Pediat. Res. 12: 992-997 (1978) p-Aminohippuric acid organic acid transport developmental physiology proximal tubules kidney Maturation of p-Aminohippuric Acid Transport in the Developing Rabbit Kidney: Interrelationships of the Individual Components BARBARA R. COLE, 1291 J. TREVOR BROCKLEBANK, ROBERT G. CAPPS, BRENDAN. MURRAY, AND ALAN M. ROBSON The Edward Mallinckrodt Department of Pediatrics, Washington University, and the Renal Division, St. Louis Children's Hospital, St. Louis, Missouri, USA Summary and in immature animals ( 11 ), and in vitro studies of intracellular PAH accumulation by proximal renal tubular cells have supported The excretion of both endogenous organic (aryl) acids, such as the in vivo observations (10, 12). benzoic acid, and exogenous ones, such as p-aminohippuric acid Since aryl acids are excreted by the kidney in part by glomerular (PAH), penicillin, and furosemide, is reduced in the human neonate filtration, the decreased glomerular filtration rates in the neonate and other immature animals. The unique developmental pattern of (2) will contribute to the decreased excretion of these acids. aryl acid transport in immature rabbit kidney cortex slices is However, the principal route for excretion of these aryl acids is by produced by the interrelationships of P AH uptake, efflux, and active transport in the proximal renal tubule (22). This involves amount of intracellular binding protein, all of which reach mature active transport across the basolateral membranes into the proxi­ levels at different ages. mal tubular cell, intracellular concentration with protein binding to ligandin (13), and subsequent passive diffusion into the tubular Speculation lumen (22). Thus a reduced renal secretion of aryl acids could result from 1) immature active transport mechanisms; 2) a decrease The clinical relevance of immature mechanisms of aryl organic in ligandin levels; 3) a decrease in the affinity ofligandin for PAH acid secretion by the proximal tubules of the kidney of the human decreasing the intracellular ability to concentrate P AH; 4) a infant will be recognized increasingly in the future. Increasing knowledge of the limited ability of the immature kidney to excrete decreased diffusive capacity across the luminal membrane; 5) an increased permeability of the basolateral membranes permitting exogenous organic acids such as penicillin and furosemide should increased back leak of P AH; or 6) a combination of these changes. aid the clinician in the management of the premature human The present study was undertaken to examine systematically newborn infant. In addition, it will help in understanding syndromes associated with organic acidemia syndromes that present in the the contribution of the individual cellular mechanisms to the young. Finally, it may well be that the cellular dysfunction of reduction in aryl acid excretion in the immature rabbit kidney, uremia will also be determined to be related in large part to the P AH being used as the test aryl acid. The results indicated that the fetal kidney is unable to generate an intracellular P AH con­ high concentrations of aryl acids produced by the kidney's inability centration gradient comparable to that seen in the mature animal to excrete them. due principally to a decrease in the active uptake of P AH. Passive efflux of P AH from fetal kidney cells was decreased, The clinical importance of mechanisms responsible for the helping to maintain definite, although low, intracellular accumu- transport of the aryl organic acids is becoming increasingly ap­ 1-ation of P AH. Ligandin concentrations in the immature kidneys parent. The renal transport system for these acids is responsible were also decreased. Maturation of the several components of not only for the excretion of endogenous aryl acids but also for a P AH accumulation occurred at different ages. The interrelation­ wide variety of exogenous substances including the penicillins, the ships between these maturational rates resulted in the pattern of cephalosporins, furosemide, and ethacrynic acid (3, 23, 25). In­ maturation of intracellular P AH accumulation that has been deed, the observation that a wide variety of organic acids is described previously in the developing rabbit and rat kidneys (9, excreted by the same transport system has been used to clinical 12). advantage with the simultaneous administration of probenicid and penicillin, reducing the excretion of the antibiotic by compet­ itive inhibition and thus prolonging its biologic half-life. More MATERIALS AND METHODS recently, significant elevations in the concentrations of aryl acids have been found in both the serum and cerebrospinal fluid of PAH KINETICS uremic subjects (19). This accumulation may be responsible for some of the altered proximal tubule functions that occur in uremia The renal tubular handling of PAH was studied by three (8) and general organ dysfunction in uremia may result from the techniques using cortical slices prepared from kidneys obtained aryl acids' potential to act as competitive inhibitors of organic from New Zealand White rabbits. Results from fetal animals and anion transport (19). Finally, abnormal urinary excretion of aryl from those 1-10 weeks of age were compared to values obtained acids, most commonly benzoic acid, has been observed in patients from young adults 6 months of age and from pregnant animals with mental retardation (24) and schizophrenia and other psy­ near term. The three methods of study were 1) measurement of choses (17, 18), but the clinical relevance of these observations, if steady state intracellular/extracellular P AH concentration ratios any, has not been defined. to evaluate ability to concentrate PAH at different ages; 2) mea­ A variety of studies has shown that the aryl organic acid surement of rates of PAH uptake to determine the rate of matu­ transport system is incompletely developed in the young. In vivo ration of the active transport mechanisms for the entry of organic the renal extraction of PAH is low in both the human neonate ( 4) acids into cells; and 3) measurement of rates of PAH efflux to 992 p-AMINOHIPPURIC ACID 993 indicate permeability of the cell membranes to PAH at different 60 ages. SLICE/ MEDIUM (S/M) RATIOS • Steady state P AH concentrations were measured by methods 50 Mature Rabbit similar to those published previously (6), and only the variations "'Q 3 3 from this basic method are detailed. To obtain kidneys from fetal >< y = 1.46 X 10 x + 19.4 X 10 animals, pregnant rabbits of 29 days of gestation were killed by a (/) r = 0.98 blow on the head and immediately delivered by cesarean section 0 ...J 40 of their fetuses which were quickly beheaded. Kidneys from these 0 animals, from the pregnant mothers, and from all older rabbits, (/) removed quickly uJ which were killed by a blow on the head, were ::, from the body, decapsulated, quartered, and placed in an iced (/) Ringer's solution with the following composition: NaCl 13 lmM, (/) 30 KCI 10 mM, Na acetate lOmM, phosphate buffer 3mM, CaCb 2.5 I- (!) mM, MgSO4 2 mM. Cortical slices of0.3 to 0.4 mm thickness were then cut using a Stadie-Riggs microtome. 0 Slices from any individual kidney were distributed randomly O 20 among several Erlenmeyer flasks containing 4 ml of the Ringer's I ._ solution to which were added inulin (1300 mg/ dl) and PAH (2 '::E ii. • mg/dl) so that each flask contained approximately 150 mg tissue. u • • In the case of the fetal animals, sufficient tissue could be obtained 10 Fetal Rabbit only by combining cortical slices from both kidneys of each rabbit 3 3 in a single flask. Flasks were incubated at 27° for 60 min in 10% y = 0 .22 X 10 x + 11.3 X 10 oxygen using a Dubnoff shaking metabolic unit. The tissue was r = 0.95 then removed from each flask, blotted on filter paper, weighed, and homogenized in 10% trichloroacetic acid (TCA). Contents of the homogenizer, including washings, were filtered as was the 4 8 12 16 20 24 incubation medium. Tissue and medium filtrates were analyzed TIME OF INCUBATION (MINUTES) for inulin using the anthrone method (27) and for P AH using the Fig. I. Uptake of [14 C]PAH by renal cortical slices from a mature modified Bratton-Marshall reaction (21). In each experiment, at rabbit (e) and fetal rabbit (II). · · · ·, rapid uptake, presumably into the the end of incubation, tissue from representative flasks was blotted, extracellular fluid space; --, slower movement into the intracellular weighed, then oven-dried at 95° for 24 hr and reweighed, the space. difference in weights representing total tissue water. The inulin values were used as an estimate of extracellular fluid (ECF); subtraction of this value from the value for total tissue water constant for PAH uptake was calculated from the slope of this yielded an estimate of the intracellular fluid space (ICF). The line using the formula of Foulkes and Miller (7). amount of P AH calculated to be in the tissue ECF was subtracted from the total tissue P AH content to obtain intracellular PAH PAH RUNOUT content. The intracellular/ extracellular PAH concentration ratio Measurements of rates of efflux of PAH from cortical slices pre­ was calculated by dividing intracellular P AH concentration (mil­ incubated in a Ringer's solution containing [14C]PAH were per­ ligrams per dl ICF) by the concentration of P AH in the incubation formed using the method of Welch and Bush (26). The only medium, also expressed as milligrams per dl. For simplicity this variation from this method was the measurement of efflux at 30- ratio is termed the S/M ratio. The four to eight results from any sec intervals for the first 6 min, and subsequently at 1-min that individual animal were averaged to obtain a final value for intervals, compared to the I-min intervals employed by Welch animal.