Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules

To examine directly in real time the efflux of organic compounds [e.g., org anic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approa ch utilizing a recently developed epifluorescence microscopy system for con tinuous monitoring of fluorescence in the collected perfusate. To illustrat e this approach, we measured the luminal efflux rate of FL in mineral oil-c overed, isolated, perfused S2 segments of rabbit renal proximal tubules. Th e washout profile of FL showed a deviation from linearity at time 0 when pl otted on a semilog scale, indicating that the luminal efflux of FL was a sa turable process. We were able for the first time to determine the kinetic p arameters of luminal efflux [FL concentration at one-half maximal FL efflux (K-t(lumen)) of similar to 560 muM and maximal rate of FL efflux across th e luminal membrane (J(max)(lumen))of similar to 635 fmol . min(-1). mm(-1)] . From the present study, we conclude that the transport step for OAs acros s the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organi c compound that is sufficiently fluorescent to be measured with commonly av ailable, highly sensitive optical equipment.