The renal effects and initial of venom nattereri Steindachner, 1870

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Toxicology

Reports

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / t o x r e p

The

renal

effects

and

initial

characterization

of

venom

from

Philodryas

nattereri

Steindachner,

1870

Marinetes

Dantas

de

Aquino

Nery

,

Natacha

Teresa

Queiroz

Alves

,

Renata

de

Souza

Alves

_,

_Daniel

_Freire

_de

_Sousa

_,

Dalgimar

Beserra

de

Menezes

_,

_Erik

_de

_Aquino

_Nery

_,

Hermano

Damasceno

de

Aquino

_,

_Rayane

_de

_Tasso

_Moreira

_Ribeiro

_,

Helena

Serra

Azul

Monteiro

a_{DepartmentofPhysiologyandPharmacology,DepartmentofBiology,FederalUniversityofCeará,Fortaleza,Ceará,Brazil}

b_{DepartmentofPhysiologyandPharmacology,FederalUniversityofCeará,Fortaleza,Ceará,Brazil}

c_{FacultyofPharmacy,FederalUniversityofCeará,Fortaleza,Ceará,Brazil}

d_{DepartmentofPathology,FederalUniversityofCeará,Fortaleza,Ceará,Brazil}

e_{GeneralHospitalofFortaleza,Fortaleza,Ceará,Brazil}

f_{DepartmentofBiology,FederalUniversityofCeará,Fortaleza,Ceará,Brazil}

g_{InstituteofBiosciences,UniversityofSãoPaulo,SãoPaulo,SãoPaulo,Brazil}

h_{DepartmentofPhysiologyandPharmacology,FacultyofMedicine,FederalUniversityofCeará,Fortaleza,Ceará,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28June2014

Receivedinrevisedform4September2014 Accepted9September2014

Availableonline2October2014

Keywords:

Philodryasnattereri

Proteincontent Renalfailure Cytotoxicactivity

Chemicalcompoundsstudiedinthisarticle:

Beta-mercaptoethanol(PubChemCID: 1567)

Serumalbumin(PubChemCID:16132) MMTtetrazolium(PubChemCID:64965) Trishydrochloride(PubChemCID93573) 2-Propenamide(PubChemCID:6579) Saline(PubChemCID5234)

a

b

s

t

r

a

c

t

ThevenomofthesnakePhilodryasnattereriisamixtureofproteinsandtoxicpeptideswith severalimportantlocalandsystemicactions,whicharesimilartothoseoccurringin Both-ropssnakebites.Themechanismsinvolvedinthelocalandsystemicactionsofthisvenom areunknown.TheaimsoftheworkweretoinitialcharacterizationofP.nattererivenom andinvestigatetheeffectsofthepoisonintherenalperfusionsystemandinculturedrenal tubularcellsofthetypeMDCK(Madin–Darbycaninekidney).TheP.nattererivenomis composedmajorityofproteins(86.3%)andthispoisonpromotedchangesinallthe evalu-atedrenalparameters,mainlydecreasingrenalperfusionpressure(PP)andrenalvascular resistance(RVR)andincreasingurineflow(UF)andglomerularfiltrationrate(GFR).The mostrelevantresultwasthatthisvenomwashighlydetrimentaltotherenaltubules inde-pendentofthePPreduction,whichwasshownbyadecreaseinsodium(Na+_),potassium (K+_{)andchloride(Cl}−_{)electrolytetransportinthestudiedconcentrations.Theglomeruli} andtubulescontainproteinbodiesandbloodextravasation,whichwereobservedby his-tologicalanalysis.ThevenomofP.nattererireducedviabilityoftheMDCKcellsonlyathigh concentrations(50and100␮g/mL)withanIC50of169.5␮g/mL.

©2014TheAuthors.PublishedbyElsevierIrelandLtd.Thisisanopenaccessarticleunder theCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).

∗ Firstandcorrespondingauthor.Tel.:+558533669834.

E-mailaddress:[email protected](M.D.d.A.Nery).

1. Introduction

Philodryas nattereri Steindachner, 1870 of the family Dipsadidae[1],commonlycalledthebrownracer,hasan olivegreencolorationwiththefinalportionofitsbody col-oredbrown.Thissnakeis1.20–1.60mlong,haslargeeyes

http://dx.doi.org/10.1016/j.toxrep.2014.09.005

withroundpupils,isfastandhasanintensedailyactivity (VittandColli[27]).Thesnakes’habitatisrelatedtothe environment’sphysicalstructure,foodavailability, pres-enceofpredatorsandthephysiologyofthesesnakes,which arediurnal,arborealandsemi-arboreal.Thesesnakesfeed onsmallmammals,birdsandlizards[25],areoviparous andlayfrom6to20eggs.Thesnakes’dentitionis opistho-glyphousandconnectedtotheDuvernoy’sgland.

P.nattereriisdistributedinaridandsemiaridregionsof SouthAmericaandismostcommoninnortheasternBrazil (CearáandRioGrandedoNorte).

Duringevolution,snakeshavespecializedinaffecting thevitalfunctionsoftheirpreybyreleasingalarge num-ber oftoxins (enzymes, proteinsand peptides) through venom that destabilize the physiological levels of hor-mones, alter the activity of enzymes, receptors or ion channels,andpromotecardiovascularandnervoussystem imbalanceintheirprey.Theuseofsnaketoxinsas phar-macologicaltoolsandprototypesfordrugdevelopmentis increasing[2].

Itisalsoimportanttoemphasize thattheseverityof symptoms after poisoning is related to the amount of venom inoculated, which depends on the snake’s size, ageandtime itwasfed.Poisoningcausedbyspecies of

Philodryasischaracterizedbylocalsymptomssuchaspain, swelling, erythema, bruising, renal failure and regional lymphadenopathywithnormalcoagulation[3].

The pathogenesis of the renal alterations following envenomationby Philodryasspecies isnot welldefined. Thus, this study aimed to evaluate the renal effects of

P.nattererivenom ina perfusionsystemusingdifferent concentrations of venom, to characterize possible his-tological alterations promotedbyvenom in isolated rat kidneysand tostudyvenom-inducedchangesinculture ofMadin–Darbycaninekidney(MDCK).

2. Materialandmethods

2.1. Animalsandvenomextraction

P.nattererisnakeswerecapturedonAroeirasFarminthe municipality of Upanema(5◦₃₈′₃₂′′_{Sand 37}◦₁₅′₂₇′′_W), stateofRioGrandedoNorte andtransportedtoNUROF (OphiologyRegionalNucleusofCeará).

Theanimalsweremaintainedinindividualcageswith freeaccesstowaterandfedwith15gmiceevery30days.

Venompools weremadefrommore than40 individual

snakesandcollectedfromthevenomglandintocapillary tubestopreventcontaminationwithsaliva.Afterthe out-flowofthevenomintothecapillarytube,thevenomwas frozenandlyophilized.

2.2. Analysisofproteincontent

Thevenom ofP.nattereri(1mg)waslyophilizedand resuspendedin1mLofsalinesolution.Analiquotofvenom (100␮L)wastakenforthequantificationofproteinsbythe methodofBradford[4]usingBio-Radreagentsandbovine serumalbumin(BSA)asastandard.

2.3. Sodiumdodecylsulphate-polyacrylamidegel electrophoresis(SDS-PAGE)

Sodium dodecyl sulphate-polyacrylamide gel elec-trophoresis(SDS-PAGE)wasperformedaccordingtothe methodof Laemmli[5].SDS-PAGE wascarried outin a 2mmverticalslabgel(10cm×8cm)consistingof stack-inggel mix, 5%total acrylamide, andmain running gel mix, 17.5% acrylamide, prepared in 3.0M Tris-HCl, pH 8.8.Samples(20␮g)weredissolvedinTris–HCl0.0625M, pH 6.8, containing 1% SDS and 1% ␤-mercaptoethanol andincubated at100◦_{Cfor 10min.Electrophoresis was} carriedoutat20mAperplatefor1.5hours.Protein mark-ers employed were myosin (212kDa), ␤-galactosidase (116kDa),phosphorylaseB(97.4kDa),BSA(66.2kDa),egg albumin(45kDa),carbonicanhydrase (31kDa),soybean trypsininhibitor(21.4/19.7kDa)andlysozyme(14.2kDa) (AMRESCOInc.,Ohio,USA)[6,28].

2.4. Nuclearmagneticresonancespectroscopy(NMR)

For1_{HNMRanalysis,spectrawererecordedonaBruker}

DRX-300MHzFTNMRspectrometer.VenomsamplesofP. nattereri(5mg/mL)werelyophilizedandpreparedusing dimethylsulphoxide(DMSO)asasolvent.Thespectrawere obtainedat85◦_{Cusingarelaxationdelayof1sandapulse} widthof90◦_{toreachtheconditionsofquantitative} anal-ysis,accordingwithAhmadetal.[7].SilicaGel60(Merck, 70–230mesh)wasusedforanalyticalTLC.Column chro-matographieswereperformedoversilicagel(Merck,60 F254230–400mesh).

2.5. Kidneyperfusion

AdultmaleWistarrats(260–320g)werefastedfor24h withfreeaccesstowater.Theratswereanesthetizedwith sodiumpentobarbitone(50mg/kg, i.p.)andaftercareful dissectionoftherightkidney;therightrenalarterywas cannulatedviathemesentericarterywithoutinterrupting thebloodflowasdescribedbyBowman[8].

The perfusion fluid consisted of modified Krebs– Henseleit solution (MKHS) of the following composi-tion (in mmol/L): 114.00 NaCl, 4.96 KCl, 1.24 KH2PO4,

0.5 MgSO4·7H2O, 2.10 CaCl2 and24.99 NaHCO3.Bovine

serumalbumin(BSA6g%;fractionV),urea(0.075g),inulin (0.075g) and glucose (0.15g) were added to the solu-tion,resultinginafinalperfusatevolumeof100mL.The pHwas adjusted to7.4. In each experiment, 100mLof MKHSwererecirculatedfor120min.Theperfusion pres-sure(PP)wasmeasuredatthetip ofthestainless steel cannula in the renal artery.Samples of urine and per-fusionfluidwerecollectedat10minintervalsforanalysisof thesodium,potassiumandchloridelevelsbyion-selective electrodes(Rapidchem744,BayerDiagnostic,UK);inulin, asdescribedbyWalseretal.[9]andmodifiedbyFonteles etal.[10];andosmolality,whichwasmeasuredinvapor pressureosmometer(Wescor5100C,USA).ThevenomofP. nattereri(10mg/mL)wasaddedtothesystem30minafter thebeginningofeachperfusion.

thepercentageofsodium(%TNa+_{),potassium(%TK}+_)and

chloride(%TCl−_{)tubulartransportweredetermined[11].} Theresultswerecomparedtothecontrolgroup,at30min earlyineachexperiment(n=6).Theexperimental proce-dureswereconductedaccordingtoguidelinesforthecare anduseoflaboratoryanimalsasapprovedbytheEthical Committee(68/08)fromFederalUniversityofCeará(UFC).

2.6. Renalhistologicalevaluation

Aftertherenalperfusionexperiment, both rightand left kidneys were removed and fixed in 10% formalde-hyde for histological processing. Kidney tissue was embedded in paraffin, cut into 5␮m sections, stained withhematoxylin–eosinand furtherprocessed for light microscopy(OlympusBX41,USA).Thephotomicrographs weretakenbymeansofadigitalcamera(NikonCoolpix 885,Japan).

2.7. Cellculture

EpithelialMadin–DarbyCanineKidney(MDCK)was cul-tivatedinRPMI1640medium(MEM)supplementedwith 10%fetalbovineserum,1%penicillin(10000IU/mL)and streptomycin(10mg/mL).Foreachexperiment,cellswere removed and incubated with trypsin-EDTA (0.25/0.02%, v/v)at37◦_{Catabout5min.}

Afterthis,thecellswerecountedinaNeubauerchamber andsuspendedinculturemedium(1×105_cells)and24h

laterusedfortheexperiments.

2.8. Cytotoxicassay

CellviabilitywasassessedbyMTT (4,5-dimetilazil-2-il)-2,5diphenyltetrazolium)assayasdescribedbyMosmann

[12]. The MDCK cells are plated in 96-well plates at a densityof105_{cellsandtreatedwithdifferent}

concentra-tionsofP.nattererivenom(1.56,3.12,6.25,12.5,25,50, 100␮g/mL).After24hoftreatment,thecellswere incu-batedwith0.5mgofMTT/mLfor4h.Theformazancrystals thatresultedfromMTTreductionweredissolvedbyadding SDS(10%)toeach wellfollowedby incubationfor 17h. Theabsorbancewasreadat570nminamicroplatereader, andcellviabilitywascalculatedbycomparingthe result-ingabsorbanceswiththemeanabsorbanceofthecontrol wells(withoutvenom,consideredtobe100%viable).

2.9. Statisticalanalysis

Theresultswereexpressedasmeans±SEM(n=6). Sta-tisticalevaluationwasdeterminedbyanalysisofvariance (ANOVA)andcorrectedbytheBonferrnitest.Statistical sig-nificancewassetat5%.Theprogramsusedtoperformthe statisticalanalysiswereMicrosoftExcel2007and Graph-PadPrism5.0.

3. Results

3.1. Proteincontentofvenom

Inthepresentstudy,thevalueoftotalproteinofthe venom from P.nattereri wasof 863.9␮g/mg of venom,

Fig.1.ElectrophoreticprofilesofP.nattereri(P.n.)andB.jararaca(B.j.) venoms.Eachlanewasloadedwith20␮Lofvenom.Proteinsweresilver stained.

corresponding in 86.3% of total content of venom this species. In comparisonwithvenom ofothers species of the generaPhilodryas, suchas P.olfersii(923␮g/mg),P. patagoniensis(814␮g/mg)andP.nattereri(847␮g/mg)and

Bothropsjararaca(799␮g/mg)[13,14].ThevenomofP. nat-tereriwassimilaramongtheothersvenoms.

3.2. SDS-PAGEandNMRspectrum

SDS-polyacrylamidegelelectrophoresiswascarriedout accordingtoLaemmli[5]andshoweddistinctprotein pat-terns among the venomsof P.nattereri and B. jararaca. ThevenomofP.nattererishowedmultipleproteinbands, rangingfrom45kDato100kDa,whileinB.jararacahave proteinbandsrangingfrom45to210kDa(Fig.1).

The1_{HNMR spectrumof P.nattereri venomshowed}

thepresence ofpeptides, amino acidssimple (phenolic, aromaticsandaliphatic)andaminoacidderivatives rep-resentedthemajorcomponentsofthisvenom(Fig.2).The peaksgeneratedcorrespondtoaminoacidsoftheprotein constituentsofthepoison,whichcomprise86.3%ofthis.

3.3. EffectsoftheP.nattererivenomintheisolatedrat kidney

Fig.2.Detailed1_{HNMR-spectroscopic(500MHz,CD}

3OD)analysisofP.nattererivenom.(A)The1HNMRspectrumrevealingahighlycomplexcomposition withdifferentgroupsofaminoacids(B)Cozyspectrum(CD3OD)withdifferentgroupsofaminoacids.

andtothe3mg/mLreducedsignificantlyat60and90min forbothparameters,butat120minincreasesignificantly in UF, while GFRreturned tonormal perfusion(Fig.3C andD).

Na+_,K+_{, Cl}− _{electrolyte transport wasaltered in the} perfusedkidney,regardlessofthevenomconcentration. Regardingthepercentofsodiumtubulartransport(%TNa+₎

wasreducedat60,90and120minfor1and3mg/mL con-centrationsof venom and percentof potassium tubular transport(%TK+_{)wasreducedat60,90and120minonlyin}

3mg/mLconcentration,whencomparedincontrolgroup (Fig.4AandB).

Thepercentofchloridetubulartransport(%TCl−_)was reducedat90and120minfor1mg/mL,whilein3mg/mL

Fig.4. EffectsofP.nattererivenominconcentrationsof1and3mg/mLonsodium(A)potassium(B)chloride(C)tubulartransportpercentandosmotic clearance.Dataareexpressedasmean±SEMfromsixdifferentanimals.*p<0.05comparedtothecorrespondingcontrolgroupforeachinterval(ANOVA andBonferronitest).Thevenomwasaddedtothesystem30minafterthebeginningofeachperfusion.

concentrationat60, 90and 120min.Osmoticclearance showedincreaseat90and120minin1mg/mL concentra-tionofvenomofP.nattereri(Fig.4CandD).

3.4. Renalhistologicalevaluation

Inthecontrolgroupcomposedofleftkidneysperfused withKrebs solution(MKHS),thekidneyshowednormal structures(glomerulus, tubule,vesselsand interstitium) (Fig.5A).

The right kidney perfused with 1mg/mL of venom

showedtubulardilation,mainlyinthedistalconvoluted tubuleandHenle’sloop,proteinaceousmaterial(PM)and bloodaccumulation(BA)withinthetubules,normalvessels andinterstices(Fig.5BandC).

Kidneysperfusedwith3mg/mLofvenomshowedan obvioustubutardegeneration,presenceofproteinaceous

material within the tubules and the Bowman spaces,

glomeruliwithslightalterationsandtubuleswith mod-eratedilatation(TD)(Fig.5DandE).

3.5. CytotoxiceffectoftheP.nattererivenomonMDCK cells

TheP.natterericytotoxicitywasassessedintherenal tubularcells(MDCKcellculture)after24hofexposureto

variousvenomconcentrations(3.125,6.25,12.5,25,50and 100␮g/mL).Thevenomsignificantlyreducedtheviability of theMDCK cells in the 50 and 100␮g/mL concentra-tionscomparedtothecontrolwithanIC50of169.5␮g/mL

(Fig.6).

4. Discussion

ToinitialcharacterizationofvenomfromP.nattereri,we analyzedtotalproteincontentandprofilebySDS-PAGEand NMRanalysisoflyophilizedvenom.ThepoisonofP. nat-tereriexhibited86.3%oftotalproteincontent.Thisvalueis similartothatfoundbyZelanisetal.[14]forothersspecies of thegeneraPhilodryas,suchasP.olfersii(923␮g/mg),

P.patagoniensis(814␮g/mg)andP.nattereri(847␮g/mg), andBothropsjararaca(Viperidaefamily)with799␮g/mg

[13].

RegardingSDS-PAGEanalysis,ThevenomofP.nattereri

showedalesscomplexprofileincomparisontotheother twocongenericspecies(P.olfersiiandP.patagoniensis)[14], withmajorproteinbandsrangingfrom45kDato100kDa, whileinB.jararacahavealargenumberofproteinbands rangingfrom45to210kDa.

Theseresultsmaystrengthenthehypothesesofclose similarity regardingthe actionsand activitiesof venom

Fig.6.ViabilityofMDCKcellsincubatedwithdifferentconcentrationsof

P.nattererivenomfor24h.

corroborate the eletrophoretic analysis performed by RochaandFurtado[26].

Toinvestigatetheeffectof theP.nattererivenom in thekidneywithoutinterference ofsystemic factors,we usedperfusionintheratkidney.Inthepresentstudy,we observeda decrease in perfusionpressure,renal vascu-larresistance,urinaryflowandglomerularfiltrationrate, aswell asa decrease in sodiumtransport and chloride afterthekidneywasperfusedbythevenom,whereasthe clearanceosmoticwashighercomparedwithcontrol.This agreeswithfindingsforBothropsmarajoensis[15],Bothrops insularis[24],Bothropsjararaca[16] andBothrops jarara-cussu[17].

Bothrops venom is characterized for promoting hypotensionthroughmechanismsthatpromotesystemic vasorelaxation[18].These effects are likely identical to thoseaffectingrenalPP,asweobservedinthisstudyforP. nattererivenom.

However, the reduction in almost renal parameters observedintheexperimentscannotberelatedtoa spe-cificcomponentbecausetheoverallcompositionofcrude

P.nattereri venom is unknown. Despite this limitation, mediators,suchas tumornecrosisfactor-alpha(TNF-␣), interleukin-1(IL-1),IL-6,IL-10andgammainterferon (IFN-Y),canbeinvolvedinrenalparametersdecreases(PP,GFR, RVR,TNa+_,TK+_andTCL−_{),becausevenomtypicallyinduces} therelease of these substances, ashas beenpreviously reported[19].Inaddition,theremightbecompoundsthat exertdirectactiononkidneytissues.Theseinflammatory mediators,withrelaxationpotential,mayhavecontributed totheRVRreductionobservedinthisstudy.

The histological alterations promoted by P.nattereri

venomintwoconcentrationsof1 and3mg/mL, respec-tively showedinitially blood accumulation, glomerulus, tubuleswithproteinaceousmaterialanddilatationtending towarddegeneration,reversiblelesionsandthe accumula-tionofproteinsmaterialreleasedintothedamagedcells cytoplasmreflectedtheearlystagesofvenomtoxic agres-sionandthehistologicalanalysiscorroboratetheresults forthegenusBothrops[17,20,24].

ThecytotoxicpotentialofP.nattererivenomwas eval-uatedusingMDCKcells,whichisacelllinewithsimilar

morphologicalandfunctionalcharacteristicstocellsinthe distalcollectingtubulesofmammals[21].

Thresultsofcytotoxiceffectsshowedthatthevenom wastoxictocellsbecauseitsignificantlyreducedviability inconcentrationsof50and100mg/mLcomparedtothe controlwithanIC50of169.5␮g/mL.ThisvalueforIC50is

higherinrelationofothersspeciesofsnakes,suchas Both-ropsleucurusand Crotalusdurissuscumanensiswith1.25 and5.38␮g/mL,respectively[22,23].

5. Conclusions

TheP.nattererivenomwascomposedandcaused tox-icityinkidneyisolatedandinducedcelldeathoncultured MCDKcells.WedemonstratedP.nattererivenomis com-posed for proteins(86.3%) and capable ofchanging the kidneyfunctionalparameters(PP,RVR,GFR,UF,osmotic clearance, percent of sodium, potassium and chloride transport) in the isolated rat kidneys. Furthermore,the venom promotedmorphologicalalterations in therenal tubules,suchasbloodaccumulation,glomerulus,tubules withproteinaceousmaterial.Itissupposedthatthisrenal changeoccurs probablybydamagingboth vascular and glomerularsites.Regardingtheeffectsofvenominviability oftheMDCKcellswasobservedonlyathighconcentrations (50and100␮g/mLofvenom)withanIC50of169.5␮g/mL.

Thesefindingsmaybeimportantaspectsoftheprocessof toxicitymediatedbyP.nattererivenom.

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