1

AssociationofElevatedUrinarymiR-126,miR-155andmiR-29bwithDiabeticKidneyDiseaseCristinaBeltrami,*KateSimpson,*†MarkJesky,‡AlexaWonnacott,*

ChristopherCarrington,*PeterHolmans,§LucyNewbury,*†RobertJenkins,*ThomasAshdown,*ColinDayan,¶SimonSatchell,||PeterCorish,**

PaulCockwell,‡DonaldFraser,*†‡‡andTimothyBowen*†‡‡§§* Wales Kidney Research Unit, Division of Infection and Immunity, School of Medicine,College of Biomedical and Life Sciences, Cardiff University, Heath Park Campus, CardiffCF144XN,UK

† CardiffInstituteofTissueEngineeringandRepair,CardiffUniversity,MuseumPlace,CardiffCF103BG,UK

‡ Departmentof RenalMedicine,QueenElizabethHospital Birmingham,MindelsohnWay,BirminghamB152GW,UK

§ MRC Centre for Neuropsychiatric Genetics and Genomics, Division of PsychologicalMedicine andClinicalNeurosciences, School ofMedicine, College of Biomedical and LifeSciences,CardiffUniversity,MaindyRoad,Cathays,CardiffCF244HQ,UK

¶ DiabetesResearchGroup,DivisionofInfectionandImmunity,SchoolofMedicine,CollegeofBiomedicalandLifeSciences,CardiffUniversity,HeathParkCampus,CardiffCF144XN,UK

||Bristol Renal, Bristol Medical School, University of Bristol, Dorothy Hodgkin Building,WhitsonStreet,BristolBS13NY,UK

**BBIGroup,TheCourtyard,TyGlasAvenue,CardiffCF145DX,UK‡‡TheseauthorscontributedequallytothisworkRunningtitle:UrinarymiRNAsinDiabeticNephropathy§§Towhomcorrespondenceshouldbeaddressed:TimothyBowen,WalesKidneyResearchUnit,SchoolofMedicine,CollegeofBiomedicalandLifeSciences,CardiffUniversity,HeathParkCampus,CardiffCF144XN,UK.Tel.:44-29-2074-8389;Fax:44-29-2074-8470;E-mail:bowent@cf.ac.uk

CountsAbstract:215words;Text:15pages;Tables:3;Figures:4

Support This isapublicationof independent research fundedby theNational Institute for

HealthResearch(NIHR)InventionforInnovation(i4i)Programme(GrantReferenceno.II-LA-

0712-20003).ThePrincipalInvestigatorforthegrantisTB.Theviewsexpressedinthispaper

arethoseoftheauthorsandnotnecessarilythoseoftheNHS,theNIHR,ortheDepartment

ofHealth,UK.TheauthorsalsoacknowledgesupportfromKidneyResearchUKProjectGrant

AwardsRP44/2014(TB)and IN4/2013(SS).TheJABBSFoundationfundedcollectionofthe

RenalImpairmentinSecondaryCare(RIISC)cohort(PC).TheWalesKidneyResearchUnitis

fundedbycoresupportfromHealthandCareResearchWales(DJF).

Declarationof interestsCD:NovoNordiskAdvisor,SanofiGenzymeandAstraZenecaservices(2015-2017);SS:BoehringerIngelheimTravelSupportandUCBUKGrantSupport(2013-2015);PCor:LifeSciencesBridgingFundWalesConsultancy(2015-2017);PC:JABBSFoundationGrantSupport (2014-2015); DF and TB: Patent application PCT/GB2017/050195: Kidney diseasediagnostic(2017).

2

ABSTRACT1

Effective diabetic kidney disease (DKD) biomarkers remain elusive, and urinary2

microRNAs (miRNAs) represent a potential source of novel non-invasive disease3

sentinels.Weprofiled754miRNAsinpooledurinesamplesfromDKDpatients(n=20),4

detectingsignificantlyincreasedmiR-126,miR-155andmiR-29bcomparedtocontrols5

(n=20).Theseresultswereconfirmedinan independentcohortof89DKDpatients,6

62 diabetic patients without DKD and 41 controls: miR-126 (2.8-fold increase;7

p<0.0001),miR-155 (1.8-fold; p<0.001) andmiR-29b (4.6-fold; p = 0.024). Combined8

receiveroperatingcharacteristiccurveanalysisresultedinanareaunderthecurveof9

0.8.A relativequantification threshold equivalent to 80% sensitivity for eachmiRNA10

gaveapositivesignalfor48%ofDKDpatientscomparedto3.6%ofdiabeticpatients11

without DKD. Laser capture microdissection of renal biopsies followed by RT-qPCR12

detectedmiR-155 in glomeruli, proximal and distal tubules,whilemiR-126 andmiR-13

29b were most abundant in glomerular extracts. Subsequent experiments showed14

miR-126andmiR-29benrichmentinglomerularendothelialcells(GEnCs)comparedto15

podocytes,proximaltubularepithelialcellsandfibroblasts.SignificantlyincreasedmiR-16

126andmiR-29bweredetectedinGEnCconditionedmediuminresponsetotumour17

necrosis factor-alpha and transforming growth factor-beta 1, respectively. Our data18

revealanalteredurinarymiRNAprofileassociatedwithDKDandlinkthesevariations19

tomiRNAreleasefromGEnCs.20

21

Keywords: microRNAs, urine, biomarker, diabetic kidney disease, chronic kidney22

disease23

24

3

Abbreviations:25

ACR albumin:creatinineratio26

CKD chronickidneydisease27

DKD diabetickidneydisease28

eGFR estimatedglomerularfiltrationrate29

GEnC glomerularendothelialcell30

KDIGO kidneydisease:improvingglobaloutcomes31

NKFKDOQI NationalKidneyFoundationkidneydiseaseoutcomesqualityinitiative32

LCM lasercapturemicrodissection33

MDRD modificationofdietinrenaldisease34

miRNA microRNA35

PTC proximaltubularepithelialcell36

RIISC renalimpairmentinsecondarycarestudy37

38

39

4

Introduction40

Recentestimatessuggestthat1in12oftheglobalpopulationsuffersfromdiabetes41

mellitus,approximately40%ofthoseaffectedwillgoontodevelopdiabetickidney42

disease(DKD).1DKDistheleadingcauseofend-stagerenaldiseaseandpredisposing43

factors include genetic causes, ethnicity, hyperglycaemia, insulin resistance,44

intraglomerularhypertensionandhyperfiltration.2,345

Hyperglycaemia results in numerous deleterious consequences including46

upregulated cytokine synthesis, renin-angiotensin system activation, generation of47

advancedglycationendproductsandreactiveoxygenspecies,andincreasedprotein48

kinase C activity.4,5 Nitric oxide andNF-κB pathway-driven loss of endothelial and49

vascularmodulationhavebeen implicated in insulinresistance,andearlyDKDmay50

beassociatedwith insulinsignalingdefects specific to thepodocyte.6These insults51

result in loss of glomerular filtration rate and ultimately to renal failure from52

mesangial hyperexpansion, nodular glomerulosclerosis and tubulointerstitial53

fibrosis.754

Detection of urinary microalbuminuria currently forms the basis of DKD55

progressionmonitoring, varying from normalmean albuminuria values around 1056

mg/daytoadiagnosisofmicroalbuminuriaat30–300mg/dayandmacroalbuminuria57

above 300 mg/day.8 Prognosis is complicated, since not all microalbuminuric58

patientsprogress toovertnephropathy.Anumberofnovelbiomarkershavebeen59

assessedforutility inDKDbutnonearebeingusedasroutineclinicalmarkers,and60

theymaylackspecificityandsensitivitytopredictindividualDKDpatientoutcomes.61

In light of the above, novel markers that can discriminate aetiology, progression62

and/orresponsetotreatmentremainhighlydesirable.63

5

MicroRNAs (miRNAs)areubiquitously-expressedshortnoncodingRNAsthat64

regulate the expression ofmost protein coding genes in the human genome, and65

detectionofmiR-192,miR-194,miR-215,miR-216,miR-146a,miR-204andmiR-88666

iselevatedinthekidney.9UrinarymiRNAsrepresentahighlypromisingnovelsource67

of non-invasive biomarkers that are stabilised via argonaute 2 protein/exosome68

associationandarerapidlyandpreciselydetectedbyRT-qPCR.1069

Reports have suggested a role for miRNAs in the pathology of DKD,11,1270

includingpreviousworkfromthislaboratoryshowingdecreasedmiR-192inbiopsies71

from late-stage DKD patients with diminished renal function.13 However,72

comparatively little is known about the abundance of urinary miRNAs in DKD73

patients.74

We hypothesised that alterations in urinary miRNA profiles would be75

associatedwithDKD.WeidentifiedcandidateDKDbiomarkersbycomparingmiRNA76

profilesinurinesamplesfromapatientdiscoverycohortwiththosefromunaffected77

controls.Selectedcandidateswerethenmeasured ina larger, independentcohort.78

Subsequently,lasercapturemicrodissectionofrenalbiopsiesandinvitrocellculture79

were used to investigate the sources of our candidate urinary miRNA DKD80

biomarkerswithrespecttonephrondomainandcelltype.81

82

6

MaterialsandMethods83

StudyParticipants84

DKDwasdefinedinaccordancewiththeNationalKidneyFoundationKidneyDisease85

Outcomes Quality Initiative (NKF KDOQI) Clinical Practice Guidelines and Clinical86

Practice Recommendations for Diabetes and Chronic Kidney Disease (CKD).1487

Accordingly, CKD should be attributable to diabetes in the presence of88

macroalbuminuria (in the absence of urinary infection), or in the presence of89

microalbuminuriawithconcomitantdiabeticretinopathy,orintype1diabetesofat90

least10yearsduration.14Theinitialprofilingstudycohortof20DKDpatientsand2091

healthy controls was obtained from the Wales Kidney Research Tissue Bank,92

UniversityHospitalofWales,Cardiff.TheDKDgroupwaspredominantlymale(85%),93

meanage72years(SD+/-8.7).DKDpatientswereCKDstage3-5(pre-dialysis),with94

meaneGFRof29ml/min/1.73m2(SD+/-8.5)andameanurinaryAlbumin:Creatinine95

ratio(ACR)of13.5mg/mmol(SD+/-14.5).Thecontrolgroup(n=20)intheprofiling96

cohortwere50%male,meanage47years (SD+/-11.0)withnomicroalbuminuria97

(ACR<3mg/mmol).ForfurtherdetailsonACRcategoriesseeTable1.98

99

The confirmation cohortwas drawn from two secondary care facilities: theWales100

KidneyResearchTissueBank(asabove)andtheRenalImpairmentInSecondaryCare101

(RIISC) study, University Hospital of Birmingham, UK.15 89 patients with DKD,102

including 3 patients with type 1 diabetes, and 41 healthy controls were recruited103

acrossthetwosites.Anadditionalcontrolgroupof62diabeticswithoutDKDwere104

recruitedfromCardiff, including17patientswithtype1diabetes. Ethicalapproval105

7

wasgrantedbytheWalesKidneyResearchTissueBankGovernanceCommitteeand106

theSouthBirminghamLocalResearchEthicsCommittee,respectively.107

108

Patient demographics and clinical parameters are shown in Table 1. All patients109

wererecruitedfromspecialistnephrologyanddiabetescareservicesatthetwosites110

during the period spanning autumn2010 to autumn2013.DKDpatients from the111

RIISCstudycohortwerepredominantlyadvancednephropathsasperRIISCprotocol112

inclusioncriteria:briefly,patientswithCKDstages4-5(pre-dialysis),orCKDstage3113

and accelerated progression and/or proteinuria as defined by the UK National114

Institute for Health and Care Excellence 2008 CKD guideline for secondary care115

review.Thediabeticpatientcontrolgroupallhadadiagnosisofdiabetesbystandard116

American Diabetes Association criteria,16 but without evidence of DKD (i.e. not117

fulfillingtheKDOQIcriteria).118

119

At initial clinic visit, renal function was recorded using estimated glomerular120

filtration rate (eGFR), calculated using the modification of diet in renal disease121

(MDRD)equation17.Urinesampleswerealiquotedforalbumin:creatinineratio(ACR)122

assessment and for RNA extraction (see below). ACR cut-offs for disease severity123

were defined as per Kidney Disease: Improving Global Outcomes (KDIGO) 2012124

guidelines.18125

126

UrineCollection,RNAIsolationandRT-qPCRanalysis127

UrinesampleswerecollectedandRNAextractionfrom350µlofurine,generationof128

cDNA from equal volumes of RNA extracts and RT-qPCRwere then carried out as129

8

described in detail elsewhere.10 TaqMan assays (Thermo Fisher Scientific, Paisley,130

UK) used in this study were: hsa-miR-29b-3p (ID 000413); hsa-miR-126-3p (ID131

002228); hsa-miR-155-5p (ID 002623); hsa-miR-191-5p (ID 002299). Relative132

quantities were calculated using the 2-ΔΔCt method, and miRNA expression was133

normalizedtohsa-miR-191-5p.10134

135

MiRNAprofilingbyTaqManArrayHumanMicroRNACards136

UrinarymiRNAswerereversetranscribedusingtheMegaplexPrimerPools(Human137

Pools A v2.1 and B v3.0, Thermo Fisher Scientific) with a predefined pool of 381138

reversetranscription(RT)primersforeachMegaplexPrimerPool.Afixedvolumeof139

3μlofRNAsolutionwasusedas input ineachRTreaction,andRTreactionswere140

performedaccordingtothemanufacturer’srecommendations.RTreactionproducts141

wereamplifiedusingMegaplexPreAmpPrimers(PrimersAv2.1andBv3.0,Thermo142

Fisher Scientific), the samples were then diluted to a final volume of 100 μl and143

controlsubjectandDKDpatientproductswerepooledasfollows.144

145

Toexcludethepossibilitythatgender,ageandeGFRstatushadextremeeffectson146

miRNAexpressionprofiles,thefollowingpoolingstrategywasfollowed.Controlpool147

(CP)1:urinesamplesfrom5femalesofaverageage44.8years;CP2:5females,57.6;148

CP3: 5 males, 35.2; CP4, 5 males, 53.2. Patient Pool (PP)1: urine samples from 5149

CKD3patientswithaneGFRbetween43.3and36mL/minper1.73m2;PP2:5stage3150

patients,35-31;PP3:5stage4/5patients,27.3-23;PP4:5stage4/5patients,22-151

12.9.152

153

9

TaqManArrayHumanMicroRNACardsAv2.1andBv3.0 (ThermoFisherScientific)154

wereusedtoquantify754humanmiRNAs.Eacharrayincluded377testmiRNAs,3155

endogenouscontrolsandanegativecontrol.Quantitative(q)PCRwascarriedouton156

an Applied Biosystems 7900HT thermo cycler (Thermo Fisher Scientific) using the157

manufacturer’srecommendedprogram.158

159

LaserCaptureMicrodissection(LCM)fromRenalBiopsySamples160

Glomeruli,proximaltubularanddistaltubularprofilesweremicrodissectedfrom6-161

μmsectionsobtainedfromfiveFFPEarchivedrenalbiopsiesfromunaffectedpeople162

using the Arcturus Pixcell IIe infrared laser enabled LCM system (Thermo Fisher163

Scientific).164

165

CellCulture166

Human conditionally immortalised glomerular endothelial cell (GEnC) and human167

podocyte(ATC)celllineswerepropagatedat33°Casdescribedpreviously.19,20After168

5(GEnC)and14(ATC)days,cellsweretransferredto37°C incubationto inactivate169

the SV40 T antigen and permit differentiation, prior to experimental use. Where170

stated, GEnCs were growth arrested for 24 h and then treated with TNF-α (10171

ng/mL)orTGF-β1(1ng/mL)ateithernormoglycaemic(5mM)orhyperglycaemic(25172

mM)D-glucoseconcentrationsfor24h.Proximaltubularepithelialcell(PTC)lineHK-173

221 and fibroblast22 cultures were maintained as described elsewhere. Cells and174

culturemediumobtainedfromeachwellwereusedforRNAextractionasdescribed175

above.176

177

10

Statisticalanalysis178

MiRNA profiling data were analysed using Thermo Fisher Scientific’s DataAssist179

Software (version 3.01), NormFinder Software (http://moma.dk/normfinder-180

software; last access 21/02/18) and GraphPad Prism 6 (version 6.0d). Pearson181

CorrelationCoefficientswasusedtodetectclustersofsimilarityinmiRNAthreshold182

cyclevaluesbetweeneachpoolgroupinpatients,andbetweeneachpoolgroupin183

controls. To identify a suitable reference gene for the normalization of miRNA184

expression in this study, the NormFinder algorithmwas applied to the expression185

dataobtained from theHumanTaqManmiRNAArrays.Analysis comparingmiRNA186

levelsbetweensubjectswithDKDandcontrolswascarriedoutusingGraphPadPrism187

version 6 version 6.0d. Values for p below 0.05 were considered statistically188

significant. MiRNA profiling data sets can be found in Gene Expression Omnibus189

(https://www.ncbi.nlm.nih.gov/geo;accessionnumberGSE114477). 190

191

11

Results192

AlteredurinarymiRNAdetectioninDKDpatients193

ToselectcandidatemiRNAsthatmayactasDKDbiomarkers,wefirstcompareddata194

from unbiased expression profiling of 754miRNAs in urine samples from 20 DKD195

patients and20unaffected controls. Analyseswereperformedon4 patient and4196

controlpools,eachcomposedofurinesamplesfrom5individualsasrecommended197

byZhangandcolleagues.23Sampleswerepooledprior toprofiling tominimize the198

contributionofsubjecttosubjectvariationandtomakesubstantivefeatureseasier199

to find, and thereby identify biomarkers common across individuals.24 Previous200

analysis suggested that40 individualsmightoptimallybepooledacross8arrays,23201

whichwasourchosenpoolingapproach.202

In Figure 1A the 12 data points in the upper right quadrant of the plot203

representthosemiRNAsforwhichstatisticallysignificantfold-changeincreaseswere204

detected inpatienturinecomparedtocontrol samples, the35points in theupper205

left quadrant the corresponding downregulatedmiRNAs. The fold-change data for206

these47miRNAsaresummarisedinFigure1B,andthe8miRNAsexhibiting>5-fold207

change were subsequently selected as potential candidate biomarkers for further208

analysis.209

Specific RT-qPCR assays were then used to analyse these miRNAs in each210

component urine sample pooled for profiling analysis. Statistically significant211

differences in miRNA detection between DKD patient and control urine samples212

werereplicatedformiR-126(4.3-fold increase;p=0.0087),miR-155(22.9-fold;p=213

0.0024)andmiR-29b(4.9-fold;p=0.0002)(Figure1,C-E).214

12

ElevatedurinarymiR-126,miR-155andmiR-29bdetectioninanindependentDKD215

patientcohort216

To test the above findings, miR-126, miR-155 and miR-29b were quantified in217

samples from an independent cohort of patients with established DKD from the218

Renal Impairment in Secondary Care Study (RIISC).15 Samples from 89 patients219

meeting the criteria established in the UK National Institute for Health and Care220

Excellence 2008 criteria were available. An additional cohort of 62 patients with221

diabetesmellitusbutwithoutproteinuriaorotherevidenceofDKDwereincluded,as222

were samples from41 peoplewithout evidence of diabetes orDKD (Table 1).We223

includeddiabetespatientswithoutDKDasa thirdgroup in thisanalysis to identify224

DKD-specificmiRNAdetectionchangesandnotpurelyhyperglycaemia-driveneffects225

fromourprofilingcomparisonofDKDpatientswithcontrolindividuals.226

Significant differenceswere again seenbetweenDKDpatients and controls227

for miR-126 (2.8-fold increase; p<0.0001; Figure 2A), miR-155 (1.8-fold; p<0.001;228

Figure2B)andmiR-29b(4.6-fold;p=0.024;Figure2C).ComparisonofDKDpatients229

withdiabeticpatientswithoutDKDwasstatisticallysignificantformiR-126(3.1-fold230

increase;p<0.0001)andmiR-155(1.6-fold;p=0.024)withatrendtoincreasedmiR-231

29b(4.1-fold;p=0.121)(Figure2A-C).232

RT-qPCR data for all 3 miRNAs were used to compare DKD patients and233

diabetic patients without DKD in the combined receiver operating characteristic234

(ROC) curve analysis shown in Figure 2D, giving an area under the curve (AUC) of235

0.80.ToanalysethecontributionsofeachmiRNAtotheaboveROCcurve,individual236

specificity and likelihood ratioswere calculated for relative expression (RQ) values237

13

equivalent to a sensitivity of 80%.25,26 Data displayed in Table 2 illustrate the238

magnitude of corresponding specificity values wasmiR-126 >miR-155 >miR-29b,239

and that combined miRNA data resulted in a ≥6.5% increase in specificity and240

likelihoodratiocomparedwithindividualmiRNAs.TheseRQdatawerethenusedas241

consecutive threshold values to discriminate between DKD and diabetic patients242

withoutDKD(D inTable3) fromthe independentcohort.Thediscriminatoryorder243

was miR-29b (DKD/D = 5.62) > miR-126 (3.48) > miR-155 (2.23), and RQ values244

exceedingall3thresholdswereobtainedfor48.0%ofDKDpatientscomparedwith245

3.6%ofdiabeticpatientswithoutDKD(Table3).246

247

Lasercapturemicrodissectionshows increasedglomerularabundanceofmiR-126248

andmiR-29bthatisreplicatedinGEnCculture249

Previous reports have linked changes inmiRNA expression to DKD pathology, but250

have focused on whole tissue studies. For example, we showed association of251

decreasedmiR-192 expressionwith disease progression in DKD biopsies by in situ252

hybridisation.13253

Inthepresentstudyweused lasercapturemicrodissection(LCM)to isolate254

glomeruli, proximal and distal tubules (Figure 3A) from histologically normal255

formalin-fixed, paraffin-embedded (FFPE) renal biopsy samples, and analysedmiR-256

126, miR-155 and miR-29b expression by RT-qPCR. In Figure 3B, a typical CD10-257

stained FFPE biopsy section is seen before and after LCM to isolate glomeruli,258

proximalanddistalrenaltubules.MiR-126,miR-155andmiR-29bweredetected in259

extracts from all three nephron regions (Figure 3, C-E). Increased glomerular260

14

abundanceswereobserved formiR-126 (Figure3C)andmiR-29b (Figure3E),while261

miR-155wasmostabundantinthedistaltubule(Figure3D).262

Conclusions regarding nephron region-specific miRNA expression from the263

aboveanalysesare inherently limited,however,sincetissueextractsaresubjectto264

tracecontaminationbycellsfromothernephrondomains.Therefore,cellularmiRNA265

localisationwithineachnephron regionwas subsequently investigatedbyRT-qPCR266

analysisofpodocyteandendothelialcell(GEnC)culturesfromtheglomerulus,renal267

proximal tubular epithelial cells (PTC) and fibroblasts. Detection of miR-126 was268

significantlyhigher inGEnCscomparedwithothercell types(Figure3F).MostmiR-269

155wasdetected inPTCsand least inGEnCs (Figure3G),whilemiR-29bwasmost270

abundantinGEnCs(Figure3H).271

272

GEnC release ofmiR-126 andmiR-29b in an in vitromodel of hyperglycaemia is273

drivenbyTNF-αandTGF-β1,respectively274

The above data localized themajority of miR-126 andmiR-29b expression to the275

GEnC.We next sought stimuli by whichmiRNAs are released into the glomerular276

ultrafiltrate, and hence the urine. Data from animal models of diabetes show277

increasedglomerularandPTCTNF-αexpression,andrenoprotectiveeffectsofTGF-β278

inhibitors have also been reported.27,28 GEnC expression of our candidatemiRNAs279

wasthusanalysedinvitroinresponsetoTNF-αandTGF-β1innormoglycaemiaand280

hyperglycaemia(Figure4,A-F).281

The presence of TNF-α led to significantly increased miR-126 detection in282

GEnCconditionedmediumat5mMand25mMD-glucose(Figure4B),apatternalso283

seen for miR-29b following TGF-β1 addition (Figure 4D). These cytokines did not284

15

increaseGEnCexpressionofmiR-126(Figure4A),ormiR-29b(Figure4C),apattern285

consistentwithincreasedrelease,butnotexpression,ofmiRNAs.286

NosignificantchangesinmiR-155weredetectedinresponsetoelevatedD-287

glucose with either cytokine, and data for TNF-α are shown (Figure 4, E and F).288

Similarly, changes in miR-126 following TGF-β1 addition, and for miR-29b in the289

presenceofTNF-α,werenotobserved (datanotshown).ElevatedD-glucosealone290

didnotchangemiRNAexpressioninGEnCsorconditionedmedium(Figure4,A-F).291

292

16

Discussion293

Diabetickidneydisease (DKD) is the leadingcauseofkidney failure requiring renal294

replacement therapy worldwide, but effective methods to identify and halt295

progression of disease-specific pathophysiological changes remain elusive. Current296

effective interventions such as control of blood glucose and blood pressure are297

challenging to achieve, costly and time intensive. Existing tests track DKD from298

diabetic diagnosis to kidney failure, but do not allow accurate prognosis for the299

individual patient. In addition, the absence of treatment response biomarkers300

hindersdevelopmentofemergingDKDtherapies.There is thusanunmetneed for301

additionalDKDbiomarkerstotargetinterventionandfollowresponsetotherapy.302

InthisstudywesetouttoidentifyurinarymiRNADKDbiomarkers.Increased303

detection of miR-126, miR-155 and miR-29b was observed in the urine of DKD304

patients in comparison with both unaffected individuals and diabetic patients305

without DKD. MiRNA localization and release studies further suggested specific306

releaseofmiR-126andmiR-29bfromGEnCs.Thisraisedthepossibilitythaturinary307

miRNAquantificationmightprovidedataonongoingpathologicalprocesses,andso308

aidpatientstratificationandmeasurementofresponsetotherapy.309

UrinarymiRNAbiomarkershaveseveralpotentialsignificantadvantagesover310

circulatingmiRNAsforadoptionintoexistingtreatmentpathwaysalongsidecurrent311

biomarkers,includingspeedandcostofnon-invasivesampleaccess.29However,few312

urinarymiRNADKDbiomarkerdatahavesofarbeenreported.Previousstudieshave313

focusedoncirculatingmiRNAs,andhavegeneratedconflictingdatawithrespectto314

associationofmiR-126withdiabetesmellitusand/orDKD.A recentcross-sectional315

analysis of type 2 diabetes mellitus patients found a negative association with316

17

plasma miR-126,30 and similar findings have been reported for type 1 diabetes317

mellitus and all complications.31 By contrast,miR-126 detection did not change in318

whole blood from type 2 diabetes mellitus patients and control subjects, but319

decreasedinDKDpatientsamples.32Furthermore,nochangeinplasmamiR-126was320

observedinastudyofpaediatrictype1diabeticpatients.33Theseanalysesprovide321

inconsistentdataforthebiomarkerutilityofcirculatingmiR-126, incontrasttothe322

significantandreproducibleincreaseswedetectedinmiR-126,miR-155andmiR-29b323

inDKDpatienturineinthepresentstudy.324

TheDKD-specificalterationsinurinarymiRNAprofilesdetectedinthisstudy325

mayhave functional significance.Our in vitro analyses localizedmiR-126 andmiR-326

29bprincipally to theGEnC,withmiR-155expressiondistributedevenlyacross the327

nephron.GlomerularendotheliallocalizationofmiR-126mayreflecttheroleofthis328

transcript in vascular regulation. Targeted mouse miR-126 deletion resulted in329

vascular abnormalities by removing inhibition of sprouty-related EVH1 domain-330

containing protein 1 expression, thereby enhancing vascular endothelial growth331

factor (VEGF) function.34 A role in DKD pathology for VEGFA signalling between332

GEnCs and podocytes has been proposed.35 In addition, miR-126 repression of333

vascular cell adhesion molecule 1 expression in human umbilical vein endothelial334

cells regulates their response to pro-inflammatory adhesionmolecules.36MiR-126335

has also been implicated in the heterogenic inflammatory response of renal336

microvascularendothelialcells.37337

Increased expression of miR-155 has been observed in DKD patient renal338

biopsies, in close correlationwith increased serumcreatinine.38 Furthermore,miR-339

155deficiencyattenuatedrenaldamageandIL-17expressionwasdownregulatedin340

18

streptozotocin-induced DKD mice.39 Together with miR-126, miR-155 has been341

implicated in multiple forms of vascular remodelling and associated with342

cardiovasculardisease.40343

DecreasedmiR-29bhasbeenreportedinearlyandadvancedanimalmodels344

ofdiabeticrenalfibrosis.41ChenandcolleaguesfoundthatlossofrenalmiR-29bin345

db/db mice led to increased albuminuria, TGF-β-mediated fibrosis and immune346

injury,whilerestoredmiR-29bexpressioninhibitedrenalinjury.42Indeed,whilewe347

have focused on upregulatedmiRNAs in this study,we acknowledge the potential348

importanceofmiRNAdownregulationsthatwedetected.349

In the present studywe localizedmiR-29b to the glomerular endothelium.350

ReductionofcollagenandlamininsynthesishasbeenreportedfollowingforcedmiR-351

29bexpressioninhumancornealendothelialcells.43InapoEknockoutmice,miR-29b352

inducedaorticendothelialpermeability in response toahigh fatdiet,andbrought353

aboutaorticapoptosisbydirecttargetingofmelatoninreceptormt1.44 Inaddition,354

upregulated miR-29b expression has been observed in human umbilical vein355

endothelialcellsexposedtohyperglycaemia.45356

The cytokine-driven release fromGEnCs observed formiR-126 (TNF-α) and357

miR-29b (TGF-β1) reported here suggests that these cells may be the principal358

sourceofelevatedurinarymiR-126andmiR-29bdetectedinDKD.Wespeculatethat359

thisconstitutesevidencefordisease-relatedsignallingdownthenephronthatwillbe360

interesting to test in future studies. Indeed,we have demonstrated association of361

urinary miRNAs with exosomes10 and exosomal transport, which might facilitate362

passageofmiRNAsthroughthenephron,hasbeenreportedforallthreecandidate363

biomarkermiRNAs.364

19

Exosome-mediated release of miR-126 from CD34+ peripheral blood365

mononuclear cells is proangiogenic, and decreased miR-126 was detected in366

elevatedglucosecellcultureanddiabeticpatients.46MiR-155isdepletedinurinary367

exosomes frommicroalbuminuric type 1 diabetesmellitus patients.47 Endogenous368

miR-29b, spontaneously released frombeta-cellswithinexosomes, stimulatesTNF-369

αsecretionfromspleencellsisolatedfromdiabetes-proneNODmiceinvitro.48370

Insummary,wehaveusedunbiasedprofilingapproachestoidentifyaurinary371

miRNAsignatureassociatedwithDKD,andhavesubsequentlyconfirmed increased372

miR-126,miR-155andmiR-29binanindependentpatientcohort.MiR-126andmiR-373

29bwereidentifiedasenrichedinGEnCs,andreleasedfromthesecellsinresponse374

to DKD-related cytokines. Urinary miR-126, miR-155 and miR-29b are therefore375

promising DKD biomarkers, and the potential pathological significance ofmiR-126376

andmiR-29breleasefromGEnCsmeritsfurtherevaluation.377

378

20

Acknowledgments379

Wethankcontrolsubjectsandpatientsforthedonationofurinesamplesincluding380

those samples kindly provided by co-authorsDrMarkD. Jesky and Professor Paul381

Cockwell(QueenElizabethHospitalBirmingham,Birmingham,UK).382

C.B.andK.S.performed theexperimentalwork,generatedandanalyzeddata,and383

helped draft the manuscript. A.W., C.C., L.N., R.J. and T.A. performed the384

experimentalwork,generatedandanalyzeddata.M.J.,P.H.,C.D.,S.S.,P.CorandP.C.385

discussedelementsofexperimentaldesignand/orcohortcomposition.D.F.andT.B.386

designed the research. T.B. wrote themanuscript, which was edited by D.F. then387

amendedandapprovedbyeachauthor.388

389

21

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28

Figurelegends555

Figure 1UrinarymiRNAdetection inurine samples fromDKDpatientsandcontrol556

subjects.A:Volcanoplotshowingthedetectionprofileofthe377urinarymiRNAsin557

TLDACardAinDKDpatients(n=20;fourpoolsoffivepatients)andcontrols(n=20;558

fourpoolsoffivecontrols).Thedottedhorizontallinerepresentsapvalueboundary559

of 0.05. B: Fold change of miRNA detection between DKD patients and controls.560

DataAssist Software (Thermo Fisher Scientific) was used to perform relative561

quantificationforsamplecomparison,toperformt-testsamplegroupcomparisons,562

andtoproducethegraphicoutputshown. (C-E)RT-qPCRanalysisshowssignificant563

differences in detection of (C) miR-126, (D) miR-155 and (E) miR-29b between564

patients and control urine in the component urine samples pooled for profiling565

analyses(AandB).DKDpatientsversuscontrolsforC:miR-126,D:miR-155,E:miR-566

29b(n=20foreachgroup).Analysiswascarriedoutbyunpaired2-tailedt-testwith567

Welch’scorrection.ProfilingdataanalysisusingtheNormFinderalgorithmidentified568

miR-191asoptimalfornormalisationofourRT-qPCRdata.Datawerenormalizedto569

endogenouscontrolmiR-191andarepresentedasmean+/-SEM.**P<0.01,***P<570

0.005and*****P<0.0005.571

572

Figure 2 RT-qPCR detection of selected miRNAs in patients and control subjects.573

Relativeexpressionwassignificantlydifferentin89DKDpatientscomparedwith62574

diabeticpatientswithoutDKDand41controlsfor(A)miR-126and(B)miR-155,and575

significantly different in DKD patients compared with controls for (C) miR-29b. A:576

DKDpatientsversusdiabeticpatientswithoutDKDandcontrols;B:DKDpatientsand577

diabeticpatientswithoutDKD;DKDpatientsversuscontrols;C:DKDpatientsversus578

29

controls (n = 89 DKD patients, 62 diabetic patients without DKD, 41 controls).579

Analysis was carried out by unpaired 1-tailed t-test withWelch’s correction. Data580

were normalized to endogenous control miR-191 and are presented as mean +/-581

SEM.(D)CombinedreceiveroperatingcharacteristiccurveanalysisformiR-126,miR-582

155andmiR-29b,areaunderthecurve(AUC)=0.80.Dataweregeneratedusingthe583

pROCpackageinR-3.2.3.*P<0.05,****P<0.001and******P<0.0001.584

585

Figure 3 Localization ofmiRNA expression by laser capturemicrodissection (LCM)586

andcellculture.A:Keyfunctionalnephrondomainsincludetheglomerulus(G),the587

proximaltubule(PT)andthedistaltubule(DT).B:ACD10-stainedFFPErenalbiopsy588

samplebeforeandafterexcisionofglomerulibyLCM.Bars=100µm.(C-E)Relative589

expressionofmiR-126,miR-155andmiR-29b, respectively, inLCM-isolatedGs,PTs590

andDTsfrom5renalbiopsiesofhealthyindividuals.C:GversusPT;GversusDT(n=591

5biopsies).(F-H)RelativeexpressionofmiR-126,miR-155andmiR-29b,respectively,592

in in vitro cultured HK-2 renal proximal tubular epithelial cells (PTCs), fibroblasts,593

podocytes and conditionally immortalized glomerular endothelial cells (GEnCs). F:594

GEnCsversusfibroblasts;GEnCsversusPTCsandpodocytes;G:PTCversusGEnCs;H:595

GEnCsversuspodocytes(n=4).Analysiswascarriedoutbyone-wayANOVAanalysis596

withTukey’smultiplecomparisontest.Datawerenormalizedtoendogenouscontrol597

miR-191 and are presented asmean +/- SEM. *P < 0.05, **P < 0.01 and ****P <598

0.001.599

600

601

30

Figure4MiRNAexpressioninGenCsandGenCconditionedmediuminresponseto602

hyperglycaemia and DKD-related cytokines. Following 24 h culture in 5mMor 25603

mM D-glucose, relative expression in GEnCs and GEnC conditioned medium,604

respectively,of(AandB)miR-126inresponseto10ng/mlTNF-α,(CandD)miR-29b605

inresponseto1ng/mlTGF-β1and(EandF)miR-155inresponseto10ng/mlTNF-α,606

andinuntreatedcells.B:5mMD-glucoseversus5mMD-glucoseplusTNF-α,and25607

mMD-glucoseversus25mMD-glucoseplusTNF- α;C:5mMD-glucoseversus5mM608

D-glucoseplusTGF-β1,and25mMD-glucoseversus25mMD-glucoseplusTGF-β1;609

D:5mMD-glucoseversus5mMD-glucoseplusTGF-β1;25mMD-glucoseversus25610

mM D-glucose plus TGF-β1 (n = 4). Analysis was carried out by one-way ANOVA611

analysis with Tukey’s multiple comparison test. Data were normalized to612

endogenouscontrolmiR-191andarepresentedasmean+/-SEM.*P<0.05and**P613

<0.01.614

615

616

617

618

619

620

621

622

623

624

625

31

Table 1Demographicandclinicalparametersofpatients recruited from2centres:626

Wales Kidney Research Tissue Bank, Cardiff (University Hospital Wales) and627

Birmingham (UniversityHospital Birmingham, Renal Impairment in Secondary care628

(RIISC)studycohort)(n=151)629

630

Feature

Patients(n=151)

Diabetic(n=62)

DKD(n=89)

Controls(n=41)

Malen(%) 37(58) 55(62) 18(44)Non-Caucasiann(%) 13(21) 33(37) MeanAge(years,SD) 52+/-16.1 62+/-13.6 55+/-15.4 eGFRml/min/1.73m2 Mean(SD) 78+/-16.3 30+/-20.9 Median(IQR) 84(72-90) 22(17-38) CKDstagen(%) NoCKD/CKDG1(eGFR≥90) 23(37) 2(2) CKDG2(eGFR=60-89) 32(52) 10(11) CKDG3(eGFR=30-59) 5(8) 17(19) CKDG4(eGFR=15-29) 2(3) 45(51) CKDG5(eGFR<15) 0 15(17) ACR*mg/mmoln(%) A1-Normal-highnormal(ACR<3) 54(87.1) 15(16.9) A2-Moderatelyincreased(ACR3-30) 8(12.9) 25(28.1) A3-Severelyincreased(ACR>30) 0(0) 49(55.0) *Albumin:Creatinine ratio (ACR) group cut-offs and nomenclature derived from631

KDIGO2012recommendations.632

633

634

635

32

Table2Specificityvalues,likelihoodratiosandRQthresholdsformiR-126,miR-155,636

miR-29bandallthreemiRNAsabovean80%ROCcurvesensitivitythreshold637

638

Sensitivity(%)

Specificity(%)

LikelihoodRatio

RQThreshold

3miRNAs 80.21 63.64 2.206 >1.148

miR-126 80.41 57.14 1.876 >0.6762

miR-155 80.61 52.00 1.679 >0.9110

miR-29b 80.61 40.00 1.344 >0.8058639

Table3DKDanddiabeticpatientswithoutDKD(D)patientnumbersandpercentages640

abovean80%ROCcurvesensitivitythresholdformiR-126,miR-155,miR-29bandall641

threemiRNAs642

643 Patientsabove80%sensitivitythreshold

miR-126miR-155miR-29b3miRNAsTotal

D 23 30 13 2 55

DKD 80 67 73 47 98

Percentageofpatientsabove80%sensitivitythresholdmiR-126miR-155miR-29b3miRNAs

D 41.8 54.6 23.6 3.6

DKD 81.6 68.4 74.5 48.0

644

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GlomerularcapsuleDistaltubule

Proximaltubule

Collec>ngduct

CORTEX

MEDULLA

A B

C D

**

E F

**

***

Figure4

CulturedGEnCs GEnCcondi7onedmedium

Rela7v

eexpression

miR-126

Rela7v

eexpression

miR-126

Rela7v

eexpression

miR-29b

Rela7v

eexpression

miR-29b

Rela7v

eexpression

miR-155

Rela7v

eexpression

miR-155

5mM 5mM+TNF-α

25mM 25mM+TNF-α

5mM 5mM+TNF-α

25mM 25mM+TNF-α

5mM 5mM+TNF-α

25mM 25mM+TNF-α

5mM 5mM+TNF-α

25mM 25mM+TNF-α

5mM 5mM+TGF-β1

25mM 25mM+TGF-β1

5mM 5mM+TGF-β1

25mM 25mM+TGF-β1