A mathematical model of rat collecting duct II. Effect of buffer delivery on urinary acidification

A mathematical model of the rat collecting duct (CD) is used to examine the effect of delivered load of bicarbonate and nonbicarbonate buffer on urinary acidification. Increasing the delivered load of HCO[Formula: see text] produces bicarbonaturia, and, with luminal carbonic anhydrase absent, induces a disequilibrium luminal pH and a postequilibration increase in urinary Pco 2. At baseline flows, this disequilibrium disappears when luminal carbonic anhydrase rate coefficients reach 1% of full catalysis. The magnitude of the equilibration Pco 2 depends on the product of urinary acid phosphate concentration and the disequilibrium pH. Thus, although increasing phosphate delivery to the CD decreases the disequilibrium pH, the increase in urinary phosphate concentration yields an overall increase in postequilibration Pco 2. In simulations of experimental HCO[Formula: see text] loading in the rat, model predictions of urinary Pco 2 exceed the measured Pco 2 of bladder urine. In part, the higher model predictions for urinary Pco 2 may reflect higher urinary flow rates and lower urinary phosphate concentrations in the experimental preparations. However, when simulation of CD function during HCO[Formula: see text] loading acknowledges the high ambient renal medullary Pco 2 (5), the predicted urinary Pco 2 of the model CD is yet that much greater. This discrepancy cannot be resolved within the model but requires additional experimental data, namely, concomitant determination of urinary buffer concentrations within the tubule fluid sampled for Pco 2 and pH. This model should provide a means for simulating formal testing of urinary acidification and thus for examining hypotheses regarding transport defects underlying distal renal tubular acidosis.

Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct

The isolated perfused tubule technique was utilized to determine whether endogenous luminal carbonic anhydrase is present in the initial or terminal parts of the inner medullary collecting duct (IMCD) of the rat. This was accomplished by measuring the luminal disequilibrium pH in the presence of a large luminal proton source created by perfusing the lumen with a solution containing 10 mM NH4Cl. (NH3 efflux causes H+ to be released from NH+4 in the lumen). The disequilibrium pH was calculated by subtracting the equilibrium pH from the measured pH at the end of the tubule lumen. The end-luminal equilibrium pH was calculated from the total CO2 concentration in the collected fluid, as measured by microcalorimetry. The end-luminal pH was determined by measuring the fluorescent signal from the the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which was added to the luminal perfusate in its nonesterified form. In the initial IMCD, there was no measurable disequilibrium pH. With the addition of the carbonic anhydrase inhibitor acetazolamide to the luminal fluid, a significant acidic pH disequilibrium was elicited. In the terminal IMCD under control conditions a statistically significant acidic disequilibrium pH was measured. The disequilibrium was obliterated when exogenous carbonic anhydrase was added to the luminal perfusate. These findings were verified by measuring total ammonia flux by ultramicrofluorometry. The results demonstrate endogenous luminal carbonic anhydrase activity in the initial IMCD but a lack of enzyme activity in the terminal IMCD.

Na+-H+ exchange and pH regulation in red blood cells: role of uncatalyzed H2CO3 dehydration

Erythrocytes of rainbow trout respond to adrenergic stimulation by activation of a Na+-H+ exchange. When red blood cells are suspended in their own plasma and equilibrated with a convenient gas mixture in a tonometer, the extrusion of H+ induces a fast, very strong acidification of the blood (by 0.5-0.7 pH units), explained as follows. Excretion of H+ into a medium containing HCO3- causes the formation of H2CO3. The uncatalyzed dehydration of H2CO3 is slow so that H+ accumulates above the level that would prevail at equilibrium, promoting a strong acid disequilibrium pH. Then the blood pH progressively returns to a value close to its initial value because of the slow uncatalyzed dehydration of H2CO3 and washout of the CO2 so produced. The period of acid disequilibrium pH, however, is lengthened because part of the CO2 generated by the spontaneous dehydration is not washed out by tonometry but diffuses into the red cells where it is rapidly converted into HCO3- and H+ by carbonic anhydrase and then excreted by Na+-H+ and Cl-HCO3- exchangers. This recycling process "refuels" the ionic reaction, increasing the time needed to reach equilibrium. The anion exchanger does not sense this strong acid disequilibrium pH, since the external HCO3- concentration is practically unchanged at that time. During the extracellular pH (pHe) recovery period, simultaneously extracellular HCO3- content decreases and intracellular Cl- content increases. Thus intracellular pH and pHe appear to be uncoupled. This overall interpretation is confirmed by experiments using carbonic anhydrase and drugs such as propranolol and amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Disequilibrium pH and ammonia transport in isolated perfused cortical collecting ducts

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.

Bicarbonate and ammonia transport in isolated perfused rat proximal straight tubules

Bicarbonate, ammonia, and fluid transport were studied in isolated perfused proximal straight tubules from rats. The mean rate of fluid absorption (0.77 nl X min-1 X mm-1) and the mean rate of total CO2 absorption (42 pmol X min-1 X mm-1) exceeded corresponding rates measured previously in rabbit proximal straight tubules. The limiting total CO2 concentration when the tubules were perfused at slow flow rates was 5 mM, a value similar to those reported previously for rat proximal convoluted tubules and thick ascending limbs. When rat proximal straight tubules were perfused and bathed with solutions containing 1 mM total ammonia at slow perfusion rates, the measured total ammonia concentration in collected fluid rose to a level predicted by the diffusion trapping model of ammonia secretion in the absence of a luminal disequilibrium pH. We conclude the proximal straight tubule of the rat can absorb bicarbonate at a rate that can account for a large portion of the bicarbonate absorption measured in vivo between the late proximal convoluted tubule and the early distal tubule, the rat proximal straight tubule is capable of transepithelial ammonia secretion, most likely by NH3 diffusion down a concentration gradient generated by luminal acidification, and the rat proximal straight tubule apparently does not generate a luminal disequilibrium pH despite the occurrence of proton secretion, implying the presence of endogenous luminal carbonic anhydrase.

Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct

Pages F1148–F1157: Robert A. Star, Maurice B. Burg, and Mark A. Knepper. We regret that an error was made in the title of this article. The correct title and the article are reprinted here.

Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct

We measured bicarbonate, ammonia, and luminal pH in segments of the rabbit outer medullary collecting duct (OMCD) to determine the relationship between luminal pH and ammonia transport. Both the inner-stripe and outer-stripe portions of the OMCD absorbed bicarbonate at high rates. The outer stripe OMCD generated an acidic pH disequilibrium that was reversibly dissipated by exogenous luminal carbonic anhydrase. In contrast, the inner stripe OMCD did not generate a spontaneous pH disequilibrium unless perfused with the carbonic anhydrase inhibitor acetazolamide. Ammonia secretion was three times more rapid in the outer stripe OMCD than in the inner stripe OMCD. We conclude the following. 1) Both the inner-stripe and outer-stripe portions of the rabbit OMCD secrete protons at substantial rates. 2) Functional luminal carbonic anhydrase is present in the inner stripe OMCD but absent from the outer stripe OMCD. 3) Ammonia secretion occurs predominantly by NH3, diffusion in both portions. 4) The luminal pH disequilibrium, which is normally present in the outer stripe OMCD, enhances ammonia secretion. ammonia; ammonium; bicarbonate; diffusion trapping; nonionic diffusion; carbonic anhydrase; disequilibrium pH; fluorescence spectroscopy; hydrogen ion secretion; carbonic acid; acetazolamide; heterogeneity Submitted on September 15, 1986 Accepted on January 15, 1987

Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct

Page F1148: Robert A. Star, Maurice B. Burg, and Mark A. Knepper. “Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct.” We regret that an error was made in the title of this article. The correct title and the article are reprinted in the August 1987 issue of this journal.

Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct [corrected and issued with original paging in Am J Physiol 1987 Aug;253(2 Pt 2)]

We measured bicarbonate, ammonia, and luminal pH in segments of the rabbit outer medullary collecting duct (OMCD) to determine the relationship between luminal pH and ammonia transport. Both the inner-stripe and outer-stripe portions of the OMCD absorbed bicarbonate at high rates. The outer stripe OMCD generated an acidic pH disequilibrium that was reversibly dissipated by exogenous luminal carbonic anhydrase. In contrast, the inner stripe OMCD did not generate a spontaneous pH disequilibrium unless perfused with the carbonic anhydrase inhibitor acetazolamide. Ammonia secretion was three times more rapid in the outer stripe OMCD than in the inner stripe OMCD. We conclude the following. 1) Both the inner-stripe and outer-stripe portions of the rabbit OMCD secrete protons at substantial rates. 2) Functional luminal carbonic anhydrase is present in the inner stripe OMCD but absent from the outer stripe OMCD. 3) Ammonia secretion occurs predominantly by NH3 diffusion in both portions. 4) The luminal pH disequilibrium, which is normally present in the outer stripe OMCD, enhances ammonia secretion.