Abstract
This study aimed to investigate the underlying molecular mechanism of ouabain-induced apoptosis and inhibited viability of tubulointerstitial cells in lupus nephritis (LN). Serum ouabain expression in 21 LN patients and 20 healthy controls (HCs) was detected by ELISA. Na-K-ATPase α1 (NKA) expression in kidney tissue from 21 LN patients and 6 controls who underwent nephrectomy was determined by immunohistochemistry assay. HK-2 cells were treated by ouabain with concentration 0 nM (control), 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM for 72 h; MTT assay was performed at 24 h, 48 h and 72 h to detect cells viability, AV/PI assay was conducted at 24 h to detect the cell apoptosis. NKA expression was detected by immunofluorescence assay and western blot (WB) while pSrc, pERK, pAkt, pS6K and caspase 3 expression was detected by WB after 100 nM ouabain treatment. Serum ouabain was elevated in LN patients compared to HCs, while kidney NKA was reduced in LN patients compared with controls. HK-2 cell viability by MTT assay was decreased while cell apoptosis by AV/PI was increased at each time point after ouabain 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM treatment compared to control group. Both immunofluorescence and WB disclosed that NKA expression was decreased at 12 h and 24 h compared to 0 h after 100 nM ouabain treatment. Moreover, pSrc, pERK, pAkt, pS6K and caspase 3 expressions were elevated after 100 nM ouabain treatment. In conclusion, ouabain may contribute to LN etiology by inhibiting human proximal tubular cell viability and promoting cells apoptosis through regulating NKA, pSrc, pERK, pAkt, pS6K and caspase 3.