Renal Oxidative Stress in Medullary Thick Ascending Limbs Produced by Elevated NaCl and Glucose

Abstract

The effects of NaCl, glucose, and thyroid hormone on the production of superoxide (O 2 ·− ) within the renal medulla of Sprague-Dawley rats were examined. Responses of intracellular superoxide [O 2 ·− ] i in isolated medullary thick ascending limbs (mTALs) were studied using real-time fluorescent microscopy with measurement of the dehydroethidium (DHE) to ethidium (Eth) conversion ratio (Eth/DHE ratio unit). The results demonstrated that elevations of extracellular NaCl (from 152 to 252 mmol/L), d -glucose (from 5 to 25 mmol/L), and triiodo-thyronine (T3; 10 μmol/L) significantly increased [O 2 ·− ] i levels. Preincubation with superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (1 mmol/L) significantly inhibited these responses. Stimulation with equamolar amounts of choline chloride or l -glucose failed to increase [O 2 ·− ] i , indicating that these O 2 ·− responses were not determined by changes in osmolality. The responses to NaCl, d -glucose, and T3 were abolished by pretreatment with the Na + /K + -ATPase pump inhibitor ouabain (4 mmol/L) and with Na + /H + -exchanger inhibitor dimethylamiloride (100 μmol/L). We conclude that elevations of extracellular NaCl, d -glucose, or T3 levels can activate both the Na + /K + -ATPase pump and Na + /H + exchanger in mTAL, which, in turn, is associated with increased intracellular concentrations of superoxide.