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

R Juncos, JL Garvin - American journal of physiology-renal …, 2005 - journals.physiology.org
R Juncos, JL Garvin
American journal of physiology-renal physiology, 2005journals.physiology.org
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 …
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.
American Physiological Society