Cystatin-C and TGF-β levels in patients with diabetic nephropathy

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w w w . r e v i s t a n e f r o l o g i a . c o m

RevistadelaSociedadEspañoladeNefrología

Original

article

Cystatin-C

and

TGF-

␤

levels

in

patients

with

diabetic

nephropathy

Mumtaz

Takir

a,∗

_,

_Aslı

_Dogruk

_Unal

b

_,

_Osman

_Kostek

c

_,

_Nilufer

_Bayraktar

b

_,

Nilgun

Guvener

Demirag

b

a_Division_of_{Endocrinology}_and_Metabolism,_Department_of_Internal_Medicine,_Goztepe_Training_and_Research_Hospital,_Istanbul

MedeniyetUniversity,Istanbul,Turkey

b_Division_of_{Endocrinology}_and_Metabolism,_Department_of_Internal_Medicine,_Bas¸kent_University_{Hospital, ˙Istanbul,}_Turkey c_Department_of_Internal_Medicine,_Goztepe_Training_and_Research_Hospital,_Istanbul_Medeniyet_University,_Istanbul,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10September2015

Accepted25June2016

Availableonline13October2016

Keywords: Diabeticnephropathy CystatinC TGF-␤1 Microalbuminuria Normoalbuminuria

a

b

s

t

r

a

c

t

Objective:Toevaluaterenalimpairmentintype2diabeticpatientswithnormoalbuminuria

ormicroalbuminuriabydetectionofserumcystatinCandserumandurinaryTGF-␤levels.

Methods:Cross-sectionalstudyconductedattheDepartmentofEndocrinologyinBaskent

UniversitySchoolofMedicine.Patientswithtype2diabetesmellituswithoutknownovert

diabetic nephropathywereincludedinthestudy.Recruitedpatientswerestratiﬁedinto

fourgroups,matchedintermsofage,gender,microalbuminurialevelandestimatedGFR

calculatedwithMDRD.

Results:78patientswereenrolled.Theywerecategorizedintofourgroupsdependingon

theirurinaryalbuminexcretionandestimatedglomerularﬁltrationrate.

Macrovascularcomplicationwasfoundtobehigherinpatientswithmicroalbuminuria

thaninotherpatients(p<0.01),buttherewerenodifferencesintermsofotherdiabetic

complications.SerumcystatinClevelwassigniﬁcantlyhigherinnormoalbuminuricgroup

onepatients,whileserumandurinaryTGF-␤1levelswerehigherinmicroalbuminuricgroup

twopatients.TheserumlevelofcystatinCwasfoundtonegativelycorrelatewitheGFRin

grouptwopatients(r=−0.892,p<0.001).Finally,therewasanegativecorrelationbetween

eGFRandcystatinCinallthepatientgroups(r=−0.726,p=0.001).

Conclusions: Althoughurinaryalbuminexcretionisrecommendedforthedetectionoftype

twodiabeticnephropathy,thereisagroupofpatientswithdecreasedeGFRbutwithout

increasedurinaryalbuminexcretion,inwhichserumcystatinClevelwasindicatedtobe

usedasanearlybiomarkerofdiabeticnephropathy.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.

org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author.

E-mailaddress:mumtaztakir@yahoo.com(M.Takir).

http://dx.doi.org/10.1016/j.nefro.2016.06.011

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Concentración

de

cistatina

C

y

TGF-

␤

en

pacientes

con

nefropatía

diabética

Palabrasclave: Nefropatíadiabética CistatinaC TGF-␤1 Microalbuminuria Normoalbuminuria

r

e

s

u

m

e

n

Objetivo:Evaluarlainsuﬁcienciarenalenpacientescondiabetestipo2con

normoalbumin-uriaomicroalbuminuriamedianteladeteccióndelaconcentracióndecistatinaCensuero

ydeTGF-␤ensueroyorina.

Métodos:EstudiotransversalrealizadoenelDepartamentodeEndocrinologíadelaFacultad

deMedicinadelaUniversidaddeBaskent.Enelestudio,seincluyóapacientescondiabetes

mellitustipo2sinnefropatíadiabéticamaniﬁestaconocida.Lospacientesseleccionadosse

estratiﬁcaronen4grupos,agrupadosentérminosdeedad,sexo,gradodemicroalbuminuria

yﬁltraciónglomerularestimada(FGe)calculadamediantelafórmulaMDRD.

Resultados:Seincluyóa78pacientes.Seclasiﬁcaronen4gruposdependiendodelaexcreción

urinariadealbúminaydelaFGe.

Seobservóquelacomplicaciónmacrovasculareramayorenlospacientescon

microalbu-minuriaqueenotros(p<0,01),peronohubodiferenciasconrelaciónaotrascomplicaciones

diabéticas. Laconcentración sérica de cistatinaC fuesigniﬁcativamentemayor enlos

pacientesdelgrupo1connormoalbuminuria,mientrasquelasconcentracionesdeTGF-␤1

ensueroyorinafueronmayoresenlospacientesdelgrupo2conmicroalbuminuria.Se

observóunacorrelaciónnegativaentrelaconcentraciónséricadecistatinaCylaFGeenlos

pacientesdelgrupo2(r=−0,892,p<0,001).Porúltimo,seobservóunacorrelaciónnegativa

entrelaFGeylacistatinaCentodoslosgruposdepacientes(r=−0,726,p=0,001).

Conclusiones:Aunqueserecomiendalaexcreciónurinariadealbúminaparaladetección

delanefropatíadiabéticatipo2,hayungrupodepacientescondisminucióndelaFGe,

perosinaumentodelaexcreciónurinariadealbúmina,enlosqueestabaindicadousar

laconcentracióndecistatinaCensuerocomounbiomarcadortempranodenefropatía

diabética.

Nefrolog´ıa.Esteesunart´ıculoOpenAccessbajolalicenciaCCBY-NC-ND(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Diabetic nephropathy(DN) is the leading cause of chronic

kidneydisease.1,2_Albumin_excretion_rate_(AER)_and

glomeru-larfiltrationrate(GFR)areusedtostageDN.However,these measurementsarenotsufficientinearlydiagnosisand mon-itoringtheprogressionofDN.Thereforeearlymarkersshould bedevisedfor thesepurposes. Althoughmicroalbuminuria is accepted as a risk factor and marker for DN and pro-gressiverenalinsufficiency,3_some_patients,_especially_type₂ diabetics, may have normoalbuminuric chronic kidney disease.4

Thisshowsthatbothnormoalbuminuricandalbuminuric pathwaysareatwork.5_While_some_patients_with_MA_present withnormalkidneystructure,someothernormoalbuminuric diabeticshavewell-determinednephropathiclesion.6,7

Cystatin C which is used for measurement of GFR, is producedinall nucleatedcells and ﬁlteredfreelyfrom the glomerulus. Itis reabsorbed totally and catabolised in the proximaltubules.CystatinCisasuitablemarkerforGFR mea-surementowingtonotbeingaffectedbyage,weight,gender andproteinintake.8–10

Transforming growth factor-beta (TGF-␤1) is one of the growth factors which are implicated in DN pathogenesis. TGF-␤1causesmesangialextensionbypromotingrenal cel-lular hypertrophy and byinducingthe extracellularmatrix

expansion.11_Urinary_TGF-␤1_levels_are_increased_as propor-tionaltotheseverityofthenephropathy.12

WeaimedtodeterminethevalueofserumCystatinCand TGF-␤1inearlydetectionofdiabeticnephropathyin normoal-buminuricpatientswithtype-2diabetesmellitus.

Material

and

methods

Patients

Thiswasacross-sectionalstudywhichwasconductedatthe

Department ofEndocrinology inBaskent University School

ofMedicine,Istanbul,Turkey,during2009–2011,whoagreed

toparticipateinthestudy.BaskentUniversityEthical

Com-mitteeapprovedthestudyprotocol(Approvalno:KA09/308).

Patientswithtype2diabetesmellituswithoutknownovert

diabetic nephropathywere includedinthestudy.Recruited

patientswerestratiﬁedintofourgroups,matchedintermsof

age,gender,microalbuminurialevelandestimatedGFR

cal-culatedwithMDRD.Wetriedtoexcludeapparentcausesof

nondiabetic renaldisease basedondataobtainedwith

uri-nary system ultrasonography, urine microscopy and urine

culture.Patientswhohavehistoryofnondiabeticrenal

dis-ease, uncontrolled hypertension, liverdisease,NYHA stage

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wereexcludedfromthestudy.Allparticipantswereincluded inthestudyaftersigninginformedconsentforms.

Laboratoryevaluation

Following thorough medical history and detailed physical examination,bloodsamplesweretakenfromallparticipants formeasurementoffastingbloodglucose,HbA1c,bloodurea, creatinineandelectrolytes,lipidproﬁle,serumalbumin,high sensitive C reactive protein (hsCRP). Freshly voided early morning urine samples were also collected for urinalysis. Patientsalsoprovided24-hcollectedurinespecimensfor mea-surementofmicroalbumin.

Serumglucose,creatinine,totalcholesterol,HDLandLDL cholesterol, triglyceride were studied by enzymatic colori-metric;serumalbumin;bromocresolgreen(BCG),ure;(BUN) urease, HbA1c; immune-examination (C4000, Abbott,USA), andhsCRPwasstudiedwithimmunoturbidimetricmethods (C8000,Abbott,USA). Microalbuminlevelin24-hurinewas determined withimmunoturbidimetricmethod (C4000 sys-tem,Abbott,USA).

EstimatedGFRwascalculatedwith6variableMDRD equa-tion using age, race, gender, serum creatinine,blood urea nitrogenandalbuminvalues.13

Theserums ofpatientswere stored in−20◦_C _until

lab-oratoryanalysis.SerumcystathionineC,serumTGF-␤1and urine TGF-␤1 levels were determined with enzyme linked immunosorbent assay (ELISA) method. BioVendor (Czech Republic)ELISA kit and eBioscience (North America) ELISA wereusedforCystatinCandTGF-␤1measurements, respec-tively.

Statisticalanalysis

For statisticalevaluation, NCSS(Number Cruncher Statisti-calSystem)2007andPASS(PowerAnalysisandSampleSize) 2008Statistical Software(Utah, USA) programs were used. Oneway Anova testwas used in intergroupcomparison of theparametersshowingnormaldistributionandTukeyHDS testwasusedinthedeterminationofthegroupcausingthe difference.Kruskal–Wallistestwasusedinintergroup com-parisonoftheparametersnotshowingnormaldistribution andMann–WhitneyUtestwasusedinthegroupcausingthe difference.Incomparisonofthequalitativedata,Chi-square testwasused.Thesigniﬁcancewasacceptedasp<0.05.

Results

Atotalof78patientswithtype2DMwereincludedinthe

study(meanage57±8.Fourgroupsweredescribed;patients

with GFR value<60ml/min/1.73m2 _and _MA _<30_mg/day _as

group1;patientswithGFRvalue<60ml/min/1.73m2_and_MA

between30mgand300mg/dayasgroup2;patientswithGFR

value>120ml/min/1.73m2_and_MA_<30_mg/day_as_group_3,_and

patientswithGFRvaluebetween90and120ml/min/1.73m2

andMA<30mg/dayasgroup4.Therewere 10menand10

womeningroup1and4whereas10menand11womenin

group2and15menand2womeningroup3.Thelaboratory

parametersofthe groupsare showninTable 1. Signiﬁcant

eGFR (mL/min/1.73 m2) 120 80 40 120 80 40 120 80 40 Cystatin C (mg/dL) 3000 2500 2000 1500 1000 500 0 120 80 40 Group 4 Group 3 Group 2 Group 1 r=–0.236 r=–0.047 r=–0.892 r=–0.211 P=.372 P<.001 P=.857 P=.317

Fig.1– CorrelationofserumcystatinCwitheGFRinallfour studygroups.

differencewaspresentamonggroupswithregardstomean

age,fastingbloodglucose,HbA1candserumcreatininelevel.

Nosigniﬁcantdifferencewasfoundamonggroupsinterms

ofthepresenceofHTwhichhasamajorroleinDN

patho-genesis.Theevaluationofthegroupsintermsofmicroand

macrovascularcomplicationsofdiabetesisdepictedinTable2.

Highestrateofcoronaryarterydisease(CAD)wasseenGroups 1and 2.Nosigniﬁcancedifferencewas evidentamongthe groupsregardingprevalenceofretinopathy,diabeticfootand neuropathy(p>0.05).

ResultsofGFR, cystatinC,serum andurineTGF-␤1,MA measurements are shownin Table3. Significant difference waspresentamongthegroupsintermsofcystatinC,serum andurineTGF-␤1(p<0.05).PostHocTukeyHSDtestwhichwas performedtodeterminetheintragroupsignificance,showed thatcystatinClevelofgroups1and2wassignificantlyhigher comparedwiththoseofgroups3and4(forGroup1;p=0.001,

p=0.001; forGroup2;p=0.002, p=0.01).For the serum and urineTGF-␤1,wefoundthatthelevelsingroup2were signif-icantlyhigherthangroup4(p=0.012;p=0.012).Nosigniﬁcant associationwasdeterminedbetweenTGF-␤1andanyofthe studiedvariables.

Cystatin Cshowed asigniﬁcant andpositive correlation withage,durationofdiabetes,andserumuricacidingroup 2(forage:r=0.477,p=0.029;fordurationofdiabetes:r=0.437,

p=0.048;forserumuricacid:r=0.465,p=0.039).Asexpected, serum cystatinClevel wasnegativelyassociatedwith esti-matedGFRlevel(r=−0.892,p=0.001)onlyingroup2(Fig.1). Ingroup3,onlyanegativesigniﬁcantcorrelationwaspresent betweenLDLandcystatinC(r=−0.72,p=0.002).Ingroup4, cystatin C had a positive correlation withduration of dia-betes(r=0.453,p=0.045)andanegativecorrelationwithMA (r=−0.520,p=0.019)(Fig.2).

When all thepatients whichare taken tothe studyare evaluated,statisticallysignificantrelationinnegative direc-tion and in % 72.6 level is detected (r=−0.726, p=0.001). SignificantnegativecorrelationisdetectedbetweenGFRand creatinine(r=−0.806,p=0.001).Positivesignificantcorrelation

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Table1–Laboratoryparametersofthestudygroups.

Group1(n=20) Group2(n=21) Group3(n=17) Group4(n=20) p

Age,years 62.9(5.8) 59.7(6.9) 49.0(8.0) 53.7(8.3) 0.001

DurationofDiabetesMellitus,months 116.4(100.1) 127.4(96.7) 96.0(71.8) 78.0(45.1) 0.243

BMI,kg/m2 _29.6_(4.2) _32.0_(5.3) _32.2_(6.6) _31.6_(5.4) _0.430 Glucose,mg/dL 118.8(26.6) 152.1(62.7) 143.9(42.6) 121.1(32.7) 0.043 HbA1c,% 6.1(0.7) 6.9(1.2) 7.3(1.1) 6.7(1.3) 0.028 Creatinine,mg/dL 1.2(0.2) 0.9(0.3) 0.6(0.1) 0.6(0.1) 0.001 Albumin,g/dL 4.2(0.3) 4.2(0.2) 4.3(0.3) 4.3(0.3) 0.559 HDL-Cholesterol,mg/dL 41.2(10.1) 38.4(7.8) 39.8(10.9) 40.5(5.7) 0.774 LDL-Cholesterol,mg/dL 102.6(35.5) 115.3(34.3) 129.6(30.3) 105.9(29.6) 0.079 Triglyceride,mg/dL 148.3(91.1) 174.8(79.9) 175.9(50.9) 184.6(68.1) 0.074

Sedimentationrate,mm/hour 22.3(14.8) 15.1(10.8) 13.4(5.2) 14.2(8.0) 0.412

hsCRP,mg/dL 5.1(4.5) 6.0(7.7) 5.5(5.5) 5.6(5.2) 0.969

Bodymassindex(BMI),hemoglobinA1c(HbA1c),highdensitylipoprotein(HDL),lowdensitylipoprotein(LDL),highsensitivecreactiveprotein (hsCRP).

Mean(standarddeviation).

Table2–Evaluationofmicroandmacrovascularcomplicationsaccordingtogroups.

CoronaryArteryDisease 9(45%) 10(47.6%) 1(5.9%) 2(10%) 0.003

Retinopathy 5(25%) 7(33.3%) 2(11.8%) 4(20%) 0.454

DiabeticFoot 0(%0) 1(4.8%) 0(%0) 0(%0) 0.432

Neuropathy 9(45%) 9(42.9%) 2(11.8%) 5(25%) 0.096

isdetectedbetweenage,hypertensionandCADandcystatin

Clevels(forager=0.534,p=0.001;forhypertensionr=0.347,

p=0.002;forCADr=0.382,p=0.001,respectively).

When60ml/min/1.73m2_is_selected_for_GFR_as_the_cut-off

value,itisobservedthatthecystatinClevelsaresigniﬁcantly

differentbetween the patientswho have eGFRvaluesover

andbelowthiscut-offpoint(GFR<60ml/min:1548.6±613.8;

GFR≥60ml/minmean:816.8±310.8;p=0.001).ROCanalysis

forcystatinC,inthecaseswherecystatinCisC≥1064mg/L,

catch-upsensitivityofGFRin<60ml/mwascalculatedas80%,

speciﬁcityas86.21%;positiveconjecturevalueas66.67%and

negativeconjecturevalueiscalculatedas92.59%(Fig.3).The

areaunderthecurveinROCanalysiswasfoundas85.4%and standarderrorwascalculatedas6%.

Discussion

Thealbumin excretionrate (AER)which isused instaging

ofDNand GFR,isnot sufﬁcientforexplainingthe disease

progress.Whilealbuminexcretionthroughurineisnormal,

GFRcandecreasewithoutanyincreaseinAER.Byconsidering

thatDNmayhavenormoalbuminuricstage,11_this_situation_is

reportedintype1diabeticpatientswhoshowtypical speciﬁ-cationsinfavorofDNinrenalbiopsy.11_In_our_study,_when detecting the early stage diabetic nephropathy, cystatin C levelsaredetectedashigherinthegroupswhichhave nor-moalbuminuriceGFR<60ml/m/1.73m2 _and_as_independent

fromeGFRserumandurineTGF-␤1levelisdetectedashigher inmicroalbuminuriagroup.

In AER and GFR relation age is an important factor. GFR istype2diabetics over 70 yearsold,is approximately 60ml/m/1.73m2_._In_our_study,_eGFR_was_the_lowest_and_the

agewastheoldestinnormoalbuminuricgroup(average62±5 years).

Today,GFRisacceptedasthebestindexreﬂectingkidney function.13_GFR_can_be_directly_measured_with_the_infusion_of theexogenousmarkerssuchasinulinor51_Cr-EDTA._However

thesemethodsarenotpracticalandnotusedindailypractice. Therefore, for the sake of practicality, American Diabetes Association (ADA)andNational KidneyFoundation suggest theformulaofCockcroft-Gault(CG)andModiﬁcationofDiet inRenalDiseasefour-variable(MDRD)equationstoestimate

Table3–EstimatedGFR,cystatinC,serumandurineTGF-␤1,andMAlevelsinstudygroups.

eGFR,mL/m/1.73m2 _54.7_(7.6) _78.2_(23.5) _125.7_(6.9) _106.1_(29.8) _0.001

CystatinC,mg/L 1.4(0.6) 1.1(0.4) 0.6(0.1) 0.7(0.1) 0.001

sTGF-␤1,ng/mL 16.7(6.9) 18.9(3.0) 16.1(6.1) 13.2(6.4) 0.022

uTGF-␤1,pg/mgcre 558.6(23.0) 633.2(100.6) 539.3(204.3) 442.2(214.3) 0.022

MA,mg/24h 18.0(5.9) 89.8(37.7) 22.2(8.9) 16.3(5.1) 0.001

Estimatedglomerularﬁltrationrate(eGFR),serumtransforminggrowthfactor-beta1(sTGF-␤1),urinetransforminggrowthfactor-beta1 (uTGF-␤1),microalbuminuria(MA).

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Cystatin C (mg/dL) 3000 2500 2000 1500 1000 500 0 Microalbuminuria (mg/24 hour) 200 150 100 50 0 Group 4 Group 3 Group 2 Group 1 Group 4 Group 3 Group 2 Group 1

Fig.2–CorrelationofserumcystatinCwith microalbuminuriainallfourstudygroups.

GFR.14,15_Particularly_MDRD_equation_is_accepted_as_superior

thanCGowingtonotdependonbodyweightmeasurement.13 Serumcreatinineconcentrationisanimperfectmeasure ofGFR.Inourstudy,thecreatininelevelinthepatientgroups whoseGFRwas<60ml/m/1.73m2_,_has_not_increased_yet_and

thepatientswerenormoalbuminuric.Sinceserumcreatinine levelisaffected bymusclemassanddiet,itisnotanideal markerforGFR calculation. Novel methodsare needed for earlyandaccuratedetectionofDN.

CystatinCisanendogenicmarkerusedforGFR determina-tionbecauseitsserumlevelisdependenttoalmostwholeof eGFRparticularlyitisseenmorevaluableinnormalandmild decreasedeGFR.8 1-specificity 1.0 0.8 0.6 0.4 0.2 0.0 Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 ROC Curve AUC=85.4 % SE=6.0 %

Fig.3–ROCcurvewhichisobtainedforcystatinC.

Laterzaetal.5_have_attributed_cystatin_C_to_be_an_important

reagent inearlystagekidneydamagetoitsstabile produc-tion,musclemass, notbeing dependenttoageandgender ornotshowingreversetendencytokidneysecretionorblood circulation.

Inourstudy,thedifferenceincystatinClevelsshowedthat eveninnormoalbuminuriastage,thediabeticrenal disease canbeidentiﬁed.

In earlydiabetic kidney disease,thereare contradictory resultsrelatedtotheroleofcystatinC.Aspursuanttoour study,two studiesshow that cystatinCisnot more sensi-tivethancreatinine.16,17_Contrary_to_our_results,_in_the_study wherecystatinCbasedequationsandcreatininebased equa-tions like Schwartz,MDRD and CGformulasare compared and inameta-analysiswhere46studies included,cystatin C is considered superior than creatinine in determination ofdecreasedGFR.18,19 _Although_a_negative_weak_relation_is detectedincystatinCandeGFRinourstudy,itwasnot statis-ticallysigniﬁcant.Wethinkthatthissituationmightbedueto thelimitednumberofcasesinthepatientgroup.Inthestudy whereMussapandcolleagueshaveexamined52type2 dia-beticpatients,20_eGFR_has_decreased_from₁₂₀_ml/m/1.73_m2_to

20ml/m/1.73m2_and_cystatin_C_has_increased_more_than_the

serumcreatininehas.TheassociationbetweenCystatinCand eGFRwasdetectedstrongerthantheserumcreatinine.

Inthestudy ofSurendaretal.inIndianswhohave glu-coseintolerance,itwasdeterminedthatcystatinClevelshave increasedascomparative withglucoseintolerance and the highestcystatinClevelswerefoundinthegroupwhichhad microalbuminuriaandretinopathy.21_In_our_study,_no differ-encewasdetectedintermsofretinopathyamongthegroups andcystatinClevelswerefoundhighestinthe normoalbu-minuricgroup.

Inthepreviousstudy,ROCanalysismadeforidentifying thediagnosticproﬁleofserumcystatinCinpredictingeGFR <60ml/dk/1.73m2_in_the_type₂_diabetic_patients_and_as_similar

toourstudy,cut-offvalueisdetectedas1.06mg/dl,the sen-sitivityofserumcystatinClevelis81%andthespeciﬁcityis detectedas.22_As_pursuant_to_our_study,_serum_cystatin_C_level ofthenormoalbuminuricisdetectedassigniﬁcantlyhigher thanthepatientswithGFRof≤60ml/dk/1.73m2_._This_makes

usthinkthatcystatinClevelsofserumandurinearerelated with the subclinicalfailure and may bethe kidney uptake reagentswhicharemeasurableintheperiodearlierthanthe onsetofalbuminuria.InthestudyofYangandfriends,serum cystatinClevelshowedcorrelationwithMAlevel.23

WhenweconsidertherelationbetweenserumcystatinC andserumcreatininelevelsandMDRDinwholestudy pop-ulation wedetermined that the predictiveabilityof serum creatinineforGFRwashigher.Thisresult,whichisdiscordant withthemajorityoftheliterature,canbeconnectedtothe insufﬁciencyofthecasesinpatientgroups,selectingthecase fromthe patientswho haveearlyperiodDNandformation of25%ofthetotalpatientpopulationbymicroalbuminuric patientgroup.

Inthemicroalbuminuricpatientgroupandinthepatient groupwithGFR<60ml/m/1.73m2_or_>60_ml/m/1.73_m2_,

nega-tiveandsigniﬁcantassociationwasfound.Thispatientgroup wasthe solepatient groupwithMA.Thisgroupwasmade upfromolderpatientsthannormoalbuminuricandtwoother

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patientgroupswithGFR>90ml/m/1.73m2_._It_is_known_that

theserumcystatinClevelincreaseswithadvancingage.The presenceofsigniﬁcantdifferenceamongthepatientgroupsin termsofage,canbeseenasanotherfactorwhichlimitsour study.

Inbothstudies,highserumcystatinClevelisassociated withtheprevious coronaryheartdisease.24,25 _As_similar _to thesestudies,wehavefoundpositivesigniﬁcantcorrelation between CAD and serum cystatin C in the whole patient cohort.

OneofthecytokineswhichisresponsibleforDNinitiation andprogressionisTGF-␤.ItisproventhatTGF-␤intervenesto allpathologicalchangeswhichdependonthediabetickidney disease.26 _TGF-_␤_causes _kidney_cell_hypertrophy _and exces-siveextracellularmatrixproductionininterstitialﬁbroblasts, glomerularandtubularcells.

In experimental DM, glomerular and tubule-interstitial compartments, expresses TGF-␤ or TGF-␤ type II receptor. Althoughthekidneyofthenon-diabeticsubjectremoves TGF-␤1fromtheblood,kidneyofadiabeticpatientreleasesTGF-␤1 proteinintothecirculation.TGF-␤1levelwhichhasincreased inurineisassociatedwithadverseclinicaloutcomes.27_In₃ patientgroupswhichdonothaveMAinourstudy,wehave foundtheserum andurineTGF-␤1levelswere signiﬁcantly higherinthegroupswithMA.

Increased kidney TGF-␤1 production in the diabetic patients was examined and renal vein TGF-␤1 concentra-tionwasfoundpositiveindiabeticpatientsandnegativein non-diabetic patients.27 _Urine _TGF-_␤1 _level _in _the _diabetic patientsincreasedfourfoldthanthenon-diabeticpatientsand ascontrarytoourstudy,increasedurineTGF-␤1excretionwas presentinalldiabeticpatientsirrespectiveofMAstatus.

Therearesomelimitationsofourstudythatneedtobe mentioned.The mostimportant limitation ofour study is theestimation ofGFRmeasurements. Allestimates ofGFR basedonserumcreatininewillbeaffectedfromphysiologic and/orpathologiclimitations28_and_be_far_from_ideal_GFR._On theotherhand,recentstudiesshowedtheCKD-EPIcreatinine equationhassimilarGFRpredictionindifferentpatient pop-ulationcomparedwiththeMDRDStudyequation,eveninthe earlierCKDstages.29,30 _In_addition,_the _CKD-EPI creatinine-cystatinCequationismoresuitablecomparedwithstandard reference for cystatin C and creatinine based equations.31 However, the cost effectivenessof the CKD-EPI creatinine-cystatinCequationforclinicaluseshouldbeconsidered.The secondimportantlimitationisthesamplesizeofthestudy groups.Butwewantedtobesureaboutpatientswithtype2 diabetesmellituswithoutknownovertdiabeticnephropathy. Largernewclinicaltrialsarerecommendedtodeterminethe roleofserumcystatinC,serumandurineTGF-␤1levelasan earlybiomarkerofdiabeticnephropathy.Thethird,diabetic footulcerwithorwithoutconcomitantperipheralarterial dis-easeis considered astwo separatedisease.32 _In_our _study, wedidnotexamthepatientswhethertheyhaveperipheral vasculardisease.

Conclusion

Althoughurinaryalbuminexcretionissuggestedfordetection

oftype2diabeticnephropathy,thereisgroupofpatientswith

decreasedeGFRbutwithoutincreasedurinaryalbumin

excre-tion,inwhichserumcystatinClevelwasindicatedtobeused

asanearly biomarkerofdiabetic nephropathy.In addition,

despitethatserumandurineTGF-␤1levelswerealso

signiﬁ-cantlyhigherinpatientswithMA,onlyincreasedurineTGF-␤1

excretionwaspresentinalldiabeticpatientsirrespectiveof

MAstatus.

Conﬂict

of

interest

Theauthorsdeclarenoconﬂictofinterest.

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