Experimental model of lead nephropathy

ENVIRONMENTAL RESEARCH 58, 3 5 - 5 4 (1992)

Experimental Model of Lead Nephropathy I1. Effect of Removal from Lead Exposure and Chelation Treatment with Dimercaptosuccinic Acid (DMSA) FARHAD K H A L I L - M A N E S H , * HARVEY C . GONICK,* ARTHUR C O H E N , ~ ENRICO BERGAMASCHI,~ AND ANTONIO

MUTTI~

*Departments of Medicine, Trace Element Laboratory, Cedars-Sinai Medical Center, Los Angeles, California 90048; ~'Departmentof Pathology, UCLA-Harbor General Hospital, Torrance, California 90509; and ~Laboratory of lndustrial Toxicology, University of Parma, Parma, Italy Received September 3, 1991 Male Sprague-Dawley rats were exposed to high-dose (0.5%) lead acetate for periods ranging from l to 9 months; then lead exposure was discontinued, and animals were sacrificed after 12 months. Controls were pair-fed. Two additional groups of low-dose (0.01%) and high-dose (0.5%) rats were exposed to lead for 6 months, then lead was discontinued and the rats were treated with three 5-day courses of 0.5% DMSA (dimercaptosuccinic acid) over the next 6 months. Controls were rats exposed to lead for 6 months, then removed from exposure for 6 months without receiving DMSA. Low-dose lead-treated rats showed no significant pathological changes with or without DMSA treatment, but exhibited a significant increase in GFR after DMSA. High-dose lead-treated animals showed no functional or pathological changes when lead exposure was discontinued after 1 month. However, when duration of exposure was 6 or 9 months, GFR was decreased and serum creatinine and urea nitrogen were increased as compared to controls. Tubulointerstitial disease was severe. Administration of DMSA resulted in an improvement in GFR and a decrease in albuminuria, together with a reduction in size and number of nuclear inclusion bodies in proximal tubules. However, tubulointerstitial scarring was only minimally reduced. It may be concluded that, except for brief initial exposure, discontinuation of high-dose lead exposure fails to reverse lead-induced renal damage. Treatment with the chelator, DMSA, improves renal function but has less effect on pathological alterations. As GFR improved after DMSA treatment in both low-dose and high-dose lead-treated rats, irrespective of the degree of pathological alterations, it may be concluded that the DMSA effect is most likely mediated by hemodynamic changes. © 1992 Academic Press, Inc.

INTRODUCTION There has been mounting evidence for a relationship between lead exposure and kidney disease. Numerous studies involving occupationally exposed workers have provided evidence for lead-induced chronic nephropathy, at blood leads ranging from 40 to more than 100 ~zg/dl (Campbell et al., 1977; Emmerson, 1973; Batuman, et al., 1981). Others have suggested that renal damage may occur even at levels below 30 ixg/dl (Suketa et al., 1979; Mouw et al., 1978). Parallel animal studies have reinforced the findings in humans. A number of transient effects on human and animal renal function are consistent with pathological findings in experimental animals of reversible lesions such as nuclear inclusion bodies, cytomegaly, swollen mitochondria, and increased numbers of iron-containing lysosomes in proximal tubule cells (Fowler et al., 1980; Spit et al., 1981). Irreversible lesions

35 0013-9351/92 $5.00 Copyright © 1992by Academic Press, Inc. All rights of reproduction in any form reserved.

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such as interstitial fibrosis have also been well documented in both humans and animals following long-term exposure to lead (Goyer, 1971; Cramer et al., 1974). In industry, lead-exposed individuals who are at risk are removed from further exposure. The present Occupational Safety and Health Administration (OSHA, 1978) recommendations are to remove a worker from the exposure environment when blood lead exceeds 50 txg/dl and to not permit return to lead exposure until blood lead is less than 40 ~g/dl. However, removal from exposure alone may not be sufficient to lower blood or tissue lead to an acceptable level or to reverse preexistent renal damage. Along with removal from exposure, chelating agents such as dimercaprol (BAL) (Foreman, 1961), calcium disodium ethylenediaminetetraacetic acid (CaNa2 EDTA) (Foreman, 1961), o-penicillamine (Chisholm, 1968), 2,3,-dimercapto-l-propane sulfonic acid (DMPS) (Chisholm, 1985), and meso-2,3-dimercaptosuccinic acid (DMSA) (Friedheim, et aI., 1978; Aposhian, 1983) have been used to mobilize tissue lead and to increase the excretion of lead in human and animal subjects. The purpose of the present study was to examine: (1) whether simple removal from lead exposure would allow reversal of lead-related changes in pathology and markers of renal dysfunction in rats treated with high doses of lead for varying time periods; and (2) the effect of the chelating agent, DMSA, in improving renal function as well as hematopoietic markers of toxicity in animals treated with either high (0.5%) or low (0.01%) doses of lead. MATERIALS AND METHODS

Animals Male Sprague-Dawley rats were given a semipurified low-calcium diet (ICN Biochemicals, Cleveland, OH) and either 0.0I% (experimental discontinuous low dose) or 0.5% lead acetate (experimental discontinuous high dose) in their drinking water starting at 2 months of age. Lead administration was discontinued at 1, 6, and 9 months in high-dose animals (at 6 months only in low-dose animals), and all animals were sacrificed after 12 months of study. All experimental animals had age-matched pair-fed controls. Another two groups of rats were given either 0.01% lead acetate or 0.5% lead acetate for 6 months and then treated with DMSA. Three 5-day courses of DMSA (0.5% in drinking water) were given 2 months apart (beginning after cessation of lead) to each group. The animals were sacrificed at 12 months, i.e., 2 months after the final course of DMSA. Animals were weighed before entry into the study and at the end of the study. Kidneys were removed and weighed after determination of glomerular filtration rate and removal of blood from femoral artery. The initial kidney weight ("wet weight") was obtained immediately after removal. A small slice of the kidney was taken and dried at 70°C for 24 hr to determine "dry weight." Determination of Glomerular Filtration Rate (GFR) GFR was measured directly by using a modified plasma isotope-clearance tech-

D M S A C H E L A T I O N IN L E A D N E P H R O P A T H Y

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nique described by Bryan et al. (1972) and indirectly by measurement of serum true creatinine (Hare, 1950; Lauson, 195 I) and serum urea nitrogen (SUN) (Tietz, 1987). Collection of Urine and Blood Two days prior to sacrifice, rats were individually placed in metabolic cages during the day and urine samples were collected in plastic tubes on ice. Urine samples were centrifuged and aliquots (1 ml) were removed and frozen at -80°C until required for examination. Blood (10 ml) was drawn from the femoral artery in a heparinized syringe and red blood cells (RBCs) were separated from plasma by centrifuging 8 ml of whole blood at 3000 rpm for 10 rain. Red blood cells were used for determination of membrane Na,K-ATPase activity and plasma was used for measurement of serum true creatinine and SUN. An aliquot of whole blood (1 ml) was frozen at -80°C for determination of lead content, while the remainder was used for zinc protoporphyrin (ZPP) measurements. Measurement of Urinary Markers Urinary N-acetyl-13-D-glucosaminidase (NAG) and glutathione S-transferase (ligandin or GST) were measured according to the methods described by Yuen et al. (1984) and Feinfeld et al. (1977), respectively. Urine creatinine was determined colorimetrically (Bonsnes and Taussky, 1945). All urinary markers were expressed as units per gram creatinine. Urine albumin was measured by the ELISA technique (Valcavi et al., 1986). Determination of Trace Elements in Blood, Urine, and Kidney Cortex Lead content of whole blood and urine was measured using an atomic absorption spectrophotometer (Perkin-Elmer Model 305 with graphite furnace). Whole blood lead values were expressed as micrograms per deciliter and urine lead as micrograms per gram creatinine. Slices of kidney cortex were dried at 70°C for 24 hr, samples were weighed, and trace element content (lead, silicon, copper, and zinc) was determined by emission spectroscopy (Indraprasit et al., 1972). Kidney trace element content was expressed as parts per million dry weight. Determination of Red Blood Cell Membrane Na,K-ATPase Red blood cells were washed with 150 mM NaC1 solution. Cells were lysed by freezing and thawing and membrane fragments were washed again with the same NaCI solution. Ouabain-sensitive Na,K-ATPase activity was measured according to the method described by Khalil-Manesh et al. (1987). The protein content of samples was determined by the method of Lowry et al. (1951). Inorganic phosphate was measured as described by Fiske and Subbarow (1925). Na,K-ATPase activities were expressed as micromoles of inorganic phosphate liberated per milligram protein per hour.

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Measurement of Zinc Protoporphyrin Zinc protoporphyrin was measured with a hematofluorometer as described by Peter and Strunc (1983). ZPP concentrations were expressed as milligrams per deciliter.

Pathology Kidneys were removed and blot dried and 1- to 2-mm slices were dissected out and fixed for electron and light microscopy. Electron microscopy staining was performed with uranyl acetate and lead citrate after fixation in glutaraldehyde. The semithin sections (3 txm), stained with toluidine blue from the tissue fixed in glutaraldehyde and embedded in plastic, were utilized for light microscopic evaluation.

Statistical Treatment of Data Statistical analysis of the data was performed using the t test. Results are expressed as mean + standard deviation. Statistical significance for t-test was assessed using a two-tailed probability level of 0.05, and computing was conducted using the Biomedical Computer Programs (BMDP) (Dixon, 1988). RESULTS

High Lead Discontinuous Animals One month. One-month high-lead discontinuous animals showed no functional or pathological changes as compared to control animals (Figs. 1 and 2, Table 1), although blood lead was approximately twice normal (experimental, 7.9 -+ 1.1 vs control, 3.3 --- 0.4 ixg/dl; P < 0.001) (Fig. 1). Urine lead was also increased 100

150

High Lead

p