A Novel Amiloride-Sensitive H+ Transport Pathway Mediates Enhanced Superoxide Production in Thick Ascending Limb of Salt-Sensitive Rats, Not Na+/H+ Exchange
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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.13BN 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.13BN 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.13BN 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.13BN, 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.
- Received April 15, 2009.
- Revision received May 5, 2009.
- Accepted May 28, 2009.