Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology.
Comparison of approaches to isolate extracellular vesicles from the urine of healthy individuals and from patients with nephrotic syndrome
Urine is collected as a spot or timed urine sample (part a). The process of urinary extracellular vesicle isolation begins with a low speed and/or low centrifugal force (3,000 g) centrifugation step for a short time (≤10 min) and at low temperature (4oC) to clarify the urine (that is, remove the flocculent material, which can include bacteria and cells). Ideally, the urine is carried forward (part b) through to the urinary extracellular vesicle isolation step. This step represents a dynamic field of investigation, and includes methods such as differential centrifugation or ultracentrifugation, single step centrifugation using density gradient material (sucrose, Percoll), filtration or ultrafiltration, precipitation (for example, Exoquick), immunoaffinity capture and hydrostatic dialysis. The complex composition of urine from patients with nephrotic syndrome interferes with the isolation of urinary extracellular vesicles, and additional steps (such as density gradient centrifugation or size exclusion chromatography (SEC)) are required (part c) to further purify the urinary extracellular vesicles from contaminating high-molecular-weight protein complexes (such as albumin) that co-isolate with the urinary extracellular vesicles. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; D2O, deuterium oxide; DTT, dithiothreitol; HPLC, high performance liquid chromatography; MWCO, molecular weight cut-off; Q, Qiagen; SB, Systems Biosciences; TFS, ThermoFisher Scientific; VVLP, hydrophilic polyvinylidene difluoride.