Markers to aid in the diagnosis of nephrotic syndrome and uses thereof

Abstract

Provided is a use of ephrin-B as a marker for assisting in the diagnosis of nephrotic syndrome. A nephrotic syndrome testing method comprises a step for measuring the level of expression of ephrin-B in the urine of a subject. A reagent for nephrotic syndrome diagnosis includes a substance that specifically binds to ephrin-B.

Description

[Technical Field]

[0001] The present invention relates to a marker for assisting in the diagnosis of nephrotic syndrome and uses thereof. Specifically, the present invention relates to the use of the marker for assisting in the diagnosis of nephrotic syndrome, a method for testing nephrotic syndrome, and a diagnostic reagent for nephrotic syndrome. This application claims priority based on Japanese Patent Application No. 2021-004184, filed on January 14, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] Currently, the number of potential chronic kidney disease patients is estimated to be approximately 13 million, half of whom are thought to require treatment. As kidney disease progresses, renal failure occurs, resulting in the near-elimination of kidney function. The only treatment options for patients with renal failure are dialysis, such as hemodialysis (artificial dialysis), or kidney transplantation. However, kidney transplantation is only performed in limited cases, and most patients with renal failure undergo hemodialysis. More than 330,000 patients currently undergo hemodialysis, with total medical costs reaching approximately 2 trillion yen per year. Proteinuria is the most important factor contributing to the progression of kidney disease. The development of new drugs to suppress proteinuria is highly desirable, as they could prevent or delay the progression to renal failure and potentially reduce the enormous medical costs associated with dialysis.

[0003] Nephrotic syndrome is diagnosed when urinary protein levels are 3.5g or more per day and blood albumin levels are 3.0g / dL or less. Nephrotic syndrome is a condition in which excessive protein excretion in the urine reduces blood protein levels (hypoproteinemia), resulting in swelling (edema). In severe cases of nephrotic syndrome, fluid accumulates in the lungs, stomach, heart, and scrotum. Hypoproteinemia also increases blood cholesterol, increasing the risk of renal failure, thrombosis (e.g., pulmonary infarction, myocardial infarction, cerebral infarction), and infections.

[0004] In previous basic research, the inventors have demonstrated that the slit diaphragm, an intercellular adhesion mechanism of kidney glomerular epithelial cells (podocytes), functions as the final barrier to prevent proteinuria (see, for example, Non-Patent Document 1). Furthermore, the inventors have used next-generation sequencer analysis, subtraction / differential display studies, and other methods to identify molecules whose gene expression changes immediately before the onset of proteinuria and during the pathogenesis stage, and have analyzed their expression patterns, localization, and molecular functions to search for and identify molecules involved in the onset of proteinuria (see, for example, Non-Patent Document 1). Through a series of studies, they have demonstrated that ephrin-B1 is a membrane protein that constitutes the slit diaphragm and plays an important role in maintaining the barrier function of the slit diaphragm (see, for example, Non-Patent Document 2).

[0005] Non-Patent Document 2 shows that in various pathological model animals, the expression of ephrinB1 decreases before that of other functional molecules in glomerular epithelial cells, and that the decrease in expression (function) of ephrinB1 is extremely important as an early change in the onset of pathological conditions. However, no investigation has been conducted into the leakage of ephrinB1 into urine or the role of ephrinB1 as a diagnostic marker. There is also a need for markers that are useful for pathological differentiation and prognosis diagnosis of nephrotic syndrome, which is characterized by severe proteinuria. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Miyauchi N et al., “Synaptic vesicle protein 2B is expressed in podocyte, and its expression is altered in proteinuric glomeruli.”, J Am Soc Nephrol., vol.17, p2748-2759, 2006. [Non-patent document 2] Fukusumi Y et al., “Nephrin-Binding Ephrin-B1 at the Slit Diaphragm Controls Podocyte Function through the JNK Pathway.”, J Am Soc Nephrol., vol. 29, Issue 5, pp. 1462-1474, 2018. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above circumstances, and provides a novel marker molecule useful for assisting in the diagnosis of nephrotic syndrome, as well as a test method and a diagnostic reagent for nephrotic syndrome using the marker molecule. [Means for solving the problem]

[0008] That is, the present invention includes the following aspects. (1) Use of urinary ephrin B as a marker to aid in the diagnosis of nephrotic syndrome. (2) The use described in (1), wherein the ephrin B is ephrin B1. (3) A method for testing for nephrotic syndrome, comprising a step of measuring the expression level of ephrin B in the urine of a subject. (4) A diagnostic reagent for nephrotic syndrome, which comprises a substance that specifically binds to ephrin B and is used on a urine sample. (5) The diagnostic reagent for nephrotic syndrome according to (4), wherein the substance that specifically binds to ephrin B is an antibody specific to ephrin B or Eph. [Effects of the Invention]

[0009] According to the above aspect, a novel marker molecule useful for assisting in the diagnosis of nephrotic syndrome can be provided. The test method and diagnostic reagent for nephrotic syndrome of the above aspect utilize the marker molecule and are useful for diagnosing the pathology of nephrotic syndrome. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows fluorescent images of ephrin B1 expression detected in normal (proteinuria-negative) human kidney specimens and human kidney specimens with nephrotic syndrome in Example 1. [Figure 2] FIG. 1 shows the results of detecting ephrin B1 in the urine of rats with adriamycin (ADR) nephropathy, a rat model of focal segmental glomerulosclerosis, in Example 2. [Figure 3] FIG. 1 shows the results of detecting ephrin B1 in the urine of rats with adriamycin (ADR) nephropathy, a rat model of focal segmental glomerulosclerosis, in Example 2. [Figure 4] FIG. 1 shows the results of detecting ephrin B1 in urine from mesangial proliferative nephritis model rats in Example 2. [Figure 5] FIG. 1 shows the results of detecting ephrin B1 in the urine of rats with anti-nephrin antibody (ANA)-induced nephropathy (ANA nephropathy), a rat model of specific slit diaphragm injury in Example 2. [Figure 6] FIG. 1 shows the results of detection of ephrin B1 in urine samples provided by human patients with mild proteinuria in Example 3. [Figure 7] FIG. 1 shows the results of detecting ephrin B1 in urine samples provided by human patients diagnosed with nephrotic syndrome at the time of remission (when proteinuria becomes mild) and at the time of relapse in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Markers to aid in the diagnosis of nephrotic syndrome> In one embodiment, the present invention provides the use of ephrin B as a marker to aid in the diagnosis of nephrotic syndrome.

[0012] In this embodiment, ephrin B is useful as a marker for assisting in nephrotic syndrome.

[0013] A healthy person's urine contains about 0.15g of protein per day, and in a urine protein test, a urine protein concentration of less than 15mg / dL is considered to be no particular problem and is diagnosed as negative (-). On the other hand, in a urine protein test, a urine protein level of 15mg / dL to less than 30mg / dL is considered a false positive (±), 30mg / dL to less than 100mg / dL is considered a positive (1+), 100mg / dL to less than 300mg / dL is considered a positive (2+), 300mg / dL to less than 1000mg / dL is considered a positive (3+), and 1000mg / dL or more is considered a positive (4+).

[0014] In general, "nephrotic syndrome" is a disease in which excessive protein is excreted in the urine, resulting in a decrease in blood protein concentration (hypoproteinemia), resulting in swelling (edema). Nephrotic syndrome is diagnosed when urinary protein levels are 3.5g or more per day and blood albumin levels are 3.0g / dL or less. In severe cases of nephrotic syndrome, fluid accumulates in the lungs, stomach, heart, and scrotum. Hypoproteinemia also increases blood cholesterol, increasing the risk of complications such as renal failure, thrombosis (e.g., pulmonary infarction, myocardial infarction, cerebral infarction), and infections.

[0015] As shown in the Examples below, ephrinB is detected in urine even at the stage of mild proteinuria in various pathological models, and thus ephrinB is useful as a marker for the early diagnosis of nephrotic syndrome before it becomes severe. EphrinB is a membrane protein that constitutes the slit diaphragm and plays an important role in maintaining the barrier function of the slit diaphragm. Therefore, ephrinB is useful as a marker for the pathological diagnosis of nephrotic syndrome (proteinuria) caused by slit diaphragm damage. Furthermore, as shown in the Examples below, in various pathological models, the amount of ephrin B detected in urine tends to increase with an increase in the amount of protein in urine, i.e., with the progression of nephrotic syndrome. Therefore, ephrin B is useful as a marker for diagnosing the progression of nephrotic syndrome and for prognostic diagnosis.

[0016] [Ephrin B] Ephrin-B is a single-pass transmembrane protein with a molecular weight of approximately 30 to 45 kJ. It consists of an extracellular domain, a transmembrane domain, and an intracellular domain with a C-terminal PDZ-binding sequence. It is expressed in renal glomerular epithelial cells and, together with nephrin, constitutes the slit diaphragm.

[0017] Ephrin B is classified into three subclasses: ephrin B1, ephrin B2, and ephrin B3. Among them, ephrin B1 is preferably used as a marker because it is expressed prominently in renal glomerular epithelial cells.

[0018] As used herein, the use of ephrin B as a marker to aid in the diagnosis of nephrotic syndrome includes the following embodiments. (1) Detecting the expression of ephrin B in urine or measuring the expression level. (2) Detecting or measuring the presence of mRNA encoding ephrin B or a nucleotide fragment having a partial sequence thereof in urine.

[0019] To diagnose nephrotic syndrome by detecting or measuring the expression level of ephrinB in urine, immunological methods such as Western blotting using a substance that specifically binds to ephrinB, ELISA (Enzyme-Linked ImmunoSorbent Assay), CLEIA (Chemiluminescent Enzyme Immuno Assay), immunoprecipitation, and immunohistological staining can be used.

[0020] Western blotting is a method in which, for example, ephrinB from a urine sample containing ephrinB is separated by acrylamide gel electrophoresis, transferred to a membrane, and reacted with an antibody (primary antibody) that can recognize ephrinB. After removing the primary antibody that has not bound to ephrinB by washing or other methods, the resulting immune complex between ephrinB and the primary antibody is detected using a labeled secondary antibody. The amount of ephrinB present can be measured by measuring the amount of label in the labeled secondary antibody that has bound to the resulting immune complex.

[0021] Immunohistological staining involves reacting tissue sections or cells fixed on a glass slide with an antibody to form an immune complex, removing the primary antibody that has not bound to ephrin B by washing, and then detecting the immune complex with a labeled secondary antibody that has bound to it. This method is effective for analyzing the expression site of ephrin B in tissues or cells.

[0022] The substance that specifically binds to ephrin B, such as an antibody specific to ephrin B (hereinafter sometimes referred to as "ephrin B-specific antibody"), may be the same as those exemplified in the section "Diagnostic Reagents for Nephrotic Syndrome" described below.

[0023] Furthermore, the use of ephrin B as a marker to assist in the diagnosis of nephrotic syndrome also includes methods for detecting mRNA encoding ephrin B or a nucleotide fragment having a partial sequence thereof. More specific examples of such methods include, but are not limited to, quantitative RT-PCR, Northern blotting, and in situ hybridization. Such detection methods can use probes or primers prepared based on the known base sequence information of ephrin B, or a combination of these.

[0024] The presence of a polynucleotide encoding ephrin B can also be detected by examining whether or not mRNA encoding ephrin B contained in urine, or a nucleotide fragment having a partial sequence thereof, is amplified by polymerase chain reaction (PCR) using specific primers.

[0025] Herein, the subject of diagnosis using an ephrin B marker to assist in the diagnosis of nephrotic syndrome is not particularly limited as long as it is an animal that develops nephrotic syndrome, but mammals are preferred. Examples of mammals include, but are not limited to, humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, and monkeys. Humans are preferred.

[0026] <Testing methods for nephrotic syndrome> In one embodiment, the present invention provides a method for testing for nephrotic syndrome, comprising the step of measuring the expression level of ephrin B in the urine of a subject.

[0027] According to the testing method (method for obtaining test data) of this embodiment, it is possible to diagnose whether or not a subject has a condition that leads to nephrotic syndrome.

[0028] [Measurement process] Methods for measuring the expression level of ephrin B in urine obtained from a subject include immunological methods such as Western blotting using a substance that specifically binds to ephrin B, ELISA (Enzyme-Linked ImmunoSorbent Assay), CLEIA (Chemiluminescent Enzyme Immuno Assay), and immunoprecipitation. Details of the Western blotting method are as explained above in <Markers for assisting the diagnosis of nephrotic syndrome>.

[0029] The substance that specifically binds to ephrin B, such as an ephrin B-specific antibody, that can be used includes the same substances as those exemplified in the section below entitled "Diagnostic Reagents for Nephrotic Syndrome."

[0030] Examples of substances that specifically bind to ephrin B include a method for detecting ephrin B in urine, in which a substance that specifically binds to ephrin B is immobilized on a support, a urine sample collected from a subject is added to the support, and the sample is incubated to allow the substance that specifically binds to ephrin B to bind to ephrin B, followed by washing.The ephrin B bound to the support via the substance that specifically binds to ephrin B can then be detected.

[0031] In the method for measuring ephrinB, a control sample may be prepared in addition to a urine sample from a subject in which ephrinB is to be detected. Examples of control samples include a negative control sample containing no ephrinB and a positive control sample containing ephrinB. In this case, ephrinB in a urine sample from a subject can be detected by comparing the results obtained from the negative control sample containing no ephrinB with the results obtained from the positive control sample containing ephrinB. Alternatively, a series of control samples with gradually varying concentrations can be prepared, and the detection results for each control sample can be obtained as numerical values ​​to create a standard curve. EphrinB in the urine from the subject can then be quantitatively detected based on the standard curve and the numerical values ​​of the urine sample from the subject.

[0032] Methods for measuring the expression level of ephrin B in urine obtained from a subject include, for example, methods for detecting mRNA encoding ephrin B or nucleotide fragments having a partial sequence thereof. More specific examples include, but are not limited to, quantitative RT-PCR, Northern blotting, and in situ hybridization. Such detection methods can use probes or primers prepared based on the known base sequence information of ephrin B, or a combination of these.

[0033] In the testing method of this embodiment, a test subject is determined to be suffering from nephrotic syndrome or to be at risk of developing nephrotic syndrome based on the measured expression level of ephrin B. Specifically, when the expression level of ephrin B exceeds a predetermined reference value, it is possible to evaluate or predict that the subject has nephrotic syndrome or is likely to develop nephrotic syndrome. On the other hand, when the expression level of the protein is equal to or less than the predetermined reference value, it is possible to evaluate or predict that the subject does not have nephrotic syndrome or has not developed nephrotic syndrome. Therefore, the testing method of this embodiment can also be referred to as a method for diagnosing nephrotic syndrome. Alternatively, when a subject has urinary protein but less than 3.5 g per day and has not been diagnosed with nephrotic syndrome, the testing method of this embodiment can also be referred to as a method for early diagnosis of nephrotic syndrome.

[0034] The reference value here is a reference value for distinguishing between a group of patients with nephrotic syndrome and a group of patients without nephrotic syndrome.

[0035] Alternatively, the reference value is a reference value for distinguishing between a group of patients with nephrotic syndrome caused by slit diaphragm injury and a group of patients with nephrotic syndrome caused by a condition other than slit diaphragm injury. When this reference value is used, the testing method of this embodiment can also be said to be a method for distinguishing between nephrotic syndrome caused by slit diaphragm injury and nephrotic syndrome caused by other conditions.

[0036] The reference value can be experimentally determined as a threshold value that can distinguish between a nephrotic syndrome patient group and a non-nephrotic syndrome patient group by measuring the ephrin B concentrations in urine of the patient group, or can be experimentally determined as a threshold value that can distinguish between the two groups by measuring the ephrin B concentrations in urine of the patient group with nephrotic syndrome caused by slit diaphragm injury and the patient group with nephrotic syndrome caused by a condition other than slit diaphragm injury.

[0037] In this embodiment, the method for determining the reference value of urinary ephrin B concentration is not particularly limited, and can be determined using, for example, a general statistical method.

[0038] Specifically, the reference value can be determined by measuring the ephrinB concentration in urine collected from patients who have been diagnosed with nephrotic syndrome by other methods, such as commonly performed tests for the amount of protein in urine, before diagnosis based on urinary protein concentration (e.g., at the time of hospitalization). After measuring multiple patients, the ephrinB concentration in their urine can be calculated from the average or median, and a value including this can be used as the reference value.

[0039] Alternatively, for example, for patients who have been diagnosed with nephrotic syndrome caused by slit diaphragm injury by other methods, such as commonly performed diagnosis using renal samples, the ephrin B concentration is measured in urine collected in advance (at the time of hospitalization, etc.) before diagnosis using renal samples. After measuring multiple patients, the ephrin B concentration in their urine can be calculated from the average or median, and a value including this can be used as the reference value.

[0040] Furthermore, the ephrin B concentration in urine collected from multiple nephrotic syndrome patients and multiple non-nephrotic syndrome patients before diagnosis based on urinary protein concentration (e.g., at the time of hospitalization) can be measured, and the urinary ephrin B concentrations and their variability for the nephrotic syndrome patient group and non-nephrotic syndrome patient group can be calculated from the mean or median, etc., and a threshold value that allows the two values ​​to be distinguished, taking the variability into consideration, can be determined and used as the reference value.

[0041] Alternatively, the ephrin B concentration in urine collected before diagnosis using kidney samples (e.g., at the time of hospitalization) can be measured for patients with nephrotic syndrome caused by multiple slit diaphragm lesions and patients with nephrotic syndrome caused by multiple conditions other than slit diaphragm lesions. The urinary ephrin B concentrations and their variability can be calculated from the mean or median values ​​for the group of patients with nephrotic syndrome caused by slit diaphragm lesions and the group of patients with nephrotic syndrome caused by conditions other than slit diaphragm lesions. A threshold value that allows the two values ​​to be distinguished, taking the variability into consideration, can then be determined and used as the reference value.

[0042] Furthermore, for example, when the expression level of Ephrin B in the urine of a subject who is a nephrotic syndrome patient is higher than the expression level of Ephrin B in the urine of the subject at the time of the previous measurement, it can be evaluated or predicted that nephrotic syndrome is progressing or becoming more severe. Alternatively, when the expression level of Ephrin B in the urine of a subject who is a nephrotic syndrome patient is lower than the expression level of Ephrin B in the urine of the subject at the time of the previous measurement, it can be evaluated or predicted that the symptoms of nephrotic syndrome are tending to improve or that the subject is tending to recover from nephrotic syndrome. Therefore, the testing method of this embodiment can also be referred to as a method for predicting the progression of nephrotic syndrome. Alternatively, the testing method of this embodiment can also be referred to as a method for diagnosing the prognosis of a patient with nephrotic syndrome. Furthermore, the need for treatment can be determined based on the results of the testing method of this embodiment. That is, if the expression level of ephrin B exceeds a predetermined reference value, treatment such as medication is administered to the subject. Furthermore, a treatment for nephrotic syndrome can be selected. Treatment for nephrotic syndrome mainly involves the use of corticosteroids, immunosuppressants, and renin-angiotensin-aldosterone system inhibitors such as angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors, either alone or in combination. The dosage and duration of treatment are determined appropriately depending on the subject's symptoms and disease state. In treatment-resistant cases, biologics (such as rituximab) and LDL apheresis therapy are also used.

[0043] <Diagnostic reagent for nephrotic syndrome> [First embodiment] In one embodiment, the present invention provides a diagnostic reagent for nephrotic syndrome, comprising a substance that specifically binds to ephrin B.

[0044] The diagnostic reagent of this embodiment can diagnose whether a subject has nephrotic syndrome or not. The diagnostic reagent of this embodiment is also useful for early diagnosis of nephrotic syndrome before it becomes severe, diagnosis of the progression of the condition, pathological differentiation, prognosis, etc.

[0045] The substance that specifically binds to ephrinB contained in the diagnostic reagent of this embodiment may be one type or two or more types, as long as it allows the expression level of ephrinB to be measured.

[0046] Substances that specifically bind to ephrin B include, for example, ephrin B-specific antibodies, aptamers, and Eph, which is a ligand for ephrin B. Among these, ephrin B-specific antibodies or Eph are preferred.

[0047] The Ephrin B-specific antibody may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody, but is preferably a monoclonal antibody because of its high specificity and excellent quantitation ability.

[0048] The ephrin B-specific antibody may be commercially available or may be prepared by the method described below. Examples of commercially available ephrin B-specific antibodies include rabbit polyclonal anti-human ephrin B1 antibody (isotype: IgG) manufactured by Santa Cruz Biotechnology, rabbit polyclonal anti-human ephrin B1 antibody (isotype: IgG) manufactured by Abcam, goat polyclonal anti-mouse ephrin B1 antibody (isotype: IgG) manufactured by R&D Systems, mouse monoclonal anti-human ephrin B1 antibody (isotype: IgG) manufactured by Santa Cruz Biotechnology, mouse monoclonal anti-human ephrin B1 antibody (isotype: IgG) manufactured by Thermo Fisher Scientific, rabbit polyclonal anti-ephrin B antibody (isotype: IgG) manufactured by GeneTex, rabbit polyclonal anti-ephrin B antibody (isotype: IgG) manufactured by Proteintec, and rabbit monoclonal anti-ephrin B antibody (isotype: IgG) manufactured by Cell Signaling Technology.

[0049] As used herein, "specific binding" means that an antibody binds only to a target protein (antigen), and can be quantified, for example, by the binding of an antibody to an epitope of the antigen in an in vitro assay, preferably a plasmon resonance assay using purified wild-type antigen (e.g., BIAcore, GE-Healthcare Uppsala, Sweden, etc.). The affinity of binding can be defined by ka (rate constant for antibody binding from the antibody-antigen complex), kD (dissociation constant), and KD (kD / ka). When an antibody specifically binds to an antigen, the binding affinity (KD) is 10 -8 mol / L or less, and -13 mol / L or more 10 -9 It is more preferable that the concentration is 100 mol / L or less.

[0050] In this embodiment, the term "polyclonal antibody" refers to an antibody preparation containing different antibodies against different epitopes. That is, when the antibody of this embodiment is a polyclonal antibody, it may contain different antibodies that specifically bind to ephrin B.

[0051] Furthermore, the term "monoclonal antibody" refers to an antibody (including antibody fragments) obtained from a population of substantially homogeneous antibodies. In contrast to polyclonal antibodies, monoclonal antibodies refer to antibodies that recognize a single determinant on an antigen.

[0052] In this embodiment, the term "functional fragment" of an antibody refers to a portion (partial fragment) of an antibody that specifically recognizes a target protein. Specific examples include Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabodies, multispecific antibodies, and polymers thereof.

[0053] Eph is known as a ligand for ephrin. There are no particular limitations on the Eph as long as it binds to ephrin B, but EphB is preferred. Six subclasses of EphB have been identified: EphB1, EphB2, EphB3, EphB4, EphB5, and EphB6. Of the above EphB subclasses, any one may be used alone, or two or more may be used in combination. Of the above EphB subclasses, it is particularly preferred to use EphB2, as it has the strongest binding affinity to ephrin B1.

[0054] Ephs may be commercially available, chemically synthesized, or produced by known genetic engineering techniques based on the known nucleotide sequence information of ephrin B. Examples of commercially available Ephs include mouse EphB2-Fc chimeric recombinant protein and mouse EphB4-Fc chimeric recombinant protein manufactured by R&D Systems; human Fc-tagged human EphB4 protein manufactured by Sino Biological Company; mouse EphB2-Fc chimeric recombinant protein and rat EphB1-Fc chimeric recombinant protein manufactured by Sigma; and mouse EphB2-Fc chimeric recombinant protein, human EphB2-Fc chimeric recombinant protein, and rat EphB1-Fc chimeric recombinant protein manufactured by Novus.

[0055] An aptamer is a substance that has the ability to specifically bind to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers that have the ability to specifically bind to an antigen can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers that have the ability to specifically bind to an antigen can be selected, for example, by the two-hybrid method using yeast.

[0056] Furthermore, the substance that specifically binds to ephrin B may be bound to a labeling substance or a modifying substance. Examples of the labeling substance for the antibody include stable isotopes, radioisotopes, fluorescent substances, enzymes, and magnetic materials. Among these, fluorescent substances or enzymes are preferred as the labeling substance for the antibody because they are easily detectable and highly sensitive. By providing a labeling substance to a substance that specifically binds to ephrinB, ephrinB can be detected and quantified simply and with high sensitivity.

[0057] Examples of stable isotopes include 13 C. 15 N, 2 H, 17 O. 18 Examples include, but are not limited to, O. Radioisotopes include, for example: 3 H, 14 C. 13 N, 32 P, 33 P, 35 Examples include, but are not limited to, S. Examples of fluorescent substances include, but are not limited to, cyanine dyes (such as Cy3 and Cy5), rhodamine 6G reagents, and other known fluorescent dyes (such as GFP, FITC (Fluorescein), and TAMRA).

[0058] Examples of enzymes include alkaline phosphatase and peroxidase (HRP). When the labeling substance is an enzyme, it is preferable to use an enzyme substrate. For alkaline phosphatase, p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP), etc. can be used as the enzyme substrate. For peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), etc. can be used.

[0059] Examples of magnetic substances include, but are not limited to, gadolinium, Gd-DTPA, Gd-DTPA-BMA, Gd-HP-DO3A, iodine, iron, iron oxide, chromium, manganese, or complexes or chelate complexes thereof.

[0060] Examples of modifiers include biotin, Fc fragments, etc. By providing a modifier to a substance that specifically binds to ephrinB, ephrinB can be detected and quantified simply and with high sensitivity using a substance that specifically binds to the modifier and that is bound to a labeling substance (for example, a specific antibody, or avidin or streptavidin if it is a substance that specifically binds to biotin).

[0061] (Antibody manufacturing method) When the ephrin B-specific antibody is a polyclonal antibody, it can be obtained by immunizing an animal with an antigen (e.g., ephrin B such as syntaxin or a fragment thereof, or cells expressing these, etc.), and purifying it from the antiserum by conventional means (e.g., salting out, centrifugation, dialysis, column chromatography, etc.). EphrinB or a peptide fragment having a partial amino acid sequence thereof to be used as an antigen may be chemically synthesized or may be produced by known genetic engineering techniques based on the known base sequence information of ephrinB.

[0062] Furthermore, when the ephrin B-specific antibody is a monoclonal antibody, it can be produced by the hybridoma method or recombinant DNA method.

[0063] Examples of the hybridoma method include the method of Kohler and Milstein (see, for example, Kohler & Milstein, Nature, 256:495 (1975)). Examples of antibody-producing cells used in the cell fusion step in this method include spleen cells, lymph node cells, and peripheral blood leukocytes of animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats, etc.) immunized with an antigen (ephrin B or a fragment thereof, or cells expressing these, etc.). Antibody-producing cells obtained by reacting the antigen in a culture medium with the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be derived from different animal species, as long as they are fusible, but are preferably derived from the same animal species. Methods for obtaining hybridomas include, for example, a method in which hybridomas are produced by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then, by screening, hybridomas that produce monoclonal antibodies specific to a target protein are obtained. Methods for obtaining monoclonal antibodies produced by hybridomas include, for example, a method in which monoclonal antibodies against a target protein are obtained by culturing hybridomas or from the ascites of a mammal to which a hybridoma has been administered.

[0064] Examples of recombinant DNA techniques include cloning DNA encoding the above-mentioned antibody or a functional fragment of the antibody from a hybridoma, B cell, or the like, incorporating it into an appropriate vector, and introducing it into host cells (e.g., mammalian cell lines, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of this embodiment as a recombinant antibody (see, for example, P.J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme, A.M. et al., Eur. J. Biochem. 192:767-775 (1990)).

[0065] In expressing DNA encoding an antibody, DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector and used to transform host cells, or DNA encoding the heavy chain and light chain may be incorporated into a single expression vector and used to transform host cells (see, for example, International Patent Application No. 94 / 11523). The antibody of this embodiment can be obtained in a substantially pure and homogeneous form by culturing the host cells and isolating and purifying it from within the host cells or from the culture medium. Methods used for ordinary polypeptide purification can be used for antibody isolation and purification. Examples of methods using transgenic animal production technology include producing transgenic animals (e.g., cows, goats, sheep, pigs, etc.) into which antibody genes have been incorporated, and then obtaining large quantities of monoclonal antibodies derived from the antibody genes from the milk of the transgenic animals.

[0066] The antibody may be an amino acid sequence variant, so long as it can specifically bind to ephrin B. Amino acid sequence variants can be produced by introducing mutations into DNA encoding the antibody chain or by peptide synthesis. The site of the antibody amino acid sequence modification may be the constant region of the heavy or light chain of the antibody, or may be the variable region (framework region and CDR), as long as the antibody has activity equivalent to that of the antibody before modification. Alternatively, a technique may be used in which amino acids in the CDR are modified to screen for antibodies with increased affinity for the antigen (see, for example, PNAS, 102:8466-8471 (2005); Protein Engineering, Design & Selection, 21:485-493 (2008); WO 2002 / 051870; J. Biol. Chem., 280:24880-24887 (2005); Protein Engineering, Design & Selection, 21:345-351 (2008)).

[0067] The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (for example, within 2 amino acids, 1 amino acid). The amino acid modification is preferably a conservative substitution.

[0068] As used herein, the term "conservative substitution" refers to a substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar side chains are well known in the art. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, and histidine), and neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, and proline), amino acids with hydroxyl groups (serine and threonine), sulfur-containing amino acids (cysteine ​​and methionine), amino acids with amide groups (asparagine and glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, and tryptophan). Preferably, the amino acid sequence variant has higher antigen-binding activity than the reference antibody.

[0069] In this embodiment, the binding activity of an antibody (including functional fragments and amino acid sequence variants of the above-mentioned antibodies) to an antigen can be evaluated by, for example, ELISA, Western blotting, immunoprecipitation, immunostaining, etc.

[0070] (Labeling Reagent) The diagnostic reagent for nephrotic syndrome of this embodiment may further comprise, in addition to the substance that specifically binds to ephrin B, a labeled reagent in which a labeling substance is bound to the substance that specifically binds to ephrin B (for example, a labeled substance is bound to an ephrin B-specific antibody), or a labeled substance is bound to an antibody against the substance that specifically binds to ephrin B (for example, a labeled substance is bound to an antibody against an ephrin B-specific antibody).

[0071] For example, when an ephrin B-specific antibody bound to a labeling substance is used as a labeling reagent, specific ephrin B can be detected and quantified using a sandwich ELISA (Enzyme-Linked ImmunoSorbent Assay) method or a chemiluminescent enzyme immunoassay (CLEIA) method using an antigen measurement system.

[0072] Furthermore, for example, when a labeled reagent is prepared by binding a labeled substance to an antibody against an ephrin B-specific antibody, specific ephrin B can be detected and quantified using an antibody measurement system such as ELISA, indirect fluorescent antibody assay, or CLEIA.

[0073] The antibody against the Ephrin B-specific antibody is preferably an antibody against the animal from which the Ephrin B-specific antibody is derived (e.g., mouse, rat, hamster, rabbit, monkey, goat, etc.). For example, when the Ephrin B-specific antibody is derived from a mouse, the antibody is preferably an anti-mouse antibody. Furthermore, the antibody against the Ephrin B-specific antibody to which a labeling substance is bound includes all classes and subclasses of immunoglobulins.

[0074] The diagnostic reagent for nephrotic syndrome of this embodiment may further comprise a reaction stop solution, if necessary. Examples of the reaction stop solution include sulfuric acid and sodium hydroxide.

[0075] The diagnostic reagent for nephrotic syndrome of this embodiment may further comprise a buffer solution. The buffer solution may be appropriately selected from those known in the art, such as phosphate buffer, Tris-HCl buffer, citrate buffer, carbonate buffer, borate buffer, succinate buffer, acetate buffer, etc. The buffer solution may contain at least one of NaCl, a surfactant (e.g., Tween 20, Triton X-100, etc.), and a preservative (e.g., sodium azide, etc.), as needed. Specific examples of the buffer solution include phosphate buffered saline (PBS), PBS-T (PBS-Tween 20), Tris-buffered saline (TBS), and TBS-T (TBS-Tween 20).

[0076] In the diagnostic reagent for nephrotic syndrome of this embodiment, the substance that specifically binds to ephrin B (specific antibody, Eph, etc.) and the labeled reagent may be in a dry state or may be dissolved in the above-mentioned buffer solution. Of these, it is preferable that these antibodies be in a dry state from the viewpoint of storage stability.

[0077] [Second embodiment] In one embodiment, the present invention provides a diagnostic reagent for nephrotic syndrome, comprising a random primer for a reverse transcription reaction, and a forward primer and a reverse primer for amplifying a cDNA encoding ephrin B from the reverse transcription product obtained by the reverse transcription reaction using the random primer.

[0078] The diagnostic reagent for nephrotic syndrome of this embodiment can be used to measure the expression level of the gene encoding ephrin B (i.e., the expression level of mRNA) by quantitative RT-PCR. The diagnostic reagent for nephrotic syndrome of this embodiment can easily and accurately diagnose whether or not a subject has nephrotic syndrome.

[0079] In this embodiment, the random primer for the reverse transcription reaction may be, for example, a mixture of 6-mer or 9-mer deoxyribonucleotides having a random sequence, and it is preferable that the 5' ends of the deoxyribonucleotides are phosphorylated.

[0080] The forward primer in this embodiment can have a sequence specific to the nucleotide sequence of the 3'-terminal region of cDNA derived from mRNA encoding ephrin B, among the reverse transcription products obtained by the random primer for the reverse transcription reaction. Furthermore, the reverse primer in this embodiment can have a sequence specific to the nucleotide sequence of the 5'-terminal region of cDNA derived from mRNA encoding ephrin B, among the reverse transcription products obtained by the random primer for the reverse transcription reaction. By using the forward primer and reverse primer with the above sequences, cDNA encoding ephrin B can be more specifically amplified in a real-time PCR reaction from the reverse transcription products obtained by the reverse transcription reaction using the random primer.

[0081] The nucleotide sequences of forward and reverse primers for amplifying cDNA derived from mRNA encoding human ephrin B1 or B2 are shown in Table 1 below. The nucleotide sequence of mRNA encoding human ephrin B1 is disclosed under GenBank accession number NM_004429.5, and the nucleotide sequence of mRNA encoding human ephrin B2 is disclosed under GenBank accession number NM_004093.4. Therefore, forward and reverse primers for amplifying cDNA derived from mRNA encoding human ephrin B1 or B2 are not limited to those listed in Table 1 below, and can be appropriately designed by those skilled in the art based on the nucleotide sequences of the mRNAs with the above GenBank accession numbers.

[0082] [Table 1]

[0083] The diagnostic reagent for nephrotic syndrome of this embodiment may further comprise a labeled oligonucleotide probe. The labeled oligonucleotide probe comprises a base sequence complementary to at least three bases of the base sequence of cDNA derived from mRNA encoding ephrinB. By including this base sequence, the probe can specifically hybridize to the amplification product of the forward primer and reverse primer. As a labeled probe of this embodiment, for example, one having a quencher attached to the 5' end and a labeling substance attached to the 3' end can be used. By providing a quencher at the 5' end and a labeling substance at the 3' end, during the extension reaction by DNA polymerase in real-time PCR, the 5'-3' exonuclease activity of the polymerase degrades the probe hybridized to the cDNA derived from mRNA encoding ephrinB among the reverse transcription products, thereby releasing the inhibition by the quencher and allowing the labeling substance to be detected.

[0084] Examples of the labeling substance bound to the 3' end of the probe include fluorescent dyes, fluorescent beads, quantum dots, biotin, antibodies, antigens, energy absorbing substances, radioisotopes, chemiluminescent materials, and enzymes.

[0085] Examples of fluorescent dyes include FAM (carboxyfluorescein), JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoramidite), Cy3, Cy5, Alexa568, and Alexa647.

[0086] When the labeling substance attached to the 3' end of the probe is a fluorescent dye, the combination of the labeling substance attached to the 3' end of the probe and the quencher attached to the 5' end of the probe is preferably a combination of labeling substances that can cause FRET (Fluorescence (Förster) Resonance Energy Transfer). Specific examples include a combination of a fluorescent dye with an excitation wavelength of about 490 nm (e.g., FITC, rhodamine green, Alexa (registered trademark) fluor 488, BODIPY FL, etc.) and a fluorescent dye with an excitation wavelength of about 540 nm (e.g., TAMRA, tetramethylrhodamine, Cy3), or a combination of a fluorescent dye with an excitation wavelength of about 540 nm and a fluorescent dye with an excitation wavelength of about 630 nm (e.g., Cy5, etc.).

[0087] The diagnostic reagent for nephrotic syndrome of this embodiment may contain one or more of nucleotide triphosphates as substrates, nucleic acid synthesizing enzymes, and amplification reaction buffer solutions, although this may vary depending on the nucleic acid amplification method used. The nucleotide triphosphates are substrates (dNTPs, rNTPs, etc.) appropriate for the nucleic acid synthesizing enzymes. The nucleic acid synthesizing enzymes are enzymes appropriate for the nucleic acid amplification method used, and examples thereof include DNA polymerase, RNA polymerase, and reverse transcriptase. Examples of amplification reaction buffer solutions include Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, and Good's buffer solution, and the pH is not particularly limited. [Example]

[0088] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0089] [Example 1] (1) Sample preparation With permission from the donors, samples obtained by kidney biopsy (a test in which a small piece of tissue is taken from the kidney) from healthy individuals and patients with nephrotic syndrome were used as glomerular epithelial cell samples. The obtained samples were immediately frozen at -70°C using n-hexane. Next, multiple 3 μm-thick frozen sections were prepared from each sample from a healthy individual and a patient with nephrotic syndrome.

[0090] (2) Detection of ephrin B1 The sections were then incubated at 4°C for 16 hours using anti-ephrin B1 antibody (rabbit anti-human ephrin B1 antibody, manufactured by Santa Cruz Biotechnology). After appropriate washing, the sections were stained with FITC-conjugated anti-rabbit IgG antibody (manufactured by DAKO). The sections were then observed using an IF microscope (BX50, manufactured by Olympus) (magnification: 400x). The results are shown in Figure 1. In Figure 1, "normal" refers to samples from healthy individuals, and "nephrotic syndrome cases" refers to samples from patients with nephrotic syndrome.

[0091] As shown in Figure 1, ephrinB1 was observed in a linear pattern along the capillary walls of the glomerulus in normal human kidney samples with negative proteinuria, whereas ephrinB1 expression was significantly reduced in human kidney samples with severe proteinuria and nephrotic syndrome.

[0092] [Example 2] (1) Preparation of various pathological model rats of nephrotic syndrome Various pathological model rats of nephrotic syndrome were prepared using known methods. As model rats of various pathological conditions of nephrotic syndrome, rats with adriamycin (ADR) nephropathy, rats with mesangial proliferative nephritis, and rats with anti-nephrin antibody (ANA) induced nephropathy (hereinafter sometimes referred to as "ANA nephropathy") were used. ADR nephropathy rats are a model that exhibits persistent and progressive proteinuria and ultimately leads to renal failure. On the other hand, mesangial proliferative nephritis rats and ANA nephropathy rats exhibit significant proteinuria (equivalent to or greater than that seen on day 20 in ADR nephropathy) that peaks 10 and 5 days after the induction of the lesion, respectively, but proteinuria normalizes 3 to 4 weeks after the induction of the disease.

[0093] Specifically, in ADR nephropathy rats, adriamycin (6.0 mg / kg body weight, dissolved in saline) was intravenously injected into the rats under anesthesia to induce ADR nephropathy. Urine samples were collected from the rats before adriamycin injection and 1, 2, and 4 weeks after adriamycin injection. Note that the urine samples collected before adriamycin injection and 1 and 2 weeks after adriamycin injection were from separate independent studies, and the urine samples collected 4 weeks after adriamycin injection were from separate independent studies. Urine samples were collected for 24 hours using a metabolic cage.

[0094] For mesangial proliferative nephritis in rats, rats were anesthetized and intravenously injected with a monoclonal anti-Thy-1.1 antibody (mAb 1-22-3; see Reference 1 (Ikezumi Y et al., "FK506 ameliorates proteinuria and glomerular lesions induced by anti-Thy 1.1 monoclonal antibody 1-22-3," Kidney International, Vol. 61, pp. 1339-1350, 2002); 500 μg / rat; dissolved in saline) to induce mesangial proliferative nephritis. Urine samples were collected from rats before and 1, 3, 5, 7, and 10 days after anti-Thy-1.1 antibody injection. Urine samples were collected for 24 hours using a metabolic cage.

[0095] For ANA nephropathy rats, rats were anesthetized and intravenously injected with a monoclonal anti-nephrin antibody (mAb5-1-6, see Non-Patent Documents 1 and 2; 10 mg / rat; dissolved in saline) once to induce ANA nephropathy. Urine samples from rats 5 days after anti-nephrin antibody injection were used. Urine samples were collected for 24 hours using a metabolic cage.

[0096] Next, ephrin B1 in each of the obtained urine samples was detected by Western blotting using an anti-ephrin B1 antibody (rabbit anti-human ephrin B1 antibody, manufactured by Santa Cruz Biotechnology). The results for rats with ADR nephropathy are shown in Figure 2 (after 2 weeks) and Figure 3 (after 4 weeks). In Figure 3, "Normal" refers to a urine sample from a normal rat in which ADR nephropathy had not been induced. "Normal Rabbit Serum" (NRS) is a negative control in which a urine sample from a rat with ADR nephropathy was reacted with normal rabbit serum instead of an antibody. The results in rats with mesangial proliferative nephritis are shown in FIG. The results in rats with ANA nephropathy are shown in FIG.

[0097] As shown in Figures 2 and 3, ephrinB1 was detected in the urine of ADR nephropathy rats 2 weeks after pathology induction. Furthermore, 4 weeks after pathology induction, ephrinB1 expression in the urine was significant, suggesting that the amount of ephrinB1 detected in the urine increased with the progression of the disease. This suggests that ephrinB1 may be a useful marker for diagnosing the progression of nephrotic syndrome.

[0098] As shown in Figure 4, in rats with mesangial proliferative nephritis, proteinuria peaked 10 days after the induction of the lesion, whereas ephrinB1 was detected in the urine as early as 3 days after the induction of the pathology. This suggests that ephrinB1 may be a useful marker for the early diagnosis of nephrotic syndrome before it worsens.

[0099] As shown in Figure 5, ANA was also detected in the urine of rats with ANA nephropathy 5 days after the induction of the condition.

[0100] [Example 3] With permission from the donors, ephrin B1 was detected in urine samples (10 samples) from human patients who showed mild proteinuria during medical examinations by Western blotting using an anti-ephrin B1 antibody (R&D Systems, goat anti-ephrin B1 antibody (isotype: IgG)). The results are shown in Figure 6.

[0101] As shown in Figure 6, ephrin B1 was detected in some of the human urine samples.

[0102] [Example 4] With permission from the donor, ephrin B1 was detected in urine samples taken from a human patient diagnosed with nephrotic syndrome during remission (when proteinuria became mild) and relapse by Western blotting using the same antibody as in Example 3. The results are shown in Figure 7.

[0103] As shown in Figure 7, ephrin B1 was detected in human urine samples taken immediately after and at the time of relapse of nephrotic syndrome (when proteinuria became prominent). [Industrial Applicability]

[0104] According to this embodiment, a novel marker molecule useful for assisting the diagnosis of nephrotic syndrome can be provided. The test method and diagnostic reagent for nephrotic syndrome of this embodiment utilize the marker molecule, and are useful for diagnosing nephrotic syndrome, specifically, for example, early diagnosis, pathological differentiation, prognosis, etc.

Claims

1. Use of urinary ephrin B as a marker to aid in the diagnosis of nephrotic syndrome.

2. The use according to claim 1, wherein the ephrin B is ephrin B1.

3. A method for testing for nephrotic syndrome, comprising a step of measuring the expression level of ephrin B in the urine of a subject (excluding humans).

4. A diagnostic reagent for nephrotic syndrome, which is used on a urine sample and contains a substance that specifically binds to ephrin B.

5. The diagnostic reagent for nephrotic syndrome according to claim 4, wherein the substance that specifically binds to ephrin B is an antibody specific to ephrin B or Eph.

Images