JP5777149B2 - Method for testing diseases associated with glomerular filtration membrane abnormality, and test kit for use therewith - Google Patents

Description

  The present invention relates to a method for examining a disease associated with an abnormality in a glomerular filtration membrane, and a test kit for use therein.

  A group of renal diseases that cause hypoproteinemia due to high proteinuria is collectively called nephrotic syndrome. Nephrotic syndrome is mainly caused by primary glomerular disease, diabetic nephropathy, IgA nephropathy and the like, accounting for 75% or more of the total. Among these, primary glomerular disease accounts for 61% of the causes of nephrotic syndrome. Examples of primary glomerular diseases include minute change nephrotic syndrome, membranous nephropathy, membranous proliferative glomerulonephritis, and mesangial proliferative glomerulonephritis. Of these, minimal change nephrotic syndrome (38.7%) and membranous nephropathy (37.8%) alone account for 76% of all primary glomerular diseases. Minimal change nephrotic syndrome and membranous nephropathy are both diseases associated with abnormalities in the membrane that filters blood in the glomeruli (glomerular filtration membrane).

  Minimal change nephrotic syndrome is a disease that accounts for about 80% of nephrotic syndrome in pediatric areas. Almost no pathological changes are observed by observation with an ordinary optical microscope. Can be confirmed.

  On the other hand, membranous nephropathy is a disease that occurs frequently in men aged 40 to 70 years, and is the most frequent of the primary diseases of nephrotic syndrome. When observed with an optical microscope, the glomerular basement membrane is thickened, and when observed with an electron microscope, adhesion of deposits to the glomerular basement membrane epithelium can be confirmed. Among patients with membranous nephropathy, those who are resistant to the administration of steroids have a poor prognosis, and about one third of all patients in Japan have end-stage renal disease.

In recent years, it has been observed that substances contained in vesicles called exosomes are used as communication tools between cells, and attention has been focused on substances contained in exosomes (for example, non-patent documents). 1). The information carriers that have been recognized in the past have been hormones, cytokines, neurotransmitters, and the like. However, since it was discovered that exosomes selectively contain abundant membrane proteins and non-coding RNA (ncRNA; non-coding RNA), they have been regarded as vesicles that carry intercellular signaling substances. . The name “exosome” was derived from the fact that vesicles were observed to be secreted from sheep reticulocytes in 1983 and the vesicles were named exosomes. Later, it became clear that exosomes were secreted from various cells, and now exist in blood, urine, milk, saliva, etc., with a diameter of 100 nm or less and a specific gravity of 1.10 to 1.19 g / cm. ³ and is recognized as a vesicle containing RNA.

  In the past, the present inventors have focused on urinary exosomal proteins in relation to renal diseases, and one of them, aquaporin-1 (aquaporin-1), is specifically expressed in proximal tubule cells. In the case of acute renal failure, the activity of aquaporin 1 excretion in urinary exosomes may change in acute renal failure. I made a hypothesis and started the experiment. As a result, it was observed that the amount of aquaporin 1 excreted in urinary exosomes decreased from the very early stage of acute renal failure, and a diagnostic value was found for this phenomenon (see Non-Patent Document 2 and Patent Document 1). . It has also been found that aquaporin 2 is a diagnostic index in acute renal failure (see Patent Document 2).

  By the way, PDCD6IP (programmed cell death 6 interacting protein, also known as: ALG-2 interacting protein, Alix) protein is a 95 kDa cytoplasmic protein, and is a molecule having an important function in endocytosis, cell death, cell-cell adhesion and the like. It is thought that there exists (refer nonpatent literature 3 and nonpatent literature 4). The reason why the functions of PDCD6IP are so diverse is that there is a region containing many proline residues at the C-terminus, and there are many proteins that are considered to bind to this region (for example, TSG101, CIN85, Src, endophilin A1, ALG-2). PDCD6IP is also known as a protein contained in urinary exosomes. However, there has been no report on whether PDCD6IP contained in urinary exosomes is useful as a biomarker for diagnosis / testing of renal diseases.

JP 2008-175630 A International Publication No. WO2010 / 150613 Pamphlet

Raimondo F, Morosi L, Chinello C, Magni F, Pitto M. (2011). Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery.Proteomics. 11, 709-720. Sonoda H, Yokota-Ikeda N, Oshikawa S, Kanno Y, Yoshinaga K, Uchida K, Ueda Y, Kimiya K, Uezono S, Ueda A, Ito K, Ikeda M. (2009). Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 297, F1006-F1016. Trioulier Y, Torch S, Blot B, Cristina N, Chatellard-Causse C, Verna JM, Sadoul R. (2004) .Alix, a protein regulating endosomal trafficking, is involved in neuronal death.J Biol Chem 279, 2046-2052. Okumura M, Ichioka F, Kobayashi R, Suzuki H, Yoshida H, Shibata H, Maki M. (2009). Penta-EF-hand protein ALG-2 functions as a Ca2 + -dependent adaptor that bridges Alix and TSG101. Biochem Biophys Res Commun 386, 237-241.

  In chronic renal failure including nephrotic syndrome, since the treatment policy differs depending on the type of renal disease, development of a diagnostic method for discriminating the type is required. However, currently available diagnostic methods are only microscopically diagnosed by an invasive technique called renal biopsy, which imposes a burden on patients. In addition, since hospitals where renal biopsy can be performed are limited, the widespread diagnosis is also a major problem. In order to solve these problems, it is important to develop a technique that can easily and accurately (that is, noninvasively) and accurately determine and diagnose the type of kidney disease.

  In particular, a non-invasive method capable of specifically diagnosing and examining diseases associated with glomerular filtration membrane abnormalities such as minimal change nephrotic syndrome and membranous nephropathy has not yet been established.

  Therefore, an object of the present invention is to provide a means that can easily (i.e., noninvasively) and accurately diagnose and test a disease related to glomerular filtration membrane abnormality.

  The inventors of the present invention have intensively studied to search for a novel biomarker of kidney disease. In the process, an attempt was made to measure the amount of PDCD6IP in the urine of a renal disease patient. As a result, surprisingly, the amount of PDCD6IP excreted in the exosome fraction in urine is affected by diseases associated with glomerular filtration membrane abnormalities such as membranous nephropathy and microvariable nephrotic syndrome. Found to be increased in patients who had Based on the results, puromycin aminonucleoside (PAN) was administered to the rat to create a micro-change nephrotic syndrome model rat, and the amount of PDCD6IP contained in the urinary exosome fraction was examined. As a result, in the PAN administration group, a significant increase in the amount of PDCD6IP excreted into the urinary exosome fraction was observed to confirm the above findings. Based on these findings, the present inventors have completed the present invention.

  That is, according to one aspect of the present invention, a method for examining a disease associated with an abnormality in a glomerular filtration membrane is provided. The test method is characterized in that it includes a step of measuring the amount of PDCD6IP (programmed cell death 6 interacting protein) protein in a urine sample collected from a subject.

  Moreover, according to the other form of this invention, the kit for a test | inspection for using for the inspection method mentioned above is provided. The test kit is characterized in that it contains an antibody that specifically binds to PDCD6IP protein.

  ADVANTAGE OF THE INVENTION According to this invention, the means which can diagnose and test | inspect easily easily (that is, noninvasively) the disease relevant to abnormality of a glomerular filtration membrane can be provided.

7 is a table comparing the amino acid sequences of PDCD6IP proteins from seven different species. 7 is a table comparing the amino acid sequences of PDCD6IP proteins from seven different species. 7 is a table comparing the amino acid sequences of PDCD6IP proteins from seven different species. Healthy subjects, patients with kidney disease (IgA nephropathy, non-IgA mesangial proliferative nephritis, membranous nephropathy, focal glomerulosclerosis, diabetic nephropathy, minimal change nephrotic syndrome), or diseases other than kidney disease ( It is the graph which compared the result of having measured the amount of PDCD6IP protein in the urinary exosome fraction sample prepared from the patient of the kidney transplant donor and the lung cancer by the Western blot method as described in (II-b) of an Example. It is the graph which showed the measurement result in the rat disease model created by (Ib) of an example compared with a control group. FIG. 3A is a graph comparing the amount of urinary total protein excretion. FIG. 3B is a graph comparing the amount of PDCD6IP protein in the urinary exosome fraction sample.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the technical scope of this invention is not limited only to the following form.

  One embodiment of the present invention is a method for examining a disease associated with an abnormality of a glomerular filtration membrane, which includes a step of measuring the amount of PDCD6IP (programmed cell death 6 interacting protein) protein in a urine sample collected from a subject. .

≪Subject≫
In the present invention, the “subject” is not particularly limited as long as it is an animal that can develop a disease associated with an abnormality of the glomerular filtration membrane, 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. Information (determination results) obtained when an animal other than a human (non-human animal) is used as a subject can also be used for examination of a disease associated with an abnormality of the glomerular filtration membrane of the non-human animal. However, it is also useful in that the determination result can be used for research and development for establishing a method for diagnosing and examining diseases associated with glomerular filtration membrane abnormalities in humans.

  The test method of the present invention is characterized in that the amount of PDCD6IP protein in a urine sample collected from a subject is measured as described in detail below. Here, as shown in FIGS. 1-1 to 1-3, the amino acid sequence of the PDCD6IP protein to be measured is highly conserved across species. In addition, changes in urinary PDCD6IP protein levels observed in disease model animals such as rats are also observed in patients. Therefore, it can be said that the experimental results in non-human animals can be extrapolated to humans.

<< Sample >>
In the examination method of the present invention, urine collected from a subject is used as a sample. For example, a sample obtained by performing the following treatment on collected urine can be used to measure the amount of PDCD6IP protein.

  The collected urine is first centrifuged at 1,000 g for 10 minutes, and then the supernatant is centrifuged at 17,000 g for 15 minutes. The supernatant is centrifuged at 200,000 g for 60 minutes, and the precipitate is separated as an exosome fraction. The obtained exosome fraction was mixed with 4 × 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 minutes. Solubilize to obtain an exosome fraction sample.

The use of a sample obtained by such a treatment (urinary exosome fraction sample) as a sample in the inspection method of the present invention has an advantage that a highly accurate diagnosis and inspection can be performed. Of course, as long as accurate and accurate measurement is possible, the collected urine may be used as the sample of the test method of the present invention without preparing the urinary exosome fraction sample. Examples of the sample preparation method include a filter method, an ultracentrifugation method, and a column separation method. Further, it is possible to use these treatments without performing these treatments, for example, in the case of carrying out measurement by an immunological technique using an antibody having a high titer. A proteolytic enzyme inhibitor may be appropriately added to the collected urine for the purpose of preventing the occurrence of measurement errors due to protein degradation in the sample. Whole urine ≪PDCD6IP protein≫
In the present specification, the PDCD6IP protein typically means a mature or complete PDCD6IP protein, but is a concept that includes a sugar chain-modified PDCD6IP protein molecule. At this time, the type of sugar chain and the binding position thereof are not limited. The amino acid sequence of PDCD6IP protein varies slightly depending on the animal species of the subject. As an example, the sequence listing includes the amino acid sequence of PDCD6IP protein derived from mouse (Mus musculus) as SEQ ID NO: 1, the amino acid sequence of PDCD6IP protein derived from rat (Rattus norvegicus) as SEQ ID NO: 2, and the human ( The amino acid sequence of PDCD6IP protein derived from Homo sapiens), the amino acid sequence of PDCD6IP protein derived from dog (Canis lupus familiaris) as SEQ ID NO: 4, the amino acid sequence of PDCD6IP protein derived from bovine (Bos taurus) as SEQ ID NO: 5, The amino acid sequence of PDCD6IP protein derived from frog (Xenopus laevis) is shown as No. 6, and the amino acid sequence of PDCD6IP protein derived from Danio rerio is shown as SEQ ID No. 7, respectively. All of these amino acid sequences are those of isoform 1 of PDCD6IP protein. FIGS. 1-1 to 1-3 are tables comparing the amino acid sequences of PDCD6IP proteins derived from these seven different species. As shown in FIGS. 1-1 to 1-3, human PDCD6IP protein (isoform 1) has the highest homology with dogs (97%), followed by bovine (96%), rats (94%) , Mouse (94%), frog (76%), fish (72%). As is clear from this, it can be said that the PDCD6IP protein is well conserved across species. In addition, it has been confirmed that the antibodies used in Examples described later can specifically recognize PDCD6IP protein in human, rat and mouse kidneys and urine.

≪Measurement of PDCD6IP protein content≫
The amount of PDCD6IP protein in the sample is preferably measured by an immunological measurement method using an antibody (anti-PDCD6IP antibody) that specifically binds to PDCD6IP protein. As such an antibody, an antibody prepared by a conventional method using a PDCD6IP protein of the animal species to be measured or a polypeptide containing a partial sequence thereof as an antigen can be preferably used. This antibody may be a polyclonal antibody or a monoclonal antibody. An antibody can also be used as a fragment as long as it specifically binds to PDCD6IP protein. 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.

  First, the above-mentioned antigen (polypeptide containing the PDCD6IP protein of the animal species to be measured or a partial sequence thereof) is added to the animal at a site where the antibody can be produced by administration of the antigen, together with the carrier or diluent. Administer. 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 an individual having an antibody titer from an animal immunized with the antigen, collecting the spleen or lymph node 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 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.

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

  On the other hand, a polyclonal antibody can be prepared, for example, by the following procedure.

  Polyclonal antibodies, for example, create a complex of an antigen and a carrier, immunize a mammal in the same manner as the above-described monoclonal antibody production method, collect 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 kind of carrier and the mixing ratio of the antigen and the carrier can be any type of antigen as long as the antibody can be efficiently produced against the antigen cross-linked to the carrier. You may make it bridge | crosslink in a ratio. Examples of the carrier 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 active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, dithiobilidyl group, and the like are used.

  The complex of an antigen and a carrier is administered to a immunized animal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration can usually be carried out about once every 2 to 6 weeks, 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.

≪Evaluation of PDCD6IP protein content≫
The test method of the present invention includes the step of measuring the amount of PDCD6IP protein in a urine sample collected from a subject, but it is not always necessary to measure the absolute amount of PDCD6IP protein, and the amount of PDCD6IP protein in a control urine sample is not necessarily measured. If the relative relationship with the amount of PDCD6IP protein can be clarified, it is sufficient for evaluation.

  A typical example of a method for evaluating the amount of PDCD6IP protein is an immunological assay using the above-described anti-PDCD6IP 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, turbidimetry for measuring turbidity associated with antigen-antibody complex formation, potential change due to binding to antigen using antibody solid phase membrane electrode An enzyme sensor electrode method for detection, immunoelectrophoresis, and the like can be used. Among these, the EIA method or the Western blot method is preferable. The EIA method includes a competitive enzyme immunoassay method, a sandwich enzyme-linked immunosolid phase assay method (sandwich ELISA method), and the like.

  When the western blot method is employed as an immunological assay for carrying out the present invention, for example, PDCD6IP protein in a urine sample can be detected as follows.

  First, a measurement sample is added to a sodium decylsulfate-containing polyacrylamide gel (SDS-PAGE), electrophoresis is performed with a certain voltage applied, and proteins separated on the gel by electrophoresis are subjected to PVDF (polyvinylidene fluoride). After electrically transferring to a blotting membrane such as a membrane and blocking this membrane with skim milk or the like, the above-mentioned anti-PDCD6IP antibody (primary antibody) is reacted with the membrane, and then chemiluminescent, fluorescent, or A secondary antibody labeled with an enzyme (such as horseradish peroxidase) is bound, and a detection operation is performed according to the labeling substance. Thereby, when PDCD6IP protein exists on a film | membrane, the said protein can be detected and quantified. In addition, the specific detection method by Western blotting is described in the Example mentioned later.

≪Inspection method≫
In the present invention, the amount of PDCD6IP protein in a urine sample collected from a subject by the method as described above is measured, and a disease related to glomerular filtration membrane abnormality is examined based on the measurement result. More specifically, a disease related to glomerular filtration membrane abnormality is examined using an increase in the amount of PDCD6IP protein in the urine sample to be measured as an index.

  In the present invention, “examination” typically means determination (in a narrow sense) whether or not a subject suffers from a disease associated with an abnormality of the glomerular filtration membrane. It is not limited. The “examination” in the present invention refers to, for example, an examination of the severity of a disease associated with an abnormality of the glomerular filtration membrane, an examination of a therapeutic effect for a disease associated with the abnormality of the glomerular filtration membrane, and a glomerular filtration membrane after the treatment It is a concept (in a broad sense) that also includes a test of whether or not there is a risk of re-affection (recurrence) of a disease associated with an abnormality in the above. Since the amount of PDCD6IP protein in urine is significantly increased even in the early stage of the disease associated with glomerular filtration membrane abnormality, the test method of the present invention is suitable for the disease associated with glomerular filtration membrane abnormality. It can be said that it is useful for early diagnosis and examination (in this case, the term “diagnosis” is used in a narrow sense). Moreover, since urine is used as a sample, it can be said that this is a very preferable method with little load on the subject.

  When determining whether or not a subject suffers from a disease associated with abnormal glomerular filtration membrane, the amount of PDCD6IP protein in a urine sample collected from a healthy sample can be used as a reference. Further, when determining the therapeutic effect, the amount of PDCD6IP protein in a urine sample collected from a subject before treatment can be used as a reference. In the present invention, these samples to be compared are also referred to as “control samples”.

  In the test method of the present invention, typically, the amount of PDCD6IP protein in a urine sample collected from a subject is measured, and the measured amount is compared with the amount of PDCD6IP protein in a urine sample collected from a control sample. However, when the former is more (increased) than the latter, the subject suffers from a disease associated with glomerular filtration membrane abnormalities, or suffers from the disease more severe than the control specimen. Judge that

  The measurement of the amount of PDCD6IP protein in the urine sample collected from the control sample can be performed in the same procedure as the measurement of the amount of PDCD6IP protein in the urine sample collected from the subject. The amount of PDCD6IP protein in the urine sample collected from the control sample may be measured every time urine is collected from the subject, or may be measured in advance.

≪Test kit≫
According to another aspect of the invention, a test kit is also provided. This test kit includes reagents used in the above-described test method. Specifically, for example, when the test kit is for measuring the amount of PDCD6IP protein in a urine sample by the above-described immunological measurement method, an antibody that specifically binds to PDCD6IP protein is used. contains. In this case, the test kit includes a buffer solution, a washing solution, a secondary antibody, a fluorescent dye, a reaction vessel, a positive control, a negative control, an instruction document describing the test protocol, etc. These components may be further included. Further, these components can be mixed or combined in advance as required. By using this test kit, it is possible to easily test a disease associated with an abnormality of the glomerular filtration membrane according to the present invention, and it is very useful for determining an early treatment policy.

  EXAMPLES Hereinafter, although an Example and a reference example demonstrate this invention more concretely, the technical scope of this invention is not limited to these.

≪I. Subject >>
(Ia) Human Patient Specimen Urine was collected from a human patient for whom consent was obtained under the approval of the Ethics Committee and used as a urine sample.

(Ib) Rat disease model causing abnormalities in glomerular filtration membrane Puromycin aminonucleoside (PAN) (150 mg / kg, ip) was administered to rats, and abnormalities were observed in the glomerular filtration membrane. A rat disease model with proteinuria was created. On the other hand, a group of rats administered with the same volume of physiological saline was used as a control group.

<< II. Quantification of PDCD6IP protein in exosome fraction of urine samples >>
(II-a) Preparation of exosome fraction sample Urine collection and blood collection were performed at arbitrary time points from the rat disease model prepared by the method of (Ib) above. Urine was collected using a metabolic cage and urine volume was measured. Thereafter, exosomes were separated. In addition, separation of the exosome fraction from urine by the following method was performed on urine containing a certain amount of creatinine.

  Specifically, the collected urine was first centrifuged at 1,000 g for 10 minutes, and then the supernatant was centrifuged at 17,000 g for 15 minutes. The supernatant was centrifuged at 200,000 g for 60 minutes, and the precipitate was separated as an exosome fraction. The obtained exosome fraction was mixed with 4 × 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 minutes. Solubilized to obtain an exosome fraction sample.

  In addition, when preparing samples from human patient specimens, early morning urine was obtained from patients for whom consent was obtained. Using this urine, an exosome fraction sample was prepared using the same procedure as that for animal model urine.

(II-b) Quantification of PDCD6IP protein in exosome fraction sample PDCD6IP protein in exosome fraction sample was detected and quantified by Western blotting by the following method.

  First, proteins in the exosome fraction sample prepared in the above (II-a) were first separated on the basis of molecular weight by SDS-PAGE according to a conventional method. Thereafter, the protein in the gel was transferred to a polyvinylidene fluoride (PVDF) membrane using a semi-dry type (KS-8460, Marisol, Tokyo) blotting apparatus. The transferred PVDF membrane was washed in Tris buffered saline (TBS) while shaking with a rotator (twice for 15 minutes), and with TBS containing 5% skim milk and Tween-20 (5% SM TTBS). Blocking (overnight, 4 ° C.). After blocking, a primary antibody (anti-Alix antibody, Santa Cruz Biotechnology, CA) diluted 1: 100 with 1.6% SM TTBS was reacted for 60 minutes (room temperature). After the reaction, the primary antibody was washed with TTBS (twice for 5 minutes), reacted with a secondary antibody (anti-goat IgG antibody) diluted 1: 4000 with 1.6% SM TTBS, and then washed with TTBS. (5 minutes, 4 times), and finally washed with TBS (10 minutes, once). When visualizing the band, the band was emitted using SuperSignal (registered trademark) West Femto Maximam Sensitivity Subatrate (PIERCE, IL) and photographed with a polaroid camera (ECLTM-mini Camera, Amersham International plc., Tokyo).

  In addition, when quantifying protein amount, it quantified as a relative value when the average value of the value of a control group is set to 100%.

(II-c) Measurement of plasma creatinine concentration Whole blood was collected from a subject, and after turbidity with heparin, the plasma was separated by centrifugation at 12,000 rpm for 10 minutes. Using this plasma, the creatinine concentration in the plasma was measured by Fuji Dry Chem Slide (CRE-PIII, Fuji Photo Film Co., Ltd., Tokyo).

(II-d) Measurement of urinary protein concentration and calculation of protein excretion rate The urinary protein concentration was measured by a Bradford protein assay kit (Bio Rad Laboratories, Tokyo). Moreover, the protein excretion rate was calculated as the total excretion amount per fixed time by multiplying the obtained measured value by the urine amount.

<< III. Result >>
(III-a) Comparison of PDCD6IP protein levels in urinary exosome fraction samples in patients with renal diseases or other diseases FIG. 2 shows healthy subjects, renal diseases (IgA nephropathy, non-IgA mesangial proliferative nephritis, membranes) PDCD6IP in a urinary exosome fraction sample prepared from patients with diabetic nephropathy, focal glomerulosclerosis, diabetic nephropathy, minimal change nephrotic syndrome) It is the graph which compared the result of having measured the protein amount by the Western blot method as described in said (II-b). Here, the data shown in FIG. 2 is a relative value when a control sample prepared by mixing urine of three healthy persons is analyzed and the amount is set to 100% (broken line in the graph). The vertical axis of the graph is represented on a logarithmic scale.

  As shown in FIG. 2, among 40 patients who collected samples in this experiment, membranous nephropathy (5 cases) and minimal change nephrotic syndrome (2 cases), which are diseases related to glomerular filtration membrane abnormality ), The amount of PDCD6IP protein in the urinary exosome fraction sample was significantly increased.

  This suggests that the amount of PDCD6IP protein in the urinary exosome fraction sample can be a useful marker for diagnosing and examining diseases associated with glomerular filtration membrane abnormalities. In addition, among individuals with non-IgA mesangial proliferative nephritis and focal glomerulosclerosis, individuals with an increased amount of PDCD6IP protein were observed. The reason for this may be that the glomerular filtration membrane had an abnormality in these patients.

(III-b) Examination in Rat Disease Model FIG. 3 is a graph showing the measurement results in the rat disease model prepared in (Ib) above compared with the control group. Specifically, FIG. 3A is a graph comparing the urinary total protein excretion. As shown in FIG. 3A, in the rat disease model, a significant increase in protein excretion (proteinuria) was observed after 5 days after PAN administration. FIG. 3B is a graph comparing the amount of PDCD6IP protein in the urinary exosome fraction sample. As shown in FIG. 3B, in the rat disease model, urinary exosome fraction samples in 2 out of 3 individuals on day 4 after PAN administration, and in all 3 individuals on day 5 after PAN administration. An increase in the amount of PDCD6IP protein was observed. From this, the amount of PDCD6IP protein in the urinary exosome fraction sample discussed in (III-a) above can be a useful marker for diagnosing and examining diseases associated with glomerular filtration membrane abnormalities. This hypothesis was supported.

  In addition, the value of the blood creatinine concentration in a rat disease model is shown as the numerical value of the upper stage of the photographed photograph of each western blot in B of FIG. As shown in FIG. 3B, no significant change in blood creatinine concentration was observed even when PAN was administered.

  From the above results, it is considered that the rat disease model prepared in this experiment is a model in which glomerular blood filtration membranes are abnormal and exhibit proteinuria, although renal failure has not occurred.

≪Summary≫
According to the present invention, diseases associated with abnormalities in blood filtration membranes in glomeruli, such as membranous nephropathy and minimal change nephrotic syndrome, by an extremely simple method of examining only the amount of a specific protein (PDCD6IP) in urine Can be diagnosed. Therefore, it is possible to avoid invasive diagnosis and testing methods that cause patient pain such as renal biopsy and blood sampling (non-invasive), and if widespread, can reduce the mortality due to the above diseases and the number of dialysis patients. It is expected to contribute greatly to the improvement of human health.

[SEQ ID NO: 1]
It is an amino acid sequence of PDCD6IP protein derived from mouse (Mus musculus).
[SEQ ID NO: 2]
It is an amino acid sequence of PDCD6IP protein derived from rat (Rattus norvegicus).
[SEQ ID NO: 3]
It is an amino acid sequence of PDCD6IP protein derived from human (Homo sapiens).
[SEQ ID NO: 4]
It is an amino acid sequence of PDCD6IP protein derived from a dog (Canis lupus familiaris).
[SEQ ID NO: 5]
It is an amino acid sequence of PDCD6IP protein derived from bovine (Bos taurus).
[SEQ ID NO: 6]
It is an amino acid sequence of PDCD6IP protein derived from a frog (Xenopus laevis).
[SEQ ID NO: 7]
It is an amino acid sequence of PDCD6IP protein derived from fish (Danio rerio).

Claims (6)

A method of measuring an increase in the amount of PDCD6IP protein as an indicator of membranous nephropathy or microvariable nephrotic syndrome, which comprises the step of measuring the amount of PDCD6IP (programmed cell death 6 interacting protein) protein in a urine sample collected from a subject . Measuring the amount of PDCD6IP protein in a urine sample collected from a subject;
Comparing the amount of PDCD6IP protein in a urine sample collected from a subject with the amount of PDCD6IP protein in a urine sample of a healthy sample ;
Including, methods who according to claim 1. The urine sample is a Ekisozomu fraction urine sample, methods who according to claim 1 or 2. The subject is a human, method towards according to any one of claims 1-3. The measurement of the amount of PDCD6IP protein in the urine sample is performed by immunoassay using an antibody or fragment thereof that specifically binds to PDCD6IP protein, how according to any one of claims 1-4 . Comprising the antibody or fragment thereof that specifically binds to PDCD6IP protein, kit for use in the way described in claim 5.