Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb

Superoxide (O(2)(-)) enhances Na reabsorption by the thick ascending limb (THAL). Na absorption in this segment involves the Na-K-2Cl cotransporter, K channel, and Na-K-ATPase. We hypothesized that O(2)(-) stimulates NaCl absorption primarily by enhancing Na-K-2Cl cotransport. First, we measured steady-state intracellular Na (Na(i)) and chloride (Cl(i)). Xanthine oxidase (XO; 0.75 mU/ml) and hypoxanthine (HX; 0.125 mM) were added to the bath to increase O(2)(-). During the control period, Na(i) was 12.2 +/- 1.9 mM. After treatment with O(2)(-), it rose to 20.9 +/- 3.3 mM, a 71% increase (P < 0.01). Cli also increased (P < 0.01). Neither XO nor HX alone had a significant effect on Na(i) or Cl(i). Next, we tested cotransport activity by measuring the initial rate of increase in Nai caused by changing luminal Na-Cl-K from 50/0/0 to 140/134/4 mM. During the control period, the initial rate of increase was 0.13 +/- 0.02 arbitrary units ( AU)/min. After treatment with O(2)(-), it was 0.22 +/- 0.04 AU/min (P < 0.025), a 69% increase. Neither XO nor HX alone had a significant effect. Furosemide completely blocked the increase in intracellular Na in the control and O(2)(-) treatment periods. Next, we studied K channel activity by measuring the depolarization caused by increasing luminal K from 1 to 25 mM using a voltage-sensitive dye. During the control period, maximum depolarization was 7.31 +/- 0.77 AU. After O(2)(-) treatment, it was 6.18 +/- 0.90 AU (P < 0.05), a 15% decrease. Finally, we assessed the effects of O(2)(-) on Na-K-ATPase activity in THAL suspensions by measuring ATP hydrolysis. V(max) and K(1/2) for Na were not affected by O(2)(-). We concluded that O(2)(-) stimulates THAL NaCl absorption primarily by enhancing Na entry via Na-K-2Cl cotransport.