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NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs

Saez, F;Hong, NJ;Garvin, JL;

Luminal flow augments sodium reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of sodium, playing a key role in its homeostasis. Whether flow elevations enhance Na/K/2Cl cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing O2- production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of O2- production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8{plus minus}0.9 to 6.3{plus minus}1.2 arbitrary fluorescent units/second (AFU/s). Maintaining flow at 5 nl/min didn't alter NKCC2 activity. The O2- dismutase mimetic MnTBAP blunted NKCC2 activity from 3.5{plus minus}0.4 to 2.5{plus minus}0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2{plus minus}1.1 to 4.5{plus minus}0.8 AFU/s, but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating O2- production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevent flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased O2-. NKCC2 activation is primarily due to insertion of new transporters in the membrane.