Alteration of renal amino acid transport system in cadmium-intoxicated rats

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

Alteration

KYOUNG

106,

102-l

l l (1990)

of Renal Amino Acid Transport in Cadmium-Intoxicated Rats

System

RYONG KIM,* HAE YOUNC; LEE,* CHOON KWANG AND YANG SAENG PARK*.’

KIM,~

Alteration of Renal Amino Acid Transport System in Cadmium-Intoxicated Rats. KIM. K. R.. H. Y.. KIM, C. K., AND PARK. Y. S. (I 990). Tctrico/. .1pp/. Phurtnuwl. 106, 103-l I I. Effects of cadmium intoxication on renal transport systems for various amino acids were studied. Subcutaneous injections of CdClz, at a dose of Z mg Cd/kg. day for 2 weeks. resulted in polyuria. proteinuria, glycosuria, phosphaturia, and aminoacidutia. as observed in chronic cadmium-intoxicated humans and experimental animals. The nature of aminoaciduria was nonspecific. including iminoacid as well as almost all species of neutral. acidic, and basic amino acids. In renal cortical brush border membrane vesicles isolated from cadmium-intoxicated rats, Na’-dependent transpor? of L-proline. L-alanine, and I.-lysine was markedly attenuated. whereas the amino acid transport in the basolateral membrane vesicle was not significantly affected. Similar results were obtained in the normal membrane vesicles directly exposed to inorganic cadmium. These results indicate that cadmium intoxication impairs various Nat-amino acid cotransport systems in the renal brush border membrane. which leads to panaminoaciduria. ir I990 Academic Prcs Inc I-E,

Chronic exposure to cadmium results in various renal functional defects. One of the typical renal functional changes documented in cadmium-intoxicated humans (Kazantzis et al., 1963; Adams et al., 1969; Goyer et ul., 1972) and experimental animals (Gieske and Foulkes, 1974; Nomiyama and Nomiyama. 1982; Nomiyama et al., 1975. 1982) is aminoaciduria. The mechanism by which cadmium induces aminoaciduria has not been fully elucidated. Under normal conditions, amino acids in the glomerular filtrate are mostly reabsorbed in the renal proximal tubule by Na’ -dependent transport processes(Burg, 1986). Amino acids first enter the tubular cell by the Na’amino acid cotransport system in the brush border membrane (BBM) and then diffuse across the basolateral membrane (BLM) by facilitated diffusion mechanisms. We, there’ To whom 0041.008X/90

correspondence

should

$3.00

Copyright C 1990 by Academic Press. Inc. All rights of reproductmn 8” any form reswved

be addressed.

fore, questioned if cadmium intoxication impaired one of thesetransport steps.The present seriesof study was undertaken to evaluate this possibility. using renal cortical plasma membrane vesicles isolated from cadmium-intoxicated rats. Since renal BBM contains several different classesof amino acid transport systems specific for neutral amino acids, acidic amino acids, basic amino acids, iminoacids. and others (Ullrich, 1983; Burg, 1986), we have attempted to test the cadmium effect on each of these transport systems. The present paper describes the results for iminoacid (Lproline) and neutral (L-alanine) and basic (I lysine) amino acids; the results for acidic amino acid (L-glutamate) were reported elsewhere (Lee et al., 1990). METHODS .~l~~inzu/.c Sprague-Dawlcy maintained under standard IO?.

malt rats (X0-300 laboratory conditions

g) were with ud

(‘ADMIIJM

AND

RENAI.

/i/?i/lrr?t access to food and water. unless otherwise mandated by experimental protocol. Cadmium intoxication was induced by daily subcutaneous injections of CdC& at a dose of 2 mg Cd/kg body wt. day for 2-3 weeks. Saline was injected into the control rats. (:r-iriu/j:cic. At I-week intervals, animals were kept rn metabolic cages and were denied food and water for 24 hr. Urine was collected under mineral oil. and was analyzed for creatinine (Wako Technical Bulletin No. 271-10509, Wako Pure Chemical Ind.. Osaka. Japan), glucose (Wako Tcchnicdl Bulletin. No. 270.66509, Japan). protein (Lowry LPIcl/.. 195 I ). phosphate (Fiske and SubbaRow. 1925). and amino acids (LKB alpha plus amino acid analyzer). In sonic animals, blood samples were collected from the tail or heart and analyzed for creatinine. I’rcpirul~~~n

o/

hrmh

hdcr

und

husrdalcwl

r~cwhnw

re\ic Irs /row hrdnc,~. rovfc:~. Renal plasma membrane vesicles were isolated by a procedure similar to those described by Kinsella c’/ u/. ( 1979) and Scalera c/ u/ ( I98 I ). Kidneys rcmovcd from animals were perfused through the renal &cry with an ice-cold solution containing 140 rnM NaCI, IO mM KC]. and 1.5 mM CaQ. The cortex was cut OIL minced. and placed in 250 mM sucrose-10 mM triethanolamine hydrochloride. pH 7.6, at 4°C (sucrose buffer). The cortical tissue was homogenized with 20 strokes in a motor-driven glass homogenizer with a tight-fitting Teflon pestle (clearance 0.15 mm) at I800 rpm. The tissue homogenate was centrifuged at 1075g for 10 min in a Sorvall refrigerated centrifuge. The supernatant was saved. and the pellet was suspended again in half the original volume of sucrose buffer and homogenized with IO strokes at 1800 r-pm. The homogenate was centrifuged as above for 10 min at 1075,~ The supcrnatant was decanted and combined with the previous supernatant (Fraction I). Fraction I was centrifuged at 14.460~ for I5 min; the resulting suprrnatant and the soft light portion of the pellet were taken and pooled (Fraction 2). Fraction 7 was then centrifuged at 47,X00x for 30 min: the supernatant and the lower dark pellet were discarded. and the upper huffy layer of the pcllct was suspended in sucrose buffer to a total volume of 26.5 ml (Fraction 3). This constituted the mcrosomal fraction. Plasma membranes in this fraction w’ere purihed further by centrifugation on a gradient of Percoll. To fraction 3. 3.5 ml stock solution of Percoll (Pharmacia 1:inc Chemicals, Ilppsala, Sweden) was added; the solution was mixed by inversion and ccntrifugcd at 47.800~ for 40 min. The spontaneously formed Pcrcoll gradient was fractionated from the top by careful pipetting and was collected in I -ml fractions. Initially. each fraction was assayed for Na’-K’-ATPase (a marker enzyme of basolateral mcmhranc) (Kinne and Schwartz. 197X: Sachs and Kinne. 1980) and alkaline phosphatase (a marker enzyme of brush border membrane) (Kinnc and Schwartz. 1978; Sachs and Kinne, 19X0) by the method of Jorgensen and Skou ( I97 I ) and Waho Technical Bulletin No. 270-04609, respectively. Routinely thereafter. fractions wcrc pooled according to the distribution of marker enzymes to obtain aliquots cnriched in either RI-M or BBM. Typically. the first 4 ml

AMINO

ACID

TRANSPORT

103

was discarded: 5- I5 ml were pooled as BLM and I6630 ml were pooled as crude BBM. Each pooled fraction was mixed with an equal volume of sucrose buffer, and the Percoll was removed by centrifuging at 100,000~ for I hr in an ultracentrifuge (Sorvall OTD-75). In the case of basolateral membranes. they were resuspended in an appropriate vesicle buffer by passing the membrane pellet through a 25-ga needle several times. The protein concentration of the vesicle fraction (measured by the method of Lowry PI al.. 1951) was adjusted to 6-8 mg/ml. Unless stated otherwise, the composition of the vesicle buffer was 300 mM mannitol, 20 mM Hepes. pH 7.4. with Tri-base. Brush border membranes were further purified from a fraction of the Percoll gradient by the magnesium precipnation method (Booth and Kenny, 1974). After removal of the Percoll. the crude BBM was suspended in IO ml of vesicle buffer solution containing IO mM MgCIZ, stirred on ice for 60 min. and then centrifuged at 107513 for IO min. The supernatant was saved, and the pellet was resuspended in the same buffer and centrifuged again at 1075gfor IO min. The resulting supematant was combined with the first supernatant. The BBM contained in the combined supernatants was packed by centtifugation at lOO,OOO~e for I hr. The resulting pellet was suspended in the vesicle bulfer solution as described above. The BLM and BBM vesicles (BLMV and BBMV. respectively) were incubated for 30 min at 37°C to facilitate buffer loading. and were stored on ice or at 4°C until used. The specihc activities of the marker enzymes in each fraction were described in a previous paper (Lee 6’1 al.. 1990). In the control group, the Na+-K’-ATPase activity in the BLM was 15.7 pmol p,/mg protein. hr, approximately IO-fold that in the cortical homogenate and the BBM preparations were enriched in alkaline phosphatase, with a specific activity of 2 I .3 K-A units/mg protein . hr, 7.6-fold greater than that of the homogenate value. However. Na+-K’-ATPase in the BBM fraction was 1.9.fold enriched and alkaline phosphatase activity in the BLM fraction was 3.2-fold enriched. indicating that each fraction was slightly contaminated with opposite side membranes. Similar results were obtained in the cadmium group. although the alkaline phosphatase activity m each fraction was significantly lower than that of the corresponding value in the control group. Sidedness of vesicles was determined for BLM preparations by a methodology similar to those employed by Kinsella t? u/. ( 1979) and Windus t’l oi. ( 1984). The sidedness was inferred from the asymmetry of the Na’-K+ATPase such that ouabain acts on the enzyme from the exterior of the intact vesicle whereas ATP acts from the interior. It was assumed that ATP and ouabain are not permeable to sealed vesicles. BLM preparations, suspended in 25 tIIM imidazole (pH 7.4) and 2 mM EDTA. were disrupted with 0.6 mg/ml deoxycholate at 25°C for 30 min. Total ATPase and ouabain (I mM)-sensitive ATPase activities were measured before and after disruption. The fraction of membranes for which orientation was not assignable (i.e.. open vesicles) was determined by measuring

104

KIM

ET AL.

TABLE

1

EFFECTOFCADMIUM TREATMLNTON URINE FLOW.CREATININECLEARANCEANDURIYARY EXCRETIONOF GLUCOSE. PROTEIN, AND PHOSPHATE Days of treatment

Control (N = IO)

Cadmium (iv= IO)

P

~Vorc~. Cadmium group animals were subcutaneously injected with CdCl? at a dose of 2 mg Cd/kg. day for I3 days and the control animals were treated with the same volume of saline. Values represent means * SE.

the quotient of ouabain-sensitive ATPase before disruption and the quotient of ouabain-sensitive ATPase after disruption. The fraction of inside out Lesicles (IOV) was assessed by subtracting the fraction not assignable from the quotient of total ATPase before disruption to total ATPase after disruption. The fraction of right-side out vesicles (ROV) was determined by calculation I -fraction not assignable-fraction everted. Such determinations revealed that the fractions of ROV. IOV. and open vesicles were 0.23. 0.07. and 0.70 in the control group and 0.25. 0.10. and 0.65 in the cadmium group; thus more than 70% ot the sealed vesicles were oriented right-side out in both groups. Dt~lerr~~imriot~

of.c~rt~.srrale

trc~t~sporl

it2 tnemhrmr

pmol/mg protein for a given time. Since the absolute uptake rate varied somewhat from preparation to preparation. the results were expressed zs a percentage ofthe equilibrium level. which was determined by the amount of uptake at 60 min. All the radioactive compounds used in this study were purchased from Amersham International (Amersham. UK). S’m~is~itd arml~:vi.c. Statistical evaluation of the data was done using the Student t test (unpaired comparison) and all results were presented as the means t SE.

I’(‘\-

W/~Y Transport of amino acids in membrane vesicles was determined using a rapid liltration method (Hopfer e/ (I/.. 1973). For the amino acid uptake. an aliquot of membrane vesicles was incubated in 8 vol of incubation medium ( 100 mM NaCI. 100 mM mannitol. and 20 mM Hepes, pH 7.4. with Tris) containing 20 PM ‘“C-amino acid (t-proline. Ialanine. or to-lysine) at 25°C. At appropriate intervals. a IOO-~1 aliquot was removed and quickly filtered through a Millipore filter (Type HA, pore size 0.45 wm). which was soahed overnight in distilled water prior to use. The filter was washed with 5 ml of ice-cold stop solution ( 100 mM NaCl-100 mM mannitol). r4C compound in the filter was dissolved into 10 ml of scintillation fluid. and the 14C activity was counted on a liquid scintillation counter (Packard Tricarb 3000 C). Nonspecitic binding of the radioactive material to the plasma membrane was determined by incubating vesicles in distilled water containing 0. I B deoxycholate and 14C amino acid for I hr. An aliquot of 100 ~1 was filtered through the millipore filter, and the filter was rinsed with the stop solution as above. The value of nonspecihc binding was subtracted from the experimental value. and the vesicular uptake was calculated as

RESULTS

Renal Fmctions Table 1 summarizes the effect of cadmium treatment on urine flow, creatinine clearance, and urinary excretion of glucose, protein, and phosphate. Subcutaneous injections of CdCl, at a dose of 2 mg Cd/kg * day for 2 weeks resulted in a marked polyuria. The creatinine clearance was not significantly changed; thus, the glomerular filtration rate remained unchanged. However, excretion of glucose, protein, and phosphate appeared to be significantly increased after 2 weeks of cadmium treatment, as observed in another study (Kim of al., 1988). These results indicate that the cadmium-treated rats in the present study also

(‘.ADMIUM

AND

RENAL

AMINO

ACID

105

TRANSPORT

developed renal functional changes typical of chronic cadmium intoxication. Table 2 shows the increase in renal amino acid excretion after 14 days of cadmium treatment. Although the most substantial increases were observed for threonine. proline, and glutamic acid. the cadmium treatment enhanced excretion of all 15 amino acids tested. The plasma levels of these amino acids were not elevated by cadmium treatment (Table 3). These results indicate that the renal transport systems for neutral, acidic, and basic amino acids were universally impaired by cadmium.

and absence of a 100 mM inwardly directed Na’ gradient. In the control vesicle, the Natdependent proline uptake increased rapidly during the initial period and then declined to the equilibrium value, showing the characteristic “overshoot” phenomenon of a Na’ gradient-dependent transport process(Fasset ul., 1977: Hammerman and Sacktor, 1977; Slack et ul.. 1977). The level of the proline uptake at 30 set was approximately 1.8 times greater than the equilibrium (60 min) level. In the vesiclesof cadmium-treated animals, however, the initial rate of the proline uptake was markedly reduced and the overshoot phenomenon was much lessapparent. There was no difference in the Na’-independent uptake of L-proline between the vesicles of controls and those of cadmium-treated animals. The Figure I illustrates the time course of the Lsteady-state uptake was similar, but the initial prolinc uptake by the BBMV in the presence 5-set uptake was 20% (control)-50% (cadTAB1.E

EFECI

OFCADMII~M

TRFATMENTON Renal

Before Amino

Alanine \ aline lroleucine Seri ne l.hreonine Kcutral aromatic Phenylalaninc I J rwnc Neutral sulfur (‘ystine Mcthionlne Neutral imlno Proline Acidic Aspark Glutamlc Basic Histidine Lysine :I’~J/<,. In each Lg. day). Values

acid acid

R~NALEXCRFTI~NOFAMINOACIDS

excretion

( pmol/kg

treatment

. day)

Cd treatment

[Al

acid

Neutral aliphatlc <;lvcine

2

15.54 2.66 0.98

i 7.40 t 0.62 + 0.70

3.65 5.64 1.3x

+ 0.x3 t 2.05 rf 0.5Y

( I4 days)

PI

[Al/PI

118.‘) I i 45.29 27.19 t 9.63 I I .03 + 5.99 10.44 43.01 44.45

7.7 10.2 11.3 5.6 7.6 IX.7

k 5.47 t 19.07 i 2 I .43

I .7h ri- 0.40 7.32 t 0.x5

IO.8 I + 4.3fI 8.01 k ?.I9

6. I 3.3

I .36 k 0.71 I .30 t 0.70

5.03 12.83

f I.81 t 4.6 I

3.4 9.9

0.70

i 0.2 I

15.06

-t 6.62

31.5

I.32 2.05

f 0.78 -t 0.73

9.24

+ 4.89

37.75

7.0 18.4

I.71 1.71

-+ 0.8 +- 0.71

7.56 4.68

animal renal excretion of amino acid was compared represent means + SE of four determinations.

before

t

18.13

t 2.38 k 2.14 and

after

4.4 1.7 cadmium

treatment

(2 mg Cd/

106

KIM

mium) lower than that with the Na+-gradient. Qualitatively similar results were obtained with L-alanine (Fig. 2) and L-Lysine (Fig. 3). These results indicate that the Na+-dependent transport systems for neutral and basic amino acids were seriously impaired by cadmium treatment. as was that for acidic amino acid (Lee et ul.. 1990). Figures 4 and 5 illustrate. respectively. the t--proline and L-alanine uptake by the BLMV in the presence of a 100 mM Nat gradient (medium > vesicles). In both cases. the uptake was not significantly different between the control and cadmium groups. The slight tendency to overshoot observed for L-alanine uptake (Fig. 5) might be associated with contamination with BBM (see Methods). In a separate series of experiments. in which L-alanine uptake and D-glucose uptake were simultaneously determined in the same batch of BLMV preparation, we have observed a small, but significant, component of Nat-dependent transport for both I.-alanine and u-glucose (Data not shown).

In the next series of experiments, we investigated the effect of direct exposure of the membrane to CdCl? on transport systems for amino acids in the renal brush border membrane. For comparison, the effect of cadmium bound to metallothionein (CdMt) was also tested. BBMVs isolated from normal animals were preincubated for 60 min at 37°C in a medium containing an appropriate concentration of CdCl, (or CdMt), and the 30-set uptake of an amino acid was determined in the cadmium-free medium at 25°C. Figure 6 depicts changes in the Nat-dependent L-proline uptake as a function of cadmium concentration in the preincubation medium. The uptake was drastically inhibited by free cadmium of above 10 pM, showing the maximum inhibition at about 100 pM. For the same concentration range, CdMt showed no significant effect on the uptake. On the basis

ET

AL. TABLE EFFECTOFCADMILIM

3

TREAIMHGTON

CONCENTRATION

OF AMINO Control (n;3)

Glycine Alanine Valine Isoleucine Serine Phenvlalanine Tyro&e Cystine Methionine Prolinc Glutamic acid Histidine Lysinc

322 565 260 136 70’ 7x I’4 35 70 361 362 87 39x

PL~sM.~

ACIDS (GM) Cadmium (*V ‘I 374 5(l7 107 145 ‘09 92 IO3 7 64 I I’) 33x 70 350

,Yotc,. Cadmium group animals were subcutanrousl~ injected with CdCI, (2 mg Cd/kg. day) for 14 days and the control animals were injected with saline.

of these results, we fixed the free cadmium concentration at 50 pM in the following experiments. Figures 7 and 8 illustrate, respectively. the time course of I.-proline and L-alanine uptake by the BBMV with and without CdCI, (50 pM Cd) pretreatment. In both cases, the Na ’ -dependent uphill transport was signficantly attenuated, but the Na+-independent transport was not apparently changed by the cadmium treatment. These results indicate that free cadmium ions directly alter the renal brush border membrane function. DISCUSSION Although chronic exposure to cadmium induces abnormalities in various organs in the body. the kidney is known to be the most critical organ (Nordberg, 1976). The events between cadmium ingestion and development of renal dysfunction can be summarized as follows (Friberg, 1984: Kjellstrom, 1986): The cadmium ingested is bound to metallothionein (Mt). a Cd-binding low molecular weight protein (Foulkes. 1982: Elinder and Nordberg,

CADMIUM

/\ND

RENAI.

1986). The Cd-Mt complex is transported via blood to the kidney, filtered through glomeruli, and absorbed into proximal tubular cells by endocytosis (Fowler and Nordberg, 1978: Squibb e/ al.. 1979). After entering lysosomes, the complex is catabolized and free Cd released is bound to newly synthesized Mt in the tubular cell. It has been proposed that kidney damage is prevented until a stage is reached at which the kidney can no longer produce enough Mt. and at this time the non-Mtbound Cd ions become nephrotoxic (Nordberg, 1978; Nomiyama and Nomiyama, 1982). The critical concentration of Cd in renal tissue was estimated to be 100-300 mg/ kg wet weight (Friberg, 1984). The time to reach this point appears to depend on the form of cadmium given and the dosage schedule. Renal functional changes observed in chronic cadmium-intoxicated patients and experimental animals include polyuria (Suzuki. 1974: Kazantzis, 1978; Nomiyama et trl., 198 1). proteinuria (Axelson and Piscator, 1966: Piscator, 1966; Nordberg and Piscator. 1 E

BBMV

i

0 a l

Cont. Cont. Cd

Na-gradient No Na Na.gradient

AMINO

ACID

BBMV

Time

0 Cont. b Cont. l Cd A Cd

-2

i

,I!

0

1 Incubation

Na-gradient No Na Na-gradient No Na

2

‘, Time

(min)

FIG. 3. Time course of Nat-dependent and Na’-indepcndent t-alanine uptakes by renal cortical BBMV of control and cadmium-treated rats. The conditions 01 incubation except amino acid were identical to those described in Fig. I. The medium contained X pM of L[“C]alanine. Values are expressed as a percentage ofequilihrrum (60 min) level. Values are means t SE of six deierminations from the same batch of vesicles in each group.

1972: Bernard et al.. 1981; Nomiyama et al.. 1982). glycosuria (Kazantzis et al.. 1963; Adams et al.. 1969; Nomiyama et ~11..1975) aminoaciduria (Kazantzis et (I/.. 1963; Adams et al.. 1969: Goyer et ul.. 1972; Gieske and

z

Incubation

107

TRANSPORT

300

c

BBMV

o b

Cont. Cont.

Na-gradient No Na

(min)

FIN;. I. Trme course of Nat-dependent and Nat-independent I -prolinc uptakes by renal cortical BBMV of control and cadmium-treated rats. Vesicles containing IOU rnst mannitol. 100 mM KCI. and 70 mM Hepes-Tris (pH 7.1) were incuhatcd in a medium containing Xl pM L[“C]prolinc. IO0 rn~ mannitol. IO0 IIIM NaCl (or KCI in the case of Nat-independent uptake). 70 mM Hepes-Tris (pli 7.4). and 7 PM valinomycin at 25°C. Values are espressed as the percentage of t-prolinc taken up by vesicles after a hO-mm mcuhation. Each datum represents the mean i SF of siy determinations from two different hatches of vesicle preparations in each group.

,

0

2

4 Incubation

10 Time

+,il (min)

FIG. 3. Time course of Nat-dependent and Na’-independent I.-lysine uptakes by renal cortical BBMV ofcontrol and cadmium-treated rats. The conditions of incubation except amino acid were identical to those described in Fig. I. The medium contained 70 pM of L-[rJC]lysine. Values are expressed as a percentage ofequilibrium (60 min) level. Values are means -t SE of six determinations from the same batch of vesicles in each group.

108

KIM ELMV,

Na

Gradient 0 l

Incubation

Time

Control Cadmium

(min)

FIG. 4. Trme course of L-proline uptake by renal cortical BLMV of control and cadmium-treated rats. The conditions of incubation were identical to those described in Fig. I, Values are expressed as a percentage ofequilibrium (60 min) level. Values are means i SE ofsix determinations from the same batch of vesicles in each group.

Foulkes. 1974: Nomiyama et al.. 1975; Bernard rf LJ., 1979) phosphaturia (Adams et ul., 1969; Kazantzis. 1978) hypercalciuria (Scott et al.. 1976) and hyposthenuria (Saito et ~1.. 1977; Kazantzis. 1978). Our previous study (Kim et al.. 1988) using male Sprague-Dawley rats demonstrated that subcutaneous injections of CdCll at a dose of 2 mg Cd/kg. day for 12-16 days induced typical renal functional defects and, consequently. produced a model suitable for study of renal tubular functions in chronic cadmium intoxication. In agreement with this study. subcutaneous administration of 2 mg Cd/kg - day of CdCl? for 7 weeks in the present study resulted in various renal functional changes, characteristic of chronic cadmium intoxication. The animal developed marked polyuria, proteinuria, glycosuria. and phosphaturia (Table 1) as well as various aminoacidurias (Table 2). Although there was some quantitative difference, renal excretion appeared to be significantly increased in all 15 amino acids measured, in the absence of plasma level change, indicating that entire classes of amino acid transport systems in the renal proximal tubule were impaired by cadmium intoxication. This finding is consistent with the notion that cadmium may induce

ET

AL

a panaminoaciduria due to inhibition of renal tubular reabsorption (Gieske and Foulkes. 1974). The results of vesicle studies indicate that the first step ofamino acid reabsorption in the kidney is impaired by cadmium intoxication. In the renal cortical BBMV prepared from cadmium-treated rats, Nat gradient-dependent uphill transport of L-proline (Fig. I ), I.alanine (Fig. 3). and L-lysine (Fig. 3) appeared to be markedly attenuated, as observed for L.glutamic acid (Lee cl al.. 1990). Since the Natamino acid cotransport depends on the electrochemical potential gradient for Na* (Fass ef al.. 1977: Hammerman and Sacktor, 1977). it would decrease if the Na’ gradient is more rapidly dissipated in the vesicles of cadmiumtreated animals. However, as described in a previous paper (Lee et ul.. 1990) the Na’ uptake itself by the BBMV was not apparently changed by cadmium intoxication. Thus. the reduction of amino acid uptake was not due to alterations in the Na’ permeability of the membrane. It is. therefore, apparent that the process of amino acid transport coupled with Na ’ transport in the renal BBM was impaired in cadmium-intoxicated animals. The mechanism underlying this change is not entirely BLMV,

Na Gradient 5 l

2 a L

O--!I

0

1 2 Incubation

Control Cadmium

-.--Al0 Time

(min)

Frc;. 5. Time course of L-alanine uptake by renal cortical BLMV of control and cadmium-treated rats. The conditions of incubaiion were identical to those described in Fig. 2. Values are expressed as a percentage ofequilibrium (60 min) level. Values are means i- SE ofsix determinations from the same batch of vesicles in each group.

CADMIUM

AND

RENAL

AMINO

ACID

BBMV

BBMV f

I”

f a

0 Cont. A Cont. l Cd . Cd

CdMt

Jo-----

5

I\ o’5:

109

TRANSPORT

Na-gradient No Na Na-gradient No Na

\

\

CdC,*

n .I -

50

0

Cadmium FIG. 0. L-proline

100

Concentration

uptake

(MM)

by BBMV

as ;I function

ofcad-

m~um (CdC‘lz or CdMt) concentration in the premcubation medium. BBMVs isolated from normal animals were prelncuhatrd in a Cd (or CdMt)-containing medium for 30 min and I -[‘4C]proline uptake was determined in Cd lor (‘dMt)-free medium for 30 SK. I.he conditions of incubation mere identical to those described in Fig. I. Data rcprcsent e0ec.t and

the mean rf SE of six determinations three determinations for CdMt etfect.

for Cd

clear at present. However. the fact that direct exposure of normal membrane vesicles to free Cd-containing medium induced similar

BBMV 0 n

incubation

Cont. Cont.

Time

Na-gradlent No Na Na-gradlent No Na

(min)

FIG. 7. Time course of Na’-dependent and Na’-independent I -proline uptakes by the BBMV with and without cadmium (SO MM) pretreatment. BBMVs isolated from normal ammals w’ere prcincuhated in a Cd-containing medium for 30 min. The conditions of incubation were Identical to those described in Fig. 1. Values are expressed as a percentage of equilibrium (60 min) level. Values are means & SE of six determinations from the same hatch of vcsiclcs.

Incubation FIG. 8. Time pendent cadmium normal medium

course

Time

of Na+-dependent

(min) and

Na+-inde-

L-alanine uptakes by the BBMV with and without (50 pM) pretreatment. BBMVs isolated from animals were preincubated in a Cd-containing for 30 min. The conditions of incubation were

identical to those described in Fig. 2. Values are expressed as a percentage of equilibrium (60 mm) level. Values are means i SE of six determinations from the same batch of vesicles.

changes in amino acid transport (Figs. 6, 7, and 8) strongly suggests that in long-term cadmium-exposed animals free Cd ions liberated in the renal tubular cytoplasm directly impair BBM function. and not through alterations of certain intracellular processes. Bevan rf (11. ( 1989) have also proposed in a recent study involving renal BBMV of the winter flounder that the Cd ion directly inhibits the Na’-alanine cotransport by binding to sites at the brush border membrane. In any event, defect in BBM function by cadmium does not seem to be confined to the amino acid transport. Previous studies on renal BBMV (Lee et al.. 1990) showed that Na+-glucose cotransport and H’-organic cation (TEA) antiport systems are all impaired in cadmium-intoxicated animals. Whether other Na+-dependent transport systems in the BBM, such as Na+-phosphate and Nat-sulfate cotransports, are also affected by cadmium intoxication has not been determined. The uptakes of L-proline and L-alanine by BLMV were not inhibited by cadmium intoxication (Figs. 4 and 5). These amino acids are

110

KIM

known to be transported in the BLM by facilitated diffusion (Silbernagl, 1988; Zelikovic and Chesney, 1989); thus, the present results seem to suggest that cadmium intoxication does not impair amino acid carriers mediating facilitated diffusion. The effect of cadmium on basolateral uptakes of several other amino acids (such as glutamine. glycine, taurine, and acidic amino acids), which involve Na’-cotransport carriers (Silbernagl, 1988; Zelikovic and Chesney. 1989), has not been determined in the present study. It is, however, anticipated that the inhibition of these transport processes. if it occurred, would reduce, rather than accelerate, urinary excretion of the amino acids in intact animals.

E-i- AL. EI.lNDER. C. G.. AND NORDBERG, M. (1986). Metallothionein. In Cudtniutn uttd Hdth: .-I To.xitrtlo~i~~trl utxl Epid~,tttio(o,gic,u/ .+lpprui.sd. (L. Friberg, C. G. Elinder. T. Kjellstrbm. and G. F. Nordherg. Eds.). Vol. I. pp. 65-79. CRC Press. Boca Raton. FL. Fnss. S. J., HAMMERMAN. M. R.. AND SAC’KIOR. B. ( 1977). Transport of amino acids in renal brush border membrane vesicles. J. Bid C’hctn. 252, 583-590. FISKE. C. H.. AND SUBBAROW, Y. ( 1925). The colorimetnc determination of phosphorus. J. Btd ~‘hwt 66. 37.5% 400.

FOIILKES. t. C. (1981). Role of metallothionein in transport of heavy metals. In Bw/o~icu/ Roles q/ ~Ilctulk~tlriotwrn (G. F. Foulkes, Ed.). pp. I3 I-140. Elsevieri North Holland. New York. FOWLER. B. A.. AND NORDBER~;. G. F. (1978). The renal toxicity of cadmium metallothionein: Morphometric and X-ray microanalytical studies. Taxid lpp/. I’lrurrnuc~d.

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L. ( 1984). Cadmium and the Kidney. Etwirotr 54. l-l I. GIESKE. T. H.. .AND FOuLhES. E. C. ( 1974). Acute erects of cadmium on proximal tubular function in rabbits. Ilealllr

ACKNOWLEDGMENTS This study was support by a grant from Korea Science and EngineeringFoundation (87 l-0408-0 I G- 1). We are indebted to Dr. S. K. Hong for encouragement and support in the conduct of these experiments.

Per.spc~~l.

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57, 635-642.

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