ArchToxicol(1992) 66: 107-111

Archives of

Toxicology 9 Springer-Verlag 1992

Acetaminophen nephrotoxicity in male Wistar rats Laura Trumpet, Guillermina Girardi, and Maria M6nica Elias Farmacologfa.Facultad de Ciencias Bioquimicasy Farmaceuticas de la Universidad Nacional de Rosario. Consejo Nacional de Investigaciones Cientificasy Tecnicas(CONICET) - Consejo de Investigacionesde la UniversidadNacional de Rosario (CIUNR), Republica Argentina Received25 March 1991/Receivedafter revision 7 August 1991/Accepted8 August 1991 Abstract. Acute acetaminophen (APAP) nephrotoxicity was studied in male Wistar rats 1 h after different APAP single doses (200, 500 and 1000 mg/kg body wt, i.p.). Significant impairments in glomerular filtration rate (GFR) and clearance of p-aminohippuric acid (C1pAH) were observed in a dose-dependent way, although tubular parameters measured, water and electrolyte fractional excretion, remained at control values, while the urine to plasma osmolality ratios (Uosm/Posm) were diminished in APAP-1000 rats (control = 2.93___0.20, APAP1000 = 1.40 + 0.04). The time course of renal function was also studied in APAP-1000 mg/kg-treated animals; parallel impairments were observed in GFR, CIpAH and tubular functions. Maximal alteration was observed at 16 h and restorement began at 24 h post-injection. Glucose renal handling, either at low or at high tubular glucose loads, remained at control values. Thus, our data suggest that the early stage of acetaminophen nephrotoxicity might be due to renal hemodynamic changes which might induce an alteration in tubular function principally in distal structures of medullary tissue, as shown by the Uosm/Posm results. These effects occurred coupled with a diminution in hepatic glutathione (GSH) levels at every APAP dose and in renal GSH levels in APAP-1000 mg/kg-treated rats. Moreover, renal damage was observed both in the presence or absence of hepatic damage. Key words: Acetaminophen - Acute nephrotoxicity Glutathione

renal toxicity both in man (Boyer and Rouff 1971; Prescott et al. 1971) and laboratory animals (McMurtry et al. 1978; Tarloff et al. 1989). The appearance of renal lesions is variable. It may occur coupled with hepatic damage (Newton et al. 1982b), or in its absence suggesting different susceptibility of the kidney and the liver to APAP toxicity (Peter 1969; Cobden et al. 1982). It has been described that a single large dose of APAP administered to male Fisher 344 rats induced necrosis of the proximal tubules (McMurtry et al. 1978). Other evaluations of APAP-induced renal functional changes were elevation in blood urea nitrogen (Newton et al. 1982b; Tarloff et al. 1989) or plasma creatinine (Siegers and Moller-Hartman 1989). An impairment in p-aminohippurate accumulation by renal cortical slices has also been reported (Newton et al. 1982b). Although these data concluded that APAP is a nephrotoxicant, little is known about the sequential aspects of the development of the APAP-induced nephrotoxicity in vivo. On the other hand, McMurtry et al. (1978) postulated that APAP toxicity could be due to a chemically reactive arylating metabolite which could bind to cellular macromolecules after a substantial loss of glutathione (GSH). Toxic metabolites could be formed both in liver (Mitchell et al. 1973) and kidney (Newton et al. 1982a). The aim of this study was to examine in rats the effects of a single dose of APAP on renal physiology in order to determine the primary renal target structure to the development of APAP nephrotoxicity. Liver and kidney GSH levels were also measured as an indirect index of the production of APAP toxic metabolites.

Introduction

Materials and methods

Acetaminophen (APAP) is a widely used antipyretic-analgesic drug. Following an overdose it causes hepatic and

Animals and treatments

Offprint requests to: Maria M6nica Elias, Farmacologia - Facultad de Ciencias Bioquimicas y Farmaceuticas, Suipacha 531 - 2000 Rosario, RepublicaArgentina

Male Wistar rats (3 months; 250-350 g body wt) were used. They were housed in rooms with controlled temperature(21-23 ~C), humidity and light (0600- 1800 hours), and maintainedon a standard diet and water ad libitum. They were fasted 18 h before the experiment,but were allowed free access to water. Several experimentalgroups were studied: (i) ani-

108 mals that received different single doses of APAP as follows: 200 (n = 10), 500 (n = 7) and 1000 (n = 10) mg/kg body weight, i.p. and were used 1 h later for renal clearance studies. The highest dose assayed was previously described as nephrotoxic (Tarloff et al. 1989); (ii) animals injected with the single maximal dose of APAP used in this study (1000 mg/kg body wt, i.p.) that were prepared for clearance studies 1 (n = 10), 6 (n = 4), 16 (n = 6), 24 (n = 4) and 48 (n = 6) h post-injection; (iii) animals injected with the single maximal dose that were prepared to study the maximal tubular transport capacity (Tin) of glucose 1 h (n = 6) and 16 (n = 4) h after drug administration. For each experimental group appropriate control animals were studied (n = 10). APAP was dissolved in 0.15 M NaCI and 1 M NaOH to give a final pH of 10. Control rats received an equivalent volume of NaCI 0.15 M, pH = 10. At the end of the experiments the kidneys and the livers were promptly removed and the ghitathione (GSH) content was assayed in whole tissue homogenates. GSH results were expressed as gmol/g wet tissue.

Rosario, Argentina) and the urinary proteins with the EDTA/Cu reag~: (Biuret's method, Henry et al. 1980b) (Wiener Lab., Rosario, Argentina SGPT assays were performed according to the method developed t Reitman and Frankel ( 1957; Wiener Lab. Rosario, Argentina).

Statistical analysts Statistical analyses were performed using the unpaired Student t-tes: multiple comparisons were made by the one-way analysis of variant: The 0.05 level of significance was used as the criterion of significanc~ All data are expressed as means • SEM.

Results

Experimental procedures

Effect of different doses of APAP on renal and hepatic" glutathione content

Clearance studies. Animals were anesthetized with urethane (1.2 g/kg body wt, i.p.). The femoral vein and femoral artery were cannulated and a bladder catheter (3 mm i.d.) was inserted through a suprapubic incision. Clearance studies were performed conventionally as previously reported (Torres et al. 1986; Girardi et al. 1989). Briefly, a water solution containing inulin (0.9 g/ml), p-aminohippuric acid (PAH) (0.3 g/ml) and D-mannitol (5 g/ml) was infused through the venous catheter, employing a constant infusion pump (Unita Braun Melsungen, FRG) at a rate of 5 ml/h. After equilibration for 45 min periods, urine was collected during two 30-min periods. Blood from the femoral artery was obtained at the midpoint of each clearance period. This experimental protocol induced osmotic diuresis in the rats. Arterial blood pressure was measured throughout the experiments with a manometer inserted in the femoral artery. The glomerular filtration rate (GFR) was calculated from the clearance of inulin. Renal plasma flow was estimated by the clearance of p-aminohippuric acid (Clean). The fractional excretion of water (FE % H20); sodium (FE % Na) potassium (FE % K) and glucose (FE % Glu) were calculated by conventional formulae for each animal. The ratio urine to plasma osmolality (Uo~m/po~m)was calculated. Urine protein excretion was also determined. Serum glutamic-pyruvic transaminase (SGPT) was measured in all the animals as an index of hepatocellular damage.

Maximal glucose reabsorptionrate (Tm glucose). Rats (APAP-1000 and controls) were prepared for clearance studies 1 h and 16 h post injection as described above. A 25% glucose solution in water containing inulin (10 mM) was infused i. v. as described above (Westenfelder et al. 1980; Grosman et al. 1983). The glucose infusion was increased in a stepwise fashion (1.75, 3 and 5 ml/h) allowing stabilization of plasma glucose and inulin at increasing concentrations. At each plasma glucose concentration, two clearance samples were obtained. GFR was calculated from the clearance of inulin, and tubular glucose reabsorption was calculated from the difference between the amounts of glucose filtered and excreted, and was expressed relative to GFR.

Analytical methods Serum and urinary inulin concentration were determined by Roe's procedure (Roe et al. 1949) and PAH concentrations in the same samples were determined by Brun's method as modified by Waugh and Beall (1974). Sodium and potassium were measured by flame photometry. The volume of urine was measured gravimetrically. Osmolality was determined in a vapor pressure osmometer (Wescor 5100 C, USA). Determination of renal and liver non-protein sulfllydryls (roughly representing GSH) was carried out in whole tissue homogenates prepared in cold 5% trichloroacetic acid in 0.01 M HC1 and measured as described by Ellman (1959). The plasma and urinary glucose were measured by the colorimetric-enzymatic method according to Henry et al. (1980a) (Wiener Lab.,

A l l the A P A P d o s e s a s s a y e d r e s u l t e d in a d i m i n i s h e d hepatic G S H l e v e l in a d o s e d e p e n d e n t w a y (Control -3.75+0.16, A P A P - 2 0 0 = 3 . 1 4 + 0 . 1 5 , A P A P - 5 0 0 -2.30+0.13 a n d A P A P - 1 0 0 0 = 1 . 7 4 + 0 . 1 5 g m o l / g we: tissue). A l t h o u g h m u l t i p l e c o m p a r i s o n s o f renal GSH l e v e l s d i d n o t s h o w statistical s i g n i f i c a n c e , APAP-10C( t r e a t e d rats p r e s e n t e d a s i g n i f i c a n t d e c r e a s e ( 1 5 % ) of thek renal G S H c o n t e n t ( 1 . 7 0 + 0 . 0 8 g m o l / g w e t tissue) wkr. c o m p a r e d w i t h c o n t r o l rats ( 2 . 0 1 + 0 . 0 8 g m o l / g w~ tissue).

Effect of different doses of APAP on renal function, 1 h after injection T h e m e a s u r e d p a r a m e t e r s o f renal f u n c t i o n are presentedin Fig. 1. It c a n b e s e e n (Fig. 1A) that the G F R and the C1pA~ d e c r e a s e d in p a r a l l e l w i t h A P A P d o s a g e i n c r e m e n t as was c o n f i r m e d b y the c a l c u l a t e d v a l u e s o f the filtration fraction (FF). F F in all the A P A P g r o u p s (data n o t shown)are s i m i l a r to c o n t r o l v a l u e s ( 3 1 . 8 % + 3 . 7 4 ) . O n the other hand, t u b u l a r f u n c t i o n s (Fig. 1 B) w e r e n o t m o d i f i e d except the F E % K, w h i c h w a s i n c r e a s e d in A P A P - 1 0 0 0 rats Uosm/posm v a l u e s w e r e s i g n i f i c a n t l y d i m i n i s h e d in A P A P - 1000 rats ( c o n t r o l = 2.93 + 0.20, APAP1000 = 1.40 + 0.04). T h i s d a t a s u g g e s t e d that the pattern in F E % H 2 0 s h o w e d an actual t r e n d to b e a u g m e n t e d in A P A P - 1 0 0 0 rats. N o d i f f e r e n c e s w e r e o b s e r v e d in the a m o u n t o f p r o t e i n s e x c r e t e d (data n o t s h o w n ) . B l o o d press u r e d i d n o t c h a n g e d u r i n g the e x p e r i m e n t s , and there were n o d i f f e r e n c e s b e t w e e n g r o u p s . A s f r a c t i o n a l excretion of g l u c o s e r e m a i n e d u n c h a n g e d w i t h A P A P administration (control =0.25%+0.05, n = 10), the p r o x i m a l tubular t r a n s p o r t c a p a c i t y w a s c h a l l e n g e d b y i n c r e a s i n g glucose s e r u m c o n c e n t r a t i o n . D a t a w e r e a d j u s t e d to a MichaelisM e n t e n k i n e t i c u s i n g a m u l t i p l e n o n - l i n e a r r e g r e s s i o n analysis b y m e a n s o f a c o m p u t e r p r o g r a m ( E n z F i t t e r ) . There w e r e n o s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s a m o n g the para m e t e r s o b t a i n e d in c o n t r o l a n d A P A P - 1 0 0 0 treated rats, e i t h e r at 1 h o r at 16 h a f t e r t r e a t m e n t . T h e "apparenf' Tmax/GFR were 8.57+1.22 m g / m l for c o n t r o l rats, 8.95 + 1.32 m g / m l for A P A P - 1000 t r e a t e d rats 1 h post-in-

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Acetaminophen nephrotoxicity in male Wistar rats.

Acute acetaminophen (APAP) nephrotoxicity was studied in male Wistar rats 1 h after different APAP single doses (200, 500 and 1000 mg/kg body wt, i.p...
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