Ouabain-induced Apoptosis In Renal Tubular Epithelial Cells

Objective To evaluate the serum ouabain level in patients with lupus nephritis(LN)and the effects of ouabain on renal tubular epithelial cell injury.Methods 1.ELISA was applied to measure the level of endogenous ouabain(EO)in serum of 20 healthy controls and 21 LN patients respectively;2.Expression of the Na/K-ATPase ? 1(NKA ? 1)was evaluated in renal biopsy samples from LN patients and normal renal tissues from patients with renal tumor after resection by immunohistochemical staining.3.After HK-2 cells were treated with different concentrations of ouabain for different time period,MTT assay and flow cytometry were performed to evaluate the cell proliferation.Western blot and immunofluorescence assay were used to measure the expression of NKA a 1 and p-Src/Src,p-ERK/ERK,p-Akt(473)/Akt,p-S6K/S6K and Caspase3.4.After HK-2 cells were pretreated with PP2(1 uM)or rapamycin(10 ug/1)for one hour and then treated with ouabain(100 nM)for 0.5,1 or 24 hours,flow cytometry was performed to evaluate the cell apoptosis and Western blot was used to measure the expression of p-Src/Src,p-ERK/ERK,p-Akt(473)/Akt,p-S6K/S6K and Caspase3.Results 1.Serum EO was increased in patients with LN(p<0.001);2.Expression of NKA a 1 in the renal tissue of LN patients was significantly lower than that in the healthy controls(p<0.05).3.Ouabain induced HK-2 cell apoptosis in a dose-and time-dependent manners.The NKA a 1 expression was increased and then reduced after exposure to ouabain with different concentrations of ouabain for different time period.Expression of p-Src,p-ERK,p-Akt,p-S6K and Caspase3 was increased after exposure to ouabain in a dose-and time-dependent manners.4.Pretreatment with pp2 or rapamycin reversed ouabain-induced HK-2 cells apoptosis.Conclusion Serum ouabain was increased in patients with LN and ouabain induced HK-2 cells apoptosis through NKA/Src/ERK/Akt/mTOR signaling pathway.