The Study Of Screening And Validation Of EGF As A Biomarker In Diabetic Kidney Disease And Its Underlying Mechanism

Objective:Diabetic kidney disease(DKD)is one of the major diabetic microvasucular complications,and the leading cause of end-stage renal disease(ESRD),seriously affect the quality of patient's life,it also has an increased prevalence in China.Many patients suffered a severe diabetic kidney disease at the time of diagnosis.Due to the lack of effective criterions to diagnose this disease,it does count much to search specific biomarkers.Proteomics are regular technologies to detect the expressions of proteins among different diseases,and urine plays an important role since it is convenient to collect and noninvasive.This study aimed to select discrepancy proteins by detected the different expressions of urinary proteins during different stages in diabetic kidney disease,and validated among patients groups to find more valid biomarkers,and further cell experiments were conducted for the underlying mechanism.Methods: 1.Proteomics Study: 18 type 2 diabetes patients including 9 males and 9 females were selected according to the ACR,and divided into three groups: normalalbuminuria,microalbuminuria and proteinuria,6 healthy controls were matched as normal control group.The middle morning urine was collected and mixed,and then centrifuged.After cold acetone precipitation,the urinary proteins were collected and removed high abundant proteins.The proteins which expression folds above 1.5 or below 0.667 were defined discrepancy proteins after mass spectral analysis.2.Population Validation: 90 patients with type 2 diabetes matched with age and gender were enrolled and divided into three groups: normalalbuminuria,microalbuminuria and proteinuria according to the ACR.30 healthy controls were matched.The proteins in middle morning urine were detected by Elisa Kits.3.Cell Experiments: Human messangial cells were cultured in normal glucose concentration(5.6mM)and high glucose concentration(25mM)for 24 hours,qPCR and Western Blot were performed to detect expressions of mRNAs and miRNA as well as proteins.Results: 1.Proteomics Study: Total 537/467/398/439 proteins were detected in urine from normal controls,normalalbuminurial group,microalbuminurial group and proteinurial group.12 up-regulated proteins and 19 down-regulated proteins in diabetic kidney disease were screened out.Bioinformatics analysis showed that EGF and THBS1 might be the significant proteins involve the pathogenesis of diabetic kidney disease.2.Population Validation:(1)The expressions of urinary EGF and THBS1 in diabetic kidney disease groups were lower compared with normal controls,and decrease followed the progress of DKD.(2)EGF showed a negative correlation with disease course,BMI,family history,HbA1 C and uMA(r=-0.121,-0.155,-0.380,-0.125,-0.281,p<0.05).THBS1 was negative correlated with disease course,family history,HbA1 C,SBP and uMA(r=-0.109,-0.274,-0.150,-0.821,-0.283,p<0.05)adjusted by age and gender.(3)The AUC-ROC for microalbuminuria of urine EGF is 0.804(95%CI:0.708-0.882),which has a sensitivity of 81.2% and specificity of 46.3%.The AUC-ROC of urine EGF in the diagnosis of early stage of DKD is 0.876(95%CI:0.729-0.982),the sensitivity and specificity were 96.6% and 65.1%.3.Cell Experiments:(1)The expression of miR-192 in human kidney messangial cells cultured in high glucose concentration was higher than normal glucose concentration.The CAV1 showed a lower expression and EGF and COLI as well as FN were up-regulated and the cell migration and proliferation were increased.(2)The expression of CAV1 was up-regulated after cultured with miR-192 inhibitors.EGF expression was lower,companied with less extracellular matrix and cell migration and proliferation compared with controls,while cultured with miR-192 mimics showed an opposite phenomenon.Conclusion: 1.This study demonstrated a different urinary protein profiles,urinary proteomics can be an important source of diagnosis markers in DKD.2.Urinary EGF and THBS1 were closely correlated with DKD progression.Urinary EGF showed a favorable value to diagnose early diabetic kidney disease from clinical diabetic kidney disease or non-DKD,showed that EGF could be a valuable diagnosis biomarker of DKD.3.The EGF expressed differently in stages of DKD.miR-192 and CAV1 can involved in DKD pathogenesis by regulated EGF expression and influenced the extracellular matrix as well as cell migration and proliferation,which induced the different expressions of EGF.4.High glucose can regulated the formation of extracellular matrix in messangial cells through miR192-CAV1-EGF pathway.