Rat model of perchloroethylene-induced renal dysfunctions

ENVIRONMENTAL RESEARCH 59, 427--439 (1992)

Rat Model of Perchloroethylene-lnduced Renal Dysfunctions

ENRICO BERGAMASCHI, ANTONIO M U T T I , MARIA CONCETTA BOCCHI,

ROSSELLA ALINOVI, GIORGIO OLIVETTI,* GIAN MARCO G H I G G E R I , t

AND INNOCENTE FRANCHINI

Institute of Clinical Medicine & Nephrology and *Department of Pathology, University of Parma, Via A. Gramsci 14, 1-43100 Parma, Italy; and ?Laboratory of Nephrology, G. Gaslini Institute,

Via 5 Maggio, 1-16148 Genova, Italy

Received April 12, 1992

To investigate whether perchloroethylene (PCE) can induce renal disturbances and to compare morphological alterations with functional data, two groups of 12 male and female Fischer-344 mature rats were treated daily with PCE (500 mg/kg body wt in corn oil, p.o.) for 4 weeks. Sex- and age-matched control groups received corn oil only. Weekly, the urinary excretion of albumin (Alb), c~2~-globulin (c~2~) and retinol-binding protein (RBP) was measured in 24-hr urine samples using immunoassays specific for rat proteins. N-acetylglucosaminidase (NAG) activity was measured by a colorimetric assay. Electrophoretic analysis of proteinuria included SDS-PAGE and isoelectric-focusing of Alb purified from serum and urine. Weekly histopathology comprised light and electron microscopy. In the male rat, a trend toward progressive albuminuria (up to 15 times the pair-fed controls) was observed, together with transient increases in e~2¢ and NAG; RBP showed a twofold increase at the end of treatment. Histopathology failed to demonstrate glomerular changes, whereas it dis- played tx2~ accumulation and mild lesions in the $2 segment of proximal tubules. Thus, in the male rat, the selective damage to $2 was associated with "glomerular" proteinuria, the c~2~ cortical content being closely correlated with albuminuria (n = 9, r = 0.92, P < 0.001). In the female rat, only minor, although statistically significant (P < 0.05), increases were recorded for Alb, whereas urinary ct2~ reached up to four times the control values. As a whole, these findings suggest that PCE, like other hydrocarbons, selectively affects the tubular segment $2 in the rat. A competition with a2~ for tubular uptake could explain enhanced albuminuria. Owing to the species specificity of ~2~, caution should be exercised in extrapolating these findings to man. © 1992 Academic Press. Inc.

INTRODUCTION

A number of organohalogenated chemicals have been reported to cause nephrotoxicity in experimental animals and, in some accidental cases, in man. Owing to a low vapor pressure (14 mmHg), a high boiling point (121°C), and poor me- tabolization, perchloroethylene (PCE) is thought to be a relatively safe chemical of low acute toxicity and is widely used in dry-cleaning shops, in metal degreasing operations, and as a heat exchange fluid (WHO, 1984).

The body of evidence of renal effects by PCE exposure in both animals and humans is limited. In general, experimental data suggest that renal dysfunctions and/or damage occurs at very high doses, whereas such effects at low doses are controversial. Histopathology on renal tissues of animals acutely treated with PCE seems to suggest renal tubular changes at doses approaching the observed LDs0 of 2,9 ml/kg body wt for mice (Klaasen and Plaa, 1965). Experimental studies also reported negative findings for renal effects from lower doses in rats (13 days, 7-hr exposures to 2500 ppm) (Rowe et al., 1952).

427

0013-9351/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

428 BERGAMASCHI ET AL.

A limited number of studies have been carried out in male rats given oral doses of PCE and these studies focused on renal tumorigenesis (Schumann et al., 1980; Goldsworthy et al., 1988). Recently, Green et al. (1990) showed an increase in urinary enzymes N-acetylglucosaminidase (NAG) and ALP activities in rats given oral doses of 1500 mg/kg body wt for 42 days, thus suggesting mild proximal tubular damage.

In humans, two cross-sectional studies carried out on dry cleaners occupationally exposed to PCE reported contradictory results. Using sensitive urinary indicators of renal dysfunction Franchini et al. (1983) found urinary excretion of renal tubular enzymes [3-glucuronidase and lysozyme significantly elevated compared to that of workers not exposed to solvents. Such effects were not confirmed in a small group of workers examined by Lauwerys et al. (1983).

Recent findings among workers occupationally exposed to PCE in dry-cleaning shops showed a high prevalence of renal dysfunctions as assessed by sensitive immunochemical methods (Mutti et al., 1992). These abnormalities consisted of increased excretion of high molecular weight proteins, namely albumin, transfer- tin, and IgG, associated with enhanced excretion of kidney-derived antigens, mainly of tubular origin, and minor changes in low molecular weight proteinuria, thus suggesting that PCE may induce glomerular dysfunction and/or mild tubular damage.

In the present study we describe a rat model set up to investigate whether PCE-induced renal disturbances can be reproduced in rats and whether these disturbances are associated with morphological alterations. The results are dis- cussed with regard to the possible significance of this response to PCE exposure and with the aim of comparing animal responses to human responses by using the same indicators.

MATERIAL AND METHODS

Experimental protocol. Two groups of 12 male and female Fischer-344 mature rats, 3 months old (Charles River Laboratories, Calco, Como, Italy) were treated daily with PCE (99 + %, epoxide stabilizer-free, Aldrich Chemical Co., Inc., Ger- many) 500 mg/kg body wt for 4 weeks. The chemical was dissolved in corn oil (Sigma Chemical Co., St. Luis, MO) up to 0.8-1 ml final volume and administered by gavage. Sex- and age-matched control groups were dosed with corn oil only. All animals had free access to laboratory chow, with a protein content of 19% (4 RF21, Italiana Mangimi, Calco, Como, Italy) and filtered tap water. Weekly, the animals were housed in individual metabolic cages provided with urine-feces separators and 24-hr urine samples were collected in tubes immersed in cooling ice.

Urinary protein measurements. Albumin (Alb), as a marker of increased glomerular permeability, and low molecular weight proteins ~2~-globulin (~2~) and retinol-binding protein (RBP), as indicators of tubular dysfunction, were determined by immunochemical methods using mono-specific antisera and rat proteins (Lucertini et al., 1984; Alinovi et al., 1988). Rat albumin was purchased from

PERCHLOROETHYLENE AND KIDNEY DYSFUNCTION 429

Calbiochem (La Jolla, CA) and rabbit anti-rat albumin from Bio-Science Products (Emmenbrucke, Switzerland). e~2~, purified according to the method described by Stonard et al. (1986), was a gift from Dr. E. Lock (ICI, Alderley Park, UK). Anti-et2~ antiserum was a kind gift of Dr. A. Bernard (UCL, Bruxelles, Belgium). RBP was measured by a recently developed "sandwich" ELISA (Biagini et al., 1992).

N-acetyl-[3-D-glucosaminidase (NAG) was determined by a colorimetric assay using a commercially available kit (Boehringer, Mannheim).

Electrophoretic techniques. SDS-PAGE of pooled urine samples collected at various times throughout the study was carried out on prepacked microgels with a 8-25% acrylamide gradient using a semiautomatic PhastSystem apparatus (Phar- macia, Uppsala, SW) and double silver-staining procedure (Heukenshoven and Dernick, 1988).

To better understand the meaning of albuminuria, rat urinary (RUA) and serum albumin (RSA) were purified by pseudo-ligand chromatography on Affi-Gel Blue (Bio-Rad, Richmond, CA) according to the procedure described by Ghiggeri et al. (1987) and analyzed by analytical isoelectric focusing (IEF) in ultrathin gels and double silver staining (Merril et al., 1982). Briefly, a 0.5-ml volume of serum diluted in 200 ml of 0.05 M Tris-HCl, pH 7.0, or 10-15 ml of urine diluted with the same buffer to 400 ml was applied to Affi-Gel Blue packed columns (7 x 1.5 cm i.d.) equilibrated with 0.05 M Tris-HC1-0.25 M potassium chloride, pH 7.0. Chro- matography was carried out at room temperature following the method described by Ghiggeri et al. (1985a). The albumin fraction was concentrated through Amicon PM 30 membranes using an inert gas atmosphere. Ultrathin-layer (240 p~m) IEF was performed in polyacrylamide slab gels (T = 5%, C = 3%) cast on silanized glass plates, the standard polymerization solution containing 2.5% v/v carrier ampholytes in a nonlinear pH 4-7 range. The run was performed at 10°C by applying 2000 V (13 W) for 6 hr, with prefocusing for 1 hr at 500 V (13 W). Constant amounts of protein (25 mg in 20-30 ml) were directly applied to gel in preformed pockets. Silver staining was performed by the photochemical method of Merril et al. (1982).

Morphology. Histopathological evaluations were carried out in four animals per group in the middle and the end of the study period and comprised light and electron microscopy. The fixation procedure was performed as follows: after anesthesia with 50 mg/kg ip sodium thiopenthale (pentothal sodium, Abbott), the abdomen was opened and the abdominal aorta below the renal arteries isolated and cannulated with a polyethylene catheter (PE200) filled with phosphate buffer (0.2 M, pH 7.4) and heparin (100 IU/ml). When the tip of catheter was just below the level of the left renal vein the cannula was sealed in place with a ligature and connected to a rotary pump to maintain the perfusion pressure equal to the in vivo values. The aorta below the renal arteries was then clamped and the renal vas- culature perfused with buffer for 3 min and then for 15 min with fixative (2% p-formaldehyde and 2.5% glutaraldehyde). The kidneys were then excised and cut into I-ram-wide strips perpendicular to the long axis. Ten to fifteen tissue blocks from each kidney containing the full cortex and the outer medulla were then

430 BERGAMASCHI ET AL,

obtained in each rat. The specimens were kept for 3 hr in fresh fixative at room temperature, washed in buffer, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in araldite. One-micrometer-thick sections of each block were cut with a Reichert OMU-2 ultramicrotome and stained with methylene blue and safranin.

Renal ~2~-globulin concentration. The concentration of ~2p~ was measured in aliquots of kidney homogenates (33% w/v in phosphate-buffered saline (PBS) pH 7.2) from male rats. After dilution (50- to 100-fold) and centrifugation at 3000g at 4°C for 10 rain, an aliquot of supernatant was taken for protein determination by the method of Lowry (1961) and 0.1-ml aliquots were assayed in triplicate using a sandwich enzyme-linked immunosorbent assay with a double rabbit polyclonal antibody against rat o~2~. Calibration curve for ~2p~ (0.5 to 50 ng/ml) was prepared in PBS; ~2~ cortical concentration was measured and the results were expressed as mg ~2~/g protein in the crude homogenates.

Statistical analysis. All urinary parameters showed a skewed distribution. Sta- tistical analysis was thus based on log-transformed values, which showed a normal distribution (Kolmogorov-Smirnow nonparametric test). Differences between groups were assessed by Student's test for independent samples, whereas any correlations between variables were assessed by Pearson's correlation coef- ficient.

RESU LTS

Morphology. Morphologic examination of electron microscopic preparations failed to demonstrate any changes in the basement membrane or in other glomerular structures (Fig. 1).

In male control rats, the cells of P2 segment showed small apical vacuoles and dark basophilic protein droplets, considered to be phagolysosomes; the majority of these inclusion bodies were small in diameter and predominantly round (Fig. 2A). Male rats dosed with PCE showed a progressive increase in number and size of phagolysosomes (hyaline droplets) in proximal convoluted epithelial cells over the treatment period (Fig. 2B). Subcellular analysis revealed that many of these cell-filling phagolysosomes were angular and had cross-sectional diameters vary- ing from 0.5 to 5 ixm (Fig. 2C). Occasionally, individual cells within this segment showed high vacualotion accompanied by intratubular cell debris. Similar, though much less severe, abnormalities were found increased also in female-treated rats at the late stage of treatment (Fig. 2D).

Proteinuria. The results of protein determination on urine samples in male rats are summarized in Table 1. A trend toward progressively increasing albuminuria was observed in treated male rats, with values raising up to 15 times those of pair-fed controls; transient increases in urinary OLZp ~ were also found. RBP showed a twofold increase in comparison to controls toward the end of the study period. NAG excretion rate transiently, but significantly, rose in the early stages of the treatment.

Table 2 shows the results of protein determination in urine of female rats. Minor though statistically significant increases were recorded for Alb, whereas Ot.2~ pro-


FIG. 1. Transmission electron micrograph of a glomerulus from PCE-treated male rats demonstrat- ing the structural integrity of the basement membrane, of the capillary endothelium (C) and the mesangial area (M). (4050× ; uranyl acetate and lead nitrate).

gressively rose up to four times the control values. RBP slightly increased over baseline and control values.

In male rats, a weak correlation was found between Alb and oL2~ excretion rates (r = 0.30, P < 0.05). Urinary Alb and RBP were also correlated (r = 0.37, P < 0.05). In female rats, a significant relationship between az~ and RBP was found (r = 0.68, P < 0.01), whereas no correlation occurred between albumin and markers of tubular dysfunction.

Electrophoresis. In male rats, SDS-PAGE of urine samples revealed qualitative changes in the protein excretion pattern. Whereas the low molecular weight (LMW) range remained substantially unchanged, high molecular weight (HMW) proteins (i.e., Alb, transferrin, IgG, and other unidentified proteins) tended to increase over the study period (Fig. 3A). In female rats, while the HMW proteins remained stable during the whole period, there was a progressive increase in LMW proteins, namely az, and RBP (Fig. 3B).

Rat Alb purified from sera of normal and PCE-treated rats analyzed by IEF showed a highly microheterogeneous pattern with pI's ranging from 4.7 to 5.8. The IEF pattern of urinary Alb was less heterogeneous, consisting of major bands with pI around 4.7 both in treated and control animals (Fig. 4).

FIG. 2. Light microscopy of kidney cortex from a male control rat (A). The epithelial cells of proximal tubules contain various amount of small predominantly round, protein droplets (arrowheads I ). (B) Light microscopy of kidney cortex from a PCE-treated male rat in which larger number of epithelial cells from the proximal tubules contain an increased number of small to large protein droplets (arrowheads ~ ). (C) At higher magnification, the protein droplets appear to be more angular and irregular, some crystalloid figures ( i ) also being visible. (D) Light microscopy of kidney cortex from a PCE-treated female rat in which the epithelial cells of proximal tubules contain only a few protein droplets in comparison with that seen in male. (A, B = 130×; C, D = 660×, methylene blue and safranin).

432


FIG. 2--Continued.

Renal oL2~-globulin concentration. The kidney concentration of o~2~., determined in male rats at various stages of treatment, showed a progressive increase and appeared to be strongly correlated with the onset and significance of albuminuria (r 2 = 0.85, P < 0.001, Fig. 5).


TABLE 1 URINARY EXCRETION OF SINGLE PLASMAPROTEINS IN PCE-TREATED MALE RATS IN COMPARISON

WITH UNTREATED CONTROL RATS DURING THE WHOLE STUDY PERIOD

Baseline Day 7 Day 15 Day 21 Day 28 GM (GSD) GM (GSD) GM (GSD) GM (GSD) GM (GSD)

PCE Albumin (ng/min) 43.3 (1.8) 64.5 (1.2) 568.9 (4.6)*** 390.6 (4.1)*** 1050.9 (1.5)*** Otzp " 0xg/min) 6.52 (0.9) 5.74 (1.6) 8.23 (2.3)* 4.62 (1.4) 5.13 (1.4) RBP (pg/min) 78.4 (1.2) 65.5 (1.6) 79.7 (2.3) 138.2 (2.3)* 185.4 (3.6)* NAG (IxU/min) 63.0 (1.2) 72.2 (1.2) 110.6 (0.9)* 61.2 (1.1) 92.5 (1.3)

Controls Albumin (ng/min) 49.8 (1.8) 68.9 (3.2) 56.1 (4.6) 48.7 (3.5) 70.6 (4.2) a2~ (p.g/min) 4.58 (0.8) 6.73 (1.8) 5.64 (1.3) 4.28 (1.4) 4.46 (1.4) RBP (pg/min) 65.3 (1.3) 55.8 (1.4) 77.3 (1.8) 82.1 (4.3) 71.7 (2.2) NAG (p~U/min) 58.2 (1.3) 75.2 (0.9) 64.3 (1.6) 66.5 (1.3) 71.5 (1.6)

Note. All variables are expressed as excretion rate. * P < 0.05; ***P < 0.001 as compared to pair-fed controls (Student t test on log-transformed data).

DISCUSSION

Male rats exposed to structurally diverse hydrocarbons develop hyaline droplet nephropathy (HDN), a condition characterized by accumulation of proteins in epithelial cells of convoluted proximal tubules, mainly in the $2 tract, with min- imal abnormalities in renal function (Alden et al., 1984; Viau et al., 1986; Olson et al., 1987). The present study is thus consistent with previous observations and with those recently reported for mature male rats given oral dose of PCE (Gold- sworthy et al., 1988). We also found that high doses of PCE caused mild proximal tubular damage in male rats, together with a marked accumulation of protein droplets in the $2 segment. However, unlike other models dealing with HDN

URINARY EXCRETION OF SINGLE COMPARISON WITH UNTREATED

TABLE 2 PLASMAPROTEINS IN PCE-TREATED FEMALE RATS IN CONTROL RATS DURING THE WHOLE STUDY PERIOD

Baseline Day 7 Day 15 Day 21 Day 28 GM (GSD) GM (GSD) GM (GSD) GM (GSD) GM (GSD)

PCE Albumin (ng/min) 34.6 (1.4) 42.6 (1.8)* 28.2 (1.6) 90.3 (1.5)** 70.2 (1.5)* a2~ (rig/rain) t5.3 (0.9) 29.5 (1.9)* 58.3 (2.4)** 25.1 (2.8) 48.2 (2.5)*** RBP (pg/min) 27.3 (2.3) 30.7 (4.6) 31.1 (4.4) 20.1 (3.7) 44.2 (3.2)* NAG (~U/min) 43.8 (1.4) 46.9 (1.3) 84.2 (1.4)* 39.8 (2.0) 58.1 (1.4)

Controls Albumin (ng/min) 33.9 (1.3) 16.6 (2.0) 33.0 (1.3) 55.5 (1.6) 38.5 (1.7) tx2~ (ng/min) 13.3 (1.9) 6.55 (1.9) 14.1 (3.0) 9.53 (2.7) 13.5 (1.6) RBP (pg/min) 23.2 (2.0) 27.1 (3.2) 31.1 (4.4) 10.8 (3.1) 24.2 (3.2) NAG (txU/min) 47.5 (1.6) 58.6 (1.3) 62.9 (1.9) 28.4 (2.4) 59.7 (1.3)

Note. All variables are expressed as excretion rate. * P < 0.05; **P < 0.01; ***P< 0.001 as compared

transformed data). to pair-fed controls (Student t test on log-

A

67

43

30

20

14

e d

PERCHLOROETHYLENE AND KIDNEY DYSFUNCTION

c b a e d c

B

67

43

30

20

14

b

435

8

Kd Kd

FIG. 3. SDS-electrophoresis and silver staining of urine samples from PCE-treated male (A) and female (B) rats. (A) Whereas the pattern in LMW range remains substantially unchanged, HMW proteins (albumin, transferrin, IgG, and other unidentified proteins) tend to increase over the study period. (B) HMW proteins remain stable over time, whereas there is a progressive rise in some LMW proteins (a2~-globulin and RBP). (a, baseline; b, Day 7; c, Day 15; d, Day 28; e, molecular weight markers)

nephros is , the p resence o f these intracellular inclusions was poor ly assoc ia ted with cy to tox ic i ty or cell necrosis , but with modera te tubular dysfunct ion . Fur- the rmore , a l though to a l imited extent , in our model female rats also showed such an effect.

The in terpre ta t ion o f the funct ional correla tes o f PCE- induc e d morphologica l changes is a difficult task. On the basis o f the present s tudy, the validity o f markers o f ear ly renal damage would seem quest ionable , since bo th false posi t ive

Serum Urinary albumin albumin

a b c d e a b c d e pl

5.8

4.7

4.0 FIG. 4. Ultrathin-layer IEF of rat serum albumin (RSA) and rat urinary albumin (RUA) purified

from control (a, b) and PCE-treated male rats (c-e). Both in treated and in untreated animals, RSA shows a highly microeterogeneity with pl's ranging from 4.7 to 5.8; similarly, the IEF pattern of RUA is quite homogeneous, consisting of a main band with a pl around 4.7.


2500

C

E

2000

1500

R 2 = 0 . 8 5

p ( 0 . 0 0 1

I ! 000 ~-

i i i

500 . o

( 9 itD •

O 00 , '

, J

200 500 400 500

c~2~ globuiin (mg/g protein} FIG. 5. Relationship between ~2~-globulin accumulation in renal cortex and albuminuria in treated

male rats.

(increased albuminuria and HMW proteinuria) and false negative results (unchanged LMW proteinuria) were recorded. Whereas the former suggested glomerular impairment not revealed by histopathology, the latter failed to show PCE-induced selective lesions of the $2 tract of the proximal tubules. Thus, male rats given PCE orally for 4 weeks seem to be a model of "glomerular" proteinuria due to tubular lesions in the absence of any glomerular changes.

Two alternative or combined explanations may account for these findings: (a) glomerular changes might be functional in nature, so that early dysfunction, as revealed by albuminuria, may not be associated with structural abnormalities; such an explanation would point to a low sensitivity of histopathology, rather than to a poor specificity of HMW proteinuria; (b) the selective damage to $2 might account for the increased albuminuria, possibly due to competitive mechanism involving the altered renal handling of ~2~-

The failure of LMW proteins to show tubular damage would be consistent with the limited extension and severity of histopathological changes, not involving more proximal segments. Recently, we suggested that the renal handling of proteins is a relatively selective process, since the increase in urinary RBP excretion is greater after administration of toxins known to selectively affect the straight segment $3 (HgC12 or cisplatin) as compared to those causing more proximal lesions (NazCrO4), whereas [32-microglobulin seems to be more sensitive to S1-$2 injury. Thus, the low sensitivity of RBP observed during subacute treatment with PCE could be explained by the site selectivity of lesions involving the $2 tract of proximal tubules (Bergamaschi et al., 1992).

Both the marked changes in the HMW proteins and the slight increase in LMW proteins are also consistent with physiological studies on isolated and perfused proximal tubules, indicating that LMW proteins are almost completely reabsorbed


by S1, whereas the integrity of S1 and $2 is required to reabsorb noncationized "native" albumin (Park and Maack, 1984).

In the rat, it is well established that oL2~ is selectively taken up by the segment $2 and accumulates in the form of insoluble protein droplets affecting lysosomal function (Swenberg et al., 1989). This suggests that lysosomal impairment may represent an additional mechanism underlying increased albuminuria without con- comitant changes in LMW proteinuria.

Thus, the lack of change in the urinary activity of NAG is not surprising and is consistent with NAG activity measured following occupational exposure to PCE (Mutti et al., 1992), resembling that found in the urine of patients with subclinical diabetic nephropathy (Brogren et al., 1986). Moreover, the possible increased shedding might be balanced by a depressed activity of this lysosomal enzyme as a consequence of impaired metabolic turnover caused by fagolysosomal accumulation of the derivatized a2~.

The maintained selectivity for the urinary excretion of the most anionic isoforms of albumin would also be consistent with the hypothesis of a tubular target selectivity of PCE. Under normal conditions, only a small amount of albumin escapes the glomerular barrier and reaches the urinary space. Only the low pl isoforms (more anionic) are preferentially excreted, possibly because the effi- ciency of tubular reabsorption varies according to the molecular charge of the protein (Christensen et al., 1983). Thus, the tubular binding and uptake of posi- tively charged albumin would be proportionally higher than that of isoforms with lower pI, since the surface membrane of the luminal cells is negatively charged.

The loss of charge selectivity of the glomerular barrier observed in the early stages of diabetic nephropathy and in adriamycin-mediated nephrosis (Ghiggeri et al., 1985b, 1987) is associated with an early loss of charge selectivity of urinary albumin, this not being the case for PCE-treated rats.

As a whole, these findings suggest that PCE, like other solvents, selectively affects the tubular segment $2 in the rat. The sex-related differences, as to morphology and proteinuria, suggest that the renal handling of Or21 x plays a key role in the appearance and severity of structural and correlated functional changes. Ow- ing to species-related metabolic features and some qualitative differences revealed by IEF of albumin, caution has to be exercised in extrapolating the male rat model of "glomerular proteinuria of tubular origin" to humans. However, whereas gross changes in urinary albumin can hardly be attributed to reduced tubular uptake of the filtered protein, the latter mechanism might easily explain small changes over the reference limits observed in workers occupationally exposed to some neph- rotoxins selectively affecting the distal part of proximal tubules. Such a target selectivity would explain why "isolated" albuminuria may be a more sensitive marker of tubular dysfunction than LMW proteinuria.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the skilled technical assistance of Mrs. Claudia Biagini and Stefania Cavazzini. This study was supported, in part, by funds provided by the EEC (Early Indicators of Nephrotoxic Effects Resulting from Exposure to Environmental Pollutants-Contr. EV4V-0190-I-A).

438 B E R G A M A S C H I ET AL.

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Rat model of perchloroethylene-induced renal dysfunctions

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