Probenecid Protects against In Vivo Acetaminophen-InducedNephrotoxicity in Male Wistar Rats1

LAURA TRUMPER, LILIANA A. MONASTEROLO and M. MONICA ElIAS

Farmacologıa, Facultad de Ciencias Bioquımicas y Farmaceuticas, Universidad Nacional de Rosario, Republica Argentina

Accepted for publication October 27, 1997 This paper is available online at http://www.jpet.org

ABSTRACTRenal effects of acetaminophen (APAP) were studied in ratspretreated with probenecid to analyze whether acute APAP-induced nephrotoxicity could be related to a probenecid-sen-sitive transport system for APAP or its S-derived conjugates.The administration of probenecid (200 mg/kg b.wt. i.p.) 30 minbefore APAP administration (1000 mg/kg b.wt. i.p.) improvedurine flow rate and protected against the alterations on glomer-ular filtration rate and urea and creatinine plasma levels inducedby APAP. Fewer epithelial cells and granular casts and a de-crease in the urinary excretion of protein and glucose wereobserved in rats pretreated with probenecid. Probenecid pre-treatment promoted an elevation in the urinary 16-hr excretionof APAP and a diminution in the plasma levels attained by

APAP. These results suggest that protection afforded by pro-benecid in vivo could be a consequence of the inhibition ofAPAP S-conjugate renal uptake and/or an increase in APAPrenal clearance. The effects of APAP in presence of probenecidwere studied with the isolated perfused kidney model. Perfu-sion with probenecid (0.1 mM) before APAP (10 mM) did notchange APAP direct renal effects, APAP urinary excretion, orAPAP renal clearance relative to glomerular filtration rate. Ourresults suggest that protection afforded by probenecid in vivocould be the result of the inhibition of the uptake of nephrotoxicAPAP metabolites and/or a diuresis-induced enhanced APAPrenal excretion.

APAP is a widely used analgesic/antipyretic drug. Eventhough APAP is considered a safe drug, in overdose situa-tions it produced hepatic necrosis and renal failure in bothhumans (Boyer and Rouff, 1971; Cobden et al., 1982; Prescottet 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 neph-rotoxic dose used produced a diminution in hepatic GSHlevels (Trumper et al., 1992), so important formation of GSHconjugates could be assumed. The contribution of the GSH-derived APAP metabolites formed in the liver and the in-volvement of the renal gGT-dependent transport of theseconjugates also were reported. However, despite a significantinhibition of renal gGT activity by acivicin pretreatment,only a partial protection of APAP renal effects was observed(Trumper et al., 1996). This could be the result of othermechanisms participating in the renal incorporation of theGSH conjugates. In this regard, Lash and Jones (1984) char-

acterized a PROB-sensitive transport system for GSH andGSH conjugates in renal basolateral vesicles. Proximal tubu-lar toxicity of hexachloro-1,3-butadiene to the rat kidney wasrelated to a PROB-sensitive transport process (Lock andIshmael, 1985). PROB also protected against acute toxicity ofhexachloro-1,3-butadiene and methyl mercury to the mousekidney (Ban and de Ceaurriz, 1988).

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

MethodsAnimals

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

Received for publication May 27, 1997.1 This work was supported by grants from Consejo Nacional de Investiga-

ciones Cientıficas y Tecnicas (CONICET) and Consejo de Investigaciones de laUniversidad Nacional de Rosario (CIUNR) (L.T.).

ABBREVIATIONS: APAP, acetaminophen; IPK, isolated perfused kidney; GSH, glutathione; GFR, glomerular filtration rate; gGT, g-glutamyltranspeptidase; PAH, p-aminohippuric acid; CPAH, clearance of p-aminohippuric acid; CAPAP, clearance of acetaminophen; FENa, fractionalexcretion of sodium; FEH2O, fractional excretion of water; FEGlu, fractional excretion of glucose; UFR, urine flow rate; PROB, probenecid; PF,perfusion flow; PP, perfusion pressure.

0022-3565/98/2842-0606$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 2Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.JPET 284:606–610, 1998

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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 theexperiments. They were always allowed free access to water. Theywere kept singly in stainless steel metabolic cages (La Tecnica 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 regimenwas allowed before initiation of the experiment. On the third day,several experimental groups were studied: (1) animals that receiveda single dose of APAP (Sigma Chemical, St. Louis, MO), 1000 mg/kgb.wt. i.p. at 5 ml/kg in propylene glycol (APAP, n 5 4), before thecollection period; this dose of APAP was described previously asnephrotoxic in male Wistar rats (Trumper et al., 1992); (2) animalsinjected with PROB (Sigma Chemical), 200 mg/kg b.wt. i.p. at 10ml/kg in isotonic saline (the solution was made alkaline and thenadjusted to pH 7.4), 30 min before the administration of APAP(PROB-APAP, n 5 4); (3) animals injected with PROB, 200 mg/kgb.wt. i.p., 30 min before the collection period (PROB, n 5 4); and (4)animals that received the corresponding volume of APAP vehicle(control, n 5 5). The dose of PROB used is similar to the used byothers (Ban and de Ceaurriz, 1988; Fowler et al., 1993).

At the end of the 16 hr-collection period, animals were anesthe-tized with sodium thiopental (70 mg/kg b.wt. i.p.), and blood wascollected from inferior vena cava into a syringe containing heparin.Livers were promptly removed to study the GSH content on tissuehomogenates as an index of S-conjugate formation. Plasma wasseparated immediately through centrifugation for the determinationof creatinine, urea, glucose and APAP concentrations. Urine volumewas recorded before urine centrifugation. Urine sediment was exam-ined by light microscopy. The activity of gGT was determined onfresh urine samples. Urinary creatinine, protein, glucose and APAPalso were determined. Hepatic GSH content was measured on tissuehomogenates.

Based on previous experiments, we found that plasma creatininelevels (control 5 3.5 6 0.2 mg/ml) were elevated after 1 hr of APAPtreatment (10.4 6 0.3 mg/ml) and these levels remained elevated forthe following 16 hr (10.6 6 1.4 mg/ml), GFR was estimated as thecreatinine clearance.

Effects of APAP in the Presence of PROB in the IPK

Perfusion procedure and apparatus. Rats were anesthetizedwith sodium thiopental (70 mg/kg b.wt. i.p.). The right kidney wasprepared and perfused as described previously (Elıas et al., 1981;Trumper et al., 1995). The perfusion medium (pH 7.4) consisted ofRinger-Krebs solution containing dextran 2% (Sigma Chemical; av-erage 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 0.5mM cysteine, 0.5 mM glutamic acid and 2.3 mM glycine to preventthe loss of GSH from specific regions of the kidney (Brezis et al.,1983; Epstein et al., 1982; Torres et al., 1986). The medium wasconstantly bubbled with 95% O2/5% CO2. The whole system operatedunder thermostatic control at 37°C. The total volume of perfusateused in the recirculating system of each experiment was 100 ml. PFthrough the isolated kidney in situ was performed with a peristalticpump (Masterflex; Cole-Parmer Instrument) at a constant rate mea-sured with a flowmeter (Gilmont Instruments, Barrington, IL) in-serted in the arterial line. PP was continuously measured at the tipof the arterial cannula by a pressure transducer (P231D; Gould, GlenBurnie, MD) and recorded with a multipen recorder (Rikadenki;Kogyo Co., Tokyo, Japan).

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

Four experimental groups were studied. (1) Kidneys were per-

fused with Ringer-Krebs solution to assess the viability of the prep-arations during the experimental time (control, n 5 7). (2) Kidneyswere perfused with APAP (perfusate concentration 5 10 mM)(APAP, n 5 7). APAP was dissolved in a small volume of perfusateand added to the recirculating medium (total volume, 100 ml) at theend of the second period after the equilibration time. The concentra-tion of APAP examined is in the range of the concentration obtainedin vivo 1 hr after the administration of APAP 1000 mg/kg (Trumperet al., 1995). (3) Kidneys were perfused with 0.1 mM PROB from themoment of isolation (PROB, n 5 4). The concentration of PROB inthe perfusate was used by others (Newton et al., 1986) to inhibit thesecretion of APAP mercapturate in the IPK. (4) Kidneys were per-fused with APAP (10 mM) added as stated in (2) to the perfusionmedium containing 0.1 mM PROB (PROB-APAP, n 5 4).

Functional data from the two first experimental periods after theequilibration time were averaged and considered to be control valuesfor each preparation. All the subsequent results were expressed aspercentage change relative to control values.

The following parameters were evaluated as a criteria of overallkidney function during the course of the perfusion: PF, PP, UFR,GFR (estimated on the basis of the clearance of creatinine), Cpah,FEGlu, FENa, and FEH2O.

Analytical Methods

The volume of urine was estimated gravimetrically. Creatininewas determined by the Jaffe reaction with a commercial kit (Henryet al., 1980a; Wiener Laboratories, Rosario, Argentina), glucose bythe glucose oxidase method (Henry et al., 1980b; Wiener Laborato-ries), 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 pho-tometry. Determination of liver nonprotein sulfhydryls (roughly rep-resenting GSH) was carried out in whole tissue homogenates pre-pared in cold 5% trichloroacetic acid in 0.01 M HCl and measured asdescribed by Ellman (1959). Total urinary proteins were measuredwith the EDTA/Cu reagent (Biuret’s method, Henry et al., 1980d;Wiener Laboratories). APAP was determined by a spectrophotomet-ric method (Glynn and Kendal, 1975). Urinary gGT activity wasdetermined by use of g-glutamyl-p-nitroanilide as substrate with acommercial kit according to the manufacturer’s directions (WienerLaboratories).

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 clear-ance relative to GFR also was calculated.

Statistical Analysis

Results are expressed as mean 6 S.E.M. In vivo data were ana-lyzed using the one-way analysis of variance followed by Newman-Keuls comparisons. IPK data were analyzed by two-way analysis ofvariance and Bonferroni’s comparisons. The .05 level of probabilitywas used as the criterion of significance in all cases.

ResultsEffects of PROB on APAP-Induced Nephrotoxicity In Vivo

Tissue measurements. APAP administration induced asignificant diminution in hepatic GSH content, whereas dualtreatment did not produce any change in the APAP-induceddiminution of hepatic GSH levels (control 5 3.83 6 0.1,PROB 5 3.48 6 0.2, APAP5 2.51 6 0.3,* PROB-APAP 52.63 6 0.5* mmol/g wet tissue; *P , .05 compared withcontrol).

Renal function. PROB treatment promoted an increasein UFR. UFR showed a trend to decrease in APAP-treatedrats. In rats that received the dual treatment PROB-APAP,

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UFR was improved. PROB did not alter GFR, plasma creat-inine or urea levels compared with control values. APAPtreatment significantly decreased GFR, associated with anincrease in creatinine and urea plasma levels. Prior treat-ment with PROB followed by APAP protected against APAP-induced diminution of GFR. Plasma creatinine levels of ratsthat received the dual treatment PROB-APAP were signifi-cantly lower than those of animals treated with APAP aloneand were not different from control values. Urea plasmalevels, although different from control values, were signifi-cantly lower than those observed in APAP-treated rats. Alldata are shown in figure 1.

APAP promoted a marked proteinuria and glucosuria anda significant increase in the urinary excretion of gGT (fig. 2).PROB treatment had no effect on protein and glucose excre-tion and urinary gGT activity. Total urinary protein, glucoseand gGT excretion of rats that received the dual treatment ofPROB and APAP were significantly decreased comparedwith animals treated with APAP alone and did not differfrom control levels.

Urinary sediments of APAP-treated animals containedmany granular casts and epithelial cells. In the urinary sed-iment of PROB-pretreated rats, very few casts or cells wereobserved. These data combined could provide evidence thatPROB protects against APAP-induced nephrotoxicity.

Plasma APAP levels 16 hr after APAP administration weresignificantly higher in rats treated with APAP alone than inrats pretreated with PROB. APAP urinary excretion duringthe 16-hr urinary collection period was higher in rats pre-treated with PROB (fig. 3). These results suggest that PROB

increases APAP renal clearance, and this could explain itsprotective effects.

Effects of APAP in the Presence of PROB in the IPK

Perfusion with PROB decreased the CPAH in the IPK (table1), showing an important level of inhibition of the organicanion transport system. Other functional parameters evalu-

Fig. 1. Effects of APAP and the dual treatment PROB-APAP on renalfunction. Rats were treated with PROB 200 mg/kg b.wt. i.p. or saline 30min before the administration of APAP 1000 mg/kg b.wt. i.p. (PROB-APAP or APAP, respectively) or vehicle (PROB or CONTROL, respective-ly). Urine was collected during the 16 hr after the last dosing. After theurinary collection period, rats were anesthetized and then exsangui-nated. Each result is the mean 6 S.E.M. of four or five experiments.**P , .01 compared with control. ƒƒ P , .01 compared with APAP-treated rats.

Fig. 2. Effects of APAP and the dual treatment PROB-APAP on urineparameters measured 16 hr after dosing. Rats were treated with PROB200 mg/kg b.wt. i.p. or saline 30 min before the administration of APAP1000 mg/kg b.wt. i.p. (PROB-APAP or APAP, respectively) or vehicle(PROB or CONTROL, respectively). Urine was collected during the 16 hrafter the last dosing. Each result is the mean 6 S.E.M. of four or fiveexperiments. **P , .01 compared with control. ƒƒ P , .01 compared withAPAP-treated rats. ND, not detected.

Fig. 3. Plasma APAP levels and urinary APAP excretion in rats treatedwith PROB 200 mg/kg b.wt. i.p. (PROB-APAP) or vehicle (APAP) 30 minbefore the administration of APAP 1000 mg/kg b.wt. i.p. Urine was collectedduring the 16 hr after the last dosing. After the urinary collection period,rats were anesthetized and then exsanguinated. Each result is the mean 6S.E.M. of four or five experiments. **P , .01 compared with APAP.

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ated in control experiments did not differ from PROB perfu-sions. Functional parameters remained unchanged duringthe whole perfusion time. Renal function data during controlperiods in all the experimental groups did not differ fromdata observed in control experiments presented in table 1.

Figure 4 shows that in the IPK, APAP (10 mM) promotesan increase in FEH2O, FENa, and FEglu and a decrease inGFR. Changes promoted by APAP occurs mainly during thefirst 20 min after APAP addition; thereafter, functional pa-rameters remained stable. Perfusion with PROB beforeAPAP did not change the increase in FEH2O, FENa, and FEglu

and the decrease in GFR induced by APAP.No differences were observed in APAP urinary excretion

among the groups (APAP 5 0.99 6 0.1 mmol/min/g; PROB-APAP5 0.84 6 0.2 mmol/min/g). APAP renal clearance rela-tive to GFR (CAPAP/GFR) was not modified by PROB

(APAP 5 0.55 6 0.07; PROB-APAP 5 0.59 6 0.07). Underboth experimental conditions, the fractional clearance islower than unity, so tubular reabsorption probably is thepredominant pathway for the renal transport of APAP.

DiscussionBased in our previous work, we can postulate that APAP-

induced nephrotoxicity may involve the contribution of atleast two components: direct effects of APAP, which wereconfirmed with the IPK model (Trumper et al., 1995), and theeffects of GSH-derived APAP metabolites (Trumper et al.,1996). In this regard, S-conjugates in systemic circulationmay be accumulated in the kidney via several carrier-medi-ated mechanisms, including the organic anion transporterand degradation by membrane bound gGT (Commandeur etal., 1995; Koob and Dekant, 1991). In our previous work, weshowed that an almost complete inhibition of renal gGTactivity only partially protected against APAP renal effects.

In the present work, PROB was used as another possibletool 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 thenephrotoxicity of S-(2-chloroetyl) GSH (Kramer et al., 1987).

This study presents evidence that PROB pretreatment af-fords protection of APAP renal effects in vivo. PROB treat-ment before APAP intoxication protected against APAP-in-duced decrease of GFR and elevations in creatinine and ureaplasma levels. PROB pretreatment also protected against theelevations of the urinary excretion of protein, glucose andgGT induced by APAP. Fewer epithelial cells and granularcasts were observed in rats that received PROB pretreatmentcompared with the abundant cells and casts observed in ratstreated with APAP alone. It is noticeable that PROB alonepromotes an enhanced diuresis. The trend to decrease UFRby APAP treatment was blunted when rats were pretreatedwith PROB. Moreover, APAP plasma levels 16 hr after APAPadministration were significantly higher in rats treated withAPAP alone compared with the level attained in rats pre-treated with PROB. APAP urinary excretion during a 16 hrurinary collection period was higher in rats pretreated withPROB. It is noteworthy that the colorimetric method forAPAP determination is specific for unchanged drug (Glynnand Kendal, 1975).

APAP administration induced a significant diminution inhepatic GSH content, while dual treatment PROB-APAP didnot produce any change in the APAP-induced diminution ofhepatic GSH levels, so we could assume the same level ofhepatic GSH derived conjugate formation in both experimen-tal conditions.

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

The effects of APAP in presence of PROB were studied withthe IPK model to assess whether PROB protects againstdirect renal effects of APAP or modifies its renal clearance.The IPK provides a suitable experimental model in which nohepatically derived metabolites are present. Our clearancestudies in the IPK suggest that APAP is reabsorbed from thetubular lumen. In this regard, it is known that APAP excre-tion involves filtration and reabsorption by passive diffusion

TABLE 1Functional parameters of control periods of IPK

Control(n 5 7)

PROB(n 5 4)

CPAH (ml/min/g) 2.5 6 0.3 0.9 6 0.4**UFR (ml/min/g) 0.11 6 0.02 0.07 6 0.02GFR (ml/min/g) 0.30 6 0.03 0.23 6 0.06FEH2O (%) 22.0 6 5 22.3 6 5FENa (%) 18.0 6 4.6 22.2 6 5.0FEGlu (%) 11.3 6 2.6 6 6 2PP (mm Hg) 124 6 3.4 129 6 4.8PF (ml/min) 22 6 0.3 21 6 0.37

Kidneys were perfused with Ringer-Krebs alone (Control) or with the addition ofprobenecid 0.1 mM from the moment of isolation (PROB). Values are expressed asmean 6 S.E.M. of control periods.

** P , .01 compared with control preparations.

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 5 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. Valuesare expressed as percentage change relative to control period. Each pointrepresents mean 6 S.E.M. *P , .05 compared with previous time. ƒP ,.05 compared with time 5 5 min.

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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 aniontransport system, as PROB and PAH share a common mech-anism in isolated kidney cells (Lash and Anders, 1989).

Perfusion with PROB before APAP did not change theincreases in FEH2O, FENa, and FEGlu or the decrease in GFRinduced by APAP. These results show that in this model,PROB does not modify APAP renal effects, so it could besuggested that the delivery of APAP to the renal cells is notmodified. This suggestion is reinforced by the fact that APAPurinary excretion and APAP renal clearance relative to GFRwere not modified by PROB. This suggests that a PROB-sensitive system does not participate in the accumulation ofAPAP in the renal cell. However, UFRs attained in the IPKmodel may be too high to test the hypothesis of a PROB-sensitive reabsorption system.

In summary, protection afforded by PROB in vivo could bethe result of the inhibition of a PROB-sensitive transportsystem for GSH-derived APAP conjugates. PROB did notmodify APAP renal uptake as shown in the IPK experiments.PROB in vivo promoted an enhancement of APAP urinaryexcretion. Walker and Duggin (1988) stated that diuresisresults in an increased clearance of APAP. In our study, theimprovement in urine flow occurred together with an en-hancement in APAP urinary excretion. The enhanced APAPrenal excretion also could account for the protection ob-served. The participation of a PROB-inhibitable reabsorptionsystem should not be disregarded.

Acknowledgments

The authors wish to thank Wiener Laboratories (Rosario, Argen-tina) for the gift of analytical reagents.

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