Erythropoietin increases asymmetric dimethylarginine in endothelial cells:: Role of dimethylarginine dimethylaminohydrolase

Recombinant human erythropoietin therapy frequently causes hypertension in humans and animals with chronic renal failure. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase, and its accumulation has been associated with reducing NO bioavailability and increasing superoxide generation. Whether epoetin beta (EPO) or darbepoetin alpha (NESP) can modify the levels of ADMA in endothelial cells was investigated. Endothelial cells from the third passage were incubated for 24 h in the presence of various concentrations of EPO or NESP (0, 0.1, 1, 10, 50, 100, and 200 U/ml). The levels of ADMA, allantoin, nitrate, and nitrite in conditioned media and the activity of dimethylarginine dimethylaminohydrolase (DDAH), the content of thiols and reactive oxygen species in endothelial cells, were determined. When endothelial cells were exposed to EPO or NESP, ADMA concentration in the cell culture medium increased significantly in a dose-dependent manner versus control. This effect was associated with a reduced activity of DDAH, the enzyme that degrades ADMA. Furthermore, EPO- or NESP-induced accumulation of ADMA was accompanied by a significant reduction of NO synthesis and an increase in oxidative stress. Both allantoin, a marker of oxygen free radical generation, and reactive oxygen species increased significantly after EPO or NESP treatment compared with control. The antioxidant pyrrolidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation in the same way as the co-incubation with anti-EPO neutralizing antibody. EPO and NESP posttranslationally impair DDAH activity via increased oxidative stress, causing ADMA as an important cardiovascular risk factor to accumulate and inhibit NO synthesis.