Research Article Assessment of Urinary Osteopontin...

Research ArticleAssessment of Urinary Osteopontin in Associationwith Podocyte for Early Predication of Nephropathy inDiabetic Patients

Abdulrahman L Al-Malki

Biochemistry Department Faculty of Science King Abdulaziz University PO Box 80203 Jeddah 21589 Saudi Arabia

Correspondence should be addressed to Abdulrahman L Al-Malki alalmalkikauedusa

Received 9 February 2014 Revised 15 April 2014 Accepted 16 April 2014 Published 4 May 2014

Academic Editor Vincent Sapin

Copyright copy 2014 Abdulrahman L Al-Malki This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objectives Microalbuminuria has been clinically used for noninvasive evaluation of renal dysfunctions However it is a nonspecificmarker of diabetic nephropathy (DN)MethodsThis studywas conducted fromMarch 2012 toApril 2013 at BiochemistryUnit KingFahd Medical Research Center (KFMRC) In this study urinary osteopontin podocytes number and levels of immunoglobulin M(IgM)were determined in 60 patients (diabetic normoalbuminuria diabeticmicroalbuminuria andnephritic syndrome) comparedwith healthy subjects Results It was found that in diabetic microalbuminuria patients have a highly significant increase in urinaryIgM osteopontin and podocyte levels as compared to other groups Nephrotic syndrome patients showed a moderate significantelevation of these parameters compared to control subjects At a given specificity of 97 podocytes yielded the highest sensitivityof all markers 955 The sensitivity was considerably higher compared to IgM and osteopontin Podocyte number was positivelycorrelated with serum IgM and osteopontin (119903 = 063 and 056) respectively Its cutoff corresponding to the 10 coefficient ofvariation was most appropriate for early diagnosis of DN Conclusion Monitoring urinary podocyte may provide a noninvasivetool that is a sensitive accurate and specific biomarker of glomerular injury and can be used in combination with osteopontin andIgM to more reliably detect and monitor prognosis

1 Introduction

Diabetesmellitus (DM) is considered one of themost chronicdiseases affecting people worldwide and the incidence rises to500million by 2030 [1]The diabetic complications includingneuropathy cardiovascular diseases and nephropathy whichis one of the most complications developed in late stageincrease the morbidity and mortality Diabetic nephropathyoccurs in approximately 30 of diabetic patients and leads torenal failure in most countries The first sign of nephropathywas detected by microalbuminuria (presence of albumin inurine) but doesnrsquot cover all patients with renal impairment[2]

Microalbuminuria was detected in patients of severe kid-ney impairment as hypertension and glomerular basementimpairment Prediabetic hypertensive patients may developnephropathy in noninsulin-dependent diabetic subjects [3]Glycemic control is the most important for glucose level

in diabetes and the most important is glycated hemoglobin(HA1C) as a good index of glucose level [4]

Once diabetic nephropathy was well detected accordingto the therapeutic protocols including glycemic and bloodpressure controls were followed up Diabetic nephropathycan be prevented to a significant degree by early detectionusing novel biomarkers in body fluids [5]

Podocytes are cells present outside the basement mem-brane of nephron In pathological cases podocytes areexpected to be easily found in urine of diabetic patientswith micro and macroalbuminuria [6] Human podocyteshave been found to be damaged in diabetic nephropathypatients [7] An elevation in foot-process pores has beendetected in diabetic patients and correlated directly withmicroalbuminuria [8] A decreased number of podocytes(podocytopenia) in diabetic patients were detected [9 10]

Urinary excretion of immunoglobulin M (IgM) wasfound to be a better index of impaired kidney function than

Hindawi Publishing CorporationDisease MarkersVolume 2014 Article ID 493736 5 pageshttpdxdoiorg1011552014493736

2 Disease Markers

albuminuria in diabetic patients However it has not beenregarded as an early biomarker of end stage renal disease [11]while plasma cystatin C levels could be a useful marker forrenal dysfunction in diabetic patients with normoalbumin-uria [12]

Osteopontin (OPN) is a calcium binding protein whichis expressed in bone endothelial cells and glomerularbasement membrane [12] It is involved in bone turnoverand inflammation In diabetic patients plasma osteopontinlevel was significantly correlated with the degree of diabeticnephropathy [13] In urine increased OPN excretion levelwas detected in animal model of acute renal disease [14]

This study was designed for evaluation of urinarypodocyte in combination with osteopontin and plasma cys-tatin C to explore the early more specific and sensitivebiomarkers for nephropathy in diabetic patients This is tohelp the physicians in controlling the occurrence of renalfailure

2 Subjects and Methods

21 Subjects This study was conducted from March 2012to April 2013 at the Biochemistry Unit King Fahd MedicalResearch Center (KFMRC) Informed consent was obtainedfrom patients and healthy subjects before starting the exper-iment The study was approved by the Ethics Committee ofKing Abdulaziz University Hospital

This study was conducted on 60 patients (mean age37 plusmn 7 years 40 males and 20 females) and 20 age-and-gender matched healthy subjects (mean age 40 plusmn 8 years12 males and 8 females) with normal GFR cutoff value ge90 Patients were admitted to outpatientrsquos King AbdulazizUniversity Hospital King Abdulaziz University The healthysubjects did not have any systemic diseases as diabeteshypertension cardiovascular disease or renal insufficiency

The patients were divided into three groups as follows InGroup I the diabetic patients for 10 years were not sufferingfrom nephropathy (normoalbuminuria) In Group II thediabetic patients for 15 yearswerewith nephropathy (microal-buminuria urinary albumin excretion (20ndash200 ugmin))In Group III there were nondiabetic nephrotic syndromepatients (microalbuminuria)

Blood samples were collected after an overnight fastPlasma samples were separated and stored at minus80∘C untilassay Urine samples were collected from all subjects in themorning and were stored at minus80∘C within 2 h of collectionuntil analysis

22 Methods Plasma samples were subjected to analysis ofglucose glycated hemoglobin and creatinine levels by spec-trophotometer method using kit (Bioline England) Plasmacystatin C level was measured by the latex agglutination test(ALPCO Diagnostics) assay In addition urine samples weresubjected to analysis of immunoglobulin M and osteopontinby ELISA kit from ALPCO Diagnostics (Catalog number 41-OPNHU-E01) and detecting podocyte by immunofluores-cence using monoclonal antibody against podocalyxin onthe surface of podocytes (Table 2) After centrifugation urine

sediment was incubated with anti-humanmono podocalyxinantibody (Bioline England)

23 Statistical Analysis Results were expressed as meanplusmn SD Sensitivity was calculated as follows patients withpositive markersall patients specificity was calculated asfollows patients with negative markersall patients Resultswere compared using Studentrsquos 119905-test for continuous variablesand chi-square analysis A 119875 value le 005 was consideredsignificant One-way ANOVA was additionally used as aconfirmatory test complemented by Tukeyrsquos test to discoversignificant intergroup differences Wilcoxon test was alsoapplied as a nonparametric significance test ROC curveanalysis was used to calculate the diagnostic accuracy ofeach marker the ldquobest cutoffrdquo value and the correspondingclinical sensitivity and specificity

3 Results

Table 1 showed the results of plasma glucose glycatedhemoglobin creatinine and the cystatin C levels Therewas a significant elevation in plasma glucose and glycatedhemoglobin in diabetic normo- and microalbuminuria ascompared with nephrotic syndrome and control groups (119875 lt0001) for both Plasma creatinine and cystatin C were sta-tistically significantly elevated in diabetic microalbuminuriaand nephrotic syndrome patients compared with diabeticnormoalbuminuria and control groups (119875 lt 0001) for bothANOVA analysis showed nonsignificant changes that wereobserved between nephrotic syndrome and diabetic microal-buminuria Plasma cystatin C level showed a significantincrease in albuminuric level (119875 lt 0001)The level of plasmacystatin C positive correlated with increase in microalbu-minuria (normoalbuminuria versus microalbuminuria 119875 lt001)

Table 2 referred that urinary IgM was increased about15-fold in microalbuminuric diabetic patients and nephroticsyndrome patients and compared with control group andnormoalbuminuric group However osteopontin was ele-vated about 3-fold in diabetic microalbuminuric group and15-fold in nephrotic syndrome group compared with othergroups

The number of urinary podocytes was significantlyelevated in diabetic patients with microalbuminuria andnephrotic syndrome as compared with the diabetic normoal-buminuria (119875 lt 0001) The AUC increased for selected cutoff points for podocytes with sensitivity 955 and specificity97 Positive correlations were observed between urinarypodocytes and urinary osteopontin (119903 = 056) and IgM(119903 = 063) while urinary podocytes and urinary osteopontinwere not correlated with other laboratory markers such asplasma glucose creatinine and HA

1C In multiple regression

analysis plasma cystatin C was affected by creatinine andpodocyturia

Receiver operating curve (ROC) analysis was performedto define the diagnostic profile of urine level markers amongsubjects with diabetes The serum level of IgM showed anarea under curve (AUC) of 090 with sensitivity of 890

Disease Markers 3

Table 1 The levels of fasting plasma glucose glycated hemoglobin creatinine and cystatin C in all studied groups (mean plusmn SD)

ParametersPatients groups

Healthy control119899 = 20

Diabetic(normoalbuminuria)119899 = 20

Diabetic(microalbuminuria)119899 = 20

Nephrotic diseases119899 = 20

Glucose (mgdL)Mean plusmn SD 908 plusmn 8 140 plusmn 16 1497 plusmn 11 1093 plusmn 821198751value mdash lt0001 lt0001 NS1198752value mdash mdash lt0001 lt005

Glycated hemoglobin ()Mean plusmn SD 41 plusmn 03 75 plusmn 05 97 plusmn 08 52 plusmn 021198751value mdash lt0001 lt0001 NS1198752value mdash mdash lt0001 lt0001

Creatinine (mgdL)Mean plusmn SD 08 plusmn 002 09 plusmn 006 19 plusmn 009 21 plusmn 0071198751value mdash NS lt0001 lt00011198752value mdash mdash lt0001 NS

Cystatin C (mgdL)Mean plusmn SD 08 plusmn 002 09 plusmn 002 13 plusmn 003 14 plusmn 0041198751value mdash NS lt001 lt00011198752value mdash mdash lt0001 NS

119899 = number of cases 1198751 value diabetic normoalbuminuria and nephritic syndrome versus control 1198752 value diabetic microalbuminuria versus diabeticnormoalbuminuria

and specificity of 90 Also the urine level of osteopontinsupported the diagnostic profile showing an AUC of 073with sensitivity of 923 and specificity of 899 In additionthe urine number of podocytes supported the diagnosticprofile showing an AUC of 092 with a cutoff value of 10(sensitivity of 955 specificity of 97) (Table 3)

4 Discussion

Diabetic nephropathy (DN) is the most common complica-tion of diabetes and leads to renal failure Here we will tryto find a sensitive biomarker for diagnosis of early phaseof diabetic nephropathy Microalbuminuria is a result ofimpairment of the filtration of the glomerular basementmembrane in diabetes and is used as a predictor of DN Earlydiagnosis of nephropathy in diabetic patients is necessary toinitiate appropriate treatment

Cystatin C is a protease inhibitor freely filtrated by renalglomeruli and used as a good marker of renal failure [15]Cystatin C is produced at a constant rate and releasedinto bloodstream Its level is mainly dependent on clear-ance efficiency of the kidney Patients were categorized into3 groups depending on their different degrees of kidneydamage (normoalbuminuria diabetic microalbuminuria ornephrotic syndrome) Plasma cystatin C level was signifi-cantly increased in patients with diabetic microalbuminuriaand nephritic syndrome compared with other groups It wasthought that this increment was probably due to the tubularphase before glomerular manifestationThis suggests that thecystatinC levels of plasmawere related to tubular impairment

and can be an earliermeasurablemarker of renal involvementbefore onset of albuminuria This finding indicated that thecystatin C could be an index reflecting renal tubular epithelialcells

Elevation of serum creatinine and microalbuminuriawere the common markers used in renal impairment diag-nosis Recent studies have indicated that serial changes incystatin erythropoietin and collagen can improve sensitivityfor early diagnosis In the present study serum creatinine andcystatin and urinary IgM osteopontin and podocyte levelswere determined and correlated to identify the good indexfor renal impairment

This study revealed that plasma cystatin C was elevatedin association with diabetic microalbuminuria Howeverplasma endogenous creatinine depends on creatinine syn-thesis metabolism to creatinine and tubular clearance [16]Moreover several tubular proteins are excreted even beforethe detection of microalbuminuria and elevation of plasmacreatinine [17] Therefore other biomarkers for evaluation ofrenal function have been found and one of themwas cystatinC [18] It was suggested that cystatin C may be one of theadditional tubular markers which represent kidney state ofdiabetic patients

Results obtained found that renal function of diabeticpatients was inversely associated with urine IgM excretionwhich indicated that increased urinary IgM excretion wasa better predictor of declining kidney function than albu-minuria [19] This is in agreement with the present studythat showed a positive correlation betweenDN and nephroticsyndrome with albuminuria Since its excretion in urine isassociated with nephrotic syndrome it is a good predictor

4 Disease Markers

Table 2 The levels of urinary immunoglobulin M osteopontin and podocyte in all studied groups (mean plusmn SD)


Normal control119899 = 20




IgM (ngL)Mean plusmn SD 21 plusmn 006 3 plusmn 03 317 plusmn 28 263 plusmn 221198751value mdash NS lt0001 lt00011198752value mdash mdash lt0001 NS

Osteopontin (UL)Mean plusmn SD 1208 plusmn 23 130 plusmn 56 3127 plusmn 258 1793 plusmn 2821198751value mdash NS lt0001 lt0011198752value mdash mdash lt0001 lt0001

Podocyte (cellsmL)Mean plusmn SD Nil Nil 39 plusmn 007 23 plusmn 021198751value mdash mdash mdash lt00011198752value mdash mdash mdash lt001


Table 3 Receiver operating curve (ROC) analysis of investigatedurine parameters as a test for diagnosis of diabetic nephropathy

Variable IgM Osteopontin PodocyteAUC 09 073 092Sensitivity () 89 923 955Specificity () 904 899 97

which may detect the eventual need for renal functionmonitoring [20]

In the present study increased OPN level was detectedin urinary diabetic albuminuric patients and nephroticsyndrome compared with control and normoalbuminuricpatients United States Food andDrugAdministration (FDA)used this marker in order to improve drug discovery [21] Inthis study diabetic patients with renal impairment showedsignificantly elevated urinaryOPN level comparedwith othergroups [22]

This finding suggested that urinary OPN might act as apredictive diagnosticmarker because of its further prognosticvalue The trend towards elevated urinary OPN in diabeticsubjects resulted from enhanced glomerulus dysfunctionHowever elevated urinary OPN level as a result of diabeticrenal injury [23] may be important for evaluation of kidneyefficiency

Podocyte impairment was found in renal insufficiencyPodocytes in urine can be found in diabetic patients withmicro- and macroalbuminuria [24]

Urinary podocytes were not detected in the normoalbu-minuric subjects 95 at the diabetic microalbuminuric stageand 87 at the nephrotic syndrome which indicated thaturinary podocytes might be a useful biomarker for detectingearly glomerular injury in diabetic patients [25]

Damage to the glomerulus is a good measure of kidneydysfunction Such damage invariably leads to protein loss

known as proteinuria and might possibly result in cellsediment shedding Intermediate-to-high molecular weightproteins such as albumin if found in urine and not causedby pre- or postrenal factors are indicative of kidney damageAdditionally special glomerular cells known as podocytesare also thought to bemarkers of glomerular damage if foundin urineThe podocyte layer comprises one of the three maincomponents of the glomerular filtration barrier

In conclusion these biomarkers showed good predictivevalues with regard to ROC-AUC (021 and 065 for podocyteand osteopontin resp) and higher sensitivity and specificity

Disclosure

Thisworkwas not supported or funded by any drug company

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author would like to acknowledge with thanks ProfessorAhmed Khalil consultant of internal medicine for selectionand diagnosis of the studied cases

References

[1] M E Hellemons J Kerschbaum S J L Bakker et al ldquoValidityof biomarkers predicting onset or progression of nephropathyin patients with type 2 diabetes a systematic reviewrdquo DiabeticMedicine vol 29 no 5 pp 567ndash577 2012

[2] B Mackinnon L Shakerdi C J Deighan J G Fox D SJ OrsquoReilly and M Boulton-Jones ldquoUrinary transferrin high

Disease Markers 5

molecular weight proteinuria and the progression of renaldiseaserdquo Clinical Nephrology vol 59 no 4 pp 252ndash258 2003

[3] T Narita M Hosoba M Kakei and S Ito ldquoIncreased urinaryexcretions of immunoglobulin G ceruloplasmin and transfer-rin predict development of microalbuminuria in patients withtype 2 diabetesrdquo Diabetes Care vol 29 no 1 pp 142ndash144 2006

[4] H-J Bangstad A Kofoed-Enevoldsen K Dahl-Jorgensen andK F Hanssen ldquoGlomerular charge selectivity and the influenceof improved blood glucose control in type 1 (insulin-dependent)diabetic patients with microalbuminuriardquo Diabetologia vol 35no 12 pp 1165ndash1169 1992

[5] T Gohda W H Walker P Wolkow et al ldquoElevated urinaryexcretion of immunoglobulins in nonproteinuric patients withtype 1 diabetesrdquo American Journal of PhysiologymdashRenal Physi-ology vol 303 pp F157ndashF162 2012

[6] O Bakoush J Tencer J Tapia B Rippe andO Torffvit ldquoHigherurinary IgM excretion in type 2 diabetic nephropathy comparedto type 1 diabetic nephropathyrdquoKidney International vol 61 no1 pp 203ndash208 2002

[7] C E Mogensen ldquoMicroalbuminuria as a predictor of clinicaldiabetic nephropathyrdquo Kidney International vol 31 no 2 pp673ndash689 1987

[8] A Patari C Forsblom M Havana H Taipale P Groop andH Holthofer ldquoNephrinuria in diabetic nephropathy of type 1diabetesrdquo Diabetes vol 52 no 12 pp 2969ndash2974 2003

[9] G Cheung A Sahai M Billia P Dasgupta and M S KhanldquoRecent advances in the diagnosis and treatment of bladdercancerrdquo BMCMedicine vol 11 article 13 2013

[10] MCThomasWC Burns andMECooper ldquoTubular changesin early diabetic nephropathyrdquo Advances in Chronic KidneyDisease vol 12 no 2 pp 177ndash186 2005

[11] CM Giachelli ldquoVascular calcification in vitro evidence for therole of inorganic phosphaterdquo Journal of the American Society ofNephrology vol 14 pp S300ndashS304 2003

[12] L A Fitzpatrick A SeversonW D Edwards and R T IngramldquoDiffuse calcification in human coronary arteries Associationof osteopontin with atherosclerosisrdquo The Journal of ClinicalInvestigation vol 94 no 4 pp 1597ndash1604 1994

[13] C Zhou J Wu L Torres et al ldquoBlockade of osteopontininhibits glomerular fibrosis in a model of anti-glomerularbasement membrane glomerulonephritisrdquo American Journal ofNephrology vol 32 no 4 pp 324ndash331 2010

[14] X Yan M Sano L Lu et al ldquoPlasma concentrations of osteo-pontin but not thrombin-cleaved osteopontin are associatedwith the presence and severity of nephropathy and coronaryartery disease in patients with type 2 diabetes mellitusrdquo Cardio-vascular Diabetology vol 9 article 70 2010

[15] R L Rouse J Zhang S R Stewart B A Rosenzweig PEspandiari andNK Sadrieh ldquoComparative profile of commer-cially available urinary biomarkers in preclinical drug-inducedkidney injury and recovery in ratsrdquo Kidney International vol79 no 11 pp 1186ndash1197 2011

[16] M Kestila U Lenkkeri M Mannikko et al ldquoPositionallycloned gene for a novel glomerular protein-nephrin-is mutatedin congenital nephrotic syndromerdquo Molecular Cell vol 1 pp575ndash582 1998

[17] V Ruotsalainen P Ljungberg J Wartiovaara et al ldquoNephrinis specifically located at the slit diaphragm of glomerularpodocytesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 96 no 14 pp 7962ndash7967 1999

[18] O Beltcheva P Martin U Lenkkeri and K TryggvasonldquoMutation spectrum in the nephrin gene (NPHS1) in congenitalnephrotic syndromerdquo Human Mutation vol 17 no 5 pp 368ndash373 2001

[19] N Kotajima T Kimura T Kanda et al ldquoType IV collagenas an early marker for diabetic nephropathy in non-insulin-dependent diabetes mellitusrdquo Journal of Diabetes and its Com-plications vol 14 no 1 pp 13ndash17 2000

[20] S M Titan J M Vieira W V Dominquez et al ldquoUrinaryMCP-1 and RBP independent predictors of renal outcome inmacroalbuminuric diabetic nephropathyrdquo Journal of Diabetesand its Complications vol 26 pp 546ndash553 2012

[21] T Narita H Sasaki M Hosoba et al ldquoParallel increase inurinary excretion rates of immunoglobulin G ceruloplasmintransferrin and orosomucoid in normoalbuminuric type 2diabetic patientsrdquo Diabetes Care vol 27 no 5 pp 1176ndash11812004

[22] L Qin X Zeng and G Huang ldquoChanges in serum and urineceruloplasmin concentrations in type 2 diabetesrdquo Zhong NanDa Xue Xue Bao vol 29 no 2 pp 208ndash211 2004

[23] FWK Tam B L Riser KMeeran J Rambow CD Pusey andA H Frankel ldquoUrinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) asprognostic markers for progression of diabetic nephropathyrdquoCytokine vol 47 no 1 pp 37ndash42 2009

[24] T Iijima S Suzuki K Sekizuka et al ldquoFollow-up study onurinary type IV collagen in patients with early stage diabeticnephropathyrdquo Journal of Clinical LaboratoryAnalysis vol 12 pp378ndash382 1998

[25] K Broedbaek A Weimann E S Stovgaard and H E PoulsenldquoUrinary 8-oxo-78-dihydro-21015840-deoxyguanosine as a biomarkerin type 2 diabetesrdquo Free Radical Biology and Medicine vol 51no 8 pp 1473ndash1479 2011

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

albuminuria in diabetic patients However it has not beenregarded as an early biomarker of end stage renal disease [11]while plasma cystatin C levels could be a useful marker forrenal dysfunction in diabetic patients with normoalbumin-uria [12]

Osteopontin (OPN) is a calcium binding protein whichis expressed in bone endothelial cells and glomerularbasement membrane [12] It is involved in bone turnoverand inflammation In diabetic patients plasma osteopontinlevel was significantly correlated with the degree of diabeticnephropathy [13] In urine increased OPN excretion levelwas detected in animal model of acute renal disease [14]

This study was designed for evaluation of urinarypodocyte in combination with osteopontin and plasma cys-tatin C to explore the early more specific and sensitivebiomarkers for nephropathy in diabetic patients This is tohelp the physicians in controlling the occurrence of renalfailure

2 Subjects and Methods

21 Subjects This study was conducted from March 2012to April 2013 at the Biochemistry Unit King Fahd MedicalResearch Center (KFMRC) Informed consent was obtainedfrom patients and healthy subjects before starting the exper-iment The study was approved by the Ethics Committee ofKing Abdulaziz University Hospital

This study was conducted on 60 patients (mean age37 plusmn 7 years 40 males and 20 females) and 20 age-and-gender matched healthy subjects (mean age 40 plusmn 8 years12 males and 8 females) with normal GFR cutoff value ge90 Patients were admitted to outpatientrsquos King AbdulazizUniversity Hospital King Abdulaziz University The healthysubjects did not have any systemic diseases as diabeteshypertension cardiovascular disease or renal insufficiency

The patients were divided into three groups as follows InGroup I the diabetic patients for 10 years were not sufferingfrom nephropathy (normoalbuminuria) In Group II thediabetic patients for 15 yearswerewith nephropathy (microal-buminuria urinary albumin excretion (20ndash200 ugmin))In Group III there were nondiabetic nephrotic syndromepatients (microalbuminuria)

Blood samples were collected after an overnight fastPlasma samples were separated and stored at minus80∘C untilassay Urine samples were collected from all subjects in themorning and were stored at minus80∘C within 2 h of collectionuntil analysis

22 Methods Plasma samples were subjected to analysis ofglucose glycated hemoglobin and creatinine levels by spec-trophotometer method using kit (Bioline England) Plasmacystatin C level was measured by the latex agglutination test(ALPCO Diagnostics) assay In addition urine samples weresubjected to analysis of immunoglobulin M and osteopontinby ELISA kit from ALPCO Diagnostics (Catalog number 41-OPNHU-E01) and detecting podocyte by immunofluores-cence using monoclonal antibody against podocalyxin onthe surface of podocytes (Table 2) After centrifugation urine

sediment was incubated with anti-humanmono podocalyxinantibody (Bioline England)

23 Statistical Analysis Results were expressed as meanplusmn SD Sensitivity was calculated as follows patients withpositive markersall patients specificity was calculated asfollows patients with negative markersall patients Resultswere compared using Studentrsquos 119905-test for continuous variablesand chi-square analysis A 119875 value le 005 was consideredsignificant One-way ANOVA was additionally used as aconfirmatory test complemented by Tukeyrsquos test to discoversignificant intergroup differences Wilcoxon test was alsoapplied as a nonparametric significance test ROC curveanalysis was used to calculate the diagnostic accuracy ofeach marker the ldquobest cutoffrdquo value and the correspondingclinical sensitivity and specificity

3 Results

Table 1 showed the results of plasma glucose glycatedhemoglobin creatinine and the cystatin C levels Therewas a significant elevation in plasma glucose and glycatedhemoglobin in diabetic normo- and microalbuminuria ascompared with nephrotic syndrome and control groups (119875 lt0001) for both Plasma creatinine and cystatin C were sta-tistically significantly elevated in diabetic microalbuminuriaand nephrotic syndrome patients compared with diabeticnormoalbuminuria and control groups (119875 lt 0001) for bothANOVA analysis showed nonsignificant changes that wereobserved between nephrotic syndrome and diabetic microal-buminuria Plasma cystatin C level showed a significantincrease in albuminuric level (119875 lt 0001)The level of plasmacystatin C positive correlated with increase in microalbu-minuria (normoalbuminuria versus microalbuminuria 119875 lt001)

Table 2 referred that urinary IgM was increased about15-fold in microalbuminuric diabetic patients and nephroticsyndrome patients and compared with control group andnormoalbuminuric group However osteopontin was ele-vated about 3-fold in diabetic microalbuminuric group and15-fold in nephrotic syndrome group compared with othergroups

The number of urinary podocytes was significantlyelevated in diabetic patients with microalbuminuria andnephrotic syndrome as compared with the diabetic normoal-buminuria (119875 lt 0001) The AUC increased for selected cutoff points for podocytes with sensitivity 955 and specificity97 Positive correlations were observed between urinarypodocytes and urinary osteopontin (119903 = 056) and IgM(119903 = 063) while urinary podocytes and urinary osteopontinwere not correlated with other laboratory markers such asplasma glucose creatinine and HA

1C In multiple regression

analysis plasma cystatin C was affected by creatinine andpodocyturia

Receiver operating curve (ROC) analysis was performedto define the diagnostic profile of urine level markers amongsubjects with diabetes The serum level of IgM showed anarea under curve (AUC) of 090 with sensitivity of 890

Disease Markers 3













4 Discussion







4 Disease Markers





















Disclosure




Acknowledgment


References



Disease Markers 5






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3













4 Discussion







4 Disease Markers





















Disclosure




Acknowledgment


References



Disease Markers 5






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers





















Disclosure




Acknowledgment


References



Disease Markers 5






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Assessment of Urinary Osteopontin...

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