JP7405399B2 - How to determine moisture status - Google Patents

Description

TECHNICAL FIELD The present invention relates to a water state determination method that can be used to determine the water state of a body, such as a dehydrated state or a water-filled state.

In the body, several mechanisms work together to maintain water balance. One mechanism for maintaining water balance involves the pituitary gland, located at the base of the brain, and the kidneys. When the body becomes dehydrated, vasopressin is secreted into the blood from the pituitary gland. Vasopressin works by stimulating the kidneys to retain water and produce less urine. When there is too much water in the body, the pituitary gland secretes little vasopressin, and the kidneys excrete the excess water in the urine.

When the concentration of vasopressin in the blood increases due to an increase in blood osmotic pressure due to dehydration or a decrease in body fluid volume, vasopressin receptors (V2 receptors) present in the principal cells of renal collecting ducts become activated. As a result, protein kinase A (PKA) is activated within the cell, and the 256th, 264th, and 269th amino acid residues present at the C-terminus of aquaporin 2 (AQP2) are phosphorylated. Of these three positions, the 269th amino acid residue is most significantly phosphorylated. Phosphorylated AQP2 rides on the vesicular transport system and moves to the cell membrane on the side that is in contact with urine (luminal membrane), and water flows into the cell through AQP2 expressed on the luminal membrane. do. The inflowing water molecules are reabsorbed into the blood vessel via AQP3 and AQP4 expressed on the opposite cell membrane (blood vessel side membrane). In other words, it can be said that when the 269th amino acid residue in AQP2 is phosphorylated, AQP2 becomes able to exert its physiological functions.

By the way, the amount of water filtered by the glomerulus per day for an adult is 180 liters, and the daily urine output is around 1.8 liters. Approximately 99% of the filtered water is reabsorbed in the kidneys. What ultimately determines the amount of renal reabsorption is the vasopressin-AQP2 system mentioned above. As mentioned above, vasopressin secretion from the pituitary gland that activates this system is caused by an increase in blood osmolality and a decrease in blood volume. Therefore, in a wide variety of diseases such as hemorrhage, edema, heart failure, hypertension, liver cirrhosis, pulmonary disease, renal disease, central disease, inflammatory disease, sepsis, and tumors, the vasopressin-AQP2 system is activated and a compensatory mechanism occurs.

If compensation cannot be achieved, the above-mentioned diseases will worsen. Therefore, there is a strong need for the development of a measurement system that can accurately assess the status of the vasopressin-AQP2 system in clinical settings and accurately diagnose the hydration status of patients.

Up to now, in order to understand the status of the vasopressin-AQP2 system, efforts have been made to develop a method for measuring blood vasopressin concentration. However, because vasopressin has a short half-life of 24 minutes and more than 90% of its half-life is bound to platelets, accurate measurement results cannot currently be obtained and it is hardly used in clinical practice.

In addition, Non-Patent Document 1 states that urinary exosomes from rats with acute kidney injury due to renal ischemia reperfusion contain AQP2 in which the 269th amino acid residue is phosphorylated, and that the AQP2 was successfully measured. It is disclosed that what has happened. That is, Non-Patent Document 1 discloses that the effects of damage directly applied to the kidney can be monitored using AQP2, which is phosphorylated at the 269th amino acid residue contained in urine exosomes.

Am J Physiol Renal Physiol 314: F584-F601, 2018

However, there is no known method for determining the body's water status, such as dehydration or water overflow, by determining the vasopressin-AQP2 system, and the present invention aims to identify biomarkers that can be used to determine the water status. The purpose of this invention is to provide a method of determining the moisture state using this.

In order to achieve the above-mentioned purpose, the present inventors conducted intensive studies and found that aquaporin 2 contained in urine samples, especially aquaporin 2 in which the 269th amino acid residue is phosphorylated, is useful as a biomarker for understanding water status. The inventors have discovered that this is the case, and have completed the following invention. The present invention includes the following inventions.

(1) A method for determining moisture status, including a step of measuring aquaporin 2 in a urine sample of a subject.
(2) The method for determining water status according to (1), which comprises measuring aquaporin 2 present in exosomes contained in the urine sample.
(3) The method for determining water status according to (1), wherein the aquaporin 2 has a 269th amino acid residue phosphorylated.
(4) Measuring aquaporin 2 in the urine sample of the subject; comparing the amount of aquaporin 2 in the urine sample of the subject with the amount of aquaporin 2 in the urine sample of the control sample; and The subject is dehydrated when the amount of aquaporin 2 in the subject's urine sample is greater than the amount of aquaporin 2 in the control urine sample. The method for determining the water state according to (1), comprising the step of determining that the subject is in a water-filled state when the amount of aquaporin 2 is small compared to the amount of aquaporin 2.
(5) The method for determining moisture status according to (1), wherein the subject is a human.
(6) The method for determining water status according to (1), wherein the measurement of aquaporin 2 is performed by an immunoassay using an antibody and/or a fragment thereof that specifically binds to aquaporin 2.

According to the present invention, the vasopressin-AQP2 system of a subject can be determined based on aquaporin 2 in a urine sample, and the water status can be determined. Therefore, by applying the present invention, it is possible to contribute to simpler and more accurate diagnosis of the moisture status of a subject.

FIG. 2 is a characteristic diagram showing the analysis results of pS269-AQP2 by Western blotting. FIG. 2 is a characteristic diagram showing the analysis results for total AQP2 by Western blotting.

1. Subject In the present invention, the "subject" is not limited as long as it is an animal whose water status is to be ascertained, but humans are particularly preferred. Examples of non-human subjects include non-human primates such as monkeys and chimpanzees, and other mammals such as dogs, cows, mice, rats, and guinea pigs.

In addition, the subject may include a patient suffering from a disease or a patient suspected of having the disease. Diseases include, but are not limited to, bleeding, edema, heart failure, hypertension, liver cirrhosis, pulmonary disease, renal disease, central disease, inflammatory disease, sepsis, tumor, heat stroke, and various other diseases for which it is important to understand the hydration status. can be mentioned. Note that these diseases are merely listed as examples, and patients suffering from or suspected of suffering from diseases other than these are also subjects.

2. Sample In the present invention, a urine sample from a subject is used as a sample. The urine sample can be obtained by subjecting collected urine to, for example, the following treatments. That is, first, a protease inhibitor solution is added to urine on ice, and then centrifuged (1,000 g, 10 minutes, 4°C) to obtain a supernatant. After diluting a portion of the supernatant 10 times, measure the creatinine concentration in the urine using a chemical analyzer. Urine proteins are separated from the remaining supernatant using two methods, a filter method and an ultracentrifugation method, as described below.

Place urine containing 3 mg of creatinine in a centrifugal filtration tube (Millipore.Carrigtwohill.co.) and centrifuge (4,000 g, 4°C) until the volume is less than 200 μl. Then, add 4x Laemmli sample buffer (62.5mM Tris-HCl, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 0.4 M DTT) to a total volume of 300μl and incubate at 37℃ for 30 minutes. Incubate and use as a sample (filter method).

Centrifuge the urine containing 3 mg of creatinine (200,000 g, 60 min, 4°C). Add 50 μl of a 5-fold diluted protease inhibitor solution to the pellet, dissolve it, and add 25 μl of 4× Laemmli sample buffer (62.5 mM Tris-HCl, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 0.4 M DTT). ) and incubate at 37°C for 30 minutes to prepare a sample (ultracentrifugation method).

In this method, urine samples treated by the above two methods can be used, but if the EIA method using a high titer antibody is used, a solution obtained by simply adding a protease inhibitor solution to urine can be used. The present invention can also be carried out using the following methods.

Moreover, in the present invention, it is particularly preferable that the urine sample contains a fraction containing exosomes (also referred to as exosomes). Exosomes are one of the organelles secreted by cells, and are membrane vesicles with a diameter of approximately 50 nm to 150 nm.

The fraction containing exosomes is not particularly limited, and can be collected by conventionally known techniques. For example, methods for collecting exosome fractions include methods using stepwise centrifugation or ultracentrifugation. Examples of the ultracentrifugation method include a method that combines a sucrose density gradient solution and a sucrose cushion solution, and can separate exosomes, which have a relatively low density, from other particles such as endoplasmic reticulum. . More specifically, a urine sample prepared according to a standard method is centrifuged at 1000 x g for 30 minutes. Collect the supernatant after centrifugation, add protease inhibitors, and centrifuge at 17,000 x g for 15 minutes. Thereafter, collect the supernatant and centrifuge at 200,000 x g for 1 h. The resulting precipitate can be collected as an exosome fraction.
Furthermore, exosome fractions can also be collected using a commercially available reagent kit.

3. Aquaporin 2
In this specification, aquaporin 2 typically refers to a mature or complete aquaporin 2, and also includes aquaporin 2 molecules modified with sugar chains. The type of sugar chain, the bonding position, etc. are not limited. The amino acid sequence of aquaporin 2 differs slightly depending on the animal species tested. As an example, aquaporin 2 derived from human (Homo sapiens) is shown as SEQ ID NO: 1 in the sequence listing.

In particular, Aquaporin 2 to be measured is preferably one in which at least one amino acid residue selected from the group consisting of amino acid residues 256, 264, and 269 in its amino acid sequence is phosphorylated. Furthermore, it is more preferable that the aquaporin 2 to be measured has the 269th amino acid residue in its amino acid sequence phosphorylated.

4. Anti-Aquaporin 2 Antibody Aquaporin 2 is preferably measured using an antibody that can specifically bind to Aquaporin 2. As such an antibody, an antibody produced by a conventional method using as an antigen aquaporin 2 of the animal species to be measured or a polypeptide containing a partial sequence thereof can be suitably used.

The antibody may be a polyclonal antibody or a monoclonal antibody. Antibodies can also be used as fragments as long as they can specifically bind to aquaporin-2. Examples of antibody fragments include Fab fragments, F(ab)'2 fragments, single chain antibodies (scFv), and the like.

Monoclonal antibodies can be produced, for example, by the following procedure. That is, first, the above-mentioned antigen is administered to an animal by itself or together with a carrier and a diluent at a site where antibody production is possible by administration of the antigen. In order to increase antibody production ability during administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Examples of animals used include mammals such as monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats. The antibody titer in antiserum can be measured by a conventional method.

By selecting individuals with a recognized antibody titer from animals immunized with the antigen, collecting their spleens or lymph nodes 2 to 5 days after the final immunization, and fusing the antibody-producing cells contained therein with myeloma cells. Monoclonal antibody-producing hybridomas can be prepared. The fusion operation can be carried out according to known methods, for example, the method described in Nature 256: 495 (1975). Examples of the fusion promoter include polyethylene glycol (PEG). Examples of myeloma cells include NS-1, P3U1, and SP2/0.

Selection of monoclonal antibodies can be carried out according to known methods or similar methods, and usually can be carried out using an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium may be used as long as it allows hybridomas to grow. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in antiserum.

Separation and purification of monoclonal antibodies can be carried out in the same manner as the usual separation and purification of polyclonal antibodies, such as salting-out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, adsorption-desorption method using an ion exchanger (e.g. DEAE), ultraviolet This can be carried out according to immunoglobulin separation and purification methods using centrifugation, gel filtration, antigen-binding solid phase, or specific purification using protein A, protein G, or the like.

On the other hand, polyclonal antibodies can be produced, for example, by the following procedure. That is, polyclonal antibodies can be produced by, for example, making a complex of an antigen and a carrier, immunizing a mammal in the same manner as the above-mentioned monoclonal antibody production method, and collecting a substance containing antibodies against activated haptoglobin from the immunized animal. , can be produced by separating and purifying antibodies. The antigen used for producing polyclonal antibodies is the same as for producing monoclonal antibodies. When forming a complex between an antigen and a carrier, the type of carrier and the mixing ratio of the antigen and the carrier should be determined so that antibodies can be efficiently produced against the antigen cross-linked to the carrier. It may be crosslinked with As the carrier, for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. are used. Further, various condensing agents can be used for coupling the antigen and the carrier, and examples include glutaraldehyde, carbodiimide, maleimide active ester, active ester reagents containing a thiol group, and a dithioviridyl group.

The antigen-carrier complex is administered to the animal to be immunized at a site where antibody production is possible, either by itself or together with a carrier or diluent. In order to increase antibody production ability during administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Administration can be carried out usually once every 2 to 6 weeks, about 3 to 10 times in total. The animals used include the same mammals as in the case of monoclonal antibody production. The polyclonal antibody can be collected from the blood, ascites, etc. of an animal immunized by the above method, preferably from the blood. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of polyclonal antibodies can be carried out in the same manner as for the separation and purification of monoclonal antibodies described above.

5. Method for evaluating the amount of aquaporin 2 Although the method of the present invention includes a step of measuring aquaporin 2 in a urine sample from a subject, it is not necessary to measure the absolute amount of aquaporin 2, and urine as a control is used. It is sufficient for evaluation if the relative relationship with the amount of aquaporin 2 in the sample can be clarified.

A typical method for evaluating the amount of aquaporin 2 includes immunoassay using the above-mentioned anti-aquaporin 2 antibody. There are no particular restrictions on the immunoassay, and conventionally known methods such as enzyme immunoassay (EIA), latex agglutination, immunochromatography, Western blotting, radioimmunoassay (RIA), and fluorescence can be used. Immunoassay method (FIA method), luminescence immunoassay method, spin immunoassay method, nephelometric method that measures the turbidity associated with the formation of antigen-antibody complexes, and uses antibody solid-phase membrane electrodes to measure potential changes due to binding with antigen. An enzyme sensor electrode method for detection, an immunoelectrophoresis method, etc. can be employed. Among these, EIA method or Western blotting method is preferred. Note that the EIA method includes a competitive enzyme immunoassay method, a sandwich enzyme-linked immunosorbent assay method (sandwich ELISA method), and the like.

When Western blotting is employed as the immunoassay method of the present invention, aquaporin 2 in a urine sample can be detected, for example, as follows. A protein sample derived from a urine sample as a specimen is added onto a polyacrylamide gel containing sodium dodecyl sulfate, electrophoresis is performed by applying a constant voltage, and the proteins separated on the gel are separated using PVDF (polyvinidene difluoride). After electrically transferring to a blotting membrane such as a membrane and blocking this membrane with skim milk etc., the anti-aquaporin 2 antibody is reacted with the membrane, and then a chemiluminescent substance, a fluorescent substance, or an enzyme (horseradish perilla) is reacted with the membrane. A secondary antibody labeled with oxidase, etc.) is bound to the antibody, and further detection operations are performed depending on the labeling substance. Aquaporin 2 can be detected if present on the membrane. Note that a specific detection method using Western blotting is described in Examples below.

In particular, the method of the present invention determines the relative relationship between the amount of aquaporin 2 in a urine sample and the amount of aquaporin 2 in a control urine sample (ie, whether aquaporin 2 is high or low). Therefore, when comparing the amount of aquaporin 2 between urine samples, it is preferable that the total amount of protein contained in the urine samples to be compared be approximately the same, or that the amount of aquaporin 2 be corrected based on the total amount of protein.

In addition, in the method of the present invention, when measuring the amount of aquaporin 2 in exosomes, the total amount of exosomes contained in the urine samples to be compared is made approximately the same, or the amount of aquaporin 2 is corrected based on an index of the amount of exosomes. It is preferable to do so. Here, examples of indicators of the amount of exosomes include Alix protein and TSG101 protein, which are known as marker molecules of exosomes that exist inside exosomes. That is, it is preferable to confirm that the amounts of exosomes in urine samples to be compared are approximately the same, or to correct the measured amount of aquaporin 2 based on the amounts of these Alix proteins and TSG101 proteins.

6. Diagnostic method In the present invention, the amount of aquaporin 2 in a urine sample is measured, and the water status is determined using the amount of aquaporin 2 as an index. In the present invention, "diagnosis" typically refers to determining the water status (dehydration or overflowing state) of a subject (diagnosis in a narrow sense), but is not limited to this. This is a concept (diagnosis in a broad sense) that also includes the determination of the severity of water damage (or flooding).

When determining the water state of a subject, for example, the amount of aquaporin 2 in a urine sample of a healthy subject can be used as a reference. When determining the effectiveness of treatment, the amount of aquaporin 2 in the urine sample of the subject before treatment can also be used as a reference value. In the present invention, these samples to be compared are referred to as "control samples."

Here, the moisture condition can also be referred to as moisture balance. Hydration status includes dehydration due to water loss or water shortage, flooding status where excess water is stored in the body, and edema which is excessive retention of water in the interstitium.

In the method of the present invention, the subject is dehydrated when the amount of aquaporin 2 in the urine sample of the subject is large compared to the amount of aquaporin 2 in the urine sample of the control sample. When the amount of aquaporin 2 is small compared to the amount of aquaporin 2 in the urine sample of the control specimen, it is determined that the subject is in a water-filled state.

The amount of aquaporin 2 in the urine sample of the control specimen can be measured using the same procedure as the measurement of the amount of aquaporin 2 in the urine sample of the subject. The amount of aquaporin 2 in the urine sample of the control specimen may be measured each time urine is collected from the specimen, or may be measured in advance.

In the method of the present invention, the amount of aquaporin 2 is measured in a urine sample rather than a blood sample of a subject. Compared to blood collection, urine collection is extremely less invasive to the patient and easier. Therefore, the method of the present invention can easily measure the amount of aquaporin 2 without placing a burden on the patient, and can contribute to accurate and rapid determination of hydration status.

7. Diagnostic agent or diagnostic kit The present invention also relates to a diagnostic agent for hydration status, which comprises an antibody capable of specifically binding to aquaporin-2.

The diagnostic agent of the present invention can also be made into a kit together with necessary reagents. For example, a diagnostic kit may include necessary components such as an anti-aquaporin 2 antibody, a labeled secondary antibody, and a reagent for detection.
The kit may further contain buffer solutions, washing solutions, instructions for use, and the like.

These diagnostic agents or antibodies in the diagnostic kit are used for the method for determining water status using the amount of aquaporin 2 in a urine sample as an index, as disclosed herein.

1. Experimental procedure
Eight-week-old male SD rats were divided into a control group under free drinking conditions, a dehydration group under water deprivation conditions, and an overflowing group under free drinking conditions with 20% sucrose solution, and kept for 3 days. Blood and urine were collected from each group on the third day and tested according to standard methods. In addition, exosomes were separated from urine by stepwise centrifugation, and AQP2 in urine exosomes was examined by Western blotting (WB).
Table 1 shows the results of urine tests in each group.

Furthermore, the results of blood tests in each group are shown in Table 2.

As shown in Tables 1 and 2, the results of urine osmolality measurements showed a significant increase in the dehydrated group, a significant decrease in the overflow group, and a significant increase in hematocrit value in the dehydrated group. These results indicate that each group is valid as a model.

Next, the exosome fraction was separated from the urine collected in a container containing a protease inhibitor as follows. First, the cells were centrifuged at 1,000 x g for 15 minutes to remove nuclei and the like, and then the supernatant was centrifuged at 17,000 x g for 15 minutes. Thereafter, the supernatant was centrifuged at 200,000×g for 1 hour, and the precipitate was collected as an exosome fraction.

Aquaporin 2 protein was quantitatively analyzed by Western blotting using the obtained exosome fraction according to the following procedure. In this example, Aquaporin 2 in which the 269th amino acid group was phosphorylated (pS269-AQP2) was analyzed with and without sugar chain modification. Furthermore, in this example, total AQP2, which is a combination of phosphorylated AQP2 and non-phosphorylated AQP2, was analyzed for those that had undergone sugar chain modification and those that had not. Each sample was mixed with 4x sample buffer (0.5 M Tris-HCl, 8% SDS, 50% glycerol, 0.01% bromophenol blue, 0.2 M DTT) and incubated at 37°C for 30 min to prepare SDS-HCl as a protein sample. PAGE was performed, and the proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-dry type (KS-8 460, Marisol, Tokyo) blotting device. After the transfer, it was washed in TBS with shaking on a rotator (5 minutes, twice), and blocked (overnight, 4°C) with TBS containing 5% skim milk and Tween-20 (5% SM TTBS). After blocking, the primary antibody (anti-pS269-AQP2 antibody, provided by Gunma University; anti-AQP2 antibody, manufactured by Alomone, catalog number AQP-002) diluted 1:100 with 1.6% SM TTBS was reacted for 60 minutes ( room temperature). After the reaction, wash the primary antibody with TTBS (5 minutes, 2 times), react with the secondary antibody (anti-rabbit IgG antibody) diluted 1:1000 with 1.6% SM TTBS, and wash with TTBS (5 minutes). 4 times for 1 minute) and finally washed with TBS (1 time for 10 minutes). The band was visualized using SuperSignal (registered trademark) West Femto Maximum Sensitivity Substrate (PIERCE, IL), photographed using ImageQuant LAS 4000mini (GE Healthcare Japan), and using the attached software to visualize the band with a molecular weight of around 25 kDa. AQP2 without sugar chains was quantified from the band, and sugar chain AQP2 was quantified from the band with a molecular weight in the range of 30 to 37 kDa.

2. Experimental Results Figure 1 shows the analysis results for pS269-AQP2 by Western blotting. FIG. 1 shows an electrophoretic photograph of SDS-PAGE and data obtained by quantifying sugar chain pS269-AQP2 and sugar chain-free pS269-AQP2 based on the image. Furthermore, Figure 2 shows the analysis results for total AQP2 by Western blotting. FIG. 2 shows an electrophoretic photograph of SDS-PAGE and data obtained by quantifying total sugar chain AQP2 and total AQP2 without sugar chains based on the electrophoretic photograph.

In this example, three rats were used in each group (n=3). The lower graphs of FIGS. 1 and 2 show each data as mean±SE and the distribution of values for each individual.

The results shown in Figure 1 revealed that pS269-AQP2 significantly increased in the dehydrated group, and pS269-AQP2 significantly decreased in the flooded group. Furthermore, the results shown in Figure 2 revealed that no significant change was observed in total AQP2 in the dehydrated group, and that total AQP2 tended to decrease in the flooded group.

From these results, by measuring pS269-AQP2 in urine, it is possible to determine the state of the vasopressin-AQP2 system, that is, the water state (dehydration state, overflow state), and also to measure total AQP2 in urine. It was shown that the state of the vasopressin-AQP2 system, especially the overflow state, could be determined by doing so.

Claims (5)

A method for determining the water status of a subject, the method comprising the step of measuring aquaporin 2 , in which the 269th amino acid residue is phosphorylated, contained in a urine sample of the subject. The method according to claim 1, characterized in that aquaporin 2 present in exosomes contained in the urine sample is measured. a step of measuring aquaporin 2 in which the 269th amino acid residue contained in the urine sample of the subject is phosphorylated ;
The amount of aquaporin 2 phosphorylated at the 269th amino acid residue contained in the urine sample of the subject is compared with the amount of aquaporin 2 phosphorylated at the 269th amino acid residue contained in the urine sample of the control sample. The process of
The amount of aquaporin 2 in which the 269th amino acid residue is phosphorylated in the urine sample of the subject is compared with the amount of aquaporin 2 in the control sample in which the 269th amino acid residue is phosphorylated. When the test subject is dehydrated, the amount of aquaporin 2 in which the 269th amino acid residue contained in the test subject's urine sample is phosphorylated is higher than the 269th amino acid residue contained in the control test sample's urine sample. determining that the subject is in a flooded state when the residue is small compared to the amount of phosphorylated aquaporin 2;
The method according to claim 1. 2. The method according to claim 1, wherein the subject is a human. The measurement of aquaporin 2 in which the 269th amino acid residue is phosphorylated is an immunoassay method using an antibody and/or its fragment that specifically binds to aquaporin 2 in which the 269th amino acid residue is phosphorylated. 2. The method according to claim 1, which is carried out by.