Endogenous flow-induced superoxide stimulates Na/H exchange activity via PKC in thick ascending limbs

Luminal flow stimulates Na reabsorption along the nephron and activates protein kinase C (PKC) which enhances endogenous superoxide (O(2) (-)) production by thick ascending limbs (TALs). Exogenously-added O(2) (-) augments TAL Na reabsorption, a process also dependent on PKC. Luminal Na/H exchange (NHE) mediates NaHCO₃reabsorption. However, whether flow-stimulated, endogenously-produced O(2) (-) enhances luminal NHE activity and the signaling pathway involved are unclear. We hypothesized that flow-induced production of endogenous O2 (-) stimulates luminal NHE activity via PKC in TALs. Intracellular pH recovery was measured as an indicator of NHE activity in isolated, perfused rat TALs. Increasing luminal flow from 5 to 20 nl/min enhanced total NHE activity from 0.104 ± 0.031 to 0.167 ± 0.036 pH U/min, 81%. The O(2) (-) scavenger tempol decreased total NHE activity by 0.066 ± 0.011 pH U/min at 20 nl/min but had no significant effect at 5 nl/min. With the NHE inhibitor EIPA in the bath to block basolateral NHE, tempol reduced flow-enhanced luminal NHE activity by 0.029 ± 0.010 pH U/min, 30%. When experiments were repeated with staurosporine, a nonselective PKC inhibitor, tempol had no effect. Because PKC could mediate both induction of O2 (-) by flow and the effect of O(()-) on luminal NHE activity, we used hypoxanthine/xanthine oxidase to elevate O(2) (-). Hypoxanthine/xanthine oxidase increased luminal NHE activity by 0.099 ± 0.020 pH U/min, 137%. Staurosporine and the PKCα/β1-specific inhibitor Gö6976 blunted this effect. We conclude that flow-induced O(2) (-) stimulates luminal NHE activity in TALs via PKCα/β1. This accounts for part of flow-stimulated bicarbonate reabsorption by TALs.