Intracellular pH regulates superoxide production by the macula densa

We hypothesized that elevated macula densa intracellular pH (pH(i)) during tubuloglomerular feedback enhances O-2-production from NAD(P) H oxidase. Micro-dissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O-2-production increased from 0.53 +/- 0.09 to 2.62 +/- 0.54 U/min (P < 0.01). To determine whether inhibiting the Na/H exchanger blocks O-2-production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O-2-production induced by NaCl was blunted by 40%. To study the effect of pH(i) on O-2-in intact macula densa cells, we measured O-2-while pH(i) was changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, O-2-production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of O-2(-), we used the NAD(P) H oxidase inhibitor apocynin. In the presence of apocynin (10(-5) M), O-2-production induced by elevating pHi was blocked. Finally, we measured the optimum pH for O-2-production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pH(i) is similar to 8 for O-2-production. We found that elevated pH(i) enhances O-2-production from NAD(P) H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and O-2-production is pH sensitive, with an optimum pH(i) of 8.