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

Abstract

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 (H(2)O(2)) treatment to human renal proximal tubular (HK-2) cells.

Materials and methods: HK-2 cells were incubated with H(2)O(2) (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-3beta (GSK-3beta), mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) were also analysed.

Results: Cells incubated with H(2)O(2) 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-3beta, mTOR and ERK1/ERK2 of H(2)O(2)-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 H(2)O(2) 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.

Figure 1

(a) Effect of erythropoietin (EPO) at concentrations of 100 and 400 U/ml on viability of HK‐2 cells treated with hydrogen peroxide (H₂O₂). Columns represent chemical reduction of MTT by cultured HK‐2 cells either untreated (Control, made equal to 100%) or treated with H₂O₂ (2 mm) alone (H₂O₂) or H₂O₂ (2 mm) and EPO (H₂O₂ + EPO 100 U) added at the same time to cell cultures, or H₂O₂ (2 mm) and EPO (H₂O₂ + EPO 400 U) added also in this case at the same time. H₂O₂ caused a significant decrease in cell viability. No change was observed when the cells were treated with H₂O₂ and EPO at concentration of 100 U/ml compared with cells treated with H₂O₂ alone, indicating no significant effect of EPO (100 U/ml) on cell viability. However, addition of EPO at concentration of 400 U/ml caused further (and significant) decrease in cell viability compared to cells treated with H₂O₂ alone (as shown by the further and significant decrease in MTT chemical reduction). Data are representative of three separate experiments. H₂O₂ vs. Control; P < 0.0001 (°). H₂O₂+ EPO 100 U vs. Control; P < 0.0001 (^). H₂O₂+ EPO 400 U vs. Control; P < 0.0001 (*). H₂O₂+ EPO 100 U vs. H₂O₂; P > 0.05 (+). H₂O₂+ EPO 400 U vs. H₂O₂; P < 0.0001 (#). (b) Effect of EPO alone at concentrations of 100 and 400 U/ml on cell viability of H₂O₂‐untreated HK‐2 cells. Columns represent chemical reduction of MTT by cultured HK‐2 cells either untreated (Control, made equal to 100%) or treated with EPO alone at concentrations of 100 and 400 U/ml (EPO 100 U and EPO 400 U). Addition of EPO alone at concentrations of 100 and 400 U/ml did not cause, in both cases, any significant change in cell viability (P > 0.05). Data are representative of three separate experiments. EPO 100 U vs. Control; P > 0.05 (°). EPO 400 U vs. Control; P > 0.05 (^).

Figure 2

Effect of hydrogen peroxide (H₂O₂) alone and H₂O₂ + erythropoietin (EPO) at concentrations of 100 and 400 U/ml on phosphorylation of Akt, GSK‐3β, ERKs and mTOR in HK‐2 cells. Samples of lysed HK‐2 cells treated with H₂O₂ (2 mm) alone and H₂O₂ (2 mm) and EPO at concentrations of 100 and 400 U/ml added at the same time, were immunoblotted using the appropriate antibody against the phosphorylated forms of the kinases Akt, GSK‐3β, ERK1/ERK2 and mTOR (‘pAkt,’‘pGSK‐3β’, ‘pERK1/2’ and ‘p mTOR’). There was a clear effect of EPO (at a concentration of 400 U/ml) in decreasing phosphorylation levels of all analysed kinases. This same effect of decreasing phosphorylation status was also observed for the transcription factor FKHRL1. Lane for β‐actin also shows that loading was even for all time points. Data are representative of three separate experiments.

Figure 3

Effect of erythropoietin (EPO) alone at concentrations of 100 and 400 U/ml on phosphorylation of Akt, GSK‐3β, ERK1/ERK2 and mTOR in HK‐2 cells. Samples of lysed HK‐2 cells treated with EPO at concentrations of 100 U/ml and 400 U/ml were immunoblotted using appropriate antibody against phosphorylated forms of the kinases Akt, GSK‐3β, ERK1/ERK2 and mTOR (‘pAkt,’‘pGSK‐3β’, ‘pERK1/2’ and ‘p mTOR’). There was an evident effect of EPO at concentrations of 100 and 400 U/ml on phosphorylation levels of ERKs and Akt, whose phosphorylation increased after 10 min incubation with EPO. Lane for β‐actin shows that loading was even for all time points. Data are representative of three separate experiments.