Abstract
Background Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear.
Methods Urine 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.
Results Urine 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.
| Original language | English |
|---|---|
| Pages (from-to) | 1210-1226 |
| Number of pages | 17 |
| Journal | Journal of the American Society of Nephrology |
| Volume | 32 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 2021 |
Bibliographical note
Funding Information:This work was supported by the Dutch Kidney Foundation (16OI04 and CP1805).
Funding Information:
C.A. Carvajal and E.R. Barros are supported by the Agencia Nacional de Investigación y Desarrollo - Fondo Nacional de Desarrollo Científico y Tec-nológico (ANID-FONDECYT) Grants 1160695, 1212006, and ANID-Millennium Science Initiative Program - IMII P09/016-F, ICN09_016 ICM (Iniciativa Científica Milenio), outside the submitted work. T.A. Hartjes and M.E. van Royen are supported by the Dutch Cancer Society, the Innovative Measurements and Markers for PROstate cancer prognosis using extracellular VEsicles (IMMPROVE) Alpe d’HuZes grant (EMCR2015-8022) and Prostate Cancer UK (grant G2012-36), outside the submitted work. E. Hoorn reports receiving honoraria from UpToDate; being a scientific advisor or member of the editorial boards of JASN, Journal of Nephrology, and Frontiers in Physiology, Member of the Scientific Council of the Dutch Kidney Foundation, and being a board member of Dutch Federation of Nephrology. G. Jenster has a license agreement with Cell GS for the CD9 and CD63 TR-FIA (TRIFic). J. Hoenderop reports being a scientific advisor or editorial board member of European Journal of Physiology. M. Clahsen-van Groningen reports receiving research funding from Astellas Pharma B.V. R. Bindels reports being a scientific advisor or member of Regenerative Medicine Crossing Borders and member of the Scientific Council of the Dutch Kidney Foundation. R.A. Fenton reports being a scientific advisor or member as the associate editor for the American Journal of Physiology - Renal Physiology, and as editorial board member of JASN, PLOS One, andNature Scientific Report; and is supported by the Novo Nordisk Foundation and the Leduq Foundation, outside the submitted work. All remaining authors have nothing to disclose.
Publisher Copyright:
© 2021 by the American Society of Nephrology