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.

Unless otherwise stated, all authors have no conflicts of interest. D.A. Hesselink reports receiving lecture and consulting fees from Astellas Pharma, Chiesi Pharma, Medincell, Novartis Pharma, and Vifor Pharma and receiving grant support from Astellas Pharma, Bristol-Myers Squibb, and Chiesi Pharma [paid to his institution], without employment or stock ownership at any of these companies, nor does he have patents or patent applications.

This study was funded by the China Scholarship Council, grant number 202008430154.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study including human participants was performed in line with the ethical principles of the Declaration of Helsinki and its later amendments. Approval was granted by the Ethics Committee of Erasmus Medical Center (Ethics approval number: 2018-035). Informed consent regarding participating and publishing data was obtained from all individuals included in this study.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The data generated during this study are not publicly available due to concerns regarding participant privacy. Data can be accessed from the corresponding author upon reasonable request. The uEV experimental parameters were submitted to the EV-TRACK knowledgebase (ID: EV240051) and are available at the following URL: https://evtrack.org/.

https://evtrack.org/