Urinary Extracellular Vesicle Whole-Transcriptome Analysis in Diabetic Nephropathy. Urinary Extracellular Vesicle Whole-Transcriptome Analysis in Diabetic Nephropathy

Enrolled 24 participants, including twelve renal biopsy-proven T2DN and twelve T2DM, and isolated uEV s by ultracentrifugation to perform adequate parallel microarrays for mRNAs, lncRNAs and circRNAs and sequencing for miRNAs.One hundred mL of first-morning urine was collected.All urine samples were retained and immediately transferred to the laboratory and centrifuged at 2500 x g at 4°C for 15 min using a horizontal rotor (Dynamica V14R Pro, UK) and at 17,000 x g at 4°C for 40 min using a fixed angle rotor (FA15C rotor, Dynamica, UK). Supernatants were filtered using 0.22µm PES filters (Jet Bio-Filtration, Guangzhou, China) and transferred to new centrifuge tubes and stored at -80 degrees. After overnight melting through a 4 degree, ultracentrifuged at 200,000 xg at 4°C for 2h in a fixed angle P45AT rotor (k factor= 130, CP100NX, Hitachi, Tokyo, Japan) and repeat again after resuspending the pellet in PBS. Supernatants were discarded and the pellets were washed in PBS to obtain the uEVs. Overall design: 12 T2DN vs. 12 T2DM urinary extracellular vesicle samples