Toxicology, 64 (1990) 235--243 Elsevier Scientific Publishers Ireland Ltd.

Delayed nephrotoxic effects of cadmium and their reversibility by chelation

J.Y. Zhao a*, E.C. F o u l k e s a a n d M . J o n e s b

*Departments of Environmental Health and Physiology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0056 (U.S.A.) and bChemistry Department, Vanderbilt University,

Nashville, TN 37235 (U.S.A.)

(Received May 14th, 1990 ; accepted June 19th, 1990)

Summary

A single injection of CdCI 2 (15 tamol/kg) together with 300 tamol/kg mercaptoethanol into adult male rats induced renal dysfunction only after a delay of at least 12 h; in contrast, the peak value of cadmium in renal cortex (34 4- 3 tag/g) was reached within 2 h. The chelator methoxyben- zylglucaminedithioate (MBGDT) rapidly reduced the renal Cd level by 2/3, primarily by extracting the metal from the sedimentable rather than the cytosolic fraction. MBGDT prevented the subsequent onset of malfunction, but only when given at an early stage after Cd administration (2 h). It follows that the renal damage seen after the initiation period is independent of the Cd concentration at that time. The changes occurring during initiation remain unexplained.

Key words: Cadmium; Rats; Delayed nephrotoxicity; Indirect effects; Chelation; Subcellular distribu- tion

Introduction

C a d m i u m is well known to be highly nephrotoxic and its ma jor public health impor tance has led to extensive studies [1]. Many details of its mechanism of

action, however, still remain unclear. After chronic exposure, cadmium leads to aminoac idur ia , p ro te inur ia and other signs of tubu la r mal func t ion . In contrast , acute loading of the kidney causes no immedia te inhib i t ion of amino acid reab-

sorpt ion [21. In other words, the metal is not a direct inhibi tor of carrier- mediated t ranspor t . Instead, it appears to trigger some pre l iminary act ion which, after an ini t ia t ion delay, becomes reflected indirectly in aminoac idur ia . The pur-

pose of the present experiments was to explore the na ture of this triggering

Address all correspondence to: Dr. E. C. Foulkes, Department of Environmental Health, University of Cincinnati, College of Medicine, 3223 Eden Avenue, Cincinnati, OH 45267-0056, U.S.A. *Present address: Research Center of Occupational Medicine, Third Hospital, Beijing Medical Univer- sity, Beijing 100083, People's Republic of China.

0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Prihted and Published in Ireland

235

action. Specifically, we wished to determine whether removal of cadmium during the initiation period would prevent the subsequent appearance of overt renal mal- function. This work was made possible by the introduction of chelators able to reduce the renal Cd load in Cd-treated animals [3].

Materials and methods

Adult male Sprague--Dawley rats, 250--400 g, maintained on commercial rat chow, were used for these experiments. They were injected i.p. with cadmium chloride and mercaptoethanol (ME) [4]. In experiment A, six groups of 5 rats each were given a single injection of 15 /~mol/kg CdCI 2 mixed with 300/Jmol/kg of ME and 1.5 gCi of ~°gCdCl2 in a final volume of 0.5 ml saline. In experiment B, six similar groups of rats were injected as in A; 2 h later they were further treated i.p. with sodium-N-[4-methoxybenzyl]-o-glucamine-N-carbodithioate (MBGDT), 1 mmol/kg. The compound was prepared as described by Jones et al.

[3]. In experiment C, three further groups of cadmium-treated rats were treated as

in B; the chelator was given immediately after injection of cadmium (group 1), or 4 h (group 2) and 12 h (group 3) later.

Rats were placed in individual metabolic cages. After a 6--8 h stabilization period, control urine samples were obtained; further samples were collected at 2, 4, 12, 24, 36 and 48 h after Cd injection. In experiments A and B, rats were killed under pentobarbital anesthesia (i.p.); blood (by cardiac puncture), liver and kidney samples were then obtained. In experiment C, all the rats were killed at 48 h after injection of cadmium.

Urines were analyzed for total amino acids by the simplified method of Lor- entz and Flatter [5]; results are expressed as mmol /mg creatinine [6]. Protein content was determined with the Coomassie brilliant blue G-250 method [7], using the protein assay kit (Bio-Rad Labs, Rockville Center, NY).

Glomerular filtration rate (GFR) was equated to the clearance of endogenous creatinine. Urinary concentrating ability was measured with a vapor pressure osmometer (model 5100C, Wescor Inc., U.S.A.). A modified fluorimetric method was used for measurement of N-acetyl-/~-glucosaminidase (NAG) activity [8]; l unit of activity = 1 nmol 4-methylumbelliferone produced/h /mg creatinine at 37°C, pH 4.5. Cadmium concentrations in heparinized plasma, in tissues and in urine were calculated from ~°gCd counts as determined on a gamma spectrometer (Packard auto-gamma 800C).

Kidney cortex and livers were cut into slices and weighed prior to counting. For separation of cytosolic from particulate Cd, the slices were homogenized in 3 ml of 0.9% saline with a motor-driven Teflon glass homogenizer, 10--20 strokes [9]. Homogenates were centrifuged at 12 000 g for 20 min (2--4°C) in a Beck- man model J2-21 refrigerated centrifuge. Sodium and potassium contents of renal cortex, plasma and urine were measured using a digital flame analyzer (Cole- Parmer 2655-00), after extraction of the tissues with 10% TCA at 100°C for 3 h.

All the results are presented as means ± S.D. Student's t-test (two-tailed) for independent data was used as a test of significance between means [10].

236

8

=,

E ._= E

" o

50

40

30

20

10

0

0 0 N o C h e l a t o r

H C h e l a t o r A f te r 2 h

x l x

l

i 2 ~ i i la2 i i i 4 6 24 36 48

f

H o u r s Af te r In ject ion of C a d m i u m

Fig. 1. Renal conical cadmium levels. All animals were injected at zero time with Cd (15 t~mol/kg) + mercaptoethanol, as described in the text; chelator (I mmol/kg) was administered i.p. after 2 h, as shown. Each point represents the mean _+ S.D. from 5 rats. *Different from non-chelated group (P < 0.001); xSignificantly different from preceding value.

Results

Levels o f cadmium in renal cortex Figure I shows the changes in the renal cortical concentration of cadmium

with time after injection. Within 2 h the renal cadmium reached its peak, 34 _.+ 3 /ag/g wet weight, corresponding to 7.7 _+ 0.907o of injected dose/g. Subsequently, the concentration fell, reaching about 4007o of peak concentration after 48 h.

Injection of the chelator caused a fast reduction of renal cortical cadmium to about 1/3 of the peak level within 2 h; little further change occurred over the next 44 h. Whether chelation at time points later than 2 h leads to the same loss of cortical Cd was not determined.

Distribution of cadmium between cytosol and particulate matter The distribution of cadmium between cytosol and particulate matter in renal

cortex and the effect of chelation on this distribution are shown in Table I. Initially, about 60°70 of renal cadmium was contained in the sediment. The Cd

content of cytosol, present presumably to a significant extent in metallothionein, fell only slowly over the next 46 h, in contrast to the much faster decrease in the sedimented Cd. The sedimented Cd fraction was also more sensitive to MBGDT than was cytosolic Cd.

Levels of cadmium in plasma and urine As illustrated in Fig. 2, the peak concentration of cadmium in plasma was

reached one hour after injection; 4 h later a lower steady level was achieved. Chelation increased plasma Cd only for a few hours. The peak of urinary Cd appeared at the second hour after injection. Chelation greatly increased the uri-

237

T A B L E 1

D I S T R I B U T I O N OF C A D M I U M B E T W E E N C Y T O S O L A N D S E D I M E N T A B L E F R A C T I O N OF R E N A L C O R T E X O F C d - T R E A T E D RATS, A N D E F F E C T S OF C H E L A T I O N A F T E R 2 h

T ime af ter Cd in jec t ion (h)

C h e l a t o r C a d m i u m levels

Sed iment Cy toso l

/~g/g - A % /ag/g

Renal cor tex

- AOTo iJg/g - A %

2

12

24

48

- 18.8 ± 2.0 - 15.1 .+ 2.9 - 34,4 ± 2.9 -

- 9.5 ¢ 3.7 49.5 14.5 ± 2.3 4.0 24,3 ± 2.5 29.4

+ 3.3 ± 0.9 82.4 9.7 ± 2.2 35.8 12.9 ± 2.4 62.5

- 6.4 ± 0.7 66.0 14.2 ± l . l 6.0 20.6 ± 2.0 40.1

+ 3.1 • 0.6 83.5 9.1 ± 1.2 37.1 13.1 ¢ 2.5 61.9

- 3.1 ± 0.1 83.5 l l . 3 ± ! .6 25.2 14,5 ± 2.0 57.8 + 2.2 ± 0.9 88.3 8.1 ± 3.9 46.4 10.3 ± 4,3 70.1

-A°70 represents the reduc t ion in c a d m i u m concen t r a t ion

to results f rom n o n - h o m o g e n i z e d tissue. W h e r e ind ica ted ,

(1 m m o l M B G D T / k g ) 2 h fo l lowing Cd admin i s t r a t i on .

be low the value at 2 h. Renal cor tex refers the an ima l s were injected wi th the chela tor

nary excretion of cadmium; this presumably reflects in part mobilization of renal Cd.

Changes in renal function Renal function in the rats from experiments A and C are shown in Figs. 3, 4,

5 and Table II. The injection of cadmium clearly increased urinary amino acid

v

6 A == . _ 5 X

. jO )

x .

! 1246 12 24 36 48 01246 12 24

HOURS AFTER INJECTION OF CADMIUM

O No Chelator

36

Chelator After 2 h

Fig. 2. C a d m i u m levels in p l a sma and ur ine. For detai ls , see Fig. 1.

B

48

238

A No Chelator Chelator at Time

f f l " O

"~ .~. ,~ ,,.. Ot~

~E m E

0.25"--'~

0.2---~

0.15---~

0 .1 "~

o.o5--~ --4

0 '

- r -T-

T -I-

0 12 24 36 48

0

7 ~ -T- !, -r- T {{ . . . . . .

12 24 36 48

4h 12h

12 24 36 48 12 24 36 48

._=

o o f l , ,01

._= E i , . v :3

B No Chelator

2,~ T 7- .-4 "-4

2o--I

lo--1 T - i

3-r- o l

0 12 24 36 48

Chelator at Time 0

12 24 36 48

4h 12h

t l 12 24 36 48 12 24 36 48

~,,2. = i P

:3

C

3000-~j

2500.~

2000-~

1500-~

1000-~

500--.-] 0 1

No Chelator

T T

T

Chelator at Time 0 4h 12h

0 12 24 36 48 12 24 36 48 12 24 36 48 12 24 36 48

Hours After Injection of Cadmium

Fig. 3. Changes in amino acid, protein and NAG excretion after cadmium treatment. Each column represents the mean ± S.D. from 5 rats; base line values are shown as the zero time point in non- chelated animals. Chelator (1 mmol /kg ) was administered together with Cd (time 0), or 4 or 12 h later.

239

C

~ 0 "6E E v U) 0

1200

1000

800

600

400

200

0

~ T , ~ . . ~ ~ 'Tc '

I I I I I I I

0 2 6 12 24 36 48

Hours After Inject ion of Cadmium

Fig. 4. Changes in urine osmolality following Cd treatment. Each point represents mean :t: S.D. for 5 animals. Rats received 15 ~mol Cd/kg plus mercaptoethanol. A: No further treatment; C,, C 2 and C~ were treated in addition with 1 mmol chelator at 0, 4 and 12 h following Cd injection. Statistical significance as for Fig. 1.

excretion (Fig. 3a), with a m a x i m u m 24 h after injection. The chelator, if given

within 2 h, prevented most of this increase, but later admin is t ra t ion was less effective. Similar results are shown with ur inary protein excretion (Fig. 3b) and the activity of N A G in urine (Fig. 3c). Fig. 4 shows the drop in urine osmolali ty,

again prevented by chelat ion only if applied within 2 h after cadmium injection. The same conclusion can be drawn from Fig. 5 which illustrates the well known effect of cadmium in depressing GFR; this is at least partially prevented by chela-

t ion within 2 h, but not at later times.

E

E. ( -

'-,t ID O

60

50

4 0 :

30

20

10

o ~ Control

T il

A C 1 C 2 C 3

A

30 I T . 20 ~ ~i~ 1 0 ~ • 0

Control A

B 1

Fig. 5. Glomerular function in Cd-treated rats. Each column represents mean _ S.D. for 5 rats stud- ied 48 h after Cd injection; control animals received no Cd. Groups A, C t, C 2 and C 3 see Fig. 4. *Significantly different from control (P < 0.001).

240

TABLE II

SODIUM AND POTASSIUM CONTENTS OF RENAL CORTEX, AND FRACTIONAL ELEC- TROLYTE EXCRETION IN RATS 24 h AFTER INJECTION OF CADMIUM

Control Cadmium- P rats treated rats

Renal Cortex Na 59 ± 6 54 ± 10 >0.05 ~Eq/g) K 70 ± 6 75 __. 10 >0.05

Plasma Na 138 ± 5 137 ± < 1 >0.05 OEq/g) K 5.2 ± 0.6 5.5 ± 0.5 >0.05

Urine Na 0.6 ± 0.12 0.05 ± 0.02 <0.001 (Fractional K 16.0 ± 2.2 11.20 ± 4.3 <0.05 excretion, %)

Four samples were taken for each measurement from each rat, and results represent means (± S.D.) from 5 animals. Urinary excretion is expressed as percent of filtered load.

Levels of sodium and potassium in renal cortex, plasma and urine In the search for ear ly toxic d a m a g e in the cor tex, the concen t ra t ions o f Na

and K were de t e rmined (Table II). C a d m i u m caused no s ignif icant changes; net f rac t iona l excret ions o f f i l tered Na and K were bo th decreased.

Discussion

The results presented in this pape r con f i rm the existence o f an in i t ia t ion de lay preceding the appea rance o f over t ma l func t ion fo l lowing acute admin i s t r a t ion o f

c a d m i u m . As prev ious ly po in ted out [2], the de lay canno t be adequa te ly expla ined ei ther in terms o f c a d m i u m - m e t a l l o t h i o n e i n fo rma t ion , o r by a slow diss ipa t ion o f m e r c a p t o e t h a n o l , or ig ina l ly a d d e d to permi t renal up take o f the

meta l . A fur ther obse rva t ion in the present work is that r emova l o f the metal by che-

la t ion dur ing the in i t ia t ion pe r iod will not prevent the subsequent onset o f mal- funct ion except at an ear ly stage. The inabi l i ty to prevent damage is in s t rong cont ras t with the s ignif icant reduc t ion achieved in the concen t ra t ion o f c a d m i u m in the t issue (Fig. 1). The che la to r used ( M B G D T ) was in t roduced by Jones et al. [3], who repor ted that repea ted admin i s t r a t i on reduced renal Cd content in Cd- t rea ted mice by 80°70.

Whe the r che la to r t r ea tmen t at later t imes fo l lowing exposure to Cd removed as much metal as the in jec t ion af te r 2 h was not de te rmined in these rats. In a series o f rabbi t s (unpubl i shed exper iments ) i .p. in ject ion o f 0.25 mmol M B G D T / kg 3 - - 5 h af te r i.v. in jec t ion o f 15/Jmol C d / k g (in presence o f mercap toe thano l ) removed as much as 8007o o f renal cor t ica l Cd wi thout prevent ing comple te renal shu tdown 45 h later ; this c lear ly dissociates the final lesion f rom the con t inued

241

presence of Cd. In any case, if one assumed that in the rats Cd becomes more resistant to chelation at some time point later than 2 h from its administration, one would imply that a critical change in tissue Cd must have occurred at that time. This implication would fully support the conclusion that there is a critical time during the initiation delay after which the effect of Cd becomes irreversible under the present conditions.

Prolonged treatment of rats with a similar chelator (N-benzylglucam- inedithiocarbamate) in the hands of Kojima et al. [11] led to a partial reversal of the renal effect of cadmium. This was not our experience after only one injection of the methoxy derivative employed here. In our rats, MBGDT reversed the effects of Cd only when given within 2 h of acute exposure.

The fact that chelation tended to reduce the sedimentable Cd more so than that in cytosol (Table I) may reflect the accumulation of the metal in cytosolic metallothionein. The additional finding that damage is prevented by chelation only at the time when sedimentable Cd is still relatively high suggests the possibil- ity that the toxic species of the metal at that early stage is in the sediment. The toxic action of cadmium, prevented by early chelation as in Fig. 3, may therefore involve primarily the particulate fraction.

The finding that Cd exerts its effects on renal function not at the time of its maximum tissue concentration but only much later, at the end of a relatively pro- longed initiation period, could be interpreted in agreement with Yuile [12] to indicate that the metal does not directly inhibit tubular function.

Such an explanation agrees with the interpretation of recent observations on the basolateral transport of amino acids by the renal tubules of the Cd-poisoned rabbit [13]. Absence of early overt effects on the kidney was confirmed not only by the unchanged tubular transport capacity for aspartic acid [2], and the rela- tively insignificant fall in the urinary concentrating ability (Fig. 4), but also by the normal electrolyte content of the tissue (Table II). An early increased frac- tional reabsorption of filtered Na in the dog following Cd administration [14] probably reflects circulatory changes. Further work will be required to clarify the nature of changes occurring in the renal parenchyma during the initiation period.

Acknowledgments

We thank Dr. Minnema for making available the fluorimeter used in this study, Ms. Blanck for her helpful assistance in the study and Ms. Townsley for her excellent secretarial assistance. The research was supported by NIH grants ES-02453 and ES-00159.

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