Acidification in proximal tubule of the isolated rat kidney, perfused in vitro, was studied by stopped-flow microperfusion techniques, using Sb microelectrodes to measure luminal pH. The kidney was perfused with mammalian Ringer's solution at pH 7.4 buffered by 20 mmol/l phosphate and containing 7.5 g/100 ml bovine albumin, equilibrated with air. Final urine pH was 6.88 +/- 0.5. Steady-state pH in proximal segments was 6.81 +/- 0.03 (n = 80), and acidification half-time (t/2) 7.25 +/- 0.33 (80) s, giving a net secretory H+ ion flux of 0.51 +/- 0.05 nmol . cm-2 . s-1. This flux was about 70% of "in vivo" (blood perfused kidneys). During luminal perfusion with solutions at pH 6.2, back-flux of H+ was 0.82 +/- 0.08 nmol . cm-2 . s-1, with an alkalinization t/2 of 6.33 +/- 0.34 (34) s. The difference between acidification and alkalization t/2 was not significant. This is compatible with a pump-leak system of H+ transport. This is compatible with a pump-leak system of H+ transport. The back flux of H from the lumen was markedly reduced in low Na+ perfused kidneys in the presence of 10(-4) mol/l amiloride in the lumen, indicating that this process is mediated by the luminal Na/H exchanger. Observations in the presence of high K levels suggest that it may have also a charged component. 10(-4) mol/l acetazolamide added to the kidney perfusate reduced acidification to 0.5% of control, and 10(-6) mol/l SITS to 25% of control. Thus, despite the low pCO2 (0.1-0.4 kPa, or 1-3 mm Hg), the CO2/HCO-3 buffer system still plays an important role in tubular acidification in this preparation.
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The effect of temperature on proximal tubular acidification was studied in isolated rat kidney, perfused with 20 mM phosphate Ringer's containing 7.5 g/100 ml bovine albumin, equilibrated with air. Tubular pH was measured with Sb microelectrodes during stopped-flow microperfusion. The temperature of the kidney was varied between 10 and 46 degrees C. At 10 degrees C the proximal tubule was still able to maintain pH gradients of about 0.7 pH units. However, half-times (t/2) of both acidification and alkalinization were markedly increased, from 6-7 s at 37 degrees C to 27-30 s at 10 degrees C. In consequence, net H+-ion flux into the tubule was reduced to 26% of that at 37 degrees C. In this system, in the absence of exogenous HCO-3 and CO2, t/2 of acidification and alkalinization were very similar at 37 degrees C and below. Above 37 degrees C alkalinization t/2 fell markedly to 1.43 +/- 0.09 (11) s at 46 degrees C, while acidification t/2 stayed at about 7 s. H+-ion back-fluxes increased progressively from 10-46 degrees C, while secretory JH reached a maximal value at 37 degrees C and fell at higher temperatures. Apparent activation energies calculated from rate coefficients were 8.48 kcal . mol-1 for acidification, and 9.30 for alkalinization, and those calculated from JH were 6.30 and 9.55 respectively. These data indicate that both H-ion secretion and back-flux are carrier-mediated, probably flowing through the Na/H exchanger in the luminal membrane, since their activation energies are of the same order of magnitude and markedly higher than those for protons in solution.
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