Maturational changes in rabbit renal brush border membrane vesicle urea permeability

Abstract

Urea transport in the proximal tubule is thought to occur by passive diffusion through the lipid bilayers of the cell membranes. The lipid composition of cell membranes changes during maturation and may directly affect urea permeability of proximal tubule membranes. The present study examined the maturation of urea transport in rabbit renal brush border membrane vesicles (BBMV). BBMV from adult and neonatal (9- to 11-d-old) New Zealand white rabbits were loaded with 500 mM urea and mixed with an iso-osmotic mannitol solution using a stop-flow instrument. Vesicle shrinkage, due to efflux of urea, was followed with light scattering and urea permeability was calculated from an exponential fit of the data. Urea permeability was significantly lower in the neonatal BBMV than the adult at 25 degrees C (0.34+/-0.04 x 10(-6) versus 0.56+/-0.03 x 10(-6) cm/sec;p < 0.001, n=7) and 37 degrees C (0.45+/-0.04 x 10(-6) versus 0.66+/-0.03 x 10(-6) cm/sec; p=0.001, n=7). There was no effect of 250 microM phloretin on urea permeability in either adult or neonatal BBMV at either temperature. The activation energy for urea diffusion was higher in the neonatal than the adult BBMV. Because the maturational increase in urea permeability could potentially be due to a sodium-dependent urea transporter in the adult BBMV, the sodium dependence of urea uptake in adult BBMV was examined. There was no difference in urea permeability in the presence or absence of 20 mM NaCl. Permeability of the lipid-soluble molecule, glycerol, was also found to be the same in the neonatal and adult BBMV. Urea transport in the apical membrane of neonatal and adult proximal tubules is not phloretin sensitive, a finding consistent with diffusion of urea via the lipid bilayer. The rate of urea diffusion is lower in neonatal membranes and may be an important factor in overall urea excretion. This may also play a role in developing and maintaining a high medullary urea concentration and thus the ability to concentrate the urine during renal maturation.

Figure 1

Representative tracings from experiments with adult BBMV (top curve) and neonatal BBMV (bottom curve). The data were fit with an exponential curve and P_urea calculated from the rate constant.

Figure 2

P_urea for neonatal and adult BBMV at 25° and 37°C. At both temperatures, the adult BBMV P_urea is higher than the neonatal BBMV. * p < 0.001 vs. adult at 25°C. ** p < 0.001 vs. neonatal at 25°C and adult at 37°C. † p < 0.001 vs. adult at 25°C.

Figure 3

Effect of phloretin on P_urea in neonatal BBMV. At both 25° and 37°C, phloretin (250 µM) had no effect on urea transport in the neonatal BBMV. Both control and phloretin-treated BBMV had a higher P_urea at 37°C than 25°C. * p < 0.001 vs. control at 25°C. ** p < 0.001 vs. phloretin at 25°C; p = NS vs. control at 37°C.

Figure 4

Effect of phloretin on P_urea in adult BBMV. At both 25° and 37°C, phloretin (250 µM) had no effect on adult BBMV urea transport. P_urea was higher in both control and phloretin-treated adult BBMV at 37°C than at 25°C. *p < 0.001 vs. control at 25°C. ** p < 0.001 vs. phloretin at 25°C; p = NS vs. control at 37°C.

Figure 5

Effect of sodium on urea uptake in adult BBMV. Addition of 20 mM NaCl to the extravesicular solution had no effect on urea permeability. p = NS.

Figure 6

Glycerol permeability in neonatal and adult BBMV. No difference was found in neonatal and adult BBMV glycerol permeability. p = NS.