Differential Analysis Of Urinary Proteomics In Patients With Mild-to-moderate Type 2 Diabetic Nephropathy By Dapagliflozin

Objective:In this study,patients with early type 2 diabetic nephropathy（T2DN）who used sodium-dependent Glucose Transporters 2 inhibitor（SGLT2-i）as a representative drug,dapagliflozin,were studied.We examined the differences in protein expression in the urine of early T2DN patients compared with normal controls by proteomic analysis to investigate the biomarkers of early DN damage and the possible molecular mechanisms of dapagliflozin action in early T2DN patients.Methods:1.Five patients with early T2DN treated in the Department of Endocrinology,from October 2020 to May 2021 were selected as the study subjects.They were given dapagliflozin in addition to basic insulin hypoglycemic therapy.The basic treatment was named DN group,and the addition of dapagliflozin was named DN-DAPA group.At the same time,5 healthy subjects in the physical examination center of our hospital were selected as blank controls and named as NC group.The urine samples and clinical data of the patients were collected.2.Urine samples were identified and screened by i TRAQ non-standard quantitative proteomics method in DN/NC group and DN-DAPA/DN group.3.The differential proteins were analyzed using genetic Ontology（GO）,Kyoto Encyclopedia of Genes and Genomes（KEGG）and other bioinformatics analysis methods.4.Based on bioinformatics analysis methods and searching related literatures on DN,five differential proteins were screened out which were significantly expressed in DN/NC group and DN-DAPA/NC group with opposite trends.Further discussion was conducted on the five differential proteins to explore the molecular mechanism of dapagliflozin effect on early T2DN.Results:1.Statistical analysis was performed on baseline data of patients.Body Mass Index（BMI）and Glycosylated Hemoglobin Type A1C%（HbA1c%）in DN group and DN-DAPA group.HbA1c%,Fasting Plasma Glucose（FPG）,Serum Creatinine（Scr）,Systolic Blood Pressure（SBP）,Diastolic Blood Pressure（DBP）,and Estimate The Glomerular Filtration Rate（e GFR）were higher than those in NC group.NC group Uric Acid（UA）,Total Cholesterol（TC）,Total triglyceride（TG）,Low Density Lipoprotein Cholesterol（LDL-C）was higher than DN group.Only HbA1c% had statistical significance（P < 0.05）.2.193 differentially expressed proteins were detected with P<0.05 and|log2FC|>1.5 as the cut-off criteria.Compared with NC group,91 expressions were up-regulated and 72 expressions were down-regulated in DN group.Compared with DN group,11 expressions were up-regulated and 26 expressions were down-regulated in DN-DAPA group.3.GO enrichment analysis showed that 60 items were obtained in the two comparison groups（P < 0.05）.There were 14 Biological processes（BP）in DN/NC group.Cell Component（CC）involves 8 items;Molecular Function（MF）involves 8items.14 BP items were involved in DN-DAPA/DN group.CC involves 8 items;MF involves 8 items.The subcellular localization of differential proteins in DN/NC group was mainly located in extracellular,cytoplasm,plasma membrane and mitochondria.BP was mainly involved in cellular process,biological regulation,metabolic process and response to stimulation.CC is mainly cellular,intracellular and protein-containing complex;MF mainly has catalytic activity,molecular function regulation and molecular sensor activity.The subcellular localization of differential proteins in DN-DAPA/DN group was mainly located in cytoplasm,extracellular and nucleus,and BP was mainly involved in cellular process,biological regulation and response to stimulation,etc.CC is mainly cellular,intracellular and protein-containing complex;MF mainly has binding function and catalytic activity.4.KEGG pathway analysis showed that 13 signaling pathways were enriched in DN/NC group,and up-regulated proteins were involved Complement and coagulation cascades、Staphylococcus aureus infection、Systemic lupus erythematosus、Pertussis、Vitamin digestion and absorption、Down-regulated proteins are involved in2-oxocarboxylic acid metabolism,Glyoxylate and dicarboxylate metabolism,Biosynthesis of amino acids,Glycerolipid metabolism and Cell adhesion Molecules,Phosphatidylinositol Signaling System,Arginine biosynthesis,Pentose and glucuronate interconversions.DV-DAPA /DN group was enriched in 8 signaling pathways,and the up-regulated proteins were involved in the Transcriptional mis-regulation in cancer signaling pathway.Down-regulated proteins are involved in Pathways of neurodegeneration-multiple diseases,Huntington disease,Prion Disease,Amyotrophic lateral sclerosis,Synaptic vesicle cycle,Rheumatoid arthritis Arthritis,Parkinson disease,Spinocerebellar ataxia.5.Through bioinformatics analysis,differentially expressed proteins in DN/NC group and DN-DAPA/DN group with opposite expression trends were screened,and 5protein molecules were screened: SOD1,SLC25A6,PPP2R2A,S100A13 and CXCL12,combined with searching related literatures of DN.Conclusion:1.DN-DAPA /DN group and DN/NC group had a large number of significantly different proteins,and urinary protein changed at the early stage of T2DN2.SOD1,SLC25A6,PPP2R2A,S100A13 and CXCL12 are involved in the pathogenesis of early T2DN and may serve as potential biomarkers for early T2DN diagnosis.The application of dapagliflozin affected the expression levels of five protein molecules,down-regulate their expression.3.The possible molecular mechanism of dapagliflozin effect on early T2DN and reversal of microalbuminuria is that PPP2R2A and CXCL12/S100A13 are down-regulated by ROS dependent mechanism.