Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

Abstract

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. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

Extracellular vesicles: the growth as diagnostics and therapeutics; a survey

“…EVs are lipid-based membrane vesicles of endocytic origin that are released by both healthy and cancerous cells [1]. The term “exosomes” is often used for a nano-scale subpopulation of EVs, presumably of endocytic origin, although this last point is rarely demonstrated.…”

The Authors Reply

“…The focus of our study was to explore the functional cargo of exosome CCL2 mRNA in mediating the crosstalk between tubular cells and macrophages, and abundant protein did not seem to influence RNA profiling of this cargo of extracellular vesicles. 5 Nevertheless, we agree that when evaluating exosomes in proteinuric urine, the potential influence of protein on the exosome purification should be considered.…”

Extracellular vesicles in urologic malignancies—Implementations for future cancer care

“…The advent of density gradient ultracentrifugation increases the efficiency of particle separation according to their buoyant density. However, an important disadvantage of differential centrifugation cannot thoroughly separate protein and other non‐exosomal particles from EVs, limiting its efficacy and usage in clinical studies for diagnosis, to large extent, the advent of density gradient ultracentrifugation that reverses the poor separation efficiency due to their buoyant density, and it is frequently used for EV isolation though with a considerable loss of EVs …”