Enhanced amiloride-sensitive superoxide production in renal medullary thick ascending limb of Dahl salt-sensitive rats

The aims of the present study were to determine whether superoxide (O ₂^-) production is enhanced in medullary thick ascending limb (mTAL) of Dahl salt-sensitive (SS) rats compared with a salt-resistant consomic control strain (SS.13^BN) and to elucidate the cellular pathways responsible for augmented O₂^- production. Studies were carried out in 7- to 10-wk-old male SS and SS.13^BN rats fed either a 0.4% NaCl diet or a 4.0% NaCl diet for 3 days before tissue harvest. Tissue strips containing mTAL were isolated from the left kidney, loaded with the O₂^--sensitive fluorescent dye dihydroethidium, superfused with modified Hanks' solution, and imaged at X60 magnification on a heated microscope stage. O₂^- production was stimulated in mTAL by incrementing superfusate NaCl concentration from 154 to 254 to 500 mM. O₂^- production was enhanced in mTAL of SS rats compared with SS.13^BN rats in response to incrementing bath NaCl. Addition of N-methyl-amiloride (100 μM) or inhibition of NAD(P)H oxidase reduced O ₂^- production in SS mTAL to levels observed in SS.13 ^BN rats. Both amiloride- and ouabain-sensitive pathways of O ₂^- production were elevated following 3 days of high (4.0%) NaCl feeding in mTAL of SS and SS.13^BN rats. We conclude that mTAL from SS rats exhibit enhanced amiloride-sensitive O₂^- production. The amiloride-sensitive O₂^- response in mTAL is independent of active Na⁺ transport and appears to be mediated by NAD(P)H oxidase. Amiloride-sensitive O₂^- production is likely to contribute to augmented outer medullary O₂^- production observed in SS rats during both normal and high NaCl diets.