KIF3A在单侧输尿管梗阻小鼠肾脏中的表达及意义

Expression of kinesin KIF3A in the kidney of mice with unilateral ureteral obstruction

目的通过研究KIF3A在单侧输尿管梗阻(UUO)小鼠和TGF-β1诱导的NRK-52E细胞中的表达情况,探讨KIF3A在肾间质纤维化中的作用。方法将36只C57BL/6J小鼠随机分为假手术组(Sham,n=18)和单侧输尿管梗阻组(UUO,n=18),分别于术后7、14、21 d处死小鼠。苏木素-伊红(HE)染色、马松(Masson)三色法染色和天狼星红(Sirius Red)染色观察小鼠肾间质纤维化程度,RT-PCR、免疫组织化学(IHC)和免疫印迹(Western blot)检测小鼠肾组织KIF3A的表达和分布。免疫印迹检测小鼠肾组织KIF3A、E-cadherin和α-SMA蛋白的表达。TGF-β1诱导NRK-52E细胞转分化后,免疫印迹检测KIF3A、E-cadherin、α-SMA、Wnt4和β-catenin蛋白的表达。结果与Sham组相比,UUO小鼠肾间质纤维化程度随着梗阻时间延长而增加,表明造模成功。同时,KIF3A mRNA和蛋白表达均增高,于21 d达到高峰,α-SMA蛋白表达显著上调,E-cadherin蛋白表达明显降低。NRK-52E细胞发生转分化时,KIF3A蛋白的表达增多(P<0.001),同时wnt/β-catenin信号通路中Wnt4和β-catenin蛋白表达增加(P<0.05,P<0.0001)。结论KIF3A可能通过wnt/β-catenin信号通路介导的上皮细胞-间充质转化参与肾纤维化的发生发展过程。

Objective To investigate the expression of KIF3A in mice with unilateral ureteral obstruction(UUO)and TGF-β1-induced NRK-52E cells and the role of KIF3A in renal tubular epithelial cell transdifferentiation.Methods Thirty-six C57BL/6J mice were randomly divided into the sham group(n=18)and UUO group(n=18).Six mice in each group were sacrificed at 7,14 and 21days after the operation.The degree of renal tubulointerstitial fibrosis of the mice was observed by HE staining,Masson trichrome staining and Sirius red staining.The expression and distribution of KIF3A in the kidney of the mice was detected using RT-PCR,Western blotting and immunohistochemistry.Western blotting was used to detect the expression of KIF3A,E-cadherin andα-SMA proteins in the renal tissue of the mice.The expressions of KIF3A,E-cadherin,α-SMA,Wnt4 andβ-catenin proteins in NRK-52E cells with TGF-β1-induced transdifferentiation were detected by Western blotting.Results Compared with the sham-operated mice,the mice with UUO showed worsened renal interstitial fibrosis with the increase of obstruction time,indicating successful modeling.The expressions of KIF3A mRNA and protein increased progressively and reached the peaked level at 21 days after UUO.The expression ofα-SMA protein was significantly increased while E-cadherin protein expression was significantly reduced after UUO.The transdifferentiated NRK-52E cells showed significantly increased expressions of KIF3A(P<0.001),Wnt4(P<0.05)andβ-catenin proteins(P<0.0001).Conclusions KIF3A may participate in the development of renal fibrosis through epithelial-mesenchymal transition mediated by wnt/β-catenin signaling pathway.