Enhancement in cellular Na+K+ATPase activity by low doses of peroxynitrite in mouse renal tissue and in cultured HK2 cells.

Abstract

In the normal condition, endogenous formation of peroxynitrite (ONOOˉ) from the interaction of nitric oxide and superoxide has been suggested to play a renoprotective role. However, the exact mechanism associated with renoprotection by this radical compound is not yet clearly defined. Although ONOOˉ usually inhibits renal tubular Na+K+ ATPase (NKA) activity at high concentrations (micromolar to millimolar range [μM–mM], achieved in pathophysiological conditions), the effects at lower concentrations (nanomolar range [nM], relevant in normal condition) remain unknown. To examine the direct effect of ONOOˉ on NKA activity, preparations of cellular membrane fraction from mouse renal tissue and from cultured HK2 cells (human proximal tubular epithelial cell lines) were incubated for 10 and 30 min each with different concentrations of ONOOˉ (10 nmol/L–200 μmol/L). NKA activity in these samples (n = 5 in each case) was measured via a colorimetric assay capable of detecting inorganic phosphate. At high concentrations (1–200 μmol/L), ONOOˉ caused dose‐dependent inhibition of NKA activity (−3.0 ± 0.6% and −36.4 ± 1.4%). However, NKA activity remained unchanged at 100 and 500 nmol/L ONOOˉ concentration, but interestingly, at lower concentrations (10 and 50 nmol/L), ONOOˉ caused small but significant increases in the NKA activity (3.3 ± 1.1% and 3.1 ± 0.6%). Pretreatment with a ONOOˉ scavenger, mercaptoethylguanidine (MEG; 200 μmol/L), prevented these biphasic responses to ONOOˉ. This dose‐dependent biphasic action of ONOO − on NKA activity may implicate that this radical compound helps to maintain sodium homeostasis either by enhancing tubular sodium reabsorption under normal conditions or by inhibiting it during oxidative stress conditions.