Toxicology, 76 (1992) 219-231 Elsevier Scientific Publishers Ireland Ltd.

219

Incorporation of [35S]sulfate into glomerular membranes of rats chronically exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria Algvaro C~irdenas, Alfred Bernard and Robert Lauwerys Industrial Toxicology and Occupational Medicine Unit, Faculty of Medicine, Catholic University of Louvain, 30.54. Clos Chapelle-aux-Champs, 1200 Brussels (Belgium) (Received March 9th, 1992; accepted September 8th, 1992)

Summary The aim of the present work was to assess the effects of long-term exposure to Cd on the sulfatation of glomerular membranes and their relation with proteinuria and urinary glycosaminoglycans (GAG). For this purpose the in vitro incorporation of [35S]sulfate was investigated in female Sprague-Dawley rats given 100 ppm of Cd in drinking water for 7 months. When compared with their controls, glomeruli from Cd-treated rats showed a 12.8% decrease in the incorporation of the label into glomerular membranes. This effect, which was not explained by differences in viability or in sulfate uptake by the giomeruli, suggests that sulfatation of glomerular membranes is impaired in Cd-treated rats. In support of this, in another independent experiment, a decrease, 17.4% on average, of the sulfate content of glomerular membranes was observed in long-term Cd-treated rats (100 ppm in drinking water for 4 months). This effect was significantly correlated with albuminuria and transferrinuria but not with f12" microglobinuria, suggesting that a loss of heparan sulfate of the glomerular capillary wall could be involved in the Cd-induced glomerular proteinuria. On the other hand an enhanced urinary excretion of GAG, negatively correlated with the sulfate content of glomerular membranes, was also observed in Cd-treated rats. Moreover GAG excretion was associated with tubular and glomerular proteinuria, which suggests that GAG might be a useful marker of Cd-induced nephrotoxicity.

Key words: Cadmium; Rat; Kidney; Proteinuria; Glomerular basement membrane; Sulfatation; Glycosaminoglycans

Introduction T h e a n i o n i c c o m p o n e n t s o f the g l o m e r u l a r c a p i l l a r y wall, k n o w n as the g l o m e r u l a r p o l y a n i o n , are r e s p o n s i b l e f o r t h e r e s t r i c t i o n to t h e p a s s a g e i n t o B o w m a n ' s s p a c e o f c h a r g e d m a c r o m o l e c u l e s [1,2]. T h e m a j o r c o n s t i t u e n t s o f the g l o m e r u l a r p o l y a n i o n a r e sialic a c i d r e s i d u e s ( s p e c i a l l y in g l o m e r u l a r e p i t h e l i a l a n d

Correspondence to: R. Lauwerys, Industrial Toxicology and Occupational Medicine Unit, Catholic University of Louvain, 30.54. Clos Chapelle-aux-Champs, 1200 Brussels, Belgium. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

220 endothelial cell membranes) and glycosaminoglycans (GAG) [3-8]. A reduction of anionic sites of the glomerular capillary wall has been observed in several conditions associated with proteinuria [2,5,9-15]. Heparan sulfate is the main GAG present in glomerular basement membrane (GBM). The sulfate and carboxyl groups of the molecule confer to the GBM a high density of negative charges, responsible for its charge selective properties [2]. Heparan sulfate is also associated with the plasma membranes of the glomerular epithelial and endothelial cells and probably contribute to the charge barrier at these locations [2,6,8]. In several experimental models of glomerular injury proteinuria is related to a loss of heparan sulfate from the GBM and other glomerular sites [2,14,15]. Glomerular and/or tubular proteinuria has been reported in both man and animal following chronic exposure to Cd [16-18]. In an earlier work, Bernard et al. [19] showed that the glomerular proteinuria induced by long-term administration of Cd in rats (100 ppm Cd in drinking water) was linked to a reduced binding of the cationic dye, Alcian blue, to the glomerular membranes of these animals. Since a negative correlation was found between the cationic dye binding to glomerular membranes and albuminuria, it was postulated that a loss of the glomerular polyanion could be involved in the enhanced trans-capillarypassage of plasma proteins observed in these animals. More recently, C~irdenas et al. [18] reported in this model of Cdglomerulopathy a decrease of the sialic acid content in glomerular membranes associated to an enhanced urinary output of albumin and transferrin. This finding suggests that the decrease of Alcian blue binding to glomerular membranes, in Cdtreated rats, observed by Bernard et al. [19] was, at least partly, caused by a loss of sialic acid in these structures. The reduction of the incorporation of [aSS]sulfate in glomeruli incubated in the presence of Cd 2÷, reported by Templeton and Chaitu [20], suggests that sulfate content could also be depleted in glomerular membranes of Cd-treated rats. A decrease of the levels of sulfate might, in addition to the decrease in the levels of sialic acid, contribute to the loss of the polyanion charge observed in Cd glomerulopathy and hence contribute to the increased glomerular permeability of proteins. The aim of this work was to test this hypothesis by evaluating the incorporation of [35S]sulfate into glomerular membranes and their sulfate content in isolated glomeruli of long-term Cd-treated rats. The relation of these effects with proteinuria and the urinary excretion of GAG was also investigated. Materials and methods

Reagents Dulbecco's modified Eagle's medium (DMEM), L-glutamine, sodium pyruvate, lactalbumin hydrolysate, heat-inactivated foetal calf serum and antibiotics were from GIBCO (Paisley, UK). Na235SO4 (5 mCi/ml, 648.65 mCi/mmol) was purchased from Du Pont de Nemours (Dreiech, Germany). Antibody against rat albumin was from USB (Cleveland, OH). Polystyrene latex particles (ESTAPOR K 109) were kindly supplied by Dr. J.C. Daniel of Rhrne-Poulenc Industries (Aubervilliers,

221

France). Sodium rhodizonate was from Sigma Chemical Co (St Louis, MO). Liquid scintillation Pico-Fluor 30 was from Packard Instrument Co. (Groningen, The Netherlands). Other reagents were from Merck (Darmstadt, Germany) and of analytical grade.

Animals and treatments Female Sprague-Dawley rats, 2 months at the onset of treatment (100-200 g) were used. Long-term exposure to Cd was achieved by the administration of 100 ppm Cd (as CdC12) in the drinking water for 7 months (incorporation of [35S]sulfate, n = 20) or for 4 months (sulfate content of glomerular membranes, n = 9). Similar numbers of control animals received only deionised water. At the end of the treatment the animals were housed in individual metabolism cages equipped with urine/feces separators. Urines were collected for a period of 24 h over 10 mg of sodium azide as preservative. No food was provided during urine collection but water was given ad libitum. Analyses of urines The urinary concentration of albumin, transferrin, immunoglobulin G (IgG) and /~2-microglobulin (/82-m) was measured by latex immunoassay according to Bernard and Lauwerys [21]. Total glycosaminoglycans (GAG) in urine were determined with the Alcian blue dye according to Whiteman [22]. Urinary activity of N-acetyl-/3-Dglucosaminidase (NAG) was assayed by the automated fluorimetric assay of Tucker et al. [23]. Incorporation of 35S04 in glomerular membranes Animals were anesthetized with pentobarbital (60 mg/kg). Kidneys were perfused in situ through the aorta with ice-cold Hank's balanced salt solution (HSS) consisting of NaCI (137 mM), KCI (5.36 mM), NaHCO3 (4.17 mM), CaC12 (1.30 mM), MgC12 (0.49 mM), KH2PO4 (0.44 mM), MgSO4 (0.41 mM), NaH2PO4 (0.34 mM) and glucose (5.55 mM). After blanching, the kidneys were excised, the capsules were removed and the cortices were dissected. Glomeruli were isolated by the sieve technique of Kreisberg et al. [24]. After abundant washing with HSS, glomeruli were redispersed in 6 ml of DMEM containing 10% of foetal calf serum, glutamine (2 mM), pyruvate (1 mM) and antibiotics (penicillin 100 units/ml and streptomycin 100/~g/ml). The glomeruli from ten control rats and ten Cd-treated rats were pooled separately and adjusted at a density of 4500 glomeruli/ml. The suspensions of glomeruli were then gently shaken and equally distributed in 10 wells (6 ml/each). Glomeruli were incubated with Na235SO4 (= 40/~Ci/ml of culture medium). Incubation was carried out at 37°C during 24 h in a humidified incubator in an atmosphere of 5% CO2. In view of the number of animals available (20 control and 20 treated), this experiment was performed twice and the results of each experiment are reported separately. Viability of the glomeruli was assessed by measuring lactate dehydrogenase activity (LDH) in the culture medium, LDH activity was measured by monitoring spectrophotometrically the oxidation of NADH (0.13 mM) in the presence of pyruvate (0.84 mM) and expressed in relation to total LDH activity obtained after cell disrup-

222 tion in 0.1% Triton X-100 and sonication, five times 10 s (setting 5) at 0-4°C, with a Virsonic-300 ultrasonicator (Virtis Co., NY). At the end of the incubation step, glomerular membranes were isolated as described previously [18], lyophilised and weighed. After hydrolysis (500/~1 H2S04 1 M, 100°C for 3 h) samples were removed and the tube rinsed twice with 250 #1 water. Sample and wash were transferred to a plastic vial containing 10 ml Pico-Fluor-30 scintillation fluid. The label was measured by scintillation counting for 10 min. Results were expressed as counts/min/mg dry weight (cpm/mg dry weight).

Sulfate content in glomerular membranes Glomerular membrane preparations of Cd-treated rats and of their controls were obtained as described above. After weighing, glomerular membranes were hydrolyzed (500/zl HCI 1 N, 100°C, for up to 3 h). The sulfate content of glomerular membranes was determined by a slight modification of the Terho and Hartiala's method [25]. Briefly, 250/~1 of samples (diluted 5 or 10 times) and standards were pipetted into test tubes and 1 ml absolute ethanol was added. After shaking, 500 #1 of BaCI~ buffer (2 ml BaC12 5 mM, 10 ml acetic acid 2 M, 8 ml NaHCO3 2 M, made up to 100 ml with absolute ethanol) were added. The tubes were shaken again and 750/zl of sodium rhodizionate solution (5 mg sodium rhodizionate dissolved in 20 ml water and 100 mg l-ascorbic acid; after dilution, volume was made up to 100 ml with absolute ethanol) was added. After shaking, the tubes were kept in the dark for 10 min at room temperature. Absorbance was measured at 520 nm. All determinations were performed at least in triplicate. The procedure was linear between 1 and 8/~g sulfate. Two experiments were carried out, labeled Expt. 1 and Expt. 2.

Statistics Means were compared by Student's t-test. Urinary variables were normalized by log-transformation before statistical analysis. Associations between variables were evaluated by Pearson's correlation. A P < 0.05 was considered as statistically significant. Results

Incorporation of sulfate in glomerular membranes From four rat kidneys we usually isolated 50 000-60 000 glomeruli, which corresponds to a yield of about 40% on the basis of a value or 30 000-35 000/kidney [26] The degree of contamination by tubular fragments was always below 5%. The viability of the preparation was assessed initially by the trypan blue exclusion test. However, the dye did not penetrate in the glomerular cells even when LDH release was found to be increased. For this reason we considered, as have other authors [27], that conventional dye exclusion assay is a not reliable test for the assessment of viability in glomerular preparations. L D H was therefore chosen as a criterion of viability. It should be noted that for the establishment of the 100% reference value for LDH, treatment with triton appeared to be insufficient to disrupt all glomerular cell membranes and sonication was also required to obtain maximal values. In our experimental conditions, L D H release increased in the first 3 h of in-

223 30-

•~ 2 0 ..j-~ ,.o

~,~10

0

0

12

24

36

48

Duration of incubation (hour) Fig. 1. Time-course of lactate dehydrogenase (LDH) activity, expressed as percentage of total activity, from incubated glomeruli. Values are means ± SEM from triplicate determinations of two independent experiments.

cubation and remained relatively stable between 6 and 48 h (Fig. 1). After 24 h incubation, viability, according to LDH activity, was estimated to be more than 75%. After 7 months of Cd administration the animals used to investigate the incorporation of sulfate to glomerular membranes showed a fourfold rise in their urinary excretion of albumin, which reflects an enhanced permeability of the glomerular filter. The effect of Cd exposure on glomerular proteinuria was similar in the two series of experiments designed to assess the incorporation of [35S]sulfate into glomerular membranes (Table I). The uptake of [35S]sulfate by isolated glomeruli was not lower in the Cd group than in the control group. During the second experi-

TABLE I EFFECT OF LONG-TERM ADMINISTRATION OF Cd (100 ppm Cd IN DRINKING WATER FOR 7 MONTHS) ON ALBUMINURIA AND ON IN VITRO INCORPORATION OF [35S]SULFATE INTO GLOMERULAR MEMBRANES (GM) OF FEMALE SPRAGUE-DAWLEY RATS Experiment 1 and Experiment 2 are two independent experiments of the same kind. Albuminuria a (#g/24 h)

[35S]Sulfate in GM b (cprn/mg dry weight)

231 (91-127) 1003 (207-6605)**

13 841 ± 492 12 319 ± 256*

242 (61-665) 960 (170-6722)**

21 229 ± 656 18 519 ± 485*

Experiment 1 Control Cd

Experiment 2 Control Cd

aGeometric means (range) of ten animals in each group. eArithmetic means ± SEM of five incubations of glomeruli (27 000 glomeruli in 6 ml of culture medium each) with 135Slsulfate (40 #Ci/ml of culture medium) during 24 h. *P < 0.05, **P < 0.01, significantly different from controls (Student's t-test).

224 ment it was estimated to be 9 cprn/glomerulus (60 000 cpm/mg dry weight) in control rats and I0 cpm/glomerulus (65 000 cpm/mg dry weight) in Cd-treated rats. In contrast, preparations from Cd-treated rats showed a reduced incorporation of [35S]sulfate into glomerular membranes (Table I), the average reduction being about 130 in Cd-treated rats compared to their controls. No differences in the cell viability of glomeruli, measured as release of LDH, was observed between both groups, control (mean ± SD: 74.5% ± 2.1) and Cd-treated rats (mean + SD: 75.3% + 2.6).

Sulfate content in glomerular membranes Female Sprague-Dawley rats given 100 ppm of Cd in drinking water showed an increased urinary output of high molecular weight proteins and a reduced content of sulfate in glomerular membranes after 4 months of treatment (Table II). Interestingly, the sulfate concentrations in glomerular membranes were correlated with the urinary excretion of albumin, transferrin and IgG (Fig. 2) but not with/32-m or NAG activity (results not shown). Unexpectedly, when compared with their controls the urinary excretion of/32-m was also enhanced in the group of rats exposed to Cd. However/32-m levels in urine were rather low in the control group and the ones observed in Cd-treated rats were similar to the values obtained previously in control rats [18]. By contrast, Cd-treated rats did not show any effect on NAG excretion (64.4 mI.U./24 h vs. 65.9 in controls). Urinary excretion of GAG was also increased in Cd-treated rats. Urinary GAG levels were positively correlated with/32-m, transferrin, albumin and IgG in urine (Table III) and negatively with the sulfate content of glomerular membranes (Fig. 3). Discussion

Incorporation of [~SS]sulfate into glomerular membranes The isolated rat glomerulus has been successfully employed to study several aspects of basement membrane metabolism, especially to compare the net synthesis of [35S]sulfated glycosaminoglycans in normal and diseased animals [20,27-321. In these studies however assessment of viability has been rarely reported, probably due to the difficulty of applying conventional methods to these complex structures [27]. In the present work we observed a pattern similar to that described by Templeton and Khatchatourian [27], i.e. a significant release of LDH in the early hours following isolation of glomeruli which subsided after - 6 h (Fig. 1). Templeton and Khatchatourian [27] showed that in spite of the increase of LDH release, sulfation of glomerular membranes was linear for up to 22 h. According to LDH activity, the viability of our glomerular preparation after 24 h incubation was near to 75% (Fig. 1). However this value may well be an underestimation of the real viability, in view of the difficulty to obtain the 100% of LDH activity. In support of this, Templeton and Chaitu [20] found, by a modification of the trypan blue test, a viability of 93% soon after isolation and of 89% after 24 h incubation. Incorporation of [35S]sulfate to glomerular membranes reflects cellular function at several levels, including de novo synthesis of core protein, glycosylation and sulfatation reaction in the Golgi apparatus, glycoprotein export and matrix or cell

284 (122-977) 595 (190-4533)

4.8 (2.3-15.5) 10.8 (3.4-72.4)*

Transferrin a (/zg/24 h) 185 (58-419) 422 (120-2332)*

IgG a (ng/24 h)

aG-eometric means (range) of nine animals in each group. ~Arithmetic means ± SEM of nine animals in each group. *P < 0.05, significantly different from controls (Student's t-test).

Control Cd

Albumin a (tLg/24 h) 5.7 (2.8-10.8) 11.8 (6.7-21.6)*

B2-m a (#g/24 h)

2.47 (1.57-3.95) 5.82 (2.02-13.5)*

GAG a (rag/24 h)

14.9 ± 0.9 12.3 ± 0.8*

Sulfate in GM b (#g/mg dry wt.)

INFLUENCE OF LONG-TERM ORAL ADMINISTRATION OF Cd (100 ppm IN DRINKING WATER FOR 4 MONTHS) ON URINARY EXCRETION OF SOME PLASMATIC PROTEINS AND NAG ACTIVITY AND ON SULFATE CONTENT OF GLOMERULAR MEMBRANES (GM), IN FEMALE SPRAGUE-DAWLEY RATS

TABLE II

q,n

226 10000y = 3.68 - 0.079 x r = -0.57,

p < 0.05

n=18

.~

1000-

E

D

100100•

y = 1.99 - 0.083 r = -0.63,

x

p < 0.01

n=18

E

1

3000 Z"

Z

y = 3.44

0.073

r = 0.57, n=18

p < 0.05

×

16

18

~.~1000

_

1O0 5O 8

10

12

14

;ulfate c o n t e n t of g l o m e r u l a r m e m b r a n e s

20

(pg/rng dry weight)

Fig. 2. Relationship between sulfate content of glomerular membranes and urinary excretion of high molecularweight proteins in nine Cd-treated rats (100 ppm in drinking water for 4 months) (I) and nine control rats (El).

membrane assembly [34]. Since no differences in viability, measured as L D H release, or in glomerular uptake of [3SS]sulfate was observed between glomeruli o f Cdtreated rats and glomeruli of control rats, the finding of a reduced incorporation of [aSS]sulfate into glomerular membranes of Cd-treated rats suggests that long-term exposure to Cd may affect one or several of the aforementioned cellular functions, among which we favour the sulfatation. This would be compatible with the results of Templeton and Chaitu [20] who showed that Cd in vitro inhibits the synthesis of

227

TABLE III CORRELATION MATRIX OF URINARY EXCRETION OF GAG, SEVERAL PLASMATIC PROT E I N S A N D N A G A C T I V I T Y I N 18 F E M A L E S P R A G U E - D A W L E Y R A T S , 9 R A T S G I V E N 100 p p m Cd I N D R I N K I N G W A T E R F O R 4 M O N T H S A N D N I N E C O N T R O L S NS, not significant.

GAG

GAG

~2-m

Transferrin

IgG

Albumin

NAG

1

r = 0.695 P < 0.005

r = 0.687 P < 0.005

r = 0.685 P < 0.005

r = 0.641 P < 0.005

r = 0.263 NS

1

r = 0.367 NS

r = 0.435 NS

r = 0.328 NS

r = 0.132 NS

1

r = 0.873 P < 0.001

r = 0.975 P < 0.001

r = 0.223 NS

1

r = 0.874 P < 0.001

r = 0.378 NS

1

r = 0.278 NS

B2,-m

Tr~ferrin

IgG

Albumin

NAG

I

15000 IIm

J~ ('4

•

10000 .

n=18 < 5000 c 0

°o ",°