the colocalization and the physical interaction between AT₂ R and TGF-βRⅡ.4. AT₂ R accelerates the degradation of TGF-βRⅡ protein in HK-2 cells. Moreover, the inhibitory effect of the AT₂ R on TGF-βRⅡ expression was blocked by the nitric oxide synthase inhibitor L-NAME, indicating that nitric oxide is involved in the signaling pathway.