Evidence for water channels in renal proximal tubule cell membranes

Abstract

Water transport mechanisms in rabbit proximal convoluted cell membranes were examined by measurement of: osmotic (Pf) and diffusional (Pd) water permeabilities, inhibition of Pf by mercurials, and activation energies (Ea) for Pf. Pf was measured in PCT brush border (BBMV) and basolateral membrane (BLMV) vesicles, and in viable PCT cells by stopped-flow light scattering; Pd was measured in PCT cells by proton NMR T1 relaxation times using Mn as a paramagnetic quencher. In BLMV, Pf (0.019 cm/sec, 23 degrees C) was inhibited 65% by 5 mM pCMBS and 75% by 300 microM HgCl2 (KI = 42 microM); Ea increased from 3.6 to 7.6 kcal/mole (15-40 degrees C) with 300 microM HgCl2. In BBMV, Pf (0.073 cm/sec, 23 degrees C, Ea = 2.8 kcal/mole, less than 33 degrees C and 13.7 kcal/mole, greater than 33 degrees C) was inhibited 65% with HgCl2 with Ea = 9.4 kcal/mole (15-45 degrees C). Mercurial inhibition in BLMV and BBMV was reversed with 10 microM mercaptoethanol. Viable PCT cells were isolated from renal cortex by Dounce homogenization and differential seiving. Impedence sizing studies show that PCT cells are perfect osmometers (100-1000 mOsm). Assuming a cell surface-to-volume ratio of 25,000 cm-1, Pf was 0.010 +/- 0.002 cm/sec (37 degrees C) and Pd was 0.0032 cm/sec. Pf was independent of osmotic gradient size (25-1000 mOsm) with Ea 2.5 kcal/mole (less than 27 degrees C) and 12.7 kcal/mole (greater than 27 degrees C). Cell Pf was inhibited 53% by 300 microM HgCl2 (23 degrees C) with Ea 6.2 kcal/mole. These findings indicate that cell Pf is not restricted by extracellular or cytoplasmic unstirred layers and that cell Pf is not flow-dependent. The high BLMV and BBMV Pf, inhibition by HgCl2, low Ea which increases with inhibition, and the measured Pf/Pd greater than 1 in cells in the absence of unstirred layers provide strong evidence for the existence of water channels in proximal tubule brush border and basolateral membranes. These channels are similar to those found in erythrocytes and are likely required for rapid PCT transcellular water flow.