Urinary extracellular vesicles in healthy individuals: positive correlation between podocyte and tubular vesicles independent of kidney function

Urinary extracellular vesicles (uEVs) are promising non-invasive biomarkers for assessing renal physiology and disease. Focusing specifically on kidney-derived uEVs (kd-uEVs) rather than the overall uEV population may offer a more precise insight into kidney health. However, research distinguishing single kd-uEVs from various nephron segments and their relationship with kidney biology remains limited. Imaging flow cytometry (IFCM) can identify single kd-uEVs by labeling them with CD63 (a uEV marker) in combination with PODXL (a podocyte marker) or AQP2 (a tubular marker). This study investigated the correlations between CD63+ AQP2+ or PODXL+ kd-uEVs and kidney function. CD63+ AQP2+ and CD63+ PODXL+ uEVs were detected in urine compared to negative controls, including urine with detergent treatment or isotype staining and reagent-containing PBS. While no significant association was found between CD63+ AQP2+ or PODXL+ uEV concentration and kidney function, a significant correlation was observed between AQP2+ and PODXL+ uEV concentrations (Rho = 0.789, p < 0.001). This correlation could be explained by the colocalization of AQP2 and PODXL on CD63+ uEVs. In conclusion, our study is the first to demonstrate the colocalization of podocyte and tubular proteins on uEVs.