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CorrelationofTypeIIDiabetesMellitusGlomerulopathywithPancreaticMorphometryLucine Papazian1 BA | Ernesto Salcedo PhD 1, 2 , Zenggang Pan MD PhD3 Francisco G. La Rosa MD3, Lisa M.J. Lee PhD1, 2
1ModernHumanAnatomyProgram,UniversityofColoradoSchoolofMedicine.Aurora,CO.USA,2DepartmentofCellandDevelopmentalBiology,UniversityofColoradoSchoolofMedicine.Aurora,CO.USA,
3UniversityofColoradoHospital.AuroraCO.USA
Results
Methods
85%
Figure1.
83% 80%
• Totalpancreaticarea(red)• Adiposearea(yellow)• Isletarea(green)
1. Investigate and correlate pancreatic morphometric alterations with glomerular pathogenesis inpatients with type II diabetes.
2. Establish a set of pancreatic histological criteria associated with the progression of type II diabetes.
• DiabeticNephropathy(DN)isaprogressivediseasecausedbypathologicglycationtotherenalglomerulus.TheprogressionofDNisclinicallyrecognizedbytheRenalPathologySociety,DNclassI-IV.(Table1,Figure1). .
• Inthepancreas,betacelldeficitsandmorphologicalalterationshavebeenobservedanddocumentedacrossthediabeticpopulation,however,nomorphometricclassificationcataloguingthestepwisechanges,orhistologicaldocumentationcorrelatingglomerularandpancreaticdiabeticpathologies,exists.
Table1.DNglomerularclassification putfourthbytheRenalPathologySocietyFigure1.HistologicalcriteriaforassessmentofDNglomerularalterations,asrecognizedbytheRenalPathologySociety*Imagesappropriated fromPathologicClassification ofDiabeticNephropathy(Tervaert etal.2014)
Introduction
Methods
Goals
Figure4.
Figure1.
• ThesystemicnatureoftypeIIdiabetesprovidesopportunitytousethealreadyestablishedDNstagingtofurtherquantifyandstagetheprogressionoftypeIIdiabetesinthepancreas.
Figure 2.PancreatictissuestainedwithH&E,[email protected],[email protected]@40x,progressivezoomofrenalcortexprovidesvisualizationofrenalglomeruli
Figure2. Figure3.91AutopsySamples• Pancreaticandrenaltissuescollected
post-mortem(COMIRB#16-1337).• 75casesdiagnosedtypeIIdiabetic
pre-mortem.• 16casesvoidofclinical,laboratorial
andhistologicaldiabeticdiagnosis.• Pancreatictissuesamplesstainedwith
HematoxylinandEosin(H&E)forhistologicalvisualization(Figure2).
• RenaltissuesamplesstainedwithPerioticAcidSchiff(PAS)forvisualizationofGBM(Figure3).RenalImaging
• RenalcortexglomeruliassessedforGBMandmesangialpathogenesiswithlightmicroscopyusingDNclassification.10glomerulifromeachsampleexamined.(Figure4).
RenalAnalysis
PancreaticAnalysis
PancreaticImaging• AperioDigitalpathology
scannercaptureddigitalimages@40x(Figure6A).
• RawPancreaticimageanalyzedfor:isletcount,isletsize,isletarea,adiposearea,totalpancreaticarea(Figure6B-C).
• RenaltissuesstagedDNI-IV,usingglomerularclassificationofdiabeticnephropathy(Figure5).
• Eachsampleassessedindependentlyofmatchedpancreaticsample.
• Matchedpancreaticsampleswereassessedforalterationsintotaladiposeareaandtotalisletarea(Figure7A-C).
• AnalyzedpancreaticsampleswereorganizedintomatchedrenalDNgroupingsandexaminedforpathogeniccorrelationsthroughoutDNprogression.
ISLET:AttheonsetofDN,diabeticsamplesexhibitanapproximate33%decreaseinpancreaticisletarea.AdvancedDNclassesexhibitrelativelystaticanddepletedtotalisletarea,statisticallydifferentiatedfromisletareainhealthysamples.AlteredpancreaticisletareaisthusavalidpredictorofDN,howeverisletareaisnotavalidmeasureofDNprogression.Isletareafollowsthegeneralizedtrend;slightlyincreasedisletareathroughDNclassIIa– IIbfollowedbyattenuateddecreaseintotalisletareathroughDNclassIII-IV(Figure8A).ADIPOSE:AttheonsetofDN,diabeticsamplesexhibitanapproximate20%increaseinpancreaticadiposearea.AdvancedDNclassesexhibit nofurthercorrelationwithpancreaticadiposeareaalterations.PancreaticadiposeareaisnotavalidmeasureforpredictionoftypeIIdiabetesdiseaseprogressionorDNclass(Figure8B).
C.
AperioScanScopeMicroscope Unanalyzedpancreassample,scanned@40x PancreassampleanalyzedwithImageScopesoftware
Figure7.
Healthy StageI StageIIa StageIIb StageIII StageIV
Figure6.
Intrapancreatic adipose,samplewithDNIII Pancreaticislets,samplewithDNIIa Healthyisletwithnormalcellmass,healthysample
Figure5.
A. B.
A.
Figure8.
Acknowledgements• UniversityofColorado,ModernHumanAnatomyprogram,fortheircontinuedsupportinthedevelopmentofthisproject.• UniversityofColorado,DepartmentofMedicine,DivisionofRenalDiseasesandHypertension(MyphoungLe,LeahVillegas,
CarlosRoncal,HeathAustinandTamaraMilagres),fortheirsupportandprovideduseoflaboratorialequipment.• PeterPapazian,forprovidinghisstatisticalanalysisanddataprocessingexpertise.
Apparentamyloidinfiltrationofpancreaticislet
Pancreaticisletwithlymphocyticinfiltration
Conclusions
Discussion/Future Directions
ISLETAMYLOIDCOMPOSITION
AttheonsetofDiabeticNephropathy,classI:• Areaofpancreaticisletssignificantlydecreases• Intrapancreatic adiposecompositionsignificantlyincreases• IsletarearemainsstaticthroughoutDNprogression
BETACELLDYSFUNCTIONANDGLYCATION• Isletbetacellsareresponsibleforinsulinproductioninresponsetometabolicdemand(Figure9A).
• Chronichyperglycemiastressesbetacells,leadingtobetacellapoptosis,depletedinsulinproductionandprolongedhyperglycemia.
• Insulininsensitivecells,suchasrenalmesangialcells,areacutelysensitivetohyperglycemia.
• Intracellularhyperglycemiapromotespathologicglycationofrenalmesangialcells,visualizedhistologicallyasadvancingDN.
• Pancreaticisletareaisnotanindicativemeasureofbetacellfunction.Tobetterassessbetacellfunctionality, immunostaining mustbeemployedinfutureanalyses.
INFLAMMATIONANDDIABETES• Chronichyperglycemiastressesbetacells,andsignalsfortheproductionofinflammatoryfactors
• Theinflammatoryresponsefavorsapoptoticprocesses,promotingbetacelldeath.
• Subsequentlymphaticinvasionislikelyassociatedwiththeremovalofapoptoticbetacells(Figure9C).
• Pancreaticisletswithlymphoidinfiltrationhaveaberrantfunctionandalteredmorphology.Immunostaining usedtovisualizefunctionalbetacells,wouldprovideinsighttobetterunderstandtheroleofinflammationinthepancreaticpathogenesisoftypeIIdiabetes.
• Amyloiddepositsintheislets,canformlargemassesandcompletelyremodelisletmorphology(Figure9B).
• Amyloiddepositionisanintracellularprocesswhichinducesbetacellapoptosis.
• Lossofbetacellmassleadstodeficientinsulinproduction,inappropriateglucosemetabolism,andaberrantglycation.
• Pancreaticisletswithapparentamyloidinfiltrationwereincludedinmeasuredisletarea.Amyloidinfiltrationrendersbetacellsnon-functional,thustoassessisletfunctionalityCongoredstainmustbeusedtoquantifyamyloidintheislet.
Apparenthealthyisletwithwelldefinedcellularmass
Figure9.
A.
B.
A. C.B.
C.
A. B.
*p=.0261*p=.0001
C.
