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Renal Failure, 33(1): 66–71, (2011)Copyright © Informa Healthcare USA, Inc.ISSN 0886-022X print/1525-6049 onlineDOI: 10.3109/0886022X.2010.541584

LRNFLABORATORY STUDY

Protective Effect of Docosahexaenoic Acid Against Cyclosporine A-Induced Nephrotoxicity in Rats: A Possible Mechanism of Action

Docosahexaenoic acid and cyclosporine A-induced nephrotoxicityAmr Darwish Mariee1,2 and Mohamed Fahmy Abd-Ellah3

1Department of Biochemistry, College of Pharmacy, Al-Azhar University, Cairo, Egypt; 2Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Al Madinah, Al Munawarah, Kingdom of Saudi Arabia; 3Department of Pharmacology, College of Pharmacy, Al-Azhar University, Cairo, Egypt

Abstract

The aim of this experimental study was to investigate whether, and how then, docosahexaenoic acid (DHA) could alle-viate the cyclosporine A (CsA)-induced nephrotoxicity. Three main groups of Sprague–Dawley rats were treated orallywith CsA (25 mg/kg), DHA (100 mg/kg), and CsA along with DHA. A corresponding control group was also used. DHAadministration significantly reduced CsA-induced nephrotoxicity and associated hyperlipidemia and proteinuria asassessed by estimating serum triacylglycerol, serum total cholesterol, serum total protein, serum urea, and creatinineclearance. Furthermore, urinary excretions of protein and N-acetyl-b-D-glucosaminidase were significantly inhibitedfollowing DHA administration. DHA supplementation slightly attenuated the oxidative damage in kidney tissues asevaluated by the levels of thiobarbituric acid-reacting substances and protein carbonyl content in the kidney homoge-nate, although there were no significant differences between CsA-intoxicated and DHA-treated animals. Moreover,DHA treatment significantly restored total nitric oxide (NO) levels in both renal tissues and urine. This study demon-strates the ability of DHA to ameliorate CsA-induced renal dysfunction, which might be beneficial to enhance the ther-apeutic index of CsA. The data suggest that the protective potential of DHA in the prevention of CsA nephrotoxicity inrats was mainly associated with the increase of total NO bioavailability in renal tissues. Nevertheless, the exact inde-pendent mechanism in which DHA exerts its beneficial effect is yet to be fully elucidated.

Keywords: CsA, DHA, nephrotoxicity, NO, rat

INTRODUCTION

Cyclosporine A (CsA) is a highly insoluble polypeptideconsisting of 11 amino acids. It is an immunosuppres-sive drug that has provided a crucial support in trans-plantation medicine and has improved survival ratesafter organ transplantation, when compared with con-ventional immunosuppressant therapy. However, therenal damage consequent to CsA administration haslimited its clinical usage.1–3

CsA nephrotoxicity is distinguished by inducing astrong vasoconstriction in the afferent preglomerulararteriole that induces a decrease in renal plasma flowand glomerular filtration rate leading to renal functionimpairment. Renal vasoconstriction is attributed to adisturbance in the complex dynamic interaction of pre-dominating vasoconstrictors with counteracting vasoac-tive substances.2,4–8 Moreover, the oxidative damage inthe body induced by reactive oxygen species and lipid

peroxidation products is frequently reported in the CsAnephrotoxicity.9–11 The involvement of free radical spe-cies in CsA nephrotoxicity was supported by the factthat many antioxidants and free radical scavengers pro-vide marked functional protection.12–15

Docosahexaenoic acid (22:6, n–3; DHA) is an essen-tial polyunsaturated fatty acid, which is predominant infish and marine oils.16 Polyunsaturated fatty acids areknown to have potential cardiovascular benefits thatinclude lowering blood pressure and improving vascu-lar tone, whereas their impact on lipid peroxidation hasbeen controversial.17–21 In view of this, we investigatedthe ability of DHA to restore the deteriorated renalfunctions induced by CsA in a rat model. Furthermore,renal tissue nitric oxide (NO) level, lipid peroxidation,protein oxidation, and urinary level of NO were investi-gated in an attempt to understand the mechanistic basisof the observed DHA activities.

Address correspondence to Amr Darwish Mariee, Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, P.O. Box 30001, Al Madinah Al Munawarah, Kingdom of Saudi Arabia. E-mail: amrmariee@yahoo.co.uk

Received 2 July 2010; revised 22 August 2010; accepted 28 October 2010

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Docosahexaenoic Acid and Cyclosporine A-Induced Nephrotoxicity 67

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MATERIALS AND METHODS

ChemicalsCsA [(Novartis Pharma AG, Basel, Switzerland)] wasdissolved in olive oil to give a final concentration of10 mg/mL. cis-4,7,10,13,16,19-DHA was purchasedfrom Sigma-Aldrich Chemical (St. Louis, MO, USA).All other chemicals used were of the highest availablecommercial grade.

AnimalsForty-eight male Sprague–Dawley rats, weighing160–180 g, were obtained from our animal facility (Al-Azhar University, Cairo, Egypt). The rats were kept inwire-floored cages under standard laboratory condi-tions of 12 h/12 h light/dark, 25 ± 2°C with free accessto food and water.

Experimental DesignThe rats were randomized and divided into 4 groups of12 animals each. Two groups were treated by gavagewith CsA at a dose of 25 mg/kg of body weight for21 days. One of the two groups was fed orally withDHA at a dose of 100 mg/kg of body weight for 4 daysbefore and for 21 days concomitant with CsA.16,11 Thethird group received DHA alone in the same dose andby the same route of administration for 25 consecutivedays. The last group of animals served as control andreceived by gavage an equivalent volume of vehicle forthe same number of days. In this experiment, 1 mL ofolive oil for each animal in all groups was used asvehicle.

On the last day of treatment, rats in all groupswere in individual metabolic cages housed for 24 h ofurine collection. During urine collection animals hadwater ad libitum, whereas food was allowed for thefirst 12 h only. The volume of collected urine wasmeasured to ascertain urine parameters [creatinine,total protein, N-acetyl-b-D-glucosaminidase (NAG),and NO]. After urine collection, and at 24 h after thelast injection, blood samples from starved animalswere taken from abdominal aorta under light etheranesthesia, and used for determination of serum tria-cylglycerol (TAG), serum total cholesterol (TC),serum total protein, serum urea, and creatininelevels. All animals were then killed by cervical dislo-cation and both kidneys from each rat were quicklyremoved, rinsed in ice-cold physiological saline,blotted dry on filter paper, weighed, and then 10%(w/v) homogenate of the kidney was made in ice-cold0.15 M KCl solution using a Potter–Elvehjemhomogenizer. Aliquots of the homogenates wereused for determination of tissue contents of thiobar-bituric acid-reacting substances (TBARS) as indexof lipid peroxidation, protein carbonyl content(PCC) as index of protein oxidation, and NO. Totalprotein content of homogenates was also determinedto ascertain tissue parameters.

Biochemical AnalysisSerum TAG, TC, and urea were assayed according tothe methods of Fossati and Prencipe22; Roeschlauet al.,23 and Hallet and Cook,24 respectively. Creati-nine clearance (Ccr) was calculated after estimation ofserum and urinary creatinine levels using the alkalinepicrate method.25 NAG activity of the collected urinesamples was measured using the methods of Mooreand Morris.26 Kidney homogenate was used for thedetermination of TBARS and PCC according to themethods of Mihara and Uchiyama27 and Levine et al.,28

respectively. Urine and tissue concentrations of NOwere measured as its stable metabolites, nitrate andnitrite. Nitrate was first reduced by nitrate reductase tonitrite and then nitrite was determined spectrophoto-metrically by the Griess reaction.29 The protein contentof the serum, urine, and homogenate samples was mea-sured by the method of Lowry et al.30

Statistical AnalysisData are expressed as mean ± SD for the groups. Dataanalysis was evaluated by one-way analysis of variance(ANOVA) followed by Tukey–Kramer test for multiplecomparisons. A 0.05 level of probability was used asthe criterion for significance.

RESULTS

The consequences of animal treatment with CsA aggra-vated an observed renal dysfunction, as evidenced bysignificant reduction in body weight gain (16%) andserum total protein (31%), whereas serum urea wassignificantly increased (169%) when compared withcontrol animals. Furthermore, apparent hyperlipidemiawas observed through the increased levels of serumTAG (47%) and serum TC (46%) as compared withthe control values in vehicle-receiving rats. The admin-istration of DHA significantly restored those modifiedvalues. Besides, an obvious reduction (79%) in Ccr wasobserved in the CsA-treated group, and it was highlyimproved upon DHA administration. Moreover, thenephrotic (CsA-treated) rats showed high urinaryexcretion of both protein (∼14-fold) and NAG (112%)compared with vehicle-treated non-nephrotic rats.A significant protection was observed upon DHAadministration through decrease of the high urinaryprotein and NAG excretion (Table 1).

In nephrotic kidneys, CsA treatment significantlyincreased TBARS and PCC levels, which werenoticeably alleviated upon administration of DHA(Figures 1 and 2).

Figure 3 indicates that CsA treatment significantlydecreased both nitrite concentration of kidneyhomogenate (Figure 3A) and urinary excretion ofnitrite (Figure 3B) when compared with the controllevels. Administration of DHA significantly increasedthe declined values of urinary and tissue nitrite almostto values obtained from the control group.

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68 A.D. Mariee and M.F. Abd-Ellah

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Table 1. Effect of DHA administration on body weight, serum total protein, serum urea, serum TAG, serum TC, urinary protein,urinary NAG, and Ccr modified by CsA treatment in rats.

Parameters Control DHACsA

nephrotoxicitya,bCsA

nephrotoxicity + DHAa,b,c

Final body weight (g) 169 ± 5.5 174 ± 6.0 142 ± 5.2 159 ± 5.0Serum total protein (g/dL) 3.9 ± 0.2 4.1 ± 0.3 2.7 ± 0.2 3.4 ± 0.4Serum urea (mg/dL) 61 ± 2.7 58 ± 2.9 164 ± 9.1 73 ± 4.6Serum TAG (mg/dL) 44.3 ± 2.7 45.7 ± 2.9 65.3 ± 4.1 49.2 ± 2.4Serum TC (mg/dL) 77.3 ± 6.4 70.1 ± 6.2 112.6 ± 8.1 93.8 ± 6.7Urinary protein (mg/mg creatinine) 2.1 ± 0.2 1.9 ± 0.2 31 ± 6.1 22 ± 4.1Urinary NAG (nmol/mg creatinine) 192 ± 21.4 205 ± 19.9 407 ± 30.3 311 ± 28.1Ccr (mL/min) 0.39 ± 0.04 0.41 ± 0.02 0.08 ± 0.01 0.27 ± 0.05

Notes: Values are expressed as mean ± SD, n = 12. Multiple comparisons were achieved using one-way ANOVA followed byTukey–Kramer as post-ANOVA test. DHA, docosahexaenoic acid; CsA, cyclosporine A; TAG, triacylglycerol; TC, total cholesterol;NAG, N-acetyl-b-D-glucosaminidase; Ccr, creatinine clearance.a, b, and c indicate significant change from control, DHA, and CsA nephrotoxicity groups, respectively, at p < 0.05.

Figure 1. Effect of DHA on the level of TBARs (index of lipid peroxidation) in the kidney of control, DHA, and CsA nephrotoxicitygroups. Data are presented as mean ± SD, n = 12. Multiple comparisons were achieved using one-way ANOVA followed by Tukey–Kramer as post-ANOVA test.Note: a and b indicate significant change from control, DHA, and CsA nephrotoxicity groups, respectively, at p < 0.05.

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Figure 2. Effect of DHA on the level of PCC (index of protein oxidation) in the kidney of control, DHA, and CsA nephrotoxicitygroups. Data are presented as mean ± SD, n = 12. Multiple comparisons were achieved using one-way ANOVA followed by Tukey–Kramer as post-ANOVA test.Note: a and b indicate significant change from control, DHA, and CsA nephrotoxicity groups, respectively, at p < 0.05.

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The solitary administration of DHA has insignificantchanges on the levels of all parameters measured com-pared with the control animals.

DISCUSSION

The results of this study showed that CsA-treated rats(25 mg/kg for 21 days) revealed a severe nephropathyas reflected by body weight loss with substantial hypo-proteinemia, high level of serum urea, hyperlipidemia,and proteinuria. This nephrotic toxicity was also asso-ciated with obvious decrease of Ccr and apparent renaltubular injury that is indicated by the increased urinaryexcretion of NAG (Table 1). This distinctive figure ofCsA-induced nephrotoxicity is comparable with thosereported by other research groups.11,13,31,32

CsA nephrotoxicity is parallel to its immunosuppres-sive property and cannot be totally avoided. The pre-cise mechanism by which CsA causes renal injury

remains obscure and is still not completely elucidated,but several studies suggest that transforming growth fac-tor-b1 expression,33 intracellular calcium handling,34

oxidative stress,35,36 vasoconstriction, and altered renalhemodynamics37–39 are involved. However, participa-tion of NO, an important renal vasodilator, is highlysuggested to participate in CsA-induced nephrotoxicity.Gossmann et al.5 reported that acetylcholine-inducedvasodilatation is impaired in vascular beds of CsA-treated animals, which may indicate deficiency ofendothelial NO synthesis. However, this conclusioncan also be referred to the enhanced generation of freeradicals in CsA-induced nephrotoxicity which is able toinactivate NO.40

DHA administration in a dose of 100 mg/kg bodyweight significantly normalized all the measuredparameters affected by CsA toxicity. DHA significantlydecreased body weight loss, serum urea, serum TAG,serum TC, and urinary excretion of protein and NAG.

Figure 3. Effect of DHA administration on the renal tissue nitrite (A), and urinary excretion of nitrite (B) modified by CsA treatment inrats. Data are presented as mean ± SD, n = 12. Multiple comparisons were achieved using one-way ANOVA followed by Tukey–Krameras post-ANOVA test.Note: a, b, and c indicate significant change from control, DHA, and CsA nephrotoxicity groups, respectively, at p < 0.05.

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It also increased Ccr and serum total protein whencompared with the untreated CsA-intoxicated animals(Table 1). This is consistent with previous findings ofSabry et al.,39 Kim and Chung, 41 and El-Meseryet al.,42 who have demonstrated the protective renaleffect of omega-3 fatty acids.

The results of this study also revealed a significantincrease in TBARS generation and the level of PCC inthe kidneys of CsA-treated animals (Figures 1 and 2).The ability of CsA-induced nephrotoxicity to modulatekidney oxidative status proves the role of increased oxi-dant stress. The role of free radicals formation and theconsequent oxidative stress in the induction of CsA-induced nephrotoxicity are widely accepted throughoutdifferent studies that support this notion9–11,32. How-ever, the contribution of DHA in prevention of renaloxidative stress induced by CsA was not that beneficial,although both lipid and protein oxidative parameterswere slightly attenuated following DHA administration(Figures 1 and 2). In contrast to our results, Kuboet al.43 reported that DHA is thought to be closelyassociated with the tissue lipid peroxide formation.More recently, Bouzidi et al.44 reported that omega-3supplementation improves oxidative stress in patientswith chronic renal failure. Nevertheless, Calvielloet al.45 stated that DHA-induced lipid peroxidation isonly dose dependant. Later, Sunada et al.,46 Taccone-Gallucci et al.,47 and Kim and Chung41 reported thatDHA neither affects susceptibility to oxidative stressnor promotes lipid peroxidation in different rat tissueseven under high oxidative stress.

Moreover, we made an attempt to define the rela-tionship between NO levels and CsA nephrotoxicity.The tissue and urine NO levels of rats intoxicated withCsA were significantly reduced (Figure 3A and 3B),which indicates that abnormal NO bioavailability playsan important role in the pathogenesis of CsA-inducednephrotoxicity. Physiological levels of NO play animportant role in the conservation of renal hemody-namics due to its vasodilator and antithrombogenicproperties. It has been reported that decrease in renalNO generation is linked to the pathogenesis of renaldisease.48–51 The decreased bioavailability of NO in thekidney as a consequence of increased oxidative stresswas also reported.52,53

Our study revealed that DHA significantly increasedNO levels in both renal tissues and urine of CsA-treatedrats (Figure 3A and 3B). DHA administration wasproved to increase the release of endothelium-derivedrelaxing factor, presumably NO, which facilitates arterialrelaxation, decreases blood pressure, improves vascular-ity, and consequently reduces the kidney functionimpairment.17,54 Similar results were reported by Robin-son and Stone,20 who observed that DHA enhanced theformation of NO, which induces its hypotensive effect.

Under our experimental conditions, DHA singletreatment of rats did not significantly affect any of thestudied parameters compared with the control animals.

In conclusion, this study provided further evidenceto support the involvement of NO in pathogenesis ofnephrotoxicity caused by CsA. These findings also sug-gested that administration of DHA exerts a renoprotec-tive effect in CsA-induced nephrotoxicity. It seems thatthe protective effect induced by DHA is most probablydue to the regulation of NO bioavailability in the kid-ney. However, the detailed mechanism by which DHAdecreased the high levels of NO in both kidney andurine of CsA-treated rats is not well defined. Therefore,further investigation is required to elucidate thedetailed mechanism.

Declaration of interest: The authors report noconflicts of interest. The authors alone are responsiblefor the content and writing of the paper.

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