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

Superoxide (O2-) 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 O2- stimulates NaCl absorption primarily by enhancing Na-K-2Cl cotransport. First, we measured steady-state intracellular Na (Nai) and chloride (Cli). Xanthine oxidase (XO; 0.75 mU/ml) and hypoxanthine (HX; 0.125 mM) were added to the bath to increase O2-. During the control period, Nai was 12.2 +/- 1.9 mM. After treatment with O2-, 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 Nai or Cli. 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 O2-, 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 O2- 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 O2- treatment, it was 6.18 +/- 0.90 AU (P < 0.05), a 15% decrease. Finally, we assessed the effects of O2- on Na-K-ATPase activity in THAL suspensions by measuring ATP hydrolysis. Vmax and K1/2 for Na were not affected by O2-. We concluded that O2- stimulates THAL NaCl absorption primarily by enhancing Na entry via Na-K-2Cl cotransport.