Division of Nephrology, Renmin Hospital of Wuhan University, 238 Jiefang Rd, Wuhan, 430060, Hubei, China.
J Mol Histol. 2013 Oct;44(5):597-608. doi: 10.1007/s10735-013-9505-8. Epub 2013 Mar 21.
Angiotensin II (Ang II) has been reported to cause podocyte apoptosis in rats both in vivo and in vitro studies. However, the underlying mechanisms are poorly understood. In the present study, we investigated the role of the nonreceptor tyrosine kinase c-Abl in Ang II-induced podocyte apoptosis. Male Sprague-Dawley rats in groups of 12 were administered either Ang II (400 kg/kg/min) or Ang II + STI-571 (50 mg/kg/day) by osmotic minipumps. In addition, 12 rats-receiving normal saline served as the control. Glomeruli c-Abl expression was carried out by real time PCR, Western blotting and immunolabeled, and occurrence of apoptosis was carried out by TUNEL staining and transmission electron microscopic analysis. In vitro studies, conditionally immortalized mouse podocytes were treated with Ang II (10(-9)-10(-6) M) in the presence or absence of either c-Abl inhibitor, Src-I1, specific c-Abl siRNA, or c-Abl plasmid alone. Quantification of podocyte c-Abl expression and c-Abl phosphorylation at Y245 and Y412 was carried out by real time PCR, Western blotting and immunofluorescence imaging. The nuclear c-Abl and p53 were quantified by co-immunoprecipitation and Western blotting studies. Podocyte apoptosis was analysed by flow cytometry and Hoechst-33342 staining. c-Abl expression was demonstrated in rat kidney podocytes in vivo and cultured mouse podocytes in vitro. Ang II-receiving rats displayed enhanced podocyte c-Abl expression. And Ang II significantly stimulated c-Abl expression in cultured podocytes. Furthermore Ang II upregulated podocyte c-Abl phosphorylation at Y245 and Y412. Ang II also induced an increase of nuclear p53 protein and nuclear c-Abl-p53 complexes in podocytes and podocyte apoptosis. Down-regulation of c-Abl expression by c-Abl inhibitor (Src-I1) as well as specific siRNA inhibited Ang II-induced podocyte apoptosis; conversely, podoctyes transfected with c-Abl plasmid displayed enhanced apoptosis. These findings indicate that c-Abl may mediates Ang II-induced podocyte apoptosis, and inhibition of c-Abl expression can protect podocytes from Ang II-induced injury.
血管紧张素 II（Ang II）已被报道在体内和体外研究中均可引起足细胞凋亡。然而，其潜在机制尚不清楚。在本研究中，我们研究了非受体酪氨酸激酶 c-Abl 在 Ang II 诱导的足细胞凋亡中的作用。雄性 Sprague-Dawley 大鼠分为 12 组，通过渗透微型泵分别给予 Ang II（400kg/kg/min）或 Ang II + STI-571（50mg/kg/天）。此外，12 只接受生理盐水的大鼠作为对照。通过实时 PCR、Western 印迹和免疫标记检测肾小球 c-Abl 表达，通过 TUNEL 染色和透射电镜分析检测细胞凋亡。在体外研究中，用 Ang II（10（-9）-10（-6）M）处理条件永生化的小鼠足细胞，同时存在或不存在 c-Abl 抑制剂 Src-I1、特异性 c-Abl siRNA 或 c-Abl 质粒。通过实时 PCR、Western 印迹和免疫荧光成像定量检测足细胞 c-Abl 表达和 c-Abl 在 Y245 和 Y412 的磷酸化。通过 co-免疫沉淀和 Western 印迹研究定量核 c-Abl 和 p53。通过流式细胞术和 Hoechst-33342 染色分析足细胞凋亡。体内大鼠肾脏足细胞和体外培养的小鼠足细胞均有 c-Abl 表达。接受 Ang II 的大鼠显示足细胞 c-Abl 表达增强。并且 Ang II 显著刺激培养的足细胞中 c-Abl 的表达。此外，Ang II 上调足细胞 c-Abl 在 Y245 和 Y412 的磷酸化。Ang II 还诱导足细胞中核 p53 蛋白和核 c-Abl-p53 复合物的增加以及足细胞凋亡。c-Abl 抑制剂（Src-I1）和特异性 siRNA 下调 c-Abl 表达可抑制 Ang II 诱导的足细胞凋亡；相反，转染 c-Abl 质粒的足细胞显示出增强的凋亡。这些发现表明 c-Abl 可能介导 Ang II 诱导的足细胞凋亡，抑制 c-Abl 表达可保护足细胞免受 Ang II 诱导的损伤。
