Diabetic Nephropathy1Diabeticnephropathyistheleadingcauseofchronicrenalfailureintheindustrialisedworld.Itisalsooneofthemostsignificantlong-termcomplicationsintermsofmorbidityandmortalityforindividualpatientswithdiabetes.Diabetesisresponsiblefor30-40%ofallend-stagerenaldisease(ESRD)casesintheUnitedStates.Althoughbothtype1diabetesmellitus(insulin-dependentdiabetesmellitusIDDM)andtype2diabetesmellitus(noninsulin-dependentdiabetesmellitusNIDDM)leadtoESRD,thegreatmajorityofpatientsarethosewithNIDDM.2Theglomeruliandkidneysaretypicallynormalorincreasedinsizeinitially,thusdistinguishingdiabeticnephropathyfrommostotherformsofchronicrenalinsufficiency,whereinrenalsizeisreduced(exceptrenalamyloidosisandpolycystickidneydisease).3Signs and SymptomsApproximately25%to40%ofpatientswithDM1ultimatelydevelopdiabeticnephropathy(DN),whichprogressesthroughfivepredictablestages.4Stage 1 (veryearlydiabetes)Increaseddemanduponthekidneysisindicatedbyanabove-normalglomerularfiltrationrate(GFR).Hyperglycemialeadstoincreasedkidneyfiltration(seelater)ThisisduetoosmoticloadandtotoxiceffectsofhighsugarlevelsonkidneycellsIncreasedGlomerularFiltrationRate(GFR)withenlargedkidneys5Stage 2 (developingdiabetes)ClinicallysilentphasewithcontinuedhyperfiltrationandhypertrophyTheGFRremainselevatedorhasreturnedtonormal,butglomerulardamagehasprogressedtosignificantmicroalbuminuria(smallbutabove-normalleveloftheproteinalbuminintheurine).Significantmicroalbuminuriawillprogresstoend-stagerenaldisease(ESRD).Therefore,alldiabetespatientsshouldbescreenedformicroalbuminuriaonaroutinebasis.6Stage 3 (overt,ordipstick-positivediabetes)Glomerulardamagehasprogressedtoclinicalalbuminuria.BasementmembranethickeningduetoAGEPTheurineisdipstickpositive,containingmorethan300mgofalbuminina24-hourperiod.Hypertension(highbloodpressure)typicallydevelopsduringstage3.7Stage 4 (late-stagediabetes)Glomerulardamagecontinues,withincreasingamountsofproteinalbuminintheurine.Thekidneysfilteringabilityhasbeguntodeclinesteadily,andbloodureanitrogen(BUN)andcreatinine(Cr)hasbeguntoincrease.Theglomerularfiltrationrate(GFR)decreasesabout10%annually.Almostallpatientshavehypertensionatstage4.8Stage 5 (end-stagerenaldisease,ESRD)GFRhasfallento10ml/minandrenalreplacementtherapy(i.e.,haemodialysis,peritonealdialysis,kidneytransplantation)isneeded.9101112CAPILLARY ENDOTHELIUMBASEMENT MEMBRANEFOOT PROCESSES OF PODOCYTESFILTRATION SLITFENESTRATION13NORMAL GBM. LEFT-asingleglomerulus.Thereareonemillionoftheseineachkidney.RIGHT-acloseupoftheGBM(G)aroundpartofonetinybloodvesselinaglomerulus(redcircleinlefthanddiagram)14Glomerular Histology:Theglomerularcapillarywalliscomposedofanendothelialcelllayer(bloodside),athickbasementmembrane,andepithelialcelllayer(urineside).(i) Glomerular EndotheliumThe glomerularendotheliumisfenestrated.Thefenestrae(0.07to0.1mm-micrometers-inmaximaldiameter)allowthepassageofelectrolytes,proteins,andglobulin.However,platelets(3mm),redcells(7mm)andneutrophils(15mm)cantpassthroughtheendotheliallayer.15(ii)Glomerular Basement Membrane (GBM):TheGBMisatri-laminarstructure,0.3micronsinthickness,composedofcollagen,proteoglycansandlaminin.Itisproductofthefusionoftheendothelialandepithelialbasementlaminae.ThedensecentralGBMarea,orlaminadensa,isduetotheoverlappingofthetwolaminae.Around50%oftheGBMiscollagenIV.16ThenegativechargeoftheGBMhasbeenattributedtothepresenceoftheheparansulphateproteoglycan(HSPG)calledperlecan.Thesenegativelychargedmoleculesaregeometricallyarrangedinclustersseparatedbyabout0.003mfromeachother.Thisanionicmolecularsieverestrictsthepassageofmoleculesaccordingtosizeandcharge.Water,salts,glucose,aminoacidsandneutral,orcationic,moleculeswithradiilessthat0.0035marefilteredwithrelativeease.Thealbuminmoleculemeasures0.0035mandisnegativelycharged.Thereforeitsfiltrationisrestricted.17Presenceofproteinintheurineisasignthateitherthechargeorthedistancebetweentheanionicclusters,orboth,arepathologicallyaltered.Thepresenceofredcellsintheglomerularurine,iscertainindicationofGBMruptures.OtherclassicalconstituentsofthebasementmembranearetypeIVcollagen,laminin,andentactin.18Glomerular mesangium:Theintra-capsularglomerularcapillarynetworkiskepttogetherbythemesangium thatisiscomposedofmesangialcellstypeIandII,andothertissuematrix.MesangialtypeIcellsaremonocyteswithphagocyticfunctions.Thesecellscanextendcytoplasmicprojectionsintotheglomerularcapillary.Theyalsocleanthemesangiumofmaterialsthatleakfromthecapillarylumenintothematrix.Thesecellsarestimulatedbycytokinestoproducefreeradicalsandcytotoxicpeptides.19MesangialtypeIIcellsaremyofibroblastswiththeabilitytocontractuponADHandangiotensinstimulation.Theircontractioncausesareductionoftheeffectiveglomerularfiltrationarea.MesangialMatrixisatissuemeshcomposedofdifferenttypesofcollagens(I,III,IV),lamininandproteoglycans.20Threemajorhistologicchangesoccurintheglomeruliofpersonswithdiabeticnephropathy.1.Mesangialexpansionisdirectlyinducedbyhyperglycemia,perhapsviaincreasedmatrixproductionorglycosylationofmatrixproteins.2.GBMthickeningoccurs.3.Glomerularsclerosisiscausedbyintraglomerularhypertension(inducedbyrenalvasodilatationorfromischemicinjuryinducedbyhyalinenarrowingofthevesselssupplyingtheglomeruli).21Glomerular Hyper filtrationGlucoseprovidesanosmoticdiureticeffectResultisincreasedrenalfiltration,leadingtoglomerularhypertrophyGlomerularpressureincreasesKidneyrespondswithhypertrophyofepitheliumandendotheliumAcceleratesglomerularcellfailureResultisprematureglomerulosclerosis22Metabolic Perturbations1.OxidantStress-relatedtoglomerularhypertrophyandabnormalmetabolism2.Non-enzymaticglycosylationofmacromolecules-particularlybasementmembrane(BM)3.Activationofglucosemetabolizingenzymes4.Cytokineandotherhumoralimbalances23Non enzymatic GlycosylationBiochemicalstudieshaveshownthatbasementmembranesindiabetesincludeexcessamountsoftypeIVcollagen,themaincomponentofbasementmembranes,anddecreasedamountsofproteoglycansBothchangesdecreasethepermeabilityofcapillariesanddisturbleukocytediapedesis,oxygendiffusion,nutritionandmetabolicwasteremoval.1.AlteredchargeonBMmayexplainalbuminuria2.MacrophagereceptoractivationleadstoIL1,TNFproductionwhichstimulatesmatrix3.AGEPformationleadstoabnormalcollagen,increasedtoxicoxygenspecies24Humoral Imbalances in DM Nephropathy1.InsulinDeficiency2.ElevatedGlucagonConcentrations3.IncreasedTransforming Growth Factor(TGF)-4.IncreasedangiotensinII5.Abnormallyregulatedthromboxanesandendothelins6.Abnormalinsulinlikegrowthfactor(IGF)-17.Elevatedplateletderivedgrowthfactor(PGDF)25Role of TGF-1.Stimulatesextracellularmatrixsynthesis2.Inhibitsextracelluularmatrixdegradation3.Upregulatesproteaseinhibitors;downregulatesmatrixdegradingenzymes4.Stimulatessynthesisofintegrins(matrixreceptors)5.Keyroleinglomerularandtubuloepithelialhypertrophy,basementmembranethickening,andmesangialmatrixexpansion6.TGF-hasbeenimplicatedinanumberofchronic,scarringdiseases2627AngiotensinIIandThrombospondin(TSP1)canbothstimulatetheproductionoftransforminggrowthfactor-(TGF-)bytubuloepithelialcellsandfibroblasts.TGF-,inturn,causesproliferationoffibroblastsandtubuloepithelialcells.TGF-ultimatelyincreasesextracellularmatrixproteins,likelybyseveralmechanisms.TGF-stimulatesproductionofseveralgrowthfactorsincludingbasisfibroblastgrowthfactor(bFGF)andplateletderivedgrowthfactor(PDGF)thatstimulatetheformationofextracellularmatrix(ECM)proteins.28Ultrastructural changes of the glomerular basement membrane in diabetic nephropathy revealed by newly devised tissue negative staining method.ThenormalhumanGBMshowedafinemeshworkstructureconsistingoffibrilsformingthesmallpores.Thediameteroftheseporeswasslightlysmallerthanthatofhumanalbuminmolecules.TheGBMinpatientswithdiabeticnephropathyshowedirregularthickening.Athighermagnification,unknowncavitiesandtunnelstructures,whichwerenotseeninnormalcontrols,wereobservedinthethickenedGBM.29Insomeportions,thesecavitiespresentedahoneycomb-likeappearance.Thediametersofthecavitiesandtunnelswerefarlargerthanthedimensionsofalbuminmolecules.TheseenlargedstructuresarebelievedtoallowserumproteinmoleculestopassthroughtheGBMfromthecapillarylumentotheurinaryspace.TheseresultssuggestthatthecauseofmassiveproteinuriaindiabeticnephropathyisthedisruptionofthesizebarrieroftheGBM.30Glomerular and vascular pathology is linked to hyperglycemia.Changesinglomerularbasementmembranestructureoccurveryearlyindiabeticnephropathy,beforeevenmicroalbuminuriaisapparent.CollagenIVdepositionisdirectlystimulatedbyhyperglycaemiaandincreasedurinarylevelsindicatechangesintheglomerularbasementmembrane.Contributingfactorsincludetheformationofadvancedglycosylationendproducts(AGEs)duetonon-enzymaticglycosylationofcapillarybasementmembranes,asaconsequenceoflong-termhyperglycaemia.31Non-enzymaticglycosylationhasrecentlyattractedincreasinginterestasacrucialpathophysiologiceventbehindallthesehyperglycaemia-relatedalterationsandinthepathophysiologyofthedevelopmentofdiabeticcomplications.Proteinsandlipidsexposedtoaldosesugarsgothroughreactionswhicharenotenzyme-dependent,andgenerationofreversibleSchiffbasesorAmadoriproductstakeplace.Later,throughfurthermolecularrearrangements,irreversibleadvancedglycosylationendproducts(AGEs)areformed.Thisprocessalsotakesplaceduringnormalageing,butindiabetestheirformationisacceleratedtoanextentrelatedtothelevelanddurationofhyperglycaemia.32Hencelargestudieshaveshownadelayinonsetorslowingoftheprogressionofthesecomplicationsifnearnormo-glycaemiacanbemaintained.Theglycatedproteinscross-link,contributingtobasementmembrane(andmesangial)thickening,(culminatinginthekidneyinnodularglomerulosclerosis),aswellaslossofthenormalselectivepermeability(leadingtoproteinuria,retinalhardexudatesandmicrohaemorrhages).33ThepotentialpathophysiologicalsignificanceofAGEsisassociatedwiththeiraccumulationinplasma,cellsandtissuesandtheircontributiontotheformationofcross-links,generationofreactiveoxygenintermediatesandinteractionswithparticularreceptorsoncellularsurfacesAGEshavedirecteffectsonthehostresponsebyaffectingtissuestructures,e.g.byincreasingcollagencross-links,whichisfollowedbychangesincollagensolubilityandturnover.ThickeningofbasementmembranesispartlyduetoglycosylationofmembraneproteinsorentrapmentofglycosylatedserumproteinsintobasementmembraneItisevidentthatAGEscaninteractwithcellfunctions,tissueremodellingandinflammatoryreactionsinseveraldifferentways.343536WhenAngIIisincreased,greaterAT1receptor-mediatedconstrictionofefferentthanafferentarteriolesincreasessinglenephronglomerularfiltrationrateandraisesintraglomerularpressure,causingglomerularhypertension.SustainedorsevereincreasesinintraglomerularpressurecanleadtoGBMdamage,glomerularendothelialdysfunction,andultimately,extravasationofproteinintoBowmanscapsule.Inadditiontohypertension,conditionslikediabetesthatareassociatedwithincreasedoxidativestress(increasedformationofreactiveoxygenspecies)independentofhypertensionandglyco-oxidativemodificationofproteins(AGEs)comprisingtheglomerularbasementmembranecanleadtoextravasationofprotein.3738Glomerularhypertensioncanleadtoinjurytotheglomerularbasementmembranecausingittoleakplasmaproteinsintotheurine.Attemptsbytheproximaltubulestoreabsorbthisfilteredproteincausesinjurytothetubularcells,activatesaninflammatoryresponse,andisassociatedwiththedevelopmentoflipidmetabolicabnormalitiesthatcreatefurtheroxidativestressonthealreadycompromisedglomerulus.Theresultanttubularinflammatoryresponseandrenalmicrovascularinjuryactivatepathwaysthatleadtofibrosisandscarringofbothglomerularandtubularelementsofthenephron.39Anadditionalconsequenceofglomerularhypertensionandresultantreductioninglomerularfiltrationrate(GFR)activatesgrowthfactorsandcytokinesthatpromoteaninfluxofmonocytesandmacrophagesintothevesselwallandintotherenalinterstitium,andalsocausesthedifferentiationofrenalcellsintofibroblasts.Monocytes,macrophagesandfibroblastsarecapableofproducingthosegrowthfactorsandcytokinesthatactivatepathwaysleadingtoexpansionofextracellularmatrix,fibrosisandlossofbothtubularandglomerularstructures.40CollagenIVistheprincipalcomponentoftheglomerularbasementmembraneanditisreleasedintotheurineduringitsturnover.IncreasedurinarylevelsofcollagenIVarefoundinseveralconditionswhereglomerularinjuryisfound,particularlyindiabeticnephropathy.CollagenIVistoolargetocrosstheglomerularmembrane(MW540000)andsourinarycollagenIVisaspecificsensitiveindicatorofchangestothestructureofextracellularmatrixofthekidney.Unlikeserumcreatinine,thatmeasureschangesinglomerularfunction,increasedlevelsofurinarycollagenIVindicatethatdamageisoccurringtotherenaltissue.UrinarycollagenIVisaveryearlyandspecificbiomarkerforpathologicalchangestotheglomerularmembrane,particularlyindiabeticnephropathy.41