Activation of NAD(P)H oxidase by outward movements of H+ ions in renal medullary thick ascending limb of Henle

The present study was designed to test the hypothesis that the production of superoxide (O-2(-center dot)) by NAD(P)H oxidase is coupled to tubular metabolic activity through ionic activation mediated by H+ movement across cell membrane. Using dual fluorescent microscopic imaging analysis, intracellular O-2(-center dot) levels and pH (pHi) in renal medullary thick ascending limb of Henle (TALH) cells were simultaneously measured. It was found that intracellular O-2(-center dot) levels in these cells were increased in parallel to the elevation of pHi by outflow of H+ induced via NH4Cl loading followed by rapid removal. This increase in intracellular O-2(-center dot) levels was substantially blocked by an inhibitor of Na+/H+ exchanger, methylisobutyl-amiloride (MIA; 100 mu M), a chemical SOD mimetic, Tiron (1 mM) or an inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI; 100 mu M). In additional groups of TALHs, a proton ionophore, carbonylcyanide m-chlorophenylhydrazone (10 mu M) was used to produce H+ conductance, leading to H+ flux across cell membrane depending on extracellular pH. The efflux of H+ increased both pHi and intracellular O-2(-center dot) levels, but the influx of H+ did not increase intracellular O-2(-center dot) levels. The H+ efflux-induced increase in intracellular O-2(-center dot) levels was completely blocked by DPI and another NAD(P)H oxidase inhibitor, apocynin (100 mu M). In in invo experiments, renal medullary infusion of MIA ( 100 mu M) was found to significantly decrease the concentrations of H2O2 in the renal medullary interstitium. These results suggest that it is the outward movements of H+ ions that activates NAD(P)H oxidase to produce O-2(-center dot) in TALH cells. This H+ outflow-associated activation of NAD(P) H oxidase importantly contributes to tissue levels of reactive oxygen species in the renal medulla.