Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles

Abstract

BackgroundUrinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear.MethodsUrine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization.ResultsUrine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47–0.95) and patients (R2 0.41–0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers.Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment–specific EVs.

Urinary Extracellular Vesicles for Renal Tubular Transporters Expression in Patients With Gitelman Syndrome

“…Finally, the specificity and sensitivity of the antibodies used for this study might affect expression of renal transporters between immunoblotting and immunofluorescence (for example NKCC2). Using the detergent for immunoblotting is another approach to enhance intracellular epitope recognition in uEVs ( 22 ).…”

“…For immunoblotting, the loading volume of each uEVs sample was adjusted so that the loaded amount of creatinine was constant ( 21 , 22 ). SDS/PAGE was carried out on an 8% polyacrylamide gel, and proteins were transferred to Immobilon®-P membranes (Millipore, Amsterdam, The Netherlands).…”

Urinary Extracellular Vesicles Are a Novel Tool to Monitor Allograft Function in Kidney Transplantation: A Systematic Review

“…According to the newest guidelines published by the Urine Task Force of the International Society for Extracellular Vesicles, variable dilution of urine is a major challenge in uEV research [ 76 ]. Whether in healthy or unwell subjects, the concentration of uEV is highly influenced by water-loading [ 77 ]. For this, two methods are commonly recommended to realize normalization: (1) the relative excretion rate is based on the ratio between target-marker and other markers of uEV (e.g., numbers, the total yield of protein or RNA, tetraspanin, prostate-specific markers, etc.…”

Serum Alpha-1-Acid Glycoprotein-1 and Urinary Extracellular Vesicle miR-21-5p as Potential Biomarkers of Primary Aldosteronism

“…Urinary EVs (uEVs) were isolated by a sequential ultracentrifugation protocol previously described by Barros et al (26). Urinary creatinine was used to normalize samples of uEVs (59,60). Isolated uEVs were characterized as previously described (26,31) and according to the International Society for Extracellular Vesicles guidelines (27) using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot with characteristic EV proteins (61).…”