Cystine and lysine transport in cultured human renal epithelial cells

Cystine and Lysine Transport Beatrice States,

John Foreman,

in Cultured Human Renal Epithelial Judithann

Lee, Dorothy

Harris, and Stanton

Cells

Segal

The transport of the amino acids, cystine and lysine. was studied in epithelial cell lines propagated from human kidney cortex. Cystine uptake data were reproducible in different cell lines and did not vary over several cell passages of an individual cell line. The transport of this disulfide amino acid was sodium-dependent with kinetic analysis showing one apparent K, system of 0.09 mmol/L and V,, of 0.054 mmol/L cell water/min. Studies of the kinetics of lysine transport, however, revealed two uptake systems with apparent high and low affinities with K, of 0.14 mmol/L and 5 mmol/L and V,, of 0.041 and 0.167 mmol/L cell water/mitt. respectively. Glutamate appeared to be the most potent inhibitor of cystine uptake by these cultured human renal cells and this interaction was competitive. Although cystine did not inhibit lysine uptake, arginine and ornithine were shown to be major inhibitors, thus providing evidence for the presence of a shared dibasic amino acid transport system. o 1987 by Grune & Stratton, Inc.

0

NE of the major difficulties in studying the underlying abnormalities of inherited human renal transport disorders such as cystinuria is the unavailability of human kidney samples for in vitro investigation. An approach to obviate this difficulty is the establishment of human kidney epithelial cells in culture. Such cell propagation is possible.‘*’ Human renal cortical epithelial cells cultured in our laboratory have characteristics in common with proximal tubule cells as demonstrated by their response to PTH but not AVP, the presence of similar enzyme activities, and the capacity to transport amino acids and alpha-methylglucoside. Studies with these cultured cells could provide insights into the fundamental nature of amino acid transport in the human kidney. Two amino acids of particular interest are cystine and lysine since their renal reabsorption is defective in human cystinuria.3q4 Extensive studies of the characteristics of cystine and lysine transport in rat tubules’ and renal brush-border membrane vesicles6 have been reported. However, our understanding of the transport of these amino acids in human tissue is limited. Previous studies using human renal cortical slices have provided some information, but the slice is not an optimal model system for study.’ We undertook, therefore, the assessment of cystine and lysine transport into cultured human renal cortical epithelial cells. Our results form the basis of this report. MATERIALS AND METHODS Dulbecco’s Modified Eagle Medium (DME), Ham’s F-12, Medium 199, 20 mmol/L glutamine, 7.5% sodium bicarbonate, 2.5% trypsin and penicillinstreptomycin (5,000 IU/mL: 5,000 rg/ mL) were purchased from Flow Laboratories Inc, McLean, VA.

From the Division of Biochemical Development and Molecular Diseases, ChildrenS Hospital of Philadelphia and the Departments of Pediatrics and Medicine, University of Pennsylvania School of Medicine Philadelphia. Supported by Grant AM IO894 from the National Institutes of Health, Bethesda, MD and Grant 841122 from the American Heart Association. Dallas. Address reprint requests to Beatrice States, PhD. Division of Biochemical Development and Molecular Diseases, Children’s Hospital of Philadelphia, 34th & Civic Center Blvd. Philadelphia. PA

19104. Q 1987 by Grune & Stratton, Inc. 0026.0495/87/3604-OOIO.$O3.00/0 356

CLS collagenase (c. histolyticum collagenase Type III) was obtained from Worthington, Freehold, NJ. L-[“5-S]cystine (331.7 mCi/mmol), L-[U-“C]lysine, HCI (348 mCi/mmol), [14C]urea (57 mCi/mmol), and D[l’4C]mannitol (55 mCi/mmol) were purchased from Amersham Corporation, Arlington Heights, IL. L-cystine, cysteine-HCI, N-ethylmaleimide (NEM), reduced glutathione (GSH), oxidized glutathione (GSSG), and L-lysine were purchased from Calbiochem, San Diego. An L-amino acid kit was obtained from Sigma Chemical Company, St Louis. Biofluor and Liquifluor were purchased from New England Nuclear, Boston. Thin-layer cellulose plates were obtained from Eastman Kodak Co, Rochester, NY. Primaria Falcon tissue culture flasks, tissue culture cluster dishes, and all high quality reagents were obtained from Fisher Scientific, King of Prussia, PA.

Human Kidney Cells Cell lines were initiated from small pieces of unaffected cortex taken from specimens removed surgically for neoplasia of the kidney and from autopsy material usually within several hours of death as reported previously.’ Renal cortical epithelial cell lines from four children and four adults were successfully propagated in 25 cm2 Falcon Primaria flasks containing 5 mL of either medium 199 supplemented with 2 mmol/L glutamine and 3% fetal calf serum or the hormonally defined Ham’s F-12:DME medium described by Taub et al,8 depending on which media resulted in maximal cell proliferation. The hormonally defined medium contained 5 pg/mL insulin, 25 ng/mL prostaglandin E,, 5 x 10-s mol/L hydrocortisone, 5 x lo-” mol/L triiodothyronine, 5 pg/mL transferrin, and 5 x lo-’ mol/L sodium selenite in a 1:l mixture of Ham’s F-12:DME medium.

Transport Studies In transport studies, 2 x 10’ human kidney cells were suspended in 1 mL of medium 199 supplemented with 2 mmol/L glutamine and 3% fetal calf serum. Cells were seeded onto either 2 cm2 or 8 cm2 Costar cluster wells. When using 2 cm” wells, 0.4 mL of the cell suspension was added to each of the 24 wells contained in a cluster tray and an additional 2 mL of complete medium 199 was added. Unless otherwise stated, confluent cell monolayers with scattered domes (generally ten days after seeding) were studied. Cells were refed on the ninth day with media containing 5% serum to stimulate dome formation. Prior cell feedings with complete medium 199 containing 3% serum were on the third and sixth days after seeding. All cells were incubated in a water-jacketed incubator at 37OC under compressed air:CO, (9O:lO). To begin transport studies, each well was washed three times with 2 mL of sterile PBS pH 7.4. Monolayers of cultured cells were incubated at 37OC with gentle shaking in PBS pH 7.4 supplemented Metabolism, Vol36,

No 4 (April), 1987: pp 356-362

357

CYSTINE AND LYSINE TRANSPORT

with 0.1% glucose

and radioactive

substrates.

To determine

the

dependence of low Nat on uptake, choline chloride replaced NaCl in the PBS incubation medium. Studies were terminated at timed intervals by removing the media containing the labeled substrate and washing the cells three times with cold PBS as described by States et al.9 Radioactivity was extracted with 0.5 mL 50% tricholoroacetic acid (TCA) according to the method of Gazzola et al.” To determine intracellular fluid space (ICF), extracellular fluid space (ECF), and uptake of radioactive amino acids, the entire extract was added to 5 mL of Biofluor scintillation fluid and counted. The procedure for determining ICF and ECF was according to the method described by States et a1.9 The TCA precipitated protein in each well was solubilized by adding 0.5 mL of 1N NaOH as described by Gazzola et aI.“and the protein content per well wasdetermined by the Biorad method.” Uptake of radioactivity, corrected for retention of a trace amount of label on the cell monolayer, was based on calculations of distribution ratio, the ratio of cpm/mL ICF to cpm/mL ECF, as described by States et al.9 The distribution ratios reported represent the mean of three to four determinations. Incubations for determinations of the concentration dependence of L-cystine and t_-lysine were for 30 minutes where uptake of the amino acid continued to be linear and the variability of counts among triplicate or quadruplicate determinations was minimal. Since the distribution ratios in these experiments were much higher than 1. the highest expected for diffusion, no correction was made for the diffusion component.

Kature of the Intracellular

‘-‘S Compounds

Intracellular labeled cystine and the incorporation of j5S from cystme into its metabolites, cysteine and glutathione, were determined after the cells in several wells were ruptured by scraping into two 2 mL portions of 40 mmol/L NEM in 0.01 mol/L phosphate buffer, pH 7.4. The suspension was transferred to a small tube and mixed for one minute using a Vortex mixer. Each sample was deproteinized immediately with cold 50% TCA and concentrated by lyophilization. The percentage of label in the cellular extract recovered as cystine was calculated after isolation by high-voltage electrophoresis using a Camag apparatus. The run was made at 95 V/cm for 35 minutes in 6% formic acid pH 1.7. Cysteine-NEM and glutathione-NEM were isolated by thin-layer chromatography as described by States and Sega1.r2 Nonradioactive standards of cystine. cysteine-NEM, and glutathione-NEM were included in all high-voltage electrophoresis and thin-layer chromatography runs. The high-voltage chromatogram was cut into l-cm strips and the thin-layer chromatogram into 0.5-cm strips. These strips were counted in 2 mL of Packard Phosphor scintillator prepared by

adding

I60 mL of Liquifhror

to 3.9 L of Fisher

Scintanalyzed

toluene. The percentages of label appearing in intracellular free cystine, cysteine-NEM, and glutathione-NEM were converted to nanomoles of initial L-[35S]cystine. RESULTS

Uptake of ,s13’S]Cystine The uptake of 0.025 mmol/L L-[35S]cystine by one line of cultured human renal epithelial cells in passages 1.2, and 7 is plotted in Fig 1A. Cystine uptake was linear over the initial 40 minutes of incubation. Further, there was little difference in cystine transport among the three passages; therefore, uptake appeared to be independent of the cell passage. The second parameter which could possibly influence amino acid transport was variability in different cell lines in the same passage. Figure 1B shows that the uptake of 0.02 mmol/L L-[3sS]cystine by two different cultured kidney cell lines in passage 3 was superimposable. Concentration

Dependence of Cystine Uptake

The concentration dependence of cystine transport was evaluated in three cell lines over the concentration range of 0.02 to 0.2 mmol/L. High medium concentrations of cystine were not possible because of the limited solubility of cystine and the difficulty in maintaining the medium pH at 7.4 when the cystine concentration exceeded 0.2 mmol/L. Figure 2 is a Lineweaver-Burk plot of the data obtained with one of these lines and is similar to that of the other two lines. This plot shows that the uptake process for cystine adhered to saturation kinetics. From Lineweaver-Burk plots of the transport data obtained with each of three cell lines, there appears to be one saturable transport system for cystine uptake with an apparent K, which ranged from 0.09 to 0.17 mmol/L and a V mar which ranged from 1.62 to 2.5 mmol/L cell water/30 min. Nature of Intracellular

‘jS Compounds from /3SS]Cystine

Previous studies9.‘3 of cystine uptake into cultured cells showed a rapid reduction of cystine to cysteine and incorporation of label from cystine into intracellular glutathione. The intracellular distribution of 35S in cystine, cysteine, and

20

k A0

IO

20 TIME (mm)

30

40

40

20

B0

TIME

60

(min)

Fig 1. (A). Uptake of 0.025 mmol/L L-[“S]cystine by cultured human renal epithelial cells originating from child no. 1 in passage 1 (0). passage 2 (ml, and passage 7 (@I. Incubations were in triplicate at 37°C. (61, Uptake of 0.02 mmol/L L-[%]cystine by human renal epithelial cells in passaga 3 originating from child no. 1 (0) and child no. 2 (0).

STATES ET AL

358

“1 _!_

3.

V

0

20 TIME

1

-20

-10

0

IO

20

30

40

40 (min.)

60

Fig 3. Uptake of 1 mmol/L L-[U”C]lysine by cultured human renal epithelial cells in passage 3 originating from child no. 2. Incubations were in quadruplicate at 37°C.

50

+I Fig 2. Lineweaver-gurk plot of the concentration dependence of cystine uptake. Incubations were in triplicate for 30 minutes at 37°C. This renal epithelial cell line originated from child no. 2 and was in passage 2.1 /V is the reciprocal of mmol/L/BO min and 1 /S is millimolar cystine-‘.

reduced glutathione, therefore, was determined in human renal cortical cells. Incubations were with 0.02 mmol/L t_-[35S]cystine for 30 and 60 minutes. The results are shown in Table 1. Values represent the calculated nanomoles of 35S from exogenous labeled cystine found in intracellular cystine, cysteine, and glutathione per mg cell protein. The data show clearly that only a small percentage, 2% to 3%, of the intracellular label remains as cystine with approximately 40% as cysteine and more than 50% of the 35S incorporated into reduced glutathione. Thus, the distribution ratio used as a measure of L-[“Slcystine uptake represents a radioactivity ratio and not a cystine concentration gradient. Uptake of L-[U-‘~C] Lysine

Because lysine is considered to be transported by the same system as cystine in the human kidney in vivo,3,4,‘4L-lysine uptake with time was determined in the third passage of kidney cells obtained from an adult donor. Figure 3 shows that the uptake of 1 mmol/L L-lysine was linear over the initial 30 minutes of incubation and then approached a steady state after 40 minutes of incubation. Since lysine is not metabolized readily by the kidney,15 the distribution ratios represent the intracellular levels of the exogenous labeled lysine over a 60-minute incubation period.

mmol/L and V,,, after 30 minutes of 1.22 mmol/L cell water and the other with an apparent high K, of 5 mmol/L and V,, of 5 mmol/L cell water over the same time period. In light of the above data, the uptake of 1 mmol/L lysine, which was used in the timed uptake studies, represents transport by both systems. Sodium Dependence of Cystine and Lysine Transport

Sodium has been shown to be an important ion for the uptake of cystine in isolated renal tubules’ and brushborder membrane vesicles.‘6 Lysine transport, however, was independent of sodium in renal cortical slices from the rat.” Because cystine transport is sodium-dependent and lysine transport sodium-independent in other uptake systems in vitro ” the effect of reducing the medium sodium to 8 mmol/L on the uptake of 0.02 mmol/L L-cystine and 0.05 mmol/L L-lysine by an adult kidney cell line was examined. The results appear in Fig 5. The data clearly show that by reducing the medium sodium concentration cystine uptake is markedly lowered to approximately 10% of the control level under low-sodium conditions, indicating sodium dependence. In contrast, lysine uptake is diminished by only about 55% after 30 and 60 minutes incubation. Interactions of Cystine and Lysine With Other Amino Acids

The effects of several amino acids on the uptake of cystine after 10 and/or 60 minutes incubation were examined. Table 2 shows that at the physiologic level of 0.02 mmol/L cystine, a number of amino acids, except AIB, inhibited cystine uptake. Of these, glutamate clearly was the most potent.

Concentration Dependence of Lysine Uptake

The kinetics of lysine uptake for 30 minutes over a range of 0.025 mmol/L to 10 mmol/L were studied in kidney cells from a child donor. The data in Fig 4 show the presence of two saturable systems, one with the apparent low K, of 0.14 Table 1. Nature of Intracellular ‘% in Cultured Child Kidney Cell Line No. 1, Passage 4

Nanomoles 0.02 mmol

L-[“SjCystine

Converted/mg Protein

30 min

60 min

Cystine

0.04

0.07

Cysteine

0.44

1.34

0.65

1.73

Reducedglutathione Incubation

of cells were

at 37’C.

Fig 4. Lineweaver-gurk plot of the concentration dependence of lysine uptake. Incubations were in triplicate for 30 minutes at 37°C. This renal epithelial cell line originated from child no. 4 and was in passage 3.1 IV is the reciprocal of mmol/L/30 min and 1 IS is millimolar lysine-‘.

CYSTINE

AND

LYSINE

359

TRANSPORT

8o1I _/ 0 c

60. /’

d

/’

I’

,’

/

I4

.’

,’

/’

16 1

T

_/*A

12 IO 1

/’

60

40

20

0

-20

I/El

60

40

20

0

(min)

TIME

Fig 5. Sodium dependence of cystine and lysine transport by human renal epithelial cells in passage 1 originating from adult no. 8. Lysine transport in complete medium (A). and in medium containing 8 mmol/L sodium (A). Cystine uptake in complete medium (0). and in medium containing 8 mmol/L sodium (0). Bars above average values represent SEM of triplicate determinations.

Inhibition of cystine uptake by glutamate also has been shown in the cultured Iibroblast” and hepatocyte.20.2’ The nature of this interaction in the cultured human renal epithelial cell was therefore examined. The results, shown in Fig 6, are a Lineweaver-Burk plot of the data obtained after 30 minutes incubation of cells over a concentration range of 0.02 mmol/L to 0.2 mmol/L L-[35S]cystine in the presence and absence of 1 mmol/L glutamate. The data show that glutamate is a competitive inhibitor of cystine uptake with a change in apparent K, of transport from 0.09 mmol/L to 0.22 mmol/L cystine and a calculated apparent Ki22 of 0.66 mmol/ L. Table 3 reveals the results of experiments to determine the interactions of lysine with other amino acids during the uptake process. Cystine did not inhibit lysine uptake. Other amino acids inhibited to variable extent, the major inhibitors Table 2. Effect of Amino Acids on 0.02 mmol/L “S Cystine Transport*

Child

kidney

no.

1

Lysme

(3 mmol/L)

Adult

kidney

no. 2

Lysine

13 mmol/L)

Adult

kidney

no. 3

Lysine

(3 mmol/L)

Adult

kidney

no. 9

Lysine

Child

kidney

no. 5

Arginine

Child

kidney

no. 5

Ornithine

-

(1 mmol/L) (1 mmol/L)

kidney

no. 9

Al8

no. 9

Glycine

(1 mmol/L)

29

I1 mmol/L)

(1 mmol/L)

43

Adult

kidney

no. 9

Leucine

kidney

no. 4

Glutamate

(1 mmol/L)

Child

kidney

no. 6

Glutamate

(1 mmol/L)

tAlB

stimulated

at 37’C.

The percent

determinations

cystine

uptake.

inhibitions

of distribution

were

Amino Acid

11

Concentration Cell Line

based

ratios.

(0.5 mmol/L)

% Inhibition lOmr7

60 min

23

Adult

kidney

no. 9

Cystine

0

2

33

Adult

kidney

no. 9

Glycine

7

13

Adult

kidney

no. 9

Leucine

36

55

Glutamate AI9

36

- 13

11

Adult kidney no. 9 Adult kidney no. 9

-5ot

-13t

95

Adult kidney no. 9

Argimne

90

87

90

Adult kidney no. 9

Ornithine

92

90

33

76

‘*C

Lysine Transport*

24

- 20t

-23t

Child

were

Table 3. Effect of Amino Acids on 0.02 mmol/L

11

29

(1 mmol/L)

kidney

of quadruplicate

DISCUSSION

Our studies show that cultured renal epithelial cells originating from human kidney cortex are capable of transporting cystine and lysine. Cystine uptake is reproducible over a number of cell line passages with no significant differences between two cell lines studied simultaneously. Cystine uptake by cultured human kidney epithelial cells originating from renal cortex occurs via a single transport system in the concentration range studied. Rosenberg et al’ also observed a single transport system for cystine when they used a more extended range of concentrations in their studies with human kidney cortex slices, although the slice technique appears to be a less than optimal method for studying cystine transport. Indeed, only one cystine transport system was noted in rat cortical slices,’ but studies with isolated rat renal tubules’ and rat brush-border membrane vesicles6 show cystine transport to occur via low and high K, systems. The apparent K, for cystine uptake by cultured cells of 0.09 mmol/L cystine corresponds to the low K, (0.033 mmol/L)

28

Adult

*Incubations

60 min

10min

Adult

average

being arginine and ornithine thus providing evidence for the presence of the shared dibasic amino acid transport system.

% Inhibition

Amino Acid Concentration

Cell Llm?

Fig 6. Lineweaver-Burk plot of the interaction of cystine with 1 mmol/L glutamate. Triplicate wells of adult kidney epithelial cell line no. 8 in passage 2 were incubated for 30 minutes at 37°C with 0.02 to 0.20 mmol/L cystine with (m) and without (0) 1 mmol/L glutamate. 1 /V is the reciprocal of mmol/L/BO min and 1 /S is millimolar cystine-‘.

on the

*Incubations were at 37°C. The percent inhibitions were based on the

average of quadruplicate tstimulation

of lysine

determinations uptake.

of Distribution

Ratios

STATES ET AL

system found in rat renal tubules’ and isolated rat brushborder membrane vesicles (0.031 mmol/L), the system shown to be shared with lysine.6 Unfortunately, no data from studies with isolated tubules and brush-border membrane vesicles from human kidney are available for comparison with studies with cultured human renal cells. There are a number of possible explanations as to why only one cystine transport system was observed in cultured human renal epithelial cells: (1) The cultured human renal epithelial cells may reflect the function of only a portion of the proximal tubule which contains only one of the two cystine transport systems, while isolated rat renal cortical tubule and rat brush border membrane vesicle preparations come from the entire proximal tubule. Barfuss and Schaferz3 have shown that the glycine transport systems are arranged such that the early portion of the proximal tubule contains one and the late portion the other. (2) The low affinity system may have been lost because of dedifferentiation of these cells with culturing.’ (3) The limited solubility of cystine in the incubation media permitted the discernment of only one system, but with higher concentrations used in rat vesicle studies a high K, system would be seen. It is the low K, system of rat renal tubules and isolated rat brush-border vesicles that is shared with lysine and this may be the important system with regard to the defect in human cystinuria.24 The high K, unshared system is of unknown biologic importance. (4) Although two cystine transport systems were observed in the rat kidney, it is conceivable that only one exists in the human. Another characteristic of cystine transport is the rapid reduction of cystine to cysteine, which has been noted previously in renal cortical slices from the human, and the dog and rat and in rat tubules. Our studies show reduction of cystine to cysteine with rapid incorporation of label from cystine into glutathione. Crawhall and Segal” reported that cysteine accounted for 100% of the total intracellular cyst(e)ine after incubation of human kidney cortex slices with labeled cystine. The apparent difference between the above data and our data showing 39% to 57% incorporation of exogenous label from cystine into intracellular cysteine and glutathione, respectively, could be explained both by the fact that the technique used by Crawhall and Segal does not adequately separate the two NEM adducts and the greater viability and tenfold increased cystine uptake by the intact cultured cell as compared with the human kidney cortical slice. This reduction step does not appear to be necessary for transport since rat renal brush-border membranes take up cystine in the absence of any reduction to cysteine.6 Efforts in recent years have been made to determine which side of the plasma membrane predominates in amino acid uptake into epithelial cells. Rabito et a126*27 have shown that specific transport systems for neutral amino acids only reside in the basolateral membrane of LLC-PK, cells. Aspartate, however, appears to be taken up by both membrane species, but mainly across the apical membrane. We have not yet determined the sideness of cystine uptake in cultured human renal cells, but Mullin et al** have called into question the validity of these studies in a confluent layer of cultured epithelial cells where substrates may easily diffuse between the cells.

Our data show clearly a sodium requirement for cystine transport by human renal cells in primary culture and low passage. Sodium also has been shown to be important for the uptake of cystine into noncultured rat renal tubule cells and isolated brush-border membranes. Further, a sodium-dependent cystine transport system has been described for isolated rat hepatocytes. The activity of this system diminishes as the latter cells are cultured and a sodium-independent cystine transport system emerges. ” Cultured fibroblasts, whether from fetal lung” or skin,29 possess only a sodium-independent system for cystine uptake. Whether this sodium-dependent system found in our cultured human renal cells will be lost after their ability to be passed beyond the sixth passage is established remains to be seen. The uptake of lysine was partially sodium-dependent. Whereas the sodium dependence of cystine transport in these cells parallels the observations made in other tubule cell preparations, the presence of a sodium-dependent component of lysine uptake differs from that of renal and other cell preparations. There is no dependence of lysine uptake on sodium by rat kidney cortex slices” or rat renal brush-border membrane vesicles. 3oAlso lysine uptake by membrane vesicles from cultured human fibroblasts is not sodium-dependent,” nor is the dibasic amino acid entry in intact human fibroblasts.32 No information is available, however, on the sodium requirement of human renal cortical cells for comparison with our data on cultured renal cells. Glutamate, not lysine, had the greatest effect on cystine uptake by these cultured renal cells. Indeed, the dibasic amino acids had no greater effect than leucine and glycine. This is in contrast to cystine uptake by isolated rat renal tubules and brush-border membrane vesicles in which glutamate had no effect but lysine did.33 Culturing these rat tubule cells led, however, to the emergence of a cystineglutamate interaction, although the cystine-lysine interaction remained. Glutamate is an important inhibitor of cystine transport in other cultured cells such as skin fibroblasts,29 fetal lung fibroblasts,i3 and hepatocytes.*’ In these cultured cells there appears to be a common transport system for these two amino acids. Lysine, on the other hand, had no effect on cystine uptake by any of these cells. In the present studies, cystine did not affect lysine uptake but arginine and ornithine were potent inhibitors supporting the presence of a lysine system shared with other dibasic amino acids. The data suggest the independence of the cystine and lysine transport processes, but the lack of a cystine effect could be due to the cystine level used as an inhibitor of lysine uptake. This was not as great as the other dibasic amino acids due to the limited solubility of cystine. Several possibilities arise to explain these results. First, a cystine-glutamate transport system exists in the human kidney in vivo, but is masked by other cystine transport systems. This system is then unmasked by culturing or the cell type with this system predominates in the culture. A more likely explanation, which is supported by previous work showing the appearance of a glutamate effect with culturing,*’ is that with culture the human kidney cells become dedifferentiated leading to the emergence of a cystineglutamate interaction. It may be possible to preserve the

361

CYSTINE AND LYSINE TRANSPORT

lysine-cystine transport system, which appears to exist in the human kidney in situ with agents that enhance differentiation, such as hexamethylene bisacetamide.34 Finally, it is conceivable that a cell type is present within the heterogeneous cell population that makes up these cultures which retains the cystine-dibasic amino acid transport system. Cloning individual cells from this mixture could then bring out this system. The cultured human renal cortical cell offers a potentially

useful model system. Although not completely reflecting human cortical cell functions in vivo, cultured human renal epithelial cells are in a cellular environment which is capable of manipulation and control, especially in the presence of completely defined media. These advantages have been elegantly demonstrated with MDCK3St36and LLC-PK,37t38 cells originating from dog and pig kidney, respectively. Cells arising from a human source also should offer insights into the complexities of human renal function.

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