Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles
Copyright © 2021 by the American Society of Nephrology..
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.
| Errataetall: |
CommentIn: J Am Soc Nephrol. 2021 May 3;32(5):1013-1015. - PMID 33782170 |
|---|---|
| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2021 |
|---|---|
| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:32 |
|---|---|
| Enthalten in: |
Journal of the American Society of Nephrology : JASN - 32(2021), 5 vom: 03. Mai, Seite 1210-1226 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Blijdorp, Charles J [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
AYI8EX34EU |
|---|
| Anmerkungen: |
Date Completed 01.10.2021 Date Revised 03.02.2023 published: Print-Electronic CommentIn: J Am Soc Nephrol. 2021 May 3;32(5):1013-1015. - PMID 33782170 Citation Status MEDLINE |
|---|
| doi: |
10.1681/ASN.2020081142 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM323379702 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM323379702 | ||
| 003 | DE-627 | ||
| 005 | 20231225184044.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2021 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1681/ASN.2020081142 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1077.xml |
| 035 | |a (DE-627)NLM323379702 | ||
| 035 | |a (NLM)33782168 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Blijdorp, Charles J |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles |
| 264 | 1 | |c 2021 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 01.10.2021 | ||
| 500 | |a Date Revised 03.02.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a CommentIn: J Am Soc Nephrol. 2021 May 3;32(5):1013-1015. - PMID 33782170 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2021 by the American Society of Nephrology. | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a aquaporin-2 | |
| 650 | 4 | |a biomarker | |
| 650 | 4 | |a creatinine | |
| 650 | 4 | |a exosomes | |
| 650 | 4 | |a normalization | |
| 650 | 4 | |a osmolality | |
| 650 | 4 | |a particles | |
| 650 | 4 | |a tetraspanin | |
| 650 | 4 | |a urinary extracellular vesicles | |
| 650 | 4 | |a uromodulin | |
| 650 | 7 | |a Biomarkers |2 NLM | |
| 650 | 7 | |a Creatinine |2 NLM | |
| 650 | 7 | |a AYI8EX34EU |2 NLM | |
| 700 | 1 | |a Tutakhel, Omar A Z |e verfasserin |4 aut | |
| 700 | 1 | |a Hartjes, Thomas A |e verfasserin |4 aut | |
| 700 | 1 | |a van den Bosch, Thierry P P |e verfasserin |4 aut | |
| 700 | 1 | |a van Heugten, Martijn H |e verfasserin |4 aut | |
| 700 | 1 | |a Rigalli, Juan Pablo |e verfasserin |4 aut | |
| 700 | 1 | |a Willemsen, Rob |e verfasserin |4 aut | |
| 700 | 1 | |a Musterd-Bhaggoe, Usha M |e verfasserin |4 aut | |
| 700 | 1 | |a Barros, Eric R |e verfasserin |4 aut | |
| 700 | 1 | |a Carles-Fontana, Roger |e verfasserin |4 aut | |
| 700 | 1 | |a Carvajal, Cristian A |e verfasserin |4 aut | |
| 700 | 1 | |a Arntz, Onno J |e verfasserin |4 aut | |
| 700 | 1 | |a van de Loo, Fons A J |e verfasserin |4 aut | |
| 700 | 1 | |a Jenster, Guido |e verfasserin |4 aut | |
| 700 | 1 | |a Clahsen-van Groningen, Marian C |e verfasserin |4 aut | |
| 700 | 1 | |a Cuevas, Cathy A |e verfasserin |4 aut | |
| 700 | 1 | |a Severs, David |e verfasserin |4 aut | |
| 700 | 1 | |a Fenton, Robert A |e verfasserin |4 aut | |
| 700 | 1 | |a van Royen, Martin E |e verfasserin |4 aut | |
| 700 | 1 | |a Hoenderop, Joost G J |e verfasserin |4 aut | |
| 700 | 1 | |a Bindels, René J M |e verfasserin |4 aut | |
| 700 | 1 | |a Hoorn, Ewout J |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Journal of the American Society of Nephrology : JASN |d 1990 |g 32(2021), 5 vom: 03. Mai, Seite 1210-1226 |w (DE-627)NLM012606502 |x 1533-3450 |7 nnns |
| 773 | 1 | 8 | |g volume:32 |g year:2021 |g number:5 |g day:03 |g month:05 |g pages:1210-1226 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1681/ASN.2020081142 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 32 |j 2021 |e 5 |b 03 |c 05 |h 1210-1226 | ||