Macula densa Na+/H+ exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms

Functional and immunohistochemical studies were performed to localize and i dentify Na+/H+ exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparatio n dissected From rabbit kidney, intracellular pH (PHi) was measured with fl uorescence microscopy by using 2',7'-bis-(2-carboxyethyl)-5-(and -6) carbox yfluorescein. NHE activity was assayed by measuring the initial rate of Na-dependent pH(i) recovery from an acid load imposed by prior lumen and bath Na+ removal. Removal of Na+ from the bath resulted in a significant, DIDS- insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pH(i). This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC50 = 9.0 and 247 mu M, respectively, consistent with NHE4). The rec ently reported apical NHE was more sensitive to inhibition by these drugs ( IC50 = 0.86 and 7.6 mu M, respectively, consistent with NHE2). Increasing o smolality, a known activator of NHE4, greatly stimulated basolateral NHE. I mmunohistochemical studies using antibodies against NHE1-4 peptides demonst rated expression of NHE2 along the apical and NHE4 along the basolateral, m embrane, whereas NHE1 and NHE3 were not detected. These results suggest tha t macula densa cells functionally and immunologically express NHE2 at the a pical membrane and NHE4 at the basolateral membrane. These two isoforms lik ely participate in Nat transport, pH(i), and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.