Effect of PACAP treatment on kidney morphology and cytokine expression in rat diabetic nephropathy

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Peptides

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

Short communication

Effect of PACAP treatment on kidney morphology and cytokine expression in rat diabetic nephropathy

E. Banki

^a

, P. Degrell

^b

, P. Kiss

^a

, K. Kovacs

^c

, A. Kemeny

^d

, K. Csanaky

^a

, A. Duh

^a

, D. Nagy

^d

, G. Toth

^e

, A. Tamas

^a

, D. Reglodi

^a,∗

aDepartmentofAnatomyPTE-MTA“Lendulet”PACAPResearchTeam,UniversityofPecs,Pecs,Hungary

bInternalMedicine2/NephrologyCenter,UniversityofPecs,Pecs,Hungary

cDepartmentofBiochemistryandMedicalChemistry,UniversityofPecs,Pecs,Hungary

dDepartmentofPharmacologyandPharmacotherapy,UniversityofPecs,Pecs,Hungary

eDepartmentofMedicalChemistry,UniversityofSzeged,Szeged,Hungary

a r t i c l e i n f o

Articlehistory:

Received2January2013

Receivedinrevisedform4February2013 Accepted4February2013

Availableonline13February2013

a b s t r a c t

Pituitaryadenylatecyclaseactivatingpolypeptide(PACAP)isaneuropeptide,exertingdiverseeffects.One ofitsfrequentlyexaminedfunctionsiscellprotection,whichisachievedmainlyviainhibitingapoptotic, inflammatoryandoxidativeprocesses.Allitsthreereceptors(PAC1,VPAC1,VPAC2)areexpressedinthe kidneyandPACAPhasbeenshowntohaveprotectiveeffectsagainstdifferentrenalpathologies.Diabetic nephropathyistheleadingcauseofendstagerenaldisease.Theaimofthepresentstudywastoinvestigate thepossibleameliorativeeffectofPACAPinstreptozotocin-induceddiabeticnephropathyandtoevalu- ateitsanti-inflammatoryeffectinthismodel.Diabeteswasinducedbyasingleintravenousinjectionof streptozotocin(65mg/kg)inmaleWistarrats.PACAP-treatedanimalswereadministeredip.20␮gPACAP everysecondday,whileuntreatedanimalsweregivenvehicle.Kidneyswereremovedafter8-weeks survival.Besidesthecomplexhistologicalanalysis(glomerularPASpositivearea/glomerulusarea,tubulardamage,arteriolarhyalinosis),expressionofseveralcytokineswasevaluatedbycytokinearrayand Luminexassay.Histologicalanalysisrevealedseverediabeticchangesinkidneysofcontroldiabeticani- mals(glomerularPAS-positiveareaexpansion,tubulardamage,Armanni-Ebsteinphenomenon).PACAP treatmentsignificantlydiminishedthedamage.Diabetickidneysshowedsignificantcytokineactivation comparedtotheirhealthycontrols.PACAPwaseffectiveindownregulationofseveralcytokinesincluding CINC-1,TIMP-1,LIX,MIG,s-ICAM.Toconclude,PACAPiseffectiveinamelioratingdiabeticnephropathy atleastpartlythroughitswell-knownanti-inflammatoryeffect.Theseresultsraisetheopportunityfor theuseofPACAPasapossibletherapeuticorpreventivemethodintreatingthecomplicationsofdiabetes.

1. Introduction

Pituitaryadenylatecyclaseactivatingpolypeptide(PACAP)isa neurotrophicandneuroprotectiveneuropeptide,whichexistsin twoformswith38and27aminoacidresidues:PACAP-38and- 27,respectively[42].PACAPactsthroughthreeGprotein-coupled receptors:thespeciﬁcPAC1andthenon-speciﬁcVPAC1andVPAC2 receptors.Highestlevelsof PACAPoccurinthecentralnervous systemandendocrineglands[42].However,lowerexpressionhas alsobeenshowninthegastrointestinal,respiratory,cardiovascular andurogenitalsystems[13,42].Basedonthewidespreaddistribu- tion,PACAPhasbeenfoundtohavediverseeffectsinthenervous

∗Correspondingauthorat:DepartmentofAnatomy,UniversityofPecs,7624Pecs, Szigetiu12,Hungary.Tel.:+3672536001x35398;fax:+3672536393.

E-mailaddress:dora.reglodi@aok.pte.hu(D.Reglodi).

systemandperipheralorgans.Oneoftheearlydescribedeffects ofPACAPisitsstrongneuroprotectivepotential.Severalstudies haveproventhatPACAPisageneralcytoprotectivepeptide,exert- ingcell-survivalpromotingeffectsinnumeroustissuesandcells, including endothelialcells, lymphocytes, liver,lungs and ovary [11,28,45,47].

Actionsof PACAP in thekidney are less known.PACAP and itsreceptors(PAC1,VPAC1 andVPAC2)areallexpressedin the human, rat and chicken renal tissues [27]. Some effects have alsobeendescribedinthekidney,suchasvasodilationandrenin secretion[27].However,alargebodyofexperimentaldatasup- portstheprotectiveeffectsofPACAPinthekidney.Itiseffective againsthydrogenperoxide-inducedoxidativestressinvitro[15].In vivo,PACAPisprotectiveagainstischemia/reperfusioninjuryand improvesthesurvivalrateofratsundergonerenalvesselclamping [17,38].PACAPisalsorenoprotectiveagainstmyelomanephropa- thy,subsequentlyevenconﬁrmedinasinglepatientstudy[2,20].

http://dx.doi.org/10.1016/j.peptides.2013.02.002

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SimilarnephroprotectiveeffectcanbeobservedincyclosporineA andgentamicin-inducedinjuries[2,18,20,27].

Based on thesedata, the nephroprotectiveeffects of PACAP arewell-established.However,littleisknownabouttherelation- shipbetweenPACAPandoneofthemostcommonnephropathies, thediabeticnephropathy.Amongalltheclassicaldiabeticcompli- cations(retino-,nephro-andneuropathy),diabeticnephropathy causesthehighestincreaseinmorbidityandmortality.Diabetic nephropathy affects approximately 30–40% of all the diabetic patientsanditistheleadingcauseofchronickidneydiseaseand endstagerenalfailure.Patientssufferingfromdiabeticnephropa- thyshow15–20-foldhighermortalityratecomparedtohealthy subjects.Thediseaseischaracterizedbybothfunctionalandstruc- turalchangesofthekidney.Glomerularhypertrophy,glomerular basement membrane thickening, apoptosis and desquamation of the podocytes are already present in the early stage of the disease. Mesangial matrix expansion, diffuse and nodular glomerulosclerosis,tubulointerstitialﬁbrosis,tubularatrophyand glycogen-accumulationalsodevelopwiththeprogressionofthe disease(Armanni-Ebsteinphenomenon)[40,48].Inrats,diabetic nephropathycanbemimickedbystreptozotocin-inducedpancre- aticbetacellloss[41].Anearlierstudyhasdescribedprotective effectsofPACAPinearlydiabeticnephropathy[20]andwehave recentlyproventhatPACAPisprotectiveinanothercommoncom- plicationofdiabetes,thediabeticretinopathy[36].Theaimofthe presentstudywastoinvestigatetheprotectivepotentialofinvivo PACAPtreatmentin8weekdiabetes-inducednephropathyandto studytheinvolvementof anti-inﬂammatorypathwaysbyusing cytokinemeasurements.

2. Materialsandmethods 2.1. Animals

AdultmaleWistarrats(n=19)weighing250–300gwerehoused underlight/darkcyclesof12:12handreceivednormalratchow and drinkingwater ad libitum. Rats were randomlydivided in 4 groups: (1) untreated control (saline i.v. and i.p., n=4); (2) PACAP-treatedcontrol(salinei.v.and20␮gPACAP1-38i.p.every second day, n=4); (3) untreated diabetic (65mg/kg streptozotocin(Sigma,Hungary)i.v.andsalinei.p.,n=6);(4)PACAP-treated diabetic(65mg/kg streptozotocin i.v.and 20␮g PACAP1-38i.p.

every second day, n=5). Rats were weighed and their blood glucose concentration was measured weekly by blood glucose monitor(Accu-CheckActive,Roche,Hungary).Animalswerecon- sidereddiabeticincasestheyshowedelevatedbloodglucoselevels (>11mmol/l) and theclassical symptoms of diabetes including polyuria,polydipsia,polyphagiaandexcessiveweightloss.After 8weeks,animalsweresacriﬁcedwithanoverdoseofanesthetics andkidneyswereremovedandweighed.Experimentalprocedures werecarriedoutinaccordancewithapprovedprotocols(University ofPecs;BA02/2000-15024/2011).

2.2. Renalhistologyandmorphometricanalysis

Kidneyswerefixedin10%formalin,embeddedinparaffinand 5␮mthinsectionswerecutwithmicrotome.Sectionswerestained withperiodicacid-Schiffbase(PAS)or withhematoxylin–eosin anddigitalphotosweretaken.DiastasedigestedPASreactionafter absolutealcoholfixationwasalsoperformedtoprovetheglycogen contentof tubulargranules.Renalhistologicalanalysiswascar- riedoutbyanephropathologistblindedtothetreatmentgroups (PD).Atleast10glomerulioneachslidewereexaminedfordeter- minationofglomerularchanges[40].Thedegreeofaccumulated glycogengranules(Armanni-Ebsteinphenomenon)wasevaluated

asanindicatoroftubulointerstitiallesions.Analysiswascarriedout at400×magnification.MagentastainingofintraglomerularPAS positiveareashowsthemesangialmatrixand basalmembrane, whiletubularPASpositivegranules,Armanni-Ebsteinlesionsare thesignoftubulardamage.AdobePhotoshopprogramwasused tomarktheglomerulusmanually,thenitwasconvertedtoblack, whiletheremainingareatowhite.Thepropermagentaofintra- glomerularPASpositiveareaandtubularglycogengranuleswere alsomarkedandconvertedtoblackwiththesamemethod.Finally in every picture the “area of interest”was converted to black withtheremainingareabecomingwhite,thuscreatingblackand white pictures. Black areas in the vision fields were measured usingScionImageprogram.Finally,theintraglomerularPASpos- itiveareawasexpressedagainsttheareaofthesameglomerulus, whileglycogengranulesagainstthevisionfield.Arteriolarhyali- nosis,alackofsmoothmuscleinthearteriolarwall,wasevaluated onhematoxylin-eosinsectionsaccordingtothefollowingscore:“0”

arteriolewithoutanysignofhyalinosis,“1”,“2”,“3”,“4”withhyali- nosisaffecting¼,½,

¾

ortheentirecrosssectionofthearteriole, respectively.

2.3. Cytokinemeasurements

Thesemiquantitativecytokinearraywasperformedfromkid- ney homogenates using Rat Cytokine Array kit (R&D Systems;

BiomedicaHungaria,Hungary).Thesearraysarebasedonbinding between sample proteinsand carefully selected captured anti- bodiesspottedonnitrocellulosemembranes.Weexaminedtissue samples fromall the 4 groups:control, PACAP-treated control, diabeticandPACAP-treateddiabeticgroups.Thearraywasper- formedasdescribedbythemanufacturer,similarlytoourprevious study[25].Briefly,kidneysampleswerehomogenizedinPBSwith proteaseinhibitor.TritonX-100wasaddedtothefinal concen- trationsof 1%.Afterblocking thearraymembranes for 1hand addingthereconstituteddetectionantibodycocktailforanother 1hat roomtemperature, themembranes wereincubated with 1ml of tissue homogenates at 2–8^◦C overnight on a rocking platform. After washing with buffer three times and addition ofhorseradishperoxidase-conjugatedstreptavidintoeachmem- brane,weexposedthemtoachemiluminescentdetectionreagent (AmershamBiosciences,Hungary),thensideuptoanX-rayfilm cassette.Thearraywasrunin duplicatesets.Resultsarerepre- sentedinpixel densityand arecomparedin asemiquantitative fashion.

LuminexMultiplexImmunoassaywasperformedasdescribed previously[25].Briefly,soluble intercellularadhesionmolecule- 1 (sICAM1) and L-selectin levels were determinated using customizedFlurokineMAPRatBaseKit(R&DSystems).Theexper- imentwasperformedaccordingtothemanufacturer’sinstructions (R&DSystems).Followingpreviousoptimizations,allsampleswere testedundilutedinablind-fashion.Luminex100devicewasused fortheimmunoassayandLuminex100ISsoftwarefortheanalysis ofbeadmedianfluorescenceintensity.Sampleswerehomogenized withRPMI-1640(GIBCO)containing1%proteaseinhibitorcock- tail,sampleswereusedin20mg/mlconcentrations.Allthetests wereruninduplicate.50␮lvolumeofeach sample,control,or standardwasaddedtoa96-wellplate(providedwiththekit)containing 50␮lofantibody-coated fluorescentbeads.Biotinylated secondaryandstreptavidin-PEantibodieswereaddedtotheplate withalternateincubationandwashingsteps.Afterthelastwash- ingstep,100␮lofthebufferwasadded tothewells;theplate wasincubatedand readontheLuminex100arrayreader,using afour-PLregressioncurvetoplotthestandardcurve.Datawere subsequentlyanalyzedusingtheLuminex100managersoftware.

ResultsaregiveninpgL-selectinors-ICAM/gwettissue.

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Fig.1.Histologicalanalysisofcontrolanddiabetickidneys.Representativehistologicalsectionsofglomeruli(rowA)andrenaltubules(B).Sectionsofcontrol(AandB), PACAP-treatedcontrol(AandB),diabetic(AandB),PACAP-treateddiabetic(AandB)animals.ControlandPACAP-treatedcontrolanimalsshowednormalglomerular andtubularstructure.DiabetickidneysshowedsevereintraglomerularPASpositiveareaexpansionandglomerulardamage(A)andseveretubularatrophyanddeposition ofglycogengranules,calledArmanni-Ebsteinphenomenon(B,arrows).PACAP-treatmentwaspreventiveagainstdiabeticchanges,milderdamagewasobservedinthese sections(AandB).Periodicacid-Schiff(PAS)reaction.Calibrationmark:20␮m.Representativehistologicalsectionsofarterioleswithaseverityscoreof0,1,2,3,4 (C,C,C,C,Crespectively).Hematoxylin–eosinstaining.Calibrationmark:20␮m.PASpositiveareaperglomerulararearatio(D)***p<0.0001vs.allothergroups (control,control+PACAP,diabetes+PACAPgroups).Glycogengranules(Armanni-Ebsteinphenomenon)pervisionﬁeldratio(E)***p<0.0001vs.allothergroups(control, control+PACAP,diabetes+PACAPgroups).Vascularhyalinosisscore(F).Signiﬁcanthyalinosiswasobservedindiabeticanimals,whileinthePACAP-treateddiabeticanimals therewerenosignsofthePASpositivearteriolarwallthickening.***p<0.0001diabetesvsallothergroups(control,control+PACAP,diabetes+PACAPgroups).

2.4. Statisticalanalysis

Statistical analysis wasperformed by Microsoft Office Excel andGraphPadsoftware.Repeatedmeasuresanalysisofvariance (ANOVA)withBonferronicorrectionwasusedtodetectsignificant differencesbetweengroups.p-Valuelessthan0.05wasconsidered tobestatisticallysignificant.

3. Results

3.1. Bodyweight,totalkidneyweighttobodyweightratioand bloodglucoselevels

Thebodyweightof theanimalsafter8 weeksoftreatments was365±29gintheintactcontrolsand351±31ginthePACAP- treatedgroup.Diabeticanimalsshowedasignificantdecreasein theirweight:wemeasured230±17ginthediabeticand260±19g inthePACAP-treateddiabeticgroup.However,theslightdifference betweendiabeticandPACAP-treateddiabeticratsdidnotresultto bestatisticallysignificant.PACAPtreatmentalonedidnotresultin significantchangeinkidney/bodyweight(6.44±0.13)compared totheuntreatedcontrolkidneys(6.68±0.09).Indiabeticanimals, wecouldobserveasignificantincreaseinthisratio(13.80±1.36), similarlytoobservationsbyothers[6].PACAPtreatmentindia- beticanimalsslightlydecreasedthisratio,however,thiswasnot statisticallysignificant(12.33±0.37).Therewasnodifferencein

the weekly blood sugar level in the intactcontrol and PACAP- treatedgroupsorbetweenthediabeticcontrolandPACAP-treated groups.Attheendoftheexperiment(atweek8)thebloodglucose levelwas7.85±0.42and7.55±0.36mmol/Lintheintactcontrol andPACAP-treatedintactanimals,respectively.Inthediabeticani- mals,thesevalues were30.13±1.75mmol/Linthediabeticrats and32.42±0.69mmol/LinthePACAP-treateddiabeticanimals.

3.2. PACAPattenuateddiabeticnephropathy:renalhistologyand morphometricanalysis

In control animals, normal glomerular structure was found withoutanytubularor glomerularinjury and PACAPtreatment for8weeksdidnotcauseanychanges.Kidneysfromdiabeticani- malsshowedsignsofseverenephropathy,withmesangialmatrix expansionandabundantglycogengranules(Fig.1A–D).Ahallmark of diabeticchangesindicating theprogression ofthemesangial changes indiabeticnephropathy is theratioof intraglomerular PASpositiveareaperglomerulararea.Thissigniﬁcantlyincreased in the diabetic rats, while PACAP treatment could effectively counteract this lesion: PACAP-treated diabetic animals showed no signiﬁcant differencecompared with intact control kidneys (Fig.1AandD).Anotherveryimportantcharacteristicsofdiabetic nephropathyistheappearanceofglycogendepositsinthetubules, alsocalledArmanni-Ebsteinphenomenon[48].Thisfeaturewas completelymissingfromcontrolkidneys,whilethepresenceof

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Fig.2.DataofthesemiquantitativecytokinearrayofCINC-1,TIMP-1,LIX,MIG,RANTES,MIP-3␣,CNTF(A)–(G).Dataarepresentedasmeanpixeldensity.Measurement ofL-selectinands-ICAMbyLuminexassay(H)and(I).Dataarepresentedasmeanpg/gwettissue±SEM.*p<0.05,**p<0.001,***p<0.0001vs.control,^###p<0.0001vs.

diabetes+PACAP.

glycogendepositsmarkedlyincreasedindiabeticrats.Incontrast, theamountofglycogen depositswassigniﬁcantlylower inthe PACAP-treateddiabeticgroupthanintheuntreateddiabeticgroup (Fig.1BandE).Acharacteristicfeatureofmicroangiopathyisvas- cularhyalinosis,whichissparinglypresentinthecontrolgroups.

Thedegreeofarteriolarhyalinosiswassigniﬁcantlyhigherinthe diabeticgroupcomparedtocontrolanimals,whilePACAPtreat- mentwasabletocompletelypreventthedevelopmentofhyalinosis (Fig.1CandF).

3.3. Cytokinemeasurements

PACAP treatment had no effect on the level of numerous cytokines in control animals, however, it elevated the expres- sionof afew cytokines: tissue inhibitorof metalloproteinase 1 (TIMP-1),monokineinducedbygammainterferon(MIG/CXCL9), macrophage inﬂammatory protein 3␣ (MIP-3␣), regulated and normalTcellexpressedandsecreted(RANTES,CCL5),L-selectin (CD62L/LECAM-1)anddecreasedlipopolysaccharide-inducedCXC chemokine(LIX/CXCL5)andciliaryneurotrophicfactor(CNTF)levels.Diabetesstronglyincreasedtheexpressionofcytokineinduced neutrophilchemokine(CINC-1),TIMP-1,LIX,MIG,MIP-3␣,RANTES, CNTF and L-selectin. Treatment with 20␮g PACAP markedly decreasedtheabove-mentionedcytokinesandchemokines,with someofthemreachingcontrollevels(Fig.2A–I).Nochangeswere observedinotherspots:Fractalkine,thymuschemokineandinter- leukins(datanotshown).

4. Discussion

In the present study we showed that in vivo PACAP treat- mentisprotectiveindiabeticnephropathyandthisameliorative effect is at least partly due to the anti-inﬂammatory effect of PACAP.Ourobservationscompletethelistofkidneypathologies againstwhichPACAP hasprotective effects.Among others,it is

effectiveagainstoxidativestress,gentamicin-inducednephropa- thy, myeloma and ischemia-induced injuries and cyclosporine A-inducedlesion[2,15,18,20,38].Anearlierstudyhasevenmen- tionedthatPACAPhasprotectiveeffectsinearly(2weeks)diabetic nephropathy in rats [20]. Our present results, with prolonged survival time, are thus in accordance with these observations and complete these ﬁndings withdetailed histologicalanalysis andcytokinemeasurements.Furthermore,weappliedlong-term PACAPadministrationfortheﬁrsttimeinthetreatmentofrenal diseases,provingthatPACAPtreatmentcanbeeffectiveevenin chronictherapy.

OurstudyshowedthatPACAPmarkedlyattenuatedthecharac- teristicpathologicalalterationsobservedindiabeticratswithout alteringtheblood glucoselevels.IncreasedintraglomerularPAS positiveareaisareliableindicatorofbasementmembranethick- ening and glomerular mesangial matrix expansion, which are characteristicfordiabeticglomerulopathy[40].Inthepresentstudy wefoundsignificantexpansionofglomerular PAS-positive area caused byhyperglycemia aftera singlestreptozotocin injection similarlytoothers[22].Hyperglycemiastimulatesthepatholog- icalaccumulationoftheotherwise physiologicalcomponentsin the extracellular matrix, like laminin, fibronectin, collagene IV throughoverexpressionofTGF-␤1[12].Changesinthetubuloint- erstitiummaypossessevenhigherimportanceintheprogression ofdiabetickidneydisease[39].Tubularvacuolationonautopsyis consideredtobepathognomictodeathduetodiabeticcoma.In ourstudy,tubulardamagewasevaluatedbasedontheamountof glycogengranuleswithintheproximaltubules,calledArmanni- Ebsteinphenomenon[19,48].Arteriolarhyalinosisoccursinboth afferentand efferentarteriolesin thediabetickidney,however, efferentarteriolarhyalinosisisconsideredtobecharacteristicfor diabeticnephropathy[35].KidneysofPACAP-treateddiabeticani- malsshowedsignificantlymilderglomerular,tubularandvascular changes.

Asfarastheprotectivemechanismisconcerned,weinvesti- gatedtheanti-inﬂammatoryeffectsofPACAPinthepresentstudy.

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PACAPiswell-knownforitsanti-inflammatoryactionsalsoinsome oftheabove-mentionedrenalpathologicalconditions[27].Recent studiesindicatethatinflammationisoneofthekeymechanisms inthedevelopmentofdiabeticnephropathyinadditiontohyper- glycemia and hemodynamicfactors [24]. Mostrenal cells, such asendothelial,mesangial,epithelialcellsandpodocytesbecome activated and produce proinflammatory cytokines, chemokines and adhesion molecules upon injury. These chemoattractant moleculesinduceinfiltrationofseveralimmunecells,likemono- cytes, neutrophils and lymphocytes intothe kidney, leadingto tubulointerstitialfibrosis,glomerulosclerosis,tubularatrophyand vasculardamage[1,46].PACAPhasbeenfoundtoaltercytokine expressioninnumerousstudies.Wehavedescribed earlierthat PACAPcounteractedseveralchangesincytokineandchemokine expressioninducedbyintestinalandkidneyischemia/reperfusion andretinalischemia[15,25,37].Inthepresentstudywefoundthat PACAPreducedthediabetes-inducedelevationofCINC-1,TIMP-1, LIX,MIG,MIP-3␣,RANTES,L-selectinands-ICAM.Allthesefactors playapotentialroleindiabeticnephropathy.Forexample,CINC-1, oneofthemajorneutrophilchemoattractantsstimulatedbyoxida- tivestress and ischemia/reperfusion, isalso present in diabetic nephropathy[7,23].LevelsofTIMP-1,LIX,MIGands-ICAM,arealso elevatedintheurineandserumofdiabeticnephropathypatients [4,5,14,16].MIP-3␣mRNA and proteinwere foundtobe upre- gulatedbyhyperglycemiabothinvivoandinvitroandelevated urinaryMIP-3␣aftertransplantationmightcontributetoallograft rejection[26,43].TheeffectsofPACAPontheinjury-inducedupre- gulatedexpressionofinflammatorycytokinesseemtobeamore generalphenomenon,sincewefoundsimilareffectsintheretina andintestinaltissue[25,37].

We found an interesting pattern of RANTES and L-selectin expression in our treated groups. PACAP alone increased the expression of both, while it descreased theirlevels in diabetic animals.TheeffectofPACAPonsomecytokinesandchemokines iscontradictoryandbothstimulatingandinhibitingeffectshave beendescribeddependingonthecelltypeinjury [9,37]. Earlier studieshaveshownupregulatedRANTESindiabetickidneys,cor- relating withproteinuriaand expressionofNF␬B, suggestiveof progressivediabeticnephropathy.Earlierstudieshavefoundthat PACAPcaninducethesecretionofRANTESinastrocytesandcortical neurons[3,31],mostprobablyassociatedwithincreasedneuro- protection.However,inotherstudies,PACAPreducedtheelevated expressionofRANTESintheretinaandinmicroglialcells[9,37].

The exact role of PACAP in renal RANTES expression needsto befurtherinvestigated,buttheﬁndingthatPACAPcouldreduce thediabetes-inducedelevationsuggestsanephroprotectiveaction.

Arapid increasein L-selectin,a celladhesionmoleculerespon- siblefor therenalmononuclear infiltration,hasbeendescribed afterrenalischemia/reperfusioninjury[10,32,34].Thefindingthat PACAPreducedthediabetes-inducedelevationofL-selectinsug- gestsaprotectiveeffect,buttheinitialstimulatoryeffectofPACAP incontrolanimalsneedstobefurtherclarified.Incontrasttoother cytokines,PACAPstimulatedtheexpressionofthetrophicfactor CNTFindiabetickidneys,indicating anotherpossibleprotective mechanismsimilarlytoearlierdescriptions[29].

Insummary,ourpresentstudyshowedtheprotectiveeffects ofinvivoPACAPtreatmentindiabeticnephropathybyinvolving anti-inﬂammatoryactions.Ourstudyfurthersupportstheimplica- tionthatPACAPmighthaveprotectivepotentialindiabetesandits complications.PACAPisabletoincreasepancreaticbetacellpro- liferation,enhanceinsulinsecretionandithasbeenshowntobe protectiveintwomajordiabeticcomplications:retinopathyand nephropathy[30,33,36,44].Thus, PACAPisagood candidatefor therapeuticinterventionsindiabetesatmultiplelevels.Oneofthe drawbacksofsystemicPACAPtreatmentistherapiddegradation ofthepeptideinthebloodbydipeptidylpeptidase-IV[DPP-IV,42].

Inhibitorsofthisenzymearebeingusedinthetreatmentofdia- beticpatientsfortheincreasedlevelofglucagon-likepeptide-1[8].

Protectiveeffectshavebeendescribedevenindiabeticnephropa- thy[21].Arecentpaperhassuggestedthatthetherapeuticeffects ofDPP-IVinhibitorscouldinvolvesofarunknownmechanisms, suchasincreasedPACAPlevels[8].Whethertheprolongedpres- enceofPACAPinthecirculationisprotectiveindiabetes-related complications,istobedeterminedbyfutureinvestigation.

Acknowledgements

This study was supported by OTKA K104984, TAMOP (4.2.1.B-10/2/KONV-2010-002, 4.2.2.B-10/1-2010-0029, 4.2.2.A- 11/1/KONV-2012-0024),ArimuraFoundation,PTE-MTA“Lendület”

Program.

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