低密度脂蛋白诱导大鼠肾小管上皮细胞转分化的作用机制研究

Study of the mechanism of low density lipoprotein-induced renal tubular epithelial-to-mesenchymal transition in rats

目的探讨低密度脂蛋白(LDL)对大鼠近端肾小管上皮细胞(NRK-52E)转分化的影响及其作用机制。方法将体外培养的NRK-52E细胞分为4组:正常对照组、洛伐他汀(Lov,1μmol/L)组、LDL(250 mg/L)刺激组、LDL(250mg/L)+Lov(1μmol/L)组。应用倒置显微镜观察细胞形态学改变;RT-PCR检测NRK-52E细胞α-平滑肌肌动蛋白(α-SMA)、E-钙粘蛋白(E-cadherin)、转化生长因子β1(TGF-β1)、Smad2、Smad3和整合素链接酶(ILK)mRNA表达;West-ern blot检测α-SMA、E-cadherin及磷酸化Smad2/3(p-Smad2/3)蛋白表达;ELISA检测上清液TGF-β1。结果加入LDL培养24 h后,NRK-52E细胞由立方形或多角形变为长梭形,α-SMA、TGF-β1、Smad2、Smad3和ILK mRNA的表达上调,E-cadherin mRNA的表达下调,与对照组比较差异均有统计学意义(P<0.01);α-SMA和p-Smad2/3蛋白表达量显著高于对照组(P<0.01),E-cadherin蛋白表达量显著低于对照组(P<0.01);细胞上清液TGF-β1浓度显著高于对照组(P<0.01)。LDL+Lov组与LDL组比较,NRK-52E细胞α-SMA、TGF-β1、Smad2、Smad3、ILK mRNA的表达及α-SMA、p-Smad2/3的蛋白表达均明显降低,E-cadherin mRNA和蛋白表达明显增强。结论LDL可诱导肾小管上皮细胞转分化,其机制可能是激活TGF-β1/Smads/ILK信号通路,而Lov能部分阻断LDL诱导的小管上皮细胞转分化。

Objective To investigate the effect and the mechanism of low density lipoprotein （LDL） on induced epithelial-to-mesenchymal transition （EMT） in cultured proximal tubule epithelial ceils （NRK-52E） from rats. Methods The cultured NRK-52E ceils were divided into four groups, a control group, a group treated with lovastatin （1 μmol/L）, a group treated with LDL（250 mg/L）, and a group treated with LDL（250 mg/L） plus lovastatin （1 μmol/L）. Cell morphological changes were observed with an inverted microscope. Expression of a-smooth muscle actin （α-SMA）, E-cad- herin, transforming growth factor beta 1 （TGF-β1）, Smad2, Smad3, and integrin-linked kinase （ILK） mRNA was assessed by RT-PCR. Expression of α-SMA, E-cadherin and phosphorylated Smad2/3（p-Smad2/3） proteins was detected by Western blotting. The level of TGF-β1 in the supernatant of cultured cells was measured by enzyme-linked immunosorbent assay （ELISA）. Results After incubation with LDL for 24 hours,the typical cube morphology or polygonal morphology acquired a spindle-shaped morphology in NRK-52E cells. LDL up-regulated the expression of α-SMA, TGF-β1, Smad2, Smad3, and ILK mRNA and down-regulated the expression of E-cadherin mRNA. There were statistically significant differences in comparison to the control group （P〈0.01）. Compared to the control group, α-SMA and p-Smad2/3 protein expression increased significantly and E-cadherin protein expression decreased markedly （P〈0.01）. The level of TGF-β1 increased markedly in the supernatant of cultured cells （P〈0.01）. In the LDL plus lovastatin group, the expression of α-SMA, TGF-β1, Smad2, Smad3, and ILK mRNA and the expression of α-SMA and p-Smad2/3 proteins were markedly down-regulated while E-cadherin mRNA expression and protein expression were statistically up-regulated in comparison to the LDL group. Conclusion LDL is able to induce tubular EMT and does so by activation of the TGF- β1/Smads/ILK signaling pathway in NRK-52E cells, but lovastatin may partially inhibit LDL-induced tubular EMT.