Real-time measurement of transepithelial secretion of fluorescein in isolated, perfused rabbit proximal tubules

To measure the transepithelial secretion of the organic anion fluorescein (FL) in real time, we developed a tubule perfusion setup designed to monitor simultaneously the fluorescence of FL and a volume marker (tetramethylrhodamine-dextran, TMRD) in the collection pipette. During perfusion experiments, FL is added to the bath and TMRD is added to the perfusate. Both fluorescent dyes are measured continuously by focusing excitation light (490 ± 10 nm) into the middle of a modified collection pipette positioned at the bottom of a custom chamber on the stage of an epifluorescence microscope. The fluorescent emission is first separated by a dichroic mirror (DRLP 540), and each beam is appropriately filtered (520 ± 10 nm for Fl; 580 ± 30 nm for TMRD) and counted by a separate photomultiplier in photon-counting mode. In initial studies, rabbit renal S2 proximal tubules were perfused at 10-15 nl/min in a bathing medium continuously exchanged at ∼ 3 ml/min at 37°C. TMRD was present continuously in the perfusate and FL was added to the bath to study its transepithelial secretion. The design of the collection pipette reduced background fluorescence from the bath sufficiently to permit a simple correction. With 500 nM FL in bath, the collected perfusate-to-bath FL concentration ratio was ∼4 and the rate of net FL secretion was ∼24 fmol/min/mm. Addition of 1 mM PAH to the bath inhibited the rate of FL secretion by about 90%, confirming that observed FL transport was via the organic anion system. The results demonstrate that this system can be used to study transepithelial organic anion secretion in real-time in isolated. perfused renal tubules.