H⁺ secretion in rabbit mesonephric collecting tubule

The mesonephros, the precursor of the metanephros, the definitive kidney, is the functional excretory organ in the 12- to 20-day-old rabbit fetus. It is believed to acidify allantoic fluid. To determine whether H⁺ excretion occurs in the distal nephron, we examined isolated perfused mesonephric collecting tubules by microcalorimetry and pH-sensitive fluorescent dyes. Collecting tubules secreted H⁺ (absorbed HCO₃^-) at rates twice those observed in the mature outer medullary collecting duct (OMCD) of the metanephric kidney. H⁺ secretion was not inhibited by ouabain (18.5 ± 2.2 vs. 16.7 ± 4.0 pmol·min^-1·mm^-1; n = 7, P = NS) but was reversibly inhibited by removing Cl^- from the bathing solution (15.1 ± 2.3 to -0.6 ± 3.7 to 15.5 ± 1.1 pmol·min^-1·mm^-1; n = 5, P < 0.05); luminal application of N-ethylmaleimide (NEM), an inhibitor of the H⁺-ATPase, also inhibited H⁺ secretion (n = 2). These results suggested that H⁺ secretion in the mesonephric collecting tubule is mediated by transporters similar to those of the OMCD. To test this hypothesis, we stained collecting tubules from 15-20 day embryos with 6-carboxyfluorescein (6-CF) diacetate to identify intercalated-like cells or perfused them with 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) to measure intracellular pH (pH_i). We found that 139 ± 15 cells/mm concentrated 6-CF or BCECF, consistent with the phenotype of metanephric intercalated cells. Na⁺-independent pH_i recovery from an acid load (NH₃/NH₄⁺) was rapid (0.53 ± 0.14 pH U/min; n = 5) and could be inhibited by luminal application of H⁺ pump inhibitors, NEM, N,N′-dicyclohexylcarbodiimide, or bafilomycin (0.08 ± 0.08 pH U/min; P < 0.05). Thus H⁺ secretion in the mesonephric collecting tubule occurs at high rates and is mediated by cells possessing polarized H⁺/ HCO₃^- transporters in a distribution similar to that of the metanephric α-intercalated cell, with apical H⁺-ATPase and basolateral Cl^-/HCO₃^- exchangers.