Temporal Dynamics of Urinary Extracellular Vesicle Excretion and Cargo in Healthy Subjects over 24 Hours

Urinary extracellular vesicles (uEVs) are promising non-invasive biomarkers of renal physiology and pathology; however, their temporal excretion patterns and optimal normalization strategies remain incompletely defined, particularly because urine concentration varies diurnally. This basic physiology study used a uniquely designed protocol of urine collection to characterize 24-hour variation in uEV excretion and cargo in thirteen healthy adults collected every void over 24 hours. Each void and a representative 24-hour collection sample were analyzed. uEVs were enriched by low-speed centrifugation with three buffer washes to deplete uromodulin. uEV size, concentration, morphology and cargo were characterized with nanoparticle tracking analysis, cryo-electron microscopy, immunoblotting and single EV flow cytometry. uEV concentrations varied up to 100-fold between individuals, ranging from 6.2 * 10⁶ to 4.5 * 10⁹ particles/mL. The relationship between urine flow and uEV excretion varied significantly across the day. uEV concentration was positively associated with urine creatinine, specific gravity, and TSG101 EV cargo signal, allowing normalization of uEVs to urine creatinine and TSG101. Normalized uEV cargo markers exhibited substantial within individual variation without consistent interindividual trends. When voids were grouped into predefined time periods, sodium chloride cotransporter (NCC) normalized to TSG101 showed a transient dip at noon followed by a significant increase in the next period, indicating a temporal excretion pattern. TSG101 signal didn't vary significantly over the day and was notably positively correlated with uEV excretion rate. These findings offer insight into uEV excretion patterns and highlight the importance of normalization, methodological design, and temporal patterns in advancing uEV biomarker research.