Alkali treatment stabilizes fluctuations of urine AQP2 values measured by ELISA.

Sei Sasaki,Daisuke Koga,Kiyonori Katsuragi,Yoko Saijo,Fusako Iwata,Yasukazu Ohmoto

doi:10.1007/s10157-015-1176-1

Sei Sasaki, Daisuke Koga + Show 4 more

Open Access

https://doi.org/10.1007/s10157-015-1176-1

Copy DOI

Abstract

BackgroundAquaporin-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.MethodsHuman 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.ResultsUrine 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.ConclusionPretreatment 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.

Highlights

Aquaporin-2 (AQP2) is a water channel protein in the kidney collecting duct that determines the urine concentrating ability of kidneys [1, 2]
Urine AQP2 is present in exosome membranes and we speculated that this structural organization causes the fluctuations
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

Summary

Introduction

Aquaporin-2 (AQP2) is a water channel protein in the kidney collecting duct that determines the urine concentrating ability of kidneys [1, 2]. AQP2, is deeply involved in water-balance disorders, and in turn, it could serve as a useful biomarker for diagnosis and prognosis of such diseases. Urine AQP2 has been measured in a wide variety of clinical disorders [2, 4, 5]. Initially, it was a mystery how a membrane protein, like AQP2, could be excreted into the urine. Aquaporin-2 (AQP2) in urine is measured in many water-balance disorders and regarded as a useful biomarker for diagnosis and prognosis. Urine AQP2 is present in exosome membranes and we speculated that this structural organization causes the fluctuations

Methods

Results

Conclusion