Renal Glutathione Depletion Research Articles

Farnesoid X receptor agonist obeticholic acid inhibits renal inflammation and oxidative stress during lipopolysaccharide-induced acute kidney injury

It is increasingly recognized that farnesoid X receptor (FXR) has anti-inflammatory and antioxidant activities. The present study investigated the effects of obeticholic acid (OCA), a novel synthetic FXR agonist, on renal inflammation and oxidative stress in a model of sepsis-induced acute kidney injury. All mice except controls were intraperitoneally injected with lipopolysaccharide (LPS, 2.0 mg/kg). In the OCA + LPS group, mice were orally pretreated with three doses of OCA (5 mg/kg) at 48, 24 and 1 h before LPS injection. Interestingly, OCA pretreatment alleviated LPS-induced renal dysfunction and pathological damage. Moreover, OCA pretreatment repressed renal inflammatory cytokines and chemokines during LPS-induced acute kidney injury. In addition, OCA blocked nuclear translocation of nuclear factor kappa B (NF-κB) p65 and p50 subunits in tubular epithelial cells of renal cortex. Additional experiment showed that OCA pretreatment attenuated LPS-induced renal glutathione depletion, lipid peroxidation and protein nitration. Moreover, OCA pretreatment inhibited the upregulation of renal NADPH oxidase and inos genes during LPS-induced acute kidney injury. In conclusion, OCA pretreatment protects against sepsis-induced acute kidney injury through inhibiting renal inflammation and oxidative stress. These results provide evidence for roles of FXR as an important regulator of inflammation and oxidative stress in the kidney.

Crepidiastrum denticulatum Extract Ameliorates Kidney Ischemia-Reperfusion Injury in Mice

BackgroundCrepidiastrum denticulatum (CD) is a well-known, traditionally consumed vegetable in Korea, which was recently reported to contain bioactive compounds with detoxification and antioxidant properties. Ischemia-reperfusion injury (IRI) is a major problem after renal transplantation. Furthermore, inflammatory responses to IRI exacerbate the resultant renal injury. In the present study, we investigated whether CD extract exhibits renoprotective effects against IR-induced acute kidney injury in mice. Materials and methodsRenal IRI was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 minutes followed by reperfusion for 48 hours. CD extract (75 mg/kg) was administered orally 5 days before IRI. ResultsTreatment with CD extract significantly decreased blood urea nitrogen and serum creatinine levels as well as kidney tubular injury. CD also prevented IRI-induced renal glutathione depletion and increased malondialdehyde levels. Western blotting and reverse transcriptase polymerase chain reaction indicated that CD extract significantly attenuates inducible nitric oxide synthase and toll-like receptor 2/4 protein levels 48 h after IRI. The expression of tumor necrosis factor-α and interleukin-1β was significantly decreased in the CD extract treatment group. ConclusionCD extract improved acute renal IRI through its antioxidant and anti-inflammatory effects. These findings suggest that CD extract is a potential therapeutic agent for acute ischemia-induced renal damage.

The oxidative stress induced in vivo by Shiga toxin-2 contributes to the pathogenicity of haemolytic uraemic syndrome

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)-producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in-vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti-oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti-oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro-oxidative state that contributes to kidney failure, and exogenous anti-oxidants could be beneficial to counteract this pathogenic pathway.

Pluchea lanceolata protects against Benzo(a) pyrene induced renal toxicity and loss of DNA integrity.

Evidence from epidemiological, experimental and clinical trial data indicates that a plant based diet can reduce the risk of chronic diseases and reduces toxic effects. In the present study, we report the antioxidant and anticlastogenic activity of Pluchea lanceolata (PL), an important medicinal plant, in both in vitro and in vivo model. Benzo(a)pyrene (B(a)P) administration leads to depletion of renal glutathione and its metabolizing enzymes. Pretreatment with PL (100 and 200 mg /kg b.wt) restored renal glutathione content and its dependent enzymes significantly (p<0.001) with simultaneous increase in catalase(CAT), quinone reductase(QR) in mouse kidney. Prophylactic administration of PL prior to B (a) P administration significantly decreased the malondialdehyde(MDA), H2O2 and xanthineoxidase (XO) levels at a significance of p<0.001, at both the doses. PL extract pretreated groups showed marked inhibition in B(a)P induced micronuclei formation in mouse bone marrow cells with simultaneous restoration of DNA integrity, viz. alkaline unwinding assay and DNA damage shown by gel-electrophoresis. HPTLC confirms the presence of quercetin in plant extract which could be responsible for PL protecting efficacy. In conclusion, the present findings strongly support the antioxidant efficacy of PL, possibly by modulation of antioxidant armory.

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet's effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet's effects on genomic stability and DNA methylation.

Pluchea lanceolata attenuates cadmium chloride induced oxidative stress and genotoxicity in Swiss albino mice

Cadmium intoxication induces lipid peroxidation and causes oxidative damage to various tissues by altering antioxidant defence system enzymes. At 24 h after treatment with a single intraperitoneal dose of cadmium chloride (5 mg kg-1), Swiss albino mice showed a significant increase in the levels of malanodialdehyde and xanthine oxidase (P<0.001), and a concomitant depletion of renal glutathione, catalase (P<0.001) and other antioxidant enzymes. CdCl2 also led to a simultaneous increase in micronuclei formation (P<0.001) and chromosomal aberrations (P<0.05) in mouse bone marrow cells. Oral pre-treatment with Pluchea lanceolata extract at doses of 100 and 200 mg kg-1 for 7 consecutive days before CdCl2 intoxication caused a significant reduction in malanodialdehyde formation and xanthine oxidase activity (P<0.001). A significant restoration of the activity of antioxidant defence system enzymes such as catalase, glutathione peroxidase (P<0.05), glutathione-S-transferase and glutathione reductase (P<0.001) was observed. A significant dose-dependent decrease in chromosomal aberrations and micronuclei formation was also observed (P<0.05). The results indicate that pre-treatment with P. lanceolata attenuates cadmium chloride induced oxidative stress and genotoxicity by altering antioxidant enzymes and reducing chromatid breaks and micronuclei formation.

Propylthiouracil attenuates acetaminophen‐induced renal damage in the rat

To date, there is no specific antidote to acetaminophen poisoning. Propylthiouracil (PTU) has been shown to be protective against acetaminophen (APAP)-induced liver damage in rats; however, the nephroprotective effect of propylthiouracil has not been studied yet. In order to verify this, rats were given different doses of PTU (100, 200 or 400 mg/kg per body weight, orally) 1 h before a nephrotoxic dose of APAP (1,000 mg/kg per body weight, intraperitoneally (i.p.)). Propylthiouracil pretreatment significantly reduced APAP-induced nephrotoxicity in a dose-dependant manner, as evidenced by reduction in plasma creatinine and by amelioration of renal pathology (interstitial congestion, tubular cell degeneration and necrosis). The mechanism of protection by PTU is probably not due to the sparing effect of non-protein thiol (approximately 95% of which is reduced glutathione), as similar depletion of renal glutathione was observed regardless of PTU pretreatment; other mechanisms are suggested.

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity II. Possible involvement of the γ-glutamyl cycle

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl- p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid γ-glutamyl acceptor substrates for γ-glutamyl transpeptidase (γ-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the γ-glutamyl cycle through interaction with γ-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the γ-glutamyl transpeptidase (γ-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a γ-glutamyl acceptor for both murine and bovine renal γ-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a γ-glutamyl-cysteinyl-acetaminophen metabolite. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the γ-glutamyl cycle.

Glyceryl trinitrate, a nitric oxide donor, abrogates ferric nitrilotriacetate-induced oxidative stress and renal damage

Ferric nitrilotriacetate (Fe-NTA), a common water pollutant and a known renal carcinogen, acts through the generation of oxidative stress and hyperproliferative response. In the present study, we show that the nitric oxide (NO) generated by the administration of glyceryl trinitrate (GTN) affords protection against Fe-NTA-induced oxidative stress and proliferative response. Administration of Fe-NTA resulted in a significant ( P<0.001) depletion of renal glutathione (GSH) content with concomitant increase in lipid peroxidation and elevated tissue damage marker release in serum. Parallel to these changes, Fe-NTA also caused down-regulation of GSH metabolizing enzymes including glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione- S-transferase and several fold induction in ornithine decarboxylase (ODC) activity and rate of DNA synthesis. Subsequent exogenous administration of GTN at doses of 3 and 6 mg/kg body weight resulted in significant ( P<0.001) recovery of GSH metabolizing enzymes and amelioration of tissue GSH content, in a dose-dependent manner. GTN administration also inhibited malondialdehyde (MDA) formation, induction of ODC activity, enhanced rate of DNA synthesis, and pathological deterioration in a dose-dependent fashion. Further, administration of NO inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME), exacerbated Fe-NTA-induced oxidative tissue injury, hyperproliferative response, and pathological damage. Overall, the study suggests that NO administration subsequent to Fe-NTA affords protection against ROS-mediated damage induced by Fe-NTA.

Increase in renal glutathione in cholestatic liver disease is due to a direct effect of bile acids.

Hepatic synthesis and plasma levels of glutathione are markedly decreased in chronic liver disease. Because glutathione turnover is highest in kidneys, we examined whether changes in kidney glutathione occur in chronic cholestasis and whether they are related to kidney dysfunction in liver disease. Kidney and plasma GSH and GSSG were measured 1) in bile duct-ligated (BDL) rats; 2) in healthy rats after bile acid loading to mimic cholestasis; and 3) after irreversible inhibition of glutathione synthetase with buthionine-sulfoximine (BSO), where glutathione consumption, urinary volume, and sodium excretion were also estimated. In addition, gamma-glutamylcysteine synthetase (gamma-GCS) mRNA, protein, and enzymatic specific activity were measured in kidney tissue after BDL. After BDL, kidney GSH and GSSG increased within hours by 67 and 66%, respectively. The increases were not related to plasma glutathione, which decreased below control values. Intravenous bile acid loading caused identical increases in GSH and GSSG as occurred after BDL, when glycine- or taurine-conjugated dihydroxy bile acids were administered. Glutathione consumption, as estimated after blocking of de novo synthesis with BSO, was significantly increased after BDL (127 vs. 44 nmol x g-1 x min-1). gamma-GCS mRNA and enzymatic specific activity were significantly reduced 5 days after BDL, whereas protein concentrations did not change. The urinary sodium concentration was 70% lower in BDL than in control rats. Depletion of renal glutathione normalized sodium excretion by increasing urinary sodium concentration and urinary volume. The increase in kidney glutathione after BDL seems to be mediated by an increase in plasma bile acids and is critically related to sodium retention. The increase in GSH consumption despite reduced gamma-GCS activity indicates a decreased GSH turnover tentatively due to reduced renal GSH efflux by competition with organic anions at membrane transport proteins.

Antioxidant action of bixin against cisplatin-induced chromosome aberrations and lipid peroxidation in rats

Cisplatin is one of the most active cytotoxic agents in the treatment of cancer, but has serious side effects, inducing nephrotoxicity and chromosome aberrations. In this study we evaluated the role of the carotenoid bixin on cisplatin-induced oxidative stress in Wistar rats through three markers of oxidative damage: chromosome aberrations, glutathione depletion and lipid peroxidation. The animals were divided into six treatment groups with six rats in each (n= 6). The dose of cisplatin (5.0 mg kg−1body wt.) was injected i.p. and bixin (2.5 or 5.0 mg kg−1body wt.) was given by gavage at 48, 24 h and 10 min before the cisplatin injection. The treatment with the highest dose of bixin resulted in a statistically significant reduction, by about 33%, in cisplatin-induced abnormal metaphases (P< 0.05). A single dose of cisplatin enhanced the formation of lipid peroxides in 29% and resulted in a 29% depletion in renal glutathione 24 h after cisplatin administration (P< 0.05). The pretreatment with bixin reduced the total number of chromosome aberrations, inhibited the increase in lipid peroxidation, and inhibited renal glutathione depletion induced by cisplatin. Since the pretreatment with bixin alone was safe, under the present experimental conditions, the results suggest that bixin may have future clinical application after further studies.

Effects of the antioxidants curcumin or selenium on cisplatin-induced nephrotoxicity and lipid peroxidation in rats

A large number of natural products and dietary components have been evaluated as potential chemoprotective agents. In the present investigation we report the effects of pre-treatment with two dietary antioxidants, curcumin ( 8 mgkg−1body wt.) or selenium ( 1 mgkg−1body wt.), on cisplatin-induced lipid peroxidation and nephrotoxicity in Wistar rats. Adult male rats were divided into six treatment and control groups of six rats each. The animals were pre-treated by gavage with two doses of each antioxidant, one dose 24 h and the second 10 min before cisplatin intraperitoneal injection ( 5 mgkg−1body wt.). The rats were killed 3 days after cisplatin injection and serum, urine and kidney were isolated and analysed. Cisplatin administration resulted in significant reduction of body weight and higher urinary volumes were observed in all groups treated with this antitumor drug (P< 0.05). The animals treated with cisplatin showed a depletion of renal glutathione, increased lipid peroxidation, and an increase in serum creatinine levels (P< 0.05). The administration of curcumin or selenium alone did not increase lipid peroxidation compared to the control group (P> 0.05). Three days after curcumin or selenium plus cisplatin treatments, the renal damage induced by cisplatin did not recover at a significant statistically level. This study suggests that the natural antioxidants curcumin or selenium did not offer protection against cisplatin-induced nephrotoxicity and lipid peroxidation in adult Wistar rats.

Dose‐Dependent Protection by Lipoic Acid against Cisplatin‐Induced Nephrotoxicity in Rats: Antioxidant Defense System

Abstract:This study was designed to investigate the role of graded doses of lipoic acid pretreatment against cisplatin‐induced nephrotoxicity. Male Wistar rats were divided into six groups and treated as follows: 1) vehicle (saline) control; 2) cisplatin (16 mg/kg, intraperitoneally); 3) lipoic acid (100 mg/kg, intraperitoneally); 4) cisplatin plus lipoic acid (25 mg/kg); 5) cisplatin plus lipoic acid (50 mg/kg) and 6) cisplatin plus lipoic acid (100 mg/kg). Rats were sacrificed three days after treatment, and plasma as well as kidneys were isolated and analyzed. Plasma creatinine increased (677% of control) following cisplatin administration alone which was decreased by lipoic acid in a dose‐dependent manner. Cisplatin‐treated rats showed a depletion of renal glutathione (GSH), increased oxidized GSH and decreased GSH/GSH oxidized ratio (62%, 166% and 62% of control), respectively which were restored with lipoic acid pretreatment. Renal superoxide dismutase, catalase, glutathione peroxidase (GSH peroxidase) and glutathione reductase activities decreased (62%, 75%, 62% and 80% of control), respectively, and malondialdehyde content increased (204% of control) following cisplatin administration, which were restored with increasing doses of lipoic acid. The renal platinum concentration increased following cisplatin administration, which was possibly decreased by chelation with lipoic acid. The data suggest that the graded doses of lipoic acid effectively prevented a decrease in renal antioxidant defense system and prevented an increase in lipid peroxidation, platinum content and plasma creatinine concentrations in a dose‐dependent manner.

Dose-dependent protection by lipoic acid against cisplatin-induced nephrotoxicity in rats: antioxidant defense system.

This study was designed to investigate the role of graded doses of lipoic acid pretreatment against cisplatin-induced nephrotoxicity. Male Wistar rats were divided into six groups and treated as follows: 1) vehicle (saline) control; 2) cisplatin (16 mg/kg, intraperitoneally); 3) lipoic acid (100 mg/kg, intraperitoneally); 4) cisplatin plus lipoic acid (25 mg/kg); 5) cisplatin plus lipoic acid (50 mg/kg) and 6) cisplatin plus lipoic acid (100 mg/kg). Rats were sacrificed three days after treatment, and plasma as well as kidneys were isolated and analyzed. Plasma creatinine increased (677% of control) following cisplatin administration alone which was decreased by lipoic acid in a dose-dependent manner. Cisplatin-treated rats showed a depletion of renal glutathione (GSH), increased oxidized GSH and decreased GSH/GSH oxidized ratio (62%, 166% and 62% of control), respectively which were restored with lipoic acid pretreatment. Renal superoxide dismutase, catalase, glutathione peroxidase (GSH peroxidase) and glutathione reductase activities decreased (62%, 75%, 62% and 80% of control), respectively, and malondialdehyde content increased (204% of control) following cisplatin administration, which were restored with increasing doses of lipoic acid. The renal platinum concentration increased following cisplatin administration, which was possibly decreased by chelation with lipoic acid. The data suggest that the graded doses of lipoic acid effectively prevented a decrease in renal antioxidant defense system and prevented an increase in lipid peroxidation, platinum content and plasma creatinine concentrations in a dose-dependent manner.

Oxidative stress induced by L-buthionine-( S, R)-sulfoximine, a selective inhibitor of glutathione metabolism, abrogates mouse kidney mineralocorticoid receptor function

In vitro studies have demonstrated that cysteine groups present in most of the steroid receptors play an essential role in the steroid binding process as well as in the ability of this superfamily of signaling proteins to function as transcription factors. However, there is poor experimental evidence, if any, which demonstrates that under conditions of oxidative stress the steroid receptors in general, and the mineralocorticoid receptor in particular, are affected in vivo in a similar fashion as has been described for cell-free systems or cells in culture. In the present work we report that when mice are exposed to oxidative stress by treatment with L-buthionine-( S, R)-sulfoximine ( L-( S, R)-BSO), a glutathione depleting agent, the aldosterone-dependent mineralocorticoid biological response (measured as sodium retention and potassium elimination) was diminished in a directly proportional manner with respect to the depletion of renal glutathione. Accordingly, the steroid binding capacity of the mineralocorticoid receptor was also abrogated, whereas the receptor protein level remained unchanged. The harmful effects observed in mice after glutathione depletion were efficiently prevented by co-treatment with glutathione monoethyl ester. Similar inhibition in the steroid binding capacity was also generated in vitro by receptor alkylation and receptor oxidation, an effect which was prevented in the presence of reducing agents. Since the glutathione deficit generated in vivo by treatment with L-( S, R)-BSO did not significantly affect other renal proteins which are known to be required for the mineralocorticoid mechanism of action, we suggest that in renal cells a low redox potential exerts drastic and uncompensated inhibition of the receptor-mediated mineralocorticoid biological response. This effect was ascribed to the loss of steroid binding capacity of oxidized receptor, most likely by modification of essential cysteines as supported by experiments where a decreased number of reactive thiols and reduced covalent binding of thiol-reactive ligand were evidenced on immunopurified receptor after in vivo treatment with L-( S, R)-BSO .

2-Amino-5-chlorophenol Toxicity in Renal Cortical Slices from Fischer 344 Rats: Effect of Antioxidants and Sulfhydryl Agents

2-Amino-5-chlorophenol is nephrotoxic through an unidentified mechanism. This study examined the in vitro toxicity of 2-amino-5-chlorophenol in renal cortical slices from Fischer 344 rats and specifically assessed induction of lipid peroxidation and depletion of renal glutathione. Renal cortical slices exposed to 0, 0.25, 0.5, and 1 mM 2-amino-5-chlorophenol exhibited a concentration- and time-dependent increase in lactate dehydrogenase (LDH) leakage. Pyruvate-directed gluconeogenesis was diminished in a concentration-dependent manner following a 90-min incubation with 0, 0.25, 0.5, and 1 mM 2-amino-5-chlorophenol. Lipid peroxidation was induced within 60 min by 1 mM 2-amino-5-chlorophenol in renal slices relative to control tissue. Total glutathione (GSH) levels were decreased below control values within 30 min of exposure to 0.5 and 1 mM 2-amino-5-chlorophenol. These results indicated that GSH levels were decreased prior to the appearance of increased LDH leakage and diminished membrane integrity. 2-Amino-5-chlorophenol toxicity was increased in renal slices isolated from animals pretreated with buthionine sulfoximine (BSO, 890 mg/kg ip). Pretreatment of renal slices with the phenolic antioxidant N,N′-diphenyl-1,4-phenylenediamine (DPPD, 50 μM) or the iron chelator deferoxamine did not reduce 2-amino-5-chlorophenol cytotoxicity. These results suggest that 2-amino-5-chlorophenol toxicity was not mediated through an iron-dependent mechanism. 2-Amino-5-chlorophenol cytotoxicity was reduced by a 15-min pre-incubation with 2 mM ascorbate or a 30-min preincubation with the thiol-containing agents GSH (1 mM) or dithiothreitol (1 mM, DTT). Pretreatment with GSH, DTT, or ascorbate reduced LDH leakage and lipid peroxide generation induced by 2-amino-5-chlorophenol. These results suggest that 2-amino-5-chlorophenol cytotoxicity involved free radical generation through an iron-independent mechanism. Toxicity was reduced by the presence of the antioxidant ascorbate or by addition of glutathione.

Protection by ebselen against cisplatin-induced nephrotoxicity: antioxidant system.

This study was designed to investigate the cisplatin-induced alteration in renal antioxidant system and the nephroprotection with ebselen. Male Wistar rats were injected with (1) vehicle control; (2) cisplatin; (3) ebselen; and (4) cisplatin plus ebselen. Rats were sacrificed three days post-treatment and plasma as well as kidney were isolated and analyzed. Plasma creatinine increased 598% following cisplatin administration alone which decreased by 158% with ebselen pretreatment. Cisplatin-treated rats showed a depletion of renal glutathione (GSH) levels (52% of control), while cisplatin plus ebselen injected rats had GSH values close to the controls. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities decreased 38, 75 and 62% of control, respectively, and malondialdehyde (MDA) levels increased 174% of control following cisplatin administration, which were restored to control levels after ebselen treatment. The renal platinum level did not significantly change with ebselen pretreatment. This study suggests that the protection offered by ebselen against cisplatin-induced nephrotoxicity is partly related to the sparing of antioxidant system.

Acetaminophen Nephrotoxicity in the CD-1 Mouse. II. Protection by Probenecid and AT-125 without Diminution of Renal Covalent Binding

Acetaminophen (APAP) administration (600 mg/kg, ip) to 18-hr fasted, 3-month-old male CD-1 mice results in necrosis of the convoluted renal proximal tubules with a corresponding elevation of plasma urea nitrogen (BUN). Administration of the γ-glutamyl transpeptidase inhibitor,L-(αS,5S)-α-amino-3-chloro-4,5-dihydroxy-5-isoxazoleacetic acid (AT-125) (50 mg/kg, ip), to mice 30 min before APAP significantly diminished the APAP-induced histopathologic damage and BUN elevation. Administration of the organic-anion transport inhibitor, probenecid (150 mg/kg, ip), 30 min before APAP challenge also protected against the APAP-induced elevation of BUN and detectable histopathologic changes. By contrast, pretreatment of mice with the cysteine conjugate β-lyase inhibitor, (aminooxy)acetic acid (100 mg/kg, ip), 1 hr before APAP did not alter nephrotoxicity. None of the pretreatments altered the APAP-induced elevation of plasma sorbitol dehydrogenase activity, nor were there any detectable changes in liver histopathology after APAP challenge. Despite the protective effects of both probenecid and AT-125 against nephrotoxicity, they did not affect either the level of immunochemically detectable covalent binding to protein or the depletion of renal glutathione at 4 hr after APAP. Thus, the protection appears independent of effects on renal APAP uptake or activation and indirectly suggests that an APAP–glutathione conjugate may contribute to the observed nephrotoxicity.

Tissue-specific changes in glutathione and cysteine after buthionine sulfoximine treatment of rats and the potential for artifacts in thiol levels resulting from tissue preparation

l-Buthionine- S,R-sulfoximine (BSO), a potent inhibitor of γ-glutamylcysteine synthetase, is commonly used as an experimental tool for the specific depletion of glutathione. Since cysteine is a key precursor for glutathione biosynthesis, we investigated the possibility that BSO might also affect the free cysteine pool in rat liver and kidney tissues in vivo. Male CD(SD)BR rats (150–200 g) were injected ip with various doses of BSO (0.25−4.0 mmol/kg), and glutathione and cysteine were measured in liver and kidney using HPLC with electrochemical detection and/or spectroscopic techniques. No hepatotoxicity or nephrotoxicity was observed at the highest BSO dose (4.0 mmol/kg) used. BSO caused the expected decreases of hepatic and renal glutathione at all doses, although glutathione depletion was more rapid, was achieved at a lower BSO dose, and was more sustained in kidney than in liver. Hepatic cysteine levels nearly doubled 20 min after BSO treatment (1.0 mmol/kg, ip), but were not significantly different from control at later time points. In contrast, renal cysteine was significantly depleted from 20 min to 25 hr postinjection with a time course closely paralleling that of renal glutathione depletion. These changes are discussed in the context of models for inter- and intraorgan transport of glutathione and cysteine. We also provide evidence that an artifact, most likely the γ-glutamyltranspeptidase (GGT)-initiated breakdown of glutathione, leads to a rapid postmortem increase of cysteine levels in liver and particularly in kidney of rats. Simultaneous decreases in GSH levels can be demonstrated in kidney. This artifact needs to be minimized in toxicological studies of glutathione and cysteine in kidney and other GGT-rich organs, as the measured levels of these thiols may not reflect the true concentrations occurring in vivo.

Improved use of buthionine sulfoximine to prevent cisplatin nephrotoxicity in rats.

Male Sprague Dawley rats were treated with buthionine sulfoximine (BSO) and cisplatin in different doses and schedules to optimize the chemoprotective effect of BSO against cisplatin nephrotoxicity. BSO at 4 mmol/kg, administered s.c. 2 h prior to cisplatin, resulted in normal blood urea nitrogen (BUN) levels in rats treated with 3 or 4 mg/kg cisplatin, and modestly, but significantly reduced the toxicity of cisplatin at 5 mg/kg. Administration of BSO (4 mmol/kg) at intervals ranging from 0 to 16 h prior to cisplatin (5 mg/kg) resulted in a significant reduction in BUN values. A BSO dose as low as 0.04 mmol/kg was found to be as effective as 4 mmol/kg against nephrotoxicity associated with cisplatin at 4 mg/kg. Repetitive injections of BSO (1 mmol/kg every 12 h, four times, beginning 2 h prior to cisplatin) significantly inhibited elevations of BUN associated with higher-dose cisplatin (6 mg/kg), whereas a single BSO injection of 4 mmol/kg was ineffective. The degree and duration of renal glutathione depletion was related to the dose of BSO. Renal glutathione content following 4 mmol/kg BSO was 38% of control at 2 h and 40% at 24 h; following 0.04 mg/kg, glutathione was 47% at 2 h and almost 100% at 24 h. Simultaneous in vitro administration of BSO did not inactivate cisplatin cytotoxicity as measured by the colony-forming ability of MBT-2 cells in soft agar. These data indicate that repeated injections of BSO, beginning prior to cisplatin administration, would improve the nephroprotective effect without compromising the chemotherapeutic efficacy of cisplatin. It is suggested that the ability of BSO to reduce cisplatin nephrotoxicity may not correlate with the degree of renal glutathione depletion and that the mechanism of action is unlikely to involve direct inactivation of cisplatin.