Alkali Treatment Stabilizes Fluctuations of Urine AQP2 Values Measured by ELISA

Overview

Journal Clin Exp Nephrol

Publisher Springer

Specialty Nephrology

Date 2015 Oct 15

PMID 26463736

Citations 4

Authors

Sei Sasaki

Yoko Saijo

Yasukazu Ohmoto

Fusako Iwata

Daisuke Koga

Kiyonori Katsuragi

Affiliations

Soon will be listed here.

Abstract

Background: Aquaporin-2 (AQP2) in urine is now measured in many water-balance disorders and regarded as a useful biomarker for diagnosis and prognosis. An enzyme-linked immunosorbent assay (ELISA) method has been developed for measurement of large numbers of clinical samples. However, fluctuations in the measured values were sometimes observed depending on storage conditions. Urine AQP2 is present in exosome membranes and we speculated that this structural organization causes the fluctuations.

Methods: Human urine samples from healthy subjects were measured by ELISA. Effects of maneuvers to disrupt the exosome membrane mechanically (freezing and thawing at different temperatures) and chemically (treating with alkali and detergents) prior to ELISA were examined.

Results: Urine samples stored at 4 or -80 °C did not show significant AQP2 values, whereas those stored at -25 °C for more that 2 weeks provided the values. Urine samples treated with 0.4 N NaOH and 0.5 % Triton X-305 showed the consistent and comparable values to those stored at -25 °C.

Conclusion: Pretreatment with alkali (0.4 N NaOH) to disrupt exosome membranes allowed consistent ELISA measurements of urinary AQP2. This simple method is applicable to ELISA of other membrane proteins included in exosomes.

Citing Articles

Phosphorylation profile of human AQP2 in urinary exosomes by LC-MS/MS phosphoproteomic analysis.

Sakai M, Yamamoto K, Mizumura H, Matsumoto T, Tanaka Y, Noda Y Clin Exp Nephrol. 2020; 24(9):762-769.

PMID: 32529500 PMC: 7474712. DOI: 10.1007/s10157-020-01899-4.

Involvement of Elf3 on Smad3 activation-dependent injuries in podocytes and excretion of urinary exosome in diabetic nephropathy.

Sakurai A, Ono H, Ochi A, Matsuura M, Yoshimoto S, Kishi S PLoS One. 2019; 14(5):e0216788.

PMID: 31150422 PMC: 6544199. DOI: 10.1371/journal.pone.0216788.

AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities.

Miyazawa Y, Mikami S, Yamamoto K, Sakai M, Saito T, Yamamoto T Clin Exp Nephrol. 2018; 22(4):782-788.

PMID: 29396622 DOI: 10.1007/s10157-018-1538-6.

Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies.

Nameta M, Saijo Y, Ohmoto Y, Katsuragi K, Yamamoto K, Yamamoto T Int J Mol Sci. 2016; 17(10).

PMID: 27681727 PMC: 5085667. DOI: 10.3390/ijms17101634.

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