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


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.
Andreucci
M
M
Magna Graecia University, Catanzaro, Italy. andreucci@unicz.it
Fuiano
G
G
Presta
P
P
Lucisano
G
G
Leone
F
F
Fuiano
L
L
Bisesti
V
V
Esposito
P
P
Russo
D
D
Memoli
B
B
Faga
T
T
Michael
A
A
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D004921:Erythropoietin / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D058570:TOR Serine-Threonine Kinases
D004921:Erythropoietin; D006861:Hydrogen Peroxide; D011494:Protein Kinases; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3