Protective Effect of Melatonin and 1,25-Dihydroxyvitamin D3 on Renal Ischemia–Reperfusion Injury...

Renal Failure, 2013; 35(3): 374–379Copyright © Informa Healthcare USA, Inc.ISSN 0886-022X print/1525-6049 onlineDOI: 10.3109/0886022X.2012.760409

LABORATORY STUDY

Protective Effect of Melatonin and 1,25-Dihydroxyvitamin D3 on RenalIschemia–Reperfusion Injury in Rats

Gülbüz Sezgin1, Güler Öztürk2, Şevin Güney3, Orhun Sinanoğlu4 and Matem Tunçdemir5

1Department of Internal Medicine, Faculty of Medicine, Maltepe University, Istanbul, Turkey; 2Department of Physiology,Faculty of Medicine, Maltepe University, Istanbul, Turkey; 3Department of Physiology, Faculty of Medicine, Gazi University,Ankara, Turkey; 4Department of Urology, Faculty of Medicine, Maltepe University, Istanbul, Turkey; 5Department of MedicalBiology, Cerrahpaşa Faculty of Medicine, Istanbul University, Istanbul, Turkey

Abstract

Ischemia–reperfusion (I/R) injury induces the generation of reactive oxygen species (ROS) which affect many organs. Thisstudy was designed to investigate the roles of melatonin and 1,25-dihydroxyvitamin D3 (VD3) on renal I/R injury. Thirty maleWistar albino rats were divided into five groups: group 1, control; group 2, right nephrectomy (RN) þ I/R in the contralateralkidney; group 3, melatonin þ RN þ I/R; group 4, VD3 þ RN þ I/R; and group 5, melatonin þ VD3 þ RN þ I/R. Melatonin(10 mg/kg), VD3 (0.5 μg/kg), and melatonin plus VD3 were injected intraperitoneally for 7 days before renal I/R. After 7 days,right nephrectomywas initially performed and left renal arterywas clamped for 45min. After 45-min reperfusion, the serum andkidney tissue samples were obtained for assays. Melatonin and VD3 had an ameliorative effect on biochemical parameterssuch as serum creatinine (SCr) and blood urea nitrogen (BUN). Renal tissue malondialdehyde (MDA), glutathione (GSH), nitricoxide (NO) levels, and superoxide dismutase (SOD) activitywere determined. Renal I/R decreased the kidney tissueGSH levelsand SOD activity and increased the NO levels as compared with control group. However, melatonin and VD3 and melatoninplus VD3 treatment significantly increased the tissueGSH levels andSODactivity and decreased theNO levels comparedwiththoseof I/Rgroup.Meanwhile,MDA levelswere not different between the control and I/Rgroups. But,MDA levels decreased inall treated groups compared to I/R and control groups. These data support that melatonin and VD3 have beneficial effects onrenal injury.

Keywords: melatonin, 1,25-dihydroxyvitamin D3, ischemia–reperfusion, kidney, antioxidant/oxidant status

INTRODUCTION

Cessation of kidney blood supply leads to acute renalfailure (ARF), causing failure of the kidneys over a periodof hours or days.1 ARF has relatively high incidence ratein hospitalized patients and the most prevalent causes areischemic events.2 Ischemia–reperfusion (I/R) injury is acomplex phenomenon and it leads to structural andfunctional cell damage.3 One of the most important fac-tors in pathophysiology of renal IR injury is reactiveoxygen species (ROS), which especially increases in thereperfusion phase.2 The endogenous antioxidants whichare responsible for defence against ROS during reperfu-sion have an important role in decreasing IR injury.2

ROS production is controlled by enzymatic and non-enzymatic processes.4 Superoxide dismutase (SOD)and glutathione (GSH) are, respectively, one of the

enzymatic and non-enzymatic antioxidants.4 Oxidativestress is the usual phrase used to identify the associationof the toxic free radicals with damage to cells andtissues.5 There are controversial results of antioxidantand oxidant system status after I/R injury.

The pineal hormone melatonin is a potent free radi-cal scavenger that protects the cells from damageinduced by a variety of oxidants, including hydroxylradical and lipid peroxidation products.6,7 VitaminD3 is taken in the diet or produced in the skin from7-dehydrocholesterol in the skin by ultraviolet irradia-tion.8 Vitamin D3 is transported in the circulation bythe DBP (vitamin D binding protein).9 Once deliveredto the liver, vitamin D is hydroxylated on its side chainto form 25OHD. The kidney is the primary site wherethe active form of vitamin D, 1,25(OH)2D (VD3), isproduced.9 VD3 exhibits various physiological actions,

Address correspondence to Güler Öztürk, Department of Physiology, Faculty of Medicine, Maltepe University, Istanbul, Turkey. Tel.:þ90216 370 96 15; Fax: þ90 216 399 00 60; E-mail: [email protected], [email protected]

Received 2 September 2012; Revised 4 November 2012; Accepted 9 December 2012

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including the regulation of calcium homeostasis,cellular differentiation and proliferation, immunefunctions, and reproduction.10 VD3 has been shownto induce the expression of several enzymes involved inthe antioxidant defence system.9 There is also someevidence that VD3 regulates the genes for proteinsthat protect the genome.9 Little information is availablefor the antioxidant property of VD3. In addition, thereare controversial results between studies related to anti-oxidant properties of VD3.11–14

Nitric oxide (NO) is synthesized from L-arginine bythe enzyme nitric oxide synthase (NOS).15 NO is pro-duced by any of the three NO synthases (NOS).Endothelial (eNOS) and neuronal (nNOS) isoformshave been identified in the renal vasculature andmacula densa, respectively.16,17 The third inducibleNOS (iNOS) is expressed in several segments of therenal tubule as well as in the glomerulus and interlobarand arcuate arteries of the normal rat kidney.16,18 NOis recognized as an important mediator of physiologicaland pathological processes of renal I/R injury.16

The aim of this study is to evaluate the changes in renaltissue antioxidant and oxidant status in I/R injury andwhether melatonin and VD3 or melatonin plus VD3treatment influence these parameters.

MATERIALS AND METHODS

Animals and SurgeryThe experimental protocol was approved by Animal EthicsCommittee of Maltepe University. Adult male Wistar rats,weighing approximately 200–250 g, were randomly dividedinto five groups of six rats in each group as follows: group 1,control; group 2, right nephrectomy (RN) þ I/R in thecontralateral kidney; group 3, melatonin þ RNþ I/R;group 4, VD3 (1,25-dihydroxyvitamin D3) þ RN þ I/R;and group 5, melatonin þ VD3 þ RN þ I/R. VD3 (Deva,Kocaeli/Turkey)was dilutedwith sterile saline and adjustedto a final concentration of 0.5 μg/kg. A week prior to I/R,VD3 was injected intraperitoneally for 7 days to theVD3 þ I/R group. Melatonin 10 mg/kg, (M5250 SigmaChemical, St Louis, MI, USA) was injected intraperitone-ally for 7 days to the melatoninþI/R group, and melatonin10 mg/kg þ VD3 0.5 μg/kg was injected to themelatonin þ VD3 þ I/R group. Following a 12-h fastingperiod, control, VD3,melatonin, VD3þmelatonin groupswere anesthetized with intramuscular ketamine 50 mg/kgand xylazine 10 mg/kg.

All rats had right nephrectomy, with a dorsal flankincision. In the I/R groups, the left renal pedicle isclamped with a non-crushing microvascular clamp(Bulldog Artery Clamp, Harvard Apparatus, MA,USA) for 45min and the presence of ischemia is visuallyconfirmed by observing blanching of the kidney. Forty-five minutes of reperfusion period followed the ischemiaperiod. At the end of the time, blood was collected byheart puncture formeasurement of biochemical analysis

and the kidney was removed for GSH, SOD, malon-dialdehyde (MDA), and NO measurements.

BUN and Creatinine AssaySerum samples were used for the measurement of bloodurea nitrogen (BUN) and serum creatinine (SCr) levels,which were used as indicators of impaired glomerularfunction. BUN and SCr were determined with anAbbott-Aeroset autoanalyzer (Chicago, IL, USA) usingoriginal kits.

MDA and GSH AssayLipid peroxidation was quantified by measuring theformation of thiobarbituric acid reactive substances asdescribed previously.19 Lipid peroxidation levels wereexpressed in terms ofMDA equivalents using an extinc-tion coefficient of 1.56 � 105/mol/cm. Tissue sampleswere homogenized in ice-cold trichloroacetic acid (1 gtissue plus 10 ml 10% trichloroacetic acid). The GSHlevel was determined by a modified Ellman method.20

Briefly after centrifugation at 3000 rpm for 10 min, thesupernatant was collected. Two milliliter of 0.3 MNa2HPO4�2H2O solution was added to 0.5 ml of thesupernatant. Afterwards, a 0.25 ml solution of dithio-bisnitrobenzoate (0.4 mg/mL in 1% sodium citrate) wasadded to this mixture and then mixture was vortexed.After 10 min incubation in room temperature, absor-bances of the samples were measured at 412 nm spec-trophotometrically. The GSH levels were calculatedusing an extinction coefficient of 13,000 mol/cm.

Superoxide Dismutase AssayThe SOD assay kit is used for the measurement of SODactivity in the kidney (Cayman Chemical Co., AnnArbor, MI, USA). SOD activity is assessed by measuringthe dismutation of superoxide radicals generated byxanthine oxidase and hypoxanthine.

Determining the Tissue Total NO(NOX) LevelsThe NOx levels were determined by vanadium chloride(VCl3)/Griess assay. Prior to NOx determination, thetissues were homogenized in five volumes of phosphate-buffered saline (pH ¼ 7.5) and centrifuged at 2000 � gfor 5 min. Then, 0.25 ml of 0.3 M NaOH was added to0.5 ml supernatant. After incubation for 5 min at roomtemperature, 0.25 ml 5% (w/v) ZnSO4 was added fordeproteinization. This mixture was then centrifuged at3000� g for 20 min and the supernatants were used forthe assays as described previously.21,22

Light MicroscopyRenal tissue samples were fixed in 10% neutral-bufferedformalin, followed by embedding in paraffin wax, cut into4-μmsections, and stainedwith periodic acid Schiff (PAS).

© 2013 Informa Healthcare USA, Inc.

Effects of Melatonin and Vitamin D on Kidney 375

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Evaluation was performed with light microscopy withoutknowledge of the study groups. Assessment was carriedout by expert observers.

Statistical AnalysesAll data are expressed as mean � standard error of themean (SEM). The analyses were performed usingSigmaStat 3.5 (Systat Software, Inc., Richmond, CA,USA). Differences in measured parameters among thegroups were analyzed by Kruskal–Wallis test. Dual com-parisons between groups that present significant valueswere evaluated with Mann–Whitney U test. Statisticalsignificance was accepted at a p-value of < 0.05.

RESULTSThere was a significant increase in the SCr and BUNlevels in the I/R group compared to the control rats(p ¼ 0.004). Administration of melatonin, VD3, andmelatonin þ VD3 significantly decreased the SCr andBUN levels compared to I/R group (Figures 1 and 2).

Renal I/R caused a marked decrease in kidney tissueGSH levels as compared to control group (p ¼ 0.015).However, melatonin and VD3 and melatonin plus VD3treatment significantly increased tissue GSH levels com-pared with those of I/R group (Figure 3).

Kidney SOD activity was decreased by renal I/R ascompared to control group (p ¼ 0.016). The VD3, mel-atonin, and melatonin plus VD3-treated rat kidneysshowed a significantly increased SOD activity as com-pared with I/R group without treatments (Figure 4).

Treatment with melatonin, VD3, and melatonin plusVD3 significantly reduced the tissueMDA levels as com-pared to I/R group without treatment (Figure 5). On theother hand, MDA levels were not statistically significantbetween I/R and control groups.

Kidney NOx levels were increased in the I/R groupcompared to the control group (p ¼ 0.015). NO levels

were found to be significantly lower in all treatmentgroups compared to the I/R group (Figure 6).

C IR VD3 + IR M + IR M + VD3 + IR

Cre

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mg/

dL)

0.0

0.2

0.4

0.6

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1.0

1.2

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1.6

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Figure 1. Creatinine levels. Results are expressed as amean� SEM.Note: �C versus IR and C versus VD3 p ¼ 0.004; #IR versus VD3,p ¼ 0.009; IR versus M and IR versus M þ VD3, p ¼ 0.004. C:control, I/R: ischemia–reperfusion, VD3: 1,25-dihydroxyvitaminD3,M: melatonin, M þ VD3: melatonin þ 1,25-dihydroxyvitamin D3.


BU

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g/dL

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Figure 2. Blood urea nitrogen (BUN) levels.Note: �C versus IR and C versus VD3, p¼ 0.004; #IR versus VD3,IR versusM and IR versusMþVD3, p¼ 0.002. For abbreviations,see legend to Figure 1.


GSH

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umol

/g ti

ssue

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0.0

0.1

0.2

0.3

0.4

0.5

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Figure 3. Renal tissue glutathione (GSH) levels.Note: �C versus IR and p ¼ 0.015; #IR versus VD3 and IR versusM, p ¼ 0.009. For abbreviations, see legend to Figure 1.


SOD

act

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(U

/ml)

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Figure 4. Renal tissue superoxide dismutase (SOD) activities.Note: �C versus IR, p ¼ 0.016; #IR versus VD3 and IR versus M,p ¼ 0.004; IR versus M þ VD3, p ¼ 0.03. For abbreviations, seelegend to Figure 1.

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HISTOPATHOLOGIC RESULTS (PAS)Renal morphology was normal in control group(Figure 7A). Extensive necrosis plus dilation, flatteningof epithelium, and loss of brush border in proximal tubuli(arrow) were observed in the I/R group (Figure 7B). InMel þ I/R (Figure 7C), VD3þ I/R (Figure 7D) andMel þ VD3þ I/R (Figure 7E) groups, better histopatho-logic findings compared to the I/R group were observed.

DISCUSSIONRenal ischemia is amajor cause of acute renal failure, whichdespite significant advances in critical care medicineremains amajor clinical problem, producing gravemorbid-ity and mortality that has not decreased significantly overthe last 50 years.23 Chronic kidney disease (CKD) has beenshown to result in decreased vitamin D metabolism

through multiple mechanisms.8,24 The loss of functionalrenal mass in CKD, cause of decreased production of1α(OH)ase, leads to decreased VD3 levels.8,24 In the pre-sent study, in order to evaluate the biochemical parameterswithin the reversible first phase of reperfusion injuryextending only for a few hours, the reperfusion interval islimited to 45 min. In our study, renal I/R increased serumBUN and SCr levels. Treatment with melatonin and VD3or melatonin plus VD3 prior to renal I/R significantlyreduced the serum levels of BUN and SCr. Significantincreases in the serum concentrations of BUN and Crsubsequent to I/R suggest the impairment of glomerularfunction.25,26 I/R of the kidney causes both glomerular andtubular dysfunction.25 It was reported that VD3 treatment


MD

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0

1

2

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Figure 5. Renal tissue malondialdehyde (MDA) levels.Note: �C versus VD3, p ¼ 0.002; C versus M and C versusM þ VD3, p ¼ 0.004; #IR versus VD3, p ¼ 0.009; IR versus Mand IR versusMþVD3, p¼ 0.015. For abbreviations, see legend toFigure 1.


Kid

ney

NO

x le

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ol/g

wet

wei

ght)

0

5

10

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20

25

*

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Figure 6. Renal tissue nitrite þ nitrate (NOx) levels.Note: �Cversus IR, p¼ 0.015; #IR versus VD3, IR versusMand IRversus M þ VD3, p ¼ 0.002. For abbreviations, see legend toFigure 1.

(E)(D)

(B)20 µm

20 µm 20 µm

20 µm 20 µm(C)(A)

Figure 7. PAS staining of kidney sections of control (A), I/R (B), Melþ I/R (C), VD3þ I/R (D), andMelþ VD3þ I/R (E) groups. In the I/Rgroup, extensive necrosis plus dilation, flattening of epithelium, and loss of brush border in proximal tubuli (arrow) were seen (Glomeruli;asterisk) (bar ¼ 20 μm).



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may accelerate the recovery of renal tubular cells by increas-ing the proliferation of renal tubular cells in ischemic ratkidneys.27 In this study, 0.5 μg/kg/day of VD3 was used,tolerable dose, based on the Kim et al.27 research.

Reperfusion of the kidneys causes the activation andadhesion of polymorphonuclear neutrophils (PMNLs),with the release of proinflammatory substances and theformation of free radicals, which are nitrogen-derivedreactive nitrogen species (RNS) or oxygen-derived reac-tive oxygen species (ROS) such as superoxide anion(O2

●�), hydrogen peroxide (H2O2), and hydroxyl radicals(●OH).1,28–30 Recognizing IR effects on antioxidantenzymes activities and GSH level may help to find a betterstrategy for prevention and/or therapy of ischemic ARF.2

SOD is the first line of defence of the cellular antioxidantsystem against the oxidative damage mediated by super-oxide radicals.13 It was reported that VD3 induces theexpression of several antioxidant enzymes.9 In addition,mRNA levels for the essential antioxidant enzymes SOD1and SOD2 are induced by VD3 in primary PECs9,31 andLNCaP cells,9,32 respectively.

There is little information for the antioxidant property ofVD3 and there are controversial results between studiesrelated to antioxidant properties of VD3.

11,13,14 There aremany studies concerning the antioxidant properties of mel-atonin.7,33,34 It was also reported that melatonin has aprotective effects on kidney tissue against oxidative damageand apoptosis.35,36 But for the first time, this study evalu-ated the combined use of melatonin and VD3 against renalI/R injury. Most previous studies have been shown todecrease in SOD and catalase activities and GSH levelafter IR.14,23,29,37,38 However, Rasoulian et al.2 showed adecrease in renalGSH level, increase in renal SODactivity,and unchanged MDA levels following IR. On the otherhand, Ersoz et al.1 found that SODactivity andMDA levelssignificantly increased after IR. Ekici et al.14 reported thatVD3 treatment showed no significant effect on decreasedSOD activity and GSH levels and increased MDA levelscaused by cerebral IR. However, the combination of VD3and Vitamin C treatment showed a significant decrease inMDA levels and a significant increase in SOD activity andGSH levels.14 The results of our present investigationdemonstrated that SOD activity and GSH levels decreasedafter IR comparedwith the control group. But,MDA levelsdid not change after IR. However, treatment with melato-nin and VD3 or melatonin plus VD3 prior to renal I/Rincreased the SOD activity and GSH levels. We suggestthat pretreatment with VD3 and melatonin or the combi-nation of VD3 and melatonin have a protective effect onrenal I/R injury by increasing SODactivity andGSH levels.

It is now widely accepted that the NO pathway isinvolved in the functional regulation of any organ, tissue,and cell in the body.39 Various studies have shown thatNO biosynthesis and its action are closely related to thepathogenesis of renal I/R injury.1,2,16,23 NO is producedby the endothelial cells and causes relaxation of preglo-merular arteries improving renal blood flow.40 Duringrenal I/R, ROS can disrupt the integrity of the

endothelium and can affect NO production, resulting inincrement of renal vascular resistance.40 Chander andChopra23 reported that kidney NO levels decreased inglycerol-induced acute renal failure in rats. On the otherhand, some studies showed a significant increase in NOlevels after renal I/R injury.1,16,38 In our study, renal I/Rinjury increasedNO levels in kidney tissue.Our data are inagreement with these studies,1,16,38 but contrary to theobservation of Chander and Chopra.23 Treatment withVD3 and melatonin alone or the combination of thesesubstances caused a significant decrease in renal NOlevels. Ersoz et al.1 reported that NO-induced I/R injurymay be a result of the development of ONOO�, as NOcouples with O2

�. iNOS plays a deleterious role duringrenal I/R injury. Melatonin has the ability to scavengemany of the oxygen-based and nitrogen-based radicals,including ONOO�. It was suggested that NOS activity,probably endothelial constitutiveNOS (cNOS), increasedin response to ischemia-induced renal vasoconstrictoractivity.41 Ischemic injury itselfmay increase smoothmus-cle cytosolic calcium or increase oxygen radical genera-tion, providing potent vasoconstrictor stimulation towhich NOS activation is a modulating response.41

Increased NO levels in I/R injury may be due to increasedNOS activity and ROS/RNS production. It has beenshown that VD342,43 and melatonin44,45 inhibit expres-sion of NOS. In our study, NOS was not evaluated.Treatment with these substances prior to I/R injurycould decrease NOS activity and ROS/RNS formation.For these reasons, pre-ischemic treatment with these sub-stances may reduce NO levels.

In summary, the maintaining of balance between oxi-dant and antioxidant system in preventing I/R injury maybe an important therapeutic strategy. In other words,increase in renal antioxidant enzymes and decrease inrenal production of ROS may help to prevent I/R injury.Treatment with melatonin and VD3 or melatonin combi-nation with VD3 have been shown to increase in renalSOD activity and GSH levels and decrease in NOx levels.There are many studies for melatonin to prevent I/Rdamage, but little information for using VD3. Our datasuggest that VD3 participates in the antioxidant system toprotect the renal tissue against I/R injury. Therefore, VD3should be further evaluated for use in I/R injury.

Declaration of interest: The authors report no con-flicts of interest.

This study was funded by Maltepe University(2011-04).

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Protective Effect of Melatonin and 1,25-Dihydroxyvitamin D3 on Renal Ischemia–Reperfusion Injury...

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