Proline uptake by rat renal brush border membrane vesicles from animals 7 days of age and older has been examined to delineate developmental changes in membrane function that may underlie the physiological hyperprolinuria of young animals. Although the two proline transport systems normally present in adult membranes were found in membranes from young animals, the proline "overshoot" resulting from a sodium ion gradient is minimal and increases with age of the animal from which the membranes were isolated. This is associated with a severalfold faster entry of 22Na into vesicles of the 7-day-old animal compared to entry into membranes prepared from adult kidneys. The very rapid dissipation of the sodium gradient thus diminishing the driving force for transmembrane proline movement may explain the changes in proline overshoot observed in membranes from young animals. The altered sodium permeability is consistent with the fact that young animals have a generalized inability to reabsorb other amino acids whose transport is known to be sodium gradient stimulated.Urinary hyperexcretion of proline and indeed of most amino acids is characteristic ofneonatal rat (1), dog (2), and man (3). Subsequent postnatal development of the nephron, however, reduces urinary amounts of these acids to much lower levels (4, 5). Although the subject of numerous investigations (6-9), the nature of the developmental changes occurring in the kidney that result in decreased amino acid excretion is as yet undefined. An understanding of these changes should not only increase our knowledge of normal transport processes but also provide insight into human disorders of renal amino acid transport, such as familial iminoglycinuria, in which the affected individual continues to excrete excessive quantities of certain amino acids in the urine.Previous studies of the development of renal amino acid transport in rat have relied upon the use of the cortical slice (10, 11) and isolated renal tubule fragment (1,8). Data obtained from these models, however, are influenced by factors such as bidirectional solute movement, substrate penetration, and substrate metabolism. The use of isolated membrane vesicles obviates these problems and age-related differences in solute transport by these vesicles may be examined directly.In our recent studies, proline uptake by renal brush border membrane vesicles prepared from newborn rats by free-flow electrophoresis, unlike vesicles from adult animals, did not show a Na+-gradient-stimulated overshoot but did exhibit a Na+-gradient-enhanced rate of early proline entry (12). To better understand the events accompanying the maturation of renal solute transport we have therefore examined characteristics of proline and Na+ uptake by renal brush border membrane vesicles prepared from rats of various ages ranging from 1 week oflife to adulthood. The results indicate that, although the two proline transport systems normally present in adult membranes are also found in membranes from young animals, the proline overshoot re...

A method for the isolation of brush-border membranes from newborn-rat kidney, employing centrifugation and free-flow electrophoresis, is described. The composition and purity of the preparation was assessed by determination of enzyme activities specific for various cellular membranes. Free-flow electrophoresis resolves the newborn-rat renal membrane suspension into two populations of alkaline phosphatase-enriched brush-border membranes, designated 'A' and 'B', with the A peak also showing activity of (Na+ + K+)-stimulated ATPase, the basolateral membrane marker enzyme, whereas those of the B peak were enriched 11-fold in alkaline phosphatase and substantially decreased in (Na+ + K+)-stimulated ATPase activity. Membranes in the A peak showed a 7-fold enrichment of alkaline phosphatase, and (Na+ + K+)-stimulated ATPase activity similar to that of the original homogenate. Proline uptake employed to assess osmotic dependency revealed 7% binding of proline to the B vesicles and 31% to the A vesicles. This contrasts with 60% proline binding to vesicles prepared by centrifugation alone. Unlike vesicles from adult animals, proline uptake by B vesicles did not show an Na+-stimulated overshoot, but did exhibit an Na+-gradient enhanced rate of early proline entry. proline entry.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.

The role of the enzyme, gamma-glutamyl transpeptidase on the uptake of amino acids by the brushborder membrane of the rat proximal tubule was examined by inhibiting it with AT-125 (L-[alpha S, 5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). AT-125 inhibited 98% of the activity of gamma-glutamyl transpeptidase when incubated for 20 min at 37 degrees C with rat brushborder membrane vesicles. AT-125 given to rats in vivo inhibited 90% of the activity of gamma-glutamyl transpeptidase in subsequently isolated brushborder membrane vesicles from these animals. AT-125 inhibition of gamma-glutamyl transpeptidase both in vivo and in vitro had no effect on the brushborder membrane uptake of cystine. Similarly, there was no effect of gamma-glutamyl transpeptidase inhibition by AT-125 on glutamine, proline, glycine, methionine, leucine or lysine uptake by brushborder membrane vesicles. Furthermore, the uptake of cystine by isolated rat renal cortical tubule fragments, in which the complete gamma-glutamyl cycle is present, was unaffected by AT-125 inhibition of gamma-glutamyl transpeptidase. Therefore, in the two model systems studied, gamma-glutamyl transpeptidase did not appear to play a role in the transport of amino acids by the renal brushborder membrane.

Summary a-Methyl-o-glucoside (AMG) uptake was examined in isolated renal cortical tubules from newborn, 3-month-old, and adult dogs. All three age groups demonstrated active sugar transport. The initial rate of AMG uptake was similar in the 3-month-old and . adult tubules which was twice that of the newborn. At steadystate, the adult and newborn tubules had achieved a similar intracellular AMG concentration which was 45% greater than that of the 3-month-old. Determination of the flux constants of these uptake patterns revealed that there was an age-dependent increase in both the net flux and the fractional influx rate constant. However, the 3-month-old had the highest fractional efflux rate constant and the newborn the lowest value with the adult in between. Kinetic analysis of AMG uptake showed a single saturable transport system for each age group. The newborn and adult had similar K m values but the 3-month-old had a value that was 60% higher. The 3-month-old tubules had the highest Vmax and the newborn tubules the lowest with the adult value in between. AMG uptake by tubules from each age group demonstrated a similar pattern of inhibition in a low sodium buffer and by glucose and phlorizin. This indicated that, aside from kinetic changes with maturation, the saturable transport system for AMG is similar in each age group. AbbreviationsAMG, a-methyl-o-glucoside KRB, Krebs-Ringer bicarbonate buffer DR, distribution ratio ICF, intracellular fluidThe developing kidney is characterized by a functional immaturity of both the glomerulus and tubule (35). One of the manifestations of this tubular immaturity is diminished maximal tubular reabsorption of glucose in the human (3, 6), dog (2), and sheep neonate (1). However, these studies are whole kidney clearance measurements which are influenced by numerous variables such as the state of extracellular volume, glomerular filtration rate, and the fractional excretion of sodium. Therefore, the intrinsic ability of the proximal tubule from these developing animals and the human neonate to reabsorb solute can only be approximated. Further, the mechanism of sugar transport at the level of the brush border membrane can only be inferred from such studies and not addressed directly .The use of isolated renal cortical tubule fragments allows the study of sugar transport independent of the glomerular filtration rate, sodium excretion, or extracellular fluid volume. AMG is a nonmetabolizable model for the glucose-galactose transport systern (31) which Silverman (32) has shown is transported exclusively by the brush border membrane, making it an ideal substrate for evaluating luminal sugar transport in the developing animal.A previous report of AMG transport by isolated renal cortical tubules from the newborn rat demonstrated two kinetically distinct transport systems with only the low affinity system present in the adult rat (26). However, initial uptake and steady-state values of AMG were lower in the immature rat in spite of the two systems. Since the loss of a transport system...