Downregulation of cell survival signalling pathways and increased cell damage in hydrogen peroxide-treated human renal proximal tubular cells by alpha-erythropoietin

Objective: Erythropoietin has been shown to have a protective effect in certain models of ischaemia-reperfusion, and in some cases the protection has been correlated with activation of signalling pathways known to play a role in cell survival and proliferation. We have studied whether erythropoietin would overcome direct toxic effects of hydrogen peroxide (H2O2) treatment to human renal proximal tubular (HK-2) cells. Materials and methods: HK-2 cells were incubated with H2O2 (2 mm) for 2 h with or without erythropoietin at concentrations of 100 and 400 U/ml, and cell viability/proliferation was assessed by chemical reduction of MTT. Changes in phosphorylation state of the kinases Akt, glycogen synthase kinase-3 beta (GSK-3 beta), mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) were also analysed. Results: Cells incubated with H2O2 alone showed a significant decrease in viability, which did not significantly change by addition of erythropoietin at concentration of 100 U/ml, but was further reduced when concentration of erythropoietin was increased to 400 U/ml. Phosphorylation state of the kinases Akt, GSK-3 beta, mTOR and ERK1/ERK2 of H2O2-treated HK-2 cells was slightly altered in the presence of erythropoietin at concentration of 100 U/ml, but was significantly less in the presence of erythropoietin at a concentration of 400 U/ml. Phosphorylation of forkhead transcription factor FKHRL1 was diminished in cells incubated with H2O2 and erythropoietin at a concentration of 400 U/ml. Conclusions: Erythropoietin, at high concentrations, may significantly increase cellular damage in HK-2 cells subjected to oxidative stress, which may be due in part to decrease in activation of important signalling pathways involved in cell survival and/or cell proliferation.