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 (O(2)(.-)) production in medullary thick ascending limb. We have demonstrated recently that N-methyl-amiloride-sensitive O(2)(.-) 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 O(2)(.-) production. To determine the mechanism of action of N-methyl-amiloride, we examined H(+) efflux and O(2)(.-) production in SS and SS.13(BN) 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(BN) 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 NH(4)Cl prepulse did not stimulate thick ascending limb O(2)(.-) production. In Na(+)-free media containing BaCl(2) in which Na(+)/H(+) activity was inhibited, an NH(4)Cl prepulse stimulated O(2)(.-) production in medullary thick ascending limb renal tubular segments. This response was enhanced in medullary thick ascending limb of SS rats (slope Deltaethidium/Deltadihydroethedium=0.029+/-0.004) compared with SS.13(BN) 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(BN), respectively). We concluded that only H(+) efflux through a specific, as-yet-unidentified, amiloride-sensitive H(+) channel promotes O(2)(.-) production in the medullary thick ascending limb and that this channel is upregulated in SS rats.
O'Connor
Paul M
PM
Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53202, USA. poconnor@mcw.edu
Lu
Limin
L
Liang
Mingyu
M
Cowley
Allen W
AW
D000584:Amiloride / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D004195:Disease Models, Animal; D006973:Hypertension / Q000378:metabolism / Q000503:physiopathology*; D007684:Kidney Tubules / Q000378:metabolism; D008297:Male; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress; D011336:Probability; D011897:Random Allocation; D051381:Rats; D020303:Rats, Inbred Dahl; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D012965:Sodium Chloride / Q000378:metabolism* / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D017923:Sodium-Hydrogen Exchangers; D013481:Superoxides; D009569:Nitric Oxide; D012965:Sodium Chloride; D000584:Amiloride; D019255:NADPH Oxidases