Superoxide stimulates NaCl absorption in the thick ascending limb via activation of protein kinase C

Abnormal production of superoxide (O-2(-)) contributes to hypertension, in part because of its effects on the kidney. The thick ascending limb absorbs 20% to 30% of the filtered load of NaCl. O-2(-) stimulates NaCl absorption by the thick ascending limb by enhancing Na+/K+/2Cl(-) cotransporter activity; however, the signaling mechanism is unknown. We hypothesized that O-2(-) stimulates NaCl absorption by activating protein kinase C (PKC). To test this, we measured the effect of O-2(-) on: (1) Cl- absorption in the presence and absence of PKC inhibitors, (2) total PKC activity, and (3) activation of specific PKC isoforms. Isolated perfused medullary thick ascending limbs were exposed to O-2(-) generated by xanthine oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L). O-2(-) increased Cl- absorption by 42% (from 76.2 +/- 3.6 to 108.2 +/- 11.9 pmol/min per millimeter; n=5; P < 0.05). After treatment with the general PKC inhibitor staurosporine (10 nmol/L), O-2(-) did not stimulate Cl- absorption (Delta-5.7 +/- 8.6%; n=6). In thick ascending limb suspensions, O-2(-) increased total PKC activity by 33% (from 66 +/- 11 to 88 +/- 12 mU/mg protein; n=5; P < 0.05) and increased PKC-alpha and PKC-delta activity by 1.75- and 0.37-fold, respectively. The PKC-alpha/beta-selective inhibitor Go976 (100 nmol/L) blocked the ability of O-2(-) to stimulate Cl- absorption by isolated perfused medullary thick ascending limbs (Delta 4.5 +/- 15.0%; n=5). The role of PKC-delta could not be studied because of cell necrosis caused by the selective inhibitor rottlerin. We conclude that PKC-alpha is required for O-2(-)-stimulated NaCl absorption in the thick ascending limb.