Renal oxidative stress in medullary thick ascending limbs produced by elevated NaCl and glucose

The effects of NaCl, glucose, and thyroid hormone on the production of superoxide (O2*-) within the renal medulla of Sprague-Dawley rats were examined. Responses of intracellular superoxide [O2*-]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 micromol/L) significantly increased [O2*-]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 [O2*]i, indicating that these O2*- 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 micromol/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.