Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles
Authors: Blijdorp Charles J. , Tutakhel Omar A. Z. , Hartjes Thomas A. , van den Bosch Thierry P. P. , van Heugten Martijn H. , Rigalli Juan Pablo , Willemsen Rob , Musterd-Bhaggoe Usha M. , Barros Eric R. , Carles-Fontana Roger , Carvajal Cristian A. , Arntz Onno J. , van de Loo Fons A. J. , Jenster Guido , Clahsen-van Groningen Marian C. , Cuevas Cathy A. , Severs David , Fenton Robert A. , van Royen Martin E. , Hoenderop Joost G. J. , Bindels René J. M. , Hoorn Ewout J. 
Published: March 2021

Significance Statement
Urinary extracellular vesicles (uEVs) are a promising noninvasive source of kidney biomarkers, but the optimal approaches for normalization, quantification, and characterization in spot urines are unclear. To address the hypothesis that urine creatinine can be used as a normalization variable, urine particles were quantified in dilute and concentrated urines (water deprivation–loading study) and randomly from healthy subjects and patients with kidney disease. In these various settings, urine creatinine was highly correlated with particle counts, suggesting it can be used as a normalization variable. Additional findings relevant for future uEV studies include interference of Tamm-Horsfall protein with nanoparticle tracking analysis, excretion of larger uEVs in dilute urine, and the ability to treat uEVs with detergent to enhance intracellular epitope recognition.

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 (R

2
0.96) and in random spot urines from healthy subjects (R

2
0.47–0.95) and patients (R

2
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.