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Isoparaffinic solvent-induced nephrotoxicity in the rat
Toxicology, 38 (1986) 227--240 Elsevier Scientific Publishers Ireland Ltd.
I S O P A R A F F I N I C SOLVENT-INDUCED N E P H R O T O X I C I T Y IN T H E RA T
C. VIAUa*, A. BERNARD a**, F. GUERETa, P. MALDAGUEb, P. GENGOUXc and R. LAUWERYSat aunit@ de Toxicologie Industrielle, Ddpartement de Mddecine et Hygiene du Travail, b Laboratoire de Pathologie et Cytologie Expdrimentale, cUnit@ de Dermatologie ProfessionneUe, Ddpartement de M@decine et Hygi@ne du Travail, Universit@ Catholique de Louvain (School o f Medicine), B-1200 Brussels (Belgium)
(Received June 7th, 1985} (Accepted August 29th, 1985)
SUMMARY
Prolonged inhalation exposure t o 6.5 mg/l of an isoparaffinic solvent consisting of saturated aliphatic h y d r o c a r b o n s (SAHC) resulted in b o t h functional and morphological renal changes in male rats t o the exclusion o f female or castrated rats. Functionally, the increased excretion of lactate dehydrogenase in t he absence o f an increased fl-N-acetyl-D-glucosaminidase ex cr etio n t o g e t h e r with a decreased urinary concentrating ability u p o n water deprivation and slower antinatriuretic response when the sodium intake is abruptly reduced, suggest a distal tubular alteration, fl~-Microglobulin ex cr eti on is unchanged indicating good proxi m al tubular cell function. The increased excretion of albumin and slightly lowered glomerular filtration rate suggest a m o d e r a t e glomerular impairment. Light microscopy shows p r o m i n e n t hyaline d r o p l e t accumulation in proxi m al t ubul ar cells and a few scattered foci o f regenerative epithelia in b o t h proximal and distal cells of th e deep co r t e x. T he urinary clearance o f t he major male rat urinary protein, a2u-globulin, is similar in c o n t r o l and exposed rats but the latter have a 10-fold greater renal accumulation of this protein while the hepatic levels *Research fellow from the Institut de Recherches en Sant~ et en S~curit~ du Travail du Qudbec, 505 Ouest de Maisonneuve, Montreal H3A 3C2, Canads. **Chercheur Qualifi~ of the Fonds National Belge de la Recherche Scientifique. tTo whom all correspondence should be sent: 30.54 Clos Chapelle-aux-Champs 1200 Brussels, Belgium, Abbreviations: BUN, blood urea nitrogen; CPN, chronic progressive nephrosis; GFR, glomerular filtration rate; HD, hyaline droplets; HES, hematoxylin and eosin-saffron; LDH, lactate dehydrogenase; M-H, Mallory-Heidenhain; NAG, fl-N-acetyl-D-glucosaminidase; PAH, periodic acid Shiff; PT, proximal tubular; SAHC, saturated aliphatic hydrocarbons. 0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
227
are identical in both groups. It is concluded that SAHC exposure causes moderate and reversible tubular and also glomerular changes in the male rat kidney.
Key words: a2u-Globulin; Hydrocarbon solvent; Kidney function; Kidney morphology; Proteinuria; Rat INTRODUCTION For the last few years, there has been some concern that chronic exposure to organic solvent could be associated with an increased risk of chronic glomerulonephritis or other kidney diseases [1]. Animal models have been devised in order to study that problem. In a series of articles, Carpenter et al. [2--5] have studied the toxicity of Stoddard solvent, varnish makers' and painters' naphtha, 140 ° flash aliphatic solvent and deodorized kerosene in Harlan-Wistar male rats. All these solvents contain a high percentage of aliphatic and alicyclic hydrocarbons. They found an increased blood urea nitrogen (BUN} in the case of Stoddard solvent only. In addition, various signs of tubular regeneration and occasional dilated tubules with casts were observed at the cortico-medullary junction. However, since these effects were not dose-related and t h a t t h e y even occurred in some control animals, the authors concluded t h a t t h e y were not linked to the treatment. Parker et al. [6] conducted 14-day gavage studies of up to 60 ml/kg of JP5 jet fuel in male Sprague--Dawley rats. Morphological examination of the kidneys disclosed the presence of eosinophilic hyaline droplets in the cytoplasm of proximal convoluted tubular cells, which was apparently dose~lependent. A slight increase in BUN and in serum creatinine was also noted in the exposed rats. More recently, Phillips and Egan [7,8] and Phillips and Cockrell [9] have reported on the nephrotoxicity of a C10--C12 isoparaffinic solvent in male Fisher-344 and Sprague--Dawley rats (exposure levels 1.83 and 5.48 mg/1). Their findings included a dose-related frequency of zones of regenerative epithelia and tubular dilatation with intratubular protein. Urinalysis indicated an increased giucosuria and proteinuria. BUN and serum creatinine were increased in the treated groups and a decreased creatinine clearance was noted in the high exposure group after 8 weeks of treatment. Finally, a decreased urinary concentrating ability was found to occur in both groups. Except for the morphological alterations, nearly all these manifestations were reversible after 4 weeks of non-exposure. Using decalin as a model c o m p o u n d of hydrocarbon nephrotoxicity in the rat, Alden et al. [10] pointed out to the fact that only male rats seem to be susceptible to this nephropathy. Indeed, the authors report that mice, guinea pigs, dogs and m o n k e y s of either sex and female rats are unaffected by these solvents. They indicate t h a t these renal effects appear to be related to the deficient catabolism of a sex-specific low molecular weight rat protein, a2u-globulin (a2u-G, M r 20 000). The authors state t h a t hyaline droplets are not observed
228
in exposed castrated male rats and that this is "evidence of an androgendependent inefficiency of protein reabsorption/catabolism". We have studied the effect of inhalation exposure to a C10--C12 isoparaffinic solvent in male, female and castrated male Sprague--Dawley rats using a wide variety of sensitive tests of the kidney function. Particular attention has been given to enzymuria and specific proteins excretion as used for screening purposes in humans. We have also examined the renal metabolism o f a2u-G in male and castrated male rats. All these tests were supplemented by optical microscopic examination of the kidneys and by immunofluorescence techniques to try to evaluate the implication of immunological mechanisms in the hypothetical development of glomerular alterations. MATERIALS AND METHODS
Study material and study design Four experiments were conducted in order to study various aspects of the nephrotoxicity induced in rats by the solvent (Shellsol TD, Shell company). The latter {common name: white spirit} was a mixture composed at >99% of C10--C12 saturated aliphatic hydrocarbons (SAHC) which the manufacturer claims to be almost entirely isoparaffins. All experiments were performed with Sprague--Dawley rats aged 3--4 months at the start of exposure. They were exposed in a 300-1 stainless-steel and glass whole body inhalation chamber {Young and Bertke Co, Cincinnati, OH). All exposures were 8 h/day, 5 days/week. The total air flow rate through the chamber was maintained constant at 300 1/min. The solvent was pumped at a constant rate through a flash evaporation device before the vapors were admitted to the chamber. The solvent concentration in the chamber was determined by charcoal tube sampling. The samples were desorbed with carbon disulfide and analysed by gas chromatography on a 25% carbowax 20M on Chromosorb W packed column using flame ionization detection. The system was calibrated using the crude solvent as standard. The mean {range} concentrations were 6.5 (5.8--7.5} and 0.58 {0.52--0.64} mg/1 for the high and low exposure levels respectively. The control rats were placed in an identical inhalation chamber swept by room air. The first experiment (WS-1) aimed at checking the possible long term effect of exposure to high levels (6.5 mg/1) of SAHC on some parameters of the kidney function. Male rats were divided into 24 control and 24 exposed animals and the exposure lasted for up to 16 months. Periodic urine collection was performed at t = 0, 19, 25, 30, 37 and 45 weeks. Half the animals were killed at 46 weeks, the other half at 68 weeks of exposure. Having determined what appeared to be the most sensitive tests of kidney function in this model, a second study (WS-2) was undertaken in order to check if signs of nephrotoxicity appeared earlier than 19 weeks and to check the reversibility of the functional changes. In addition, sex susceptibility was examined. Therefore 24 male and 24 female rats were equally distributed between a control and an exposed group. The latter was sub-
229
mitted to the same concentration (6.5 mg/1) of SAHC as previously for 13 weeks. The reversibility was evaluated after 6 weeks of non-exposure. In the third experiment (WS-3), 12 male rats were exposed for 16 weeks to a 0.58 rag/1 SAHC concentration which is close to the TLV for these types of compounds. A matched group of 12 controls was also examined. The effect of castration on SAHC nephrotoxicity was studied in the f o u r t h experiment (WS-4) in which 6 males and 5 castrated males were exposed to the high concentration (6.5 mg/l) of the solvent. These animals were matched to 6 normal and 6 castrated controls. In addition, WS-4 was designed to look at the metabolism of a2u-globulin in exposed vs. control rats. Urine, serum and tissue analyses All urine collections were performed from the rats placed in individual stainless-steel metabolism cages equipped with urine/feces separators. No food was given during the collection but water was provided ad libitum (except during the urinary concentration test -- see below). Ten milligrams of NaN3 was added, as a preservative, to the collection flasks. The activities of #-N-acetyl-D-glucosaminidase (NAG, EC 3.2.1.30} and of lactate dehydrogenase (LDH, EC 1.1.1.27) were determined as described previously [11,12]. The urinary concentration of albumin, the urinary and serum concentration o f #:-microglobulin (#2-m) and the urinary, serum and tissue concentration of a2u-G were measured by latex immunoassays similar to t h a t described by Bernard and Lauwerys [13]. For the determination of the tissue concentration of a:u-G, approximately 1 g of either whole kidney or liver were homogenized with 9 ml of an i c e , o l d solution containing 50 mM sodium phosphate (pH 7.4) and 100 mM NaC1. One milliliter of this 10% homogenate was diluted with 8 ml of the same buffer and 1 ml of Triton X-100 (1%}. This 1% homogenate was left for 30 min at 0°C, centrifuged and the supernatant used for the determination of a2u-G. Urinary creatinine was measured by the m e t h o d of Jaffe [14]. Blood from male and female rats of the high exposure level was also taken upon sacrifice (after 13 weeks of exposure, experiment WS-2) for the measurement of pH, PCO2 and HCO~ Functional tests Urinary concentration tests were performed after a 24-h water deprivation period (food ad lib.) followed by an 8-h urine collection and immediate osmolality measurement (Wescor vapor pressure osmometer, Logan, UT). For the urinary acidification tests, NH4CI (2 mmol/kg) was administered by gavage and the urine collected for 6 h under a layer of liquid paraffin. Net acid excretion (titratable acidity plus a m m o n i u m minus bicarbonate) was immediately measured [15]. The capacity to maximally reduce Na ÷ losses was also measured in male and female rats exposed to the high level of SAHC for 9 weeks as follows: the rats were placed on a low Na ÷ diet (150 ppm Na ÷) for 6 consecutive days and the urine collected every second day for the measurement of Na ÷ concentration. On the 6th day, blood was collected both over heparin for the determination of packed cell volume and in a dry
230
tube for the measurement of serum Na * concentration. Glomerular filtration rate (GFR} was measured after 16 weeks of the low exposure level in male rats only and after 5 weeks of the high exposure level in both normal and castrated males. This test was accomplished by the measurement of S~CrEDTA clearance according to the technique described by Provoost et al. [16].
Rats sacrifice and morphology The rats were killed by pentobarbital anesthesia and blood collection by cardiac puncture. The kidneys were removed and weighed. For WS-4, the liver and 1 kidney were kept at - 2 0 ° C for later analysis of a2u-G. One half kidney was placed in Bouin's solution. Where applicable, a small portion of the cortex was rapidly frozen in liquid nitrogen for subsequent immunofluorescence study using fluorescein-coupled goat antisera against rat IgG and rat C3. Optical microscopy was performed after hematoxylin and eosinsaffron (HES), periodic acid Shiff (PAS) and Mallory-Heidenhain {M-H) staining. In addition, 5 randomly selected exposed males and 5 controls were injected after 16 m o n t h s of exposure to the high level of SAHC, with 200 pCi of [3H]thymidine 1 h prior to sacrifice to determine the kidney cortex labelling index [17].
Statistics Student t-test was used to compare the means o f samples with equal variances. Otherwise the t-test modified after Cochran was used [18]. RESULTS Compared to their respective controls, none of the treated group showed an altered growth curve.
Enzymuria None of the exposed group showed an increased NAG excretion. However, there is a marked early increase in the urinary excretion of LDH in males exposed to the high concentration of SAHC (Fig. 1A). This sustained increase progressively declines with time as seen in the group of males exposed for almost 12 months. Also interesting is the observation that in a group of male rats removed from exposure, a complete recovery is observed. To avoid complicating the figure, the standard errors are not represented. Except at t = 0, all differences between the high exposure males and their controls are significant. A modest but significant 2-fold increase is also seen in the low exposure male group. Neither the castrated males nor the females (not shown} exposed to the high level of SAHC showed a net sustained increase in LDH excretion.
Urinary excretion of specificproteins The urinary excretion of ~2-m is shown in Fig. 2A. There is an effect of
231
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TIME (DAYS) Fig. 1. A. L D H e x c r e t i o n in male rats exposed t o 6.5 mg/l o r 0.58 mg/l o f SAJ-IC and in
castrated rats exposed to 6.5 mg/l of the solvent. B. Urinary concentration test after 24 h of water deprivation in the same animals. R.F.E. means removal from exposure.
castration on t he urinary excretion o f #=-m but no difference is seen between th e tr eated groups and their respective controls. There is a net effect of ex p o s u r e to the high c o n c e n t r a t i o n of SAHC on albuminuria in the male rats only {Fig. 2B). However, with t he onset of chronic progressive nephrosis (CPN) in th e rat [19] t he difference between t he cont rol and high exposure males diminishes with time. Since not all the rats develop CPN to the same e x t e n t and at t he same time [ 2 0 ] , this causes a greater variation in the results and we have observed that t he difference between the groups, although sustained, becomes non-statistically significant between 15 and 30 weeks of exposure. The statistical difference reappears thereafter. None of th e o t h e r tr ea t ed groups exhibits an increased albuminuria. F u n c t i o n a l tests Figure 1B shows that f r om day 25, there is a significant (P < 0.05) decrease in the capacity to p r o d u c e c o n c e n t r a t e d urine in the high exposure
232
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Fig. 3. Sodium excretion in male and female rats exposed to 6.5 mg/l of SAHC for 8 weeks and fed a low sodium diet (150 ppm) every second day. The rats were placed in metabolism cages without food on the alternate day for urine collection and they were given demineralized water ad iibitum. *P < 0.05.
233
m a l e g r o u p o n l y . As w i t h L D H e x c r e t i o n , a c o m p l e t e r e c o v e r y o f t h e u r i n a r y c o n c e n t r a t i n g a b i l i t y is o b s e r v e d in a s u b g r o u p w h i c h was r e m o v e d f r o m e x p o s u r e . Male a n d f e m a l e rats e x p o s e d t o 6.5 mg/1 o f S A H C did n o t h a v e a clear decrease in u r i n a r y a c i d i f y i n g ability u p t o 8 w e e k s o f e x p o s u r e (results n o t s h o w n ) . As s h o w n in Fig. 3, h o w e v e r , a f t e r 9 w e e k s o f e x p o s u r e b o t h t h e high e x p o s u r e m a l e a n d f e m a l e rats s h o w a slower a n t i n a t r i u r e t i c r e s p o n s e t o a low s o d i u m diet. In o t h e r w o r d s , b o t h g r o u p s w a s t e m o r e s o d i u m t h a n t h e i r m a t c h e d c o n t r o l s w h e n t h e i n t a k e is a b r u p t l y r e d u c e d . A f t e r 6 d a y s o f this diet, t h e c o n t r o l and e x p o s e d rats have r e s p e c t i v e l y m e a n h e m a t o c r i t s (-+S.E.) o f 46.5 -+ 0.8% a n d 44.8 -+ 0.4% f o r f e m a l e s and 5 0 . 6 -+ 0.6% and 49.7 +- 0.8% f o r males. T h e c o r r e s p o n d i n g values o f s e r u m s o d i u m c o n c e n t r a t i o n (+-S.E.) for c o n t r o l a n d e x p o s e d rats r e s p e c t i v e l y are 136 -+ 2.5 a n d 135 +- 1.9 m m o l / 1 f o r f e m a l e s and 142 -+ 1.9 and 144 -+ 2.0 m m o l / 1 f o r males. N o d i f f e r e n c e is t h e r e f o r e seen in e i t h e r p a r a m e t e r . F u r t h e r m o r e , w h e n t h e s e r a t s w e r e sacrificed 5 w e e k s later, i.e. a f t e r 13 w e e k s o f e x p o s u r e , n o d i f f e r e n c e b e t w e e n c o n t r o l a n d e x p o s e d rats was seen in t h e i r b l o o d p H , PCO2 o r b i c a r b o n a t e levels (results n o t s h o w n ) . T a b l e I gives the results o f t h e G F R and o f k i d n e y t o b o d y w e i g h t ratio. O n l y t h e high e x p o s u r e m a l e s s h o w e d a small b u t statistically significant r e d u c t i o n in G F R w h e t h e r e x p r e s s e d as a b s o l u t e values or p e r gram w e t weight o f k i d n e y . H o w e v e r , no increase in s e r u m ~z-m was f o u n d in a n y o f t h e t r e a t e d g r o u p (results n o t s h o w n ) . A slight k i d n e y h y p e r t r o p h y is seen b o t h in t h e high e x p o s u r e m a l e s a n d c a s t r a t e d males.
Metabolism of a~u-globulin T a b l e II s h o w s t h a t a f t e r 5 w e e k s o f e x p o s u r e , t h e high e x p o s u r e m a l e s d i s p l a y a 66% increase in t h e i r p l a s m a level o f a2u~:~ b u t t h e u r i n a r y clearTABLEI s~Cr-EDTA CLEARANCE (GFR) AND KIDNEY TO BODY WEIGHT RATIO IN MALE RATS EXPOSED TO 0.58 mg SAHC/I FOR 16 WEEKS AND IN MALE AND CASTRATED MALE (~) RATS EXPOSED TO 6.5 mg SAHC/I FOR 5 WEEKS Rats (n)
SAHC
GFR
GFR
concen-
(ml/min)
(ml/min/g
tration
Kidney weight
kidney) a
body weight (%)
1.19 1.10 1.32 1.05 1.39 1.30
0.589 0.606 0.587 0.676 0.528 0.574
(rag/l) (11)
0
(12) (6) (6) (6) (5)
0.58 0 6.5 0 6.5
3.11 3.09 3.29 2.91 2.77 2.69
_+0 . 0 6 b _+0.10 +_0.07 +_0.11" +- 0.10 +_0.09
aKidneys weighed 4 days after test. bStandard error of the mean.
*P < 0.05; **P < 0.01.
234
+ 0.03 b
_+0.04 _+0.03 +_0.04** +_0.05 _+0.03
_+0 . 0 1 0 b _+0.009 +_0.023 _+0.011"* _+0.009 _+0.013"
T A B L E II PLASMA, LIVER AND KIDNEY CONCENTRATION, URINARY CLEARANCE A N D N E T P E R C E N T A G E R E A B S O R P T I O N O F a 2 u - G L O B U L I N IN M A L E AND C A S T R A T E D (~) R A T S E X P O S E D T O 6.5 mg SAHC/I F O R 5.5 W E E K S
a~u-G in plasma (mg/l) ~2u-G in liver (mg/g wettissue) a~u-G in k i d n e y (mg/g w e t tissue) Urinary clearance (mi/min) Net reabsorption (%)b
Control d
Exposed d
Control ~
Exposed
(n= 6)
(n= 6)
(n= 6)
(n= 5)
18.8+_ 1.3 a
31.2
6.6
_+0.7
8.1
+0.9
0.11
+0.01
0.16
+0.02*
0.31
_+ 0.05
3.25
_+0.54**
0.53+0.07 2.88 + 0.31 0.24 + 0.05 92.7 _+ 1.7
_+3.7"**
0.62 + 0 . 0 4 25.35 _+ 2 . 0 7 * * * 0.27 +_ 0.05 90.8
_+ 1.6
0.028 + 0.007
0.030 +_0.009
99.0
98.9
+_0.2
-+ 0.3
aMean _+ standard error of the mean. bCalcuhted from the G F R data of Table I, assuming a sieving coefficient of 1 for a:u-G. *P < 0.05; **P < 0.01 ; ***P < 0.001 compared to matched controls.
ance and net fractional reabsorption o f t he protein is similar t o the cont rol values. No significant difference is seen on those parameters in castrated males. The high exposure male rats do not have a significantly increased hepatic level of a2u-G whereas a slight significant increase is seen in castrated male rats. Both exposed groups, however, show a dramatic 10-fold increase in th e k id n e y c o n c e n t r a t i o n o f the protein. In addition, 4 high exposure male rats were f ound to have (-+S.E.) 13.0 +- 2.0 mg a2u-G/g ki dney after 68 weeks o f exposure c o m p a r e d t o 1.44 -+ 0.82 for their m at ched controls. Note that, c o m p a r e d t o nor m al control rats, the castrated exposure rats have liver and plasma concent r a t i ons of ~2u-G which are 3 times lower, yet t h e y accumulate just as m u c h of this protein in their kidneys.
Morphology The presence of n u m e r o u s hyaline droplets (HD) was evident with the M-H staining in male rats t r e a t e d for 5.5, 46 and 68 weeks at t he high exposure level although the incidence of HD was somewhat lower in the 68 weeks of exposure group. When t he slides were examined under HE8 or PAS staining, t her e were evident zones o f t u b u l a r dilatation filled with granular material at the cortico-medullary j u n c t i o n in rats exposed for 5.5 weeks. In these animals, mainly deep cortical foci of b o t h proximal and distal tubular regeneration were encount ered. The regeneration oft en was limited to a few cells within a t ubul e while t he o t h e r cells had a normal appearance. However, the rats exposed for 46 or 68 weeks did not show these evident alterations. This was c o n f i r m e d in t he latter group by the c o r t e x labelling index which per 10 000 nuclei, a m o u n t e d to (+-S.E.) 20 +- 6
235
and 29 +-6 ( P > 0.05) labeled nuclei in the control and exposed group respectively. There is still no difference when the labelling of the outer and inner cortex are considered separately. One exposed rat in the 68 weeks group was found to have a benign tubular adenoma. In WS-1, after 46 weeks of exposure, 1 control rat had linear glomerular IgG deposits against 10 who had none while 1 exposed rat had both IgG and Ca deposits and 2 other exposed rats had IgG only glomerular deposits against 6 who had negative tests. However, in WS-1 rats killed after 68 weeks of exposure 1 control rat was positive for IgG only against 8 negative while none of the exposed rats showed either IgG or Ca deposits. SAHC exposure therefore does not induce immunologically-mediated glomerular alterations in our model. DISCUSSION
The present study clearly demonstrates t h a t only male rats show evidence of both functional and morphological renal alterations during repeated exposure to SAHC. Functionally, we have previously shown t h a t SAHC exposure for over 40 weeks induces distal tubular dysfunction in male rats [ 2 1 ] . The results reported here indicate that these reversible functional distal alterations appear early after the onset of exposure. Indeed within 10 days the male rats exposed to 6.5 rag/1 of SAHC show a large increase in LDH excretion w i t h o u t a concomitant increase in NAG. This pattern of enzymuria has been observed in rats treated with folate which is known to produce distal tubular toxicity [22]. Furthermore, after 3 weeks of exposure, male rats also have a moderate but definitive impairment of the ability to produce concentrated urine upon water restriction. The slower antinatriuretic response to a low sodium diet also reflects a diminished ability to produce a maximal sodium gradient along the nephron and also points toward a slight functional defect of the distal tubular cells. It is not known why the females had this single test altered. After 6 days of the diet, however, no repercussion is found either on the state of hydratation (hematocrit) or on the natremia further indicating the mild nature of the alteration. The defective acidifying ability, after NH4C1 administration, which we have reported in male rats exposed for over 40 weeks [21] is not observed for shorter exposure periods to the high concentration of SAHC of up to 8 weeks. It is therefore not surprising that no influence of the treatment occurred on blood pH, PC02 and bicarbonates even after an additional 5 weeks of exposure. The fact that neither NAG nor ~2-m excretion is increased in the exposed group suggests that the proximal tubular (PT) cells are functionally intact. We have indeed shown elsewhere (Viau et al., submitted for publication) that urinary ~2-m is a very sensitive test of the functional status of the PT cells and t h a t even small alterations in the pinocytic function of these cells result in an increased excretion of ~2-m. Our functional observations are in good agreement with kidney histology which despite the presence of numerous HD in the cells of the $2 segment of
236
the proximal tubules, did not show signs of tubular necrosis in rats exposed either for 5.5, 46 or 68 weeks at the high exposure level. In HES, occasional foci of regenerative proximal and distal epithelial cells were observed for a few cells per tubule in rats exposed for 5.5 weeks only. These foci were generally found in the deep cortex. The granular casts at the corticomedullary junction were only observed in the rats exposed for 5.5 weeks. The 5-fold increase in albuminuria observed after 5 weeks of exposure in male rats only could be attributed either to an increased glomerular permeability to albumin or to a decreased tubular uptake. However, since the tubular reabsorption of ~2-m is unaffected, the effect is probably glomerular. There is a 10--20% decrease in the glomerular filtration rate of the highly exposed males and a 15% increase in the kidney weights of these animals. The low exposure male group or high exposure castrated male group show almost no alteration in those parameters. The increased albuminuria and decreased G F R indicate a slight glomerular impairment. The fact that these effects are sex-linked suggest either that t h e y are secondary to the tubular alterations or t h a t SAHC exposure accelerates the development of CPN which is known to occur earlier in male than in female rats [23]. But if tubular effects to the $2 segment and to some tubular cells of the cortico-medullary junction secondarily cause some degree of damage to the glomeruli from which these tubules originate, it would remain to explain why the excretion of 5:-m and NAG are unaffected. And for that purpose, the unaffected urinary clearance of a2u-G itself indicates a formidable adaptation to the overloading of the tubular cells with this protein. Therefore, the hypothesis of a direct glomerular effect again appears more probable. Alden et al. [10] have already shown that the hyaline droplets are mainly composed of a2u-G. Our quantitative data confirm this. Indeed, highly exposed male rats accumulate some 10 times more a2u-G in their kidneys t h a n their matched controls. Lock et al. [24] have recently reported a similar 10-fold increase in male rat kidney a2u-G following 10 daffy doses of trimethylpentane (2 ml/kg, p.o.). Since in our model the hepatic levels of the protein are not increased in these exposed rats, this suggests that the accumulation of the protein would come from a deficient renal catabolism rather than from an increased hepatic synthesis. The higher serum levels of a2u-G in these rats could be the result of a greater recycling of the a ~ - G back to the circulation. Neuhaus and Lerseth [25] have indeed suggested that part of the a2u-G reabsorbed by the kidney could be returned intact to the circulation. If the kidney catabolism is much less than in control, a greater portion of the reabsorbed a2~-G could be recycled and cause slightly elevated serum levels of the protein. Our data further indicate that the urinary clearance of a2u-G is identical in both groups, also pointing toward a deficient intracellular catabolism. Our reported net fractional reabsorption (>90%} is much higher than the 57% reported by Neuhaus and Lerseth [ 2 5 ] . However, our urine collections were made without food supply t o the rats and fasting has been shown to result in a sharp decreased hepatic synthesis and increased renal uptake of a2u-G [26]. This does not invalidate
237
the control-exposed comparisons however since urine collections were made in the same conditions and at the same time for both groups. It is also interesting to compare the normal control males to the highly exposed castrated males in Table II. Indeed the castrated rats have hepatic and plasma a2u-G which are some 2--3 times lower than normal controls but they accumulate just as m u c h a2u-G in their kidneys. This also points in the direction of a renal effect. Furthermore, the exposed castrated males have 10 times more a2u-G in their kidneys than their matched castrated controls. The protein can thus accumulate in animals with low levels of circulating androgens. It appears that due to its high hepatic output, low molecular weight and peculiar renal handling and catabolism, a2u-G behaves like an amplifier-indicator of the kidney effect of hydrocarbons. However, there is no a priori reason to believe that the catabolism of other proteins is not equally affected to a lesser extent. Whether the deficient renal catabolism of a~-G comes from the formation of an undigestible complex between the protein and S A H C metabolites or from a direct effect on the renal enzymes responsible for the degradation of a2u-globulin is still unknown. However, it seems unlikely that either of these possibilities would apply exclusively to one single protein. Finally, Roy and Raber [27] have shown histochemically t h a t a2u-G is present not only in PT cells, but also in the cells of the loop of Henle and in the distal convoluted tubular cells. It seems reasonable to hypothesize that a greater accumulation of a2~-G in those distal portions of the kidney could explain the distal tubular effects reported here despite the absence of morphological evidence of hyaline droplets in these segments. In conclusion, a moderate renal toxicity of SAHC occurs only in male rats to the exclusion of female and castrated rats. There appears to be a primary effect on the proximal (hyaline droplets accumulation) and distal (functional tests and LDH excretion) tubular cells and glomerular alterations which take the form of accelerated CPN. The functional protein reabsorptive capacity seems intact from the results of the unaltered ~2-m excretion or even of the urinary clearance of azu-G. The enhanced renal accumulation of a2u-G is evident both in highly exposed male and castrated male rats. This protein seems to amplify an overall effect of the solvent on the kidney, and particularly on renal protein catabolism, but it seems unlikely that a2u-G would be the sole protein affected.
ACKNOWLEDGEMENTS The authors wish to thank F. de Cock, J. Casters, J. Gilson, D. Hermans W. Nullens and H. Bauer for skillful technical assistance and Dr. S.A. Abid for performing the labeling index measurements. The authors wish to thank Dr. J.C. Daniel from RhSne-Poulenc, France, for providing batches of polystyrene latex particles ( E S T A P O R K109).
238
REFERENCES 1 R. Lauwerys, A. Bernard, C. Viau and J.P. Buchet, Kidney disorders and hematotoxicity from organic solvent exposure. Scand. J. Work Environ. Health (in press). 2 C.P. Carpenter, E.R. Kinkead, D.L. Geary, L.J. Sullivan and J.M. King, Petroleum hydrocarbon toxicity studies. III. Animal and h u m a n response to vapors of Stoddard solvent. Toxicol. Appl. Pharmacol., 32 (1975) 282. 3 C.P. Carpenter, E.R. Kinkead, D.L. Geary, L.J. Sullivan and J.M. King, Petroleum hydrocarbon toxicity studies. II. Animal and h u m a n response to vapors of varnish makers' and painters' naphtha. Toxicol. Appl. Pharmacol., 32 (1975) 263. 4 C.P. Carpenter, E.R. Kinkead, D.L. Geary, L.J. Sullivan and J.M. King, Petroleum hydrocarbon toxicity studies. VIII. Animal and h u m a n response to vapors of 140 ° flash aliphatic solvent. Toxicol. Appl. Pharmacol., 34 (1975) 413. 5 C.P. Carpenter, D.L. Geary, R.C. Myers, D.J. Nachreiner, L.J. Sullivan and J.M. King, Petroleum hydrocarbon toxicity studies. XI. Animal and h u m a n response to vapors of deodorized kerosene. Toxicol. Appl. Pharmacol., 36 (1976) 443. 6 G.A. Parker, V. Bogo and R.W. Young, Acute toxicity of conventional versus shalederived JP5 jet fuel: light microscopic, hematologic and serum chemistry study. Toxicol. Appl. Pharmacol., 57 (1981) 302. 7 R.D. Phillips and G.F. Zgan, Effects of C10---Cll isoparaffinic solvent on kidney function in Fisher 344 rats during 8 weeks of inhalation. Toxicol. Appl. Pharmacol., 73 (1984) 500. 8 R.D. Phillips and G.F. Egan, Subchronic inhalation exposure of dearomatized white spirit and C 1 0 - - C l l isoparaffinic hydrocarbon in Sprague--Dawley rats. Fundam. Appl. Toxicol., 4 (1984) 808. 9 R.D. Phillips and B.Y. Cockrell, Kidney structural changes in rats following inhalation exposure to C10--C11 isoparaffinic solvent. Toxicology, 33 (1984) 261. 10 C.L. Alden, R.L. Kanerva, G. Ridder and L.C. Stone, The pathogenesis of the nephrotoxicity of volatile hydrocarbons in the male rat, in M.A. Mehiman (Ed.), Advances in Modern Environmental Toxicology, vol. VII, Princeton Scientific Publishers, Princeton, 1984, p. 107. 11 A. Bernard, J.P. Buchet, H. Roeis, P. Ma~son and R. Lauwerys, Renal excretion of proteins and enzymes in workers exposed to cadmium. Eur. J. Clin. Invest., 9 (1979) 11. 12 S.M. Tucker, P.J.R. Boyd, A.E. Thompson and R.G. Price, Automated assay of Nacetyl-~-glucosaminidase in normal and pathological urine. Clin. Chim. Acta, 62 (1975) 333. 13 A.M. Bernard and R.R. Lauwerys, Continuous flow-system for automation of latex immunoassay by parti~le counting. Clin. Chem., 29 (1983) 1007. 14 R.J. Henry, D.C. Cannon and J.W. Winkelman, Clinical Chemistry: Principles and Technics, 2nd edn. Harper and Row, New York, 1974, p. 552. 15 J.C.M. Chan, Renal acidosis in C.G. Durate, Renal function tests: clinicallaboratory procedures and diagnosis, Little Brown & Co, Boston, 1980, p. 262. 16 A.P. Provoost, M.H. De Jeijzer, E.D. Wolff and J.C. Molenaar, Development of renal function in the rat. The measurement of G F R and E R P F and correlation to body and kidney weight. Renal Physiol., 6 (1983) 1. 17 G. Laurent, P. Maldague, M.C. Carlier and P. Tulkens, Increased renal D N A synthesis in vivo after administration of low doses of gentamicin to rats. Antimicrob. Agents Chemother., 24 (1983) 586. 18 G.W. Snedecor and W.C. Cochran, Statistical Methods, 5th edn., Iowa State University Press, Ames, Iowa, 1966, p. 97. 19 J.E. Gray, Chronic progressive nephrosis in the albino rat. C R C Crit. Rev. Toxicol., 5, (1977) 115.
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20 O.W. Neuhaus and W. Flory, Age-dependent changes in the urinary excretion of proteins by the rat. Nephron, 22 (1978) 570. 21 C. Viau, A. Bernard and R. Lauwerys, Distal tubular dysfunction in rats chronically exposed to a "white-spirit" solvent. Toxicol. Lett., 21 (1984) 49. 22 R.G. Price, Urinary enzymes, nephrotoxicity and renal disease. Toxicology, 23 (1982) 99. 23 J.E. Gray, M.J. van Swieten and C.F. Hollander, Early light microscopic changes of chronic progressive nephrosis in several strains of aging laboratory rats. J. Gerontol., 37 (1982) 142. 24 E.A. Lock, M.D. Stonard and C.R. Elcombe, The selective induction of ~ - and #oxidation of fatty acids in the liver and kidney of male rats administered 2,2',4trimethylpentane. The Toxicologist, 5 (1985) 59. 25 O.W. Neuhaus and D.S. Lerseth, Dietary control of the renal reabsorption and excretion of a~u-globulin. Kidney Int., 16 (1979) 409. 26 O.W. Neuhaus and W. F l o w , The effect of dietary protein on the excretion of a m , the sex-dependent protein of the adult male rat. Biochim. Biophys. Acta, 411 (1975) 74. 27 A.K. Roy and D.L. Raber, Immunofluorescent localization of a2u-globulin in the hepatic and renal tissues of rat. J. Histochem. Cytochem., 20 (1972) 89.
240
I S O P A R A F F I N I C SOLVENT-INDUCED N E P H R O T O X I C I T Y IN T H E RA T
C. VIAUa*, A. BERNARD a**, F. GUERETa, P. MALDAGUEb, P. GENGOUXc and R. LAUWERYSat aunit@ de Toxicologie Industrielle, Ddpartement de Mddecine et Hygiene du Travail, b Laboratoire de Pathologie et Cytologie Expdrimentale, cUnit@ de Dermatologie ProfessionneUe, Ddpartement de M@decine et Hygi@ne du Travail, Universit@ Catholique de Louvain (School o f Medicine), B-1200 Brussels (Belgium)
(Received June 7th, 1985} (Accepted August 29th, 1985)
SUMMARY
Prolonged inhalation exposure t o 6.5 mg/l of an isoparaffinic solvent consisting of saturated aliphatic h y d r o c a r b o n s (SAHC) resulted in b o t h functional and morphological renal changes in male rats t o the exclusion o f female or castrated rats. Functionally, the increased excretion of lactate dehydrogenase in t he absence o f an increased fl-N-acetyl-D-glucosaminidase ex cr etio n t o g e t h e r with a decreased urinary concentrating ability u p o n water deprivation and slower antinatriuretic response when the sodium intake is abruptly reduced, suggest a distal tubular alteration, fl~-Microglobulin ex cr eti on is unchanged indicating good proxi m al tubular cell function. The increased excretion of albumin and slightly lowered glomerular filtration rate suggest a m o d e r a t e glomerular impairment. Light microscopy shows p r o m i n e n t hyaline d r o p l e t accumulation in proxi m al t ubul ar cells and a few scattered foci o f regenerative epithelia in b o t h proximal and distal cells of th e deep co r t e x. T he urinary clearance o f t he major male rat urinary protein, a2u-globulin, is similar in c o n t r o l and exposed rats but the latter have a 10-fold greater renal accumulation of this protein while the hepatic levels *Research fellow from the Institut de Recherches en Sant~ et en S~curit~ du Travail du Qudbec, 505 Ouest de Maisonneuve, Montreal H3A 3C2, Canads. **Chercheur Qualifi~ of the Fonds National Belge de la Recherche Scientifique. tTo whom all correspondence should be sent: 30.54 Clos Chapelle-aux-Champs 1200 Brussels, Belgium, Abbreviations: BUN, blood urea nitrogen; CPN, chronic progressive nephrosis; GFR, glomerular filtration rate; HD, hyaline droplets; HES, hematoxylin and eosin-saffron; LDH, lactate dehydrogenase; M-H, Mallory-Heidenhain; NAG, fl-N-acetyl-D-glucosaminidase; PAH, periodic acid Shiff; PT, proximal tubular; SAHC, saturated aliphatic hydrocarbons. 0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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are identical in both groups. It is concluded that SAHC exposure causes moderate and reversible tubular and also glomerular changes in the male rat kidney.
Key words: a2u-Globulin; Hydrocarbon solvent; Kidney function; Kidney morphology; Proteinuria; Rat INTRODUCTION For the last few years, there has been some concern that chronic exposure to organic solvent could be associated with an increased risk of chronic glomerulonephritis or other kidney diseases [1]. Animal models have been devised in order to study that problem. In a series of articles, Carpenter et al. [2--5] have studied the toxicity of Stoddard solvent, varnish makers' and painters' naphtha, 140 ° flash aliphatic solvent and deodorized kerosene in Harlan-Wistar male rats. All these solvents contain a high percentage of aliphatic and alicyclic hydrocarbons. They found an increased blood urea nitrogen (BUN} in the case of Stoddard solvent only. In addition, various signs of tubular regeneration and occasional dilated tubules with casts were observed at the cortico-medullary junction. However, since these effects were not dose-related and t h a t t h e y even occurred in some control animals, the authors concluded t h a t t h e y were not linked to the treatment. Parker et al. [6] conducted 14-day gavage studies of up to 60 ml/kg of JP5 jet fuel in male Sprague--Dawley rats. Morphological examination of the kidneys disclosed the presence of eosinophilic hyaline droplets in the cytoplasm of proximal convoluted tubular cells, which was apparently dose~lependent. A slight increase in BUN and in serum creatinine was also noted in the exposed rats. More recently, Phillips and Egan [7,8] and Phillips and Cockrell [9] have reported on the nephrotoxicity of a C10--C12 isoparaffinic solvent in male Fisher-344 and Sprague--Dawley rats (exposure levels 1.83 and 5.48 mg/1). Their findings included a dose-related frequency of zones of regenerative epithelia and tubular dilatation with intratubular protein. Urinalysis indicated an increased giucosuria and proteinuria. BUN and serum creatinine were increased in the treated groups and a decreased creatinine clearance was noted in the high exposure group after 8 weeks of treatment. Finally, a decreased urinary concentrating ability was found to occur in both groups. Except for the morphological alterations, nearly all these manifestations were reversible after 4 weeks of non-exposure. Using decalin as a model c o m p o u n d of hydrocarbon nephrotoxicity in the rat, Alden et al. [10] pointed out to the fact that only male rats seem to be susceptible to this nephropathy. Indeed, the authors report that mice, guinea pigs, dogs and m o n k e y s of either sex and female rats are unaffected by these solvents. They indicate t h a t these renal effects appear to be related to the deficient catabolism of a sex-specific low molecular weight rat protein, a2u-globulin (a2u-G, M r 20 000). The authors state t h a t hyaline droplets are not observed
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in exposed castrated male rats and that this is "evidence of an androgendependent inefficiency of protein reabsorption/catabolism". We have studied the effect of inhalation exposure to a C10--C12 isoparaffinic solvent in male, female and castrated male Sprague--Dawley rats using a wide variety of sensitive tests of the kidney function. Particular attention has been given to enzymuria and specific proteins excretion as used for screening purposes in humans. We have also examined the renal metabolism o f a2u-G in male and castrated male rats. All these tests were supplemented by optical microscopic examination of the kidneys and by immunofluorescence techniques to try to evaluate the implication of immunological mechanisms in the hypothetical development of glomerular alterations. MATERIALS AND METHODS
Study material and study design Four experiments were conducted in order to study various aspects of the nephrotoxicity induced in rats by the solvent (Shellsol TD, Shell company). The latter {common name: white spirit} was a mixture composed at >99% of C10--C12 saturated aliphatic hydrocarbons (SAHC) which the manufacturer claims to be almost entirely isoparaffins. All experiments were performed with Sprague--Dawley rats aged 3--4 months at the start of exposure. They were exposed in a 300-1 stainless-steel and glass whole body inhalation chamber {Young and Bertke Co, Cincinnati, OH). All exposures were 8 h/day, 5 days/week. The total air flow rate through the chamber was maintained constant at 300 1/min. The solvent was pumped at a constant rate through a flash evaporation device before the vapors were admitted to the chamber. The solvent concentration in the chamber was determined by charcoal tube sampling. The samples were desorbed with carbon disulfide and analysed by gas chromatography on a 25% carbowax 20M on Chromosorb W packed column using flame ionization detection. The system was calibrated using the crude solvent as standard. The mean {range} concentrations were 6.5 (5.8--7.5} and 0.58 {0.52--0.64} mg/1 for the high and low exposure levels respectively. The control rats were placed in an identical inhalation chamber swept by room air. The first experiment (WS-1) aimed at checking the possible long term effect of exposure to high levels (6.5 mg/1) of SAHC on some parameters of the kidney function. Male rats were divided into 24 control and 24 exposed animals and the exposure lasted for up to 16 months. Periodic urine collection was performed at t = 0, 19, 25, 30, 37 and 45 weeks. Half the animals were killed at 46 weeks, the other half at 68 weeks of exposure. Having determined what appeared to be the most sensitive tests of kidney function in this model, a second study (WS-2) was undertaken in order to check if signs of nephrotoxicity appeared earlier than 19 weeks and to check the reversibility of the functional changes. In addition, sex susceptibility was examined. Therefore 24 male and 24 female rats were equally distributed between a control and an exposed group. The latter was sub-
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mitted to the same concentration (6.5 mg/1) of SAHC as previously for 13 weeks. The reversibility was evaluated after 6 weeks of non-exposure. In the third experiment (WS-3), 12 male rats were exposed for 16 weeks to a 0.58 rag/1 SAHC concentration which is close to the TLV for these types of compounds. A matched group of 12 controls was also examined. The effect of castration on SAHC nephrotoxicity was studied in the f o u r t h experiment (WS-4) in which 6 males and 5 castrated males were exposed to the high concentration (6.5 mg/l) of the solvent. These animals were matched to 6 normal and 6 castrated controls. In addition, WS-4 was designed to look at the metabolism of a2u-globulin in exposed vs. control rats. Urine, serum and tissue analyses All urine collections were performed from the rats placed in individual stainless-steel metabolism cages equipped with urine/feces separators. No food was given during the collection but water was provided ad libitum (except during the urinary concentration test -- see below). Ten milligrams of NaN3 was added, as a preservative, to the collection flasks. The activities of #-N-acetyl-D-glucosaminidase (NAG, EC 3.2.1.30} and of lactate dehydrogenase (LDH, EC 1.1.1.27) were determined as described previously [11,12]. The urinary concentration of albumin, the urinary and serum concentration o f #:-microglobulin (#2-m) and the urinary, serum and tissue concentration of a2u-G were measured by latex immunoassays similar to t h a t described by Bernard and Lauwerys [13]. For the determination of the tissue concentration of a:u-G, approximately 1 g of either whole kidney or liver were homogenized with 9 ml of an i c e , o l d solution containing 50 mM sodium phosphate (pH 7.4) and 100 mM NaC1. One milliliter of this 10% homogenate was diluted with 8 ml of the same buffer and 1 ml of Triton X-100 (1%}. This 1% homogenate was left for 30 min at 0°C, centrifuged and the supernatant used for the determination of a2u-G. Urinary creatinine was measured by the m e t h o d of Jaffe [14]. Blood from male and female rats of the high exposure level was also taken upon sacrifice (after 13 weeks of exposure, experiment WS-2) for the measurement of pH, PCO2 and HCO~ Functional tests Urinary concentration tests were performed after a 24-h water deprivation period (food ad lib.) followed by an 8-h urine collection and immediate osmolality measurement (Wescor vapor pressure osmometer, Logan, UT). For the urinary acidification tests, NH4CI (2 mmol/kg) was administered by gavage and the urine collected for 6 h under a layer of liquid paraffin. Net acid excretion (titratable acidity plus a m m o n i u m minus bicarbonate) was immediately measured [15]. The capacity to maximally reduce Na ÷ losses was also measured in male and female rats exposed to the high level of SAHC for 9 weeks as follows: the rats were placed on a low Na ÷ diet (150 ppm Na ÷) for 6 consecutive days and the urine collected every second day for the measurement of Na ÷ concentration. On the 6th day, blood was collected both over heparin for the determination of packed cell volume and in a dry
230
tube for the measurement of serum Na * concentration. Glomerular filtration rate (GFR} was measured after 16 weeks of the low exposure level in male rats only and after 5 weeks of the high exposure level in both normal and castrated males. This test was accomplished by the measurement of S~CrEDTA clearance according to the technique described by Provoost et al. [16].
Rats sacrifice and morphology The rats were killed by pentobarbital anesthesia and blood collection by cardiac puncture. The kidneys were removed and weighed. For WS-4, the liver and 1 kidney were kept at - 2 0 ° C for later analysis of a2u-G. One half kidney was placed in Bouin's solution. Where applicable, a small portion of the cortex was rapidly frozen in liquid nitrogen for subsequent immunofluorescence study using fluorescein-coupled goat antisera against rat IgG and rat C3. Optical microscopy was performed after hematoxylin and eosinsaffron (HES), periodic acid Shiff (PAS) and Mallory-Heidenhain {M-H) staining. In addition, 5 randomly selected exposed males and 5 controls were injected after 16 m o n t h s of exposure to the high level of SAHC, with 200 pCi of [3H]thymidine 1 h prior to sacrifice to determine the kidney cortex labelling index [17].
Statistics Student t-test was used to compare the means o f samples with equal variances. Otherwise the t-test modified after Cochran was used [18]. RESULTS Compared to their respective controls, none of the treated group showed an altered growth curve.
Enzymuria None of the exposed group showed an increased NAG excretion. However, there is a marked early increase in the urinary excretion of LDH in males exposed to the high concentration of SAHC (Fig. 1A). This sustained increase progressively declines with time as seen in the group of males exposed for almost 12 months. Also interesting is the observation that in a group of male rats removed from exposure, a complete recovery is observed. To avoid complicating the figure, the standard errors are not represented. Except at t = 0, all differences between the high exposure males and their controls are significant. A modest but significant 2-fold increase is also seen in the low exposure male group. Neither the castrated males nor the females (not shown} exposed to the high level of SAHC showed a net sustained increase in LDH excretion.
Urinary excretion of specificproteins The urinary excretion of ~2-m is shown in Fig. 2A. There is an effect of
231
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castration on t he urinary excretion o f #=-m but no difference is seen between th e tr eated groups and their respective controls. There is a net effect of ex p o s u r e to the high c o n c e n t r a t i o n of SAHC on albuminuria in the male rats only {Fig. 2B). However, with t he onset of chronic progressive nephrosis (CPN) in th e rat [19] t he difference between t he cont rol and high exposure males diminishes with time. Since not all the rats develop CPN to the same e x t e n t and at t he same time [ 2 0 ] , this causes a greater variation in the results and we have observed that t he difference between the groups, although sustained, becomes non-statistically significant between 15 and 30 weeks of exposure. The statistical difference reappears thereafter. None of th e o t h e r tr ea t ed groups exhibits an increased albuminuria. F u n c t i o n a l tests Figure 1B shows that f r om day 25, there is a significant (P < 0.05) decrease in the capacity to p r o d u c e c o n c e n t r a t e d urine in the high exposure
232
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Fig. 3. Sodium excretion in male and female rats exposed to 6.5 mg/l of SAHC for 8 weeks and fed a low sodium diet (150 ppm) every second day. The rats were placed in metabolism cages without food on the alternate day for urine collection and they were given demineralized water ad iibitum. *P < 0.05.
233
m a l e g r o u p o n l y . As w i t h L D H e x c r e t i o n , a c o m p l e t e r e c o v e r y o f t h e u r i n a r y c o n c e n t r a t i n g a b i l i t y is o b s e r v e d in a s u b g r o u p w h i c h was r e m o v e d f r o m e x p o s u r e . Male a n d f e m a l e rats e x p o s e d t o 6.5 mg/1 o f S A H C did n o t h a v e a clear decrease in u r i n a r y a c i d i f y i n g ability u p t o 8 w e e k s o f e x p o s u r e (results n o t s h o w n ) . As s h o w n in Fig. 3, h o w e v e r , a f t e r 9 w e e k s o f e x p o s u r e b o t h t h e high e x p o s u r e m a l e a n d f e m a l e rats s h o w a slower a n t i n a t r i u r e t i c r e s p o n s e t o a low s o d i u m diet. In o t h e r w o r d s , b o t h g r o u p s w a s t e m o r e s o d i u m t h a n t h e i r m a t c h e d c o n t r o l s w h e n t h e i n t a k e is a b r u p t l y r e d u c e d . A f t e r 6 d a y s o f this diet, t h e c o n t r o l and e x p o s e d rats have r e s p e c t i v e l y m e a n h e m a t o c r i t s (-+S.E.) o f 46.5 -+ 0.8% a n d 44.8 -+ 0.4% f o r f e m a l e s and 5 0 . 6 -+ 0.6% and 49.7 +- 0.8% f o r males. T h e c o r r e s p o n d i n g values o f s e r u m s o d i u m c o n c e n t r a t i o n (+-S.E.) for c o n t r o l a n d e x p o s e d rats r e s p e c t i v e l y are 136 -+ 2.5 a n d 135 +- 1.9 m m o l / 1 f o r f e m a l e s and 142 -+ 1.9 and 144 -+ 2.0 m m o l / 1 f o r males. N o d i f f e r e n c e is t h e r e f o r e seen in e i t h e r p a r a m e t e r . F u r t h e r m o r e , w h e n t h e s e r a t s w e r e sacrificed 5 w e e k s later, i.e. a f t e r 13 w e e k s o f e x p o s u r e , n o d i f f e r e n c e b e t w e e n c o n t r o l a n d e x p o s e d rats was seen in t h e i r b l o o d p H , PCO2 o r b i c a r b o n a t e levels (results n o t s h o w n ) . T a b l e I gives the results o f t h e G F R and o f k i d n e y t o b o d y w e i g h t ratio. O n l y t h e high e x p o s u r e m a l e s s h o w e d a small b u t statistically significant r e d u c t i o n in G F R w h e t h e r e x p r e s s e d as a b s o l u t e values or p e r gram w e t weight o f k i d n e y . H o w e v e r , no increase in s e r u m ~z-m was f o u n d in a n y o f t h e t r e a t e d g r o u p (results n o t s h o w n ) . A slight k i d n e y h y p e r t r o p h y is seen b o t h in t h e high e x p o s u r e m a l e s a n d c a s t r a t e d males.
Metabolism of a~u-globulin T a b l e II s h o w s t h a t a f t e r 5 w e e k s o f e x p o s u r e , t h e high e x p o s u r e m a l e s d i s p l a y a 66% increase in t h e i r p l a s m a level o f a2u~:~ b u t t h e u r i n a r y clearTABLEI s~Cr-EDTA CLEARANCE (GFR) AND KIDNEY TO BODY WEIGHT RATIO IN MALE RATS EXPOSED TO 0.58 mg SAHC/I FOR 16 WEEKS AND IN MALE AND CASTRATED MALE (~) RATS EXPOSED TO 6.5 mg SAHC/I FOR 5 WEEKS Rats (n)
SAHC
GFR
GFR
concen-
(ml/min)
(ml/min/g
tration
Kidney weight
kidney) a
body weight (%)
1.19 1.10 1.32 1.05 1.39 1.30
0.589 0.606 0.587 0.676 0.528 0.574
(rag/l) (11)
0
(12) (6) (6) (6) (5)
0.58 0 6.5 0 6.5
3.11 3.09 3.29 2.91 2.77 2.69
_+0 . 0 6 b _+0.10 +_0.07 +_0.11" +- 0.10 +_0.09
aKidneys weighed 4 days after test. bStandard error of the mean.
*P < 0.05; **P < 0.01.
234
+ 0.03 b
_+0.04 _+0.03 +_0.04** +_0.05 _+0.03
_+0 . 0 1 0 b _+0.009 +_0.023 _+0.011"* _+0.009 _+0.013"
T A B L E II PLASMA, LIVER AND KIDNEY CONCENTRATION, URINARY CLEARANCE A N D N E T P E R C E N T A G E R E A B S O R P T I O N O F a 2 u - G L O B U L I N IN M A L E AND C A S T R A T E D (~) R A T S E X P O S E D T O 6.5 mg SAHC/I F O R 5.5 W E E K S
a~u-G in plasma (mg/l) ~2u-G in liver (mg/g wettissue) a~u-G in k i d n e y (mg/g w e t tissue) Urinary clearance (mi/min) Net reabsorption (%)b
Control d
Exposed d
Control ~
Exposed
(n= 6)
(n= 6)
(n= 6)
(n= 5)
18.8+_ 1.3 a
31.2
6.6
_+0.7
8.1
+0.9
0.11
+0.01
0.16
+0.02*
0.31
_+ 0.05
3.25
_+0.54**
0.53+0.07 2.88 + 0.31 0.24 + 0.05 92.7 _+ 1.7
_+3.7"**
0.62 + 0 . 0 4 25.35 _+ 2 . 0 7 * * * 0.27 +_ 0.05 90.8
_+ 1.6
0.028 + 0.007
0.030 +_0.009
99.0
98.9
+_0.2
-+ 0.3
aMean _+ standard error of the mean. bCalcuhted from the G F R data of Table I, assuming a sieving coefficient of 1 for a:u-G. *P < 0.05; **P < 0.01 ; ***P < 0.001 compared to matched controls.
ance and net fractional reabsorption o f t he protein is similar t o the cont rol values. No significant difference is seen on those parameters in castrated males. The high exposure male rats do not have a significantly increased hepatic level of a2u-G whereas a slight significant increase is seen in castrated male rats. Both exposed groups, however, show a dramatic 10-fold increase in th e k id n e y c o n c e n t r a t i o n o f the protein. In addition, 4 high exposure male rats were f ound to have (-+S.E.) 13.0 +- 2.0 mg a2u-G/g ki dney after 68 weeks o f exposure c o m p a r e d t o 1.44 -+ 0.82 for their m at ched controls. Note that, c o m p a r e d t o nor m al control rats, the castrated exposure rats have liver and plasma concent r a t i ons of ~2u-G which are 3 times lower, yet t h e y accumulate just as m u c h of this protein in their kidneys.
Morphology The presence of n u m e r o u s hyaline droplets (HD) was evident with the M-H staining in male rats t r e a t e d for 5.5, 46 and 68 weeks at t he high exposure level although the incidence of HD was somewhat lower in the 68 weeks of exposure group. When t he slides were examined under HE8 or PAS staining, t her e were evident zones o f t u b u l a r dilatation filled with granular material at the cortico-medullary j u n c t i o n in rats exposed for 5.5 weeks. In these animals, mainly deep cortical foci of b o t h proximal and distal tubular regeneration were encount ered. The regeneration oft en was limited to a few cells within a t ubul e while t he o t h e r cells had a normal appearance. However, the rats exposed for 46 or 68 weeks did not show these evident alterations. This was c o n f i r m e d in t he latter group by the c o r t e x labelling index which per 10 000 nuclei, a m o u n t e d to (+-S.E.) 20 +- 6
235
and 29 +-6 ( P > 0.05) labeled nuclei in the control and exposed group respectively. There is still no difference when the labelling of the outer and inner cortex are considered separately. One exposed rat in the 68 weeks group was found to have a benign tubular adenoma. In WS-1, after 46 weeks of exposure, 1 control rat had linear glomerular IgG deposits against 10 who had none while 1 exposed rat had both IgG and Ca deposits and 2 other exposed rats had IgG only glomerular deposits against 6 who had negative tests. However, in WS-1 rats killed after 68 weeks of exposure 1 control rat was positive for IgG only against 8 negative while none of the exposed rats showed either IgG or Ca deposits. SAHC exposure therefore does not induce immunologically-mediated glomerular alterations in our model. DISCUSSION
The present study clearly demonstrates t h a t only male rats show evidence of both functional and morphological renal alterations during repeated exposure to SAHC. Functionally, we have previously shown t h a t SAHC exposure for over 40 weeks induces distal tubular dysfunction in male rats [ 2 1 ] . The results reported here indicate that these reversible functional distal alterations appear early after the onset of exposure. Indeed within 10 days the male rats exposed to 6.5 rag/1 of SAHC show a large increase in LDH excretion w i t h o u t a concomitant increase in NAG. This pattern of enzymuria has been observed in rats treated with folate which is known to produce distal tubular toxicity [22]. Furthermore, after 3 weeks of exposure, male rats also have a moderate but definitive impairment of the ability to produce concentrated urine upon water restriction. The slower antinatriuretic response to a low sodium diet also reflects a diminished ability to produce a maximal sodium gradient along the nephron and also points toward a slight functional defect of the distal tubular cells. It is not known why the females had this single test altered. After 6 days of the diet, however, no repercussion is found either on the state of hydratation (hematocrit) or on the natremia further indicating the mild nature of the alteration. The defective acidifying ability, after NH4C1 administration, which we have reported in male rats exposed for over 40 weeks [21] is not observed for shorter exposure periods to the high concentration of SAHC of up to 8 weeks. It is therefore not surprising that no influence of the treatment occurred on blood pH, PC02 and bicarbonates even after an additional 5 weeks of exposure. The fact that neither NAG nor ~2-m excretion is increased in the exposed group suggests that the proximal tubular (PT) cells are functionally intact. We have indeed shown elsewhere (Viau et al., submitted for publication) that urinary ~2-m is a very sensitive test of the functional status of the PT cells and t h a t even small alterations in the pinocytic function of these cells result in an increased excretion of ~2-m. Our functional observations are in good agreement with kidney histology which despite the presence of numerous HD in the cells of the $2 segment of
236
the proximal tubules, did not show signs of tubular necrosis in rats exposed either for 5.5, 46 or 68 weeks at the high exposure level. In HES, occasional foci of regenerative proximal and distal epithelial cells were observed for a few cells per tubule in rats exposed for 5.5 weeks only. These foci were generally found in the deep cortex. The granular casts at the corticomedullary junction were only observed in the rats exposed for 5.5 weeks. The 5-fold increase in albuminuria observed after 5 weeks of exposure in male rats only could be attributed either to an increased glomerular permeability to albumin or to a decreased tubular uptake. However, since the tubular reabsorption of ~2-m is unaffected, the effect is probably glomerular. There is a 10--20% decrease in the glomerular filtration rate of the highly exposed males and a 15% increase in the kidney weights of these animals. The low exposure male group or high exposure castrated male group show almost no alteration in those parameters. The increased albuminuria and decreased G F R indicate a slight glomerular impairment. The fact that these effects are sex-linked suggest either that t h e y are secondary to the tubular alterations or t h a t SAHC exposure accelerates the development of CPN which is known to occur earlier in male than in female rats [23]. But if tubular effects to the $2 segment and to some tubular cells of the cortico-medullary junction secondarily cause some degree of damage to the glomeruli from which these tubules originate, it would remain to explain why the excretion of 5:-m and NAG are unaffected. And for that purpose, the unaffected urinary clearance of a2u-G itself indicates a formidable adaptation to the overloading of the tubular cells with this protein. Therefore, the hypothesis of a direct glomerular effect again appears more probable. Alden et al. [10] have already shown that the hyaline droplets are mainly composed of a2u-G. Our quantitative data confirm this. Indeed, highly exposed male rats accumulate some 10 times more a2u-G in their kidneys t h a n their matched controls. Lock et al. [24] have recently reported a similar 10-fold increase in male rat kidney a2u-G following 10 daffy doses of trimethylpentane (2 ml/kg, p.o.). Since in our model the hepatic levels of the protein are not increased in these exposed rats, this suggests that the accumulation of the protein would come from a deficient renal catabolism rather than from an increased hepatic synthesis. The higher serum levels of a2u-G in these rats could be the result of a greater recycling of the a ~ - G back to the circulation. Neuhaus and Lerseth [25] have indeed suggested that part of the a2u-G reabsorbed by the kidney could be returned intact to the circulation. If the kidney catabolism is much less than in control, a greater portion of the reabsorbed a2~-G could be recycled and cause slightly elevated serum levels of the protein. Our data further indicate that the urinary clearance of a2u-G is identical in both groups, also pointing toward a deficient intracellular catabolism. Our reported net fractional reabsorption (>90%} is much higher than the 57% reported by Neuhaus and Lerseth [ 2 5 ] . However, our urine collections were made without food supply t o the rats and fasting has been shown to result in a sharp decreased hepatic synthesis and increased renal uptake of a2u-G [26]. This does not invalidate
237
the control-exposed comparisons however since urine collections were made in the same conditions and at the same time for both groups. It is also interesting to compare the normal control males to the highly exposed castrated males in Table II. Indeed the castrated rats have hepatic and plasma a2u-G which are some 2--3 times lower than normal controls but they accumulate just as m u c h a2u-G in their kidneys. This also points in the direction of a renal effect. Furthermore, the exposed castrated males have 10 times more a2u-G in their kidneys than their matched castrated controls. The protein can thus accumulate in animals with low levels of circulating androgens. It appears that due to its high hepatic output, low molecular weight and peculiar renal handling and catabolism, a2u-G behaves like an amplifier-indicator of the kidney effect of hydrocarbons. However, there is no a priori reason to believe that the catabolism of other proteins is not equally affected to a lesser extent. Whether the deficient renal catabolism of a~-G comes from the formation of an undigestible complex between the protein and S A H C metabolites or from a direct effect on the renal enzymes responsible for the degradation of a2u-globulin is still unknown. However, it seems unlikely that either of these possibilities would apply exclusively to one single protein. Finally, Roy and Raber [27] have shown histochemically t h a t a2u-G is present not only in PT cells, but also in the cells of the loop of Henle and in the distal convoluted tubular cells. It seems reasonable to hypothesize that a greater accumulation of a2~-G in those distal portions of the kidney could explain the distal tubular effects reported here despite the absence of morphological evidence of hyaline droplets in these segments. In conclusion, a moderate renal toxicity of SAHC occurs only in male rats to the exclusion of female and castrated rats. There appears to be a primary effect on the proximal (hyaline droplets accumulation) and distal (functional tests and LDH excretion) tubular cells and glomerular alterations which take the form of accelerated CPN. The functional protein reabsorptive capacity seems intact from the results of the unaltered ~2-m excretion or even of the urinary clearance of azu-G. The enhanced renal accumulation of a2u-G is evident both in highly exposed male and castrated male rats. This protein seems to amplify an overall effect of the solvent on the kidney, and particularly on renal protein catabolism, but it seems unlikely that a2u-G would be the sole protein affected.
ACKNOWLEDGEMENTS The authors wish to thank F. de Cock, J. Casters, J. Gilson, D. Hermans W. Nullens and H. Bauer for skillful technical assistance and Dr. S.A. Abid for performing the labeling index measurements. The authors wish to thank Dr. J.C. Daniel from RhSne-Poulenc, France, for providing batches of polystyrene latex particles ( E S T A P O R K109).
238
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20 O.W. Neuhaus and W. Flory, Age-dependent changes in the urinary excretion of proteins by the rat. Nephron, 22 (1978) 570. 21 C. Viau, A. Bernard and R. Lauwerys, Distal tubular dysfunction in rats chronically exposed to a "white-spirit" solvent. Toxicol. Lett., 21 (1984) 49. 22 R.G. Price, Urinary enzymes, nephrotoxicity and renal disease. Toxicology, 23 (1982) 99. 23 J.E. Gray, M.J. van Swieten and C.F. Hollander, Early light microscopic changes of chronic progressive nephrosis in several strains of aging laboratory rats. J. Gerontol., 37 (1982) 142. 24 E.A. Lock, M.D. Stonard and C.R. Elcombe, The selective induction of ~ - and #oxidation of fatty acids in the liver and kidney of male rats administered 2,2',4trimethylpentane. The Toxicologist, 5 (1985) 59. 25 O.W. Neuhaus and D.S. Lerseth, Dietary control of the renal reabsorption and excretion of a~u-globulin. Kidney Int., 16 (1979) 409. 26 O.W. Neuhaus and W. F l o w , The effect of dietary protein on the excretion of a m , the sex-dependent protein of the adult male rat. Biochim. Biophys. Acta, 411 (1975) 74. 27 A.K. Roy and D.L. Raber, Immunofluorescent localization of a2u-globulin in the hepatic and renal tissues of rat. J. Histochem. Cytochem., 20 (1972) 89.
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