Diagnostic Markers’ Study Of Label-free-based Quantitative Proteomics In Urine Of Type 2 Diabetic Kidney Disease

Objective:Diabetic kindey disease is a common microvascular complication of diabetes.But renal impairments in patients with diabetic include DKD and NDRD,the gold standard which distinguish two diseases is Kidney biopsy.In this study,the differently expressed proteins between DKD and the other two diseases was authenticated based on label-free quantification proteomics technology and screened the candidate biomarkers by Bioinformatics Analysis.Method:1.Collecting all the patients’ morning urine with renal biopsy in department of nephrology of The First Affiliated Hospital of Kunming Medical University,The First People’s Hospital of Yunnan Province,Yan’an Hospital,Chuxiong People’s Hospital,The First Affiliated Hospital of Southwest Medical University.patients with complete medical records,laboratory and pathological data and no major chronic diseases were included and were divided into dabetic kidney disease,non-diabetic renal disease,non-diabetic renal disease with type 2 diabetes mellitus.2.The three study groups were matched by using PSM analysis based on epidemiological data such as age,gender,BMI,and 10 samples were selected forproteomics analysis in each group.3.The total protein of urine samples was quantified by Bradford method,and the integrity of urine protein was detected by gel electrophoresis.Proteomic analysis was performed with 80μg total protein in each group to identify and screen the differentially expressed proteins in DKD group compared with NDRD group and NDRD-DM group.4.Bioinformatics analysis such as GO、KEGG、PPI were performed for these differentially expressed proteins.5.The potential biomarkers were screened according to tissue localization、protein differential expression and liturature reports,and analysed differential diagnosis ability using ROC curve.Results:1.Among the 293 patients undergoing renal biopsy and puncture,134 were males and 159 were females,with an average age of(40.3±13.8)years,including 23 cases of DKD(7.85%),243 cases of NDRD(82.9%),and 27 cases of NDRD-DM(9.21%),The pathological types of NDRD are varied,including IgA nephropathy,membranous nephropathy,minor lesion nephropathy,and focal segmental glomerulosclerosis.Compared with NDRD group,patients in DKD group were older,more male,the proportion of patients with hypertension,BMI,systolic blood pressure,urea,creatinine,fasting blood glucose,urinary total protein,microalbumin and ACR were also significantly higher(P<0.05),while hemoglobin and EGFR were lower(P<0.05).Compared with the NDRD-DM group,DKD patients had a higher proportion of hypertension,more people with DR(P<0.05),longer duration of diabetes,higher systolic blood pressure and creatinine(P<0.05),and lower hemoglobin content,EGFR and urinary total protein content(P<0.05).2.After the PSM analysis of patients,there were no statistical differences in age,gender,duration of diabetes,BMI,systolic blood pressure,diastolic blood pressure,use of hypoglycemic and antihypertensive drugs,duration of hypertension,and whether the patients suffered from cardiovascular disease among the three groups(P>0.05).3.Thirty urine samples were analyzed by proteomics.The results of quantitative and qualitative protein analysis,principal component analysis,relative standard deviation analysis and Pearson correlation analysis showed that samples met the requirements of proteomics quality control.4.Proteins were identified by label-free Quantitative Proteomics Technology.193 differential expression proteins were identified between DKD group and NDRD group,of which 104 were up-regulated and 89 were down-regulated in DKD group.Compared with NDRD-DM group,210 proteins were up-regulated and 233 proteins were down-regulated.5.GO function analysis results showed that 12 biological processes,8 cell components and 7 molecular functions were annotated in DKD/NDRD group,14 biological processes,8 cell components and 6 molecular functions were annotated in DKD/NDRD-DM group.Compared with NDRD、NDRD-DM,DKD group’s differential expression proteins were involved in biological processes of biological regulation、response to stimulus and metabolic process;cellular components of cell、organelle、membrane;molecular functions of binding、catalytic activity、molecular function regulator;subcellular localization of extracellular and cytoplasm.Analysis of KEGG pathway results show that NF-Kappa B signaling pathway was common and important metabolic pathway in two comparison groups.differential expression proteins involved in this pathway included BCR、LBP、VCAM1.In addition,complement and coagulation cascades was the most important pathway in DN/NDRD-DM group.CFHR1、C6、SERPINC1、CFI、F10、PROS1、CFHR5、C8G et al were the DEPs that participate in the metabolic pathway.6.We screened out proteins that were highly expressed in the kidney in the DKD group,the results showed that the difference multiples of TDG,LRP12,DSTN,COL1A1,MTPN were large and the trend of change was consistent.In addition,We performed a literature search on the proteins that were highly expressed in the DKD group and screened out 10 proteins that were associated with diabetes or nephropathy.The ROC curve analysis showed that except for TDG(0.585)and LRP12(0.535)which are highly expressed in kidney tissue,the AUC of the other proteins are all above 0.75.Therefore,DSTN,COL1A1,MTPN,BAIAP2L1,C3,SERPIND1,A2M,FETUB,IGHV3-9,IGHV3-21,TPM1,PAM,GSTP1 may be potential differential diagnosis biomarkers for DKD.Conclusions:Biomarkers which DSTN,COL1A1,MTPN,BAIAP2L1,C3,SERPIND1,A2M,FETUB,IGHV3-9,IGHV3-21,TPM1,PAM and GSTP1 were screen out by using proteomics technology has value for the differential diagnosis of DKD.