Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

Abstract

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Keywords: biobank; biomarkers; bladder; extracellular vesicles; kidney; liquid biopsy; prostate; rigor and standardization; urine.

FIGURE 1

uEV microscopy. (a) Urinary EVs (uEVs) were isolated by centrifugation (20,000 × g pellet) and processed for cryoelectron microscopy (as described in (Musante et al., 2020)). The left image shows a wide variety of EVs in size, density and shape. In addition, polymers of uromodulin are shown which seem to entrap uEVs (see arrows). The right image shows a higher magnification of uEVs demonstrating spike like structures emerging from the phospholipid bilayer which likely represents the glycocalyx of some uEVs. (b) uEVs were isolated with ultracentrifugation (100,000 × g pellet) and processed for transmission electron microscopy (TEM) using a negative staining protocol (as described in (Puhka et al., 2017)). To the left there is a lower magnification image displaying a large number and variety of uEVs in size, shape and density. The right image shows a higher magnification demonstrating the uEV heterogeneity with differential staining densities and some spike like surface features that can be visualized despite the cup shape morphology which is due to the processing of TEM. (c) Super‐resolution images were obtained using a Nanoimager S Mark II microscope from ONI (Oxford Nanoimaging) equipped with 405 nm/150 mW, 473 nm/1 W, 560 nm/1 W, 640 nm/1 W lasers and dual emission channels split at 640 nm. The figure shows uEVs stained for CD81 (cyan) and Klotho (magenta) using primary antibodies conjugated with Alexa Fluor 555 and 647 respectively. Representative images with zoomed in insets show the expression and nanoscale distribution of the peptide and tetraspanin on the surface of two representative EVs bound to the coverslip surface. Two‐channel dSTORM data was acquired sequentially at 30 Hz in total internal reflection fluorescence (TIRF) mode. Single molecule data was filtered using NimOS (Version 1.7.1.10213, ONI) based on point spread function shape, photon count and localization precision to minimize background noise and remove low precision localizations

FIGURE 2

Origins of urinary EVs

FIGURE 3

Biogenesis pathways of urinary extracellular vesicles (uEVs). EVs are a highly heterogeneous group of membrane‐bound particles released by both healthy and malignant cells. Generation of exosomes, a specific population of small uEVs, occurs via formation and maturation of multivesicular endosomes (MVEs). Exosomes are formed as intraluminal vesicles (ILVs) in the lumen of MVEs by inward budding of the endosomal membrane. Upon fusion with the cell membrane, exosomes are released into the intercellular space. Microvesicles and ectosomes represent both small and large EVs and are formed by outward budding and scission of the plasma membrane. The process is associated with the accumulation of Ca2⁺‐dependent enzymes that change the polarity of membrane phospholipids. This causes physical bending of the cellular membrane and rearrangements in the underlying cytoskeleton, leading to the formation of microvesicles. Once released by the cell, small uEVs formed at the PM and MVB‐derived exosomes exhibit overlapping size and composition, which makes it difficult to establish their biosynthetic origin. Apoptotic bodies are formed during apoptosis (programed cell death) when cells undergo characteristic outward blebbing caused by breaks in the cytoskeleton. During this process the cellular membrane bulges outward and portions of the cytoplasm and its contents separate forming apoptotic bodies. Secretory vesicles (SV) are produced by the ER and Golgi apparatus. Most of them have specialized cargo such as hormones and neurotransmitters. SVs fuse with the cell membrane at specialized supramolecular structures (porosomes) to release their cargo in the extracellular space.

FIGURE 4

Analytical method selection in uEV research. Analytical methods used for the characterization of EVs explore their physical properties (grey) and/or molecular components (colour). Commonly studied molecular components found in EVs are proteins, nucleic acids, lipids and metabolites. Localization of these molecular components largely defines the choice of an analytical approach. Proteins (purple) can be localized in the EV membrane or lumen. EV surface proteins can be assessed specifically by antibodies, both in bulk analysis, for example, by a time‐resolved fluoroimmunoassay (TR‐FIA), Immunoblot, immuno‐bead capture‐based flow cytometry, or surface plasmon resonance imaging (SPRi) and with assays that analyse individual EVs such as fluorescent NTA, high‐resolution flow cytometry and microscopy. Analysis of luminal proteins can be performed in bulk assays, for example, immunoblot, ELISA and time‐resolved TR‐FIA after membrane permeabilization. Generally, labelling of luminal cargo can facilitate individual EV analysis through the use of membrane‐permeable fluorescent dyes that label proteins or nucleic acids such as ExoGlow™ or Syto™13. Whilst such dyes lack the specificity of more targeted approaches, they enable analysis of EVs by fluorescent microscopy, fluorescent NTA, and high‐resolution flow cytometry. Specific analyses of nucleic acids (blue) and metabolites (green), generally considered to be luminal, are usually achieved in bulk EV assays by either omics‐based approaches, or by transcript‐specific PCR based techniques. Lipids (yellow), are localized within the EV membrane and are commonly analysed in bulk assays either by mass spectrometry or colorimetric reagents, like the sulfo‐phospho‐vanillin (SPV) lipid assay

FIGURE 5

Methodological and knowledge gaps in the current uEV work flow. The urine EV task force of the International Society for Extracellular Vesicles is in the process of recruiting uEV researchers to perform collaborative studies of rigor and reproducibility to address the outlined knowledge gaps