ORIGINAL ARTICLE

Therapeutic potential of 7,8-dimethoxycoumarinon cisplatin- and ischemia/reperfusion injury-inducedacute renal failure in rats

Arunachalam Muthuraman & Shailja Sood &

Muthusamy Ramesh & Karan Deep Singh Puri &Anil Peters & Ashish Chauhan & Pradeep Kumar Arora &

Ajay Rana

Received: 2 December 2011 /Accepted: 27 March 2012 /Published online: 15 April 2012# Springer-Verlag 2012

Abstract This study was designed to investigate the role of7,8-dimethoxycoumarin on cisplatin- and ischemia/reperfusion(I/R)-induced acute renal failure in rats. Acute renal failure wasinduced in rats by administration of a single dose of cisplatin(CP) (6 mg/kg, intraperitoneally on day 6) and occlusion of theleft renal artery for 45 min (I) and opened for the next 24 h (R).The drug samples of 7,8-dimethoxycoumarin (DMC, 50, 75,and 100 mg/kg) and cyclosporin A (50 μM/kg) were adminis-tered orally for six consecutive days. Administration of a singledose of cisplatin and I/R event has significantly raised bloodurea nitrogen and creatinine, N-acetyl beta-D-glucosaminidase,and thiobarbituric acid reactive substances but decreased FrNa,creatinine clearance, reduced glutathione (GSH), mitochondrialcytochrome c oxidase, and adenosine triphosphate levels.

Further, pretreatment of DMC (50, 75, and 100 mg/kg, p.o.,for six consecutive days) has ameliorated the CP- and I/R-induced biochemical and histopathological changes in a dose-dependent manner. Furthermore, 75 and 100 mg/kg of 7,8-dimethoxycoumarin has shown to possess the significant reno-protective effect similar to that of the cyclosporin A-treatedgroup which served as positive control. Based on the results ofthe present study, it has been concluded that 7,8-dimethoxy-coumarin protects the kidney against the CP and I/R injury viaantioxidant, anti-inflammatory, and inactivation of mitochon-drial permeability transition pore opening.

Keywords 7,8-dimethoxycoumarin . Acute renal failure .

Cisplatin . Cyclosporin A .Mitochondrial permeabilitytransition pore

Introduction

The present study was designed to investigate the effect of 7,8-dimethoxycoumarin of Citrus decumana peels on cisplatin-and ischemia/reperfusion injury-induced acute renal failure inrats. Acute renal failure (ARF) occurs when kidneys are unableto excrete or discharge the daily load of waste materials andtoxins in the urine. It is characterized by a sudden decline in theglomerular filtration rate, the accumulation of nitrogenouswaste substances such as blood urea nitrogen (BUN) as wellas uric acid, and inability to regulate the electrolyte and waterbalance (Muthuraman et al. 2011a, b, c, d; Gill et al. 2005).Cisplatin (chemotherapeutic agent) is a divalent platinum com-pound with a potent nonspecific cell killing activity. It has beenemployed for the management of various types of tumors, i.e.,cancer of the testis, ovary, epithelium, bladder, endometrium,

A. Muthuraman : S. Sood :K. D. S. Puri :A. Peters :A. Chauhan : P. K. Arora :A. RanaDepartment of Pharmaceutical Chemistry,Rayat Institute of Pharmacy,Near Railmajra,Ropar 144533 Punjab, India

A. Muthuraman (*)Department of Pharmaceutical Sciences and Drug Research,Punjabi University,Patiala 147002 Punjab, Indiae-mail: arunachalammu@gmail.com

M. RameshDepartment of Medicinal Chemistry, National Instituteof Pharmaceutical Education and Research (NIPER),Mohali 160062 Punjab, India

A. Chauhan : P. K. AroraDepartment of Pharmaceutical Sciences,Lovely Professional University,Jalandhar 144402 Punjab, India

Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:739–748DOI 10.1007/s00210-012-0751-1

lung, and cervical region (Muthuraman et al. 2011a, b, c, d;Carvalho Rodrigues et al. 2010). However, the therapeuticeffect of cisplatin has been limited due to its nephrotoxicity(Carvalho Rodrigues et al. 2010; Hanigan and Devarajan2003). Mechanism of cisplatin-induced renal toxic effects hasbeen documented; it rises the free radical generation, lipidperoxidation, depletion of reduced form of glutathione(GSH), loss of adenosine triphosphate (ATP) generation, open-ing of mitochondrial permeability transition pore (MPTP), andimpairment of antioxidant defensive enzyme activities, i.e.,superoxide dismutase, catalase, and glutathione peroxidase,including DNA adduct formation (Kuriakose and Kurup2010; Muthuraman et al. 2011a, c).

In clinical setup, ischemia and reperfusion technique hasbeen employed in renal transplantation, cannulation, renal sur-gery, and cardiac bypass surgery (Rauchfuss et al. 2011; Henryand Guarrera 2011). Prolonged ischemic insult can cause an-oxic cell death via cell necrosis and/or apoptosis in varioustissue and organ system (Muthuraman et al. 2010a; 2011a, b, c,d). Recent evidence suggests that sublethal injury could beamplified by production of inflammatory mediators and acti-vation of cytotoxic cascades during the reperfusion event(Muthuraman et al. 2010a). Indeed, reperfusion injury has beenwell described in the literature to cause organ damage in thekidney, stomach, heart, lungs, liver, brain, nerve, and skeletalmuscle (Muthuraman et al. 2011a, b, c, d; 2010a; b). Numerousnumber of researcher worked on Citrus fruits, and their reportdemonstrated the potential therapeutic action to ameliorate theprogress of diseases such as cancer, inflammation, heart dis-ease, ulcers, and so on (Sood et al. 2010). Citrus juices areknown to possess a free radical scavenging molecule calledantioxidants including vitamin C, phenolic compounds, andcarotenoids (Guimaraes et al. 2010). Citrus peel extract hasalso been used traditionally in India for its beneficial effect inrelieving rheumatic pain and headache and for cosmetic prep-arations. Our previous research also demonstrated that C.decumana peel extracts possess potent analgesic, anti-inflammatory, and antiulcerative potential (Sood et al. 2009,2010). The peels of Citrus species have shown to posses theantioxidant activity due to the presence of coumarin, flavo-noids, and other active phenolic compounds (Sood et al. 2010).One of the current research reports has revealed that coumarinand coumarin derivatives possess the hepatoprotective actionagainst carbon tetrachloride (CCl4)-induced acute hepatic fail-ure in rats (Murat Bilgin et al. 2011). Our recent studies havealso documented that 7,8-dimethoxycoumarin possesses strongantioxidant, antisecretary, anti-inflammatory, and antiulcerativepotential (Sood et al. 2009, 2010). Some experimental studieshave reported that 6,7-dimethoxycoumarin is known to possesthe ameliorative potential on endotoxin-induced ARF in mice(Xing-mei et al. 1999).

Mitochondria play a vital role in both apoptosis and necro-sis processes of cell death via opening the cyclosporine A-

sensitive MPTP (Muthuraman et al. 2011a, b, c, d). Cell deaththat is associated with MPTP opening has been described asthat which is due to swelling and destruction of the outermitochondrial membrane as well as raising the energy demand(Carvalho Rodrigues et al. 2010; Muthuraman et al. 2011a; b).

The light which is interesting note is that cyclosporine Awas known to possess the potent inhibition of MPTP (Liuet al. 2011; Muthuraman et al. 2011a, b, c, d; Muthuraman etal. 2011b). Therefore, cyclosporine A has been used in thepresent study as a positive control. Based on the literaturereport, the present study was designed to investigate the roleof 7,8-dimethoxycoumarin (DMC) on cisplatin- and ischemia/reperfusion injury-induced acute renal failure in rats.

Materials and methods

Animal

Male Sprague Dawley rats (180–250 g) were procured fromPanjab University, Chandigarh. The animals were housedunder standard conditions of natural 12-h light and darkcycle, a temperature of 23±2°C, and a relative humidity of50 to 60 % with free access to food (Hindustan LeverProducts, Kolkata, India) and water ad libitum. An acclima-tization period of 7 days was allowed for the rats beforeexperimentation in the laboratory. The experimental proto-cols and surgical procedures were approved by the Institu-tional Animal Ethics Committee, and care of the animalswas carried out in accordance with the guidelines of theCommittee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA), Ministry of Environ-ment and Forest, Government of India (reg. no. 874/ac/05/CPCSEA).

Chemicals

Chemicals such as Folin–Ciocalteu's phenol reagent (MerckLimited, Mumbai), 5,5′-dithiobis(2-nitrobenzoic acid), re-duced glutathione (GSH), bovine serum albumin (BSA)(Sisco Research Laboratories Pvt. Ltd., Mumbai and S. D.Fine Chemicals, Mumbai), thiobarbituric acid, and 1,1,3,3-tetramethoxypropane (Loba Chem, Mumbai) were procuredfor the present study. Cisplatin was obtained as a gift samplefrom Ranbaxy Pharmaceuticals, Mumbai.

Plant material

The fruits of C. decumana were collected from the northernregion of India. The plant material was authenticated, and thevoucher specimen number 0353 has been deposited in theBotanical and Environmental Science Department, GuruNanak Dev University, Amritsar. The fruits were washed

740 Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:739–748

and dried properly before removing the peels. The peels werethen dried under shade at room temperature. The dried peelswere grounded into a coarse powder in a mixer. The powderwas sieved through a 1-mm metal sieve to obtain a standardparticle size.

Extraction

The dried peel powder of C. decumana (100 g) plant materialwas extracted by a maceration process using solvents of ethylacetate at room temperature over a period of 24 h, and fewprocedures were carried out thrice. The material was kept for24 h between each successive solvent for proper drying. Theextracts were filtered and concentrated under vacuum on arotary evaporator at 40°C (yield is 12.45 g) and stored in arefrigerator for further analysis.

Isolation and characterization

The compound 7,8-dimethoxycoumarin has been isolatedfrom ethyl acetate extract of C. decumana peels as describedin the procedure of Sood et al. (2010). Briefly, the ethyl acetateextract of C. decumana peels was fractionated by silica gelcolumn chromatography. The column chromatography wascarried out using toluene/acetone (9:1) as mobile phase. Elut-ing solvent fractions of 50ml were collected and monitored bythin layer chromatography (TLC) (8×3 cm). The fractionsshowing the same retention factor (Rf) value on TLC plateswere pooled, and three pools were obtained. The solvent wasremoved in vacuum, and the residues obtained were furtherpurified by preparative TLC (PTLC, 20×20 cm). Pool 2 wassubjected to PTLC using toluene/acetone (9:1) as a solventsystem. Three bands were observed under UV light. Sufficientamount of material of these bands was collected by scrappingoff the TLC plates. The material was dissolved in methanol,filtered, and then concentrated (98 % purity). The spectralanalysis of isolated compound was carried out using IR andNMR techniques. The isolated compoundwas further used foracute renal failure studies.

Induction of ARF by cisplatin

Acute renal failure has been induced by ischemia followed byreperfusion technique as described in the method of Ali et al.(2008). Briefly, single injection of cisplatin (6 mg/kg) wasadministered intraperitoneally (i.p.) at the end of the experi-mental protocol, i.e., on the sixth day.

Induction of ARF by ischemia and reperfusion

Acute renal failure has been induced by ischemia followed byreperfusion technique as described in the method of Takada etal. (1997) with slight modification of Muthuraman et al.

(2010a). Briefly, after inducing anesthesia with pentobarbital(35 mg/kg, i.p.), the abdominal cavity was exposed by open-ing the skin and muscle layer at a suitable location. Rightnephrectomy was performed via dorsal incision. The leftkidney and renal vessels were exposed followed by occlusionof the left renal artery and vein using a vascular clip for thewarm ischemia/reperfusion study. The clip was released after45 min, as the kidney does not recover if there is a prolongedinjury (60–90 min) (29). Occlusion was visually verified by achange in the color of the kidney, which became paler shade,and reperfusion was verified by the appearance of a blush.Anesthesia was maintained by administration of supplemen-tary injections of sodium pentobarbital. After performing is-chemia followed by reperfusion event, muscular and skinlayer was immediately sutured with thread, and topical anti-biotic was applied.

Experimental design

Twelve groups were employed in the present study. Each groupcomprising of six male Sprague Dawley rats rats (n06) wasinvolved in the acute renal failure study.

Group Ι (normal control group): Rats were not subjected toany drug administration and performance of surgical event.

Group IΙ (sham control group): Rats were subjected to asurgical procedure to expose the left kidneywithout performingany ischemic/reperfusion event.

Group IIΙ (cisplatin (CP) control group): Rats were sub-jected to administration of a single injection of cisplatin(6 mg/kg, i.p.) which was given on the sixth day as describedby Ali et al. (2008b).

Group IV (ischemia/reperfusion (I/R) control group):Rats were subjected to left renal ischemic/reperfusion forinduction of acute renal failure.

Group V and X (DMC+CP and DMC+I/R): Rats weresubjected to administration of DMC (50, 75, and 100 mg/kg,p.o.) for five consecutive days, and last dose (sixth dose)was administered 1 h before the cisplatin treatment andischemic/reperfusion event.

Group XI and XII (cyclosporin A (CsA)+CP and CsA+I/R): Rats were subjected to administration of CsA (50 μM/kg,p.o.) for five consecutive days, and last dose (sixth dose) wasadministered 1 h before the cisplatin treatment and ischemic/reperfusion event.

The study of the National Toxicology Program has reportedthat in the administration of 25, 50, or 100 mg/kg coumarin byoral gavage (5 days/week in F344 rats for 2 years), there wasno increase in liver tumors in any dose group of male andfemale rats (NTP 1993; Felter et al. 2006). Based on literaturereport and our previous research report (Sood et al. 2010), weselected the administration of DMC (50, 75, and 100 mg/kg,p.o.) for six consecutive days in rats. Further, we have showedthe effect of single oral administration of DMC (25, 50 and

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100 mg/kg, p.o.) before the cisplatin and I/R injury for induc-tion of acute renal failure. In addition, posttreatment study wasalso carried out by administration of DMC (25, 50, and100 mg/kg, p.o. on the seventh day) after induction of acuterenal failure by cisplatin and I/R injury.

Biochemical estimation

On the sixth day, rats were placed in the metabolic cages forthe collection of urine samples over the period of 24 h. On theseventh day, rats were anesthetized with diethyl ether. Bloodwas collected from the inferior vena cava in plain plastic tubeswith and without anticoagulant (sodium citrate), left to standat 4°C for 1 h, and centrifuged at 900×g at 5°C for 15 min toseparate serum and plasma. The serum samples obtained werestored for carrying out biochemical analyses, and the rats weresacrificed by using the method of euthanasia (overdose ofthiopental sodium 50 mg/kg, i.p.). The kidneys were removedfrom the rats and washed with ice-cold saline. A small piecefrom the left kidney was fixed in 10 % buffered formalin. Themedullary portion of the kidney was homogenized in ice-coldsaline to produce 10 % (w/v) tissue homogenate.

Evaluation of renal function

BUN and creatinine (Cr) were estimated in serum sample,whereas N-acetyl beta-D-glucosaminidase (NAG) was esti-mated in urine sample by using standard diagnostic kits (SpanDiagnostics, Gujarat, India). Abnormal changes in BUN andCr level served as an indicator of impaired glomerular func-tion, whereas changes in NAG level served as a specificindicator of tubular damage. The fractional excretion of sodi-um (FrNa0sodiumurine / sodiumplasma×creatinineplasma / crea-tinineurine×100) and the creatinine clearance (CrCl0creatinineurine / creatinineplasma×urinevolume / time) were mea-sured as index of renal function.

Estimation of tissue total protein content

Renal tissue total protein content was estimated as describedin the method of Lowry et al. (1951) using BSA as a standard.The absorbance was determined spectrophotometrically at750 nm. The concentration of tissue total protein contentwas expressed in terms of milligrams of protein per gram oftissue.

Estimation of the levels of tissue thiobarbituric acid reactivesubstances

Estimation of lipid peroxidation was performed by measur-ing the thiobarbituric acid reactive substances (TBARS) asdescribed in the method of Okhawa et al. (1979). A standardcalibration curve was prepared by using 1–10 nM of 1,1,3,3-

tetramethoxypropane. The concentration was expressed interms of nanomolars of TBARS per milligram of protein.

Estimation of tissue reduced glutathione

The endogenous antioxidant molecule, i.e., reduced gluta-thione (GSH) content, was estimated as described in themethod of Ellman (1959). A standard curve was plottedusing 5–50 μM of reduced form of glutathione. The con-centration was expressed in terms of micromolars of GSHper milligram of protein.

Estimation of tissue total calcium

Total calcium levels were estimated in the renal tissue asdescribed by Severnghaus and Ferrebee (1950) with slightmodification of Muthuraman et al. (2011c). The renal tissuehomogenate was mixed with 1 ml of trichloroacetic acid (4 %)in the ice-cold condition and centrifuged at 1,500×g for10 min. The clear supernatant was used for estimating the totalcalcium levels by atomic emission spectroscopy at 556 nm.

Preparation of mitochondria

Renal mitochondria were isolated from rat kidney tissue asdescribed in the method of Stephan et al. (1991) with slightmodification of Long et al. (2006). Briefly, tissues werewashed with saline, weighed, and put into ice-cold isolationbuffer containing 0.25 M sucrose, 10 mM Tris base, and0.5 mM EDTA (pH 7.4). Tissues were homogenized in 2.5volume of isolation buffer. The homogenate was adjusted toeight volumes with the isolation buffer and centrifuged at1,000×g for 4 min. The supernatant fraction was decantedand saved. The pellet was washed once with two volumes ofthe isolation buffer. The supernatant fractions were combinedand centrifuged at 10,000×g for 4 min. The mitochondrialpellet was washed twice with the isolation buffer. All theabove mentioned operations were carried out at 4°C. Themitochondrial protein concentration was determined as de-scribed in the method of Lowry et al. (1951) using BSA as astandard. An aliquot was used for the estimation of mitochon-dria respiration chain function (i.e., cytochrome c oxidaseactivity) and ATP content.

Estimation of cytochrome c oxidase activity and ATPcontent

The activity of cytochrome c oxidase (as an index of respiratorymarker enzymes) was measured according to the method ofRustin et al. (1994). The enzymatic activity was measured byfollowing the decrease in absorbance of the reaction mixture at550 nm with 580 nm as the reference wavelength (ε019.1 mM−1 cm−1). The activity of cytochrome c oxidase was

742 Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:739–748

measured at 25°C for 10 min, and activity was expressed interms of nanomolars of cytochrome c oxidase per minute permilligram of protein. Adenosine triphosphate was measured byusing HPLC technique as described in the method of Paroniet al. (1995) and Vecchi et al. (1998) after neutralization of theacid supernatant with sodium bicarbonate. The content of ATPwas expressed in terms of nanomolars of ATP per milligram ofprotein.

Histopathological evaluation

Samples of kidney were stored in the fixative solution (10 %formalin) and cut into 4-μm thickness size. Staining was doneby using hematoxylin and eosin as described by Girardi et al.(2011). The stained renal corticomedullary fields were ob-served at a magnification of×450. Histopathological changesin the renal tissue section were examined with the help of apathologist.

Statistical analysis

All the test results were expressed as mean±standard errorof means (SEM). The data of all biochemical estimationwere statistically analyzed by one-way ANOVA followed byTukey's multiple range tests by using Sigmastat version 2.0software. The P value <0.05 was considered to be statisticallysignificant.

Result

Effect of 7,8-dimethoxycoumarin on renal functionalmarkers

The results demonstrated that cisplatin and ischemia andreperfusion (I/R) event causes marked changes in renalfunctional markers, i.e., a rise in serum Cr, BUN, andurinary NAG and a decrease in FrNa and CrCl, as comparedto normal and sham control groups, respectively. Pretreat-ment with 7,8-dimethoxycoumarin has shown the ameliora-tive effect in a dose-dependent manner. Moreover, 7,8-dimethoxycoumarin (75 and 100 mg/kg)-treated groupshave shown a significant improvement on cisplatin- and I/R-induced alteration in the serum and urinary biomarkerchanges which were comparable with cyclosporine A-treated group (Table 1).

Effect of 7,8-dimethoxycoumarin on tissueand mitochondrial biomarker changes

The results demonstrated that cisplatin and I/R event causesa marked changes in tissue and mitochondrial biomarkers,i.e., a rise in TBARS and total calcium whereas a decrease

in reduced glutathione (GSH), mitochondrial cytochrome coxidase (Fig. 1) and ATP levels (Fig. 2), as compared tonormal and sham control groups, respectively. Pretreatmentwith 7,8-dimethoxycoumarin has shown the ameliorativeeffect in a dose-dependent manner. Moreover, 7,8-dime-thoxycoumarin (75 and 100 mg/kg)-treated groups haveshown an improvement in cisplatin- and I/R-induced alter-ation in the tissue and mitochondrial biomarker changeswhich were comparable with cyclosporine A-treated group(Table 2).

Effect of 7,8-dimethoxycoumarin on histopathologicalchanges

The histopathological changes are shown in Fig. 3. Thenormal- and sham-operated groups did not show any mor-phological changes (Fig. 3a, b). By contrast, the kidneys ofcisplatin administration- and I/R event-operated rats showedtubular cell swelling, cellular vacuolization, pyknotic nuclei,medullary congestion, and moderate to severe necrosis(Fig. 3c, d). However, cyclosporine A and 7,8-dimethoxy-coumarin (50, 75, and 100 mg/kg) pretreated rats showedthe prevention of the renal damage with slight changes inglomeruli and tubular cells (Fig. 3e–h).

Role of the pretreatment and posttreatmentof 7,8-dimethoxycoumarin in acute renal failure changes

The single-dose pretreatment and posttreatment of 7,8-dime-thoxycoumarin (25, 50, and 100 mg/kg, p.o.) did not produceany preventive and protective effect in both of the models ofacute renal failure in male Sprague Dawley rats.

Discussion

The present study demonstrated that the pretreatment of 7,8-dimethoxycoumarin (50, 75, and 100 mg/kg) significantlydecreases the blood BUN, Cr, urinary NAG, and TBARSand increases the FrNa, CrCl, GSH, cytochrome c oxidaseand ATP levels, which result in the attenuation of both renaldysfunction and mitochondrial damage as well as histopath-ological changes in rats subjected to cisplatin- and I/Revent-induced acute renal failure. This is the first report, asfar as we are aware, on the effect of 7,8-dimethoxycoumarinin cisplatin- and I/R event-induced renal injury via inactiva-tion of MPTP opening.

Coumarins are a group of natural phenolic compounds andare found in many herbal plants and food products such ascitrus fruits, tomatoes, green vegetables, and green tea. Morethan 1,300 coumarins have been identified and isolated fromnatural sources. Further, some of the coumarins and theirderivatives have been synthesized in research laboratories

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(Murat Bilgin et al. 2011). It has also been proved to havevarious pharmacological properties such as antithrombotic,antiviral, antitumor, anti-inflammatory, and ulcer-protectiveproperties (Murat Bilgin et al. 2011; Sood et al. 2010; Vooraet al. 2005). Mechanistically, 6,7-dimethoxycoumarin hasbeen reported that it possesses the renoprotective action viaenhancing the production of prostaglandin-I2 and nitric oxide(NO) (Xing-mei et al. 1999).

Several pharmacokinetic studies of coumarin derivativeshave been performed in rodents as well as in humans, whichrevealed that coumarin derivatives undergo the extensive first-pass metabolism. In F344 rats, the administration of coumarinby oral gavage has been reported to reduce the 3.5-fold level ofcoumarin in plasma than in mice, and the plasma half-life is five

times longer than that in mice (Born et al. 2000). In rat, 7-hydroxylation is a minor route of coumarin clearance in the rat,hamster, and many strains of mice. In these species, coumarin ispreferentially metabolized to a coumarin 3,4-epoxide interme-diate that rearranges to o-hydroxyphenylacetaldehyde. Furtheroxidation of this aldehyde yields o-hydroxyphenylacetic acid(o-HPAA), the major coumarin metabolite detected in rat urine(Born et al. 2000; Lake 1999). Themetabolites of coumarin, i.e.,o-HPAA, have reached the concentration of 37mg/ml in plasmaand 41 % in the urine. The Cmax of 7-hydroxycoumarin hasbeen documented to be 12 % of the coumarin treatment in ratand 7 % in mice (Born et al. 2003). In human, 7-hydroxycoumarin (7-HC) metabolite, i.e., 7-hydroxycoumaringlucuronide (7-HC-G), is formed through glucuronide

Fig. 1 Effect of DMC incisplatin- and I/R-inducedchanges in cytochrome Coxidase activity. Values inparentheses indicate the dose inmilligrams per kilogram forDMC and in micrograms perkilogram for cyclosporine A.CP, cisplatin; CsA, cyclosporineA; DMC, 7,8-dimethoxycou-marin. Values are mean±SEMof six animals (n06). aP<0.05,vs. normal control group; bP<0.05, vs. cisplatin controlgroup; cP<0.05, vs. I/Rcontrol group

Table 1 Effect of DMC in cisplatin and I/R-induced renal functional marker changes

Groups BUN (mg/dl) Cr (mg/dl) NAG (U/min/l) FrNa (%) CrCl (ml/min/100 g)

Normal 17.9±0.99 0.35±0.04 26.6±3.04 71.6±2.9 0.66±0.05

Sham 18.1±1.08 0.33±0.03 25.9±2.83 69.3±3.2 0.67±0.04

CP (6) 41.4±1.37* 0.70±0.03* 96.7±3.71* 14.74±5.8* 0.06±0.02*

I/R 44.2±1.23* 0.72±0.02* 98.2±3.49* 13.82±6.7* 0.07±0.01*

CP+DMC (50) 37.1±1.16* 0.65±0.08* 85.7±4.05* 19.54±3.9* 0.21±0.13*

CP+DMC (75) 26.5±1.19** 0.46±0.09** 56.9±3.63** 50.37±3.7** 0.44±0.03**

CP+DMC (100) 24.9±1.41** 0.41±0.06** 34.3±3.06** 56.3±5.2** 0.54±0.07**

I/R+DMC (50) 38.6±1.23* 0.62±0.05* 81.5±3.67* 19.54±3.9* 0.24±0.27*

I/R+DMC (75) 25.7±1.11*** 0.43±0.04*** 52.4±3.86*** 52.62±4.9*** 0.47±0.05***

I/R+DMC (100) 23.4±1.25*** 0.39±0.08*** 31.7±3.58*** 57.5±4.6*** 0.57±0.12***

CP+CsA (50) 20.5±0.92** 0.36±0.05** 27.5±3.46** 66.1±3.2** 0.59±0.06**

I/R+CsA (50) 22.03±1.07*** 0.35±0.03*** 26.1±3.21*** 65.3±3.7*** 0.61±0.13***

Values in parenthesis indicate the dose in milligrams per kilogram for DMC and in micrograms per kilogram for cyclosporine A. Values are mean±SEM of six animals (n06)

BUN blood urea nitrogen, CP cisplatin, Cr creatinine, CsA cyclosporine A, CrCl creatinine clearance, DMC 7,8-dimethoxycoumarin, FrNafractional excretion of sodium, NAG N-acetyl beta-D-glucosaminidase, I/R ischemia and reperfusion

*P<0.05 (vs. normal control group); **P<0.05 (vs. cisplatin control group); ***P<0.05 (vs. I/R control group)

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conjugation, and 80–90 % of the absorbed coumarin is excretedinto urine in the form of 7-HC-G by active transport processes.The remaining 10–20% of coumarin derivatives is known to bemetabolized by cytochrome P450 (CYP) enzymes in the liver(Wittgen et al. 2012). In humans, coumarin is extensivelydetoxified by CYP2A6 via the formation of 7-HC, which isfurther metabolized to its glucuronide and sulfate conjugatesprior to excretion in the urine (Wittgen et al. 2012).

Coumarin and their metabolites are of great interest in thepharmaceutical study due to their biological properties espe-cially their physiological, bacteriostatic, and antitumor activity.Coumarin and their metabolites have shown antitumour activ-ity in vivo, with the effect believed to be due to its metabolites,i.e., 7-hydroxycoumarin (Lacy and O'Kennedy 2004). A

recent study has shown that 7-hydroxycoumarin inhibits therelease of cyclin D1, which is overexpressed in many types ofcancer (Lacy and O'Kennedy 2004). Both coumarin and cou-marin derivatives have shown promise as potential inhibitorsof cellular proliferation in various carcinoma cell lines. Inaddition, it has been shown that 4-hydroxycoumarin and 7-hydroxycoumarin inhibited cell proliferation in a gastric car-cinoma cell line (Budzisz et al. 2003).

As a pharmaceutical approach, coumarin and their deriv-atives have been used in variety of disease managements.Unlike, coumarin and other coumarin derivatives have beenused as a “venotonic” to promote vein health and smallcapillaries and venule blood flow at low doses (typically7–10 mg/day). Clinically, coumarin derivatives have been

Fig. 2 Effect of DMC incisplatin- and I/R-inducedchanges in ATP content. Valuesin parentheses indicate the dosein milligrams per kilogram forDMC and in micrograms perkilogram for cyclosporineA. CP, cisplatin; CsA, cyclo-sporine A; DMC, 7,8-dime-thoxycoumarin. Values aremean±SEM of six animals(n06). aP<0.05, vs. normalcontrol group; bP<0.05, vs.cisplatin control group. cP<0.05, vs. I/R control group

Table 2 Effect of DMC in cisplatin and I/R-induced tissue biomarker changes

Groups MDA (nmol/mg of protein) GSH (μmol/mg of protein) Total calcium (ppm/mg of protein)

Normal 37.49±1.98 10.03±0.37 11.35±1.69

Sham 39.27±2.27 9.83±0.51 13.62±1.29

CP (6) 89.28±2.74* 2.94±0.37* 121.57±3.97*

I/R 92.64±2.38* 2.87±0.33* 123.32±4.04*

CP+DMC (50) 84.61±3.73* 3.73±0.42* 104.71±3.94*

CP+DMC (75) 56.04±2.85** 7.83±0.35** 56.39±3.74**

CP+DMC (100) 48.93±2.37** 8.67±0.71** 38.67±3.63**

I/R+DMC (50) 81.32±2.59* 4.36±0.82* 99.58±2.97*

I/R+DMC (75) 52.19±2.83*** 8.02±0.28*** 51.59±3.09***

I/R+DMC (100) 44.69±2.23*** 8.83±0.64*** 33.47±2.65***

CP+CsA (50) 42.27±1.93** 9.12±0.48** 25.48±2.94**

I/R+CsA (50) 40.63±2.05*** 9.84±0.92*** 24.72±3.09***

Values in parenthesis indicate the dose in milligrams per kilogram for DMC and in micrograms per kilogram for cyclosporine A. Values are mean±SEM of six animals (n06)

CP cisplatin, CsA cyclosporine A, DMC 7,8-dimethoxycoumarin, TBARS thiobarbituric reactive substances, GSH reduced glutathione

*P<0.05 (vs. normal control group); **P<0.05 (vs. cisplatin control group); ***P<0.05 (vs. I/R control group)

Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:739–748 745

used for the treatment of high-protein lymphedema at 100–800-mg/day dose. Administration of 400 mg/day coumarinhas been documented to be a successful treatment oflymphedema patients with mastectomy or other surgery(Casley-Smith et al. 1993; Chang et al. 1996; Felter et al.2006). Experimentally, coumarin and coumarin derivativeshave been evidenced for the management of lung, prostate,and kidney carcinoma (Thornes 1993; Felter et al. 2006). Inaddition, coumarin derivatives have also reported to have apotential role in gastroprotective and hepatoprotectiveactions via antioxidative, anti-inflammatory, and immuno-suppressive actions (Atmaca et al. 2011; Murat Bilgin et al.2011; Sood et al. 2010).

The present study has also proposed that herbal constitu-ents, i.e., 7,8-dimethoxycoumarin from C. decumana peelextracts, have shown to possess therapeutic potentials on thecisplatin- and I/R-induced renal injury. Mechanistically,cisplatin-induced renal injury has been well documented toinduce the generation of free radical (i.e., reactive oxygenspecies and reactive nitrogen species), rise in lipid peroxida-tion, loss of ATP generation, accumulation of calcium, dam-age renal mitochondria, opening of MPTP, and alteration ofcellular enzymatic systems (Carvalho Rodrigues et al. 2010;Muthuraman et al. 2011c). I/R event has been reported that itis inducing the potential inflammation in vascular and targetedorgan as well as in body tissue (Muthuraman et al. 2011b).Inflammatory responses activated by interaction with endo-thelial cells, leukocytes, and surrounding tissues are criticalsteps in the pathogenesis of I/R injury (Schlichting et al.2006). Ischemia followed by reperfusion has been inducingthe renal injury due to its blood flow alteration and blood andtissue component modulations (Sauriyal et al. 2011; Sehirliet al. 2008). Microvascular occlusion has been known toproduce the excess free radicals, alteration of defensive

enzymes, and modulate the expression of some specific pro-tein (Korthuis and Unthank 2000). In both of the models, freeradical and mitochondria (i.e., mitochondrial enzymes andMPTP channel) have played a major key role in the patho-genesis of acute renal failure (Muthuraman et al. 2011c;2010a). Many researchers have reported that CsA is a potentselective inhibitor of MPTP opening due to its binding affinitywith cyclophilin A protein of MPTP (Muthuraman et al.2011c; Armstrong 2006). Our previous study has also shownthe antioxidant and anti-inflammatory action of cyclosporineA in renal tissue (Muthuraman et al. 2011c; b; 2010a). Data ofthe present study revealed that 7,8-dimethoxycoumarinattenuates the cisplatin- and I/R-induced biochemical andhistopathological changes which are similar to that of thepositive control (cyclosporin A-treated group).

Conclusion

7,8-Dimethoxycoumarin was found to have an ameliorativeeffect on cisplatin- and I/R-induced acute renal failure in ratswhich was comparable to cyclosporin A. Therefore, it canbe concluded that 7,8-dimethoxycoumarin possesses thetherapeutic effect on cisplatin- and I/R-induced acute renalfailure due to its antioxidant, anti-inflammatory, and inacti-vation potential of MPTP opening.

Acknowledgments Thanks to all the faculty members of Rayat Insti-tute of Pharmacy for their encouragement and support. We are alsograteful to Rayat and Bahra Educational and Research Trust for theirunconditional help to carry out this project.

Conflict of interest There was no conflict of interest in the presentstudy.

Fig. 3 Effect of DMC in cisplatin- and I/R-induced histopathologicalchanges. a–j Transverse section of renal tissue of sham, cisplatin (CP),ischemia and reperfusion (I/R), CP+DMC (50, 75, and 100 mg/kg),I/R+DMC (50, 75, and 100 mg/kg), and cyclosporine A-pretreatedgroups. In Fig. 3b, c, black arrow shows cisplatin- and I/R-induced

tubular cell swelling, cellular vacuolization, pyknotic nuclei, medullarycongestion, and moderate to severe necrosis. In Fig. 3e, f, h–j has shownthe renoprotective effect of DMC (75 and 100 mg/kg) and cyclosporineA. Furthermore, Fig. 3d, g showsmild effect in cisplatin- and I/R-inducedrenal injury

746 Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:739–748

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