A stop-flow microperfusion technique for rapid determination of HCO₃ ^– absorption/H⁺ secretion by isolated renal tubules

Abstract.

In the present experiments on microdissected tubules of rabbit kidney we present a refined stop-flow method for determining the rate of HCO₃ ^– absorption (J _HCO3) or H⁺ secretion (J _H) that can be applied to isolated microperfused tubules. Using the pH-sensitive indicator dye BCECF (2',7'-bis [2-carboxyethyl]-5[6]-carboxyfluorescein) the luminal perfusate pH is continuously measured with a microspectrofluorometric set-up, and the pH change following a sudden stop of perfusion is analysed. Because the tubules partially collapse after stop-flow the calculation of fluxes requires a correction for volume loss. This is achieved by referring all fluxes to the remaining luminal volume, which can be estimated from the decay of the 440 nm reference fluorescence. During perfusion of the lumen with pure HCO₃ ^– Ringer solution, and of the bath with the same solution but containing 5.5 mmol/l d-glucose as metabolic substrate, J _HCO3 averaged 4.4±0.2 pmol cm^–1·s^–1 (n=40) and 13.4±0.8 pmol·cm^–1·s^–1 (n=5) in proximal straight tubules (PST) and in proximal convoluted tubules respectively. These values agree very well with data obtained in other laboratories with the picapnotherm technique. The present method has the advantage of requiring fewer micromanipulations and a shorter measuring time, thus allowing regulatory changes in J _HCO3 to be analysed. Moreover it does not involve measurements of radioactivity, and it also allows J _H to be measured in HCO₃ ^–-free solutions which in PST averaged 0.9 pmol·cm^–1·s^–1 (n=8) in the present experiments.