Abstract

Extracellular vesicles (EVs) are membranous vesicles released by various cells, and are involved in intercellular communication and disease progression. EVs that are isolated from urine are good indicators of urinary system diseases and help certain urological studies. During isolation of urine EVs, Dithiothreitol (DTT) is widely used to reduce the contamination of the major contaminant Tamm–Horsefall protein (THP),which is the most abundant protein in the human urine and the most difficult contaminant to remove in the isolation of urine EVs. Unfortunately, DTT can interfere with subsequent analysis due to its strong reducing ability and cannot completely remove THP. To optimize the urine EV isolation strategy, we compared two pretreatment protocols: incubating urine with NaCl and DTT before centrifugation. After a series of analyses by nanoparticle tracking analysis (NTA), western blotting (WB), and transmission electron microscopy (TEM), we found that NaCl removed more THP than DTT in a low-speed centrifugation step and that the residual EVs also had lower THP contamination post NaCl treatment. Remarkably, the yield of EVs obtained via the salting-out method was significantly higher than those obtained by the other methods (P = 0.001). Our study is the first to demonstrate that the salting-out method is better than the traditional DTT method in terms of efficiency in removing THP and EV yields.