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

In the normal condition, endogenous formation of peroxynitrite ( ONOO ˉ) 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 ONOO ˉ usually inhibits renal tubular Na + K + ATP ase ( NKA ) activity at high concentrations (micromolar to millimolar range [ μ M–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 ONOO ˉ on NKA activity, preparations of cellular membrane fraction from mouse renal tissue and from cultured HK 2 cells (human proximal tubular epithelial cell lines) were incubated for 10 and 30 min each with different concentrations of ONOO ˉ (10 nmol/L–200  μ mol/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  μ mol/L), ONOO ˉ 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 ONOO ˉ concentration, but interestingly, at lower concentrations (10 and 50 nmol/L), ONOO ˉ caused small but significant increases in the NKA activity (3.3 ± 1.1% and 3.1 ± 0.6%). Pretreatment with a ONOO ˉ scavenger, mercaptoethylguanidine ( MEG ; 200  μ mol/L), prevented these biphasic responses to ONOO ˉ. 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.