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

Hypertension. 2009 Aug;54(2):248-54. doi: 10.1161/HYPERTENSIONAHA.109.134692. Epub 2009 Jun 29.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amiloride / metabolism*
  • Amiloride / pharmacology
  • Animals
  • Disease Models, Animal
  • Hypertension / metabolism
  • Hypertension / physiopathology*
  • Kidney Tubules / metabolism
  • Male
  • NADPH Oxidases / metabolism*
  • Nitric Oxide / metabolism
  • Oxidative Stress
  • Probability
  • Random Allocation
  • Rats
  • Rats, Inbred Dahl
  • Sensitivity and Specificity
  • Signal Transduction
  • Sodium Chloride / metabolism*
  • Sodium Chloride / pharmacology
  • Sodium-Hydrogen Exchangers / metabolism*
  • Superoxides / metabolism*

Substances

  • Sodium-Hydrogen Exchangers
  • Superoxides
  • Nitric Oxide
  • Sodium Chloride
  • Amiloride
  • NADPH Oxidases