Mammalian Kidney* Ontogeny of Iminoglycine Transport in

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Proceedings of the National Academy of Sciences Vol. 65, No. 4, pp. 1009-1016, April 1970 Ontogeny of Iminoglycine Transport in Mammalian Kidney* Kurt E. Baerlocher,t Charles R.

1 Proceedings of the National Academy of Sciences Vol. 65, No. 4, pp , April 1970 Ontogeny of Iminoglycine Transport in Mammalian Kidney* Kurt E. Baerlocher,t Charles R. Scriver,4 and Fazl Mohyuddin DEPARTMENTS OF PEDIATRICS AND GENETICS, MCGILL UNIVERSITY-MONTREAL CHILDREN' S HOSPITAL RESEARCH INSTITUTE, MONTREAL, QUEBEC, CANADA Communicated by C. N. H. Long, December 15, 1969 Abstract. Renal tubular absorption of proline, hydroxyproline, and glycine by the newborn of most mammals is inefficient compared to that of the adult. Cortex slices from seven-day-old rat kidney also transport proline and glycine at reduced initial rates compared to mature kidney. Nonetheless, newborn slices achieve higher intracellular concentrations during prolonged incubation; the latter reflects a reduced rate of efflux, a characteristic peculiar to the membrane of postnatal kidney. The postnatal reduction of initial uptake rates is observed clearly only at substrate concentrations in or below the physiological range; it correlates with the absence of two high-affinity systems which normally serve proline and glycine transport independently at these concentrations in mature kidney, in conjunction with a "common" low-affinity system. The low-affinity system alone performs the observed uptake in the newborn kidney. Specific activity of the high-affinity systems for proline and glycine increases asynchronously after birth, suggesting independent genetic control of the three systems for iminoglycine transport in mammalian kidney. Membrane transport of amino acids in mammalian kidney is achieved by several systems' and a number of Mendelian traits are known to affect the function of these membrane "carriers."2' 3 Such observations are compatible with the current view that the migration of particular amino acids and other solutes across intact cell membranes is mediated by specific transport proteins,4 under the control of an array of independent genes. Studies on the entry of proline, hydroxyproline, and glycine into mammalian kidney cells5'-0 reveal that their uptake at physiological concentrations occurs predominantly on a common system characterized by its high capacity, low affinity (high Km), and preference for the three substrates, and on two "specific" systems, one for glycine, the other for the imino acids, both having low total capacity and high affinity (low Km) for their respective substrates. Depressed uptake of imino acids and glycine from intratubular urine, with subsequent exaggerated excretion in urine, is characteristic of postnatal renal function in man, whereas the adult human reabsorbs imino acids and glycine with great efficiency. This report presents evidence that the developmental aspects of imino-glycine transport in postnatal rat kidney, which mimic the human phenomenon, reflect an initial deficiency of the independent low-km membrane sites, and that this is overcome by a subsequent increase in their specific activities during maturation. 1009

2 1010 BIOCHEMISTRY: BAERLOCHER ET AL. PROC. N. A. S. Methods. Nonpregnant adult female Long-Evans rats weighing gm and fed freely on water and Purina rat chow, and suckling pups of both sexes, 7, 14, or 21 days old, were used in these investigations. Samples of urine and plasma were obtained from six or more animals as described previously,5 and their amino acid content was measured on a modified Beckman-Spinco amino acid analyzer." Animals were killed by decapitation and cortex slices weighing about 10 mg were obtained from mature kidneys with a chilled Stadie-Riggs microtome; slices weighing 3-7 mg were also obtained with the microtome from the kidneys of pups, avoiding inclusion of medulla and papilla. The technique of incubation, the estimation of tissue fluid spaces, and the counting of 14Clabeled substances in the intracellular and extracellular spaces, and in 14CO2 released from tissue during incubation, have been described previously.7' We used the method of Segal's group13 to determine efflux of labeled substrate from preincubated slices. The data for cellular uptake of amino acids were evaluated by the formulations described previously.'2 Net uptake velocities in kinetic studies were corrected for the nonsaturable component; distribution ratio refers to counts per milliliter ICF per unit time: counts per milliliter initial extracellular medium, unless stated otherwise. Materials. Glycine-u-14C (spec. act. 120 mci per mmole); L-proline-u-'4C (spec. act. 180 mci per mmole); a-aminoisobutyric acid-1-14c (spec. act mci per mmole) and inulin-carboxyl-'4c (2.47 mci per gram) were obtained from New England Nuclear Corporation; their radiochemical purity was reconfirmed by chromatographic or electrophoretic methods. Unlabeled amino acids were obtained from Mann Chemical Corporation. Results. Urinary excretion of imino acids and glycine: Very young rat pups excrete large amounts of proline, hydroxyproline, and glycine in their urine compared to older pups and mature animals (Table 1). Excretion of the iinino TABLE 1. Urinary excretion of imino acids and glycine in relation to their plasma concentration in the rat.* Urine Plasma,-,uMoles/gm Total Nitrogen-- -J umoles/liter Amino acid 7d 14d 21d Adultt 7d 14d 21d Adultt Hydroxyproline < Proline Glycine * Analyses were performed on samples pooled from six animals in each age group. Adult rats were fed on Purina chow; young rats were unweaned. t Values are the range, and include data of Wilson and Scriver.5 acids diminishes sharply in the second week of life; glycine excretion does not abate until the third week. This phenomenon is not the result of variation in the plasma concentrations of the respective amino acids. The postnatal hyperiminoglycinuria of rat kidney thus apparently reflects a transient impairment of net tubular absorption of these amino acids, as it does in man.'4 Investigation of renal transport of amino acids was then undertaken in vitro using the kidney slice technique. Tissue water spaces: The tissue fluid spaces in postnatal and mature kidney were calculated before proceeding with in vitro investigations. Total tissue water in the kidney of 7-day, 14-day, 21-day, and adult animals is 78.8 ± 3.7, 76.8 h 1.4, , and per cent of the wet tissue weight, respectively, while the corresponding values for the extracellular (inulin) space are , 26.0 ± 2.4, , and 25.0 ± 0.5 per cent of wet tissue weight.

3 VOL. 65, 1970 BIOCHEMISTRY: BAERLOCHER ET AL Time course of amino acid uptake: Progress curves for uptake of L-proline, glycine, and a-aminoisobutyric acid (AIB), each present at low concentrations in the initial medium, were different in newborn and mature kidney (Fig. 1). Initial rates of uptake in kidney are slower in the immature animal compared to the adult, whereas the ability to accumulate amino acids is generally much greater in the former. These observations corroborate those of Webber and Cairnsl5 who studied amino acid uptake in the kidney of the Wistar rat. We found that initial rates of uptake increased during the second and third week of life toward the adult values. L-Proline 0.051mM Glycine 0.027mM AIB 0.10mM E : E J a minutes of incubaetion FIG. 1.-Time course of amino acid uptake by kidney cortex slices obtained from adult (0-0), 7-day (4), 14-day (A-A) and 21- day (0-0) Long-Evans rats. Slices were incubated in 2.0 ml gassed (95% 02, 5% C02) Tris buffer at ph 7.4 at Data represent the mean of three or more observations. Inserts show details of initial uptake rates in kidney slices of young animals. The above-mentioned differences in initial rates of uptake between the postnatal and adult kidney were much less apparent at high concentrations of L- proline (12 mim) and glycine (6 mm). However the final distribution ratio after prolonged incubation was always per cent higher in postnatal kidney slices exposed to high concentrations of substrate. Effects of catabolism on substrate accumulaton: Catabolism and oxidation of the accumulated substrate influences the observed rate but not the Km of net uptake by mature kidney.7' 8 The influence of metabolism on transport by immature kidney was studied at low and high concentrations of proline and glycine at periods of incubation when the isotope distribution ratios are identical in postnatal and mature kidney. The gross amount of substrate which enters the tissue at low concentrations under these conditions is smaller in the immature kidney than in the mature tissue (Fig. 2); there is no obvious difference between postnatal and adult kidney under conditions of high external substrate concentrations. The different characteristics of uptake in immature and mature kidney are thus not a function of intracellular metabolism of the substance; this conclusion is corroborated by the uptake characteristics of metabolically inert AIB (Fig. 1).

4 1012 BIOCHEMISTRY: BAERLOCHER ET AL. PROC. N. A. S. Efflux of substrate: Reduced efflux accounts for the unusually high distribution ratios which are achieved by immature kidney exposed to low and high initial external concentrations of substrate. Slower efflux rates of L-proline and AIB were clearly demonstrated in the kidney of seven-day-old animals when compared with adult animals (Fig. 3); it was assured that the initial internal concentration of the effluxing substrate was identical in mature and immature tissue. The Qio for efflux is the same in newborn and adult kidney, and internal and external substrates are equally exchangeable in kidney slices from immature and mature animals. We and others'6 consider such data as evidence for a functioning membrane carrier mechanism in newborn kidney. FIG. 2.-Relationship of substrate catabolism to its uptake at high and low initial concentrations E 12 adult in the medium. Open bars indicate 10lo _ adult uptake when loss of 14C label into CO2 is considered in calculation of E8- uptake.7'8 Hatched bars indicate ae simple distribution ratio of labeled 6 1 Ik substrate and its soluble metabolites a lwk adult in slice compared with incubation U_ FL lwk adult lwk medium. In no case was there 2WI ~~~~~~~~~failureto attain concentration 2I11a1n0 X E aew Glyi j against a chemical gradient of the * l- r in L-Proline, Glcine Glycine substrate. Data reveal that gross C O.051mM 12mM 0.027mM 6mM entry of substrate at low concenat 30min at 50min at 60min at 60min trations is impeded in newborn kidney. Values are the mean of at least three observations. The difference in character of efflux between immature and mature kidney of the Long-Evans rat is found also in the Wistar'7 and Sprague-Dawley rat.'6 It suggests an altered "barrier function" of the plasma membrane in newborn kidney, but it does not explain the physiological hyperiminoglycinuria of the young kidney in vivo, nor the reduced initial rate of uptake of low substrate concentrations in vitro. The observed characteristics of influx and efflux undergo independent change at different rates in newborn kidney. Concentration-dependent uptake; Uptake of substrate by kidney slices was studied under equilibrium conditions over a wide range of initial concentration in the medium. Transport of substrate obeys Michaelis-Menten kinetics in immature kidney, as it does in adult kidney. However, whereas mediated uptake of L-proline and glycine occurs on more than one system in the mature mammalian kidney,5-'0 we were unable to demonstrate this characteristic for either substrate in the seven-day-old rat kidney (Fig. 4). The observed Km values for uptake by immature rat kidney are 2.0 mim for L-proline and 1.0 mm for glycine over a broad range of initial external concentration ( mm). Mature kidney slices yield two observed Km values (0.15 mm and 2.5 mm) for proline uptake over the same concentration range. When corrections are made for simultaneous uptake on multiple systems,9' 12, 21 the revised values are 0.09 mm and 5.0 mm.7' 8 Mature kidney also yields two observed Km values (0.25 mm and 3.5 mm) for glycine uptake; the revised

VOL. 65, 1970 BIOCHEMISTRY: BAERLOCHER ET AL. 1013 values for simultaneous uptake of glycine on multiple systems are 0.14 mimi and 2.75 mim.

5 VOL. 65, 1970 BIOCHEMISTRY: BAERLOCHER ET AL values for simultaneous uptake of glycine on multiple systems are 0.14 mimi and 2.75 mim.8 The high-km uptakes of L-proline and glycine are accommodated by a common system5-8; and low-km uptakes occur on independent systems.6-8 Newborn rat kidney appears to be deficient in the respective low-km transport systems serving -proline and glycine. Additional studies of concentrationdependent uptake by kidney of rats at different postnatal ages revealed the emergence of a low-km system for proline uptake in the second week of life and ~~~~~~5 * ~~~AlB L-Pro. 6d o ~~~~~~4- 'A5- U '0 7 ~~~~~~0 o0 6- L-Proline Glycine a 3- elwk old *l1wk old ete, o adult o adult 'IOof ' efflux period ( minutes U/S U/S FIG. 4. -Eadie-Augustinsson plots showing ivproline and glycine transport kinetics under FIG. 3. -Efflux of AIB and iproline at-) steady state conditions in 7-day-old (a 37 from kidney cortex slices of 7-day-old and adult (0-0) rat kidney cortex slices. (9 ) and adult (0-O) rats, employing The apparent Km for 0proline uptake in newth~e method of Segal's group" to determine born kidney is 2.0 mm, over the concentration rate of efflux. Data are average of tripli- range mm; two systems serve uptake cate determinations. Corresponding dif- over the same range in adult slices (see text for ferences in efflux between newborn and Km values). The apparent Km for glycine adult kidney were also evident at 17. uptake is 1.0 mm over the concentration range The initial internal concentrations of sub p mm; two systems serve uptake over strates were identical in newborn and adult this range in adult kidney (see text for Km slices. values). Of a low-km system for glycine uptake in the third week of life. These conclusions are based on the appropriate appearance of two-limbed Lineweaver- Burk and Eadie-Augustinsson plots of concentration-dependent uptake at the above-mentioned times. tnhidbition tests: Further evidence for the initial absence of the low-ttm systems serving proline and glycine uptake by immature rat kidney was sought by using competitive inhibitors of their transport. Previous studiesh e 8 show that 0.2 mml-proline uptake by mature kidney is about equally divided between a low-km system which excludes AIB, and a high-km system which does not, while 0.01 mm glycine transport in the same tissue is about equally divided between a low-km system which excludes proline and a high-km system which does not. Thus uptake of proline and glycine at these particular concentrations should be more severely n eua immature kidney which is deficient in the low-km systems.

$1014 BIOCHEMISTRY: BAERLOCHER ET AL. PROC. N. A. S. o 100- A r 90. O O\ 0 B~~~0\o.$

6 1014 BIOCHEMISTRY: BAERLOCHER ET AL. PROC. N. A. S. o 100- A r 90. O O\ 0 B~~~0\o.~~~~~~~'-~~ 8 0 o , D 0 40 C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~a60 l l 29 0-I 0~~~~~~~~ 0 4~~~~~~~~~~~~~~~ ~' io~ io i substrate (Molar inhibitor (m Molar 0' FIG. 5.-Inhibition of L-proline uptake by AIB under steady state conditions in rat kidney cortex slices of 7-day-old (0-) and adult (0-0) rats. Fig. A indicates uptake at increasing concentrations of L-proline; AIB is always present at ten times the substrate concentration (constant-ratio test). Fig. B indicates uptake of 0.2 mm L- proline in presence of various concentrations of AIB (variable-ratio test). Slices in each of the four experiments shown were obtained from a single animal. Control slices (no inhibitor) and experimental slices (plus inhibitor) were prepared from the same pair of kidneys. Uptake is expressed as percentage of control value. Uptake of L-proline at equilibrium over a wide concentration range was exposed to the inhibitor AIB, which was always present at ten times the substrate concentration (constant-ratio test, Fig. 5(A)). In a second experiment the uptake of 0.2 mm L-proline at equilibrium was exposed to increasing concentrations of AIB (variable-ratio test, Fig. 5(B)). Both types of experiment reveal greater inhibition of L-proline uptake in the immature kidney than in adult tissue. Additional studies of L-proline uptake under initial-rate conditions confirmed this finding (Table 2). When glycine was used as substrate, the constant-ratio test revealed greater inhibition of its uptake in the newborn kidney; the same characteristic was observed with the variable inhibitor test under steady-state conditions but not at initial rates (Table 2). TABLE 2. Effect of competitive inhibitors on uptake of proline and glycine by slices of immature and mature rat kidney. -Uptake: Percentage of Control* Glycine (0.012 mm) + -Proline L-Proline (0.2 mm) + AIB (100 mm) (100 mm) With 14CO2 Without "4CO2 With 14CO2 Without i4co, 5-Min incubation 7 Day old 71.3 ±1.lt 72.4 i±1.t 60.3 ± ± 5.0 Adult 83.5 ± ± ± ± 4.4 Under equilibrium conditions 7 Day old 26.4 ±t 5.5t 3.85 ± 4.9t1t 12.3 i 3.8t ± 3.8t Adult 45.5 ± ± ± ±- 5.1 * Slices were paired from the same animal. Control slices were incubated in the presence of substrate alone; the experimental slices were incubated in medium containing substrate and the inhibitor. Uptake (Cmoles/gm wet weight/5-min incubation, or gmoles/unit time/ml ICF at equilibrium) in presence of inhibitor is expressed as percentage of control uptake. Data designated "with 14CO" indicate experiments where isotope lost from slice as labeled C02 was included in calculations.7 8 Values are the mean and standard deviation of at least six paired determinations. Inhibition of uptake by slices exposed to inhibitor was significantly lower than uptake by control slices in all experiments (p <0.001 by Student's t test). t Uptake by newborn slices is significantly lower than in adult tissue (p <0.001 by Student's t test). I Several values were zero.

7 VOL. 65, 1970 BIOCHEMISTRY: BAERLOCHER ET AL Interindividual variation: We observed greater variation in the L-proline distribution ratio in seven-day-old animals than in any of the older animals. In those studies where a particular transport characteristic could be studied in different animals of the same age, we found that some seven-day-old animals appeared to have only the high-knm transport system for L-proline, while in others the low-km system was also active. A corresponding variation between individual animals was not evident for glycine uptake at 7 days but became apparent by the 14th day. This interindividual variation suggests that the time of entry of the low-km systems is a characteristic of the particular animal. Discussion. We selected iminoglycine transport as an index of postnatal developments in amino acid transport by kidney, because the former has been particularly well characterized in mature mammalian kidney in vitro.5-10 Our own survey of the Long-Evans rat, mouse, guinea pig, rabbit, cat, and man (unpublished data) reveals prominent postnatal iminoglycinuria in all these species. We selected unweaned Long-Evans rats for the present studies. We can compare our in vivo results with those for the second post-natal week in Webber's study of the Wistar rat"8 since feeding regimes in the two studies were comparable at that age. Both species of rat show post-natal hyperiminoglycinuria; our study reveals that the hyperexcretion of imino acids and of glycine subsides asynchronously, proline preceeding glycine. This suggests that independent events influence urinary excretion to these two substances in vivo. The present observations in vitro reveal post-natal emergence of two low-km membrane systems in kidney cells, an event which can explain the in vivo data. The low-km system serving uptake of L-proline and L-hydroxyproline6-8 appears during the second week of life; the other, serving glycine alone, appears during the third week. These findings indicate independent control of the specific activity of the two membrane transport sites (proteins). Evidence for differential activation postnatally of multiple systems rather than quantitative changes in the activity of a single system can be found in the earlier reports of L-cystine and ilysine transport by newborn rat kidney,"6 of ilysine and betaine transport in fetal rabbit intestine1 and of L-valine transport in rabbit yolk sac.20 In these cases and in the present instance, the emergence of a low-km system in maturing tissue would increase the efficiency of uptake at low amino acid concentrations. By contrast, rabbit reticulocytes lose their low-km concentrative systems upon maturation into circulating erythrocytes, which are metabolically less active than the reticulocytes.21 The present studies reveal another difference in the plasma membranes of postnatal and mature kidney cells. Whereas the foregoing describes a temporary deficiency of certain membrane sites for amino acid entry into newborn kidney cells, there is also a difference in the barrier function of the membrane which is identified in postnatal kidney as an augmented distribution ratio'5' 16 and a diminished efflux of amino acids once uptake has occurred.'7 This function of the membrane of kidney cells seems to be lost at a rate which is dissociated from the emergence of an increasing array of membrane sites for entry. Moreover the ability of young cells to concentrate and retain amino acids more effectively than mature tissues is not restricted to the kidney.22 The full explanation for this phenomenon is not clear but we should consider the possibility

1016 BIOCHEMISTRY: BAERLOCHER ET AL. PRoc. N. A. S. that it is, in part, yet another parameter of the membrane which is under independent genetic control.

8 1016 BIOCHEMISTRY: BAERLOCHER ET AL. PRoc. N. A. S. that it is, in part, yet another parameter of the membrane which is under independent genetic control. Agents which appear to be capable of blocking synthesis of the membrane proteins mediating amino acid entry into kidney cells23 may provide a better opportunity to study these phenomena under controlled conditions. * This work was supported by grants from the Medical Research Council (MT 1085) and the Banting Institute. t Recipient of a Medical Research Council Fellowship. $ Recipient of an Associateship of the Medical Research Council. 1 Scriver, C. R., Pediatrics, 44, 348 (1969). 2 Rosenberg, L. E., in Biological Membranes (Boston: Little, Brown & Co., 1969), p Scriver, C. R,, and P. Hechtman, Advances in Human Genetics (Plenum Press, 1970). 4Pardee, A. B,, Science, 162, 632 (1968). 6 Wilson, 0. H., and C. R. Scriver, Amer. J. Physiol., 213, 185 (1967). 6 Scriver, C. R., J. Clin. Invest., 47, 823 (1968). 7 Mohyudcdin, F., and C. R. Scriver, J3iochem. Biophys. Res. Comm., 32, 852 (1968). 8 Mohyuddin, F., and C. R. Scriver, Amer. J. Physiol., in press. 9 Hillman, R. E., I. Albrecht, and L. E. Rosenberg, J. Biol. Chem., 243, 5566 (1968). 10 Hillman, R. E., and L. B. Rosenberg, J. Biol. Chem., 244, 4494 (1969). " Scriver, C. R., E. Davies, and P. Lamm, Clin. Biochem,, 1, 179 (1968). 12 Scriver, C. R., and F. Mohyuddin, J. Biol. Chem., 243, 3207 (1968). Is Segal, S., and J. C. Crawhall, these PROCEEDINGS, 59, 231 (1968). 14 Brodehl, J., and K. Gellissen, Pediatrics, 42, 395 (1968). 15 Webber, W. A., and J. A. Cairns, Canad. J. Physiol. Pharmacol., 46, 165 (1968). 16 Segal, S., and I. Smith, Biochem, Biophys. Res. Comm., 35, 771 (1969). 17 Webber, W. A., Canad. J. Physiol. Pharmacol., 46, 765 (1968). 18 Ibid., 45, 867 (1967). 19 Deren, J. J., E. W. Strauss, and T. H. Wilson, Develop. Biol., 12, 467 (1965). 20 Deren, J. J., H. A. Padykula, and T. H. Wilson, Develop. Biol., 13, 370 (1966). 21 Winter, C. G., and H. N. Christensen, J. Biol. Chem., 240, 3594 (1965). 22 Noall, M. W., T. R, Riggs, L. M. Walker, and H. N. Christensen, Science, 126, 1002 (1957). 23 Elsas, L. J., and L. E. Rosenberg, these PROCEEDINGS, 57, 371 (1967).

Transport and Metabolism of Sarcosine in Hypersarcosinemic and Nonnal Phenotypes

Transport and Metabolism of Sarcosine in Hypersarcosinemic and Nonnal Phenotypes FRANcIs H. GLORIEUX, CHARLEs R. SCRIER, EDGARD DELVIN, and FAZL MOHYUDDIN From the debelle Laboratory for Biochemical Genetics,