JP2013130524A - Method of diagnosing end-of-life renal failure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of diagnosing an end-of-life renal failure and a transition from a chronic renal failure to the end-of-life renal failure through the use of a reliable index on which a kidney function state is further reflected.SOLUTION: A method of diagnosing an end-of-life renal failure includes the steps of: measuring the amounts of aquapolin 1 and aquapolin 2 in the urine of a subject; and comparing the amounts of aquapolin 1 and aquapolin 2 in the urine of the subject with the amounts of aquapolin 1 and aquapolin 2 in the urine of a contrast. When the amounts of aquapolin 1 and aquapolin 2 in the urine of the subject is reduced to be equal to or less than 10% when compared with the amounts of aquapolin 1 and aquapolin 2 in the urine of the contrast, it is determined that the subject suffers from the end-of-life renal failure or in a transition stage into the end-of-life renal failure.

Description

  The present invention relates to a method for diagnosing end stage renal failure using urinary aquaporin 1 and aquaporin 2 as marker proteins.

  When renal failure progresses to end-stage renal failure, treatment is limited to kidney transplantation or blood artificial dialysis. The number of dialysis patients in Japan continues to increase by 10,000 each year, 210,000 in 2000 and 260,000 in 2005. In addition, the number of patients is expected to increase further due to the westernization of the global diet and the increase in metabolic syndrome. The medical cost of dialysis is high and is said to be about 5 million yen per person per year in Japan. The total amount of medical expenses that the government spends on dialysis is over 1 trillion yen per year. Yes. Moreover, the enormous medical costs are increasing.

  Currently, the decision to introduce dialysis is based on the kidney size, blood calcium and blood bicarbonate ion concentrations, etc., as determined by ultrasonic diagnosis. However, these are only indices for estimating the renal function, and an index that more reflects the functional state of the kidney is required. In addition, blood creatinine level and blood urea nitrogen level can be an indirect indicator, but if the kidney has a dysfunction, both levels are quite good even if end-stage renal failure does not occur. Since it increases, it is insufficient as a diagnostic index for end-stage renal disease. Thus, there is currently no effective index that can grasp the functional state of the kidney at the stage of end-stage renal failure or the transition stage to end-stage renal failure.

  On the other hand, aquaporin (AQP), which was discovered as a membrane protein molecule that allows water molecules to permeate, has now been reported as 13 molecular species belonging to the same protein molecule family. In the kidney, aquaporin 1 is present in the epithelial cells of the proximal tubule and Henle's narrow descending limb, aquaporin 11 in the epithelial cells of the proximal tubule, and aquaporin 7 in the epithelial cells of the proximal straight tubule (especially the S3 segment). However, aquaporin 2, aquaporin 3 and aquaporin 4 are site-specific in the main cells of the collecting duct, aquaporin 6 in the α-interstitial cells in the collecting duct, and aquaporin 8 in the proximal tubule and epithelial cells in the collecting duct are site-specific. It is known to be expressed.

  Based on the fact that aquaporin is specifically expressed in proximal tubule cells and that the proximal tubule is selectively impaired in acute renal failure, the inventors have found that aquaporin 1 in urine Diagnosis method of acute renal failure caused by tissue damage by measurement (Patent Document 1), and drug by measurement of aquaporin 2 in urine, diagnostic method of acute renal failure caused by ischemia reperfusion (Patent Document 2) Established. However, any of these methods is a method for diagnosing acute renal failure, and does not determine end-stage renal function. Moreover, although the method of characterizing the pathological state of the kidney by a renal RNA marker is reported (patent document 3), there is no description regarding protein expression in end stage renal failure.

  End stage renal failure often progresses from chronic renal failure due to chronic glomerulonephritis, diabetes, etc., rather than from acute renal failure mainly due to ischemia reperfusion, drugs, and tissue damage. In addition, the site where the disorder is observed is different from the glomerulus in the case of chronic renal failure, whereas the acute renal failure is a tubule.

  In chronic renal failure, kidney damage progresses irreversibly, the number of tissues involved in kidney urine production decreases, the homeostasis of fluid composition cannot be maintained, and kidney function is gradually impaired over months to years, A state that is 50% or less of the normal state. Chronic renal failure is divided into stages 1 to 4 based on the results of renal function tests for serum creatinine and creatinine clearance. Most of the renal function is still compensated for in the first stage (renal reserve decline period) and the second stage (metabolic renal failure stage), but in the third stage (decompensated renal failure stage) Then, the compensation is broken, and various symptoms such as severe hypernitrogenemia, acidosis, hypocalcemia, hyperphosphatemia, hyponatremia, hyperkalemia and anemia appear. The fourth stage (uremic stage) is the end stage of renal failure, which results in uremia, necessitating the introduction of dialysis therapy, and kidney transplantation may be performed. Chronic renal failure does not recover its function, so the main treatment is to delay the progression as much as possible by dietary therapy that restricts water, salt and protein, and drug therapy that controls hypertension. In the case of the second stage chronic kidney, it has been reported that the patient can be recovered to the first stage chronic kidney in 6 years by medical intervention such as medication and diet by early diagnosis. (Fourth stage) If the transition to renal failure can be detected at an early stage, it is possible to extend the period until the introduction of dialysis by medical intervention, and it is possible to grasp the appropriate time for the introduction of dialysis.

JP2008-175630 WO 2010/150613 JP2008-504047

  The object is to provide a method for diagnosing end-stage renal failure and transition from chronic renal failure to end-stage renal failure using a reliable index that more reflects the functional state of the kidney.

  The present inventors have focused on aquaporins 1 and 2 contained in urine and examined the amounts of aquaporin 1 and aquaporin 2 contained in the exosome fraction, particularly in urine samples obtained from patients with end-stage renal failure. However, the amount of any of these was significantly decreased compared with that of a healthy person, whereas in patients with chronic renal failure exhibiting proteinuria, the decrease in the amount was not found, and the present invention was completed. .

That is, the present invention includes the following inventions.
(1) A method for diagnosing end-stage renal failure, comprising measuring the amount of aquaporin 1 and aquaporin 2 in the urine of a subject.
(2) measuring the amount of aquaporin 1 and aquaporin 2 in the subject's urine, and the amount of aquaporin 1 and aquaporin 2 in the subject's urine with the amount of aquaporin 1 and aquaporin 2 in the control urine Comparing the subject's urinary aquaporin 1 and aquaporin 2 to 10% or less as compared to the amount of aquaporin 1 and aquaporin 2 in the control urine The method according to (1), wherein it is determined that the patient has stage renal failure or is in a transitional stage to end stage renal failure.
(3) The method according to (1) or (2), wherein the amount of aquaporin 1 and aquaporin 2 in the urine is measured with an exosome fraction obtained from urine.
(4) The method according to any one of (1) to (3), wherein the subject is a human.

(5) Aquaporin 1 and aquaporin 2 are measured by immunological measurement methods using an antibody that specifically binds to aquaporin 1 and an antibody that specifically binds to aquaporin 2, respectively (1) to (4) The method in any one of.
(6) A diagnostic agent for end-stage renal disease, comprising an antibody that can specifically bind to aquaporin 1 and an antibody that can specifically bind to aquaporin 2.
(7) A diagnostic kit for end-stage renal failure, comprising an antibody that can specifically bind to aquaporin 1 and an antibody that can specifically bind to aquaporin 2.

  According to the present invention, it is only necessary to examine the amount of aquaporin 1 and aquaporin 2 in the urine of a subject, particularly in the urinary exosome fraction, or the subject has end-stage renal failure or the transition stage to end-stage renal failure Can be determined. Since the diagnostic method of the present invention is based on a reliable index that more accurately reflects the functional state of the kidney, an accurate determination of dialysis introduction and the transition stage to end-stage renal failure can be grasped early. As a result, it is possible to open up the possibility of recovery by diet and drug therapy for patients with chronic renal failure and to extend the period until dialysis is introduced by medical intervention. And support costs can be greatly reduced.

The alignment of the amino acid sequence of canine, bovine, human, rat and mouse aquaporin 1 is shown. The alignment of the amino acid sequence of human, canine (predicted), bovine, rat and mouse aquaporin 2 is shown. The amounts of aquaporin 1 (AQP1) and aquaporin 2 (AQP2) in the urinary exosome fraction of patients with end-stage renal failure are shown. The dotted line represents the level of a healthy person (control).

  The present invention relates to a method for diagnosing end-stage renal failure, comprising the step of measuring the amount of aquaporin 1 and aquaporin 2 in the urine of a subject, particularly in the urinary exosome fraction.

  In the present invention, the “subject” is not limited as long as it is an animal that can develop end-stage renal failure, but a human is particularly preferable. Examples of subjects other than humans include non-human primates such as monkeys and chimpanzees, and other mammals such as dogs, cows, mice, rats, and guinea pigs. The information (judgment results) obtained when animals other than humans are used as subjects can be used for the diagnosis of end-stage renal failure in the non-human animals. Useful in that it can be used for establishment. As shown in FIGS. 1 and 2, the amino acid sequences of aquaporin 1 and aquaporin 2 are highly conserved across species. It has also been reported that changes in urinary aquaporin excretion seen in disease model animals such as rats can also be seen in patients. Therefore, it can be said that the experimental results in non-human animals can be extrapolated to humans.

  In the method of the present invention, urine from a subject is used as a sample. Since aquaporins 1 and 2 are thought to be present in large amounts in urinary exosomes, it is preferable to prepare a sample containing a urinary exosome fraction. The sample is prepared as follows, for example. First, urine is collected from the subject, centrifuged at 1,000 g for 10 minutes, then the supernatant is centrifuged at 17,000 g for 15 minutes, and the supernatant is further centrifuged at 200,000 g for 60 minutes. Separate as an exosome fraction. The protein in the exosome fraction can be mixed with 4x sample buffer (0.5M Tris-HCl, 8% SDS, 50% glycerol, 0.01% bromophenol blue, 0.2M DTT) and incubated at 37 ° C for 30 minutes. Solubilize to make a sample.

  As used herein, aquaporin 1 is typically a mature or complete form of aquaporin 1, and also includes a sugar-modified aquaporin 1 molecule. The type of sugar chain, the binding position, etc. are not limited. The amino acid sequence of aquaporin 1 varies slightly depending on the animal species of the subject. As an example, the sequence listing includes canine (Canis familiaris) aquaporin 1 as SEQ ID NO.1, bovine (Bos taurus) aquaporin 1 as SEQ ID NO.2, human (Homo sapiens) aquaporin 1 as SEQ ID NO. No. 4 shows rat (Rattus norvegicus) -derived aquaporin 1, and SEQ ID NO: 5 shows mouse (Mus musculus) -derived aquaporin 1.

  Similarly, aquaporin 2 in the present specification is typically a mature or complete form of aquaporin 2, and includes a sugar-modified aquaporin 2 molecule. The type of sugar chain, the binding position, etc. are not limited. The amino acid sequence of aquaporin 2 varies slightly depending on the animal species of the subject. As an example, the sequence listing includes human (Homo sapiens) aquaporin 2 as SEQ ID NO: 6, canis familiaris aquaporin 2 as SEQ ID NO: 7, bovine (Bos taurus) aquaporin 2 as SEQ ID NO: 8, SEQ ID NO: 9 shows rat (Rattus norvegicus) -derived aquaporin 2, and SEQ ID NO: 10 shows mouse (Mus musculus) -derived aquaporin 2.

  Aquaporin 1 is preferably measured using an antibody that can specifically bind to aquaporin 1. Similarly, aquaporin 2 is preferably measured using an antibody that can specifically bind to aquaporin 2. As such an antibody, an antibody prepared by a conventional method using aquaporin 1, aquaporin 2 or a polypeptide containing a partial sequence thereof as an antigen can be preferably used.

  The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody can also be used as a fragment as long as it can specifically bind to aquaporin 1 or aquaporin 2. Examples of antibody fragments include Fab fragments, F (ab ′) 2 fragments, single chain antibodies (scFv), and the like.

A monoclonal antibody can be prepared, for example, by the following procedure.
The above-mentioned antigen is administered to animals at a site where antibody production is possible by administration of the antigen, together with a carrier or a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Examples of animals used include mammals such as monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and the like. The antibody titer in the antiserum can be measured by a conventional method.

  By selecting individuals with antibody titers from animals immunized with antigen, collecting spleen or lymph nodes 2-5 days after the final immunization, and fusing the antibody-producing cells contained in them with myeloma cells A monoclonal antibody-producing hybridoma can be prepared. The fusion operation can be performed according to a known method, 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, SP2 / 0, and the like.

  Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto, but can be usually performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the antiserum.

  Separation and purification of the monoclonal antibody is the same as the separation and purification of the normal polyclonal antibody, for example, salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (for example, DEAE), It can be carried out according to an ultracentrifugation method, a gel filtration method, an antigen-binding solid phase, or a method for separating and purifying immunoglobulins by a specific purification method using protein A or protein G.

On the other hand, a polyclonal antibody can be prepared, for example, by the following procedure.
For example, a polyclonal antibody forms a complex of an antigen and a carrier, immunizes a mammal in the same manner as the above-described monoclonal antibody production method, collects an antibody-containing substance against active haptoglobin from the immunized animal, It can manufacture by performing isolation | separation purification. The antigen used for the production of the polyclonal antibody is the same as that for the production of the monoclonal antibody. When forming a complex of an antigen and a carrier, the type of carrier and the mixing ratio of the antigen and the carrier can be any ratio and any ratio as long as the antibody can be efficiently produced against the antigen cross-linked to the carrier. It may be crosslinked with Examples of carriers that can be used include bovine serum albumin, bovine thyroglobulin, keyhole limpet, hemocyanin and the like. Various condensing agents can be used for coupling the antigen and the carrier, and specifically, an active ester reagent containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, or dithiobilidyl group is used.

  The complex of an antigen and a carrier is administered to a immunized animal at a site where antibody production is possible, or with a carrier or a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total. Examples of animals used include mammals similar to those used in the production of monoclonal antibodies. Polyclonal antibodies can be collected from blood, ascites, etc., preferably blood of animals immunized by the above method. 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 the polyclonal antibody can be performed by the same procedure as the separation and purification of the monoclonal antibody.

  The method of the invention comprises the step of assessing the amount of aquaporin 1 and aquaporin 2 in the urine from a subject. The amount of aquaporin 1 and aquaporin 2 may be measured in absolute amounts, for example, by comparison with a standard sample. However, it is not always necessary to measure the absolute amounts of aquaporin 1 and aquaporin 2, and it is sufficient as an evaluation if the relative relationship between the amounts of aquaporin 1 and aquaporin 2 in the control urine can be clarified. The same applies to the evaluation of the amount of aquaporin 1 and aquaporin 2 in the urinary exosome fraction.

  A typical example of the method for evaluating the amount of aquaporin 1 and aquaporin 2 is an immunological assay using the above-mentioned anti-aquaporin 1 antibody and aquaporin 2 antibody. The immunological measurement method is not particularly limited, and conventionally known methods such as enzyme immunoassay (EIA method), latex agglutination method, immunochromatography method, Western blot method, radioimmunoassay method (RIA method), Fluorescence immunoassay (FIA method), luminescence immunoassay, spin immunoassay, turbidimetric method for measuring turbidity associated with antigen-antibody complex formation, potential change due to antigen binding using antibody solid phase membrane electrode An enzyme sensor electrode method, an immunoelectrophoresis method, and the like that detect the above can be employed. Among these, EIA method or Western blot method is preferable. The EIA method includes a competitive enzyme immunoassay method, a sandwich enzyme-linked immunosorbent solid phase assay method (sandwich ELISA method), and the like.

  When the western blot method is employed as the immunological assay of the present invention, aquaporin 1 and aquaporin 2 can be detected in urine or urinary exosome fractions, for example, as follows. The sample urine or urinary exosome fraction is added onto a polyacrylamide gel containing sodium dodecyl sulfate, and electrophoresis is performed with a certain voltage applied, and the proteins separated on the gel by electrophoresis are converted into PVDF (polyvinylidene Electrically transfer to a blotting membrane such as a fluoride membrane. After blocking the membrane with skim milk or the like, the anti-aquaporin 1 antibody or anti-aquaporin 2 antibody is reacted with the membrane, and then a secondary antibody labeled with a chemiluminescent substance, a fluorescent substance, or an enzyme (such as horseradish peroxidase) is used. Then, the detection is performed according to the labeling substance. If aquaporin 1 or aquaporin 2 is present on the membrane, it can be detected. A specific detection method by Western blotting is described in Examples below.

  In the present invention, the amount of aquaporin 1 and aquaporin 2 in urine, particularly in the urinary exosome fraction, is measured, and end-stage renal failure is diagnosed by using both as a marker. Here, “end-stage renal failure” refers to a state in which the therapeutic effect of chronic renal failure is no longer recognized, and refers to renal failure that is the subject of long-term dialysis and kidney transplantation.

  In the present invention, `` diagnosis '' typically means determination of whether or not a subject suffers from end stage renal failure (diagnostic in a narrow sense), but is not limited thereto, chronic renal failure It is a concept (diagnosis in a broad sense) that includes the determination of whether or not the patient is in the transitional stage from renal failure to end-stage renal failure and the determination of the therapeutic effect of mild chronic renal failure. Preferably, “diagnosis” in the present invention means detection of end-stage renal failure in vitro. As shown in the examples, the amount of aquaporin 1 and aquaporin 2 in the urine is significantly reduced or almost zero in end-stage renal failure, so the method of the present invention is useful for early diagnosis of end-stage renal failure. (In this case, “diagnosis” is used in a narrow sense). Moreover, since urine is used as a sample, it can be said that it is a noninvasive method with little load on the subject.

  When determining whether a subject has end-stage renal failure, or whether the subject is in the transition phase to end-stage renal failure, aquaporin 1 and The amount of aquaporin 2 can be used as a reference. Alternatively, if the subject is a patient with mild to moderate chronic renal failure and the urine sample has normal amounts of aquaporin 1 and aquaporin 2 in the past examination, the urine sample in the past examination judged to be normal The amount of aquaporin 1 and aquaporin 2 in it can be based. In determining the effect of treatment, aquaporin 1 and aquaporin 2 in a urine sample collected from a subject before starting symptomatic treatment such as pharmacotherapy or diet therapy (for example, patients with stage 2 chronic renal failure) The amount can be used as a reference value. In the present invention, these samples to be compared are referred to as “controls”. Therefore, in the method of the present invention, the control includes a pool of urine obtained from normal individuals, a urine sample obtained from the subject before suspected end-stage renal failure, and a urine obtained from the subject before treatment. Samples are included.

  In a preferred embodiment, in addition to measuring the amount of aquaporin 1 and aquaporin 2 in the subject's urine, the present invention can be used to determine the amount of aquaporin 1 and aquaporin 2 in the subject's urine. And aquaporin 2 respectively, wherein the amount of aquaporin 1 and aquaporin 2 in the subject's urine is significant compared to the amount of aquaporin 1 and aquaporin 2 in the control urine, respectively. At the end of the period, it is determined that the subject has end-stage renal failure, is in transition to end-stage renal failure, or has advanced chronic renal failure compared to the control. The same applies when the amounts of aquaporin 1 and aquaporin 2 in the urinary exosome fraction of the subject are used for evaluation. Here, “remarkably decreases” specifically refers to “10% or less”, “5% or less”, “3% or less” with respect to the amount of aquaporin 1 and aquaporin 2 in the control urine. “2% or less”, “1% or less” decreases, and also includes a decrease to almost “0%”.

  That is, in the method of the present invention, typically, the amount of aquaporin 1 and aquaporin 2 in the urine of a subject is measured, and the amount is compared with the amount of aquaporin 1 and aquaporin 2 in the control urine, respectively. However, when the former is significantly less than the latter, it is determined that the patient has end-stage renal failure or is in the transition stage to end-stage renal failure. Here, in the determination of the therapeutic effect on chronic renal failure, the amount of aquaporin 1 and aquaporin 2 in the urine sample obtained from the subject after treatment using a urine sample obtained from the subject before treatment as a control Can be determined to have a therapeutic effect if increased compared to the control (ie, before treatment).

  Measurement of the amount of aquaporin 1 and aquaporin 2 in the urine of the control can be performed in the same procedure as the measurement of the amount of aquaporin 1 and aquaporin 2 in the urine of the subject. The amount of aquaporin 1 and aquaporin 2 in the control urine may be measured each time the urine of the subject is measured, or may be measured in advance.

  The present invention also relates to a diagnostic agent for end-stage renal failure, comprising an antibody that can specifically bind to aquaporin 1 and an antibody that can specifically bind to aquaporin 2.

  The diagnostic agent of the present invention can be made into a kit together with necessary reagents. For example, the detection kit may contain necessary components such as an anti-aquaporin 1 antibody and an anti-aquaporin 2 antibody, a labeled secondary antibody, a reagent for detection, and the like. The kit may further contain a buffer, a washing solution, instructions for use, and the like.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the scope of the present invention.

Example 1
1. Experimental method (1) Preparation of urine sample Under the approval of the Ethics Committee, an experiment was conducted using urine from time to time provided by a patient for whom consent was obtained. The provided urine is first centrifuged at 1,000 g for 15 minutes, then the supernatant is centrifuged at 17,000 g for 30 minutes, the supernatant is further centrifuged at 200,000 g for 60 minutes, and the precipitate is separated as an exosome fraction. did. The protein in the exosome fraction is solubilized by mixing with 4x sample buffer (0.5 M Tris-HCl, 8% SDS, 50% glycerol, 0.01% bromophenol blue, 0.2 M DTT) and incubating at 37 ° C for 30 minutes Sample. In addition, creatinine concentration in whole urine was measured using Fuji Dry Chem Slide (CRE-PIII, Fuji Photo Film Co., Ltd., Tokyo). On the other hand, the collected blood was turbid with heparin and then centrifuged at 12,000 rpm for 10 minutes to separate plasma. The plasma creatinine concentration was measured using Fuji Dry Chem Slide (CRE-PIII, Fuji Photo Film Co., Ltd., Tokyo).

(2) Protein detection in the sample The urinary exosome sample is subjected to SDS-PAGE according to a standard method, and the protein in the gel is polyvinylidene difluoride (KS-8460, Marisol, Tokyo) using a blotting apparatus (KS-8460, Marisol, Tokyo) Transferred to PVDF) membrane. After the transfer, the plate was washed in TBS while shaking with a rotator (15 minutes, twice), and blocked (overnight, 4 ° C.) with TBS (5% SM TTBS) containing 5% skim milk and Tween-20. After blocking, primary antibody (rabbit anti-aquaporin-1 antibody diluted 1: 1000 with 1.6% SM TTBS or goat anti-aquaporin-2 antibody diluted 1: 500 with 1.6% SM TTBS, Santa Cruz Baiotechnology, Inc. USA) for 60 minutes (room temperature). After the reaction, the primary antibody was washed with TTBS (5 minutes, twice), and reacted with a secondary antibody (anti-rabbit IgG antibody or anti-goat IgG antibody) diluted 1: 3000 with 1.6% SM TTBS, Washed with TTBS (5 minutes, 4 times) and finally washed with TBS (10 minutes, 1 time). Bands were visualized using SuperSignal (registered trademark) West Femto Maximam Sensitivity Subatrate (PIERCE, IL) and photographed with a polaroid camera (ECLTM-mini Camera, Amersham International plc., Tokyo).

(3) Data analysis Regarding the urinary exosome protein, the amount of protein contained in a sample containing a certain amount of creatinine was quantified, and the average value of the values in the control group was expressed as 100%. Each data was shown by the average value +/- standard error (SEM). Dunnett's method was used for the significant difference test.

2. Results FIG. 3 shows the results of summarizing the urinary aquaporin 1 or aquaporin 2 protein excretion amounts of 6 patients with end-stage renal failure. As apparent from FIG. 3, it was found that urinary aquaporin 1 and aquaporin 2 proteins were hardly detected in patients with end-stage renal failure. In addition, in a present Example, the result which remove | deviated from the other measurement result in one example of aquaporin 2 protein excretion amount is obtained. If this is regarded as an outlier, the excretion amount of the aquaporin 2 protein is almost zero, and can be made comparable to the excretion amount of the aquaporin 1 protein. On the other hand, although not shown in the figure, when the same examination was performed in 43 patients with chronic renal failure, urinary aquaporin 1 and aquaporin 2 protein excretion amounts were 550 ± 382 and 132, respectively, compared with healthy subjects. The expression level was ± 50%, and no significant difference was observed in the expression level.

  The above results indicate that when both urinary aquaporin 1 and aquaporin 2 protein excretion are significantly reduced, the kidney is hardly working, and by measuring them, end-stage renal failure can be diagnosed It was.

  The present invention can be used in the field of manufacturing diagnostic agents or kits for end stage renal failure.

Claims (7)

  A method for diagnosing end-stage renal failure, comprising measuring the amount of aquaporin 1 and aquaporin 2 in the urine of a subject.   Measuring the amount of aquaporin 1 and aquaporin 2 in the subject's urine and comparing the amount of aquaporin 1 and aquaporin 2 in the subject's urine with the amount of aquaporin 1 and aquaporin 2 in the control urine And the subject's urinary aquaporin 1 and aquaporin 2 are reduced to 10% or less compared to the amount of aquaporin 1 and aquaporin 2 in the control urine, the subject has end stage renal failure Or the method of claim 1, wherein the method is determined to be in the transitional stage to end stage renal failure.   The method according to claim 1 or 2, wherein the amount of aquaporin 1 and aquaporin 2 in the urine is measured in an exosome fraction obtained from urine.   The method according to claim 1, wherein the subject is a human.   The measurement of aquaporin 1 and aquaporin 2 is performed by an immunoassay using an antibody that specifically binds to aquaporin 1 and an antibody that specifically binds to aquaporin 2, respectively. the method of.   A diagnostic agent for end-stage renal disease, comprising an antibody that can specifically bind to aquaporin 1 and an antibody that can specifically bind to aquaporin 2.   A diagnostic kit for end-stage renal disease, comprising an antibody that can specifically bind to aquaporin 1 and an antibody that can specifically bind to aquaporin 2.

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