Direct fluorescence measurement of diffusional water permeability in the vasopressin-sensitive kidney collecting tubule

Abstract

A fluorescence method has been developed for accurate and instantaneous measurement of transepithelial diffusional water permeability (Pd) in perfused kidney tubules based on the sensitivity of the fluorophore aminonapthelane trisulfonic acid (ANTS) to solution H2O/D2O content. The fluorescence of ANTS was 3.2-fold lower in an H2O buffer than in a D2O buffer. The response of ANTS fluorescence to a change in solution H2O/D2O content occurred in less than 1 ms and was due to a collisional quenching mechanism. Isolated cortical (CCT) and outer medullary (OMCT) collecting tubules from rabbit were perfused with an isosmotic D2O buffer at specified lumen flow rates (2-100 nl/min); tubules were bathed in isosmotic H2O or D2O buffers in which vasopressin (VP) could be added rapidly. Lumen fluorescence was monitored by quantitative epifluorescence microscopy at 380 +/- 5 nm excitation and greater than 530 emission wavelengths. Pd was determined from tubule geometry, lumen flow, ANTS fluorescence, and ANTS fluorescence vs. H2O/D2O calibration relation. The instrument response time for a change in bath H2O/D2O content was less than 4 s. At 37 degrees C, Pd values (mean +/- SE in cm/s x 10(4] were 6.4 +/- 1.0 (-VP, n = 9) and 14.3 +/- 1.1 (+250 microU/ml bath VP, n = 9) in the CCT, and 5.8 +/- 1.0 (-VP, n = 6) and 15.3 +/- 2.0 (+VP, n = 6) in the OMCT; at 23 degrees C, Pd was 5.1 +/- 0.6 (-VP, n = 4) and 7.8 +/- 0.6 (+VP, n = 4) in the CCT. In response to rapid addition of 250 micro U/ml vasopressin to the bath, CCT Pd remained unchanged for 71 +/- l0s (n = 9, 37 degree C) and 170 +/- 45 s (n = 4, 23 degree C); this was followed by a slow increase in Pd(TI/2 = 91 +/- 17 s, 37 degree C; 119 +/- 31 s, 23 degree C) to the new steady-state value. These results provide a new approach for study of transepithelial water transport in kidney tubules. Compared with 3H20 methods, the fluorescence method is superior in technical simplicity, time resolution, and accuracy. The improved time resolution is important for examination of the pre-steady-state kinetics of vasopressin-induced signalling events resulting in the hydroosmotic response.