Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback

The macula densa senses increasing NaCl concentrations in tubular fluid and increases afferent arteriole tone by a process known as tubuloglomerular feedback (TGF). Nitric oxide (NO) production by macula densa neuronal nitric oxide synthase (nNOS) is enhanced by increasing NaCl in the macula densa lumen, and the NO thus formed inhibits TGF. Blocking apical Na(+)/H(+) exchange with amiloride augments TGF and mimics the effect of nNOS inhibition. We hypothesized that increasing NaCl in the macula densa lumen raises macula densa intracellular pH (pH(i)) and activates nNOS.

The thick ascending limb and a portion of the distal tubule with intact macula densa plaque adherent to the glomerulus were microdissected and perfused. Macula densa perfusate was changed from a low (10 mmol/L) to high NaCl solution (80 mmol/L) to mimic the conditions that induce TGF. Osmolality of both solutions was 180 mOsm, so that changing the solutions did not alter cell volume.

Macula densa pH(i) increased significantly from 7.0 +/- 0.5 to 7.8 +/- 0.6 when the perfusate was changed from low to high (P < 0.05; N= 5). When amiloride was added to inhibit Na(+)/H(+) exchange, the increase in pH(i) during TGF was blocked (N= 5). Fluorescence intensity of DAF-2, an NO-sensitive dye, increased by 28.8 +/- 4.1% after increasing luminal NaCl (N= 5), indicating an increase in NO production. In the presence of the Na(+)/H(+) exchanger inhibitor amiloride or the nNOS inhibitor 7-NI, the increase in NO induced by switching the macula densa perfusate from low to high was blunted. To study whether changes in pH(i) can directly alter NO production, we used nigericin, a K(+)/H(+) ionophore, to equilibrate luminal and intracellular pH. When macula densa pH was raised from 7.3 to 7.8 in the presence of 10(-5) mol/L nigericin in the low NaCl solution, fluorescence of DAF-2 in the macula densa increased by 17.9 +/- 1.3% (P < 0.01; N= 5). In the presence of 7-NI, the increase in NO induced by raising pH(i) was blocked (N= 5).

We concluded that macula densa pH(i) increases during TGF, and this increase in pH(i) activates nNos.