Superoxide stimulates PKC activity in the thick ascending limb

Superoxide (O 2 − ) is an important regulator of kidney function. Abnormal O 2 − production is a cause of or contributes to increases in blood pressure. The thick ascending limb is responsible for absorption of 20–30% the filtered load of NaCl and has been implicated in hypertension. O 2 − stimulates NaCl absorption by the thick ascending limb by enhancing Na + /K + /2Cl − cotransporter activity. 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 Cl − absorption, total PKC activity and activation of specific PKC isoforms. Medullary thick ascending limbs were exposed to O 2 − by adding xanthine oxidase (1 mU/ml) and hypoxanthine (0.5 mM) to the bath. To avoid formation of H 2 O 2 , experiments were performed in the presence of catalase (100 U/ml). We found that O 2 − increased Cl − absorption by 30% (from 112.7 ± 12.1 to 146.2 ± 13.9 pmol/min/mm) ( n=5 ; p < 0.05). After treatment with the general PKC inhibitor calphostin C (500 nM), O 2 − did not stimulate Cl − absorption (117.7 ± 10.7 vs. 108.1 ± 30.2 pmol/min/mm) ( n=5 ). O 2 − increased total PKC activity by 40% (from 0.066 ± 0.011 to 0.088 ± 0.012 U/mg of protein) ( n=5 ; p < 0.01). O 2 − treatment increased PKCα activation by 270% (from 4.73 ± 1.14 to 13.17 ± 1.75 A.U.) ( n=5 ; p < 0.01). We conclude that O 2 − stimulates NaCl absorption by the thick ascending limb by the activating PKCα.