Diffusional water permeability (PDW) of adult and neonatal rabbit renal brush border membrane vesicles

Abstract

We have shown that there is a maturational increase in osmotic water permeability (Pf) of rabbit renal brush border membrane vesicles (BBMV). The purpose of the present study was to further investigate the changes in proximal tubule water transport that occur during postnatal development. Diffusional water permeability (PDW) has not been measured directly in adult or neonatal BBMV. We validated the method described by Ye and Verkman (Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes. Biochemistry 28:824-829, 1989) to measure PDW in red cell ghosts and liposomes, to examine the maturational changes in PDW in BBMV. This method utilizes the sensitivity of 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS) fluorescence to the D2O-H2O content of the solvent. ANTS-loaded neonatal (11 days old) and adult BBMV were rapidly mixed with two volumes of isoosmotic D2O solution using a stopped-flow apparatus at 5 degrees -37 degrees C. PDW was lower in neonatal than adult BBMV at 5 degrees (3.77 +/- 0.34 vs. 5.35 +/- 0.43 mm/sec, respectively, p<0.05) and 20 degrees C (7.03 +/- 0.40 vs. 9.04 +/- 0.25 mm/sec, respectively, p<0.001), but was not different at 30 degrees and 37 degrees C. The activation energy (Ea) was higher in neonatal than in adult BBMV (9.29 +/- 0.56 kcal/mol vs. 6.46 +/- 0.56 kcal/mol, p<0.001). In adult BBMV, PDW was inhibited by 0.5 mM HgCl2 by 46.6 +/- 3.6%, while it was not affected in neonatal BBMV (p<0.001). The results indicate that PDW can be measured in rabbit renal BBMV. There are significant changes in water transport across the apical membrane during postnatal development, consistent with a maturational increase in channel-mediated water transport.

Fig. 1

Comparison of fluorescence signals from intact, ANTS-loaded adult BBMV and ANTS solution (10 mm) mixed with two volumes of iso-osmotic D₂O-resuspension buffer at 5°, 20° and 37°C. ANTS fluorescence increased as the D₂O-H₂O ratio of its solvent composition increased. Tracings were normalized to initial fluorescence and fitted to a single-exponential curve. For ANTS solution, the signal was time-independent at all temperatures. For BBMV, fluorescence was not time-independent and the rate of change increased at higher temperatures. This shows that the rate of mixing was sufficiently rapid to measure P_DW in BBMV.

Fig. 2

Typical tracings (averaged raw tracing and single-exponential fit) for adult (a) and neonatal (b) BBMV at 20°C. Curves were normalized to initial fluorescence. The bottom curves were obtained after incubation with 0.5 mm HgCl₂ at room temperature for ∼20 min. HgCl₂ reduced the initial slope by 46.6 ± 3.6% in adult BBMV, but had no effect on neonatal BBMV.

Fig. 3

Temperature dependence of P_DW of adult and neonatal BBMV. (a) P_DW of neonatal BBMV was lower at 5° and 20° C. (b) Arrhenius plot. E_a was calculated from the slope of the regression lines. E_a was 6.46 ± 0.56 kcal/mol in adult BBMV and 9.29 ± 0.56 kcal/mol in neonatal BBMV (p<0.001). * p < 0.02, ** p < 0.001 for both a and b.

Fig. 4

Mercury sensitivity of P_DW in adult and neonatal BBMV at 20°C. BBMV were incubated with 0.5 mm HgCl₂ at room temperature for ∼20 min before the experiment. The percentage inhibition of P_DW was calculated from the decrease in initial slope of the fitting single-exponential curve. * p < 0.001 vs. adult BBMV.