sodium retention in rats with cadmium-induced hypertension

The Science of the Total Environment, 22 (1981) 31--38 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

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SODIUM RETENTION IN RATS WITH CADMIUM-INDUCED HYPERTENSION*

H. MITCHELL PERRY, Jr.** and MARGARET W. ERLANGER

Medical Service, Veterans Administration Medical Center, and the Hypertension Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO (U.S.A.)

(Received April 21st, 1981; accepted in final from August 12th, 1981)

ABSTRACT

Chronic feeding of non-toxic doses of cadmium induces an average increase of 15 to 20 mm Hg in indirectly measured systolic pressure of lightly anaesthetized rats. The mechanism of this increase is not known, but cadmium has several potentially pressor effects, including increased sodium retention. This report describes both sodium balance and blood pressure in a pair of experiments where cadmium was fed and in a pair where it was injected. All four cadmium challenges induced sodium retention and also induced hypertension. Thus, rats with either low or moderate chronic exposure to fed cadmium (well below the exposures required to induce toxicity) retained more intraperitoneally injected radiosodium than controls and at the same time developed higher systolic pressures than controls. Immediately following intraperitoneally injected cadmium, rats lost a radiosodium load more slowly than controls or alternately accumulated dietary radiosodium more rapidly than controls; in both situations the blood pressure was higher than in controls. These data indicate that some of the pressor effect associated with cadmium exposure could result from its concomitant antinatriuretic effect.

INTRODUCTION

Small amounts of cadmium, administered either by injection or by feeding, can induce hypertension in rats. Intraperitoneally injected cadmium induces a prompt and marked increase in blood pressure [1, 2]. Long-term feeding of 0.1 to 5 parts per million (ppm) cadmium in drinking water induces an average increase in systolic pressure of 15 to 20 mmHg [3,4]. This exposure is well below the levels which produce any overt toxic mani- festations. Although the mechanism by which cadmium induces hypertension remains unclear, cadmium has been associated with four potentially pressor effects: (a) sodium retention [5--8] ; (b) vasoconstriction [9,10], (c) hyperreninemia [11] ; and (d) increased cardiac output [9].

* This research was supported by the Veterans Administration. ** To whom all correspondence should be addressed as follows: H. Mitchell Perry, Jr., M.D., VA Medical Center, l l l D JC, St. Louis, MO 63125, U.S.A.

0048-9697/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company

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The first of these, sodium retention, seems a particularly likely cause of at least part of the chronic hypertension induced by cadmium feeding. While investigating this possibility, Doyle and coworkers observed that cadmium feeding, using both a concentrat ion of cadmium and a duration of exposure previously reported to induce hypertension, did indeed produce sodium retention, but they observed no hypertension [5]. These investigators used both male and female Long-Evans rats which were fed 5 ppm cadmium for 5 to 10 months beginning at the time of weaning. Their demonstrat ion of sodium retention involved measuring, by whole body counting, the rate at which radiosodium was lost following the intraperitoneal injection of a single dose of sodium.

METHODS

Forty-four weanling female Long-Evans rats from Blue Spruce Farms, Altamont, New York, were randomly divided into four equal groups each of which was placed in a stainless-steel cage. All were provided ad libitum with the previously described rye-based, low-cadmium diet containing 0.014 mg cadmium and 5.9 g sodium per kg [3]. Drinking water was also provided ad libitum. Group 1 received water "for t i f ied" as described [3] , i.e. it was doubly deionized and zinc, manganese, copper, molybdenum, and cobalt were added to make the final concentrations 50, 10, 5, 1 and 1 ppm, respectively. For Groups 2 and 4, 1 ppm cadmium (as the acetate) was added to this fortified water. For Groups 3 and 4, 10,000 ppm sodium chloride (Bio- logical Grade Crystal, Fisher certified, from Fisher Scientific, St. Louis, Missouri) were added, making the fortified water 1% with respect to salt. Two and four months after weaning, each animal was individually weighed and lightly anaesthetized (25mg/kg of intraperitoneal sodium pentobarbital), after which its systolic pressure was determined indirectly by the tail cuff method of Friedman [13] as previously reported [12]. Three months after weaning the animals were again individually weighed, and each rat received, by intraperitoneal injection, 0.08 ml of 0.9% aqueous NaCI labeled with 2 pCi 22 Na (total Na injected was 0.3 pg). Five minutes after the injection, the rat was placed in an Armac-Packard Model 440 whole-body liquid scintillation detector for two minutes in order to obtain the baseline (i.e. immediate post-injection) level of 22 Na. Whole-body counting was repeated at 24-h intervals for four days and on the seventh and eleventh days after 22 Na injection, with the results being expressed as counts per minute (cpm) retained by the rat.

In a second longer experiment, the same procedures were followed with two groups of 10 rats each; for them the last radiosodium measurement was made on the tenth day. One group received fortified water and the other received fortified water plus 5 ppm cadmium for 12 months before the 22 Na injection. Indirect systolic pressures and whole body counting were obtained as before. In this experiment, sodium chloride was not added to the water of any animals.

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In the third and fourth experiments, the effect of parenteral cadmium was tested in 300-g male Wistar rats obtained from the National Laboratory Ani- mal Company of St. Louis, Missouri, and acclimated in our laboratory for four weeks to Purina Lab Chow and tap water ad libitum.

For the third experiment, eight rats received tap water ad libitum, labelled with 0 .4pCi22Na per liter ( 0 .002pgNa per liter), for 30h. At the end of this period, four animals were given intraperitoneal injections of 2 mg of cadmium (as the acetate and dissolved in 0 .1ml of 0.9% sa l ine)per kg of body weight, and four were given the 0.1 ml of saline alone. Both groups of animals continued to receive the Purina Lab Chow and 22 Na labelled water ad libitum for 22 days, at which time each animal was given a second intraperitoneal injection consisting of half of its initial injection (1 mg cadmium/ kg and/or 0.05 ml of saline). Following the second injection, the 22 Na content of the drinking water was tripled, and both groups of animals continued to receive Purina Lab Chow and the new 22 Na labelled water ad libitum for the remaining 32 days of the experiment. Baseline whole body counting for 22 Na was done on each animal immediately before the first injection of cadmium and/or saline and 35 more times during the 53-day experiment. After the last count, the rat was anaesthetized with sodium pentobarbital (50mg/kg) , and blood pressure was measured directly from the cannulated left femoral artery, as previously described [1] .

For the fourth experiment, eight rats received tap water ad libitum with 0.4 pCi 22 Na per liter for seven days. Tap water without 22 Na was then substi tuted; at the same time, four rats were given 1 mg cadmium per kg of body weight in 0.1 ml of 0.9% saline intraperitoneally, while the other four received saline alone. A baseline whole body count for 22 Na was done on each rat at the time 22 Na labelled drinking water was withdrawn and six more times during the next four days. After the last count, blood pressure was measured as in the third experiment. Standard deviations were calcula- ted for all data. Student 's t-test was used as a measure of significance.

RESULTS

Experiment 1 Figure 1 compares female Long-Evans rats given I ppm cadmium, but no

sodium, in their drinking water with control rats which received water containing neither metal. Within three months, cadmium-fed rats retained more of a tracer dose of intraperitoneally injected sodium than control animals. The differences were highly significant from the second through the fourth day (p ( 0 . 0 0 5 ) and still significant on the seventh (p ~ 0 . 0 1 ) and eleventh days (p ( 0 . 0 2 ) . The indirectly measured systolic pressures one month before the 22Na injection averaged 125 +15 for the cadmium-fed group and 105 + 11 mm Hg for the control group; the difference was significant (p 0.001). One month after the 22 Na injection, the average difference in systolic pressure was 18 mm Hg (p ~ 0.001).

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15o~]

±\ \~

1 2 4 7 11

22Na , 0 DAYS

Fig. 1. Experiment I: Retention of injected sodium in rats given 1 ppm cadmium (but no added sodium) in drinking water for three months. Average whole body 22 Na content in tens of thousands of counts per minute (cpm × 104) for two groups of 11 female Long- Evans rats. Open circles represent cadmium-fed rats and solid circles control rats. Stan- dard deviations are indicated by the half bars. The significances of the differences are indicated by the p values. The indirect systolic pressures one month before and one month after the measurement of sodium retention averaged 20 and 18 mm Hg higher, respectively, in the cadmium-exposed group than the control group, with p < 0.001 for each.

C a d m i u m did n o t a l ter sod ium r e t e n t i o n in sod ium- fed ra ts or raise the systol ic pressure. N o t u n e x p e c t e d l y , in jec ted r a d i o s o d i u m was los t m u c h m o r e rap id ly in the t w o m a t ch i ng groups of animals which had received 1% sod ium chlor ide in the i r wa t e r for th ree m o n t h s , wi th b o t h the c a d m i u m - f e d and con t ro l groups re ta in ing less than ha l f of the 22 Na a f t e r 24 h and less t han a s ixth a f t e r 48 h. The average systol ic pressures one m o n t h be fo re 22 Na in jec t ion was 139 + 22 fo r the c a d m i u m - f e d g roup and 134 + 16 m m Hg fo r the con t ro l group. Thus, the re was n o s ignif icant pressure d i f fe rence be tween these c a d m i u m - f e d and con t ro l rats and b o t h groups of sod ium- fed rats had s ignif icant ly higher average systol ic pressures t han the con t ro l animals with- o u t fed sod ium. At th ree m o n t h s , the average weight o f the rats fed cad- m i u m bu t no added s od i um was greater (300 g) t han the average weights o f the o t h e r th ree g roups ( 2 7 0 - - 2 7 2 g) (p < 0.05).

Experiment 2 Table 1 presents s imilar da t a fo r an imals fed 5 p p m c a d m i u m for 12

m o n t h s . The d i f fe rence in 22 Na r e t e n t i o n be tween the con t ro l and c a d m i u m - fed groups was smal ler t h a n in the y o u n g e r rats, and it s eemed to deve lop later , bu t it was still h ighly s ignif icant f r o m the th i rd t h rough the seven th day a f te r 22 Na in jec t ion (p < 0 .005) and less m a r k e d l y so on the e leventh day (p < 0 .025) . The average b lood pressures a m o n t h a f t e r 22 Na in jec t ion were 122 + 12 m m Hg for the c a d m i u m - f e d ra ts and 110 + 12 m m Hg for the con t ro l rats (p < 0.05). A t 12 m o n t h s there was no s ignif icant d i f fe rence in

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TABLE I EXPERIMENT 2: RETENTION OF INJECTED SODIUM IN RATS FED 5 ppm Cd FOR 12 MONTHS Average (+ standard deviation) whole body 22Na content for 10 female Long-Evans rats which received 5 ppm cadmium in water for 12 months from weaning and for 10 com- parable control rats. The indirect systolic pressures, measured a month before and a month after 22 Na injection, averaged 13 and 12 mm Hg higher in the cadmium-fed than in the control rats (p < 0.05).

Time after 22 Na injection

Day 1 Day 2 Day 3 Day 4 Day 7 Day 10

Retention of 22 Na (as percent of that present immediately after injection)

Rats fed 0 ppm cadmium 83-+2 69+3 54+-3 43+-4 25+3 16+2 Rats fed 5 ppm cadmium 84 +-3 71 +-4 60 +5 49 +4 29 +3 19 -+3 Significance of

difference (p) <0.40 <0.30 <0.005 <0.005 <0.005 <0.025

average weight be tween the groups, a l though the cadmium-fed group was heavier (375 g) than the con t ro l group (357 g).

Experiment 3 Fol lowing the in t raper i tonea l in ject ion o f 2 m g cadmium per kg b o d y

weight , f ou r male Wistar rats accumula ted more o f the 22 Na which was pre- sen ted to t hem in dr inking wate r than did four con t ro l rats w i t h o u t cadmium exposure ; a second smaller in jec t ion of cadmium (1 mg/kg) th ree weeks a f te r the first p r o d u c e d a similar bu t m o re marked increase in sod ium re t en t ion (Fig. 2). The e f fec t was delayed, with no obvious d i f fe rence be- tween the two groups in the up t ake o f fed sod ium during the first 12 days af te r the initial c admium inject ion. F r o m the twe l f th t h ro u g h the f i f t een th day af te r in ject ion, however , the cadmium exposed rats had an average o f 15 to 20% more whole b o d y 22 Na than the con t ro l animals. Even wi th the very small numbers o f animals involved, fo r those fou r days the d i f fe rence be tween the two groups was significant, with p ~ 0.05. The second in jec t ion o f cadmium p r o d u c e d a similar e f fec t which was again delayed. Thus, there was no d i f fe rence in sod ium r e t en t i on for five days, b u t t hen the d i f fe rence gradually increased, reaching a m a x i m u m of m o re than 20% some 28 days later (p ~ 0.05) a f te r which it seemed to decrease somewhat . Most o f the rela- t ively large d i f ferences were n o t significant statist ically because individual cadmium- in jec ted rats d i f fe red great ly f rom each o the r in thei r 22 Na reten- t ion. B lood pressures, measured di rec t ly in anaes the t ized rats 53 days a f te r the first c admium inject ion, averaged 1 3 0 / 1 0 4 in the cadmium-exposed group and l 1 9 / 9 2 m m H g in the con t ro l group. This d i f fe rence was n o t stat ist ically significant.

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10G

0

×

E u

v

o

© 212

A ]0 2'0 A 3'0 4'0 50 2mg/kg ip. Cd 1 mg/kg i p Cd.

DAYS

Fig. 2. Experiment 3: Uptake of fed sodium in rats injected with cadmium. Average whole body 22Na in thousands of counts per minute (cpm × 103 ) for two groups of four 300-g male Wistar rats. Open circles represent cadmium-injected rats and solid circles control rats. Significant differences in sodium retention (p < 0.05) are indicated by arrows from days 12 through 15 after the first (2 mg/kg) cadmium injection and at 28 days after the second (1 mg/kg) cadmium injection. Directly measured diastolic pressures at the end of the experiment averaged 104 +16mmHg for cadmium-injected and 92 -+2 mm Hg for control rats.

Experiment 4 Fol lowing the in t raper i tonea l in ject ion o f 1 mg cadmium per kg b o d y

weight, fou r male Wistar rats lost 22 Na, which had been fed to them during the pr ior week, less rapidly than did four con t ro l rats w i t h o u t cadmium exposure . Thus , dur ing the first 16 h fol lowing cadmium inject ion, cadmium- exposed rats lost an average o f 12% of the 22 Na present at inject ion; whereas con t ro l rats lost 23%. This cons tan t d i f fe rence of 10% o t more in 22 Na c o n t i n u e d with lit t le change (range 9 to 14%) for the 96 h t h a t the experi- m e n t con t inued . There was considerable var ia t ion among the fou r animals in each group, and the average d i f ferences were n o t statist ically significant. Direc t ly measured b lood pressures 96 h af te r cadmium injec t ion averaged 133 /110 mm Hg; whereas for the con t ro l animals i t averaged 109 /87 m m Hg. The di f ferences in pressure were statist ically significant (p < 0.05 for systolic pressures and < 0 . 0 2 5 for diastolic pressures).

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DISCUSSION

The foregoing experiments demonstrate that both fed and injected cadmium can induce the combination of hypertension and sodium retention. Moreover, the combination can be induced in male Wistar rats as well as female Long-Evans rats, so it is not limited to one strain or one sex. The two cadmium feeding experiments involved large enough numbers of rats to produce significant differences. The two 22 Na feeding experiments, on the other hand, were each limited to the eight animals that our facilities could handle. In both of them, the observed differences were in the same direc- tion and of the same order of magnitude as in the larger experiments; however, in Experiment 3 the difference in blood pressure was not significantly different and in Experiment 4 the difference in sodium retention was not significantly different.

It has long been known that chronic cadmium feeding can induce hypertension in rats [ 14] . The mechanism of this pressor effect is unknown, but it might well be related to the sodium retention induced by cadmium [ 5--8]. This antinatriuretic effect was first recognized in 1962, when Vander observed that injecting cadmium into one renal artery of a dog significantly enhanced proximal tubular reabsorption of sodium on that side [15] . Sub- sequently both Lener and Musil [7] and Doyle et al. [5] observed sodium retention following Cadmium feeding. Until the experiments presented here, however, the association of cadmium-induced hypertension and cadmium- induced sodium retention had not been reported. It therefore seemed impor- tant to confirm the association of the two effects and to demonstrate that both were general, occurring with cadmium exposures of varying intensity, with more than one route of cadmium exposure, and in rats of both sexes and of more than one strain.

When Doyle et al. induced sodium retention with cadmium, they sought -- but did not find -- hypertension [ 5], although they used cadmium exposures which we had previously reported to be pressor, i.e. 5 ppm in drinking water for 10 months [12] . Doyle 's chronic feeding experiments deviated most obviously from our standard conditions in the composit ion of the diet, which differed markedly not only in major components but also in metal content. With respect to the latter, rats exposed to our standard rye-based diet received approximately twice as much sodium and four times as much potassium and magnesium but only three-quarters as much calcium as rats fed the glucose-based diet described by Doyle et al. [5] ; they also received six times as much zinc, but only a hundredth as much chromium.

REFERENCES

1 H.M. Perry, Jr. and M. W. Erlanger, Hypertension and tissue metal levels after intraperitoneal cadmium, mercury and zinc. Am. J. Physiol., 220 (1971) 808--811.

2 H.A. Schroeder, S. S. Kroll, J. W. Little et al., Hypertension in rats from injection of cadmium. Arch. Environ. Health, 13 (1966) 788--789.

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3 H.M. Perry, Jr., M. W. Erlanger and E. F. Perry, Increase in the systolic pressure of rats chronically fed cadmium. Environ. Health Perspect., 28 (1979) 251--260.

4 H. M. Perry, Jr. and M. W. Erlanger, Metal-induced hypertension following chronic feeding of low doses of cadmium and mercury, J. Lab. Clin. Med., 83 (1974) 541-- 547.

5 J . J . Doyle, R. A. Bernhoft and H. H. Sanstead, The effects of a low level of dietary cadmium on blood pressure 22Na, 42K, and water retention in growing rats. J. Lab. Clin. Med., 86 (1975) 57--63.

6 J. D. Foulke, E. V. Kessler, G. S. Born et al., Effect of cadmium on sodium and potassium excretion and on action of hydrochlorothiazide in rats. J. Pharm. Sci., 63 (1974) 563--566.

7 J. Lener and J. Musil, Cadmium influence on the excretion of sodium by kidneys. Experimentia, 27 (1971) 902.

8 H.M. Perry, Jr., E. F. Perry and J. E. Purifoy, Antinatriuretic effect of intramuscular cadmium in rats. Proc. Soc. Exp. Biol. Med., 136 (1971) 1240--1244.

9 H. M. Perry, Jr., M. W. Erlanger, A. Yunice et al., Mechanisms of the acute hyper- tensive effect of intra-arterial cadmium and mercury in anaesthetized rats. J. Lab. Clin. Med., 70 (1967) 963--972.

10 G.J . Youkilis, R. C. Martz, P. D. Harris et al., Influence of denervation on the dilata- tion of minute blood vessels following cadmium administration. Arch. Int. Pharma- codyn., 189 (1971) 145--155.

11 H.M. Perry, Jr. and M. W. Erlanger, Elevated circulating renin activity in rats following doses of cadmium known to induce hypertension. J. Lab. Clin. Med., 82 (1973) 339--405.

12 H.M. Perry, Jr., M. W. Erlanger and E. F. Perry, Elevated systolic pressure following chronic low-level cadmium feeding. Am. J. Physiol., 232 (19770 Hl14--H121.

13 M. Friedman and S. Freed, Microphonic manometer for indirect determination of systolic blood pressure in the rat. Proc. Soc. Exp. Biol. Med., 70 (1949) 670--672.

14 H.A. Schroeder and W. H. Vinton, Jr., Hypertension induced in rats by small doses of cadmium. Am. J. Physiol., 202 (1962) 515--518.

15 A. J. Vander, Cadmium enhancement of renal tubular sodium reabsorption. Am. J. Physiol., 203 (1962) 1005--1007.

sodium retention in rats with cadmium-induced hypertension

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