摘要：
Objective Podocyte apoptosis is involved in lupus nephritis (LN).Endogenous ouabain (EO),one of cardiotonicsteroids (CTS),is markedly up-regulated in chronic kidney disease (CKD) patients and is associated with epithelial-to-mesenchymal transition during renal fibrosis in the remnant kidney of 5/6 nephrectomy rats.This study is to explore the molecular mechanism of ouabaininduced apoptosis of human podocytes in LN.Methods (1) In the in vivo experiment,the expression of nephrin protein was detected by immunohistochemistry in renal biopsy specimens from patients with active LN whose SLEDAI scores were more than 5 and those as control group from normal renal tissues in patients with renal tumor after resection of the tumor tissues.(2) In the in vitro experimenb cultured podocytes were treated with different doses of ouabain (0,0.1,1,5,10 and 100 nmol/L) for different durations (24,48 and 72 h).The MTT method was applied to assay cell proliferation and apoptosis.The apoptosis of podocytes was assessed by Hoechst 33258 staining.Western blotting was used to measure Na+-K+-ATPase α1 (NKAα1),p-Src and nephrin.The PP2 (1 μmol/L) was administered from 1 h before the addition of ouabain and continued throughout the 24 h.Western blotting was then employed to examine the expression of proteins above.Results (1) Ouabain induced human podocyte apoptosis in a dose-and time-dependent manner;(2) Nephrin staining showed an even pattern and the expression of nephrin decreased in LN as compared with controls whose nephrin showed an even and linear pattern;(3) Ouabain reduced the expression of nephrin through changing the expression of NKAα1 and p-Src;(4) Inhibition of Src kinase by PP2 increased the expression of p-Src and restored nephrin-expression.Conclusions Ouabain induced human podocyte apoptosis by NKAα1/Src complex decreasing nephrin.%目的 足细胞凋亡在狼疮肾炎(lupus nephritis,LN)发病过程中有重要作用.内源性哇巴因为强心甾类固醇中的一种,在慢性肾脏病(chronic kidney disease,CKD)患者体内表达上调,在5/6肾切除所模拟的慢性肾衰竭大鼠模型体内可诱导肾间质细胞转分化.本研究主要探讨哇巴因在LN中诱导人足细胞(human podocytecell,HPC)凋亡的作用机制.方法 ①体内实验:选取肾脏肿瘤患者被切除的正常肾组织作为对照组,系统性红斑狼疮疾病活动指数(systemic lupus erythematosusdisease activity index,SLEDAI)评分大于5分的活动性LN患者的肾组织作为研究组,通过免疫组织化学检测肾活检组织中nephrin的表达改变.②体外实验:培养HPC,以不同浓度哇巴因(0 nmol/L,0.1 nmol/L,1 nmol/L,5 nmol/L,10 nmol/L,100 nmol/L)刺激HPC不同时间(24 h、48 h、72 h)后,用MTT法观察并测定哇巴因对HPC增殖凋亡的影响;以不同浓度哇巴因处理HPC 24 h后,采用Hoechst-33258染色检测细胞凋亡;收集各个浓度哇巴因处理的细胞,用蛋白免疫印迹法检测钠钾ATP酶α1(Na+-K+-ATPase α1,NKAα1)、p-Src、nephrin的表达情况.③HPC用Src激酶抑制剂PP2(1 μmol/L)提前1h预处理,随后加入不同浓度哇巴因孵育24 h,收集蛋白,行蛋白免疫印迹,检测p-Src、nephrin的表达.结果 ①哇巴因以剂量和时间依赖方式诱导HPC凋亡.②正常对照肾组织中nephrin的表达沿1肾小球基底膜外侧呈均匀、线状分布,且表达强;LN组肾组织中nephrin在肾小球的分布不均,且表达较正常肾组织明显减弱.③随着哇巴因浓度增加,HPC细胞中NKAα1、p-Src的表达先上调再下降,同时可检测到nephrin蛋白表达逐渐减少.④PP2预处理HPC,阻断哇巴因对Src的活化,使得p-Src表达下调,逆转上述效应,逐渐恢复nephrin的表达,使nephrin表达增加.结论 哇巴因通过NKAM/Src下调nephrin蛋白的表达参与HPC凋亡.
