Disequilibrium pH and ammonia transport in isolated perfused cortical collecting ducts

Abstract

The present study was carried out to test directly whether isolated perfused rabbit cortical collecting ducts (CCDs) spontaneously generate a luminal disequilibrium pH. We determined disequilibrium pH as the difference between 1) the actual luminal pH measured by perfusing the lumen with a membrane-impermeant pH-sensitive dye [1,4-dihydroxyphthalonitrile (1,4-DHPN)] and 2) equilibrium pH calculated from the measured total CO2 concentration in fluid collected at the end of the tubule. When the peritubular bath and perfusate had the same composition, a statistically significant acidic disequilibrium pH was found (mean -0.14 units). To determine whether the disequilibrium pH is due to an absolute lack of luminal carbonic anhydrase, we measured the effective rate constant for carbonic acid dehydration in the lumen (k-1). To do this, a lumen-to-bath NH3 concentration gradient was imposed, and the luminal pH was measured along the tubule with 1,4-DHPN. NH3 absorption caused a luminal disequilibrium pH (due to dissociation of NH+4 to NH3 and H+), whose profile along the lumen is dependent on k-1 and NH3 permeability (PNH3). PNH3 and k-1 were estimated from the luminal pH profiles using a mathematical model of proton and buffer transport. The measured k-1 (37 s-1) is within the reported range of values for uncatalyzed H2CO3 dehydration. Calculations demonstrate that the measured PNH3 (2 X 10(-3) cm/s) is high enough and the measured k-1 is low enough to explain ammonia secretion rates seen in previous studies. We conclude that proton secretion in the CCD generates an acidic luminal disequilibrium pH, associated with an absolute lack of luminal carbonic anhydrase, which enhances the net rate of NH3 secretion.

Expression of the Ammonia Transporter, Rh C Glycoprotein, in Normal and Neoplastic Human Kidney

“…In animal models, luminal acidification, including the generation of a luminal disequilibrium pH, results from apical H ϩ secretion by H ϩ -ATPase and H ϩ -K ϩ -ATPase and is both necessary for and regulates transepithelial ammonia secretion (31)(32)(33)(34)(35)(36). Collecting duct ammonia transport seems to involve both diffusive and transporter-mediated components (26,37), and the transporter-mediated component of apical transport is stimulated by luminal H ϩ (37).…”

“…H ϩ secretion in the absence of luminal carbonic anhydrase activity generates a luminal disequilibrium pH. This accelerates ammonia secretion to a greater degree than is explainable by the mean luminal pH (33,35,38). Because intercalated cells are the primary mechanism of collecting duct H ϩ secretion, the luminal disequilibrium pH is greatest in the region adjacent to the intercalated cell apical membrane.…”

Expression of RhCG, a New Putative NH3/NH4 + Transporter, along the Rat Nephron

“…Final secretion of NH 4 ϩ is believed to occur mostly in the CD, where it is trapped and excreted (22)(23)(24)(25). Some pieces of evidence also exist that NH 4 ϩ secretion, albeit at low rates, occurs in the superficial late distal tubule including CNT and initial CD (26).…”

Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3)

“…Transepithelial H ϩ gradients regulate collecting duct ammonia secretion (16,25,53), which has led to the conclusion that transport involves nonionic NH 3 diffusion. However, the observation that a saturable, transporter-mediated component of ammonia transport occurs suggests other interpretations.…”

“…The presence of both apical and basolateral NH 4 ϩ /H ϩ exchange activities are well positioned to mediate coordinated mechanism of H ϩ gradient-regulated transepithelial ammonia secretion. For example, luminal acidification stimulates collecting duct ammonia secretion (30, 31,52,53 …”