α-klotho蛋白拮抗瘦素损伤小鼠足细胞的实验研究

α-klotho Antagonizes the Effect of Leptin on Mice Podocytes Injury

目的:探讨瘦素对小鼠足细胞的损伤效应及α-klotho蛋白拮抗这一效应的可能。方法:小鼠足细胞分为三组,分别为空白对照组,瘦素刺激组(瘦素15 ng/ml),α-klotho干预组(α-klotho 0.1μg/ml及瘦素15 ng/ml)。mRNA检测采用实时定量PCR方法,蛋白质检测采用免疫印迹法。结果:瘦素能显著抑制足细胞标志蛋白podocin,nephrin,podocalyxin,podoplanin表达,与对照组比较,蛋白质表达的抑制率分别为24%、25%、25%和23%;mRNA表达的抑制率分别为42%、27%、39%、24%,P均<0.05。α-klotho干预后,上述足细胞标志蛋白均被上调,与瘦素组比较,蛋白质表达分别上调1.36、1.25、1.29和1.22倍;mRNA表达分别上调1.77、1.48、1.66、1.26倍,P均<0.05。此外,瘦素能显著活化Wnt/β-catenin信号通路,与对照组比较,Wnt2b、Wnt5a、Wnt6、Wnt7a的mRNA表达分别上调2.06、1.72、2.14、1.59倍,P<0.05;β-catenin的蛋白磷酸化水平抑制率为26%,P<0.05。α-klotho干预后,上述信号通路分子均被抑制,与瘦素组比较,Wnt2b、Wnt5a、Wnt6、Wnt7a的mRNA表达抑制率分别为42%、28%、43%、30%,P均<0.05,磷酸化的β-catenin上调1.27倍,P<0.05。结论:瘦素可能通过Wnt/β-catenin信号通路损伤足细胞,而α-klotho可逆转足细胞损伤,其机制可能与抑制瘦素的作用有关。

Objective：To investigate the effect of leptin on mice podocytes injury, and α - klotho might antagonize this effect. Methods：Podocyte were incubated with medium alone, medium with leptin （15 ng/ml） or medium with α -klotho （0. 1μg/ml） and leptin （15 ng/ml）, respectively, mRNA expression was detected by realtime quantitative PCR, protein expression was detected by Western blotting. Results： The mRNA and protein expression of podocin, nephrin, podocalyxin, podoplanin were significantly down - regulated by leptin （ 15 ng/ml）. Compared with the control group, the inhibition rates of podocin, nephrin, podocalyxin, po doplanin protein expression in the leptin group were 24% ,25% ,25% and 23%, respectively （P 〈 0.05）, and the inhibition rates of their mRNA were 42% ,27% ,39% ,24%, respectively （P 〈 0.05）. α- klotho significantly up - regulated the down - regulation of pedocin, nephrin, podocalyxin, podoplanin induced by leptin. Compared with the leptin group, the protein expression of podocin, nephrin, podocalyxin, podoplanin were up - regulated to 1.36,1.25,1.29 and 1.22 times, respectively （ P 〈 0.05 ）, their mRNA expression were up - regulated to 1.77,1.48,1.66,1.26 times, respectively （ P 〈 0.05 ）. Leptin （ 15 ng/m l） can significantly acti- vate Wnt/β - catenin signaling pathway. Compared with the control group, mRNA expression of Wnt2b ,Wnt5a,Wnt6 ,Wnt7a were up - regulated 2.06,1.72,2.14,1.59 times, respectively （P 〈 0.05）. The inhibition rates of phospho - β - catenin in the leptin group was 26% （ P 〈 0.05 ）. α - klotho significantly inhibited the activation of Wnt/β - catenin signaling pathway induced by leptin. Compared with the leptin group, the inhibition rates of Wnt2b,Wnt5a,Wnt6 ,Wnt7a mRNA in theα - klotho and leptin costimulated group were 42% ,28% ,43% ,30%, respectively （P 〈0.05）. The phosphor- β-catenin in the α-klotho and leptin costimulated group were up- regulated 1.27 times （P 〈 0.05 ）. Conclusion： Leptin can cause podocyte injury through Wnt/β- catenin signaling pathway, α -klotho can reverse this effect induced by leptin.