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Uptake of glycine by human kidney cortex
Uptake of Glycine by Human Kidney Cortex Karl S. Roth, Philip Holtzapple, The transport of glycine was investigated in histologically normal adult human kidney cortical slices. Uptake occurs against a gradient and shows concentration dependence. Kinetic analysis reveals two systems for transport of glycine with apparent transport K, values of 0.511 and 34.2 mM. Glycine transport on the high-K, system is competitively inhibited by 50 mML-proline. Transport inhibition on the low-K, system could not be directly evaluated, but on theoretic grounds appears not to be inhibited by L-proline or hydroxyproline. Alpha-aminoisobutyric acid, valine. and thioproline are also shown to inhibit glycine uptake. Low medium sodium or anaerobic incubation depress the uptake of glycine. These observations are consistent with previous reports of glycine transport in rat kidney and support the proposals for the mechanism of familial iminoglycinuria based on in vivo investigations.
Myron Genel, and Stanton
in man.34 It was concluded that there were two separate transport systems for proline in human kidney and, as a result of the extensive degree of metabolism of proline by human kidney tissue, that intracellular disposition of infused proline might affect the membrane transport of this substance in a secondary fashion. Inasmuch as there are no data available in the literature relating to the transport of glycine in human kidney in vitro, we have investigated the latter process and studied the nature of the transport interactions between glycine and proline in vitro. The results of these studies constitute the basis for this report. MATERIALS
a part of a wide H YPERGLYCINURIA variety of clinical entities including the is
physiologic hyperglycinuria of the human newborn’.2 and the apparently benign condition known as familial iminoglycinuria.3 In both of these states, glycine excretion is far in excess of that seen in normal adults and is accompanied by hyperexcretion of the imino acids proline, and hydroxyproline, invariably without plasma elevations of these amino acids. Thus, the hyperexcretion of the amino acids has been attributed to alterations in uptake characteristics of the proximal tubule, since it is at this level that 95%-98’S of amino acid reabsorption normally occurs. Changes of this sort in normai function can provide enormous insight into the mechanisms of membrane transport of amino acids in the kidney, and for this reason both neonatal and familial iminoglycinuria have attracted the interest of investigators beyond their clinical importance. In vivo amino acid infusions in man3” ‘have led to the conclusion that familial iminoglycinuria results from an inherited deficiency of a shared, high-capacity transport system for glycine and the imino acids. Affected individuals retain their ability to transport these substances on a greatly reduced scale on substrate-specific, low-capacity systems also present in the kidneys of normal individuals.’ A previous publication* from this laboratory has related in vitro proline transport studies in human kidney to in vivo proline infusion studies Metabotism, Vol.28, No. 6 (June),1979
Segal
AND
METHODS
Kidneys were obtained from several adult patients undergoing nephrectomy for either localized intrarenal or extrarenal pathology. Disease-free cortical areas of the kidney (determined by histologic evaluation) were used for preparation of the slices. The techniques for determining amino acid uptake in human kidney cortical slices have been published elsewhere? Slices weighing 4-8 mg were prepared with a Stadie-Riggs microtome. Each slice was incubated in 2 ml Krebs-Ringer bicarbonate buffer (pH 7.4) in a metabolic shaker at 37°C under an atmosphere of 95% Or-5% CO, with 0.1 &i 2-r4C-glycine, specific activity 5 mCi/mmol, and appropriate amounts of unlabeled glycine. At the end of the incubation, the slices were rinsed rapidly in physiologic saline, blotted on filter paper, and weighed, and the soluble radioactivity in the slice was extracted into 1 ml water by heating in a boiling water bath for 6 min. Aliquots (0.2 ml) of the tissue extracts and the media were added to scintillation vials with 10 ml toluene-ethanol (72128) that contained 3% Liquifluor (New England Nuclear, Boston, Mass.) and counted at 90% efficiency in a liquid scintillation spectrophotometer. Total tissue water, expressed as the percentage of total tissue weight, was determined for each surgical specimen by
From the Division of Biochemical Development and Molecular Diseases, Children’s Hospital of Philadelphia. and Departments of Pediatrics and Medicine. University of Pennsylvania School of Medicine, Philadelphia. Receivedfor publication November 21. 1978. Supported by National Institutes of Health Grant No. Ah4 10894. Dr. Roth is a recipient of Research Career Development Award I KO4HDOO257-01. Address reprint requests to: Dr. Karl S. Roth, Children’s Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, Pa. 19104. 0 1979 by Grune & Stratton, Inc. 002&0495,‘79/2806--00I 2$OI .00/O
677
670
ROTH ET AL.
the difference in weight of three slices after blotting and drying at 105“C under vacuum for 24 hr. Extracellular fluid space (ECF) estimation for each specimen was determined by incubation with 0.5 pCi inulin-“C-carboxyl, specific activity 3.23 mCi/g, according to the method of Rosenberg et al.” All radioisotopes were purchased from New England Nuclear Co., Boston, Mass. All amino acids were obtained from Schwarz-Mann Research Co., New York, and were of the highest purity available. Uptake of glycine is expressed as the ratio of counts/min/ml of intracellular fluid (ICF) to counts/mitt/ml ECF, i.e., the isotope distribution ratio, corrected for the isotope trapped in the ECF.s Total tissue water averaged 82.0 + 0.4%, and ECF values averaged 49.2 + 0.5%. Because the conversion of 2-‘4C-glycine to serine and its oxidation to 14C02 has been shown to proceed very slowly,“~‘* no correction has been made for oxidative loss of isotope in the data analysis, as was done for proline uptake.’ However, data for concentration-dependent studies have been corrected for a diffusion component.”
,13c ,12c 110 100 90 60
V
70 60 50 40 30 20
RESULTS
Cellular
IO
Uptake of Glycine
-
The uptake of 0.165 and 15 mM glycine by human kidney tissue is shown in Fig. 1. At the lower substrate concentration there was rapid uptake, with achievement of a steady state by 90 min of incubation. At 15 mM glycine, a steady state was achieved by 30 min of incubation, which was one-third that reached by 90 min at
2
3
4
5
)__
6
,
7
6
9
Fig. 2. Hofstee transformation of concentrationdependent glycine uptake in human kidney cortex (apecimen Al. Each point is the mean value obtained from 6-mg slices incubated for 30 min in triplicate.
the low substrate concentration. Studies of pH dependence of glycine transport at both substrate concentrations showed relatively constant uptake over a broad pH range of 6.0-7.4, with a rapid decrease in uptake above 7.4. Substrate
Concentration
Studies
There is clear concentration-dependence of glycine uptake by human renal cortical cells. Analysis of the process indicates two separate modes, shown in Fig. 2 as a Hofstee plot (V versus V/S) of the data derived from slices of a single human kidney (specimen A). The apparent transport K, and V,,, values observed for each human specimen are shown in Table 1.
Y
I
20
1
40
I
I
60
00
TIME
1
100
4
120
(MINUTES)
Fig. 1. Time curve of 0.166 (0) and 16 fm) mM glycine uptake by human kidney alices. The symbols represent the mean uptake in five different specimens incubated in duplicate flasks. The brackets represent the SE of the meen. Distribution ratio is deffned as the ratio of cpm/ml intracellular fluid to cpm/ml extracellular fluid.
Glycine-Proline
Interactions
Figure 3 shows the Lineweaver-Burk analysis of the effect of 50 mM proline on glycine uptake over a concentration range of 5-50 mM, observed in slices from two separate specimens. Proline is clearly a competitive inhibitor of glycine uptake in this concentration range, with a calculated Ki of 26-34.7 mM.
GLYCINE UPTAKE BY KIDNEY CORTEX
Table 1. Apparent K, and V,,
679
of Glycine Transport in
Human Kidney Specimen
K ml
A
40.0
K -z
V m.w
200.0
2.22
533.3
2.00
B
62.5
0.25
C
-
0.87
D
20.0
0.286
E
14.3
0.91
Incubations were for 30
V -I
0.24
5.00 100.0
2.22
44.4
4.00
min. Radioactive amino acid was
diluted with unlabeled compound to give at least five appropriate concentrations between 5.065 and between 0.22
and 1.065
and 50 mM to determine I<,, mM to determine K,,.
In some
experiments the entire range of concentrations was examined, whereas
in others only one portion was determined.
expressed in mm/l and V_
as mm/l/30
for K,,. K,,. V,,,..,. and V,,,.,* are 34.2, respectively.
K,
is
min. The average values 0.51 1.219.4,
and 3.09, Fig. 3. Effect of 60 mM L-proline on concentrationdependent uptake of glycine by human kidney cortex. Data obtained from two specimens D W and E 10) are shown separately. Slices were incubated with glycine (6.00~60 m/W) with (W. 0) and without 10 m&f L-proline for 30 min.
The effect of the addition of proline on the high-affinity, low-capacity system could not be reliably evaluated because even at very low substrate concentrations uptake is more or less evenly divided between the two systems (Fig. 4). In an effort to circumvent this problem, the 30-min uptake of 0.065 and 0.165 mM glycine was examined with the addition of 10 and 25 mM proline. These data are compared with the control values in Table 2. Using the kinetic parameters experimentally determined for each transport system, the theoretic velocity of uptake was calculated according to the MichaelisMenten formulation, and the percentage of the total uptake contributed by the low K, system in the presence of 10 and 25 mM proline compares favorably to the expected values if no uptake were occurring via the high K, system.
Efects of Inhibitors Oxygen Derivation
and Sodium
or
The effects of other amino acids and of removal of sodium or oxygen from the system are shown in Table 3, for various samples of human kidney. In two specimens (A and D), there was significant inhibition of the 30-min uptake of 0.065 mM glycine by 25 mM LYaminoisobutyric acid (AIB) and valine, while 25 mA4 lysine did not appear to interact significantly with transport of glycine at these low substrate concentrations. In specimen D, 25 mM thioproline, a metabolizable proline analog, caused a significant decrease in 0.065 mM
100 t
Fig. 4. Comparison of theoretical velocities of glycine uptake on the high (0) and lowaffinity (0) systems in human kidney slices. Data are plotted as the percentage of the total velocity versus the various substrate concentrations, expressed as m/liter. Kinetic paremeters shown in Table 1 were inserted into the Michaelis expression for velocity.
% % w g K
0.5
I.5
I.0 GLYCINE
(mM)
2.0
660
ROTH ET AL.
Table 2.
Interactions of lmino Acids With Low-Capacity Glycine Transport
COtltCOl
0.065
mM
6.713
Prchne ( 10 mlul
Thsaetlc
DR + 0.270
0.349
146%)
Iv= 0.165
mM
6.40
+ 0.248
”
DR 3.88
? 0.225
l6
0 754 142%)
5 26 2 0.320
(33%)
DR 3.80
+ 0.527
0.247
10
1.056
OH-Pro (10 mM)
O&wed
”
132%)
DR 5.82
+ 0.095
0 376 149%)
iv=5
3 98 -t 0.52,
Observed ” 0.259
4.91
? 0 582
(34%)
,v=3
N=3
_
(57%)
OH-Pro (25 mMI DR
Observed ”
0 610(45%)
Iv=3
Uptakes w#m performed 8s descrnbsd I” text and carried out fa ccnxntrat~ons
0.252 N=
N=7
at each of the two #y&e
Prolins 125 mMl
Observed V
--
Iv=3
30 mm. Dstribution
rawx
was calculated according to the Michaelis-Mentefi
(DRI are gown as the average + SE fw N detwnmatvzms.
The theorst~c velouty of uptake
equatto”:
glycine uptake, while 5 mM thioproline had a smaller effect. Deprivation of sodium and oxygen were both inhibitory at this low glycine concentration. These observations are consistent with those made for uptake of 10 mM glycine concentration, using higher (50 mM) concentrations of the various amino acids.
iminoglycinuric syndrome is due to a homozygous state of deletion of a high-capacity, common transport site, with retention of a separate, low-capacity, substrate-specific site.’ This hypothesis was tested in human kidney, as reported in an earlier study from this laboratory, using proline as the substrate.* The results of that study led to the suggestion of extensive proline metabolism as a regulating factor in proline uptake by the human kidney. The present study thus assumes added importance in the definition of the iminoglycinuric defect. We have now shown that the uptake of giycine by human kidney is active and concentration dependent. At a substrate concentration in the physiologic range (0.165 mM), a steady-state concentration gradient of 12 is achieved by the human renal tubular cell. In the rat kidney, in vivo concentration gradients in the same range have been reported by Roth et al.” and Toback et al.‘* An intracellular:extracellular gradient in human kidney in vivo has been reported for
DISCUSSION
Early studies on renal amino acid excretion rates throughout development,’ together with the description by Joseph et aLI of a family having several members with large quantities of proline, hydroxyproline, and glycine in the urine, led Striver et al.3 to propose that these three amino acids share a common transport system in the renal tubule. This system has come to be known as the iminoglycine system, and has been the subject of numerous in vitro studies2,‘2,‘5*‘6 using rat or rabbit kidney. These observations, together with the results of amino acid infusion data in humans,‘” have led to the theory that the
Table 3. Distribution Ratios of Glycine Uptake After Addition of Various Amino Acids, Sodium Deprivation, and Anaerobiosis GlVCl”0
Th,opral,“s
CO”CWtratW” ImMl 0.065
NB +-Free Spsclllm”
ConeJo,
AI6* 2.18
w,ne*
+ 0.41
t
0.40
6.09
t
T”*
A”aWCbi0sls
5.60
-t 0.32
6
7.20
I! 0.64
3.76
t
0.43
5.33
t
C
6.22
f
2.25
i
0 37
2.57
? 0.16
D
6.13
-+ 0.52
3.71
*
0.22
2.63
+ 0.27
1.55
t
0.18
(54.81
6.4
+ 0.3,
+ 0.20
162.2)
I97
6.23 11
2.02
f
t
1.04
+ 0.07
4.10
+ 0.43
0.19
(52.7)
t
0.2,
(73.11
3.1,
t
04,
(36 31
(109.51
3.26
3.0
0.19
3 10 + 0.3,
? 0.32
+ 0.17
t
176.6,
4.72
2.16
0 29
79tO25
178.81 1.76
50 mM
1108.9)
0.48
(46 61 A
173.61
26 mM
5rnM
0.50
A
(38.91
10.065
4.12
L”W.3.
4.40
*
0.23
1107.3)
0 17
2.73
+ 0.20
1.46
+ 0 1, 1.06
2 0.02
126.9)
GLYCINE UPTAKE BY KIDNEY CORTEX
proline.’ Thus, iminoglycine accumulation by the human kidney slice reflects the in vivo state of the kidney. Analysis of the glycine entry kinetics in human kidney shows at least two transport systems, for which the apparent transport K, values differ by sixty-fold. In the concentration range of the high-capacity transport system, proline is a competitive inhibitor of glycine uptake. This observation is consistent with the proposed mechanism for the appearance of glycinuria in humans infused with large amounts of proline, whereby the high concentration of proline in the tubular lumen competitively inhibits tubular reabsorption of filtered glycine.5*7 However, even in iminoglycinuric subjects, infusion of proline cannot reduce tubular glycine reabsorption below a fixed minimum. This observation was the basis for the proposition of a glycine-specific transport system that could not be inhibited by proline or hydroxyproline, and that this accounted for the minimal tubular glycine reabsorption retained. Our data are consistent with the presence of a low-capacity, high-afficinity glycine transport system in the human kidney. The apparent transport K, of this system (0.5 1 mM) would suggest that it best subserves glycine uptake in a physiologic concentration range. However, the data derived using the Michaelis-Menten formulation indicate that in this range, both systems contribute almost equally to total uptake. Unfortunately, because of the large contribution to total uptake made at these concentrations by the high-capacity transport system, it was not possible to evaluate directly the nature of the interaction of proline with this low-capacity system. Nonetheless, using the basic assumption that high proline concentrations and low glycine concentrations might create a situation in which the observed velocity of uptake was primarily due to the low-capacity system, it was possible to show good agreement between the calculated velocity for this system and the velocity observed experimentally. Thus, it appears that the low-capacity glycine transport system is not inhibited by 10 or 25 mM proline, a conclusion that lends support to the proposed mechanism for residual glycine reabsorption by the renal tubule during proline infusion.’ Of several other amino acids tested, our data
681
suggest that only cr-aminoisobutyric acid, valine, and thioproline are consistent inhibitors of glycine uptake in human kidney. Interactions between AIB and other neutral amino acids have been reported in mammalian kidney by others.‘9920Striver and Mohyuddin demonstrated that AIB was a competitive inhibitor of proline uptake at low substrate concentrations.*’ Inhibition of proline uptake in human kidney by thioproline has been reported, and attributed to a dual effect of proline, on intracellular metabolism as well as on the membrane transport site.’ Although we did not study the effects of thioproline upon intracellular glycine metabolism, it is possible that alterations in the latter played a role in the transport inhibition of glycine as well. Valine, too, has been reported to inhibit proline uptake in human kidney.8 The similarity of glycine and proline transport in the presence of these inhibitors is further evidence in support of the hypothesis that the two share a common transport system. Equimolar substitution of the medium sodium by Tris and anerobic incubation significantly depressed glycine uptake in a fashion very similar to that reported previously for proline in human’ and in rat kidney.*’ The observations recorded here support Scriver’s contention7 that glycine reabsorption in the human kidney is subserved by a high-capacity system shared with proline and hydroxyproline, as well as a glycine-specific low-capacity system. It is noteworthy, however, that the similarity of the apparent transport K, of the high-capacity system and the calculated Ki for proline do not support the concept that glycine has the lowest affinity for this system. Although some workers have expressed concern that substrate uptake in the slice model may represent peritubular, rather than brushborder, transport events,22*23our data is completely consistent with recent observations made in adult rat renal brushborder membrane vesicles.24 In the rat vesicle, two glycine transport systems were demonstrated, with competitive inhibition by proline on the high-capacity system. These workers were able to show noncompetitive inhibition of glycine uptake by proline in the low substrate concentration range, but speculated that this might reflect the large contribution to total uptake made by the highcapacity system, even at these low concentrations. Thus, even with a subcellular purified
ROTH ET AL.
682
preparation, there is enough overlap of the two glycine transport systems so that the highaffinity mechanism cannot be studied in isolation. It is possible that clarification of the nature of the proline-glycine interactions on the lowcapacity transport site will have to await isola-
tion of the carrier molecule itself. The evidence is sufficient, however, to state that the postulated mechanism for the iminoglycinuric syndrome is probably very close to actual events taking place in the genetic direction of membrane transport.
REFERENCES 1. Brodehl J, Gellissen K: Endogenous renal transport of free amino acids in infancy and childhood. Pediatrics 42:395-404,1968 2. Baerlocher KE, &river CR, Mohyuddin F: Ontogeny of iminoglycine transport in mammalian kidney. Proc Nat1 Acad Sci USA 65:1009-1016, 1970 3. Striver CR, Efron ML, Schafer IA: Renal tubular transport of proline, hydroxyproline and glycine in health and in familial hyperprolinemia. J Clin Invest 43:37&385. 1964 4. Striver CR, Goldman H: Renal tubular transport of proline, hydroxyproline and glycine. II. Hydroxy-L-proline as substrate and as inhibitor in vivo. J Clin Invest 45:135713651966 5. Striver CR: Renal tubular transport of proline, hydroxyproline, and glycine. III. Genetic basis for more than one mode of transport in human kidney. J Clin Invest 47:823835.1968 6. Rosenberg LE. Durant JL, Elsas LJ II: Familial iminoglycinuria. An inborn error of renal tubular transport. N Engl J Med 278:1407-1413, 1968 7. Striver CR, Chesney RW, McInnes RR: Genetic aspects of renal tubular transport: Diversity and topology of carriers. Kidney Int 9149-171, 1976 8. Holtzapple P, Gene1 M, Rea C, et al: Metabolism and uptake of t_-proline by human kidney cortex. Pediatr Res 7:818-825, 1973 9. Fox M, Thier S, Rosenberg L, et al: Evidence against a single renal transport defect in cystinuria. N Engl J Med 270:556-561, 1964 IO. Rosenberg LE, Downing SJ, Segal S: Extracellular space estimation in rat kidney slices using C” saccharides and phlorizin. Am J Physiol 202:800-804, 1962 Il. Rosenberg LE, Berman M, Segal S: Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney cortex slic=es. Biochim Biophys Acta 7 1~664-675, 1963 12. Hillman RD. Albrecht 1, Rosenberg LE: Identification and analysis of multiple glycine transport systems in isolated mammalian renal tubules. J Biol Chem 243:5566557l,l968
13. Akedo H, Christensen HN: Nature of insulin action on amino acid uptake by the isolated diaphragm. J Biol Chem 237:113-117, 1962 14. Joseph iale associent
R, Ribierre M, Job JC, et al: Maladie famildes convulsions. a d&but trbs precoce, une
hyperalbuminorachie Pediatr 15:374-387,
et une hyperaminoacidurie. 1958
Arch
Fr
15. Baerlocher KE, Striver CR, Mohyuddin F: The ontogeny of amino acid transport in rat kidney. I. Effect on distribution ratios and intracellular metabolism of proline and glycine. B&him Biophys Acta 249:353-363, 1971 16. Baerlocher KE, Striver CR, Mohyuddin V: The ontogeny of amino acid transport in rat kidney. II. Kinetics of uptake and effect of anoxia. Biochim Biophys Acta 249:363372, 1971 17. Roth KS, Hwang SM, London JW, et al: Ontogeny of glycine transport in isolated rat renal tubules. Am J Physiol 233:F24-246,
1977
18. Toback FG, Mayers AM, Lowenstein LM: Alterations in renal and plasma amino acid concentrations during renal compensatory growth. Am J Physiol 225:1247-l 251, 1973 19. Wilson OH, Striver CR: Specificity of transport of neutral and basic amino acids in rat kidney. Am J Physiol 213:185-190, 1967 20. Striver CR, Mohyuddin F: Amino acid transport kidney. J Biol Chem 243:3207-3213, 1968
in
21. Mackensie S, Striver CR: Transport of L-proline and alpha-amino isobutyric acid in isolated rat kidney glomerulus. Biochim Biophys Acta 241:725-736, 1971 22. Foulkes EC: Effects of heavy metals on renal aspartate transport and the nature of solute movement in kidney cortex slices. Biochim Biophys Acta 241:815-822, 1971 23. Wedeen nonmetabolized 512, 1971
RP, Thier SO: Intrarenal distribution of amino acids in vivo. Am J Physiol 220~5077
24. McNamara PD. Ozegovic B, Pepe LM, et al: Proline and glycine uptake by renal brushborder membrane vesicles. Proc Natl Acad Sci USA 73:4521-4525, I976
Myron Genel, and Stanton
in man.34 It was concluded that there were two separate transport systems for proline in human kidney and, as a result of the extensive degree of metabolism of proline by human kidney tissue, that intracellular disposition of infused proline might affect the membrane transport of this substance in a secondary fashion. Inasmuch as there are no data available in the literature relating to the transport of glycine in human kidney in vitro, we have investigated the latter process and studied the nature of the transport interactions between glycine and proline in vitro. The results of these studies constitute the basis for this report. MATERIALS
a part of a wide H YPERGLYCINURIA variety of clinical entities including the is
physiologic hyperglycinuria of the human newborn’.2 and the apparently benign condition known as familial iminoglycinuria.3 In both of these states, glycine excretion is far in excess of that seen in normal adults and is accompanied by hyperexcretion of the imino acids proline, and hydroxyproline, invariably without plasma elevations of these amino acids. Thus, the hyperexcretion of the amino acids has been attributed to alterations in uptake characteristics of the proximal tubule, since it is at this level that 95%-98’S of amino acid reabsorption normally occurs. Changes of this sort in normai function can provide enormous insight into the mechanisms of membrane transport of amino acids in the kidney, and for this reason both neonatal and familial iminoglycinuria have attracted the interest of investigators beyond their clinical importance. In vivo amino acid infusions in man3” ‘have led to the conclusion that familial iminoglycinuria results from an inherited deficiency of a shared, high-capacity transport system for glycine and the imino acids. Affected individuals retain their ability to transport these substances on a greatly reduced scale on substrate-specific, low-capacity systems also present in the kidneys of normal individuals.’ A previous publication* from this laboratory has related in vitro proline transport studies in human kidney to in vivo proline infusion studies Metabotism, Vol.28, No. 6 (June),1979
Segal
AND
METHODS
Kidneys were obtained from several adult patients undergoing nephrectomy for either localized intrarenal or extrarenal pathology. Disease-free cortical areas of the kidney (determined by histologic evaluation) were used for preparation of the slices. The techniques for determining amino acid uptake in human kidney cortical slices have been published elsewhere? Slices weighing 4-8 mg were prepared with a Stadie-Riggs microtome. Each slice was incubated in 2 ml Krebs-Ringer bicarbonate buffer (pH 7.4) in a metabolic shaker at 37°C under an atmosphere of 95% Or-5% CO, with 0.1 &i 2-r4C-glycine, specific activity 5 mCi/mmol, and appropriate amounts of unlabeled glycine. At the end of the incubation, the slices were rinsed rapidly in physiologic saline, blotted on filter paper, and weighed, and the soluble radioactivity in the slice was extracted into 1 ml water by heating in a boiling water bath for 6 min. Aliquots (0.2 ml) of the tissue extracts and the media were added to scintillation vials with 10 ml toluene-ethanol (72128) that contained 3% Liquifluor (New England Nuclear, Boston, Mass.) and counted at 90% efficiency in a liquid scintillation spectrophotometer. Total tissue water, expressed as the percentage of total tissue weight, was determined for each surgical specimen by
From the Division of Biochemical Development and Molecular Diseases, Children’s Hospital of Philadelphia. and Departments of Pediatrics and Medicine. University of Pennsylvania School of Medicine, Philadelphia. Receivedfor publication November 21. 1978. Supported by National Institutes of Health Grant No. Ah4 10894. Dr. Roth is a recipient of Research Career Development Award I KO4HDOO257-01. Address reprint requests to: Dr. Karl S. Roth, Children’s Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, Pa. 19104. 0 1979 by Grune & Stratton, Inc. 002&0495,‘79/2806--00I 2$OI .00/O
677
670
ROTH ET AL.
the difference in weight of three slices after blotting and drying at 105“C under vacuum for 24 hr. Extracellular fluid space (ECF) estimation for each specimen was determined by incubation with 0.5 pCi inulin-“C-carboxyl, specific activity 3.23 mCi/g, according to the method of Rosenberg et al.” All radioisotopes were purchased from New England Nuclear Co., Boston, Mass. All amino acids were obtained from Schwarz-Mann Research Co., New York, and were of the highest purity available. Uptake of glycine is expressed as the ratio of counts/min/ml of intracellular fluid (ICF) to counts/mitt/ml ECF, i.e., the isotope distribution ratio, corrected for the isotope trapped in the ECF.s Total tissue water averaged 82.0 + 0.4%, and ECF values averaged 49.2 + 0.5%. Because the conversion of 2-‘4C-glycine to serine and its oxidation to 14C02 has been shown to proceed very slowly,“~‘* no correction has been made for oxidative loss of isotope in the data analysis, as was done for proline uptake.’ However, data for concentration-dependent studies have been corrected for a diffusion component.”
,13c ,12c 110 100 90 60
V
70 60 50 40 30 20
RESULTS
Cellular
IO
Uptake of Glycine
-
The uptake of 0.165 and 15 mM glycine by human kidney tissue is shown in Fig. 1. At the lower substrate concentration there was rapid uptake, with achievement of a steady state by 90 min of incubation. At 15 mM glycine, a steady state was achieved by 30 min of incubation, which was one-third that reached by 90 min at
2
3
4
5
)__
6
,
7
6
9
Fig. 2. Hofstee transformation of concentrationdependent glycine uptake in human kidney cortex (apecimen Al. Each point is the mean value obtained from 6-mg slices incubated for 30 min in triplicate.
the low substrate concentration. Studies of pH dependence of glycine transport at both substrate concentrations showed relatively constant uptake over a broad pH range of 6.0-7.4, with a rapid decrease in uptake above 7.4. Substrate
Concentration
Studies
There is clear concentration-dependence of glycine uptake by human renal cortical cells. Analysis of the process indicates two separate modes, shown in Fig. 2 as a Hofstee plot (V versus V/S) of the data derived from slices of a single human kidney (specimen A). The apparent transport K, and V,,, values observed for each human specimen are shown in Table 1.
Y
I
20
1
40
I
I
60
00
TIME
1
100
4
120
(MINUTES)
Fig. 1. Time curve of 0.166 (0) and 16 fm) mM glycine uptake by human kidney alices. The symbols represent the mean uptake in five different specimens incubated in duplicate flasks. The brackets represent the SE of the meen. Distribution ratio is deffned as the ratio of cpm/ml intracellular fluid to cpm/ml extracellular fluid.
Glycine-Proline
Interactions
Figure 3 shows the Lineweaver-Burk analysis of the effect of 50 mM proline on glycine uptake over a concentration range of 5-50 mM, observed in slices from two separate specimens. Proline is clearly a competitive inhibitor of glycine uptake in this concentration range, with a calculated Ki of 26-34.7 mM.
GLYCINE UPTAKE BY KIDNEY CORTEX
Table 1. Apparent K, and V,,
679
of Glycine Transport in
Human Kidney Specimen
K ml
A
40.0
K -z
V m.w
200.0
2.22
533.3
2.00
B
62.5
0.25
C
-
0.87
D
20.0
0.286
E
14.3
0.91
Incubations were for 30
V -I
0.24
5.00 100.0
2.22
44.4
4.00
min. Radioactive amino acid was
diluted with unlabeled compound to give at least five appropriate concentrations between 5.065 and between 0.22
and 1.065
and 50 mM to determine I<,, mM to determine K,,.
In some
experiments the entire range of concentrations was examined, whereas
in others only one portion was determined.
expressed in mm/l and V_
as mm/l/30
for K,,. K,,. V,,,..,. and V,,,.,* are 34.2, respectively.
K,
is
min. The average values 0.51 1.219.4,
and 3.09, Fig. 3. Effect of 60 mM L-proline on concentrationdependent uptake of glycine by human kidney cortex. Data obtained from two specimens D W and E 10) are shown separately. Slices were incubated with glycine (6.00~60 m/W) with (W. 0) and without 10 m&f L-proline for 30 min.
The effect of the addition of proline on the high-affinity, low-capacity system could not be reliably evaluated because even at very low substrate concentrations uptake is more or less evenly divided between the two systems (Fig. 4). In an effort to circumvent this problem, the 30-min uptake of 0.065 and 0.165 mM glycine was examined with the addition of 10 and 25 mM proline. These data are compared with the control values in Table 2. Using the kinetic parameters experimentally determined for each transport system, the theoretic velocity of uptake was calculated according to the MichaelisMenten formulation, and the percentage of the total uptake contributed by the low K, system in the presence of 10 and 25 mM proline compares favorably to the expected values if no uptake were occurring via the high K, system.
Efects of Inhibitors Oxygen Derivation
and Sodium
or
The effects of other amino acids and of removal of sodium or oxygen from the system are shown in Table 3, for various samples of human kidney. In two specimens (A and D), there was significant inhibition of the 30-min uptake of 0.065 mM glycine by 25 mM LYaminoisobutyric acid (AIB) and valine, while 25 mA4 lysine did not appear to interact significantly with transport of glycine at these low substrate concentrations. In specimen D, 25 mM thioproline, a metabolizable proline analog, caused a significant decrease in 0.065 mM
100 t
Fig. 4. Comparison of theoretical velocities of glycine uptake on the high (0) and lowaffinity (0) systems in human kidney slices. Data are plotted as the percentage of the total velocity versus the various substrate concentrations, expressed as m/liter. Kinetic paremeters shown in Table 1 were inserted into the Michaelis expression for velocity.
% % w g K
0.5
I.5
I.0 GLYCINE
(mM)
2.0
660
ROTH ET AL.
Table 2.
Interactions of lmino Acids With Low-Capacity Glycine Transport
COtltCOl
0.065
mM
6.713
Prchne ( 10 mlul
Thsaetlc
DR + 0.270
0.349
146%)
Iv= 0.165
mM
6.40
+ 0.248
”
DR 3.88
? 0.225
l6
0 754 142%)
5 26 2 0.320
(33%)
DR 3.80
+ 0.527
0.247
10
1.056
OH-Pro (10 mM)
O&wed
”
132%)
DR 5.82
+ 0.095
0 376 149%)
iv=5
3 98 -t 0.52,
Observed ” 0.259
4.91
? 0 582
(34%)
,v=3
N=3
_
(57%)
OH-Pro (25 mMI DR
Observed ”
0 610(45%)
Iv=3
Uptakes w#m performed 8s descrnbsd I” text and carried out fa ccnxntrat~ons
0.252 N=
N=7
at each of the two #y&e
Prolins 125 mMl
Observed V
--
Iv=3
30 mm. Dstribution
rawx
was calculated according to the Michaelis-Mentefi
(DRI are gown as the average + SE fw N detwnmatvzms.
The theorst~c velouty of uptake
equatto”:
glycine uptake, while 5 mM thioproline had a smaller effect. Deprivation of sodium and oxygen were both inhibitory at this low glycine concentration. These observations are consistent with those made for uptake of 10 mM glycine concentration, using higher (50 mM) concentrations of the various amino acids.
iminoglycinuric syndrome is due to a homozygous state of deletion of a high-capacity, common transport site, with retention of a separate, low-capacity, substrate-specific site.’ This hypothesis was tested in human kidney, as reported in an earlier study from this laboratory, using proline as the substrate.* The results of that study led to the suggestion of extensive proline metabolism as a regulating factor in proline uptake by the human kidney. The present study thus assumes added importance in the definition of the iminoglycinuric defect. We have now shown that the uptake of giycine by human kidney is active and concentration dependent. At a substrate concentration in the physiologic range (0.165 mM), a steady-state concentration gradient of 12 is achieved by the human renal tubular cell. In the rat kidney, in vivo concentration gradients in the same range have been reported by Roth et al.” and Toback et al.‘* An intracellular:extracellular gradient in human kidney in vivo has been reported for
DISCUSSION
Early studies on renal amino acid excretion rates throughout development,’ together with the description by Joseph et aLI of a family having several members with large quantities of proline, hydroxyproline, and glycine in the urine, led Striver et al.3 to propose that these three amino acids share a common transport system in the renal tubule. This system has come to be known as the iminoglycine system, and has been the subject of numerous in vitro studies2,‘2,‘5*‘6 using rat or rabbit kidney. These observations, together with the results of amino acid infusion data in humans,‘” have led to the theory that the
Table 3. Distribution Ratios of Glycine Uptake After Addition of Various Amino Acids, Sodium Deprivation, and Anaerobiosis GlVCl”0
Th,opral,“s
CO”CWtratW” ImMl 0.065
NB +-Free Spsclllm”
ConeJo,
AI6* 2.18
w,ne*
+ 0.41
t
0.40
6.09
t
T”*
A”aWCbi0sls
5.60
-t 0.32
6
7.20
I! 0.64
3.76
t
0.43
5.33
t
C
6.22
f
2.25
i
0 37
2.57
? 0.16
D
6.13
-+ 0.52
3.71
*
0.22
2.63
+ 0.27
1.55
t
0.18
(54.81
6.4
+ 0.3,
+ 0.20
162.2)
I97
6.23 11
2.02
f
t
1.04
+ 0.07
4.10
+ 0.43
0.19
(52.7)
t
0.2,
(73.11
3.1,
t
04,
(36 31
(109.51
3.26
3.0
0.19
3 10 + 0.3,
? 0.32
+ 0.17
t
176.6,
4.72
2.16
0 29
79tO25
178.81 1.76
50 mM
1108.9)
0.48
(46 61 A
173.61
26 mM
5rnM
0.50
A
(38.91
10.065
4.12
L”W.3.
4.40
*
0.23
1107.3)
0 17
2.73
+ 0.20
1.46
+ 0 1, 1.06
2 0.02
126.9)
GLYCINE UPTAKE BY KIDNEY CORTEX
proline.’ Thus, iminoglycine accumulation by the human kidney slice reflects the in vivo state of the kidney. Analysis of the glycine entry kinetics in human kidney shows at least two transport systems, for which the apparent transport K, values differ by sixty-fold. In the concentration range of the high-capacity transport system, proline is a competitive inhibitor of glycine uptake. This observation is consistent with the proposed mechanism for the appearance of glycinuria in humans infused with large amounts of proline, whereby the high concentration of proline in the tubular lumen competitively inhibits tubular reabsorption of filtered glycine.5*7 However, even in iminoglycinuric subjects, infusion of proline cannot reduce tubular glycine reabsorption below a fixed minimum. This observation was the basis for the proposition of a glycine-specific transport system that could not be inhibited by proline or hydroxyproline, and that this accounted for the minimal tubular glycine reabsorption retained. Our data are consistent with the presence of a low-capacity, high-afficinity glycine transport system in the human kidney. The apparent transport K, of this system (0.5 1 mM) would suggest that it best subserves glycine uptake in a physiologic concentration range. However, the data derived using the Michaelis-Menten formulation indicate that in this range, both systems contribute almost equally to total uptake. Unfortunately, because of the large contribution to total uptake made at these concentrations by the high-capacity transport system, it was not possible to evaluate directly the nature of the interaction of proline with this low-capacity system. Nonetheless, using the basic assumption that high proline concentrations and low glycine concentrations might create a situation in which the observed velocity of uptake was primarily due to the low-capacity system, it was possible to show good agreement between the calculated velocity for this system and the velocity observed experimentally. Thus, it appears that the low-capacity glycine transport system is not inhibited by 10 or 25 mM proline, a conclusion that lends support to the proposed mechanism for residual glycine reabsorption by the renal tubule during proline infusion.’ Of several other amino acids tested, our data
681
suggest that only cr-aminoisobutyric acid, valine, and thioproline are consistent inhibitors of glycine uptake in human kidney. Interactions between AIB and other neutral amino acids have been reported in mammalian kidney by others.‘9920Striver and Mohyuddin demonstrated that AIB was a competitive inhibitor of proline uptake at low substrate concentrations.*’ Inhibition of proline uptake in human kidney by thioproline has been reported, and attributed to a dual effect of proline, on intracellular metabolism as well as on the membrane transport site.’ Although we did not study the effects of thioproline upon intracellular glycine metabolism, it is possible that alterations in the latter played a role in the transport inhibition of glycine as well. Valine, too, has been reported to inhibit proline uptake in human kidney.8 The similarity of glycine and proline transport in the presence of these inhibitors is further evidence in support of the hypothesis that the two share a common transport system. Equimolar substitution of the medium sodium by Tris and anerobic incubation significantly depressed glycine uptake in a fashion very similar to that reported previously for proline in human’ and in rat kidney.*’ The observations recorded here support Scriver’s contention7 that glycine reabsorption in the human kidney is subserved by a high-capacity system shared with proline and hydroxyproline, as well as a glycine-specific low-capacity system. It is noteworthy, however, that the similarity of the apparent transport K, of the high-capacity system and the calculated Ki for proline do not support the concept that glycine has the lowest affinity for this system. Although some workers have expressed concern that substrate uptake in the slice model may represent peritubular, rather than brushborder, transport events,22*23our data is completely consistent with recent observations made in adult rat renal brushborder membrane vesicles.24 In the rat vesicle, two glycine transport systems were demonstrated, with competitive inhibition by proline on the high-capacity system. These workers were able to show noncompetitive inhibition of glycine uptake by proline in the low substrate concentration range, but speculated that this might reflect the large contribution to total uptake made by the highcapacity system, even at these low concentrations. Thus, even with a subcellular purified
ROTH ET AL.
682
preparation, there is enough overlap of the two glycine transport systems so that the highaffinity mechanism cannot be studied in isolation. It is possible that clarification of the nature of the proline-glycine interactions on the lowcapacity transport site will have to await isola-
tion of the carrier molecule itself. The evidence is sufficient, however, to state that the postulated mechanism for the iminoglycinuric syndrome is probably very close to actual events taking place in the genetic direction of membrane transport.
REFERENCES 1. Brodehl J, Gellissen K: Endogenous renal transport of free amino acids in infancy and childhood. Pediatrics 42:395-404,1968 2. Baerlocher KE, &river CR, Mohyuddin F: Ontogeny of iminoglycine transport in mammalian kidney. Proc Nat1 Acad Sci USA 65:1009-1016, 1970 3. Striver CR, Efron ML, Schafer IA: Renal tubular transport of proline, hydroxyproline and glycine in health and in familial hyperprolinemia. J Clin Invest 43:37&385. 1964 4. Striver CR, Goldman H: Renal tubular transport of proline, hydroxyproline and glycine. II. Hydroxy-L-proline as substrate and as inhibitor in vivo. J Clin Invest 45:135713651966 5. Striver CR: Renal tubular transport of proline, hydroxyproline, and glycine. III. Genetic basis for more than one mode of transport in human kidney. J Clin Invest 47:823835.1968 6. Rosenberg LE. Durant JL, Elsas LJ II: Familial iminoglycinuria. An inborn error of renal tubular transport. N Engl J Med 278:1407-1413, 1968 7. Striver CR, Chesney RW, McInnes RR: Genetic aspects of renal tubular transport: Diversity and topology of carriers. Kidney Int 9149-171, 1976 8. Holtzapple P, Gene1 M, Rea C, et al: Metabolism and uptake of t_-proline by human kidney cortex. Pediatr Res 7:818-825, 1973 9. Fox M, Thier S, Rosenberg L, et al: Evidence against a single renal transport defect in cystinuria. N Engl J Med 270:556-561, 1964 IO. Rosenberg LE, Downing SJ, Segal S: Extracellular space estimation in rat kidney slices using C” saccharides and phlorizin. Am J Physiol 202:800-804, 1962 Il. Rosenberg LE, Berman M, Segal S: Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney cortex slic=es. Biochim Biophys Acta 7 1~664-675, 1963 12. Hillman RD. Albrecht 1, Rosenberg LE: Identification and analysis of multiple glycine transport systems in isolated mammalian renal tubules. J Biol Chem 243:5566557l,l968
13. Akedo H, Christensen HN: Nature of insulin action on amino acid uptake by the isolated diaphragm. J Biol Chem 237:113-117, 1962 14. Joseph iale associent
R, Ribierre M, Job JC, et al: Maladie famildes convulsions. a d&but trbs precoce, une
hyperalbuminorachie Pediatr 15:374-387,
et une hyperaminoacidurie. 1958
Arch
Fr
15. Baerlocher KE, Striver CR, Mohyuddin F: The ontogeny of amino acid transport in rat kidney. I. Effect on distribution ratios and intracellular metabolism of proline and glycine. B&him Biophys Acta 249:353-363, 1971 16. Baerlocher KE, Striver CR, Mohyuddin V: The ontogeny of amino acid transport in rat kidney. II. Kinetics of uptake and effect of anoxia. Biochim Biophys Acta 249:363372, 1971 17. Roth KS, Hwang SM, London JW, et al: Ontogeny of glycine transport in isolated rat renal tubules. Am J Physiol 233:F24-246,
1977
18. Toback FG, Mayers AM, Lowenstein LM: Alterations in renal and plasma amino acid concentrations during renal compensatory growth. Am J Physiol 225:1247-l 251, 1973 19. Wilson OH, Striver CR: Specificity of transport of neutral and basic amino acids in rat kidney. Am J Physiol 213:185-190, 1967 20. Striver CR, Mohyuddin F: Amino acid transport kidney. J Biol Chem 243:3207-3213, 1968
in
21. Mackensie S, Striver CR: Transport of L-proline and alpha-amino isobutyric acid in isolated rat kidney glomerulus. Biochim Biophys Acta 241:725-736, 1971 22. Foulkes EC: Effects of heavy metals on renal aspartate transport and the nature of solute movement in kidney cortex slices. Biochim Biophys Acta 241:815-822, 1971 23. Wedeen nonmetabolized 512, 1971
RP, Thier SO: Intrarenal distribution of amino acids in vivo. Am J Physiol 220~5077
24. McNamara PD. Ozegovic B, Pepe LM, et al: Proline and glycine uptake by renal brushborder membrane vesicles. Proc Natl Acad Sci USA 73:4521-4525, I976