Potential urine biomarkers for the diagnosis of prediabetes and early diabetic nephropathy based on ISN CKHDP program.

BACKGROUND: Diabetic nephropathy (DN) is a major complication of diabetes mellitus (DM), and the most frequent cause of end-stage renal disease (ESRD) in many countries. Urinary extracellular vesicles (UEVs) are considered a rich non-invasive source of markers for renal diseases. In this study, UEV enrichment and analysis in diabetic nephropathy (DN) was performed in a community epidemiological survey supported through the ISN CKHDP program. MATERIALS AND METHODS: Patients were divided into five groups according to severity of kidney damage. A hydrostatic dialysis method was used for UEV enrichment followed by quantitation using Coomassie protein assays and subsequent adjustment using urinary creatinine levels. UEVs were then characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting of tumor susceptibility gene product TSG101. Two-dimensional DIGE (2D-DIGE) was used to analyze differential protein expression in the UEVs. Mass spectrometry (MS) was conducted and MASCOT search engine was used to identify potential biomarkers. RESULTS: Bradford protein assay showed that protein concentration of UEVs in diabetics with kidney injury increased significantly as compared to normal controls. UEVs present a round, cup-shaped, membrane-encapsulated structure under TEM, and the main peak of UEVs show 55 - 110 nm nanoparticles with NTA. MS and MASCOT identified 22 differential proteins, and MASP2, CALB1, S100A8, and S100A9 were selected as potential biomarkers of early DN based on bioinformatic analysis. DISCUSSION: Our results show UEV proteome changes in different stages of DN. The results of this study show four unique proteins that undergo changes in early DN. These promising discoveries may prompt a new field of research focused on improving the diagnosis of DN.