Expression and functional implications of the renal apelinergic system in rodents

15 Pages·2017·15.9 MB·English

RESEARCHARTICLE
Expression and functional implications of the
renal apelinergic system in rodents
Anne-MarieO’Carroll1*,SabrineSalih1,PhilipR.Griffiths1,AarifahBijabhai1,Mark
A.Knepper2,StephenJ.Lolait1
1 BristolMedicalSchool,HW-LINE,UniversityofBristol,Bristol,UnitedKingdom,2 EpithelialSystems
BiologyLaboratory,SystemsBiologyCenter,NationalHeart,LungandBloodInstitute,NationalInstitutesof
Health,Bethesda,Maryland,UnitedStatesofAmerica
a1111111111 *[email protected]
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a1111111111 Abstract
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ApelinbindstotheGprotein-coupledapelinreceptor(APJ;genenameaplnr)tomodulate
diversephysiologicalsystemsincludingcardiovascularfunction,andhydromineralandmet-
abolicbalance.RecentlyasecondendogenousligandforAPJ,namedapela,hasbeendis-
covered.Weconfirmthatapelaactivatessignaltransductionpathways(ERKactivation)in
OPENACCESS
cellsexpressingtheclonedratAPJ.Previousstudiessuggestthatexogenousapelais
Citation:O’CarrollA-M,SalihS,GriffithsPR,
diuretic,attributablewhollyorinparttoanactiononrenalAPJ.Thusfarthecellulardistribu-
BijabhaiA,KnepperMA,LolaitSJ(2017)
Expressionandfunctionalimplicationsoftherenal tionofapelainthekidneyhasnotbeenreported.Wehaveutilizedinsituhybridizationhisto-
apelinergicsysteminrodents.PLoSONE12(8): chemistrytorevealstrongapelalabellingintheinnermedulla(IM),withlowerlevels
e0183094.https://doi.org/10.1371/journal.
observedintheinnerstripeoftheoutermedulla(ISOM),ofratandmousekidneys.This
pone.0183094
contrastswithrenalaplnrexpressionwheretheconverseisapparent,withintenselabelling
Editor:InesArmando,GeorgeWashington
intheISOM(consistentwithvasarectalabelling)andlow-moderatehybridizationintheIM,
UniversitySchoolofMedicineandHealthSciences,
UNITEDSTATES inadditiontolabellingofglomeruli.Apelinisfoundinsparselydistributedcellsamongst
moreprevalentaplnr-labelledcellsinextra-tubularregionsofthemedulla.Thisexpression
Received:February3,2017
profileissupportedbyRNA-Seqdatathatshowsthatapela,butnotapelinoraplnr,ishighly
Accepted:July28,2017
expressedinmicrodissectedratkidneytubules.Ifendogenoustubularapelapromotes
Published:August17,2017 diuresisinthekidneyitcouldconceivablydothisbyinteractingwithAPJinvasculature,or
Copyright:Thisisanopenaccessarticle,freeofall viaanunknownreceptorinthetubules.Thecomparativedistributionofapela,apelinand
copyright,andmaybefreelyreproduced, aplnrintherodentkidneylaysthefoundationforfutureworkonhowtherenalapelinergic
distributed,transmitted,modified,builtupon,or
systeminteracts.
otherwiseusedbyanyoneforanylawfulpurpose.
TheworkismadeavailableundertheCreative
CommonsCC0publicdomaindedication.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Funding:ThisworkwassupportedbytheBritish
HeartFoundation(BHF;http://www.bhf.org.uk) Introduction
(PG/12/23/29475,A-MO’C;PG/15/14/31311,A-
MO’C,SJL).Thefunderhadnoroleinthestudy TheGprotein-coupledapelinreceptor(APJ;genenameaplnr)isactivatedbytheendogenous
design,datacollectionandanalysis,decisionto ligandapelin[1,2].Theapelinergicsystemhasbeenimplicatedinawiderangeofhomeostatic
publish,orpreparationofthemanuscript. processes,includingcardiovascularcontrol/cardio-embryogenesis,angiogenesis,hydromin-
eralbalance,hypothalamic-pituitary-adrenalaxisregulation,andmetabolichomeostasisthat
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. mayinvolveapathophysiologicalroleinobesity[2].
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Apela,apelinandaplnrinthekidney
Asahormoneintheperipheryorneuromodulatorinthebrain,apelinappearstobeco-
localizedwithAPJinsomebrainregionssuchastheparaventricularandsupraopticnucleiof
thehypothalamus,andinmanyperipheraltissuesincludinganteriorpituitary,lung,heart,kid-
ney,stomachandmammaryglands,whereitmayactinanautocrineorparacrinefashion[2].
Thetissueexpressionofaplnrislargelysupportedbythereceptorautoradiographicallocaliza-
tionofAPJ-bindingsites[3–5].
Apelinexistsasanumberofproteoforms(i.e.,[Pyr1]apelin-13,apelin-17,apelin-36)that
derivefrompost-translationalmodificationofa77aminoacidapelinpro-hormoneprecursor.
UponbindingAPJ,apelinactivatesamyriadofsignaltransductionpathwaysthatareoftenG
protein-dependent,includinginhibitionofadenylatecyclase,stimulationofMAPkinase
(ERK)phosphorylation,Aktphosphorylationthatcouldcontributetoneuroprotection,and
enhancingnitricoxidesynthaseactivity[2].APJactivationbyapelin-13mayinvolvemultiple
Gproteins[6],whilesomeactionsinvolvingAPJmaybeapelin-independent(e.g.,Gprotein-
independentmyocardialcellhypertrophyresponsetomechanicalstretch)orapelin-dependent
(e.g.,bluntingmyocardialhypertrophy)inthesametissue[7].
AsecondendogenousligandforAPJhasrecentlybeendiscovered[8,9].Originallyanno-
tatedasalongnon-coding(nc)RNA(termedEnde[10])expressedprimarilyinthedefinitive
endodermduringmouseembryogenesis,theapela(otherwiseknownaselabelaortoddler)
geneisalsotranslated[8,9].Itencodesapredicted54(58inzebrafish)aminoacidprecursor
moleculethatishighlyconservedacrossvertebratesandisstructurallydistinctfromtheapelin
prohormone[8,9].Theapelaprecursorcanbeenzymaticallyprocessedintoanumberofbio-
logicallyactiveproteoformssuchasapela-21andapela-32thatmaybesecreted[8,9].Apela-32
bindstoAPJwithanaffinity(nanomolar)similartoapelinandmaybemorepotentthanape-
lininactivatingAPJsignaltransductionpathwaysinsomeAPJ-expressingcells[11,12].Apela
isessentialforcellmovementduringgastrulationandinthedevelopmentofzebrafishheart
andvasculaturewherelossofapelafunctionusuallyresultsinembryoniclethalitythatcanbe
rescuedbyinjectionofapelamRNA[9].Zebrafishapelanullmutantsinvariablyexhibitcar-
diacdysplasia[8,9,13],reminiscentofthephenotypeobservedinthemajorityofembryonic
aplnrknockoutmice[14].Sincecardiacabnormalitiesarenotobservedinembryonicapelin
knockoutmice[14,15],itispossiblethatapelamaybethemainendogenousAPJligandduring
mousecardiovasculardevelopment.Interestinglyheartdefects(orcompleteabsenceofcardio-
myocytes)arealsoobservedwhenapelaorapelinareover-expressedinzebrafish[9,16],indi-
catingthatnormallevelsofeitherAPJligandareobligatoryforproperheartdevelopment.
Exogenouslyadministeredapelaisanorexigenic[17],andpromotesdiuresis,presumably
bydirectactivationofAPJlocatedinthekidneyand/orindirectlybyregulatingargininevaso-
pressinexpression[11]inthehypothalamus.ApelaactivatesthePI3K/Aktpathwayandis
anti-apoptoticinhumanembryonicstemcells(ESCs)eventhoughthesecellsdidnotexpress
aplnr[18],whileapela-inducedp53-mediatedapoptosisinmouseESCspointstoaRNA-regu-
latoryratherthanaprotein-codingfunction[19].Thisdependsonapelabindingheteroge-
neousnuclearribonucleoproteinL(hnRNPL),aninhibitorybindingpartnerofp53inthese
cells.Whetherapelaactsasaproteininanon-APJ-dependentmanner(e.g.,viaanunrelated
receptor)orasaregulatoryRNAinvivoisnotknown.
ApelamRNAtranscriptsappeartobemoreprevalentduringdevelopment[8,9,10].They
arepredominantlyexpressedintheheartofembryonicrodents[10]andzebrafish[8,9],and
arealsodetectedinisolatedcellsfromtheadultratheart[20],andinadultratandmousekid-
ney[10,11]byRT-PCR.Thepreciserenallocationofapelageneexpressioninthekidneyhas
notbeenreported.Evidenceforrenalapelinexpressionislessconvincing—itappearstobe
sparselydistributedintissueendothelialcellsandperhapsvascularepithelialcellsandglomer-
uli[21].Intheratandmousekidneyaplnrismainlyexpressedinvascularelements(vasa
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Apela,apelinandaplnrinthekidney
recta)oftheoutermedullaandglomeruli[5,22,23]—lowerlevelshavebeendescribedinthe
ratcollectingductsasdeterminedbyRT-PCRofisolatedtubulesegments[22].Ithasbeen
proposedthatapelin-17,actingonthecollectingductAPJ,counteractstheantidiureticeffect
ofargininevasopressinactingattheV2receptor[24].
TheaimsofthisstudyaretoconfirmapelabioactivityviaAPJinvitroandtocharacterize
theanatomicalrelationshipbetweenapelaandaplnrexpressionintheratandmousekidney
usinginsituhybridizationhistochemistry(ISHH)withgene-specificoligonucleotides.Inan
attempttogainfurtherinsightsintothepossiblefunctionsoftherenalapelinergicsystemwe
havealsoutilizedbranched-chainISHHtodeterminewhethertheapelin,apelaand/oraplnr
genesareco-expressedinthesameregionsintheratkidney.Thisdistributionissupportedby
RNA-Seqstudiesonisolatedrenaltubulesegments.Ourstudiesrevealthatwhileapelaand
aplnrexpressionoverlap,apelaismainlyexpressedinthemedullarycollectingductsandloops
ofHenlewhereasaplnrishighlyexpressedinnon-tubularstructuresintheoutermedulla.Ape-
linexpressionislargelyrestrictedtoisolatedcellsmainlyinthevicinityofaplnrcellsinthe
medulla.Thisdistributionraisesquestionsabouthowthetwo-ligandapelinergicsystemoper-
atesinregulatingrenalfunction.
Materialsandmethods
Animals
Adult(8–12weeksold)malewildtype(n=3)ornullmutants(n=2)micefromouraplnr
knockoutcolony[25](mixtureofC57BL/6x129X1/Svstrains)andadult(approx275g)male
Wistarrats(n=8)wereusedinthisstudy(Sprague-DawleyratswereusedforRNA-Seq—see
below).Animalswerehousedunderconstanttemperature(21±2˚C),light(lightsonfrom
0700to1900h)andhumidity(45–50%)regimenswithfoodandwateradlibitum.Animalcare
andmaintenancewereperformedinaccordancewiththeAnimalScientificProceduresAct
(1986)UnitedKingdomandapprovedbytheBristolUniversityAnimalWelfareandEthical
Reviewbody.
Cells
Chinesehamsterovary(CHO)cellsweretransfectedwiththerataplnrcDNA(B78[23])as
previouslydescribed[26].Stably-transfectedclonalcellswereobtainedbylimitingdilution,
andexpressionofaplnrwasdeterminedbyNortherndotblotsusingtwo32P-dATP-labelled
48bpoligonucleotidespecificfortherataplnrsequence(B78AandB78B[23];seeISHH
below).Thehighestaplnr-expressingcellcloneswereexpandedforuse,andoneline
(CHO-B78)wasusedforbranched-chaininsituhybridizationhistochemistryandERKsignal
transductionassays(seebelow).
ERKimmunohistochemistryandanalysis
CHO-B78cellsweregrownin96-wellplatesataconcentrationofapprox10,000cells/welland
serum-starved(0.1%foetalcalfserum)O/N.Dose-responsecurveswereperformedfor10min,
where[Pyr1]apelin-13stimulationofERK1/2ismaximalasestablishedinpilotstudieson
CHO-B78cells.Afterincubationwith0.01-1000nM[Pyr1]apelin-13(Bachem)orratapela-32
(cyclized,pyroglutamated;SevernBiotech,U.K.),orvehicle(dH O),thecellswereimmunos-
2
tainedfordualphosphorylated(pp)ERK1/2andtotal(t)ERK1/2—cellimageswereacquired
usingtheINCellAnalyzer1000andanalysisperformedbyInCellAnalyzerWorkstation3.5
software(GEHealthcare)aspreviouslydescribed[27,28].Thenuclearandcytoplasmicfluo-
rescenceintensitiesinindividualcellswerequantified(inarbitraryunits)—datawas
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Apela,apelinandaplnrinthekidney
normalizedtovehiclecontrolsaftersubtractionof‘noprimaryantibody’backgrounds.Ineach
experimentthedata(nuclear+cytoplasmicppERKmeasurements)ismean±SEM,n=6
(approx1200cellsimagedfrom3wellsofa96-wellplate;experimentsrepeatedtwice).
Insituhybridizationhistochemistry(ISHH)
ISHHwithradiolabelledoligonucelotides. Kidneyswerefrozenonpowdereddry-ice
andstoreddesiccatedat-80˚Cpriortoprocessing.Sections(12μm)werethaw-mountedonto
polylysine-coatedslidesandISHHperformedwithtwo35S-end-labelled48bp-oligonucleotides
targetingapela,asdescribedindetailforoligonucleotideprobes(http://www.wsyacy.com/
SNGE/Protocols/ISHH/ISHH.html).TheantisenseprobesusedinthisstudywererAP-1and
rAP-2specificforratapela(bp495-542andbp849-896,respectively,inthe3’-untranslated
regionofGenBankAccessionXM_008772035(LOC100912649)),andmAP-1andmAP-2spe-
cificformouseapela(bp481-528andbp832-879,respectively,inthe3’-untranslatedregionof
GenBankAccessionNR_040692).ISHHwithrataplnrprobes(B78AandB78Bdirectedto
bp602-649andbp886-933oftherataplnr,GenBankAccessionNM_031349,respectively[23])
wasusedtocomparetheexpressionofaplnrwithapelainratkidney.
Correspondingsenseprobesusedasnegativecontrolsgavelittle,orlow,levelsofuniform
backgroundlabelling.
SectionswereexposedtoX-rayfilm(HyperfilmMP,Amersham)for1–3monthsatRTor
apposedtoemulsion(IlfordK5)foruptoayearat4˚C(bywhichtimethesignalwassatu-
rated).FilmimageswerescannedandpseudocolouredinImageJbyinvertingLUXof‘16col-
ours’inLookupTables.Emulsion-coatedsectionsweredevelopedmanuallywithD-19
developer(AgarScientificLtd.U.K.)accordingtothemanufacturer’sinstructions,counter-
stainedwithtoluidineblueandviewedonaLeicaDM2000microscopeequippedwithaLecia
DFC70000TcameraandLeicaApplicationSuitX(LASX)workstation.
Branched-chainISHH. Tocomparetheexpressionofapela,apelinandaplnrinthesame
ratheartorkidneysectionsRNAscope(AdvancedCellDiagnostics;ACD)withtheMultiple
FluorescenceAssaykitwasused.Inthistechnique,target-specificZ-shapedprobeswithover-
hangscreatea‘bridge’thatbindsapreamplifierthatcanthenbindmultipleamplifiers.The
techniqueprovidessingleRNAcopydetection[29],typically‘dots’,andpermitsrapidvisuali-
zation(1day)oflowlevelsofmRNA(asisoftenobservedforGprotein-coupledreceptors
(GPCRs)).
Threefluorescentcolourcombinationswereavailable,Alexa488(green),Atto550(orange)
andAtto647(Farred),thecolourforeachtargetprobesetdependantonwhichfluorescent
colourmodules(Amp4AltA-C)andchannels(C1-3)areused.Theproprietaryprobeswere
designedbyACDforthefollowingratsequences:apela(GenBankAccessionXM_008772035
(LOC100912649);bp147-1053;12Zprobepairs),apelin(NM_031612;bp2-996;20Zprobe
pairs)andaplnr(NM_031349;bp147-1053;20Zprobepairs).RNAscopewasperformedon
16μmfrozensectionsthatwerepost-fixedin4%PFA,accordingtotheACDuserprotocols
(sheets#320513and320293).RNAscopedetectionofaplnrexpressioninCHO-APJ(B78)and
nontransfectedCHOcells(grownatadensityof~125,000–175,000cells/12-wellplatecontain-
ingaglass13mmcoverslip(type1)for2days)followedthetissuesectionprotocolwiththe
exceptionthatpre-treatmentwasperformedasinACDsheet#320528.Withcells,onlythe
aplnrprobe(s)(andpositiveandnegativeprobesinparallel)wereused,withablankprobein
theothertwochannels.
Positive(ratPOLR2A,PPIBandubiquitin(UBC);3-plexprobeset;ACD#407301)andneg-
ative(DapB;ACD#320871))probesobtainedfromACDwereincludedinallexperiments.Sec-
tions(andcells)werecounterstainedwithDAPIasperACDinstructions.
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Apela,apelinandaplnrinthekidney
Thefluorescencesignalinheartventriclesandthroughoutthekidneywasvisualizedunder
ax40objectiveonaLeicaSPEsinglechannelconfocallaserscanningmicroscopeattachedtoa
LeicaDMi8invertedepifluorescencemicroscopeequippedwithaLeciaDFC365FXmono-
chromedigitalcameraandLASXworkstation(UniversityofBristolWolfsonBioimaging
Centre).ZstacksweretakenofallimageswithaZstepsizeof0.5μm.Imageswereexportedto
ImageJ(whereFar-redlabellingwassometimesconvertedtowhite)orAdobePhotoshop.No
attemptwasmadetoquantitatetheamountof‘dot’labelling.
Miningoftranscriptome(RNA-Seq)dataofmicrodissectedratrenal
tubulesandglomeruli
Previouslyunpublishedhigh-throughput,‘deep’sequencing(RNA-Seq)datawasretrieved
fromadatasetonmicrodissectedratkidneytubulesfromadultmaleSprague-Dawleyrats
[30].Theprecisionofthetechniqueandaccuracyofthedissectionwasverifiedbyestablishing
thegeneexpressionlevelsofseveralwaterandsolutetransporterswithknowndistributions
acrossthetubule[30].SinceratapelaisnotannotatedintheratreferenceDNAdatabasesin
theoriginalmappingofRNA-Seqreadswelookedspecificallyinthemouseapelasequenceto
findthecorrespondingrathomologue;thedistributionofaplnrandapelininthesamekidney
structureswassimilarlyextracted.Detailsofthemethodsandanalysishavebeenoutlined[30].
Statisticalanalysis
INCellAnalyser1000experimentswereperformedintriplicatewells,withexperimentsper-
formedatleast2times.Statisticalanalysiswaswithaone-wayANOVAandposthocDunnett’s
testwithGraphPadPrismsoftware(version4.0b).p<0.05wasconsideredasstatistically
significant.
Results
StudiesonCHOcells
StudiesonCHOcellsstablytransfectedwiththerataplnrcDNA(B78)wereusedasapositive
controlfortheaplnrprobe,confirmingexpressionofAPJ,andfunctionalactivityofapela-32
atAPJ,inthiscellline.AplnrmRNAishighlyexpressedaspunctatedotsinCHO-B78cells
(Fig1A).Thedotsmergeinareasofhighaplnrexpression(e.g.,seeFig1B).Thereissomevar-
iabilityincell-cellaplnrexpressionandoccasionalcellsdonotappeartoexhibitanyaplnr
expression(<5%fromacountof81cells;seeFig1B).NosignalisdetectedinCHO-B78cells
hybridizedwithnegativecontrolprobes(Fig1C)—similarlyaplnrexpressionisnotobserved
innon-transfectedCHOcells(Fig1D).
Apela-32and[Pyr1]apelin-13stimulateERKactivation(p<0.01)inCHO-B78cellsina
dose-responsivemannerbetween10−10–10-6M(Fig2).TheEC50’sare6.9nMand15.9nMfor
[Pyr1]apelin-13andapela-32,respectively.
Apeladistributioninratandmousekidney
ISHHwithradiolabelledoligonucleotides. Themajorsiteofapelaexpressionintherat
kidneyistheinnermedulla(IM)withweakerexpressionintheinnerstripeoftheouter
medulla(ISOM)(Fig3Aand3B).Serialsectionsofthesamekidneyshybridizedwithaplnr
probesshowapatterninthemedullathatisinvertedcomparedtoapelaexpression—aplnris
morehighlyexpressedintheISOMthanIM.Aplnrisalsoexpressedinglomeruli,visualizedas
apatchy,‘speckled’patterninthecortex.
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Apela,apelinandaplnrinthekidney
Fig1.RNAscopewithaplnrprobesinCHO-B78(ratAPJ)andnon-transfectedCHOcellcultures.In
(A)clear,punctateaplnrlabellingispresentinthecytoplasmofthemajorityofCHO-B78cellswithcell-cell
variabilityinthelevelsofexpression.Strongly(openarrow),moderately(arrows)andunlabelled(arrowheads)
aplnrcellsinCHO-B78culturesareshownin(B).(C)showsnegativecontrolprobeonCHO-B78cells,while
thelackofaplnrlabellinginnon-transfectedCHOcellsisshownin(D).Scalebar=25μm.
https://doi.org/10.1371/journal.pone.0183094.g001
Inthemousekidney(Fig4Aand4B)theapelapatternoflabellingissimilartothat
observedintherat.Inthecortexofbothmouseandratkidneystherewassomelightlabelling
ofisolatedstructures.Thereisnoapparentdifferenceinthedistributionofapelabetween
aplnrwildtypeandknockoutmice(Fig4Aand4B).Senseapelaprobesgiveuniformback-
groundlabellinginthekidney(Fig4C).
Inemulsion-dippedsectionsofratandmousekidneyapelalabellingwaspronouncedin
tubules,particularlyintheinnermedullacollectingducts(Fig5A)whereasaplnrwasmost
prominentlyexpressedinscattered,intertubular(interstitial)cellsinthemedulla(Fig5B)and
inglomeruliinthecortex(Fig5C).Therewasastarktransition,fromhightolow,inthenum-
beroflabelledtubularstructuresbetweentheIMandoutermedulla(Fig5D),andouter
medullaandcortex(Fig5E).
Triplelabellingofapela,apelinandaplnrcellsusingbranched-chainISHH. Allthree
genesareexpressedintheadultratheart(Fig6A),usedaspositivetissuecontrolforaplnrand
apeladetection.Somedotsarepinpointwhileothersarelargerandappeartobeacombination
ofmorethanonedot.Aplnrishighlyexpressedincardiomyocytesthroughoutthemyocar-
diuminapatternlargelydistinctfromthatofthemoresparselydistributedapelincellsandthe
evenlessabundantapelacells(Fig6Aand6B).Themajorityofcellsappeartoexpressaplnr
alone,howeverinsomecellsaplnrandapelinappeartobecolocalized.Theoccasionalcells
expressallthreegenes,aplnrandapela,orapelinorapelaalone.Mostapela-positivecellscon-
tainonlyafewdots,incontrasttoupto20dotsobservedinaplnr-positivecells.Negativecon-
trolprobesdonotlabel(Fig6C).
Therelativenumberofapela-,apelin-andaplnr-expressingcellsintheheartsubstantially
differsfromthatobservedintheadultratkidney,wheretherearemanymorecellsexpressing
apela,particularlyintheIMandISOM,thanaplnrorapelin(Fig6D).Inagreementwithour
ISHHresultsusingradiolabelledprobes,ratkidneyapelaexpressionislargelyconfinedto
tubularelements(Fig6D–6G),andisparticularlypronouncedintheIM.Thevastmajorityof
aplnrandapelinexpressionincellsdonotoverlieapela-expressingcells(Fig6D–6G).While
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Apela,apelinandaplnrinthekidney
Fig2.[Pyr1]apelin-13andapela-32activatesERK1/2inCHO-B78cells.Cellsweretreatedfor10minat
37˚Cwiththeindicatedconcentrationofligands.RepresentativethumbnailimagesacquiredbytheINCell
Analyzer1000showimmunohistochemicalstainingforintracellularppERK1/2(green)aftertreatmentof
CHO-B78cellswithvehicle(A)or100nM[Pyr1]apelin-13(B).Theworkstationcanautomaticallydemarcate
nucleusfromcytoplasmaccordingtoDAPI(blue)nuclearstaining.Adose-responsecurve(whole-cell
immunofluorescenceexpressedinarbitraryunits)for[Pyr1]apelin-13(blue)orapela-32(green)isshownin
(C).Therearenomeasurabledose-dependentchangesintERKlevels(red)incellsstimulatedwithapela-32
—asimilarresultwasobtainedwithcellsstimulatedwith[Pyr1]apelin-13.The[Pyr1]apelin-13andapela-32
stimulationswereperformedinseparatecellwells(cellswereusedfromtheidenticalCHO-B78passage
number)andprocessedforERKimmunohistochemistryinthesameexperiment.Dataareexpressedas
mean±SEM,averagedfromtwoseparateexperiments,eachligandconcentrationorvehicle(basal)with
triplicatewells,andatleasttriplicatefieldswithinwells.**p<0.01comparingstimulationstobasalconditions.
https://doi.org/10.1371/journal.pone.0183094.g002
mostaplnr-positivecellsalsoappeartoexpressapelin,afewcellsexpressaplnrorapelinalone.
Asintheheartmanycellsexpressonlysingledigitnumbersofapela,apelinoraplnrdots.
Wedidnotobserveanyapelaorapelinlabellingofglomeruli,whileabout35%(17outof
49viewedfromkidneysof4separaterats)ofglomeruliexpressaplnr(only1–5dots/cell)ina
diffusepatterninglomerularcells(Fig6H).
Miningoftranscriptome(RNA-Seq)dataofmicrodissectedratrenaltubulesandglo-
meruli. Apela,apelinandaplnrshowdistinctpatternsofexpressionalongthetubule
(Table1).Notablyapelaisexpressedthroughoutthetubulewithstrongestexpressioninthe
LoopsofHenleandinnermedullarycollectingduct(IMCD),whereasapelinisabsentfrom
thetubules.Lowlevelsofaplnrarepresentinthedistalconvolutedtubule(DCT)andcortical
thickascendingloopofHenle(cTAL)—aplnrisnotdetectedincollectingducts.TheRNA-Seq
datadoesnotincludeextra-tubularelementssuchasvasarectawhereISHHshowsaplnrtobe
highlyexpressed.ItconfirmsandextendsthetubularexpressionofapelaobtainedbyISHH.
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Apela,apelinandaplnrinthekidney
Fig3.Representativeautoradiographicfilmimagesdemonstratingapela(A)andaplnr(B)expression
inserialsectionsofakidneyfromanadultmaleWistarrat.Apelaishighlyexpressedintheinnermedulla
(IM)whereasaplnrlabellingisprimarilyintheinnerstripeoftheoutermedulla(ISOM).Incontrasttotheapela
labelling,theaplnrprobesalsolabelasubpopulationofglomeruli(patchy‘dots’)inthecortex(CX).Thefilm
imageswereexposedtofilmfor3months,developed,scanned,exportedtoImageJandpseudocoloured.In
theseimagesyellow-reddesignateshighexpressionwhereasblue-blackrepresentsnegligibleornolabelling
(seepseudocolourscalebarin(A)).Theimagesarerepresentativeofresultsobtainedfromthekidneysof8
rats.Scalebar=500μm.
https://doi.org/10.1371/journal.pone.0183094.g003
Discussion
APJwasanorphanGPCR[23,31]untilitsendogenousligandapelinwasisolatedfromstom-
achextractsby‘reversepharmacology’almost20yearsago[32].Ithasrecentlybeenproposed
thatapela,adistinctproteinunrelatedtoapelin,andhighlyconservedacrossvertebrates[8,9],
isanewendogenousligandforAPJ.Wehaveconfirmedpreviousstudiesshowingthatapela
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Apela,apelinandaplnrinthekidney
Fig4.AutoradiographicfilmimagesofISHHwithantisense-apelaprobesinkidneysfromadultmale
aplnrwildtype(WT)(A,C)andknockout(KO)(B)mice.Asintheratkidney,apelalabellinginthemouse
kidneyismainlylocatedintheinnermedulla(IM)withweakerlabellingintheinnerstripeoftheoutermedulla
(ISOM).Thereisalsoweak,scatteredlabellinginthecortex(arrowed).Backgroundsenseapelaprobe
labellingisshownin(C),wheretheimagecontrastwasincreasedinImageJtoshowtheoutlineofthetissue.
Pseudocolourscalebarisshownin(B).Thesectionswereexposedtofilmfor5weeksandimageswere
processedasinFig3.Scalebar=500μm.
https://doi.org/10.1371/journal.pone.0183094.g004
stimulatesERKactivationinAPJ-expressingcellsinvitro[11].UsingISHHtohighlightgene
expressioninananatomicalcontextweshowthatapelamRNAisnotabundantlyexpressedin
theadultratheart,andthattherearerelativelymoreapela-expressingcellsintheinner,com-
paredtotheouter,medullaoftheratandmousekidney,whereastheconverseistrueforaplnr
inrats.Inaddition,weshowapelatobemainlyfoundintubularelementsincontrasttoaplnr
(andapelin);findingsthataresupportedbyRNA-Seqdataonmicrodissectedrattubularseg-
ments.Intherenalmedulla,apelinexpressionisalignedwithaplnrexpression,wheresome
cellsappeartoexpressbothgenes.
Fig5.BrightfieldimagesofISHHlabellingofapelaandaplnrtranscripts(blackgrains)inadultratand
mousekidney.Apelaislargelyconfinedtotubularstructures(outlineofsomearrowedin(A))whereasaplnr
ismainlyexpressedinintertubularcells(arrowedin(B))intheratinnermedulla(IM)andoutermedulla,
respectively.Approx.35%ofcorticalglomerulialsoexpressedaplnr,usuallyinauniformdistributionacross
thestructure(perimeterofglomerulusarrowed)—anaplnr-positiveratglomerulusisshownin(C)).Thereisa
cleardemarcationinoveralllabellingintubularstructuresinthetransitionfrominnermedulla(IM)toinner
zoneoftheoutermedulla(ISOM)(D)andfromtheoutermedulla(tubularstructuresarrowedintheouter
stripeoftheoutermedulla(OSOM))tothecortex(CX)(E)inmousekidneys.InDandEtheinsetsare
approximately4-foldmagnificationsofthewide-fieldimages,showingapelalabellingintubules(onestructure
arrowed)intheISOM(D)andisolatedcells(fromboxedarea)intheOSOM(E).Scalebar=50μminA-C,and
100μminDandE.
https://doi.org/10.1371/journal.pone.0183094.g005
PLOSONE|https://doi.org/10.1371/journal.pone.0183094 August17,2017 9/15
Apela,apelinandaplnrinthekidney
Fig6.Apela,apelinandaplnrexpressionintheadultratheartventricleandkidneybybranched-chain
ISHH.ThetissueswerehybridizedwithRNAscopeprobesandprocessedinoneday.Positiveapela,apelin
andaplnrandnegativecontrolstainingoftheheartisshownin(A,B)and(C),respectively.In(A)aplnr-
expressingcells(greendots;labelled1–6withgreenarrows)arewidelydistributedintheheartmyocardium.
Therarecellsexpressonlyapela(reddots;labelled7witharedarrow).Somecellsarelabelledwithboth
aplnr+apelin(whitedots)probes(labelled8–10withyellowarrows).Afewcellsexpressaplnr+apela+
apelin(labelled11withawhitearrow)oraplnr+apela(labelled12withapurplearrow).(B)isanenlargement
ofthedottedareaoutlinedin(A).Therewasnolabellingwithnegativecontrolprobesinallthreechannelsin
theheartventricles(C)where*denotesvessel.Inthekidneyinnermedulla(D)thevastmajorityofcell
expressingapela(reddots;labelled1–8withredarrows)donotexpressaplnr(greendots)orapelin(yellow
dots).Somecellsexpressbothaplnr+apelatranscripts(labelled9withapurplearrow)whileaplnr+apelin
cellsaremorecommon(labelled10–13withyellowarrows).Therarecellexhibitsapela+apelin+aplnr
labelling(14;whitearrow).RemovingtheDAPIchannelin(E)(correspondingimageof(D))highlightsthe
PLOSONE|https://doi.org/10.1371/journal.pone.0183094 August17,2017 10/15