Introduction

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APAP is a widely used analgesic/antipyretic drug. Even though APAP is considered a safe drug, in overdose situations it produced hepatic necrosis and renal failure in both humans (Boyer and Rouff, 1971; Cobden et al., 1982; Prescott et al., 1971) and experimental animals (McMurtry et al., 1978; Mitchell et al., 1973).

In previous work, we reported the development of APAP-induced acute nephrotoxicity in male Wistar rats. The nephrotoxic dose used produced a diminution in hepatic GSH levels (Trumper et al., 1992), so important formation of GSH conjugates could be assumed. The contribution of the GSH-derived APAP metabolites formed in the liver and the involvement of the renal GT-dependent transport of these conjugates also were reported. However, despite a significant inhibition of renal GT activity by acivicin pretreatment, only a partial protection of APAP renal effects was observed (Trumper et al., 1996). This could be the result of other mechanisms participating in the renal incorporation of the GSH conjugates. In this regard, Lash and Jones (1984) characterized a PROB-sensitive transport system for GSH and GSH conjugates in renal basolateral vesicles. Proximal tubular toxicity of hexachloro-1,3-butadiene to the rat kidney was related to a PROB-sensitive transport process (Lock and Ishmael, 1985). PROB also protected against acute toxicity of hexachloro-1,3-butadiene and methyl mercury to the mouse kidney (Ban and de Céaurriz, 1988).

The aim of the present study was to examine whether acute APAP nephrotoxicity could be related to a PROB-sensitive transport system. The renal effects of APAP in rats pretreated with PROB were studied in in vivo experiments. To assess whether PROB protects against direct renal effects of APAP or modifies its renal clearance, the effects of APAP in presence of PROB were studied with the IPK model.

	Methods

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Animals

Male Wistar rats (3 months; 250-350 g b.wt.) were used. They were housed in rooms with controlled temperature (21-23°C), humidity and regular light cycles (12 hr) and maintained on a standard diet and water ad libitum.

Effects of PROB on APAP-Induced Nephrotoxicity In Vivo

Rats were fasted for 17 hr (5:00 p.m. to 10:00 a.m.) before the experiments. They were always allowed free access to water. They were kept singly in stainless steel metabolic cages (La Técnica CI, Buenos Aires, Argentina) during a 16-hr (6:00 p.m. to 10:00 a.m.) urinary collection period. A 2-day acclimation period to this regimen was allowed before initiation of the experiment. On the third day, several experimental groups were studied: (1) animals that received a single dose of APAP (Sigma Chemical, St. Louis, MO), 1000 mg/kg b.wt. i.p. at 5 ml/kg in propylene glycol (APAP, n = 4), before the collection period; this dose of APAP was described previously as nephrotoxic in male Wistar rats (Trumper et al., 1992); (2) animals injected with PROB (Sigma Chemical), 200 mg/kg b.wt. i.p. at 10 ml/kg in isotonic saline (the solution was made alkaline and then adjusted to pH 7.4), 30 min before the administration of APAP (PROB-APAP, n = 4); (3) animals injected with PROB, 200 mg/kg b.wt. i.p., 30 min before the collection period (PROB, n = 4); and (4) animals that received the corresponding volume of APAP vehicle (control, n = 5). The dose of PROB used is similar to the used by others (Ban and de Céaurriz, 1988; Fowler et al., 1993).

At the end of the 16 hr-collection period, animals were anesthetized with sodium thiopental (70 mg/kg b.wt. i.p.), and blood was collected from inferior vena cava into a syringe containing heparin. Livers were promptly removed to study the GSH content on tissue homogenates as an index of S-conjugate formation. Plasma was separated immediately through centrifugation for the determination of creatinine, urea, glucose and APAP concentrations. Urine volume was recorded before urine centrifugation. Urine sediment was examined by light microscopy. The activity of GT was determined on fresh urine samples. Urinary creatinine, protein, glucose and APAP also were determined. Hepatic GSH content was measured on tissue homogenates.

Based on previous experiments, we found that plasma creatinine levels (control = 3.5 ± 0.2 mg/ml) were elevated after 1 hr of APAP treatment (10.4 ± 0.3 mg/ml) and these levels remained elevated for the following 16 hr (10.6 ± 1.4 mg/ml), GFR was estimated as the creatinine clearance.

Effects of APAP in the Presence of PROB in the IPK

Perfusion procedure and apparatus. Rats were anesthetized with sodium thiopental (70 mg/kg b.wt. i.p.). The right kidney was prepared and perfused as described previously (Elías et al., 1981; Trumper et al., 1995). The perfusion medium (pH 7.4) consisted of Ringer-Krebs solution containing dextran 2% (Sigma Chemical; average molecular weight, 82,200) as a colloid osmotic agent, glucose (10 mM), sodium pyruvate (5 mM), sodium lactate (5 mM), creatinine (400 mg/liter) and PAH (6 mg/liter). The medium also contained 0.5 mM cysteine, 0.5 mM glutamic acid and 2.3 mM glycine to prevent the loss of GSH from specific regions of the kidney (Brezis et al., 1983; Epstein et al., 1982; Torres et al., 1986). The medium was constantly bubbled with 95% O₂/5% CO₂. The whole system operated under thermostatic control at 37°C. The total volume of perfusate used in the recirculating system of each experiment was 100 ml. PF through the isolated kidney in situ was performed with a peristaltic pump (Masterflex; Cole-Parmer Instrument) at a constant rate measured with a flowmeter (Gilmont Instruments, Barrington, IL) inserted in the arterial line. PP was continuously measured at the tip of the arterial cannula by a pressure transducer (P231D; Gould, Glen Burnie, MD) and recorded with a multipen recorder (Rikadenki; Kogyo Co., Tokyo, Japan).

Experimental design. The isolated kidney was perfused for 30 min (equilibration time) until PP and PF remained constant. The subsequent experimental time was 40 min, during which urine was collected over 5-min periods. Perfusate was sampled at the midpoint of each 5-min urine collection interval. At the end of the experiment, the kidney was removed, gently blotted on filter paper and weighed.

Analytical Methods

The volume of urine was estimated gravimetrically. Creatinine was determined by the Jaffé reaction with a commercial kit (Henry et al., 1980a; Wiener Laboratories, Rosario, Argentina), glucose by the glucose oxidase method (Henry et al., 1980b; Wiener Laboratories), and PAH by Brun's method as modified by Waugh and Beall (1974). Urea was measured with a commercial kit (Henry et al., 1980c; Wiener Laboratories). Sodium was measured by flame photometry. Determination of liver nonprotein sulfhydryls (roughly representing GSH) was carried out in whole tissue homogenates prepared in cold 5% trichloroacetic acid in 0.01 M HCl and measured as described by Ellman (1959). Total urinary proteins were measured with the EDTA/Cu reagent (Biuret's method, Henry et al., 1980d; Wiener Laboratories). APAP was determined by a spectrophotometric method (Glynn and Kendal, 1975). Urinary GT activity was determined by use of -glutamyl-p-nitroanilide as substrate with a commercial kit according to the manufacturer's directions (Wiener Laboratories).

Fractional excretion of water (urine volume/min/GFR), sodium (clearance of sodium/GFR) and glucose (clearance of glucose/GFR) were calculated with the use of conventional formulae. APAP clearance relative to GFR also was calculated.

Statistical Analysis

Results are expressed as mean ± S.E.M. In vivo data were analyzed using the one-way analysis of variance followed by Newman-Keuls comparisons. IPK data were analyzed by two-way analysis of variance and Bonferroni's comparisons. The .05 level of probability was used as the criterion of significance in all cases.

	Results

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Effects of PROB on APAP-Induced Nephrotoxicity In Vivo

Tissue measurements. APAP administration induced a significant diminution in hepatic GSH content, whereas dual treatment did not produce any change in the APAP-induced diminution of hepatic GSH levels (control = 3.83 ± 0.1, PROB = 3.48 ± 0.2, APAP= 2.51 ± 0.3,* PROB-APAP = 2.63 ± 0.5* µmol/g wet tissue; *P < .05 compared with control).

Renal function. PROB treatment promoted an increase in UFR. UFR showed a trend to decrease in APAP-treated rats. In rats that received the dual treatment PROB-APAP, UFR was improved. PROB did not alter GFR, plasma creatinine or urea levels compared with control values. APAP treatment significantly decreased GFR, associated with an increase in creatinine and urea plasma levels. Prior treatment with PROB followed by APAP protected against APAP-induced diminution of GFR. Plasma creatinine levels of rats that received the dual treatment PROB-APAP were significantly lower than those of animals treated with APAP alone and were not different from control values. Urea plasma levels, although different from control values, were significantly lower than those observed in APAP-treated rats. All data are shown in figure 1.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effects of APAP and the dual treatment PROB-APAP on renal function. Rats were treated with PROB 200 mg/kg b.wt. i.p. or saline 30 min before the administration of APAP 1000 mg/kg b.wt. i.p. (PROB-APAP or APAP, respectively) or vehicle (PROB or CONTROL, respectively). Urine was collected during the 16 hr after the last dosing. After the urinary collection period, rats were anesthetized and then exsanguinated. Each result is the mean ± S.E.M. of four or five experiments. **P < .01 compared with control. P < .01 compared with APAP-treated rats.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of APAP and the dual treatment PROB-APAP on urine parameters measured 16 hr after dosing. Rats were treated with PROB 200 mg/kg b.wt. i.p. or saline 30 min before the administration of APAP 1000 mg/kg b.wt. i.p. (PROB-APAP or APAP, respectively) or vehicle (PROB or CONTROL, respectively). Urine was collected during the 16 hr after the last dosing. Each result is the mean ± S.E.M. of four or five experiments. **P < .01 compared with control. P < .01 compared with APAP-treated rats. ND, not detected.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Plasma APAP levels and urinary APAP excretion in rats treated with PROB 200 mg/kg b.wt. i.p. (PROB-APAP) or vehicle (APAP) 30 min before the administration of APAP 1000 mg/kg b.wt. i.p. Urine was collected during the 16 hr after the last dosing. After the urinary collection period, rats were anesthetized and then exsanguinated. Each result is the mean ± S.E.M. of four or five experiments. **P < .01 compared with APAP.

Effects of APAP in the Presence of PROB in the IPK

Perfusion with PROB decreased the C_PAH in the IPK (table 1), showing an important level of inhibition of the organic anion transport system. Other functional parameters evaluated in control experiments did not differ from PROB perfusions. Functional parameters remained unchanged during the whole perfusion time. Renal function data during control periods in all the experimental groups did not differ from data observed in control experiments presented in table 1.

Figure 4 shows that in the IPK, APAP (10 mM) promotes an increase in FE_H2O, FE_Na, and FE_glu and a decrease in GFR. Changes promoted by APAP occurs mainly during the first 20 min after APAP addition; thereafter, functional parameters remained stable. Perfusion with PROB before APAP did not change the increase in FE_H2O, FE_Na, and FE_glu and the decrease in GFR induced by APAP.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effects of APAP (10 mM) alone or in presence of PROB (0.1 mM) in the IPK. The time course of the fractional excretion of water (A), sodium (B) and glucose (C) and GFR (D) are shown. At time = 0 min, APAP was added as a single dose to the perfusate. In PROB-APAP, kidneys were perfused with PROB from the moment of isolation. Values are expressed as percentage change relative to control period. Each point represents mean ± S.E.M. *P < .05 compared with previous time. P < .05 compared with time = 5 min.

No differences were observed in APAP urinary excretion among the groups (APAP = 0.99 ± 0.1 µmol/min/g; PROB-APAP= 0.84 ± 0.2 µmol/min/g). APAP renal clearance relative to GFR (C_APAP/GFR) was not modified by PROB (APAP = 0.55 ± 0.07; PROB-APAP = 0.59 ± 0.07). Under both experimental conditions, the fractional clearance is lower than unity, so tubular reabsorption probably is the predominant pathway for the renal transport of APAP.

	Discussion

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Based in our previous work, we can postulate that APAP-induced nephrotoxicity may involve the contribution of at least two components: direct effects of APAP, which were confirmed with the IPK model (Trumper et al., 1995), and the effects of GSH-derived APAP metabolites (Trumper et al., 1996). In this regard, S-conjugates in systemic circulation may be accumulated in the kidney via several carrier-mediated mechanisms, including the organic anion transporter and degradation by membrane bound GT (Commandeur et al., 1995; Koob and Dekant, 1991). In our previous work, we showed that an almost complete inhibition of renal GT activity only partially protected against APAP renal effects.

In the present work, PROB was used as another possible tool to modify active tubular transport of APAP S-conjugates. PROB has shown to decrease the renal uptake of S-(1,2-dichlorovinyl) GSH (Lash and Jones, 1985) and block the nephrotoxicity of S-(2-chloroetyl) GSH (Kramer et al., 1987).

This study presents evidence that PROB pretreatment affords protection of APAP renal effects in vivo. PROB treatment before APAP intoxication protected against APAP-induced decrease of GFR and elevations in creatinine and urea plasma levels. PROB pretreatment also protected against the elevations of the urinary excretion of protein, glucose and GT induced by APAP. Fewer epithelial cells and granular casts were observed in rats that received PROB pretreatment compared with the abundant cells and casts observed in rats treated with APAP alone. It is noticeable that PROB alone promotes an enhanced diuresis. The trend to decrease UFR by APAP treatment was blunted when rats were pretreated with PROB. Moreover, APAP plasma levels 16 hr after APAP administration were significantly higher in rats treated with APAP alone compared with the level attained in rats pretreated with PROB. APAP urinary excretion during a 16 hr urinary collection period was higher in rats pretreated with PROB. It is noteworthy that the colorimetric method for APAP determination is specific for unchanged drug (Glynn and Kendal, 1975).

APAP administration induced a significant diminution in hepatic GSH content, while dual treatment PROB-APAP did not produce any change in the APAP-induced diminution of hepatic GSH levels, so we could assume the same level of hepatic GSH derived conjugate formation in both experimental conditions.

Taken together, these results may suggest that the protection afforded by PROB may be a consequence of an inhibition of the uptake of APAP-S-conjugates and/or an increase of APAP renal clearance.

The effects of APAP in presence of PROB were studied with the IPK model to assess whether PROB protects against direct renal effects of APAP or modifies its renal clearance. The IPK provides a suitable experimental model in which no hepatically derived metabolites are present. Our clearance studies in the IPK suggest that APAP is reabsorbed from the tubular lumen. In this regard, it is known that APAP excretion involves filtration and reabsorption by passive diffusion of the nonionic form (Duggin, 1980; Duggin and Mudge, 1978).

Perfusion with PROB decreased the clearance of PAH, showing an important level of inhibition of the organic anion transport system, as PROB and PAH share a common mechanism in isolated kidney cells (Lash and Anders, 1989).

Perfusion with PROB before APAP did not change the increases in FE_H2O, FE_Na, and FE_Glu or the decrease in GFR induced by APAP. These results show that in this model, PROB does not modify APAP renal effects, so it could be suggested that the delivery of APAP to the renal cells is not modified. This suggestion is reinforced by the fact that APAP urinary excretion and APAP renal clearance relative to GFR were not modified by PROB. This suggests that a PROB-sensitive system does not participate in the accumulation of APAP in the renal cell. However, UFRs attained in the IPK model may be too high to test the hypothesis of a PROB-sensitive reabsorption system.

In summary, protection afforded by PROB in vivo could be the result of the inhibition of a PROB-sensitive transport system for GSH-derived APAP conjugates. PROB did not modify APAP renal uptake as shown in the IPK experiments. PROB in vivo promoted an enhancement of APAP urinary excretion. Walker and Duggin (1988) stated that diuresis results in an increased clearance of APAP. In our study, the improvement in urine flow occurred together with an enhancement in APAP urinary excretion. The enhanced APAP renal excretion also could account for the protection observed. The participation of a PROB-inhibitable reabsorption system should not be disregarded.