ontogenetic aspects of thallium-induced nephrotoxicity in rats

JOURNAL OF APPLIED TOXICOLOGY, VOL. 16(3), 235-243 (1996)

Ontogenetic Aspects of Thallium-induced Nephrotoxicity in Rats

Dorothea Appenroth'.+ Silke Tiller,' Stepan Gambaryan? Klaus Winnefeld? Christian Fleck' and Helmut Braunlich' ' Institute of Pharmacology and Toxicology, Friedrich Schiller University, D-07740 Jena, Germany

Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, St. Petersburg, Russia Institute of Clinical Chemistry, Friedrich Schiller University, D-07740 Jena, Germany

Key words: thallium sulphate; nephrotoxicity; morphology; ontogeny; rats.

The effect of TI,S04 (TI, 20mg kg-' body wt.) on renal function was investigated in 10- and 20-day- old rats. Nephrotoxic effects were evaluated by the determination of glomerular filtration rate, urinary volume, electrolyte and protein excretion, as well as by morphological investigations.

In contrast to adult rats there were no morphological destructions in 10- and 20-day-old rats. Changes in renal function seemed to be less expressed in 10- and 20-day-old than in adult rats. The smaller nephrotoxicity in 10-day-old rats may be caused by lower TI concentration in renal tissue, whereas in 20-day-old rats decreased nephrotoxicity cannot be explained in this way.

The activity of Na+/K+-ATPase in rat renal tissues was found to be involved in the mechanisms of TI enrichment in renal tissue, being an indirect determinant of TI nephrotoxicity.

INTRODUCTION

Since the beginning of this century, thallium (Tl) toxicity to human beings has been recognized mainly from side-effects of the medical use of thallium in the treatment of syphilis, gonorrhoea, dysentery, etc. ' Acute T1 intoxication usually affects the gastrointestinal tract, the peripheral and central nervous system, the skin and the Nevertheless, nephrotoxic effects are mentioned only casually as oliguria and albuminuria in human intoxications. 1.2.4-6 Changes in kidney function and morphology in experimental animals have been documented only in a few

It is known that the renal function of rats develops until after birth,'"," and this is the reason why one could expect differences in the action of nephrotoxic compounds on renal function between immature and mature rats. Our results show age-dependent differences in the nephrotoxicity of chromatei2 and ci~platin. '" '~ Age-dependent differences of cisplatin nephrotoxicity were described also for human beings.I5

The ability of T1 to interfere with Na+/K+-ATPase was discussed to play an important role in the mechanism of TI It was shown that the activity of this enzyme in kidney develops postnatally in the rat.Ix Systematic investigations concerning T1 nephrotoxicity until now have existed only for adult rats.' Therefore, renal effects of TI,S04 were investigated in developing rats.

t Author to whom correspondence should be addressed.

MATERIALS AND METHODS

Animals The investigations were performed in young Wistar rats (Han:Wist) of both sexes from our institute's own outbreed, at the beginning of the experiments being 10 or 20 days old with average weights of 25 and 45 g, respectively. The litters were reduced to six offspring per mother. The rats were kept under standardized conditions, including standard diet and free access to tapwater for mothers.

Experimental design and methods Diuresis experiments. Diuresis experiments were performed in conscious rats without any water loading. Because of the small volume, the urine of two animals was pooled. In each experimental group six samples were investigated. Thallium sulphate (Tl, 20 mg kg-' body wt. dissolved in 50ml of 0.45% NaC1) was injected once intraperitoneally at the 10th or 20th day of life. Controls received a corresponding volume of 0.45% NaCl only.'" Diuresis experiments were performed for 1 h at 10 or 13 days after T1 up to the normalization of kidney function.

The effect of frusemide was tested after the administration of 30 mg kg-' body wt. (dissolved in 50ml of 0.45% NaCl kg-' body wt.) intraperitoneally at the beginning of the diuresis experiment. This dose was shown to be effective but non-toxic.I9 Control and T1- treated rats were loaded with the same volume of 0.45% NaCl instead of frusemide. On days 2, 5 and 10 after a single administration of 20 mg Tl,S04 kgg' body wt., urine was collected for 1 h from control, frusemide- and T1-frusemide-treated rats.

In urine samples the following parameters were determined: urinary volume, protein (according to

CCC 0260437X/96/030235-09 0 1Y96 by John Wiley & Sons, Ltd.

Received 30 Junuay 1995 Accepted 4 December 1995

236 D. APPENROTH ET AL.

Bradford),2" osmotically active substances (Semi-micro Osmometer, Knaur GmbH, Berlin, Germany) and sodium and potassium concentrations (flame photometry I").

Activity of Na+/K+-ATPase. The Na+/K+-ATPase activity was determined separately in kidney cortex and medulla according to Post and Sen" as the differ- ence between the release of inorganic phosphate from ATP in the presence of Na+, K+ and Mg'+ (total ATPase activity) and in the presence of ouabain. Inor- ganic phosphate released from kidney tissue was determined spectrophot~metrically.~~ The activity of ATPase was related to 1 mg of kidney protein.2'

Thallium concentration. The T1 concentration was determined by flameless atomic absorption spectometry with the platform technique (AAS 4 E A 4, Carl Zeiss Jena GmbH, Germany) in renal cortex and medulla. The mineralized samples were dissolved in 2 ml of 1 N nitric acid, diluted with 0.1 N nitric acid and inserted in the graphite tube. The intra-assay coefficient of variation was 5.1% (mean 70 pmol lg ' for 11 replicates). Thallium concentrations in urine and serum were determined without mineralization.

Glomerular filtration rate (GFR). The GFR was determined at days 2, 5, 10 and 13 after the administration of 20 mg Tl,S04 kg-' body wt. The rats were anaesthetized with ketamine (Unotamin@ Serumwerk, Bernburg, Germany; 75mgkg- ' body wt.) and xylaz- ine (Ursonarkon@, Serumwerk, Bernburg, Germany; 12 mg kg-' body wt.). Both substances were adminstered intramuscularly. The animals were infused with isotonic saline containing 4 g 1- ' fluorescein isothiocyanate (F1TC)-inulin (Bioflor, Uppsala, Sweden) at a priming rate of 30ml kgg' body wt. hg ' for 15 min and then at 20 ml kgg' body wt. h-' for the rest of the experiment. To minimize urine collecting periods for determination of GFR, urine was collected in three 20- min periods transurethrally via a polyethylene catheter. In previous experiments it could be shown that under these experimental conditions both the h a e m a t ~ c r i t ~ ~ and the blood pressurez5 remained nearly constant during the clearance study. In the middle of each period and at the end of the experiment, blood was collected from the retrobulbar plexus. The inulin concentration was measured fluorimetrically26 in blood and urine samples.

Morphological examinations. Morphological examinations were performed in the kidneys of rats anaesthetized with pentobarbital (50 mg kgg' body wt., i.p.). They were fixed in situ by vascular perfusion using a fixative containing 1.5% paraformaldehyde and 1 % glutaraldehyde buffered in 0.1 M sodium cacodylate (pH 7.2-7.4), then removed and tissue blocks were cut from cortex to papilla for cross-sectional orientation and left in the same fixative for 2 h. Then they were postfixed in 1% osmium tetroxide buffered in 0.1 M sodium phosphate (pH 7.2-7.4). Tissue slices were dehydrated in graded alcohol and embedded in Epon- 812 Fluka Chemie AG, Buchs). Semithin slices (1- 2 pm) were cut with an LKB Ultratome-111, stained by toluidine blue and viewed by light microscopy.

Statistics

All data are expressed as means 2 SEM. Excreted amounts were related to 100 g body wt. Statistical evaluations were made by means of the two-tailed Student's t-test (P d 0.05).

RESULTS

After the administration of 2 mg T12S0, kg-' body wt. the course of TI concentration in serum is equal in 10- and 20-day-old rats (Fig. 1). Urinary T1 excretion in 20-day-old rats is nearly double that in 10-day- old rats.

In both age groups investigated, the concentration of T1 in renal medulla was significantly higher than in cortex (Table 1). On the 2nd day after T1 administration there were the same concentrations in the cortex of 10- and 20-day-old rats but the concentration in the medulla of 20-day-old rats was twice that of 10-day- old rats. The T1 concentration at day 5 after administration is lower in cortex and medulla of 10-day-old compared with 20-day-old rats. The influence of 20 mg T1,S04 kgg' body wt. on the excretion of urine and electrolytes in 10-day-old rats is shown in Fig. 2. The decrease in the urinary volume of control rats confirms the maturation of renal function described earlier.'' With the exception of days 9 and 10 after TI administration, there was no effect on urinary volume. The excretion of osmotically active substances was significantly enhanced from day 2 to 8. From day 9 onwards it was no longer different from the values of control rats. Thallium administration in 1 0-day-old rats was followed by increased sodium excretion from day 3 to day 5, whereas potassium excretion was significantly enhanced during 7 days after TI injection-(Fig. 2).

Thallium administration in 20-day-old rats were followed by oliguria on days 5-7, whereas urinary volume was significantly enhanced during days 8-11 after T1 (Fig. 3) . Urine excretion returned to normal values on day 13. The excretion of osmotically active substances, as well as sodium and potassium, showed the same time course as urinary volume.

The excretion of urinary protein is shown in Fig. 4. It was enhanced two to four times in 10-day-old rats on days 6-8 and remained unchanged when TI was administered to 20-day-old rats. The GFR increases in control rats from the 10th to the 20th day of life, to reach adult values on the 30th day of life. The GFR was not affected by T1 administration, neither in rats treated on the 10th nor the 20th day of life (Fig. 5).

The diuretic effect of frusemide in control and T1- treated rats is shown in Fig. 6. When TI was administered on the 10th day of life (upper panel) the diuretic effect of frusemide was diminished from day 5 to 10. In 20-day-old rats (lower panel) the effect of frusemide was only slightly lowered on days 2 and 5 after the administration of T1.

The ATPase activity was higher in 20- than in 10- day-old control rats (Table 1). Its activity in 10-day- old control rats was the same in cortex and medulla but in 20-day-old rats it was higher in medulla com-

ONTOGENETIC ASPECTS OF TI-INDUCED NEPHROTOXICITY IN RATS

10

7,5 -

237

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2,5 -

0

urinary TI excretion wmol/l OOg b.wt. x 1 h]

* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I I I 1 I 1 I I

I I I I I I I I

0 6 12 18 24 30 36 42 48

time after 20 mg/kg b.wt. thallium sulfate [hours]

-TI 10 *TI 20

Figure 1. Time course of TI concentration in serum and urinary TI excretion of 10- and 20-day-old rats after one single dose of 20 mg TI,SO, kg-' body wt. * Statistically significant differences between 10- and 20-day-old rats ( P 5 0.05).

pared with cortex. Thallium did not change the ATPase activity of 10-day-old rats in renal cortex, but in the medulla it was decreased on the 2nd and 5th days after T1 administration. In 20-day-old rats the activity of Na+/K+-ATPase in both regions of the kidney was significantly decreased on the 2nd and 5th days after T1 administration.

Morphological changes are shown in Fig. 7. On the 2nd day after TI administration there were destructions in the thick ascending limb (TAL) of the loop of Henle in 55-day-old rats; these destructions were described in more detail by Appenroth et al. As shown in Fig. 7, there were absolutely no structural changes on the 2nd day after TI administration in 10- and 20-day-old rats. Morphological investigations on days 5 and 10 after T1 administration also did not show any morphological changes in 10- or 20-day-old rats (not shown).

DISCUSSION

Distinct age-dependent differences in the nephrotoxicity of metal compounds and anticancer drugs have been described p r e v i o u ~ l y . ~ ~ - ~ " ~ ' ~ In most cases there was a lower degree of nephrotoxicity in young rats compared with adults. Renal excretory processes develop after birth,'"." which was also demonstrated by the control values of 10- and 20-day-old rats in the present paper. Immature processes of enrichment of toxins into the kidney,28 a lower degree of t o~ i f i ca t ion~~ or a lower susceptibility per se may be the reasons for lower toxicity in young rats.

The exact mode of action of TI is not yet clear, although some mechanisms are considered to be prob- able. At first, because of the similarity of physiochem-

238 D. APPENROTH ET AL

Table 1. Concentration of TI (pmoyg dry wt.) and activity of Na+/K+-ATPase (nmol P/mg Prot. x min.) in renal cortex and medulla of 10- and 20-day-old rats at the 2nd and 5th day after the administration of 20mg TI,SOJkg b.wt. in comparison to control rats

Control 2nd Day after TI 5th Day after TI

Cortex Medulla Cortex Medulla Cortex Medulla

TI 10-Day-old 0 20-Day-old 0

0 0

2.26 ? 0.16 4.93 2 0.27b 1.46 2 0.10 3.14 t 0.32b 2.30 2 0.13 9.83 t 0.90b." 0.88 ? 0.14" 1.82 2 0.23b,C

ATPase 10-Day-old 34.0 i 2.0 34.2 t 1.8 33.1 2 1.9 22.4 i 1.4a,b 34.1 t 1.2 42.0 ? 1.7a,b 20-Day-old 55.7 5 2.2" 68.2 t 4.0b,C 34.6 2 2.0" 44.6 Z 1 .7a,b.c 33.2 t 1.3" 46.7 t 1.7a,b

a Statistically significant differences between TI-treated and control rats ( P s 0.05). Statistically significant differences between cortex and medulla ( P s 0.05). Statistically significant differences between 10- and 20-day-old rats (F's 0.05).

ical parameters of TI and potassium, T1 substitutes for potassium in many physiological reactions. Its affinity for Na+/K+-ATPase is 10 times higher than that of pota~sium.",'~ The activity of ATPase is lower in 10-day-old than in 20-day-old rats; ATPase activity determined in the whole kidney reached mature values already in 20-day-old rats.lx We distinguished between cortex and medula showing activity comparable to adult rats in cortex, but not in the renal medulla of 20-day-old rats (for comparison, see Ref. 9). Secondly, like other heavy metals, TI has an affinity for, and interfaces with, sulphydryl group-containing enzymes.',* Earlier investigations showed lower concentrations of GSH and an activity of glutathione reductase in renal tissue of young rats that coincides with the lower toxicity of chromate in young rats.30 Thirdly, TI combines with riboflavin, and many features of its toxicity are similar to those found in riboflavin deficiency.'

Thallium salts are rapidly absorbed, reaching highest concentrations in blood plasma 30 min after oral administration,3' which was confirmed by our results (Fig. 1). Thallium was absorbed from the intraperi- toneal depot to the same extent in 10- and 20-day-old rats, shown by equal serum concentrations 60 min after the injection of TI. The passage of T1 through the kidney is quite similar to that of potassium. It is glomerularly filtered, tubularly secreted and ca. 50% of TI was reabsorbed.34 Urinary T1 excretion during the 48-h experimental period was significant lower in 10- than in 20-day-old rats. This may be caused mainly by lower GFR in 10-day-old rats compared with 20- day-old rats (Fig. 5 ) , which was not influenced by T1 in either of the age groups investigated.

The investigation of T1 distribution in subcellular fractions of the whole kidney showed highest enrichment in the cytosol and the nuclei? Thallium concentrations in renal tissue of 20-day-old rats (Table 1 ) were equal to concentrations in adult rats' and sup- ported findings in the literature that there is a four-to fivefold higher TI concentration in renal medulla compared with cortex.33 On the 2nd day after T1 administration its concentration in medulla was significantly lower in 10- than in 20-day-old rats (Table 1). Litera- ture data showed that the activity of Na+/K+-ATPase

can be influenced by On the 2nd day after T1 injection the activity of Na+/K+-ATPase was significantly lowered in 10-day-old rats (Table I ) . Therefore, because of the similarity between potassium and thallium, less T1 is transported into the tubular cell, which may be the main reason for the lower T1 concentration in the renal medulla of these young animals. Enhanced ATPase activity on the 5th day after T1 administration may be the reason for the significantly higher concentrations remaining in the medulla of 10-day-old compared with 20-day-old rats (Table I ) .

The control values presented here (Figs 2 and 3 ) and in the literature'" showed that 10-day-old rats excrete smaller amounts of potassium than older rats. Because of the similarity between potassium and T1, we assume that the ability to excrete TI via urine, especially at high load during the first 24 h, is still lowered in 10-day-old rats. At this time the activity of the Na+/K+-ATPase was equal in rats of both age groups (Table l) , resulting in equal amounts of TI transported into the cell. Consequently, the higher TI concentration in 10-day-old rats seems to be the result of immature secretion of TI from the cell into the tubular lumen. Descriptions of human T1 intoxications and results from animal experiments show changes in renal function, such as oliguria, diminished creatinine clearance, raised blood urea nitrogen and a1buminuria.'-

Renal damage was qualified to be only slight, although there has been no systematic investigation of kidney function. Our investigations of the renal effects of T1 in adult rats' showed a distinct dose-toxicity relation. On the 2nd day after T1 administration in adult rats, strong oliguria appeared in diuresis experiments without fluid loading.9 Oliguria appeared also in 10- and 20-day-old rats, and the younger the animals the later the oliguria appeared (Figs 2 and 3 ) . In 10- day-old rats the slower elimination of T1 out of the renal tissue (Table 1 ) may at least in part be respon- sible for the more delayed occurrence of oliguria and proteinuria (Fig. 5). In 20-day-old rats oliguria was followed by significant polyuria (Fig. 3 ) . which in adult rats was the consequence of a low dose followed by lower TI concentration in renal tissue,' indicating the passage to normal renal function after 7 days in 20-day-old rats. The changes of urine volume in 20-

ONTOGENETIC ASPECTS OF TI-INDUCED NEPHROTOXICITY IN RATS 239

I

I I I I , o

6 C

3

0

._ L

'c


0 €3 0

8 0

8 O*

8 c

0- 0-

ONTOGENETIC ASPECTS OF Tl-INDUCED NEPHROTOXICITY IN RATS 24 1

urinary protein [mg/lOOg b wt x lh]

1 - 0,s

04 - -7 - 8 I----

0 1 2 3 4 5 6 7 8 9 10

time after 20 mg/kg b wt thallium sulfate [days]

con10 < ’T I10 *con20 *T I20

Figure4. Urinary protein excretion in 10- and 20-day-old rats after 20 rng TI,SO, kg-’ body wt.

Statistically Significant differences between control and TI- treated rats of the same age group ( P z 0.05).

GFR [rnl/l00 g b wl x min] 12r ~. . - ~ ~-

I I 11 ,

’, ’ I $7”

day-old rats reflect the excretion of the total amount of osmotically active substances, including sodium and potassium, excreted via urine. Glomerular filtration rate was not affected by T1 either in 10- or in 20-day-old rats. Therefore, we conclude that distinct functional disturbances of sodium and potassium transport processes may be the reason for the changed urine volume in 20-day-old rats. In 10-day-old rats the relatively slight increased excretion of the osmotically active substances sodium and potassium does not result in any corresponding change of urine excretion.

Although TI concentrations in renal tissue were the same in adult and 20-day-old rats, there were no destructions of renal structure in 20-day-old rats at any time investigated (Fig. 7). Proteinuria is a sensitive parameter of nephrotoxicity for many types of nephro- toxins, such as drugs and chemicals.’s After the dose of T1 that led to equal renal T1 concentrations, there appeared a significant proteinuria in adult’ but not in 20-day-old rats (Fig. 4). We cannot explain these sur- prising effects without assuming different susceptibility of renal tissue in 20-day-old and adult rats.

The thick ascending limb of the loop of Henle, which was destroyed in adult rats,’’ is the site of

urinary volume [rnl/lOOg b.wt. x I h J 1 O-day-old rats

*

* *

day 2 day 5

mcontrol gfu a T l + f u

20-day-old rats

* *

day 10

Figure 6. Diuretic effect of frusernide (30 mg kg-’ body wt.) in 10- and 20-day-old control and TI-treated rats. ‘ Statistically significant differences in comparison to control rats ( P c 0.05). + Statistically significant differences between frusernide (fu)- and TI + frusemide (Tl+fu)-treated rats ( P s 0.05).

action of frusemide. As a consequence, the diuretic effect of frusemide was strongly inhibited on the 2nd day after T1 administration in adult rats.’ In spite of missing morphological changes in 10- and 20-day-old rats (Fig. 7), the effect of frusemide was decreased (Fig. 6) during the time in which the oliguric effect of T1 occurred (Fig. 1). Because of the complete absence of morphological changes in 10- and 20-day- old rats, all effects on renal excretory function must be the result of functional disturbances.

Because of the similarity in renal handling of TI+ and K+, it was interesting to investigate the influence of TI on potassium excretion. It was increased significantly in 10-day-old rats from the 1st up to the 8th day after T1 administration (Fig. 2). We interpret the increased potassium excretion of 1 O-day-old rats as competition between T1+ and K+ for reabsorption in the thick ascending limb of the loop of Henle, which showed also functional changes concerning the diuretic effect of frusemide (Fig. 6).

It was discussed in the literature that the influence of TI on Na+/K+-ATPase plays an important role in the mechanism of T1 t o x i ~ i t y . ’ ~ , ’ ~ The changes shown


Figure 7. Kidney of 55-, 20- and 10-day-old rats after the administration of 20 mg TI,SO, kg-’; border between outer and inner stripe of the outer medulla: (A) 55-day-old control rats; (6) 55-day-old rat on the 2nd day after TI; (C) 20-day-old rat on the 2nd day after TI; (D) 10-day-old rat on the 2nd day after TI. Semithin section, magnification x380. TAL: thick ascending limb of the loop of Henle.

ONTOGENETIC ASPECTS OF TI-INDUCED NEPHROTOXICITY IN RATS 243

in Table 1 could not be correlated directly with the ity but as an indirect factor by influencing the concen- effect of T1 on the excretion of sodium and potassium. tration in renal tissue. In adult rats, changes of ATPase activity were in In summary, our results show that 10- and 20-day- relation to potassium ex~re t ion .~ We think that the old rats are less susceptible to T1 than adults are. influence of TI on the activity of Na+/K+-ATPase in There were no morphological destructions in renal renal medulla, at least in 10- and 20-day-old rats, was tissue and Na+/K+-ATPase activity did not seem to be not involved as a direct factor causing TI nephrotoxic- directly correlated with T1 nephrotoxicity.

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ontogenetic aspects of thallium-induced nephrotoxicity in rats

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