Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase

Superoxide (O-2(-)) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O-2(-) production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O-2(-) production by the macula densa via NAD(P) H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O-2(-) production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O-2(-) production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O-2(-), we used tempol (10(-4) M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O-2(-), we used the NAD(P) H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O-2(-) production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O-2(-) production was blocked. To test whether depolarization of the macula densa induces O-2(-) production, we artificially induced depolarization by adding valinomycin (10(-6) M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O-2(-) production. We conclude that increasing luminal NaCl induces O-2(-) production during tubuloglomerular feedback. O-2(-) generated by the macula densa is primarily derived from NAD(P) H oxidase and is induced by depolarization.