Maturational changes in rabbit renal brush border membrane vesicle osmotic water permeability

Abstract

We have recently shown that the osmotic water permeability (Pf) of proximal tubules from neonatal rabbits is higher than that of adults (AJP 271:F871-F876, 1996). The developmental change in Pf could be due to differences in one or more of the components in the path for transepithelial water transport. The present study examined developmental changes in water transport characteristics of the proximal tubule apical membrane by determining Pf and aquaporin 1 (AQP1) expression in neonatal (10-14 days old) and adult rabbit renal brush border membrane vesicles (BBMV). AQP1 abundance in the adult BBMV was higher than the neonatal BBMV. At 25 degrees C the Pf of neonatal BBMV was found to be significantly lower than the adult BBMV at osmotic gradients from 50 to 250 mOsm/kg water. The activation energy for osmotic water movement was higher in the neonatal BBMV than the adult BBMV (9.19 +/- 0.37 vs. 5.09 +/- 0.57 kcal . deg-1 . mol-1, P < 0.005). Osmotic water movement in neonatal BBMV was inhibited 17.9 +/- 1.3% by 1 mm HgCl2 compared to 34.3 +/- 3.8% in the adult BBMV (P < 0.005). These data are consistent with a significantly greater fraction of water traversing the apical membrane lipid bilayer in proximal tubules of neonates than adults. The lower Pf of the neonatal BBMV indicates that the apical membrane is not responsible for the higher transepithelial Pf in the neonatal proximal tubule.

Fig. 1

Immunoblot of AQP1 abundance in neonatal and adult BBMV. Each lane was loaded with 20 µg of total protein and probed with anti-AQP1 antibody. Densitometry demonstrated a higher abundance in the adult BBMV than the neonate.

Fig. 2

Relative fluorescence of neonatal (upper curve) and adult (lower curve) BBMV. Vesicles were loaded with 10 mm fluorescein-sulfonate in resuspension buffer (80 mOsm/kg water) and exposed to an inwardly directed osmotic gradient (250 mOsm/kg water). Vesicle shrinkage results in fluorescence quenching. Fluorescence curves were fit with a single exponential.

Fig. 3

(A) The asymptote of the exponential fit was plotted against the ratio of the solution osmalities. The linear fit suggested an inverse relation between fluorescence and relative size. (B) The asymptotes from the exponential fits are shown plotted against the relative size (assuming the vesicles to be perfect osmometers). The open circles represent the actual data and the line shows the fit equation.

Fig. 4

These curves show the same data from Fig. 2 after the relative fluorescence data has been transformed to relative size data using the transformation in Fig. 3b. The upper curve is the neonatal BBMV and the lower curve is the adult BBMV.

Fig. 5

P_f of adult and neonatal BBMV. (A) P_f was determined from a double exponential curve. (B) These values were obtained from fitting the relative size curves to second order kinetics. At all osmotic gradients tested, the P_f of neonatal BBMV was significantly lower than adult BBMV.

Fig. 6

Arrhenius plots of temperature dependence of rate constants from the raw fluorescence data. Vesicles were loaded with 10 mm fluorescein-sulfonate in resuspension buffer (80 mOsm/kg water) and exposed to an inwardly directed osmotic gradient (150 mOsm/kg water). Temperature was varied from 17 to 39°C. Activation energy, calculated from the slopes of the plots, was 9.19 ± 0.37 kcal • deg⁻¹• mol⁻¹ for the neonatal BBMV (open circles) and 5.09 ± 0.57 kcal • deg⁻¹• mol⁻¹ for the adult BBMV (closed circles) (P < 0.005).

Fig. 7

Effect of 1 mm HgCl₂ on osmotic water permeability was determined using light scattering for neonatal (B) and adult (A) BBMV. The lower curves in each panel are after treatment with 1 mm HgCl₂.