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

Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

Submitted 22 November 2004 ; accepted in final form 17 June 2005

The present study was designed to test the hypothesis that the production of superoxide (O₂^–·) 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 O₂^–· levels and pH (pH_i) in renal medullary thick ascending limb of Henle (TALH) cells were simultaneously measured. It was found that intracellular O₂^–· levels in these cells were increased in parallel to the elevation of pH_i by outflow of H⁺ induced via NH₄Cl loading followed by rapid removal. This increase in intracellular O₂^–· levels was substantially blocked by an inhibitor of Na⁺/H⁺ exchanger, methylisobutyl-amiloride (MIA; 100 µM), a chemical SOD mimetic, Tiron (1 mM) or an inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI; 100 µM). In additional groups of TALHs, a proton ionophore, carbonylcyanide m-chlorophenylhydrazone (10 µM) was used to produce H⁺ conductance, leading to H⁺ flux across cell membrane depending on extracellular pH. The efflux of H⁺ increased both pH_i and intracellular O₂^–· levels, but the influx of H⁺ did not increase intracellular O₂^–· levels. The H⁺ efflux-induced increase in intracellular O₂^–· levels was completely blocked by DPI and another NAD(P)H oxidase inhibitor, apocynin (100 µM). In in invo experiments, renal medullary infusion of MIA (100 µM) 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 O₂^–· 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.

redox signaling; renal tubules; ion transporter; Na⁺/H⁺ exchanger; superoxide

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