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. 12 T2DN vs. 12 T2DM urinary extracellular vesicle samples