Evidence for water channels in renal proximal tubule cell membranes

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Abstract
Water transport mechanisms in rabbit proximal convoluted cell membranes were examined by measurement of: (1) osmotic (P f ) and diffusional (P d ) water permeabilities, (2) inhibition ofP f by mercurials, and (3) activation energies (E a ) forP f .P f was measured in PCT brush border (BBMV) and basolateral membrane (BLMV) vesicles, and in viable PCT cells by stopped-flow light scattering;P d was measured in PCT cells by proton NMR Ti relaxation times using Mn as a paramagnetic quencher. In BLMV,P f (0.019 cm/sec, 23°C) was inhibited 65% by 5mm pCMBS and 75% by 300 μm HgCl2 (K l =42 μm);E a increased from 3.6 to 7.6 kcal/mole (15–40°C) with 300 μm HgCl2. In BBMV,P f (0.073 cm/sec, 23°C,E a =2.8 kcal/mole, 33°C) was inhibited 65% with HgCl2 withE a =9.4 kcal/mole (15–45°C). Mercurial inhibition in BLMV and BBMV was reversed with 10 μm 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,P f was 0.010±0.002 cm/sec (37°C) andP d was 0.0032 cm/sec.P f was independent of osmotic gradient size (25–1000 mOsm) withE a 2.5 kcal/mole (27°C). CellP f was inhibited 53% by 300 μm HgCl2 (23°C) withE a 6.2 kcal/mole. These findings indicate that cellP f is not restricted by extracellular or cytoplasmic unstirred layers and that cellP f is not flow-dependent. The high BLMV and BBMVP f , inhibition by HgCl2, lowE a which increases with inhibition, and the measuredP f /P d >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.