A novel amiloride-sensitive h+ transport pathway mediates enhanced superoxide production in thick ascending limb of salt-sensitive rats, not Na+/H+ Exchange

It has been reported previously that H efflux via the Na/H exchange stimulates NAD(P)H oxidase-dependent superoxide (O2) production in medullary thick ascending limb. We have demonstrated recently that N-methyl-amiloride- sensitive O2 production is enhanced in the thick ascending limb of Dahl salt-sensitive (SS) rats, suggesting that H efflux through Na/H exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na/H exchange does not mediate the ability of N-methyl-amiloride to inhibit thick ascending limb O2 production. To determine the mechanism of action of N-methyl-amiloride, we examined H efflux and O2 production in SS and SS.13 thick ascending limbs of prehypertensive, 0.4% NaCl-fed rats. Tissue strips containing the medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13 rats, loaded with either dihydroethedium or 2′,7′-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein, acetoxymethyl ester, and imaged in a heated tissue bath. In Na-replete media, activation of Na/H exchange using an NH4Cl prepulse did not stimulate thick ascending limb O2 production. In Na-free media containing BaCl2 in which Na/H activity was inhibited, an NH4Cl prepulse stimulated O2 production in medullary thick ascending limb renal tubular segments. This response was enhanced in medullary thick ascending limb of SS rats (slope Δethidium/Δdihydroethedium=0.029±0.004) compared with SS.13 rats (slope=0.010±0.004; P<0.04) and could be inhibited by N-methyl-amiloride (slope=0.005±0.002 and 0.006±0.002 for SS and SS.13, respectively). We concluded that only H efflux through a specific, as-yet-unidentified, amiloride-sensitive H channel promotes O2 production in the medullary thick ascending limb and that this channel is upregulated in SS rats.