Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb

Juncos, Ramiro, Nancy J. Hong, and Jeffrey L. Garvin. Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb. Am J Physiol Regul Integr Comp Physiol 290: R79-R83, 2006. First published August 11, 2005; doi:10.1152/ajpregu.00447.2005.-Superoxide (O-2(-)) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O-2(-) on other THAL transporters, such as Na+/H+ exchangers, are unknown. We hypothesized that O-2(-) stimulates Na+/H+ exchange in the THAL. We assessed total Na+/H+ exchange activity by measuring recovery of intracellular pH (pHi) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pHi recovery rate from 0.26 +/- 0.05 to 0.21 +/- 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state pHi from 7.52 to 7.37. Because THALs have different Na+/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na+/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na+/H+ exchange from 3.5 +/- 0.8 to 6.7 +/- 1.4 pmol (.) min(-1) (.) mm(-1) (P < 0.01) but decreased basolateral Na+/H+ exchange from 10.8 +/- 1.8 to 6.8 +/- 1.1 pmol (.) min(-1) (.) mm(-1) (P < 0.007). To ascertain whether these effects were caused by O-2(-) or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na+/H+ exchange. We conclude that O-2(-) inhibits basolateral and stimulates luminal Na+/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na+/H+ exchange may enhance stimulation of luminal Na+/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O-2(-) on Na+/H+ exchange may increase net HCO3- reabsorption by the THAL.