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RenalTubularTransportof AminoAcids John Atherton Young and Benedict Sol Freedman Cushny in 1917 first remarked on the extensive amino acid reabsorption which occurs in the nephron. Although many workers since then have studied the nature and localization of the reabsorptive mechanism, progress has been slow because of the technical difficulties of micropuncture work. The bulk of filtered amino nitrogen is reabsorbed in the proximal tubule although the possibility of there being more distal reabsorptive (or secretory) sites cannot be excluded. It is also uncertain whether all segments of the proximal tubule contribute equally to the reabsorptive process. Amino acid reabsorption is an active process involving numerous illdefined steps, the first of which is binding to the brush borders. Renal amino acid transport mechanisms are of two kinds: the high-capacity lowspecificity systems transport whole groups of amino acids-the acidic, basic, neutral, and imino-glycine groups-while the other, the low-capacity high-specificity systems, transport single or perhaps pairs of amino acids only. Although a great deal of information has been published on the mechanisms of amino acid transport in isolated cells and tissues (1), rather less work has been done on the equally important topic of transepithelial transport. Transport across the intestine is technically easy to study, so most of our knowledge of transepithelial transport comes from studies on that organ (2). The kidney is technically much more difficult to study than the intestine, so our knowledge of renal tubular transport of amino acids is scanty and comes mainly from such indirect approaches as clearance studies. With the development of sophisticated techniques for micropuncture and microperfusion of segments of kidney tubules and for microanalysis of fluid samples so obtained (3-5) it should already have proved possible to study renal amino acid transport at the tubule level. Yet it is only recently that such studies have been undertaken (6-9). From the 1)epartment of Physiology, University of Sydney, Sydney, N.S.W. 2006, Australia. This review was prepared from a paper presented at the 5th annual meeting of the Australasian Society for Nephrology and the 3rd Kanematsu Institute Conference on the Kidney, held at Sydney Hospital in October 1969. Received Nov. 17, 1970; accepted Nov. 25, 1970. Why has the tubular transport of such compounds been so long neglected? The reasons are several and they are all compelling: (a) There are a great many different amino acids, and chemically they are quite diverse. (b) Since all cells must transport amino acids for their own metabolic purposes it is difficult to distinguish between simple cell uptake and trans-cellular transport. (c) There exists not one (or even four) distinct amino acid transport mechanism, but rather a mosaic of neatly fitting processes, each with its own substrate specificity, affinity, and transport rate. (d) Amino acid transport is interlocked with the transport of electrolytes and other organic compounds. (e) The lack of specific chemical, let alone microchemical, methods for amino acid determination makes it necessary first to separate amino acid mixtures into their components before one can begin to measure them individually. Even then, the ninhydrin reaction, which one uses to measure amino acids, is not easy to work with. None of these reasons need deter one from studying amino acid transport but, taken together, they have discouraged investigation. Although it has been 53 years since it was first realized that amino acids were absorbed in the CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 245 nephron, no review of the topic has been published. At a time when techniques are at last available which permit a detailed study of the problem to be made it seems highly desirable that available knowledge be summarized. Historical Aspects \hen Ludwig, in 1844, first proposed his theory of glomerular filtration and selective renal tubular reabsorption (10-12) to explain the formation of urine he stated that plasma-water and salts dissolved in it would be filtered across the glomerular capillary walls whereas proteins and fats and minerals bound to these substances would not.’ It was not until many years later that anyone sought to explain the presence or absence of amino acids in urine in terms of his theory. Indeed, before 1900, amino acids were not known to be normal constituents of the urine (12, 13) although they had been recognized as occurring in pathological urine since 1810, when Wollaston (14) described the first recorded case of cystinuria. Wollaston did not, of course, recognize the chemical nature of the compound. Slowly, from 1820 on, when Bracannot first isolated glycine and leucine from protein hydrolysates, the realization developed that amino acids were the sole products of protein hydrolysis and that they occur in the free form widely throughout the animal kingdom (15). Eventually, in 1900, Pfaundller (16) demonstrated that small quantities of amino acids were present in normal human urine. In 1908, Sorensen (17) published a reliable titrimetric method for the determination of total a-amino-nitrogen, which was soon used to determine the normal a-amino-nitrogen excretion of humans; this was found to be about 1 to 2% of the total urinary nitrogen (18, 19). This finding was confirmed by many subsequent workers (20-24), most of whom measured the urinary aminonitrogen excretion of both normal subjects and patients suffering from a variety of diseases, especially hepatic disease. An accurate gasometric method, suitable for the determination of the amino-nitrogen content of blood plasma, was devised by van Slyke in 1912 (25, 26), who used it to determine the plasma amino-nitrogen concentration in normal human subjects. He pointed out that no previous investigator had succeeded in demonstrating that free amino acids normally circulate in the plasma, although a number of ‘“Wir nehmen nun aber hypothetisch Gefasswandungen die Eigenthumlichkeit flussigen und aufgelosten Bestandtheilen einen Theil der Extractivstoffe und die gelosten Saize durch sich hindurchtreten s#{228}mmtliche Proteinsubstanzen, die Fette Verbindung befindlichen mineralischen hindurchiassen” (10). weiter an, dass diese besitzen, von den des Blutes nur Wa.sser, freien nur im Wasser zu lassen, w#{228}hrend sie und die mit beiden in Bestandtheile nicht 246 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 workers had given amino acids parenterally to animals and demonstrated that increased amounts of amino-nitrogen appear in the urine (27-31). Similarly, Folin and Denis (32) had fed amino acids to cats and demonstrated indirectly (by measuring total plasma nitrogen and subtracting urea and protein nitrogen) that some of the aminonitrogen had been absorbed from the intestine and appeared in the plasma. By this time, Ludwig’s mechanistic theories (10-12) concerning the formation of urine were beginning to gain ascendancy over those of Heidenhain (33) who claimed that urine was formed by a process of both glomerular and tubular secretion. Ludwig’s theories, interpreted in the light of subsequent knowledge, required that if amino acids circulated freely in the plasma, unbound to protein, then they must be freely filtered across the glomerulus, and, since only a small amount appeared in normal urine, then reabsorbed in the nephron. In 1913, Abel and his colleagues (34-37), using their brilliantly devised technique of hemodialysis in the living animal, demonstrated that plasma amino acids did indeed circulate freely in the plasma and were readily filterable across a semipermeable membrane. To adherents of Ludwig’s theories this demonstration made the concept of renal tubular reabsorption of amino acids almost inescapable. Cushny (38) in the first edition (1917) of his famous monograph “The Secretion of Urine” said: “The proportion of urea to the total non-protein nitrogenous bodies is much higher in the urine than in the blood. Thus the nitrogen of urea makes up about 40% of the total non-protein nitrogen of the blood, that of the mono-amino acids rather less than 40%, while in the urine the urea nitrogen is 80% of the total nitrogen and that of the amino-acids only 3%” (footnote to p 17). and “The cells lining the tubule thus absorb from the glomerular filtrate a slightly alkaline fluid containing sugar, amino-acids and other similar food substances, and chloride, sodium and potassium in approximately the proportions in which they are present in normal plasma, or in the artificial mixtures which have been introduced for the perfusion of surviving organs” (p4fl. At the time of the second edition of his monograph (1926), little further information was to hand, and Cushny (39) merely repeated his earlier remarks, commenting that: “. . . it may be assumed that there is a high threshold for such readily available foods as the amino-acids, perhaps equal to that of dextrose” (p 12). Between 1917 and 1938, the modern theories of renal function were more adequately formulated and subjected to experimental proof. Thus gbmerular filtration and tubular reabsorption were demonstrated directly in animals by micro- puncture (40, 41) and were quantitated indirectly in man (42-44); tubular secretion of exogenous substances was established (45); the concepts of glomerular filtration rate and of renal clearance were formulated (46, 47); and the concept of a maximum (Tm) to reabsorption and excretion was formulated and established experimentally (4850). These developments [reviewed fully by Smith (51) and Pitts (52)1made it possible to re-examine the manner in which the kidney was thought to handle amino acids and to propose and test hypotheses by analogy with the renal handling of such substances as glucose. The study of amino acid transport has been greatly facilitated in recent years by a number of technical advances. With a few exceptions (53) there had been no specific methods available to determine the concentration of individual amino acids in a mixture until 1943 to 1944, when microbiological analyses and paper chromatography were introduced. Since then, methods have been developed and improved which have made it possible for individual amino acids to be studied rather than total a-amino-nitrogen: microbiological analyses (54-56), specific enzymic analysis (57), paper chromatography (58-60), ion-exchange column chromatography (61), high-voltage paper electrophoresis (62-65), thin-layer chromatography (66), and gas chromatography (67). The ready commercial availability of amino acids with ‘4C, H, and other radioactive labels has also helped to make study of amino acid transfer more specific. Site of Amino Acid Reabsorption within the Nephron It is only since the 1950’s that direct experimental evidence has been produced to localize transport of amino acids to the proximal tubule. However, clinicians, on the basis of indirect evidence, had long suspected that amino acids were reabsorbed in the same segment of the nephron as was glucose. Thus, cases of hereditary diseases had been described (associated with the names of de Toni, Fanconi, Lignac, and Debre) in which there appeared to be an associated defect in renal tubular reabsorption both of amino acids and glucose (68-74). Hence, when Walker (75) used micropuncture techniques to localize glucose reabsorption to the proximal tubule, the presumption was strong that this would also be the site of amino acid transport. Furthermore, there was a large, albeit conflicting, body of morphological data to suggest that there were anatomical abnormalities in the proximal tubules of patients suffering from the Fanconi group of syndromes (69, 76-82). A more direct attempt to localize amino acid reabsorption was made by Neame (83, 84), who demonstrated that incubated slices of kidney cortex could actually concentrate the L-forms of histidine, proline, tyrosine, and ornithine although, in comparison to other tissues such as brain and intestine, the concentration gradients established were not very remarkable. Nevertheless, since kidney cortex slices consist principally of proximal tubular segments, the presumption was that amino acid uptake was related to normal proximal tubular amino acid reabsorption. This and many subsequent studies of cortex slices suffer from the defect that one has no knowledge of the direction of the amino acid transport processes under investigation. Thus, one cannot distinguish between uptake from the luminal and from the interstitial surfaces of the cells, nor can one distinguish between simple uptake and trans-cellular reabsorption. This latter objection also applies to the histochemical studies of Lee et al. (85, 86), who demonstrated that amino acid droplets appear in the cytoplasm of proximal tubular cells after administration of amino acids to the rat. The picture emerging from these various studies has become even more confused since the demonstration that slices of kidney medulla and papilla can also concentrate amino acids (87, 88). The technique of stop-flow analysis (89) provided a tool for more direct localization of renal tubular amino acid transport. These studies demonstrated that the naturally occurring amino acids were reabsorbed in a segment of the nephron coextensive with glucose reabsorption and p-aminohippurate secretion (90-96). Although an attempt to use this technique to localize amino acid transport more precisely to various segments within the proximal tubule has been made (92, 97), it seems clear that this is not really possible with the stop-flow technique (96). Figure 1 illustrates typical amino acid stop-flow patterns for the rat kidney. By injecting so-called area-specific toxins either into the circulation or retrograde up the ureters, Wright and Nicholson claimed to be able to produce localized lesions in various segments of the nephron. From such studies they reported that amino acid reabsorption occurred principally in the more proximal parts of the proximal tubule with subsidiary reabsorptive sites located in the remainder of the proximal tubule and in the distal tubule (98-100). They also claimed to have demonstrated an amino acid secretory site in the more distal part of the proximal tubule. These studies have not met with general acceptance, however, because it is difficult to predict just what nonspecific effects the so-called “area-specific toxins” might have on kidney tubule cells. The most direct evidence for localization of amino acid reabsorption to the proximal tubule comes from a few recent micropuncture and microperfusion experiments. Bergeron (101) injected radioisotopes of leucine and lysine into the lumen CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 247 LIECTIN0 pucr cells has demonstrated that these structures can bind amino acids specifically. Similar findings for D-glucose have also been reported (105). Brush borders occur only on proximal tubule cells; this is strong evidence that the proximal tubule is the major site of amino acid reabsorption. PROXO-jAL :--C C0, 4 075 05 0?5 Active or Passive Transport TAURINE Cc’ 6 Cc _,,.j-GLUTAMICACID 100 no ALAN ME - 35 25 Cc ______,. CREATININE _ 4 60 ___ 1 CUMULATIVE 2 S URINE MASS, 6C 0 fl Fig. 1. Stop-flow analysis of renal tubular reabsorption of glycine, serine, taurine, glutamic acid, and alanine in a rat undergoing osmotic diuresis The rat was infused intravenously at a rate of 0.39 mI/mm with physiological bicarbonate-saline containing, per liter, 80 g of creatinine (as osmotic diuretic), 1.0 g of p-aminohippuric acid (PAR) and 0.03 mole each of L-phenylalanine, L-alanine, Lglutamic acid, taurine, and L-arginine. After 30 mm two clearance collections (A and B) were performed and then the ureteric catheter was obstructed for 3 mm. The obstruction was then released and the emergent urine was collected in small drops. Subsequently, two more clearance collections (C and D) were performed. The urine to plasma concentration ratio (U/Pc,) is shown to indicate the region of distal nephron water reabsorption. The PAH/creatinine clearance ratio (C,.AH/cC,) is shown to indicate the site of proximal tubular PAR secretion. The amino acid/creatinine clearance ratios are so shown; it can be seen that the four amino acids and taurine were reabsorbed in a segment of the nephron coextensive with i’.ii secretion. LTaken from Young and Edwards (95)1 of proximal tubules and, with autoradiographic techniques, demonstrated uptake of radioactivity by the cells of the entire proximal tubule. In later microperfusion studies (6, 102), labeled amino acids were injected into the proximal convoluted tubule, the pars recta, and the distal tubule, and extensive uptake of radioactivity in both the proximal convolution and the pars recta was demonstrated. The technique of microperfusion between oil blocks (3) has recently been used to quantitate reabsorption of i-histidine and glycine from the proximal tubule (7-9). Although all these studies have unequivocally identified the proximal tubule as the principal site for amino acid reabsorption, they cannot he said to have excluded the possibility that a small fraction of amino acid reabsorption occurs more distally, as is now known to occur in the case of D-glucose (103). Another very recent study (104) with isolated suspensions of brush borders from kidney tubule 248 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 The fact that a great many amino acids are present in the urine in concentrations less than those in plasma strongly suggests that there is active reabsorption of these amino acids in the nephron. Even when the urine/plasma ratios for naturally occurring amino acid are greater than one, the values, when corrected for distal tubular water reabsorption, suggest that their concentrations in the proximal tubule would be less than those in plasma (106, 107). More direct evidence of active reabsorption of amino acids comes from stop-flow studies (90, 91, 94-97). In such studies, the clearance ratio of amino acid to inulin or creatinine was shown to decrease in the proximal tubule to values less than unity, indicating reabsorption against a concentration gradient. As with other tissues, renal cortex slices can be shown to take up amino acids against a concentration gradient, which is suggestive of active transport. As mentioned above, however, the direction of amino acid transport cannot be ascertained from such studies. Slices do offer one advantage: it is possible to study the effects of metabolic inhibitors on amino acid uptake mechanisms. Thus, Schwartzman et al. (108) and Segal et al. (109) were able to inhibit the active accumulation of amino acids in kidney slices with dinitrophenol and with anaerobiosis. Using an isolated tubule preparation, Hillman (110) was able to inhibit amino acid uptake with both dinitrophenol and cyanide. More recently, he (104) studied the binding of proline to isolated proximal tubule cell brush borders (which would presumably be a step in the reabsorptive process) and found that it was partially inhibited by cyanide and dinitrophenol. The demonstration of active transport does not, of course, exclude the possibility that passive transport also contributes to reabsorption. Schwartzman et at. (111) have demonstrated autoand hetero-exchange diffusion of dibasic amino acids in rat kidney cortex slices; thus they observed increased accumulation of a labeled amino acid when the slice had been preloaded with the same amino acid (auto exchange) and with a different amino acid (hetero exchange). Such a phenomenon had been noticed much earlier in the Ehrlich ascites cell (112, 113), although none of the neutral amino acids that underwent exchange diffusion in the Ehrlich cell was able to undergo exchange diffusion in the kidney cortex slice. This exchange diffusion of the dibasic amino acid, surprisingly enough, is more susceptible to metabolic inhibition than is active transport. However, as with active transport, the direction of the exchange diffusion cannot be determined in the kidney slice. Although the above evidence points toward the existence of active and passive reabsorptive processes that require a supply of oxidative energy, microperfusion of tubules has recently provided evidence which suggests that aerobic metabolism is not always an essential prerequisite, at least for histidine reabsorption (8). In these experiments, tubular reabsorpiion of i-histidine was significantly depressed by the inclusion of 40 mmolar cyanide in the perfusion fluid. However a large part of the reabsorption remained uninhibited, suggesting the presence of some passive process, or at least one not inhibited by cyanide (see Figure 2). A similar study on glycine transport in the rat nephron showed that a component of its absorption was not inhibited by either cyanide or dinitrophenol (9). mulation and amino acid reabsorption is far from certain. Jacquez (115) postulated that a zwitterion component such as phosphatidyl choline might play a role in amino acid transport, at least in the Ehrlich ascites cell, but subsequently Schwartzman et at. (116), in work with the kidney cortex slice, excluded the possibility of phosphatidopeptides’ playing a carrier role. The most promising model that seems suited for the investigation of carrier properties is the isolated brush-border preparation. This is already being exploited for study of the glucose carrier (105), and the first amino acid studies have recently been reported (104). These membrane fragments were shown to be able to bind i-proline by a saturable and inhibitable process. More work utilizing this technique may illuminate this rather difficult problem. Carrier The renal transport of amino acids is influenced by the presence of a number of other substances such as electrolytes and sugars in the glomerular filtrate and the renal capillary blood. The extent and the nature of the interactions occurring between some of these substances and amino acids during reabsorption are discussed in this section. Our knowledge of the nature of the carrier or carriers involved in amino acid transport is rather scanty. This is largely due to the lack of a suitable experimental preparation with which to work. The kidney cortex slice technique was exploited for this purpose by Elsas and Rosenberg (114), who investigated the effect of puromycin, a protein synthesis inhibitor, on amino acid accumulation. They found that this accumulation depended on synthesis of a protein with a relatively long halflife, although the relationship between slice accu- I I I 80 0 60 1 End ni Proximol 0 Tobole 40 z 0 2(’ 100% REABSORPTION #{149} 138 picooIe$/mjn. z PERFUSED LENGTH, mm Fig. 2. The effect of sodium cyanide on proximal tubular reabsorption of L-histidine in the rat The left kidney was prepared for tubular microperfusion (3). Two techniques of microperfusion were used: microperfusion of short segments between oil blocks and microperfusion of whole nephrons. Control data from 34 animals is indicated by the shading. Approximately 80 to 90% of the infused histidine load was reabsorbed by the time the fluid reached the end of the proximal tubule. In five animals (indicated by the filled circles) 40 mmol of sodium cyanide per liter was incorporated in the perfusion fluid. Reabsorption can be seen to have been partially inhibited although about 70% of the infused load was still reabsorbed by the end of the proximal tubule (Freedman and Young, unpublished data, 1970) Interactions of Amino Acids and Other Molecules in Transport Cations The two major body cations, sodium and potassium, have been shown to influence renal amino acid transport (117), but the nature of the interaction is poorly understood. Because renal and intestinal amino acid transport mechanisms are similar in many respects (2) it may be useful to consider what is known of cation-amino acid interactions in the intestine. It has been shown quite conclusively that transintestinal amino acid flux is related to the sodium concentration of the medium bathing the intestine but is independent of the bulk cellular sodium content (118-120). Two hypotheses have been advanced to explain this interaction. The first one (119, 121, 122) is basically an extension of Crane’s ideas about sugar transport (123, 124). Sodium is thought to be involved in forming a ternary complex with the amino acid and the carrier, which leads in some way to transport of both molecules. The alternative hypothesis (125) attributes the sodium-amino acid relationship to an intracellular sodium requirement for the coupling of metabolic energy to active transport; in this model the concentration of extracellular sodium is important only because it influences the concentration of intracellular sodium. Recent data support the first hypothesis (118, 120) [similar conclusions have also been CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 249 reached from studies of the Ehrlich ascites cell (126)]. The nature and even the existence of such a relationship in the kidney is far more equivocal. As yet, only the technique in which kidney slices are studied in vitro has lent itself to the investigation of this relationship. Fox et at. (117) demonstrated a decreased amino acid accumulation in cortex slices in the presence of low Na concentrations, with complete inhibition of the accumulation of some amino acids in a Na-free medium. However, lysine accumulation could not be abolished even in Na-free media; ouabaine, which inhibited the Na-sensitive accumulation, had no effect on the Na-insensitive component of lysine accumulation. In the same study, these authors also demonstrated the dependence of amino acid accumulation on K concentration in the medium, with optimal accumulation occurring over a narrow range of K concentrations. More recent studies with isolated tubule and brush-border preparations have shown that amino acid transport involves at least two steps, binding to the brush border and subsequent active transport (104, 110). The initial binding process is much less dependent on Na ions than is the subsequent transfer of the amino acid into the cell (104). A number of subsequent studies have thrown some doubt on the interpretation of results with cortex slices. Segal and Smith (127), working with kidney slices of newborn rats, showed that a Nafree medium actually enhanced the concentrative accumulation of lysine. Such a paradoxical increase in lysine uptake in Na-free media was also reported for the toad bladder (128), but there is as yet no explanation of this phenomenon. Schwartzman (111, 116) was unable to inhibit lysine or arginine accumulation with a Na-free medium, which is at variance with the observation that lysine transport is inhibited in a Na-free medium, as was previously reported from the same laboratory (117). This discrepancy is as yet unexplained, but could be a result of the different experimental conditions used in the two series of experiments. The above studies have been concerned with the dependence of amino acid transport on cation concentrations. Some recent studies suggest that salt and water transport may depend in part on sugar and amino acid transport. Thus, the human jejunum has been shown to have a low reflection2 coefficient for sodium chloride, with the consequence that a major fraction of salt and water absorption by the jejunum is the result of solvent When a membrane separates two solutions of the same solute but of differing concentrations, the osmotic pressure difference actually developed across the membrane is usually less than that which could be expected from the osmolality gradient. The ratio of the ob8erved osmotic pressure gradient to that which could be expecied by calculation is defined as the “reflection coefficient”. 250 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 drag consequent on the active absorption of bicarbonate, sugars, and amino acids (129). Since the proximal tubule of the kidney also has a low reflection coefficient for sodium chloride (130) a similar mechanism relating salt, water, and amino acid transport may operate there. In addition to the above interactions, an interesting and seemingly unrelated phenomenon has been reported by several authors (94, 131, 132). It has been found that potassium secretion by the distal tubule can be sharply stimulated by infusing dibasic amino acids. The mechanism of this interesting occurrence is unclear. Sugars The presence of glucosuria with associated amino aciduria in the Fanconi syndrome (69, 71-73) suggested either a lesion in a common reabsorptive site or a defect in a step common to the reabsorption of both. It is interesting to note, therefore, that a number of sugars have been shown to interact with amino acids in renal transport mechanisms. Studies in vivo by use of clearance techniques have demonstrated some of these interactions. Glucose infusions impair the renal absorption of amino acids (133). Phiorizin, a competitive inhibitor of glucose transport, also inhibits amino acid absorption to some extent (134). Some amino acids-lysine, glycine, and alanine-depress glucose reabsorption; others, including aspartic acid and leucine, have no effect (135). Thier (136) used the in vitro slice technique to demonstrate that uptake of some neutral amino acids was inhibited by glucose, galactose, and fructose. These sugars were without effect during anaerobiosis or in the presence of 2,4-dinitrophenol, and had no effect on the accumulation of histidine and lysine, both of which had been shown previously to be taken up both by sodium-dependent and sodium-independent pathways (117). These findings, together with a failure to observe a hexose-induced change of affinity of amino acids for the transport sites which would be expected of competitive inhibition, led Thier (136) to suggest that the basis for the interaction was probably a common dependence on a sodium-dependent ase that provides the energy for the transport systems. Work on the glucose-amino acid interaction in the intestine has progressed a little further than in the kidney, and points to the relationship being due to some common factor, possibly the energy source required for the transport of both (137). Other Interactions A number stances have of other naturally also been shown occurring to interact subwith amino acids during their transport. Thus, the reabsorption of sulfate was found to be depressed by amino acid infusions (138), and stop-flow studies have since shown that sulfate and amino acids are reabsorbed in the same segment of nephron (93). The reabsorption of phosphate has also been linked to amino acid transport. Thus phosphate reabsorption is reduced by amino acid infusions (133, 139). Similarly vitamin D deficiency has been shown in man to impair renal amino acid reabsorption (140). Scriver (141) used rats with experimentally induced vitamin D deficiency and obtained evidence to suggest that low serum calcium concentrations stimulated the release of parathormone, which acted in some way to impair both renal amino acid and phosphate reabsorption. The dependence of amino acid reabsorption on hydrogen ion concentration has been demonstrated in the isolated perfused rat kidney by Weiss and his colleagues (142). It was found that as the urinary pH was increased from 5 to 8, amino acid reabsorption decreased for all the amino acids studied, and a few amino acids even exhibited net secretion at the higher pH values. In another study with the same technique (143), they found that as the urine pH was increased from 5 to 8, the secretion of methyl and ethyl esters of some of the amino acids used in the earlier study was greatly decreased until there was a net reabsorptive flux at the highest pH’s. The authors interpreted the results in terms of a pH-dependent, nonionic diffusion process operating for these substances. However certain reservations about the viability of their isolated perfused kidneys must be kept in mind when considering the results. For example, the histidine/inulin clearance in this isolated perfused kidney was never less than 0.25 at any urinary pH, whereas in clearance experiments in the intact rat the clearance ratio at similar histidine loads was less than 0.025 (96). In addition, there is an apparent contradiction between results for the isolated perfused kidney and the kidney cortex slice. Segal (109) demonstrated a marked inability of the slice to concentrate amino acids at pH 5 to 6, in which range the perfused kidney was reabsorbing amino acids maximally, while at pH 8 to 9 the slice showed a marked increase in concentrating ability, a range in which the perfused kidney showed a greatly diminished reabsorption to the point of a change in the net flux to secretion. This discrepancy highlights the difficulties in the interpretation of data obtained with kidney cortex slices. Renal Handlin9 of Optical Isomers of Amino Acids The possibility that the kidney might handle Dand L-isomers3 of amino acids differently did not occur to many early workers. Indeed, it was not until the studies of Gibson and Wiseman (144), who demonstrated optical specificity of intestinal amino acid transport systems, that the possibility that renal amino acid reabsorption systems might show such specificity was seriously examined. Wohlgemuth (31), in 1905, gave a racemic mixture of amino acids to a rabbit and observed that urinary amino acids during the next 24 h were predominantly of the D-form. He interpreted this as evidence for differential rates of metabolism of the two isomers and did not consider the possibility of there being differential renal handling of the isomers. Subsequent studies (28, 145-1 47) confirmed their findings and explained them in a similar fashion. However, Albanese et at. in later experiments with arginine (148) discovered that the liver could metabolize both isomers equally rapidly, so he postulated that there might be a differential renal threshold for the L- and D-forms. This conclusion was also drawn from many subsequent studies (149-153). Crampton and Smyth (57) were the first workers specifically to investigate the renal handling of the optical isomers of natural amino acids. They confirmed that the kidney of the cat did indeed have a lower threshold for the D- forms of histidine, alanine, and methionine and inclined to the view that D- amino acids were absorbed merely by passive diffusion. A number of studies since 1953 have extended the range of observations to include many other animals and a wide variety of amino acids (138, 154-157). Webber (158) studied the renal tubular reabsorption of D- and L-aspartiC acid and their influence on the excretion of other amino acids by the dog. He found that both forms were reabsorbed by ‘The nomenclature for designating the optical isomers of amino acids has changed somewhat during the last 50 years. Before the publication of a paper by Wohl and Freudenberg in 1923 (235) [cited by Vickery (236)1 it was the custom to prefix amino acids with an italicized lower case d or 1 to indicate the direction of optical rotation. Under this system a number of amino acids occurring in animal protein (the “natural” forms) were called dextro (d) amino acids although it was recognized that all the natural amino acids had the same configuration about the a-carbon atom. These amino acids were: alanine, arginine, aspartic acid, glutamic acid, isoleucine, lysine, ornit.hine, threonine, and valine. The system proposed by Wohi and Freudenberg (235) required that all the natural amino acids be designated with an italicized lower case 1 and the optical rotation was indicated by a + or - after the 1. For a number of reasons the system never worked fully and eventually Vickery (236), in 1947, proposed the system now in use. The natural and unnatural forms of amino acids are referred to not as levo and dextro but as “ell” and “dee” and they are written as small capital letters t and 1), connected to the proper or trivial names of the amino acids with a hyphen. Recently the absolute configuration of natural amino acids about the a-carbon atom has been determined and, conveniently, found to correspond to the n form which had arbitrarily been assigned to it (237). It is essential to be aware of these changes in nomenclature if older papers are to be read correctly. CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 251 a Tm-limited transport mechanism and that the Tm for D-aspartic acid was only slightly less than for the L isomer. Both isomers had similar inhibitory effects on i4-glutamic acid reabsorption. It is unfortunate, however, that Webber’s analytical technique could not distinguish between the Land D-forms of aspartic acid, thus preventing him from excluding the not unlikely possibility of conversion of the D- to the L-form in the body. Young and Edwards (96) have shown that L-a methyldopa has a competitive inhibitory effect on the reabsorption of L-histidine whereas D-methyldopa was without inhibitory effect. From the above it seems clear that the kidney has a lower threshold for the transport of most of the D-isomers than for their corresponding L-forms. However the inference drawn by most authors that D-amino acids are merely reabsorbed by passive diffusion seems open to some doubt. (a) a-amino acids can be absorbed rather more readily than one would expect for a merely passive process. Thus, for example, DL-methionine was reported to have a maximum clearance of only 5% of glomerular filtration rate (159). (b) A Tm value for D-aspartic acid has been demonstrated (160). (c) The ratio of the clearance of D-amino acids to that of inulin has been shown to increase as plasma levels of the Damino acid increase (57), which suggests that Damino acid absorption is a saturable process. (d) By analogy with the intestine one would expect at least D-methionine to be actively reabsorbed, because it has been demonstrated that rat intestinal mucosa can move this amino acid against a concentration gradient (161). Embryonic and Postnatal Development of Renal Amino Acid Transport It was known as early as 1911 that infants excreted relatively more amino acids in the urine than adults (162). This has been amply confirmed by many other workers (163-1 72). An especially complete study was that of Brodehl and Gellissen (173), who measured endogenous clearances of 17 amino acids as well as glomerular filtration rate in infants and older children and calculated the fractional reabsorption of each amino acid studied. In infants they found a decreased efficiency of absorption of threonine, serine, alanine, valine, phenylalanine, glycine, histidine, cystine, lysine, arginine, ornithine, and proline. However, methionine, leucine, isoleucine, and tyrosine were absorbed as efficiently as in older children. The authors suggest that these results are best explained by postulating a degree of glomerulo-tubular imbalance in the infant-i.e., the amount of tubular tissue had not developed sufficiently to keep pace with developing glomerular filtration. This view is supported by the finding of higher values and greater heterogeneity for the ratio of glomerular surface area to proximal 252 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 tubular volume in infants than in children or adults (174). However, the demonstration that some amino acids are more involved than others in this general decrease in reabsorption points to the implication of other factors, such as the maturation of specific transport systems. Webber (175) has observed a similarly increased amino acid excretion in immature rats, which occurred in spite of reduced filtered loads. He accounted for this in the rat in the same terms as did Brodehl and Gellissen (173) for the human (see above). Recently, Cooke and Young (1971, in preparation) studied amino acid transport in the embryonic and postembryonic chick. They found that the fractional reabsorption of the basic amino acids, arginine and lysine, and the neutral amino acid, phenylalanine, was at least as complete in the 20-day-old embryo as in older birds. On the other hand, the fractional absorption of alanine and glycine was less in the embryo than in hatched birds. The authors felt that their data suggested that the basic transport system for amino acids may have developed earlier than that for neutral amino acids. Because of the difficulty of working with embryos, a number of developmental studies on amino acid transport have been carried out with kidney cortex slices. Webber has observed a more rapid initial uptake of amino acids in mature as compared with newborn rat kidney tissue, although the final concentration ratios attained were always lower in the mature tissue (176, 177). He suggested that, despite the greater concentrative ability of newborn kidney tissue, the finding of slower initial uptake could possibly account for the decreased reabsorptive capacity of the immature whole kidney. However a later study by Segal and Smith (127), who used higher substrate concentrations, has failed to demonstrate a lower initial uptake rate for lysine in the immature kidney. Renal Amino Acid Transport Groups Although all amino acids have in common the general configuration around the a-carbon atomi.e., a hydrogen atom, an amino group, a carboxyl group, and an R-group--the differences in the Rgroup among individual amino acids allow for a useful classification to be made on the basis of structure alone. Such a classification, as shown below, provides a framework for studying transport grouping, without prejudging function. Monoaminomonocarboxylic acids with aliphatic chains (glycine, alanine, serine, and threonine). side Monoaminomonocarboxylic acids with branched chains (valine, leucine, and isoleucine). side Aromatic Heterocydic amino acids (phenyinlanine amino acids Monoaminodicarboxylic aspartic acid). (tryptophan acids and tyrosine). and histidine). (glutamic acid and Diaminomonocarboxylic ornithine). Diaminodicarboxylic acids arginine, and -9 amino (cystine). acids (cysteine, C cystine, and Amide-group amino acids (glutainine and asparagine). Imino acids (proline and hydroxyproline). On the basis of their great variety in structure, it would not seem likely that one transport mechanism could serve for the reabsorption of all of these amino acids. Indeed, evidence obtained from competition studies in vivo and in vitro, as well as “experiments of nature” involving genetic defects, suggests the existence of at least four amino acid transport systems in mammalian kidney.4 These four systems will be called, for convenience, the neutral, basic, acidic, and iminoglycine systems. The amino acids transported by each system are enumerated below and the experimental evidence for the existence of each system will be discussed. Neutral system (a) Aliphatie and branched chain amino (b) Heterocyclic amino acids (c) Aromatic amino acids (d) Amide group amino acids (e) Methionine and cysteine #{149} Basic system (a) Diaminomonocarboxylic acids (b) Diaminodicarboxylic acids (possibly) #{149} Acidic system (a) Monoaminodicarboxylic acids #{149} Iminoglycine system (a) Imino acids (b) Glycine ‘In addition to the four major transport systems, there is some evidence to suggest that there may be a fifth system for of -alanine, fl-aminoisobutyric acid, The system has been demonstrated in Ehrlich (238) and in mouse (239) and human (240) kidney. of taurine in rat kidney has also been demonstrated analysis [See Figure 1 and I (96)1. ,..-7 C, .6 ‘ .4 1 Cp 10 “cr6 -, .4 CUIAIJLATIVE Fig. 3A. Stop-flow of L-a-methyldOpa analysis URNE .5 l.A5S of renal tubular reabsorption in a rat undergoing osmotic diuresis The animal was infused intravenously at a rate of 0.39 mI/mm with a physiological salt solution containing, per liter, 80 g of creatinine, 1.0 g of p-aminohippuric acid, and 1 mmol of L-methyldopa-2-’4C. The experimental protocol was the same as that used in Fig. 1. The experiment demonstrates proximal tubular reabsorption of the synthetic a-methyl amino acid. [Taken from Young and Edwards (96)] acic1, Neutral system. Genetic evidence for the existence of a separate transport mechanism for the neutral amino acids has come from studies of the rare, recessively inherited phenotype known as Hartnup disease (106, 157, 178-183). Cusworth and Dent (180) measured amino acid clearances in this disease, and found that the clearances of all the neutral amino acids were greatly increased, except that of glycine, which was only slightly affected, and proline, which was not affected at all. Various authors have demonstrated competition for reabsorption between pairs of neutral amino acids (6, 184). More comprehensive studies have shown the effect of infusion of single neutral amino acids on the excretion (185-187) and the clearance (160, 188) of the other neutral amino acids. The evidence suggests that all the neutral amino acids the transport j -a acids Sulfur-containing methionine). (lysine, and taurine. ascites cells Reabsorption by stop-flow CUMULATIVE URINE MASS Fig. 38. The effect of L-methyldopa on proximal tubular reabsorption of L-histidine, as illustrated by stop.flow analysis experiments (for details see Figure 1) A stop-flow pattern in a control rat (broken lines) shows extensive reabsorption of L-histidine in the proximal tubule. In a rat infused with L-methyldOpa (solid lines) the proximal reabsorptive trough for L-histidine is abolished despite the much lower plasma histidine concentration. [Taken from Young and Edwards (96)] are transported by a common system. In addition, two synthetic amino acids, i.-a-methyldihydroxyphenylalanine (a-methyldopa) and a-aminoisobutyric acid, have been shown to be transported by this neutral system, competing with the natural amino acids for transport (96, 189, 190). Figure 3A shows a stop-flow pattern demonstrating proximal tubular reabsorption of methyldopa, while Figure 3B illustrates its inhibitory effect on tubular histidine reabsorption. CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 253 The studies of Oxender and Christensen (191) on neutral amino acid uptake by Ehrlich ascites cells suggested the presence of two transport mechanisms within the neutral system-an “A” or alaninepref erring system, and an “L” or leucine-pref erring system. The overlap between these two systems was so extensive that nearly all the neutral amino acids were transported by both systems, but with differing affinities for each. These findings were of particular interest, as the authors were able to interpret earlier renal competition data (184, 188) in terms of an A and L dichotomy of neutral amino acid transport in the kidney. It should be pointed out however that not all authors believe that the evidence supports the existence of the A and L systems in the Ehrlich cell (192) or in the intestine (118). Basic system. It is generally agreed that the diaminomonocarboxylic acids-lysine, arginine, and ornithine (commonly referred to as the basic amino acids)-are reabsorbed by a common transport system. This has been demonstrated unequivocally by competition studies in man, dog, and rat (6, 90, 97, 184-187, 193-1 95). However, the position of cystine in this transport group has proved an enigma, and the question is still not completely resolved. The first evidence supporting the grouping of cystine and thc basic amino acids in a common system came from a study of the hereditary disease, classical cystinuria. This disease is marked by large increases in the excretion of cystine and the basic amino acids (196, 197). Thus it was postulated that cystinuria represented a genetically determined defect of the common transport system for these amino acids. That these amino acids share a common system was further supported by clearance and competition studies in man (194) and dog (94, 160). However in the last few years much evidence, gathered mainly from in vivo and in vitro experiments in the rat, indicates that, at least in that animal, cystine is not transported by the same mechanism as the three basic amino acids (109, 111, 127, 195, 198, 199). Similarly, recent in vivo and in vitro studies of cystinuria have thrown doubt on the concept of a single transport defect for cystine and the basic amino acids. Crawhall (200) reported that while cystine clearances in cystinurics were invariably greater than the gbmerular filtration rate (GFR), indicating net secretion, the clearance of the basic amino acids was less than GFR, indicating net reabsorption. Fox et al. (117) studied uptake of amino acids into kidney cortex from cystinuric patients and found no impairment in cystine uptake, while lysine uptake was reduced by half. Thus, it would appear that in the human cystinuric kidney, cystine is handled differently from the basic amino acids, suggesting separate transport mechanisms. 254 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 Direct evidence for the presence of separate transport systems for cystine and the basic amino acids has come from the chance discoveries of some rare genetically determined aminoacidurias. Brodehi (01) reported an aminoaciduria marked by excessive cystine clearance in the presence of normal basic amino acid clearance. Such isolated cystinuria is also found normally in the Kenyan blotched genet (202, 203). Following this line of reasoning, one might also predict the occurrence of a genetic aminoaciduria involving only the three basic amino acids, but not cystine. In fact such an aminoaciduria has recently been discovered and investigated (204). It appears then, at least in man and the rat, that cystine and the basic amino acids are transported by separate mechanisms. The question of what causes competition for reabsorption between cystine and the basic amino acids in man and dog, as well as the reason for impaired handling of cystine and the basic amino acids in cystinuria, still remains unanswered. One possibility might be that the basic amino acids and cysteine (the intracellular form of cystine) share a common effiux pathway from the tubule cells. This appears to be the case in the rat (199). If such a mechanism were defective in cystinuria, there could be a “pile-up” of intracellular cysteine, resulting in the failure of reabsorbed cystine to be reduced to cysteine. A mass action effect could then prevent cystine movement across the membrane. Acidic system. The results of competition studies in the dog suggested that the two monoaminodicarboxylic acids-glutamic acid and aspartic acid-were transported together by a common system (94, 185-188). In these experiments, jul usion of one amino acid caused decreased reabsorption of the other. However, as the infusion of one of these amino acids caused a marked increase in the plasma concentration of the other (188), it was not certain whether the decreased reabsorption was the result of competition for transport, or was merely a consequence of increased filtered load. Webber (158) was able to resolve this question by determining the transport characteristics of each of the two acidic amino acids individually. He found a transport maximum (Tm) for both of these amino acids [in marked contradistinction to Gerok and Gayer (97, 193), who were unable to show a Tm for glutamic acid], which enabled him to show that the increased plasma concentration caused by infusion was not sufficient to cause saturation of the transport mechanism, and thus the decreased reabsorption observed was a direct consequence of competition for a common transport system. A different technique, which avoided the problem of increased plasma concentrations, was used to examine the acidic transport system in the rat. By microinjection of the acidic amino acids into single nephrons, Bergeron and Morel demonstrated Tm values for both aspartic and glutamic acid (102). They also showed marked mutual competition for transport between the two acidic amino acids when they were injected into the nephron simultaneously. A variety of neutral amino acids caused no inhibition of acidic amino acid transport. These data suggest strongly that glutamic and aspartic acids are reabsorbed by a single system in the rat kidney, a system which is quite distinct from that available for neutral amino acids. Iminoglycine system. The investigation of some metabolic disorders has led workers to propose the existence of a common transport system for the imino acids, proline and hydroxyproline, and the neutral amino acid, glycine. In Hartnup disease, where there is a defect in the reabsorption of all other neutral amino acids, glycine reabsorption is only slightly affected, and reabsorption of the imino acids not at all (180, 205). Scriver el al. (206) made a study of a disease characterized by high plasma proline concentrations and increased excretion of the imino acids and glycine. They found that only when the proline level exceeded its Tm was there an increased excretion of the imino acidglycine group, and this phenomenon could be mimicked in normal humans by artificially raising the plasma proline levels. In addition to this disease, the authors cited other cases of convulsive disorders accompanied by hyperexcretion of the imino acids and glycine in which there were normal plasma amino acid concentrations. These cases seemed to have impairment of a transport system common to the imino acids and glycine. Although the above studies indicate that there is a common transport system for the imino acids and glycine, this of course does not preclude the possibility of there being additional systems capable of transporting one or other of these amino acids individually. In fact there is a good deal of evidence pointing to the existence of multiple transport mediations within the iminoglycine system. Scriver et al. (206, 207), from competition studies in humans, demonstrated that some neutral amino acids that had no effect on the reabsorption of imino acids nevertheless had slight effects on glycine reabsorption. From this they suggested that the imino acids and glycine might have separate transport mediations. However in similar studies in the dog, Webber (188) demonstrated inhibition of the reabsorption of both the imino acids and glycine by the same neutral amino acids that Scriver et al. (206, 207) had used in their study. Although this discrepancy may be due to a species difference, a more probable explanation would be that the levels of the neutral amino acids achieved in Scriver’s study were not sufficient to cause inhibition of the reabsorption of the imino acids or glycine (indeed they were not sufficient to cause inhibition of the reabsorption of other neutral amino acids). Wilson and Scriver (195) used slices of rat kidney cortex to examine the affinity constants for transport and inhibition of the imino acids and glycine. On the basis of variation and diversity of these constants, the authors concluded that, rather than a single common transport system for this group, the system must be subdivided, supplemented, or both. These authors had reached much the same conclusion previously (208), believing that there were discrete catalytic sites for the individual amino acids within this system. Genetic evidence for the existence of multiple mediations within the iminoglycine system has come from studies of both heterozygotes and homozygotes with an iminoglycinuric trait (209). Scriver (209) rejected the possibility of altered affinity of a common transport system. Rather, he proposed that a high-capacity transport system, common to all these compounds, was defective and that the residual reabsorption observed was due to two other low-capacity systems, one of these being only for the two imino acids. These conclusions, with suitable modifications, are compatible with preliminary findings from isolated rabbit tubule preparations. Thus, Hillman and Rosenberg (104) demonstrated three distinct transport systems for proline: one shared with alanine, a second shared with glycine, and a third only with hydroxyproline. This tubule preparation, together with isolated brush border preparations, seems to offer the greatest promise for the elucidation of the properties of these multiple transport mediations. Intergroup interactions. Although the evidence for the existence of the four transport groups is fairly clear, some explanation for the large number of intergroup interactions that have been reported is necessary. For example, in competition studies (160, 188) in dogs it was found that the basic amino acids inhibited transport of some neutral amino acids and vice versa, and also that some neutral amino acids inhibited transport of the imino acids. Two possible theories could account for these interactions. The one theory envisages only a limited number of transport systems, which have very wide and overlapping substrate specificities. The other envisages a heterogeneity of transport systems of two basic types-namely, high-capacity low-specificity systems, which would transport groups of amino acids, and low-capacity highspecificity systems, which would transport perhaps only one or two amino acids. The existence of two general types of genetically determined aminoacidurias, one in which the defect covers a whole transport group-e.g., classical cystinuria and Hartnup disease-and the other, in which there is defective transport of only one or two amino acids-e.g., isolated hypercystinuriawould support the latter theory. Also, the investigation by Scriver (209) of the iminoglycinuric CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 255 trait provided evidence that supports the argument that there are two different types of system for the transport of the imino acids and glycine. Although only a small amount of experimental work has been directly involved with examining the possibilities of two types of transport system, a number of unrelated observations lend support to this theory. Brown (210) reported that cycloleucine, a neutral amino acid that is not metabolized, when administered to man in large doses, caused a very large increase in the excretion of the basic amino acids and cystine, with no change in their plasma levels. Christensen and Cullen (189) demonstrated a similar large increase in the excretion of the basic amino acids and cystine, as well as marked effects on neutral amino acids, after administration to rats of large doses of a-aminoisobutyric acid (AIB), another neutral amino acid that is not metabolized. Moreover, after the plasma AIB levels had dropped from their very high initial levels, the hyperexcretion of the basic amino acids and cystine disappeared, leaving a residual hyperexcretion of the neutral amino acids. These results could possibly be accounted for on the basis of the in vitro results of Segal et al. (109), who found that AIB, cycloleucine, some basic, and some neutral amino acids could be metabolized by a high-capacity system. More complete studies by these authors showed that at least lysine and glycine were both transported by two systems-one with a low capacity and high affinity, and the other with a high capacity and low affinity. Both lysine and glycine uptake by this latter system were inhibited by AIB, suggesting that this system may transport both neutral and basic amino acids. A similar nonspecific low-affinity system, known as the “lysineaccepting system,” that transports both neutral and basic amino acids, has been reported in the Ehrlich ascites cell (211). The existence of such a system could explain the interaction between the neutral and basic amino acids in Webber’s studies (160, 188) and might also explain the inhibition of the reabsorption of the basic amino acids and cystine by high levels of cycloleucine in Brown’s study (210). Similarly, the results of Christensen and Cullen (189) (see above) could mean that AlE in high concentrations inhibited the common highcapacity system shared by the neutral and the basic amino acids, while in lower concentrations, it inhibited only the low-capacity neutral system. Reabsorption Kinetics and Transport Maxima Information about reabsorption kinetics has been obtained by studying the clearance of amino acids both at endogenous plasma concentrations and after intravenous infusions of amino acids so as to produce a wide range of exogenous plasma amino acid concentrations. Endogenous amino acid clearance. Prior to the 256 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 development of chromatographic separation techniques, the plasma concentrations of individual amino acids could not be measured, and early studies were concerned only with the clearance of total a-amino nitrogen. Such measurements indicate that a-amino nitrogen reabsorption is 98 to 99% complete in normal man (180, 212). Apart from man, endogenous a-amino nitrogen clearance has been studied only in two species-the dog (98) and the alligator (sic) (213). Although reabsorption of total a-amino nitrogen is 99% complete in the human, the percentage reabsorption of individual amino acids varies widely. Cusworth and Dent (180) found that the percentage reabsorption of histidine (90 to 95%) was constantly lower than that of other amino acids. Thus glycine reabsorption ranged from 95 to 98% and serine was about 98%; for most of the other amino acids, percentage reabsorption was 99%. The relatively low histidine percentage reabsorption could be explained by its having a low affinity for the neutral transport system. This possibility is supported by findings in the dog (160, 188). In these studies, histidine reabsorption was more inhibited by alanine, glycine, and phenylalanine infusion than was the reabsorption of any other neutral amino acid. Also, histidine, as an inhibitor, was the least effective of all the neutral amino acids studied. To a lesser extent, the same is also true of glycine. Table 1 shows a more complete list of individual endogenous amino acid clearances in humans, as reported by a number of authors. In the lowest row are shown mean values ± SD for nine subjects studied with ion-exchange chromatography. It can be seen that only glycine and histidine clearances were appreciably greater than 1 mI/mn, which indicates substantially less than 99% reabsorption. In the dog (98) the percentage reabsorption of nearly every amino acid exceeded 99%. Nevertheless, the percentages of histidine and glycine reabsorbed were only 97.6 and 98.4%, respectively, which shows the same general trend as in man, allowing for the greater overall efficiency of reabsorption in the dog. Although clearance data provide the best means for comparing the renal handling of various amino acids, some information can be obtained by examination of urine/plasma concentration ratios (u/p) for the various amino acids. Evered (107) compared the u/p ratios of individual amino acids for various species. As could be expected, the u/p ratios of histidine and glycine in man were much larger than those of the other amino acids. The u/p ratios for glycine were relatively large also in the rat, sheep, and cow. However, the u/p ratio for histidine was not enhanced in rabbit, cat, rat, sheep, or cow-indicating a possible species difference in the transport of this amino acid, at least. Exogenous amino acid clearances-the titration curve and Tm. To gain a more complete picture about the kinetics of the renal handling of amino acids, it is necessary to study excretion and reabsorption patterns, not only at endogenous plasma amino acid concentrations, but also at exogenous levels. Excretion curves obtained in this way are referred to as titration curves by analogy with similar curves obtained during the study of the renal handling of glucose (48, 49). In these experiments, glucose filtered load was plotted against glucose reabsorption and the point beyond which reabsorption became constant was referred to as the transport maximum (Tm). Such curves of reabsorption vs. load were termed “titration curves” since it may be considered that the tubules have been “titrated” to a saturation value of transport by raising the load. Using a similar technique, Pitts (214, 215) infused a mixture of amino acids into dogs and obtained a titration curve for total a-amino nitrogen that had a demonstrable Tm. In man, the curve does not show a definite Tm, although a divergence in the percentage of a-amino nitrogen reabsorbed was found as the filtered load increased (216). This has been taken as indicating that reabsorption was approaching a Tm value. The titration curves for dog and man are shown in Figures 4 and 5, respectively. The apparent simplicity of the a-amino nitrogen titration curves disappears when one comes to consider the renal handling of the amino acids individually. Many authors have obtained such “titration curves” for individual amino acids; in some cases, they observed a true Tm phenomenon, in others there was no tendency toward a Tm value whatsoever, and in yet others there was a progressive decline in percentage amino acid reabsorption without, however, any tendency to reach a maximum reabsorptive rate. Table 2 summarizes the available data for a large number of amino acids whose titration curves have been studied. From an inspection of the table it can be seen that there are numerous discrepancies among the various reports. There seems to be general agreement that a transport maximum phenomenon can be observed for L-arglnine5 (in dog and rat), L-alanine (in cat and dog), L-aspartic acid (in dog and rat) and L-proline and L-hydroxyprohne (in man). The discrepancies among the various studies may partly result from difficulties in determining individual amino acids. However, even when one considers only those studies in which column chromatog- ‘It is interesting to note that Gerok and Gayer (92) found that the argilline Tm in the dog could be abolished by the simultaneous infusion of either were inclined to attribute concentration of arginine histidine or glutamic this to a metabolic within renal tubular acid. lowering cells. They of the Whatever the explanation, if the phenomenon is widespread, it may well account for the discrepancies seen among the various papers in which amino acid Tm’s have been studied. C E 0 C C LU 0 I- z 0 z AMINONITROGEN FILTERED, (rrmol/rnIE) Fig. 4. Total a-amino-nitrogen plotted against the filtered load of amino-nitrogen dog undergoing infusion reabsorption intravenous and excretion in a with gycine [Taken from Pitts (52,214)] if o E -_____ ---#{149}------ - -. -. . _______ ___________ - A ‘- .---, p 0- 5------U. - -o K U .v 0 Filtered a A: ,. - 5 - - Vv 10 15 amino -N (mg/mm/I, Fig. 5. Total a.amino-nitrogen reabsorption ‘0 25 73m2) and excretion (ordinate) plotted as a function of filtered load of aamino-nitrogen (abscissa), in human subjects undergoing infusion with casein hydrolysate Top: Filtration. Middle: reabsorption. difterent symbols are for different Stalder et al. (216)] Bottom: excretion. The individuals [Taken from raphy was used for amino acid analysis, discrepancies still exist. Thus, Gerok and Gayer (193) failed to observe a Tm for L-glutamic acid in the dog, whereas Webber (158) observed one. It is possible that Gerok and Gayer may have failed to increase the plasma concentration sufficiently, although one cannot readily calculate the filtered load from their data. Other discrepancies may be due to interspecies differences. The shape of the “titration curve.” The titration curve of total a-amino nitrogen reabsorption after glycine infusion is shown in Figure 4 [(taken from Pitts (52, 214)]; the titration curve of arginine reabsorption obtained in the chromatographic study of Gerok and Gayer (193) is shown in Figure 6. In both cases, the reabsorption curve keeps pace initially with filtration and, as the Tm is approached, the curve bends gradually. This region of bending in the titration curve, is known as CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 257 #{149} .-I (‘4 ,..4 00 (N) #{149} (‘4 (‘4 (‘4 (N) CO C) CO I (‘ #{149} o \i CO N. CO N. (4) (‘4 (‘4 (‘4 14) #{149} -‘ CO CO CO c -u UN. ci)_ 9)Cj) (4 N. ,.4 (N) = (N) (‘4 . .ic l (‘4 (N) d (“4 #{149} #{149} (N) (4) .I #{149} CO N. (‘4 #{149} . CO (.O U i (fl ‘O #{149} C) #{149} (‘4 00 O .-4 (‘4 CO (‘4 eN, (N) (‘4 E 0 N- (‘4 -4 LCO U) . -4 CO . (‘N) (N) . . . Cj)Cj) CO (‘4 . . .E (I) (‘4 V a. o (“4 CO CO (N) .1. ‘-4 -4 . C) .4 - . (N) (4) . (‘.4 (N) (‘4 (‘.4 ‘-I (‘p) (N) ,4 U) (‘4 (‘4 N. #{149} (4) . . . N. 0) U, 0)1 LC)N. o 0) 0 L4)N. E c?O) U I N. I (“400 CO - U LC)rN) CO - (Y) U) ,.-(LC) LLi) CO #{149} dd .; C) N. ,-ILO N. U) (Y)(’J . #{149} . C #{149} #{149} .b U) CO ,-( . d,- .. o - = U U 0.’; eN) (‘.4 -4 . dd ‘- ‘ (‘4 ND U) N. #{149} #{149} #{149} #{149} ,-I . ‘-I (‘4 (4) 0) (‘4 -4 L) ‘-4 (4) U E U) a. U) ,.. .-4 .-i = CO (N) #{149} V LL1) d d #{176}.‘ ,-‘i #{149} . 0#{149} ,-l .. (‘4 (N) N. (‘4 ND #{149} C -4 (‘4 #{149} I- CO 0) d - ,-iO) .. C ,-i CO (N) N. #{149} LC) ND - .5 . I(N) 0 (N, C _ C 0 .2 0 i ( #{149} .E . cC’- ow E 0 - #{149}, E w - .2 C 0 E E w b E E - C - ‘ a,-- E E Qu,EcC ,40 #{176}E#{176} CE#{176} vEcC owEcC uw 0 N. LcDU) -4 -4 G) N. - N. c E c -‘- -cCW 0 .5O cE#{176} ouEcC L6Q 0 CE#{176} ouEcC -ow dQ- 0) -4 258 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 ‘-N. 0 DcCC CE0 (“4 C 0c0 ouEcC -a,w ‘OCO -4 X a -cCs.w 0 (1) oD w 2E E fg cCE#{176} .2 ‘-4 -4 - 0 i.oC. c’4wE .o() c (‘4 -4 CO (‘4 CO I ND V C (N) U (‘4 ,-4 C U) U -4 I CO - N. 00 C . C V 5; I N. 0) C U) (‘ -4 C C 0) (N) C C N. e C 0) (N) ‘-4 C I (N) CO (N). C ‘- C - d (‘-1 r C CO ) d C CO C CO 5;c ND 1 . C N. (N) U) C C d C) (N) (‘3 I C #{149} C N. I ‘4 L I N. (‘4 C ‘-4 t4. (.0 I C d 0) C (N) 0 . 9)5; C ; U) (N) (“4 N. C) 5; 00 C #{149} N. c - C C C I(N) .N. .-4 (‘4 “l#{149} C ND C d ND - ; C C -H d . C 9; V C . C 0) C C CO C .. C C C U) #{149} d V C. U) CO U)e)) U) C . C Q. . C? c’. (‘4 0 N. -.4 C C -4 0) CO #{149} #{149} CO d Ccj (‘4 . (‘4 “ -4 ‘t V (N, 0) - C? ,i V -l eN, 4 (‘4 . C C C ND N. U) - 0) -4 U) -CO (‘4 . C ND 0) ‘- #{149} C d C (N c’.J. 5;s CO d (N) ‘ C . C C q -H ‘) ND ; C ,-4 ND -4 CO (‘4 I C (N, o 4N. (Ni) C #{149} 4 C ‘-4 . -H .-. C ND N. (N) 00 ND - V C #{149}r V ND - -l (j #{149} N. c4#{149}C9 #{149} #{176} d U) (N) ,4 . .. (N, ND -.4 C ‘ C #{176} U) CO .. C 5; d N. d d 0) CO C ND 0) , #{149} #{176} d ND CND (‘4 0) - ‘-‘ (.0 ,, -.4 (‘.4 5;5; ‘ 0) ND ,- U) “ (.0 CO 5;5; 0) C ,-4 N. U) d d d (“4 C (‘4 V 5;c; V I ND - C “ N. (N) C ‘- C (‘4 . C . 00 5; t)) CO CO 0 L0 C #{149} d OC; (N) ‘-4 C (‘4 -4 N. N. C #{149} C d V C 5;9 (.0 - #{149} C N. C (‘4 #{149} 9 -H r- . 14 (N) d d CO 0) ‘-4 0) 5; CO C C 9; (N, U) C 0 N. #{149} 5; -4 0) (‘4 0 U) . C C C (N.e V C C U V (‘4 C V (“4 C? C ND (‘4 00 -i 9;- (‘4 C ‘ C) (‘4 ( C >N C C #{149} C C C o- 0 (‘3 I#{149} IC ..!4 C4E. 00 2 aIC.C #{176} E ICN: . IC E 0 U) ND C N. U) C -I CO U) U) CO c o 9;C 00 0) ND 5;C . C #{149} - 5;’- U) -r C - 0 CE ow E co LcD-O C) -4 E t.6DC -4 0 X Q 0 CE#{176}(N) t owEcCN: - NDD(‘4 IC a. - Q CO -4 CC 5. C a, a, #{149}0 0C a, IC w ow C )< a, a, E a, C C CE#{176}(N) owE (ON: - 0 IC .0 C eli C C G) C 0 C a,IC-c CE#{176}-. 5.#{149} 0 #{149} (0th w 0 (Ca, fliL G)IC-CE owEIC EC N .2 CI U) CO E C CCE CC COC . C 0 N. C d d C 0 I. d C C?”CO-ND C) -H I N. U) CC 0. #{149} C ,-4 #{149} ND CC 0#{149} 9(N) U) C C -cC . N. C U) IC .00EC -4 U) C - .4D- (N) a, ‘-4 E ICE D- U) C) CO . - C U) . #{149} #{163} a. (‘ (0 (I) - 0.’ C 0.N. - o (ON: - a, a, .0 CCY) - ‘4 0 C (N) w - E z CO -4 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 259 Table 2. Transport Maxima in Renal Reabsorption of Amino AcidsN Amino acids DL-Alanine Tm demonstrated dog’ (215) cat’ (57) L-A Ianine Tm possibIV No Tm dog’ (233) cat’ (156) dog’ (138) D-Alanine dog’ (138) cat’ (156) L.Arginine dog’ (215) dog’ (230) dog’ (231) rJL#{149}Aspartic acid L-Aspartic acid D-Aspartic Glycine L-Glutamic acid acid dog’ (138) dog4 (97,193) rat5 (6) dog’ (23(3) dog4 (155) rat5 (102) dog4 (158) dog’ (214) dog’ (738) dog’ (233) dog’ dog’ (215) dog’ (230) dog4 (158) rat5(102) L-Histidine DL#{149}Isoleucine L-Leucine L#{149} Lysine DL-Methionine L.Methionine DL-Phenylalanine L-Proline L-Hydroxyproline DL-Threonine L-Tryptophan OL-Valine (230) rat4 (96) man4 (217) dog4 (97, 193) cat’ (156) rat’ (6) dog4 (97, 193) dog’ (231) dog4 (97, 193) dog’ (184) dog’ (232) dog’ (232) dog’ (230) dog’ (230) dog’ (231) rat5(6) rat’ (6) dog’ (184) dog’ (159) dog4 (97, 193) dog’ (231) cat’ (57) dog’ (234) rat’(6) man4 (206) man4 (209) dog’ (234) dog’ (184) dog’ (184) dog’ (232) Superscriptsindicatethe method of amino acidanalysisused: 1 = totala-amino nitrogen;2 = microbiological assay;3 = enzymic assay; 4 = paper or column chromatographic separation,followedby use of the ninhydrin reaction; 5 = radioisotopic labeling. Numbers in parentheses are reference numbers. In the case of the glucose titration curve, the splay is slight, and has been attributed to variations in the ratio of filtered load to tubule size in individual nephrons [“glomerulotubular balance” (51)]; this concept is adequate to explain the small degree of splay in the glucose titration curve. However, the much wider splay of amino acid titration curves can obviously not be fully explained on the same basis. Thus, for amino acids, some further explanation of the splay is needed. One possibility is the influence of the equilibrium constant of the transport reaction, on the shape of the titration curve. In Figure 7 [taken from Sugita (217)], the shape of the titration curve has been shown for equilibrium constants varying between zero and infinity. As the equilibrium constant ap“splay.” 260 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 infinity, there is very little splay in the titration curve. Indeed, in the case of glucose, which exhibits only minor degrees of splay, it has been shown by micropun cture studies (218) that the Michaelis-Menten constant (Km) of membrane transfer approached zero, indicative of an equilibrium constant approaching infinity. The very wide splay in the titration curve of amino acids could well be due to a relatively low equilibrium constant for membrane transfer. Such a possibility is supported by the finding of high Km (indicating low equilibrium constant) for amino acid accumulation systems in the kidney cortex slices (84, 109, 219). Bergeron and Morel (6) have recently published titration curves for neutral amino acids obtained proaches techniques are essentially different. In clearance experiments, the load of amino acid is increased by increasing the amino acid concentration of both 0 plasma and glomerular filtrate, whereas in the micropuncture experiments only the concentration .0 of intratubular fluid is raised. This would mean that the transtubular amino acid concentration a gradient is far greater in the micropuncture experiments than in the clearance experiments and could conceivably enhance the operation of any passive, gradient-dependent, reabsorptive process. The possibility of the existence of passive processes in amino acid reabsorption has been Amount filtered suggested by a number of recent mieropuncture -NH2-NI.’n/lOOnN GF studies. Freedman and Young (8) showed that a Fig. 6. Renal tubular reabsorption of L-arginine with in#{149} large component of amino acid reabsorption could creasing filtered load not be inhibited by very high concentrations (40 A dog was infused intravenouslywith L-arginine; reabsorption mmol) of cyanide in the tubule perfusion fluid. has been calculated by subtracting the amount excretedfrom Similar results were obtained by Silbernagel and the calculated filtered load (broken line). A definite Tm is shown although splay is rather wide. i-arginine was measured by the Deetjen (9) who were unable to inhibit glycine ninhydrin color reaction after separation by column chromatog. reabsorption with dinitrophenol or cyanide (partial raphy. [Taken from Gerok and Gayer (193)] inhibition was possible, 1)eetjen P., personal communication, 1970). Validity of the concept of Tm. Numerous workers have re-examined the manner in which the kidney handles glucose and have demonstrated that Tm the so-called Tm for glucose reabsorption varies according to the GFR (220-223). Furthermore, 0 many workers have shown that saline loading and N, plasma volume expansion can influence the Tm value for reabsorption of substances such as gluKrO cose, phosphate, magnesium, and p-aminohippuric acid (224-229). Although the influence of .Zm Fill. Load these factors on amino acid reabsorption has not yet Fig. 7. The influence of the equilibrium constant for been studied, it seems likely that similar anomamino acid reabsorption on the shape of the titration alies would be found. These observations call into curve question the whole concept of Tm. ‘flQ “N 0 As the equilibrium constant (K) approaches infinity the splay becomes progressively narrower. (Note that K is the reciprocal of the Michaelis-Menten constant, K.’.) Glucose, which has been shown to have a high equilibrium constant (218) has, indeed, a narrow splay (49) whereas many amino acids which have low equilibrium constants (84, 109, 219) show wide splay or no Tm at all. [Taken from Sugita et. at. (217)] by microinjection of individual nephrons. They plotted their data on log-log paper. However, when their curves are replotted on linear axes the shapes of the titration curves are found to be very similar to the slowly rising curve of an active transport process with a low equilibrium constant. Thus, for these amino acids at least, it is possible that the splay in the titration curve reflected a low equilibrium constant. On the other hand, when the basic amino acids were studied in this way, Bergeron and Morel (6) obtained titration curves with clear Tm’s and some degree of splay, indicating higher equilibrium constants for these amino acids. Of course, one must be wary of extrapolating conclusions from these micropuncture results to clearance experiments, as the conditions of the two We thank A. Cameron for his help in the preparation of the bibliography, K. Davies for her careful preparation of the typescript, and F. D. Weber for his help. We thank especially Mrs. H. Cooke for her discussions and criticisms of the manuscript. We also thank the National Health and Medical Research Council of Australia for the award of a stipend to one of us (B.S.F.) and for support of the authors’ experimental work. References 1. Johnstone, R. M., and Scholefield, P. 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