ORIGINAL ARTICLE

Nephroprotective activities of quercetin with potential relevanceto oxidative stress induced by valproic acid

Shaista Chaudhary & Pratibha Ganjoo &

Sheikh Raiusddin & Suhel Parvez

Received: 17 March 2014 /Accepted: 18 June 2014# Springer-Verlag Wien 2014

Abstract Valproic acid (VPA) is ubiquitously used as a majordrug in the intervention of epilepsy and in the control ofseveral kinds of seizures. Cellular toxicities are the seriousdose-limiting side effects of VPA when applied in the treat-ment of diseases. Oxidative stress has been proven to beinvolved in VPA-induced toxicity. Accumulating evidenceintimates that oxidative stress caused by free radicals and inkidney cells contributes to the pathogenesis of VPA-inducednephrotoxicity. The pathogenesis of these forms of VPAnephrotoxicity is still not clear. The aim of our investigationwas to evaluate the nephrotoxic potential of VPA and protec-tive effects of quercetin (QR) against VPA-induced nephro-toxicity by using rat kidney tissue preparation as an in vitromodel. Oxidative stress indexes such as lipid peroxidation(LPO) and protein carbonyl (PC) content were appraised.The levels of oxidative stress markers, LPO, and PC weresignificantly elevated. Nonenzymatic antioxidants effect wasalso demonstrated as a significant increase in reduced gluta-thione (GSH) and nonprotein thiol level (NP-SH). VPA expo-sure altered the activities of glutathione metabolizing enzymessuch as glutathione-S-transferase, glutathione peroxidase, andglutathione reductase. Pre-treatment with QR could reversethe VPA-induced effects in kidney tissue preparation of rat.Based on reno-protective and antioxidant action of QR, wesuggest that this flavonoid compound could be considered as a

potential safe and effective approach in attenuating the ad-verse effect of VPA-induced nephrotoxicity.

Keywords Nephrotoxicity . Oxidative stress . Valproic acid .

Quercetin . Antioxidants

Introduction

Valproic acid (VPA) is one of the most frequently usedantiepileptic agents. It has a potent therapeutic actionagainst an extensive approach of activity in partial andgeneralized seizures or epilepsies, bipolar psychiatric disor-ders, and migraine control (Gravemann et al. 2008). Theutility of VPA as an anticonvulsant has been supported byclinicians, which was subsequently challenged due to itsside effects and induced toxicity (Chateauvieux et al.2010). Although, VPA is a relatively safe drug, but it cancause severe side effects on biological system when it isused in higher concentrations (Pourahmad et al. 2012).There are several studies confirming that VPA induces theformation of reactive oxygen species (ROS), which isresponsible for the life-threatening side effects of VPAtherapy including hepatotoxicity (Tong et al. 2005a), neu-rotoxicity (Auinger et al. 2009), and teratogenicity (Tungand Winn 2011). Oxidative stress, as a result of ROSproduction and compromised status of antioxidants, hasbeen hypothesized to play a role in the etiology of thetoxicity (Khan et al. 2011). Numerous studies have alsodemonstrated that administration of VPA at clinically rele-vant doses to neonatal rat for anticonvulsant action inducedtoxic effects on cardiovascular (Wu et al. 2010), gastroin-testinal, hematological, and reproductive systems (Spilleret al. 2000), and several groups have also examined themechanisms of VPA toxicity in various cell lines andtissues using different schemes of administration as well

Handling Editor: Reimer Stick

Shaista Chaudhary and Pratibha Ganjoo contributed equally to the study.

S. Chaudhary : P. Ganjoo : S. Raiusddin : S. Parvez (*)Department of Medical Elementology and Toxicology, JamiaHamdard (Hamdard University), New Delhi 110 062, Indiae-mail: drsuhelparvez@yahoo.com

S. Parveze-mail: sparvez@jamiahamdard.ac.in

ProtoplasmaDOI 10.1007/s00709-014-0670-8

as distinct animal species (Fu et al. 2010; Gibbons et al.2011). VPA toxicity is associated with increased ROSformation, which in turn constitutes an important risk factorfor tissue damage and organ dysfunction (Fourcade et al.2010). A number of studies have reported the implicationof an increased generation of free radicals and oxidativestress in the toxicity induction mechanism of VPA (Kianget al. 2010; Zhang et al. 2011). Different mechanisms havebeen proposed to explain inhibition of mitochondrial func-tion by VPA (Tong et al. 2005b). It has been evident thatVPA-induced toxicity may be mediated, at least, in part, byoxidative stress which refers to the situation by which thereare enhanced level of ROS (Chang and Abbott 2006).

ROS are involved in many cellular events, includingas second messengers in the activation of several sig-naling pathways leading to the activation of transcrip-tion factors, mitogenesis, gene expression, and the in-duction of apoptosis, or programmed cell death (Birbenet al. 2012). Renal cellular oxidative stress often takesplace during the occurrence of the imbalance betweenpro-oxidants and antioxidants (Sung et al. 2013).

Flavonoids are considered as an antioxidant agentsmostly distributed in dietary plants, such as fruits, veg-etables, and teas (Mariee et al. 2012), and frequentlyconsumed by humans. Quercetin (QR) (3,5,7,3,4-pentahydroxyflavone), a member of the flavonoid fami-ly, is one of the most prominent dietary antioxidant. QRis ubiquitously contained in foods and vegetables in-cluding apples, onion, mulberry, potatoes, broccoli, tea,peanuts, soybeans, and red wine (Barcelos et al. 2011a).QR has been recognized to possess a variety of biolog-ical and pharmacological activities, including antioxi-dant, anti-inflammatory, antiallergic, antibacterial,and antitumoral (Ramos et al. 2008) activities. Thebiological action of QR has been widely studied forits antioxidant properties (Barcelos et al. 2011b). How-ever, most investigators accept that the antioxidant ac-tivity of QR conferred by its phenolic hydroxyl groupsis primarily implicated in the therapeutic potential ofthis flavonoid in different diseases (Morales et al.2006). QR has been thoroughly investigated for itsabilities to express antiproliferative and protective ef-fects in various systems. Therefore, the aim of thepresent study was to assess the toxic effect of VPA inkidney and the possible protective effect of QR tomitigate the nephrotoxicity of VPA in Wistar rat kidneypreparation as an in vitro model. Several investigationshave indicated that postnuclear supernatant (PNS) havebetter adaptive response for evaluating the toxicity ofxenobiotics (Moraes et al. 2010; Govil et al. 2012). Inour study, kidney PNS was used as an in vitro researchmodel to evaluate the effect of VPA-inducednephrotoxicity.

Materials and methods

Chemicals

Sodium salt of VPAwas obtained from ROAQ Chemicals Pvt.Ltd. (Gujarat, India). Bovine serum albumin (BSA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), oxidized glutathione(GSSG), QR, reduced glutathione (GSH), reduced nicotinamideadenine dinucleotide phosphate (NADPH), and thiobarbituricacid (TBA) were purchased from Sigma Chemicals Co. (St.Louis, MO, USA). Butylated hydroxytoluene (BHT), 1-chloro-2,4-dinitrobenzene (CDNB), 2,4-dinitrophenylhydrazine(DNPH), ethylene diamine tetraacetic acid (EDTA), orthophos-phoric acid (OPA), sulfosalicylic acid, sodium azide, and tri-chloroacetic acid (TCA) were purchased from Merck Limited(Mumbai, India).

Animals

Male Wistar rats (3–4 weeks old) weighing 100–120 g, wereused for current experiments and procured from the CentralAnimal House of Jamia Hamdard (Hamdard University), NewDelhi, India. Rats were kept at temperature 22±1 °C withrelative humidity at 65±10 % and at a photoperiod of 12-hlight/dark cycle. Food and water were supplied ad libitumprior to the start of the experiment. All the experiments werecarried out according to the standard guidelines of Institution-al Animal Ethics Committee (IAEC).

Sample preparation

PNS was prepared by differential centrifugation method withminor modifications (Govil et al. 2012). On the day of exper-iments, rats were sacrificed by cervical dislocation, the kidneywas rapidly excised on a Petri dish placed on ice, and theblood and external vessels were carefully removed. The kid-ney was weighed and kept chilled until homogenization. Thekidney was homogenized in 10 volumes (1:10, w/v) of 0.1 Msodium phosphate buffer (pH 7.4) with a Potter-Elvehjemhomogenizer. Homogenate was subjected to differential cen-trifugation in refrigerated centrifuge at temperature of 4 °Cusing a REMI C-24 centrifuge. Homogenate mixture wascentrifuged at 1,000×g for 10 min. The resulting pellet pre-pared from 10 % homogenate was the primary nuclear pellet,and the supernatant was PNS. The PNS was the biologicalfraction in which enzymatic and nonenzymatic antioxidantswere evaluated.

Experimental design

Sodium salt of VPA was used to evaluate the renal toxicityunder in vitro conditions. The PNS of kidney tissue wasincubated with the different concentrations of sodium

S. Chaudhary et al.

valproate (5, 10, and 20 mg) for 2 h at 37 °C in a temperaturecontrolled water bath. Sodium phosphate buffer was added tocontrols instead of VPA. The stock and working solution wereprepared in such a way that the same volume was added in thePNS for incubation. After evaluating the toxic effect of VPA,we designed a next set of experiments to investigate theprotective effect of QR with the higher concentration ofVPA under in vitro conditions, PNS of kidney tissue waspre-incubated with QR (0.05 mM) for 1 h; thereafter, VPA(20 mg) was added and incubated for 2 h at 37 °C.

Biochemical studies

Determination of lipid peroxidation

LPO was measured by analyzing thiobarbituric acid reactivesubstances (TBARS) as described by Uchiyama and Mihara(1978) with slight modifications. Briefly, the renal tissue washomogenized in 0.1-M chilled phosphate-buffered solution(PBS) (pH 7.4). The assay mixture contained 10 mM BHT,OPA (1 %), and TBA (0.67 %) together with 10 % homoge-nate in a total volume of 4.250 mL. The mixture was incubat-ed at 90 °C for 45 min. After cooling with tap water, themixture was centrifuged for 15 min at 1,000×g. The absor-bance was measured at 535 nm. The rate of LPO wasexpressed as micromoles of TBARS formed per hour pergram tissue based on the molar extinction coefficient of1.56×105 M−1 cm−1.

Determination of protein carbonyl content

PC content was estimated byDNPH assay as adopted from theprocedure of Floor and Wetzel (1998). The most convenientprocedure for PC estimation is the reaction between DNPHand PC content. DNPH reacts with protein carbonyls to pro-duce the corresponding hydrazone. The PNS of renal tissue(0.5 mL) was reacted with 10 mM DNPH in 2 M HCl for 1 hat room temperature and precipitated with 40 % TCA. Thepelleted protein was washed three times by resuspension inethanol/ethyl acetate (1:1) mixture. Proteins were then solubi-lized in 6M guanidine hydrochloride, formic acid (50 %), andcentrifuged at 1,600×g for 5 min to remove any trace ofinsoluble material. The carbonyl content was measured spec-trophotometrically at 340 nm. The results were expressed asnanomoles of DNPH incorporated per milligram proteinbased on the molar extinction coefficient of 2.1 ×104 M−1 cm−1.

Determination of reduced glutathione content

GSH content was estimated according to the method ofJollow et al. (1974). The reaction is based on the fact thatthe thiol group of GSH reacts with the -SH reagent

(DTNB) to form thionitrobenzoic acid. GSH content ofthe kidney was assayed by treating the PNS with 4 %sulfosalicylic acid. It was then incubated at 4 °C for aminimum time period of 1 h and then centrifuged at 4 °Cat 1,200×g for 15 min. The reaction mixture contained0.1 M phosphate buffer (pH 7.4), 10 mM DTNB, and0.4 mL PNS prepared from 10 % homogenate of ratkidney. The yellow color developed was read immediatelyat 412 nm on spectrophotometer. The GSH concentrationwas calculated as micromoles GSH per gram tissue using amolar extinction coefficient of 1.36×104 M−1 cm−1.

Analysis of nonprotein thiol

Nonprotein bound thiol (NP-SH) was determined in the tissuesamples by using the method of Sedlak and Lindsay (1968).For the determination of NP-SH, 0.5 mL of PNS was precip-itated with 0.5 mL of 40 % TCA and then centrifuged at3,000×g for 15 min. Then, the supernatant was used for themeasurement by adding 0.4 M Tris buffer (pH 8.9) and10 mM DTNB. The molar extinction coefficient of13,100 M−1 cm−1 at 412 nm was used for the determinationof thiol content. The value was expressed in micromoles pergram (μmol/g) tissue.

Determination of glutathione-S-transferase activity

The method of Habig et al. (1974) with some modificationwas used to measure the glutathione-S-transferase (GST) ac-tivity. This reaction is measured by observing the conjugationof CDNB with GSH forming a colored conjugate glutathione2,4-dinitrobenzene. For GST activity measurement, the reac-tion mixture contained 0.1 M sodium phosphate buffer(pH 7.4), 10 mM GSH, 10 mM CDNB, and 0.2 mL PNS.The enzyme activity was calculated as nanomoles of CDNBconjugate formed per minute per milligram protein using amolar extinction coefficient of 9.6×103 M−1 cm−1 at 340 nm.

Determination of glutathione peroxidase activity

Glutathione peroxidase (GPx) activity was assayed accordingto the method of Haque et al. (2003). The assay mixtureconsisted of 0.1 M phosphate buffer (pH 7.4), 1 mM EDTA,1 mM sodium azide, 1 mMGSH, 0.2 mMNADPH, 0.25 mMH2O2, and 0.1 mL PNS prepared from 10 % homogenate ofkidney tissue. Oxidation of NADPH was recorded spectro-photometrically at 340 nm. The enzyme activity was calculat-ed as nanomoles of NADPH oxidized per minute per milli-gram of protein using a molar extinction coefficient of 6.22×103 M−1 cm−1.

Nephroprotective activities of quercetin

Determination of glutathione reductase activity

Glutathione reductase (GR) activity was assayed by the meth-od of Mohandas et al. (1984). The assay system consisted0.1 M phosphate buffer (pH 7.4), 0.5 mM EDTA, 1 mMGSSG, 0.1 mM NADPH, and 0.3 mL supernatant in a totalvolume of 2.0 mL PNS. The enzyme activity was quantifiedby measuring the disappearance of NADPH at 340 nm andwas calculated as nanomoles of NADPH oxidized per minuteper milligram protein using a molar extinction coefficient of6.223×103 M−1 cm−1.

Protein determination

The protein content was determined in PNS of kidney tissueby the method of Lowry et al. (1951) using BSA as a standard.

Statistical analysis

Results were expressed as mean±standard deviation (SD). Alldata were analyzed using analysis of variance (ANOVA)followed by Tukey’s test. Values of p<0.05 were consideredas significant. All the statistical analyses were performedusing GraphPad Prism 5 software (GraphPad Software Inc.,San Diego, CA, USA).

Results

Figure 1a, b represents the effect of VPA on LPO levels inrenal PNS of rat. LPO level was markedly increased in 10 and20 mg of VPA-exposed group (p<0.01 and p<0.001) whencompared with control group (Fig. 1a). Exposure with mini-mal dose (5 mg) of VPA showed no significant change onLPO level when compared with control. On the other hand,VPA exposure (Fig. 1b) caused a significant elevation(p<0.001) on LPO level when compared with control group.Additionally, QR pre-exposure showed a significant decrease(p<0.001) on LPO level as compared to VPA-exposed group.QR-only exposure led no significant change on LPO level ascompared to control group.

Figure 1c, d also exemplifies the toxic effect of VPA on PCcontent in renal PNS of rat. VPA-exposed group has shown asignificant increase on PC levels (p<0.001) with the dosefrom 10 to 20 mg concentrations respectively in comparisonwith control group. No significant change was found in PCcontents in 5-mg VPA-exposed group when compared withcontrol (Fig. 1c). Similarly, QR pre-exposure has shown asignificant modulation (p<0.05) in PC contents when com-pared with VPA-exposed group (Fig. 1d). VPA exposurecaused a significant enhancement (p<0.001) in PC contentwhen compared with the control group. Only QR exposure

did not show any effect on PC content as compared to controlgroup.

Figure 2a, b illustrates the toxic potential of VPA as indi-cated by an increased GSH level in renal cells of rat. VPAexposure of 10 and 20 mg has depicted a significant increase(p<0.01 and p<0.001) in GSH level when compared tocontrol group. VPA exposure with low dose of 5 mg did notshow any significant change as compared to control group(Fig. 2a). For protective evaluation, QR pre-exposure hasrendered a significant alteration (p<0.05) as compared toVPA-exposed group (Fig. 2b). QR-only exposure did notexpress any effect on GSH level compared to control group.

Figure 2c, d also represents the toxic potency of VPA onNP-SH level in renal PNS preparation of rat. Exposure of 10-and 20-mg VPA-exposed group has shown a significant(p<0.05 and p<0.001) elevation in NP-SH level as comparedto control group (Fig. 2c). No significant change in the levelswas observed at 5-mg exposure of VPA when compared tocontrol. While, QR pre-exposure has shown a significantreversal (p<0.05) in NP-SH level when compared withVPA-exposed group (Fig. 2d). QR-only exposure contributedno significant change in LPO level as compared to controlgroup. VPA exposure caused a significant elevation(p<0.001) on NP-SH level when compared with control.

Figure 3a–f designates the toxic strength of VPA on GST,GPx, and GR in renal cells of rat. Doses from 10 to 20 mghave shown a significant decrease in the activity of GST(p<0.001) in kidney PNS when compared with control(Fig. 3a). No significant alteration was observed with doseof 5 mg of VPA in kidney when compared with control. Forprotection profile, pre-exposure of QR has shown no signifi-cant change in GSTactivity in kidney cells as compared to theVPA-exposed group. QR-only exposure has also shown nosignificant difference in kidney cells as compared to controlgroup. VPA exposure has shown a significant decline(p<0.01) in GST activity in kidney cells as compared tocontrol (Fig. 3b).

VPA (Fig. 3c) exposure caused a significant (p<0.05 andp<0.01) increase in the activity of GPx in 10- and 20-mgconcentration in comparison with the control group. No sig-nificant change was found in GPx activity in 5-mg VPA-exposed group when compared with control. For protectionindex, QR pre-exposure has shown a significant change inGPx activity (p<0.001) of kidney cells as compared with theVPA-exposed group. VPA exposure led to a significant rise inGPx activity (p<0.001) in kidney PNS when compared withthe control group. QR-only exposure has contributed no sig-nificant change in kidney PNS as compared to control(Fig. 3d).

VPA exposure (Fig. 3e) of both 10 and 20 mg caused amarked increase (p<0.01 and p<0.001) in GR activity inkidney as compared to control. Minimal dose of 5 mg hasshown no significant alteration in the activity of GR in kidney

S. Chaudhary et al.

PNS when compared with control. For protection visibility,QR pre-exposure has shown a significant change (p<0.05) inGR activity in kidney as compared to VPA-exposed group.VPA exposure has shown a significant enhancement

(p<0.001) in GR activity in kidney PNS as compared tocontrol (Fig. 3f). QR-only exposure has also shown no signif-icant difference in kidney PNS preparations when comparedto control group.

Fig. 1 Effect of differentconcentrations of VPA on LPO(a) and PC (c) level and protectiveeffect of QR on LPO (b) and PC(d) level in tissue of rat kidney.Values were expressed as means±S.E.M. (n=6). LPO and PC weremeasured as micromoles ofTBARS formed per hour pergram tissue and nanomoles ofDNPH incorporated permilligram protein. Significantdifferences **p<0.01 and***p<0.001 when comparedwith control and #p<0.05 and###p<0.001 when compared withVPA-exposed group

Fig. 2 Effect of differentconcentrations of VPA on GSH(a) and NP-SH (c) level andprotective effect of QR on GSH(b) and NP-SH (d) level in tissueof rat kidney. Values wereexpressed as means±S.E.M. (n=6). GSH and NP-SH weremeasured as micromoles of GSHper gram tissue and micromolesof NP-SH per gram tissue.Significant differences *p<0.05,**p<0.01, and ***p<0.001when compared with control and#p<0.05 when compared withVPA-exposed group

Nephroprotective activities of quercetin

Discussion

The important role of oxidative stress in renal toxicity and theperfect antioxidant property of QR remind us that the toxicinsult of VPA to kidney could be challenged by QR interven-tion. Therefore, the present study investigated the in vitroeffects of VPA on lipid and protein oxidative damage and onthe major enzymatic and nonenzymatic antioxidant defenseand the actual protective effect of QR on the redox imbalancein VPA-induced kidney tissue. Free-radical-induced oxidativestress has been implicated in the etiology of kidney diseases(Singh et al. 2004). LPO is probably the most largely consid-ered product produced by free radicals and hence is regardedas an excellent biomarker of oxidative stress (Messaoudi et al.2009). LPO, mediated by oxygen free radicals, is believed tobe an important cause of destruction and damage to cellmembranes and has been suggested to be a contributing factorto the development of VPA mediated tissue damage

(Chaudhary and Parvez 2012). We first observed that VPAsignificantly increased the LPO level dose dependently. Thedata obtained in our study confirm that acute intoxication withVPA can cause a significant increase of LPO level in thekidney of rats. The enhancement in LPO reported here maybe the result of increased ROS production or a decrease instatus of antioxidants and membrane fluidity (Paul et al.2012). In our study, QR pre-treatment significantly reversedthe LPO and altered status of antioxidants. It has been docu-mented that QR declined oxidative stress marker LPO byscavenging ROS and QR could reinforce a constructive actionagainst LPO, which may act as an added compensation mech-anism to retain cell integrity and protection against free radicaldamage (Renugadevi and Prabu 2010). This incited us toevaluate VPA provoked protein oxidation as observed bymarked elevation of PC formation.

PC is an important index of oxygen radical-mediated pro-tein damage under various pathophysiological conditions

Fig. 3 Effect of differentconcentrations of VPA on GST(a), GPx (c), and GR (e) activityand protective effect of QR onGST (b), GPx (d), and GR (f)activity in tissue of rat kidney.Values were expressed as means±S.E.M. (n=6). GST activity wasmeasured as nanomoles of CDNBconjugate formed per minute permilligram protein. GR and GPxactivities were measured asnanomoles of NADPH oxidizedper minute per milligram protein.Significant differences *p<0.05,**p<0.01, and ***p<0.001when compared with control and#p<0.05 and ###p<0.001 whencompared with VPA-exposedgroup

S. Chaudhary et al.

(Farombi and Ekor 2006). The increased PC contents havebeen observed in kidney tissue after VPA exposure in thepresent experiments. The increased production of PC contentswas an index of enhanced oxidative stress caused by VPA.Pre-treatment with QR prior to VPA exposure, however,prevented the enhancement in the PC contents and restoredthe renal cells to its normal physiological state. Our resultscorroborate this observation as PC content increased on beingexposed to VPA in kidney. Thereafter, we also examined thelevel of thiol profile, in which we have observed that VPAcaused a significant increase in GSH level as well as in NP-SHlevel in the nephrotic cells. This shows induction of oxidativedamage and debilitative of the cellular antioxidant defensemechanisms leading to active participation of GSH in cellulardefense against ROS. The increased values of GSH and NP-SH indicate the nonenzymatic antioxidant response to beingsubjected to oxidative stress. GSH plays a key role in cellularfunction and viability (Nabavi et al. 2012). As GSH poses ofthe majority of the pool of NP-SH, a similar trend was ob-served in both gives corroborative evidence of the possiblenephrotoxic potential of VPA. The NP-SH is an integral partof the armamentarium of antioxidant defense system whichhas direct reflection on the alteration of glutathione metabo-lizing enzymes like GST, GPx, and GR (Zhu et al. 2006). Inour results, pre-treatment with QR improved the level of thiolprofile such as GSH and NP-SH, thereby protecting againstVPA-induced oxidative damage. Our findings are in conso-nance with the other published reports which quoted that theconcentration of GSH and NP-SH can be increased duringxenobiotic intoxication (Zafeer et al. 2012; Waseem et al.2013). Therefore, protection against the VPA-induced toxicitycan be attained through the supplementation of antioxidants.The present study also confirmed that the exposure of QRsignificantly attenuated the renal oxidative stress induced bythe toxic effects exerted by VPA.

Conflicting results also exist with respect to the effect ofVPA on the levels of glutathione metabolizing enzymes ornonenzymatic antioxidants. All glutathione metabolizing en-zymes may serve as antioxidants. The nephrotoxic effect ofVPAwas demonstrated by a significant decrease in the activityof GST in tissue of rat kidney. GST is a multifunctionalenzyme, which plays an important role in the detoxificationof toxic electrophiles by catalyzing the conjugation of theseelectrophiles with GSH (Milton Prabu et al. 2010). In ourstudy, supplementation of QR along with VPA in renal tissuecould not restore the diminished activity of GST. GPx is aselenium-containing enzyme, which plays a prominent role inthe reduction of H2O2 and hydroxide and removal of excessfree radicals to nontoxic products (Abdel-Raheem et al. 2009).In our results, VPA treatment showed a significant increase inactivity of GPx in kidney cells. QR administration was able toreverse the activities of GPx in nephrotic cells of VPA-exposed tissue. So, this restoration may also be due to the

altered availability of its substrate GSH (Coskun et al. 2005).GR is the enzyme responsible for the reduction of oxidizedglutathione (GSSG) to GSH. The activity of GR directlycontributes to the protection and repair of -SH protein underoxidative stress (Tabassum et al. 2007). In our study, VPAtreatment showed increased activity of GR in kidney. It maybe due to the protection from oxidative stress formed byoverproduction of free radical (Park et al. 2010). QR signifi-cantly prevented the alteration in the activity of GR in kidneyprobably attributable to its antioxidant effect. In consistencewith this, in the present study, a significant alteration in theactivity of enzymatic and nonenzymatic antioxidants in VPAtoxicity could lead to increased susceptibility of the renaltissue to free radical damage. Most of the antioxidant enzymesbecome inactive due VPA exposure, which is associated withdirect binding of VPA to their active sites (Kiang et al. 2011).In the present study, the altered activities of the antioxidantenzymes could reflect the failure of antioxidant defense toovercome the influx of ROS on VPA exposure. Supplemen-tation of QR along with VPA significantly restored the levelsof antioxidant enzymes and nonenzymatic antioxidants,which might be due to the ability of QR to reduce the accu-mulation of free radical generation during VPA exposure.

Conclusion

In summary, the present results of these investigationsprovide us with initial impetus that VPA induces oxida-tive stress by compromising the antioxidant status of therenal tissue. Subsequently, pre-treatment of QR withVPA minimized these effects. QR may be useful inthe treatment of nephrotoxicity of VPA. QR has protec-tive effect against VPA-induced oxidative damage in thekidney tissue of rats. The mechanisms contributing toits effectiveness involve the quenching of free radicalsand ability to alter the status of antioxidants. This wasthe first study to explore the toxic effect of VPA and itsabrogation by QR in kidney of rat as an in vitro model.Recently, much attention has been focused on the pro-tective biochemical functions of naturally occurring an-tioxidants in biological systems against renal toxicity ofVPA. However, further investigations are necessary toinvestigate the exact mechanism of QR protectionagainst VPA-induced nephrotoxicity.

Acknowledgments University Grants Commission (UGC), Govern-ment of India is gratefully acknowledged for providing funding underFaculty Research Award to SP. SC was supported by a Senior ResearchFellowship of UGC-Basic Science Research.

Conflict of interest The authors declare that they have no conflict ofinterest.

Nephroprotective activities of quercetin

References

Abdel-Raheem IT, Abdel-Ghany AA, Mohamed GA (2009) Protectiveeffect of quercetin against gentamicin-induced nephrotoxicity inrats. Biol Pharm Bull 32:61–67

Auinger K, Müller V, Rudiger A, Maggiorini M (2009) Valproic acidintoxication imitating brain death. Am J Emerg Med 27(9):1177, e5-6

Barcelos GR, Angeli JP, Serpeloni JM, Grotto D, Rocha BA, Bastos JK,Knasmüller S, Júnior FB (2011a) Quercetin protects human-derivedliver cells against mercury-induced DNA-damage and alterations ofthe redox status. Mutat Res 26:109–115

Barcelos GR, Grotto D, Serpeloni JM, Angeli JP, Rocha BA, de OliveiraSouza VC, Vicentini JT, Emanuelli T, Bastos JK, Antunes LM,Knasmüller S, Barbosa F Jr (2011b) Protective properties of quer-cetin against DNA damage and oxidative stress induced by methyl-mercury in rats. Arch Toxicol 85:1151–1157

Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O (2012)Oxidative stress and antioxidant defense. World Allergy Organ J5:9–19

Chang TK, Abbott FS (2006) Oxidative stress as a mechanism of valproicacid associated hepatotoxicity. Drug Metab Rev 38:627–639

Chateauvieux S, Morceau F, Dicato M, Diederich M (2010) Molecularand therapeutic potential and toxicity of valproic acid. J BiomedBiotechnol. doi:10.1155/2010/479364

Chaudhary S, Parvez S (2012) An in vitro approach to assess the neuro-toxicity of valproic acid-induced oxidative stress in cerebellum andcerebral cortex of young rats. Neuroscience 225:258–268

Coskun O, Kanter M, Korkmaz A, Oter S (2005) Quercetin, a flavonoidantioxidant, prevents and protects streptozotocin-induced oxidativestress and beta-cell damage in rat pancreas. Pharmacol Res 51:117–123

Farombi EO, Ekor M (2006) Curcumin attenuates gentamicin-inducedrenal oxidative damage in rats. Food Chem Toxicol 44:1443–1448

Floor E, Wetzel MG (1998) Increased protein oxidation in humansubstantia nigra pars compacta in comparison with basal gangliaand pre f ron t a l co r t ex measu red wi th an improveddinitrophenylhydrazine assay. J Neurochem 70:268–275

Fourcade S, Ruiz M, Guilera C, Hahnen E, Brichta L, Naudi A, Portero-Otín M, Dacremont G, Cartier N, Wanders R, Kemp S, Mandel JL,Wirth B, Pamplona R, Aubourg P, Pujol A (2010) Valproic acidinduces antioxidant effects in X-linked adrenoleukodystrophy. HumMol Genet 19:2005–2014

Fu J, Shao CJ, Chen FR, Ng HK, Chen ZP (2010) Autophagy induced byvalproic acid is associated with oxidative stress in glioma cell lines.Neuro Oncol 12:328–340

Gibbons HM, Smith AM, Teoh HH, Bergin PM, Mee EW, Faull RL,Dragunow M (2011) Valproic acid induces microglial dysfunction,not apoptosis, in human glial cultures. Neurobiol Dis 41:96–103

Govil N, Chaudhary S, Waseem M, Parvez S (2012) Postnuclear super-natant: an in vitro model for assessing cadmium-induced neurotox-icity. Biol Trace Elem Res 146:402–409

Gravemann U, Volland J, Nau H (2008) Hydroxamic acid and fluorinatedderivatives of valproic acid: anticonvulsant activity, neurotoxicityand teratogenicity. Neurotoxicol Teratol 30:390–394

Habig WH, Pabst M, Jaoby WB (1974) Glutathione S-transferase: thefirst step in mercapturic acid formation. J Biol Chem 249:7130–7139

Haque R, Bin-Hafeez B, Parvez S, Pandey S, Sayeed I, Ali M, RaisuddinS (2003) Aqueous extract of walnut (Juglans regia L.) protects miceagainst cyclophosphamide induced biochemical toxicity. Hum ExpToxicol 22:473–480

Jollow DJ, Mitchell JR, Zamppaglione Z, Gillette JR (1974)Bromobenzene induced liver necrosis. Protective role of glutathioneand evidence for 3,4-bromobenzene oxide as the hepatotoxic me-tabolites. Pharmacology 11:151–169

Khan S, Ahmad T, Parekh CV, Trivedi PP, Kushwaha S, Jena G (2011)Investigation on sodium valproate induced germ cell damage, oxi-dative stress and genotoxicity in male Swiss mice. Reprod Toxicol32:385–389

Kiang TK, Teng XW, Karagiozov S, Surendradoss J, Chang TK, AbbottFS (2010) Role of oxidativemetabolism in the effect of valproic acidon markers of cell viability, necrosis, and oxidative stress insandwich-cultured rat hepatocytes. Toxicol Sci 118:501–509

Kiang TK, Teng XW, Surendradoss J, Karagiozov S, Abbott FS, ChangTK (2011) Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: role of biotransformation and temporalrelationship with onset of toxicity. Toxicol Appl Pharmacol 252:318–324

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein mea-surement with the folin phenol. J Biol Chem 193:265–275

Mariee AD, Abd-Allah GM, El-BeshbishyHA (2012) Protective effect ofdietary flavonoid quercetin against lipemic-oxidative hepatic injuryin hypercholesterolemic rats. Pharm Biol 8:1019–1025

Messaoudi I, El Heni J, Hammouda F, Saïd K, Kerkeni A (2009)Protective effects of selenium, zinc, or their combination oncadmium-induced oxidative stress in rat kidney. Biol Trace ElemRes 130:152–161

Milton Prabu S, Shagirtha K, Renugadevi J (2010) Quercetin in combi-nation with vitamins (C and E) improves oxidative stress and renalinjury in cadmium intoxicated rats. Eur Rev Med Pharmacol Sci 14:903–914

Mohandas J, Marshall JJ, Duggin GG, Horvath JS, Tiller DJ (1984) Lowactivities of glutathione-related enzymes as factors in the genesis ofurinary bladder cancer. Cancer Res 44:5086–5091

Moraes TB, Zanin F, da Rosa A, de Oliveira A, Coelho J, PetrilloF, Wajner M, Dutra-Filho CS (2010) Lipoic acid preventsoxidative stress in vitro and in vivo by an acute hyperphe-nylalaninemia chemically-induced in rat brain. J Neurol Sci292:89–95

Morales AI, Vicente-Sánchez C, Sandoval JM, Egido J, Mayoral P,Arévalo MA, Fernández-Tagarro M, López-Novoa JM, Pérez-Barriocanal F (2006) Protective effect of quercetin on experimentalchronic cadmium nephrotoxicity in rats is based on its antioxidantproperties. Food Chem Toxicol 44:2092–2100

Nabavi SM, Nabavi SF, Habtemariam S, Moghaddam AH, Latifi AM(2012) Ameliorative effects of quercetin on sodium fluoride-induced oxidative stress in rat’s kidney. Ren Fail 34:901–906

Park HK, Kim SJ, do Kwon Y, Park JH, Kim YC (2010) Protective effectof quercetin against paraquat-induced lung injury in rats. Life Sci 87:181–186

Paul MV, AbhilashM, VargheseMV, Alex M, Nair RH (2012) Protectiveeffects of α-tocopherol against oxidative stress related to nephro-toxicity by monosodium glutamate in rats. Toxicol Mech Methods22:625–630

Pourahmad J, Eskandari MR, Kaghazi A, Shaki F, Shahraki J, Fard JK(2012) A new approach on valproic acid induced hepatotoxicity:involvement of lysosomal membrane leakiness and cellular proteol-ysis. Toxicol in Vitro 26:545–551

Ramos AA, Lima CF, Pereira ML, Fernandes-Ferreira M, Pereira-WilsonC (2008) Antigenotoxic effects of quercetin, rutin and ursolic acidon HepG2 cells: evaluation by the comet assay. Toxicol Lett 177:66–73

Renugadevi J, Prabu SM (2010) Quercetin protects against oxidativestress-related renal dysfunction by cadmium in rats. Exp ToxicolPathol 62:471–481

Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, andnonprotein sulfhydryl groups in tissue with Ellman’s reagent. AnalBiochem 25:192–205

Singh D, Chander V, Chopra K (2004) Quercetin, a bioflavonoid, atten-uates ferric nitrilotriacetate-induced oxidative renal injury in rats.Drug Chem Toxicol 27:145–156

S. Chaudhary et al.

Spiller HA, Krenzelok EP, Klein-Schwartz W, Winter ML, Weber JA,Sollee DR, Bangh SA, Griffith JR (2000) Multicenter case series ofvalproic acid ingestion: serum concentrations and toxicity. J ToxicolClin Toxicol 38:755–760

Sung CC, Hsu YC, Chen CC, Lin YF, Wu CC (2013) Oxidative stressand nucleic acid oxidation in patients with chronic kidney disease.Oxidative Med Cell Longev 2013:301982

TabassumH, Parvez S, RehmanH,Dev Banerjee B, SiemenD, Raisuddin S(2007) Nephrotoxicity and its prevention by taurine in tamoxifeninduced oxidative stress in mice. Hum Exp Toxicol 26:509–518

Tong V, Teng XW, Chang TK, Abbott FS (2005a) Valproic acid II: effectson oxidative stress, mitochondrial membrane potential, and cytotox-icity in glutathione-depleted rat hepatocytes. Toxicol Sci 86:436–443

Tong V, Teng XW, Chang TK, Abbott FS (2005b) Valproic acid I: timecourse of lipid peroxidation biomarkers, liver toxicity, and valproicacid metabolite levels in rats. Toxicol Sci 86:427–435

Tung EW, Winn LM (2011) Valproic acid increases formation of reactiveoxygen species and induces apoptosis in postimplantation embryos:

a role for oxidative stress in valproic acid-induced neural tubedefects. Mol Pharmacol 80:979–987

Uchiyama M, Mihara M (1978) Determination of malonaldehyde pre-cursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278

Waseem M, Kaushik P, Parvez S (2013) Mitochondria-mediated mitiga-tory role of curcumin in cisplatin-induced nephrotoxicity. CellBiochem Funct 31(8):678–684

Wu G, Nan C, Rollo JC, Huang X, Tian J (2010) Sodium valproate-induced congenital cardiac abnormalities in mice are associated withthe inhibition of histone deacetylase. J Biomed Sci 17:16

Zafeer MF, Waseem M, Chaudhary S, Parvez S (2012) Cadmium-induced hepatotoxicity and its abrogation by thymoquinone.Biochem Mol Toxicol 26:199–205

Zhang M, Swarts SG, Yin L, Liu C, Tian Y, Cao Y (2011) Antioxidantproperties of quercetin. Adv Exp Biol 701:283–289

Zhu Y, Carvey PM, Ling Z (2006) Age-related changes in glutathione andglutathione-related enzymes in rat brain. Brain Res 1090:35–44

Nephroprotective activities of quercetin