Abstract 045: Endogenous Flow-induced Superoxide Stimulates Na/H Exchange Activity Via PKC In Thick Ascending Limbs
Luminal flow stimulates Na reabsorption along the nephron and endogenous superoxide (O2-) production by thick ascending limbs (TALs). The latter is due to protein kinase C (PKC) activation. Exogenously added O2- augments TAL Na reabsorption, a process also dependent on PKC. Luminal Na/H exchange (NHE) and Na/K/2Cl cotransport initiate TAL Na reabsorption. However, the role of endogenously-produced O2- in the stimulation of luminal NHE activity by flow, 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 (pHi) recovery was measured as an indicator of NHE activity in isolated, perfused rat TALs. Addition of the O2- scavenger tempol decreased total NHE activity by 39% (0.168 ± 0.035 vs 0.103 ± 0.026 pHi units/min; P < 0.01; n = 4) when luminal flow was 20 nl/min; whereas it had no effect when flow was 5 nl/min (0.131 ± 0.027 vs 0.128 ± 0.027 pHi units/min; n = 5). Using the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in the bath to block basolateral NHE so that pHi recovery only reflected flow-enhanced luminal NHE activity, tempol caused a 30% reduction in pHi recovery (0.097 ± 0.018 vs 0.068 ± 0.015 pHi units/min; P < 0.04; n = 6). When these experiments were repeated with the general PKC inhibitor staurosporine, tempol had no effect (0.136 ± 0.018 vs 0.133 ± 0.007 pHi units/min; n = 4). Because PKC could mediate both induction of O2- by flow and the effect of O2- on luminal NHE activity, we used hypoxanthine/xanthine oxidase (HX/XO) to elevate O2- and examined the role of PKC. HX/XO increased luminal NHE activity by 118% (0.140 ± 0.047 vs 0.305 ± 0.053 pHi units/min; P < 0.02; n = 5); whereas staurosporine blocked this effect (0.134 ± 0.043 vs. 0.125 ± 0.030 pHi units/min; n = 3). The PKCα/β1-specific inhibitor Gö 6976 also blunted HX/XO’s effect (HX/XO: 0.072 ± 0.023 vs 0.170 ± 0.035 pHi units/min; P < 0.008; n = 5; HX/XO in presence of Gö 6976: 0.116 ± 0.029 vs 0.137 ± 0.025 pHi units/min; n = 5). We conclude that flow-induced O2- stimulates luminal NHE activity in TALs via PKCα/β1. This process may account for part of flow-stimulated bicarbonate reabsorption in this nephron segment.
Author Disclosures: N.J. Hong: None. J.L. Garvin: None.
- © 2014 by American Heart Association, Inc.