Molecular mechanism of ouabain-induced apoptosis in human podocytes

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 ouabain-induced 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 experiment, 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.