Rac1 mediates NaCl‐induced superoxide production by the thick ascending limb

Abstract

NADPH oxidase‐derived superoxide (O2−) regulates Na absorption and renal hemodynamics. Increasing NaCl in the thick ascending limb stimulates O2− generation. NaCl activates the NADPH oxidase regulatory subunit Rac1 in other cells. However, it is unknown whether physiological changes in luminal NaCl augment O2−, nor the mediators involved in the thick ascending limb. We hypothesized that increasing luminal NaCl activates Rac1 and thereby stimulates O2− production in this segment. To test our hypothesis we increased NaCl from 10 to 57 mM in medullary thick ascending limb suspensions and measured O2− using lucigenin and Rac1 activity by translocation. Increasing NaCl stimulated O2− from 1.41±0.16 to 2.71±0.30 nmol O2−/min/mg protein (p<0.05). This was blocked by the Na/K/2Cl cotransporter inhibitor furosemide and the NADPH oxidase inhibitor apocynin. NaCl increased Rac1 in the particulate fraction from 6.8±0.8 to 11.7±2.4%, indicating Rac1 activation (p<0.05). Increasing intracellular Na with Nystatin increased Rac1 in the particulate fraction by 481±110% (p<0.01). In the presence of the Rac1 inhibitor, NaCl did not increase O2− (0.49±0.16 vs. 0.55±0.25 nmol O2−/min/mg protein). Dominant‐negative Rac1 decreased NaCl‐induced O2− from 0.81±0.17 to 0.33±0.04nmol O2−/min/mg protein (p<0.05), a 60% inhibition. We conclude that NaCl stimulates thick ascending limb O2− generation by activating Rac1.