Effect Of 1,25(OH)₂D₃ On TGF-β₁-induced Human Renal Tubular Epithelial-mesenchymal Transition And Its Mechanism

Effect of 1,25(OH)2D3 on TGF-β1-induced human renal tubular epithelial-mesenchymal transition and its mechanismObjective:Tubular interstitial fibrosis (TIF) is believed to be the common final pathway to end-stage renal failure. Recently, it has been found that renal tubular epithelial-mesenchymal transition (TEMT) plays an important role in the progress of tubular interstitial fibrosis. Transforming growth factor-β1 (TGF-β1) plays a crucial role in promoting the renal fibrosis. Smads protein are the most important cytokine in cytoplasm during signal transduction of TGF-β1, they can conduct the signal of TGF-β1 from cellular membrane into cell nucleus. The main causes of renal fibrosis are the overexpression of Smad3 and the inhibition of Smad7. 1,25-dihydroxyvitamin D3(1,25(OH)2D3)is proved to be the most important active vitamin D in vivo. The biological actions of 1,25(OH)2D3 are mediated through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily. The recent studys have showed that 1,25(OH)2D3 has effects on anti-renal fibrosis,but have no report on its molecular mechanism of improve TEMT. The objective of our research was to detect the expressions of E-Cadherin(E-Cad),a-smooth muscle actin(α-SMA),VDR,Smad3,Smad7 protein and mRNA to observe the protective effects and molecular mechanism of 1,25(OH)2D3 on TEMT in human kidney epithelial cell line (HK-2) induced by TGF-β1. Methods:Cultured normal HK-2 cells were divided into five groups:normal control group (N),TGF-β1 (5ng/ml) group (T),TGF-β1 (5ng/ml)+1,25(OH)2D3 (10-9mol/L)group (D1),TGF-β1(5ng/ml)+1,25(OH)2D3(10-8mol/L)group (D2),TGF-β1 (5ng/ml) +1,25(OH)2D3(10-7mol/L)group (D3). The morphology of cells were observed through light microscope at the time of 12h,24h,48h,72h respectively. The expression level of a-SMA,E-cadherin,VDR,Smad3,Smad7 protein were assessed by immunohistochemistry. The expression level of a-SMA,E-cadherin,VDR,Smad3,Smad7 mRNA were measured by Real time-PCR. Results:(1)Compared with the normal control groups, TGF-β1 (5ng/ml) induced many cells showed fibroblast-like in morphology under light microscope in a time-dependent manner. Immunohistochemistry showed that the expression of a-SMA,Smad3 protein were obviously increased and the expression of E-cadherin,VDR,Smad7 protein were significantly reduced in TGF-β1 induced group in a time-dependent manner (P<0.05). Real-time polymerase chain reaction showed that mRNA expression ofα-SMA,Smad3,Smad7 were increased and mRNA expression of E-cadherin,VDR were reduced in TGF-β1 induced group(P<0.05). (2) Compared with TGF-β1-induced groups, in addition to D1 12h groups and D1 24h groups, other 1,25(OH)2D3 groups could inhibit the change in morphology and significantly down-regulateα-SMA,Smad3 protein and mRNA expression and up-regulate E-cadherin,VDR,Smad7 protein and mRNA expression in a dose-dependent manner. Conclusion:(1) 1,25(OH)2D3 could prevent the TEMT induced by TGF-β1 in a dose-dependent manner. (2) 1,25(OH)2D3 improved TGF-β1 induced TEMT which may be mediated through VDR,Smad7 up-regulation and Smad3 down-regulation.