Rev. Bras. Anestesiol. vol.64 número6

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REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

OfficialPublicationoftheBrazilianSocietyofAnesthesiology

www.sba.com.br

SCIENTIFIC

ARTICLE

Effects

of

dexmedetomidine

in

conjunction

with

remote

ischemic

preconditioning

on

renal

ischemia---reperfusion

injury

in

rats

Emine

Bagcik

a

_,

_Sevda

_Ozkardesler

b

_,

_Nilay

_Boztas

b,∗

,

Bekir

Ugur

Ergur

c

,

Mert

Akan

b

_,

_Mehmet

_Guneli

d

_,

_Sule

_Ozbilgin

b

a_Gordes_State_Hospital,_{Anesthesiology}_and_Reanimation,_Manisa,_Turkey

b_Medical_Faculty_of₉_Eylul_University,_{Anesthesiology}_and_{Reanimation, ˙Izmir,}_Turkey

c_Medical_Faculty_of₉_Eylul_University,_Histology_and_{Embryology, ˙Izmir,}_Turkey

d_Medical_Faculty_of₉_Eylul_University,_Laboratory_Animal_{Science, ˙Izmir,}_Turkey

Received4July2013;accepted2January2014 Availableonline18February2014

KEYWORDS

Kidney;

Ischemia---reperfusion injury;

Dexmedetomidine; Caspase-3; Ischemic preconditioning; Apoptosis

Abstract

Backgroundandobjectives: The aim of this study was to evaluate the effects of remote ischemic preconditioning by brief ischemia of unilateral hind limb when combined with dexmedetomidineonrenalischemia---reperfusioninjurybyhistopathologyandactivecaspase-3 immunoreactivityinrats.

Methods:28Wistaralbinomaleratsweredividedinto4groups.GroupI(Sham,n=7): Laparo-tomyandrenalpedicledissectionwereperformedat65thminuteofanesthesiaandtherats wereobserved underanesthesiafor130min.Group II(ischemia---reperfusion,n=7):At65th minuteofanesthesiabilateralrenalpedicleswereclamped.After60minischemia24hof reper-fusionwasperformed.GroupIII(ischemia---reperfusion+dexmedetomidine,n=7):Atthefifth minuteofreperfusion(100␮g/kg intra-peritoneal)dexmedetomidine wasadministered with ischemia---reperfusiongroup.Reperfusionlasted24h.GroupIV(ischemia---reperfusion+remote ischemicpreconditioning+dexmedetomidine,n=7):Afterlaparotomy,threecyclesofischemic preconditioning(10minischemiaand10minreperfusion)wereappliedtothelefthindlimband after5minwithgroupIII.

Results:Histopathologicalinjury scoresand activecaspase-3immunoreactivity were signifi-cantlylowerintheShamgroupcomparedtotheothergroups.Histopathologicalinjuryscores ingroupsIIIandIVweresignificantlylowerthangroupII(p=0.03andp=0.05).Activecaspase-3 immunoreactivitywassignificantlylowerinthegroupIVthangroupII(p=0.01)andtherewas nosignificantdifferencebetweengroupIIandgroupIII(p=0.06).

∗_{Corresponding}_author.

E-mail:[email protected](N.Boztas).

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Conclusions: Pharmacologic conditioning with dexmedetomidine and remote ischemic pre-conditioning when combined withdexmedetomidine significantly decreasesrenal ischemia---reperfusioninjuryhistomorphologically.Combineduseoftwomethodspreventsapoptosisvia activecaspase-3.

PALAVRAS-CHAVE

Rim; Lesãode

isquemia-reperfusão; Dexmedetomidine; Caspase-3;

Pré-condicionamento isquêmico;

Apoptose

Efeitosdedexmedetomidinaemconjuntocomopré-condicionamentoisquêmico

remotoemlesãodeisquemia-reperfusãorenalemratos

Resumo

Justificativaeobjetivos: Avaliarosefeitosdopré-condicionamentoisquêmicoremoto, medi-antebreveisquemiademembroposteriorunilateral,emcombinac¸ãocomdexmedetomidinaem lesãodeisquemia-reperfusãorenalpormeiodehistopatologiaeimunorreatividadedacaspase-3 ativaemratos.

Métodos: Foramdivididos em quatro grupos 28 ratosmachos albinosWistar. GrupoI(Sham [cirurgia controle], n=7): laparotomia e dissecc¸ão do pedículo renal foram feitas em 65 minutos de anestesia e os ratos foram observados sob anestesia por 130minutos. Grupo II (isquemia-reperfusão, n=7): no 65◦ _minuto _de _anestesia, _os _pedículos _renais _bilaterais

foram pinc¸ados; após 60minutos de isquemia, foi feita reperfusão de 24horas. Grupo III (isquemia-reperfusão+dexmedetomidina,n=7):noquintominutodereperfusão, dexmedeto-midina (100mg/kg intraperitoneal) foi administrada ao grupo com isquemia-reperfusão. A reperfusão durou 24horas. Grupo IV (isquemia-reperfusão+pré-condicionamento isquêmico remoto+dexmedetomidina, n=7): após a laparotomia, três ciclos de pré-condicionamento isquêmico(10minutosdeisquemiae10minutosde reperfusão)foramaplicados nomembro posterioresquerdoedepoisdecincominutosaogrupoIII.

Resultados: Osescoresdelesãohistopatológicaeimunorreatividadedacaspase-3ativaforam significativamentemenoresnogrupoShamemcomparac¸ãocomosoutros.Osescoresdelesão histopatológicadosgruposIIIeIVforamsignificativamentemenoresdoqueosdoII(p=0,03e p=0,05).Aimunorreatividadedacaspase-3foisignificativamentemenornogrupoIVdoqueno II(p=0,01)enãohouvediferenc¸asignificanteentreosgruposIIeIII(p=0,06).

Conclusões: Ocondicionamentofarmacológicocomdexmedetomidinaeopré-condicionamento isquêmico remoto em combinac¸ãocomdexmedetomidina diminuem demodo significante a lesãodeisquemia-reperfusãorenalhistomorfologicamente.Ousocombinadodosdoismétodos previneaapoptoseviacaspase-3ativa.

Introduction

Ischemicacuterenalinjuryisaclinicalsyndromewithhigh mortalityandmorbidity.Duetoitshighenergyrequirements and the complex network of the renal microvasculature, thekidney is highlysensitive toischemia---reperfusion(IR) injury.1---4_The_mechanisms_of_renal_IR_injury_are multifacto-rial,includinghypoxia,free-radicaldamage,andlocaland systemicinflammatoryresponses.5_Reperfusion_of_ischemic renal tissue induces complex cellular conditions and the deathofrenalcells,duetoapoptosis.6

Apoptosisisaformofgeneticallyprogrammedcelldeath. Twomainpathwaysplayaroleinepithelialapoptosis.The first, known as the extrinsic or death receptor pathway, is stimulated by TNF-␣-family molecules bound to CD95 ligand(Fasligand=CD95)throughextracellularsignals.This pathway combines withthe second pathway, the intrinsic or mitochondrialpathway,via caspase-3 activation,which includescytochromecandtheBcl-2familyofproteins,and together,theyenhanceapoptosis.7

Renal tubular cell apoptosis is a resultof renal injury and a primary and major contributor to IR pathophysio-logy. Bothinflammation and apoptosiscoexist in renal IR injury.Duringhypoxia,caspaseactivityincreasesasaresult ofintracellular Ca2+ _{accumulation.}_Caspase_becomes

acti-vatedinischemictissuesandisanindicatorofcelldeath.8 Thesechanges,whichcanbeobservedintubularcells,may causethelossofbrushbordersofproximaltubularcells,and spilloutfromthebasementmembraneofthecellsintothe tubularlumen,witheventualtubuleobstruction.9,10

Among the methods used to reduce the effects of IR injury, remote ischemic preconditioning (RIPC) and phar-macological conditioning are the most commonly used.11 Inreviewingthe English-languageliterature,nostudy was found regarding an experimental IR model to investigate the effects of using pharmacological conditioning with dexmedetomidineadministeredincombinationwithRIPC.

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I R Group I

Group II

Group III

Group IV

I D R

D R

1565th_min

125

125130 130 65

0 0

0

0 10 20 30 40 506065 I R I R I R I

65

Figure1 Schematicrepresentationoftheexperimental pro-tocol.I,ischemia;R,reperfusion;D,deksmedetomidine.

histopathologically and to evaluate active caspase-3 immunoreactivityinaratrenalIRmodel.

Materials

and

methods

In total, 28 adult male Wistar albino rats, weighing 250---300g,wereused.Untilthebeginningoftheresearch, ratswerekeptatroomtemperature(21---22◦_C)_and_40---60% relativehumidity ona 12/12-hlight/dark cycle andwere fedwithstandard pellet diet andwater adlibitum.After approvalbytheLocalEthicsCommitteefor Animal Exper-iments at our faculty, the study was carried out in the MultidisciplinaryLaboratory of Animal Experiments. Anes-thesiawasprovidedwith50mg/kgketamineand10mg/kg xylazinehydrochlorideadministeredintraperitoneally(ip). Afteranesthesia,theanimalsweredividedintofourgroups.

Group I (Sham, n=7): After laparotomy, the left and

right renal pedicles were exposed at the 65th minute of anesthesia,andratswerekeptunderanesthesiafor130min withnootherintervention.

GroupII(IR,n=7):After60minofischemia,theclamp wasremovedandreperfusionofthekidneyswasallowedfor 24h.

Group III (IR+dexmedetomidine, n=7): After 60min

of ischemia, the clamp was removed and 100␮g/kg dexmedetomidine(Precedex100␮g/2mL,Abbott Laborato-ries,IL,USA) wasadministeredip,and reperfusionofthe kidneyswasallowedfor24h.

GroupIV(IR+RIPC+dexmedetomidine,n=7):

Follow-ing a laparotomy, after 5min of RIPC, and a subsequent 60min of renal ischemia, the clamp was removed and 100␮g/kgdexmedetomidine(Precedex100␮g/2mL,Abbott Laboratories)wasgivenandfinally24hofreperfusionwas performed(Fig.1).

ExposingrenalpediclesingroupI,andinitiatingischemia at the65th minute of anesthesiain groups II and III, was intended to synchronize all the groups to the precondi-tioning time of group IV and to standardize the start of theprocedures.Tissuesampleswereobtainedafter24hof reperfusion.

To protect the rats from hypothermia, the operating tablewasheatedwithalampheaterthroughoutthestudy andrectal body temperature wasmeasured with aprobe and maintained at 3737.5◦_C. _Hourly _subcutaneous _saline

solution,at 3mL/kgdosage, wasadministeredtoprevent dehydration. During the waiting time, the abdomen was closedwithamoiststerilepadandsurgicalforceps. Inall groups, the left kidneys were removed under anesthesia for histomorphological analyses and the rats were sacri-ficedbyacardiacpunctureexsanguinationattheendofthe study.Thekidneyswerefixedin10%bufferedformalinand embeddedinparaffinwax,cutat4---5␮m,andstainedwith hematoxylin and eosin for histological studies using light microscopy.

RenalIRmodel

Therightandleftrenalpedicleswereexposedafter laparo-tomy. Total renal ischemia in the left and right renal pedicles was maintained with atraumatic microvascular clampcompression.Adequateocclusionwasconfirmedbya lackofpulsationintherenalpediclesandpresenceofpallor inthekidneys.Aftertheischemicperiod,themicrovascular clampswereremovedandreperfusionoccurred.

RIPCmodel

ForthetourniqueteffectofRIPC,amethodthathasbeen showntobeeffectivebyperfusionscintigraphyandalaser meter wasused.12,13 _For_this_purpose, _the_left_hind_leg _of theratwasboundwithanelasticbandage(1cmwideand 30cmlong)atthegroin,applyingpressureallaround.Three cycles of 10min of ischemia wereperformed followed by 10minofreperfusion(65mintotal).Thecessationofblood flow wasconfirmedusinga lasercurrent meter(Laser Flo BPM2,Vasamedic,USA).

Histomorphologicalevaluationofrenaltissue

Renal tissue sections after IR were evaluated by light microscopybytwohistologistsblindedtotheanimalgroups intermsofstructuralchangesinproximaltubules(tubular atrophy,loss oftubularbrush border,vacuolization, tubu-lar dilatation, cast formation), mononuclear cells (MNCs) infiltration,interstitialstructuralchanges,renalcorpuscle morphology,andnecroticandapoptoticcells.

The cross-sectional images were scored semi-quantitatively in terms of tubulointerstitial damage. Scoringwasconductedasfollows:0=notatall,1=0---25%, 2=26---45%,3=46---75%,and4=76---100%.14

Immunohistochemicalmethods

Kidneytissueswerefixedin10%bufferedformaldehyde,and after routine histological follow-up procedures, paraffin-embeddedkidneytissueswerecutinto3-␮mthicksections with a microtome and collected on poly-l-lysine-coated slides.

Samples were stored in the oven at 60◦_C _for ₁₂_h. Then, a rat-specific anti-caspase-3 monoclonal antibody (RB-10287-R7 Labvision)wasusedtoassess anti-caspase-3 immunoreactivity.

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sectionsweretreatedwithanti-caspase-3antibody at4◦_C overnightandthenincubatedwithabiotinylatedsecondary antibody for 30min. Afterapplication of the Vector Elite ABC kit (Vector Laboratories Inc., Burlingame, USA), the antibody---biotin---avidin---peroxidasecomplexwasvisualized using 0.02% 3,3′_{-diaminobenzidine} _solution. _Following counterstaining with Mayer’s hematoxylin, image analysis wasperformed.

Thepositivestainingratewasevaluatedusingan indica-torofsemi-quantitativescoring (1---4),interms ofdensity anddistribution.

Exclusioncriteria

Ratsinneedofresuscitationwereexcludedfromthestudy.

Statisticalanalysis

The SPSS software (ver. 15.0; SPSS, Chicago, IL, USA) was used for statistical analyses. A Kruskal---Wallis analy-sis of variance was performed to analyze the data. The Mann---WhitneyU-testwasusedforpair-wisecomparisonsof thegroups.Alldataarepresentedasmeans±standard devi-ations.pvalues<0.05wereconsideredtoindicatestatistical significance.

Results

In total, 28 rats were included in the study group. One rat in the IR group died during the reperfusion period, andwasexcluded fromthe study.Histomorphologicaland immunohistochemicalinjuryscoresofthestudygroupsare presentedinTables1and2.

Renalhistomorphologicalinjuryscores

MNC infiltration. Histomorphological injury scores of the Sham group were significantly lowerthan those of the IR group(p=0.01).Injuryscores intheIRgroupwere signifi-cantlyhigherthanintheIR+DexandIR+RIPC+Dexgroups (p=0.04andp=0.04,respectively;Fig.2).

Structural changes in the proximaltubules. Histomor-phologicalinjuryscoresoftheShamgroupweresignificantly lower than those of the IR, IR+Dex, and IR+RIPC+Dex groups(p<0.01,p=0.02,andp=0.02,respectively).TheIR

Table2 Immunohistochemicalinjuryscoresofthegroups.

GroupI(n=7) Sham

0.85±0.37

GroupII(n=6) IR

2.50±0.54

GroupIII(n=7) IR+Dex

1.71±0.75

GroupIV(n=7) IR+RIPC+Dex

1.42±0.53

Dataarepresentedasmean±SS.

Sham

Monon

uclear cell infiltr

ation

1.8

1.6

1.4

1.2

0.8

0.6

0.4

0.2

0 1

IR IR+Dex IR+RIPC+Dex

Figure 2 Mononuclear cell infiltration scores detected by renalhistomorphological examination. *Comparisonof theIR group with the Sham group (p=0.01). =/_Comparison _of _the

IR+DexandIR+RIPC+DexgroupswiththeIRgroup(p<0.05).

groupdisplayedsignificantlyhigherscoresthantheIR+Dex and IR+RIPC+Dex groups (p=0.05 and p=0.05, respec-tively),while nosignificant differencewasfoundbetween theIR+DexandIR+RIPC+Dexgroups(p=1.00;Fig.3).

Glomerularchanges.Histomorphologicalinjuryscoresof theShamgroupweresignificantlylowerthanthoseoftheIR group(p=0.04).TheIRgroupdisplayedsignificantlyhigher scoresthantheIR+DexandIR+RIPC+Dexgroups(p=0.04 andp=0.04,respectively), while nosignificant difference wasfoundbetween the IR+Dex andIR+RIPC+Dex groups (p=1.00;Fig.4).

Totalhistopathologicalinjuryscore.Thetotal histomor-phologicalinjuryscoresoftheShamgroupweresignificantly lower than those of the IR group (p=0.003). Comparison

Table1 Histomorphologicallyinjuryscoresofthegroups.

MCI SCPD CG TSHD

GroupI(n=7) Sham 0.14±0.38 0.0±0.0 0.0±0.0 0.28±0.48

GroupII(n=6) IR 1.0±0.63 1.67±0.82 0.50±0.55 3.83±2.22

GroupIII(n=7) IR+Dex 0.28±0.49 0.71±0.75 0.0±0.0 1.42±1.27

GroupIV(n=7)IR+RIPC+Dex 0.28±0.49 0.71±0.75 0.0±0.0 1.42±1.61

pa _0.04b _0.00b _0.01b _0.008b

Dataarepresentedasmean±SS.

MCI,mononuclearcellinfiltration;SCPD,structuralchangesintheproximaltubules;CG,changesinglomerular;TSHD,totalscoreof histologicaldamage.

a _{Kruskal---Wallis}_test.

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Sham

T

ub

ular changes

3

2.5

1.5

0.5

0 1 2

Figure 3 Tubular change injury scores detected by renal histomorphological examination. *Comparison of the IR, IR+Dex and IR+RIPC+Dex groups with the Sham group (p<0.01). =/_Comparison_of_the_IR_group_with_the_IR₊_Dex_and IR+RIPC+Dexgroups(p=0.05).

Sham

Glomer

ular changes

1.2

1

0.8

0.6

0.2

0 0.4

Figure4 Glomerularchangeinjuryscoresdetectedbyrenal histomorphology.*ComparisonoftheIR groupwith theSham group(p<0.01). =/_Comparison_of_the_IR_group_with_the_IR₊_Dex andIR+RIPC+Dexgroups(p<0.05).

of the IR group with the IR+Dex and IR+RIPC+Dex groupsshowedsignificantlyhigherscoresthantheIRgroup (p=0.03 and p=0.05, respectively), while the IR+Dex andIR+RIPC+Dex groupsshowednosignificant difference (p=0.79;Fig.5).

Sham

T

otal histopathological injur

y scores

7

6

5

4

3

2

1

0

Figure 5 Total histopathological injury scores detected by renalhistomorphology. *Comparisonofthe IRgroup withthe Shamgroup(p<0.01). =/_Comparison_of_the_IR_group_with_the IR+DexandIR+RIPC+Dexgroups(p<0.05).

Sham

Glomer

ular changes

3.5

3

2.5

2

1

0 1.5

0.5

Figure6 Renalimmunohistochemicalscoring.*Comparisonof theIRgroupwiththeShamgroup(p<0.01).=/_Comparison_of_the IRgroupwiththeIR+DexandIR+RIPC+Dexgroups(p=0.01).

Immunohistochemicalinjury score. Immunohistochemi-calstainingscoresoftheIRgroupweresignificantlyhigher than in theSham group(p=0.001). Nostatistically signif-icant difference was found between the IR and IR+Dex groups(p=0.06).

In the comparison of injury scores between the IR+RIPC+Dex and IR groups, those of the IR+RIPC+Dex groupweresignificantlylower(p=0.01).Theinjuryscores of the IR+Dex and IR+RIPC+Dex groups did not differ (p=0.47;Fig.6).

Nocellinfiltrationorlossofbrushborderwasobservedin thesectionsoftheShamgroup(Fig.7AandB).However,in

B

D G

P

Group I

P G

G P

A

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A

D P

B

D

G

P P

Figure8 (AandB)SectionsoftheIRgroup.G,glomerular;P, theproximaltubule;D,thedistaltubule,( )accumulation ofproteinaceousmaterialintubules,(⋆)pouredintothelumen oftubularepithelialcells.

theIRgroup,peritubularMNCinfiltration,moreprominent inthecorticalarea,andlossofbrushborderoftheproximal tubulecells,tubularatrophy,tubulardilation,and vacuola-tionwereobserved.Insomeofthetubules,proteinaceous materialaccumulation,togetherwithcasteformation and celldebrisinthetubulelumen,wereobserved(Fig.8Aand B).

In the IR+Dex group, loss of the brush border of the tubulecells,tubularatrophy,tubulardilatation, vacuoliza-tion, and proteinaceous material accumulation and cell debrisinthetubulelumenwereobserved,albeittoalesser extentthanintheIRgroup(Fig.9AandB).

Compared withthe IR group, the IR+RIPC+Dex group displayedtubularatrophy,tubulardilatation,vacuolization, proteinaceousmaterialaccumulationandcelldebrisinthe lumenof thetubules, andloss ofthebrush borderof the tubulecells,albeittoalesserextent. However,compared withtheIR+Dexgroup,tubularchangeswerelessmarked (Fig.10AandB).

Immunohistochemicalstainingintensitywasincreasedin theactivecaspase-3-positivecellsoftheIRgroupcompared withthe Sham group,while it was decreased in thecells oftheIR+DexandIR+RIPC+Dexgroupscomparedwiththe IRgroup.Stainingintensityintheactivecaspase-3-positive cells ofthe IR+RIPC+Dex groupwasdecreased compared withtheIR+Dexgroup(Fig.11).

Group III

A

G P

P

D

B

Figure 9 (A and B) Sections of the IR+Dex group. G, glomerular; P, the proximal tubule; D, the distal tubule, ( )accumulation ofproteinaceous material to be reduced comparedtoothergroups.

Discussion

Acuterenal failure occurring asa result of ischemiamay be due to hypotension, hypovolemia, and hypoperfusion secondarytodehydrationaswellasIRdamageinmajor car-diothoracic,vascular,andtransplantsurgeries.15---18_IR_injury isone ofthe mostcommoncauses of perioperativeacute renalfailure.18

MethodssuchasRIPCandpharmacologicalconditioning withdexmedetomidine,whichareusedtopreventortreat renalIRinjury,havebeenshowntohavepositiveeffectsin termsofIRinjuryinmanypreviousstudies.11

Inaliteraturesearch,wefoundnostudythathad eval-uatedthe effects of combiningthese twomethods for IR injury.Thisexperimentalstudy,usingaratmodelofrenal IR,comparedtheeffectsof usingdexmedetomidinealone orRIPCincombinationwithdexmedetomidineagainstrenal apoptosis,assessing usingcaspase-3 immunoreactivityand histopathologicalinjuryscores.

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Group IV

A

B

D G

P

G

D

P P

D G

Figure 10 (A and B) Sections of the IR+RIPC+Dex group. G, glomerular; P, proximal tubule; D, distal tubule, and ( )accumulationofproteinaceoustobereducedcompared toother.

combined with RIPC group, suggesting that apoptosis, a majorpathwayofIRinjury,couldbereducedorprevented bythiscombination.

Variousperiods of ischemiaand reperfusion wereused inrenal IRinjury modelsinmanystudies.19---22 _The_critical ischemic period is dependent onthe organ,and ischemia lastingmorethan5minforthebrain,and15---20minforthe liverandkidneymaycauseneuronaldeathandinfarction.23 Williams et al.20 _investigated _the _effects _of _IR _injury _in bloodandtissuesamplesobtainedafterreperfusion follow-ing 45min of renal ischemia, and reported that renal IR injuryoccurred earliest,atthefourth hourandpeakedat 24h.

Organ-protectiveeffectsofdexmedetomidineagainstIR damagehavebeenshowninmanytissues,suchasthebrain, heart, and kidneys.24---27 _The _effects _of _{dexmedetomidine} inratrenalIRinjurywereinvestigatedbyKoco˘gluetal.19 Theseresearchersreportedasignificantdecreaseinscores for histopathological injury, detected at the 45th minute after ip administration of 100␮g/kg dexmedetomidine at the beginningof reperfusion. Inour study,withthe same dose of dexmedetomidine administered at the beginning of reperfusion, a renoprotective effect was achieved, as evidencedbysignificantreductionsinthehistologicalinjury scores.Thisrenoprotectiveeffectwasfoundnotonlyduring theearly periodafterreperfusioninjurybut itcontinued, evenat24h,whichwasconsideredthepeakofIRinjury.20

Althoughthemechanismunderlyingtheprotectiveeffect ofdexmedetomidineinrenalIRisunclear,itisconsidered to increase renal blood flow and glomerular filtration by reducingthereleaseofnoradrenaline.28

Villelaetal.29_reported_that_low-dose_{dexmedetomidine} administration in anesthetized dogs reduced the urinary osmolality and plasma vasopressin level and caused free-water diuresis. In patients who had no renal disease but

A

B

C

D

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experiencedthoracicsurgery, byadministeringaninfusion ofdexmedetomidine,Frumentoetal.30_showed_an improve-mentinrenalfunction,includingurineflowandglomerular filtrationinthepostoperativeperiod.

One of the most likely mechanisms of the action of dexmedetomidineisprotectingthekidneybyinhibitingthe surgicalstressresponseandpreventingadrenergic system-mediated vasoconstriction.31---34 _It _may _also _increase _renal arterialvasodilationthroughdirectvasculareffects.19 _PKC has been reported to play an important role in ischemic preconditioning and to open sarcolemmal and mitochon-drialATP-dependentK+_channels_by_stimulating_{intracellular}

transductionpathwaysandinducingsynthesisofprotective cellularproteins.Inastudyofalternative signal transduc-tionpathways, alpha 2B receptoragonists were shown to stimulatePKCactivityandtheproductionofinositol triphos-phateinthedistalrenalcollectingtubulecells,suggesting thatalpha2agonists mimiccellprotectionbyIPC.35,36 _Gu etal.18_investigated_the_mechanism_of_action_of dexmedeto-midine in vitro with a stabilized human renal proximal tubule cell culture deprived of both oxygen and glucose. They reported both a significant increase in phospho-Akt expression in cultured tubular cells after treatment with dexmedetomidine, dependent on the dose, and an alpha-2 adrenoreceptor effect. The phosphoAkt path-wayensures cellviability by inhibitingcaspase-controlled intrinsic apoptotic pathways through phosphorylation of proapoptoticBcl-2,whichtriggerscelldeath,and upregula-tionofanti-apoptoticBcl-2andBcl-XLexpression.

The neuroprotective effect of dexmedetomidine has beenreportedtobearesultoftheincreaseinanti-apoptotic Bcl-2andMdm-2expression;thisincreasehasbeen associ-atedwithadecreaseinthelevelsofproapoptoticcaspase-3 and Bax.37 _The _Akt _pathway _is _critical_in _recovering_from renalIR.Inthisstudy,asan indicatorofapoptosis, active caspase-3 immunoreactivity was evaluated. Activation of caspase-3isthefinalstepofapoptosis,whichiscommonto thetwomajorpathwaysofapoptosisandadefinitive indi-cator of cell death. Dexmedetomidine administrationwas showntodecreaseactivecaspase-3immunoreactivity,but notsignificantlyso(p=0.06),comparedwithIRinjury.

This statistically non-significant difference could be explainedbythesmallnumberofstudysubjects.Ischemic preconditioningisamethodappliedmechanicallyor phar-macologically prior to target organ ischemia to reduce the level of subsequent IR injury. The aim in ischemic preconditioningistoapplyischemiaandreperfusionto tar-get organs in short intervals, to ensure that the target organ(s)cantolerateischemiawell. Highenergydemands andtheintensemicrovascularnetworkofthekidneysmake them vulnerabletoIRinjury,which is considered amajor causeofkidney damagein renalarterystenosis andrenal microvascular surgery. Renal IR injury is the major cause ofcardiovascularmorbidityandmortality,andisassociated withpost-transplantationdelayingraftfunction,andrenal injuryoccurringincardiacandaorticsurgeryandpost-shock renalinjury.1---4,38,39

Bothexperimentalandclinical studiesinthe literature show that distant organ IPC may be protective for the kidney.40,41_Similar_to_our_study,_Wever_et_al.40_investigated theeffectof hindleg DIPConrenalIRinjury.Differently, these authors compared the effect of administering DIPC

eithercontinuouslyorperiodically,andalsotooneor two extremities.AlthoughtheyperformedthreecyclesofDIPC, theyusedI/Rperiodsof4/4minandinvestigatedthe even-tual effects of 25min of ischemia. They reported that molecule-1 expression, indicating renal tubular and renal injury,decreasedsignificantlyinthegroupwiththreecycles ofI/R;moreover,thisprotectionwasnotrelatedto adeno-sine,oneofthekeyelementsinI/Rinjury.Theunderlying mechanismof DIPCanditstransductionpathways arenot yetcompletelyunderstood.Bothneurogenicpathwaysand biochemicaltransmittersmayplayrolesinthemechanism ofDIPC.42 _These _mechanisms_may_vary, _depending _on_the targetorgan and appliedpreconditioning protocol. In the myocardialischemiamethodsusingrenalDIPC, expression ofNF-␬BproteinfollowedbytheopeningofK+_ATP_channels

havebeenreportedtobeimportant.43

AlthoughrenalIRinjuryisacommonandimportant clin-icalproblem,strategiestoreduceIRinjuryareinsufficient andnewtreatmentsareneeded.Intheliterature,noreport ofbothpharmacologicalandmechanicalprotectioninrenal IRinjuryinratscouldbefound.

Thus,twodifferentmethodsusedinratrenalIRinjury, dexmedetomidine, widely reported to be effective, and DIPC,thathasbeenshowntobeeffectiveinfewstudies,11 werecombinedandcompared.Similartodexmedetomidine, usingdexmedetomidineandDIPCincombinationprevented histopathological injury and improved IR injury scores, excepttubularchangescores, tolevelsclosetotheSham group. At the same time, significant decreases in active caspase-3 immunoreactivity compared with the IR group suggested that using dexmedetomidine and DIPC in com-binationmight preventapoptosis.Using dexmedetomidine alonedecreased activecaspase-3immunoreactivity,albeit non-significantlyso,whichsuggeststhatthesetwomethods operateviasimilarpathways.Thesetwoprotectivemethods arelikelytoincreasetheeffectofeachother.

Inourstudy,xylazine,ananestheticagenthavingalpha 2-agonistactivity,andketamine42_suggested_to_have_negative effectsonIRinjury,wereused.Histopathologicalscoresand activecaspase-3immunoreactivitywerenormalintheSham group,whichsuggeststhatketaminehadnonegativeeffect onourresults.

WedidnotshowtheeffectofDIPCaloneonapoptosis, whichplaysakeyroleinthemechanismofIRinjury;thisis alimitationofthepresentstudy.Otherlimitingfactorsare thatoxidativestressandinflammatorymediators,whichare alsoresponsibleforIRinjury,andtheneurogenicpathway, werenotexamined.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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