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, therefore, is deeply involved in water-balance disorders, and in turn, it could serve as a useful biomarker for diagnosis and prognosis of such diseases. Indeed, AQP2 was shown to be measurable in human urine soon after its cloning [
3]. Since then, urine AQP2 has been measured in a wide variety of clinical disorders [
2,
4,
5]. However, initially, it was a mystery how a membrane protein, like AQP2, could be excreted into the urine. In 2004, Pisitkun et al. reported that AQP2 is embedded in the membrane of exosomes in urine, resolving this dilemma [
6].
Exosomes are late endosomes (multiple vesicular bodies)-derived nano (40–100 nm) vesicles with a lipid bilayer membrane that are excreted from numerous types of cells [
7]. Exosomes are now attracting researchers’ intense interest because they carry microRNA, mRNA, and cytosolic and membrane proteins from their originating cells [
8]. Accordingly, exosomes could provide biomarkers for clinical diseases [
9]. Exosomes in the urine, specifically, has been shown to harbor many clinically useful biomarkers, including membrane proteins like AQP2 [
5,
6,
10,
11]. In quantitative measurements by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) of membrane proteins located in exosome membranes, antibody recognition domains of the proteins are critical. If recognition domains are inside exosomes, disruption or lysis of exosome membranes is necessary, as has been discussed recently [
5]. In this context, it should be mentioned that good antibodies against human AQP2 recognize its C-terminus that is located inside exosomes [
6].
Urine AQP2 has been measured by RIA, ELISA, and Western blotting [
3,
12‐
16]. Among these, ELISA may be most suitable for clinical use because of its high sensitivity, ease of handling (no radioisotope) and high throughput for testing a large number of clinical samples. We have developed a sensitive ELISA system for detecting urine AQP2 [
15], and the “freeze and thaw” method has been used to disrupt the exosome membranes. However, sometimes, fluctuations of measured values have been noticed depending on sample storage conditions. We speculated that this fluctuation was caused by insufficient disruption of the exosome membranes.
Materials and methods
ELISA and antibodies
The sandwich ELISA method and the antibodies were the same as reported previously (Human AQP2 ELISA kit, Otsuka Pharmaceutical Co., Ltd., Japan) [
15]. Briefly, mouse monoclonal antibody and rabbit polyclonal antibody were raised against a recombinant thioredoxin-fused human AQP2 protein (45–271 amino acid). The monoclonal antibody was used to coat a 96-well microtiter plate to trap AQP2. Then, the trapped AQP2 was sandwiched with the polyclonal antibody, followed by colorimetric detection of a horse radish peroxidase-coupled reaction. Concentrations of human AQP2 in the samples were calculated from a standard curve of the same recombinant human AQP2.
Human urine samples
This protocol was approved by the institutional ethical committee of the Research Division at Otsuka Pharmaceutical Co., Ltd. (120718) and all study participants gave written informed consent. Urine samples were obtained from healthy subjects who had no evidence of recent kidney or urinary tract disease. Urine samples were stored at 4, −25, or −80 °C until the assay. Data were shown as the mean ± SEM and the statistical analyses were performed using SAS software (R9.1, SAS Institute, Japan). The Pearson’s correlation coefficient was used to assess the correlation of AQP2 values with and without NaOH pretreatment. When the measured AQP2 values were lower than the detection limit (0.22 ng/ml), the value of the detection limit was used for analyses. P < 0.05 was considered significant.
Discussion
It is important to know that the exosome lipid bilayer membrane is surprisingly strong and stable, and may not be disrupted even after 4 weeks of storage at 4 or −80 °C, as shown by the lack of AQP2 detection by our ELISA in samples without pretreatment (Table
1). Exosome membranes can protect their cargo for a long time in usual conditions. However, this favorable characteristic can inhibit immunological detection of antigens hidden in exosomes by blocking the access of antibodies inside exosomes. This consideration only applies to antibodies that recognize intracellular domains of membrane proteins of interest, as seen in our present case. It is worth noting that in exosomes, the inside corresponds to the inside of the cell, which is opposite of typical intracellular vesicles [
6,
7].
In our previous attempts to measure urine AQP2 by RIA and ELISA [
3,
12,
15], urine samples were stored in freezers (−25 °C), which could have resulted in the disruption of exosome membranes. However, as shown in Fig.
1, the measured values of urine AQP2 can change depending on freezing temperatures and durations. The storage at −80 °C, compared to −25 °C, seems to preserve exosome structures, because little AQP2 values were detected in −80 °C samples. The difference of effectiveness of the two temperatures (−25 vs. −80 °C) may indicate that freezing temperature is critically important and thawing itself is not so. Clearly, the fluctuations in measured AQP2 values depending on storage conditions should be eliminated before applying this ELISA method to a large number of clinical samples, and our efforts to solve this problem have led to the present results.
By incubating urine samples with a strong alkali prior to ELISA, measured AQP2 values became stable, irrespective of storage temperature and duration, indicating that the alkali treatment disrupted vesicle structure of exosomes (Fig.
3). The solubilizing ability of alkali allows it to be used with many substrates, such as lipids, peptides, and celluloses. Alkali has been applied to the isolation of a hepatitis B core protein from the virus core [
17] and the recovery of functional proteins from herring [
18]. This method is superior to the freeze and thaw method because of the short incubation time (20 min vs. 2 weeks) and consistency in ELISA results (see Fig.
3). Because the use of exosomes as potential resources for clinical biomarkers is receiving increased attention, this simple and convenient method for disrupting exosome membrane could be used in a wide range of applications.
It should be mentioned that measured values of urine exosome-derived biomarkers need to be adjusted (normalized) between samples. Several methods are available for this purpose such as timed urine collection, simultaneous measurements of urine creatinine, Tamm–Horsfall protein, or household exosome proteins [
5]. For AQP2, normalization by simultaneously measured urine creatinine has been used in clinical spot urine samples [
3,
12,
13,
15,
16], but we have to be careful whether this correction accurately estimates true excretion rate of AQP2 [
19].
In conclusion, this study demonstrates that alkali treatment is valuable when proteins located in exosome membranes will be quantitatively measured by ELISA with intracellular domain-recognizing antibodies.
Acknowledgments
This study was in part supported by a grant-in-aid from the Ministry of Health, Labor and Welfare, Japan.
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