WO2015133484A1 - Kidney disease marker, and use thereof - Google Patents

Kidney disease marker, and use thereof Download PDF

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WO2015133484A1
WO2015133484A1 PCT/JP2015/056232 JP2015056232W WO2015133484A1 WO 2015133484 A1 WO2015133484 A1 WO 2015133484A1 JP 2015056232 W JP2015056232 W JP 2015056232W WO 2015133484 A1 WO2015133484 A1 WO 2015133484A1
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protein
urine
tpbg
subject
antibody
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秀斉 安部
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国立大学法人徳島大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the treatment of kidney disease.
  • Chronic Kidney Disease falls into irreversible glomerulosclerosis and eventually progresses to end stage renal failure, dialysis induction, as well as cardiovascular events such as myocardial infarction and stroke, Has become a significant risk of death.
  • CKD Chronic Kidney Disease
  • CKD goes through a common process of glomerulosclerosis, leading to chronic renal failure and dialysis.
  • CKD can prevent a decrease in renal function by treatment (microvariable nephrotic syndrome, and some nephritis and membranous).
  • nephropathy microvariable nephrotic syndrome, and some nephritis and membranous.
  • nephropathy There are two types, such as nephropathy
  • albuminuria appears and the serum creatinine level rises. Therefore, the current renal function test and urinary protein quantification are evaluated in advance in such pathological differences (ie, accompanied by decreased renal function).
  • dark cloud treatment is performed. It is also difficult to predict the prognosis of renal function in patients who are determined to be CKD.
  • kidney disease The definitive diagnosis of kidney disease is currently only by renal biopsy.
  • renal biopsies are invasive and risky and are difficult to perform repeatedly. Therefore, since the long-term progress of kidney disease is mainly evaluated by renal function and urine protein quantification, changes in the pathological condition in the kidney cannot be accurately captured.
  • nephritis even if the pathological diagnosis is the same, the response to the treatment and the prognosis of the renal function are various, and at present, there is no choice but to observe the progress of some treatment.
  • the results of the treatment of nephritis using steroids or immunosuppressants are relatively good.
  • the podocyte disorder is due to inflammation, metabolic abnormalities, hemodynamic abnormalities, or effects such as aging . Therefore, it is difficult to predict whether a treatment using a steroid or the like will be effective.
  • Trophoblast glycoprotein Tpbg protein
  • WT1 protein WT1 protein
  • kidney If the function improves, but no Tpbg protein is detected, it was confirmed that such treatment is not effective.
  • Item 1 A method of measuring Tpbg protein in urine collected from a subject in order to predict reactivity to a therapeutic agent for nephritis.
  • Item 2. Item 2. The method according to Item 1, wherein the presence of the Tpbg protein in the urine is indicative of therapeutic response.
  • Item 3. The method according to claim 1 or 2, wherein the subject is a subject whose presence of WT1 protein is confirmed in advance in the urine.
  • Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the subject is a subject suffering from chronic kidney disease.
  • Item 5. A kit for predicting responsiveness to a therapeutic agent for nephritis, comprising a reagent for detecting Tpbg protein in urine.
  • Item 6. Item 6.
  • Item 7. Item 6. The kit according to Item 5, wherein the reagent for detecting the Tpbg protein is an antibody that specifically recognizes the Tpbg protein.
  • Item 8. Item 7. The kit according to Item 6, wherein the reagent for detecting the WT1 protein is an antibody that specifically recognizes the WT1 protein.
  • Item 9. (1) collecting urine from a subject, (2) a step of measuring Tpbg protein in the urine, and (3) a step of administering a nephritis therapeutic agent to the subject in which presence of the Tpbg protein is confirmed in the urine in the step (2). For the treatment of a subject suffering from the disease.
  • Item 10 Before step (2), (4) measuring WT1 protein in the urine, Item 10.
  • Item 11. Item 10. The method according to Item 9, wherein the subject is a subject suffering from chronic kidney disease.
  • Item 10. The method according to Item 9, wherein the measurement of Tpbg protein is performed directly on the whole urine of a subject by an immunological method.
  • Item 13 Item 11. The method according to Item 10, wherein the measurement of WT1 protein is performed directly on the whole urine of a subject by an immunological method.
  • the present invention it is possible to simply and non-invasively accurately predict the presence or absence of kidney disease (particularly, progressive chronic kidney disease), the possibility of morbidity, its progress, prognosis, and / or therapeutic response. It is possible. Therefore, according to the present invention, it is possible to control the prescription of irrelevant drugs for a subject whose kidney disease is predicted not to progress (or renal function does not decrease), thereby suppressing medical costs and patient burden. Can be reduced. On the other hand, for patients whose nephropathy is predicted to progress to the 2nd, 3rd, 4th, or 5th stage, the use of a prophylactic agent for the progression of nephropathy It becomes possible to delay the timing or prevent progress.
  • the result of having measured the WT1 protein by the western blotting method using the whole urine of a healthy person and a CKD patient as a sample is shown.
  • the change of a renal function (eGFR) in a WT1 protein negative CKD patient and a WT1 protein positive CKD patient in 3 years from follow-up start is shown.
  • the unit of the vertical axis is ml / min / 1.73m 2 .
  • the amount of change in renal function ( ⁇ eGFR) for 3 years from the start of follow-up for WT1 protein negative CKD patients, CKD patients in general (all Japanese), and WT1 protein positive CKD patients is shown.
  • the unit of the vertical axis is ⁇ eGFR.
  • the result of having measured Tpbg protein by the western blotting method using the whole urine of a healthy person and a CKD patient as a sample is shown.
  • the results of measuring the amount of Tpbg protein in urine collected from patient c during the period from before treatment to 5 weeks after the start of treatment are shown.
  • the result of having measured Tpbg protein by the western blotting method using the whole urine of a healthy person and a CKD patient as a sample is shown.
  • subject includes humans and other mammals (eg, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.).
  • a preferred subject is a human.
  • the subject is preferably a human or other mammal that has a decreased or impaired renal function (eg, has been diagnosed with chronic kidney disease) or is suspected.
  • Tpbg protein is an abbreviation for Trophoblast® glycoprotein and is a 72 kDa glycoprotein. Tpbg protein has been known to be involved in the regulation of epithelial cell phenotype, and has recently been reported to be involved in podocyte damage in glomerulonephritis (Non-patent Document 1).
  • the urine subject to protein measurement is not particularly limited as long as it is normally collected in a medical institution or the like (for example, urine collection, occasional urine, early morning urine, etc.), but anytime urine from the subject. Is preferred.
  • urine may be concentrated or separated from each main component, but it is desirable that the collected urine is centrifuged at 1500 rpm for 5 minutes and the supernatant is used. From the viewpoint of providing a simpler prediction method, it is preferable to use whole urine as it is (without extracting only a specific fraction) for measurement of the target protein.
  • Tpbg protein in urine can be measured by any method.
  • Various methods for detecting a specific protein in a liquid for example, aggregation method, turbidimetric method, surface plasmon resonance (SPR) method, immunological method, etc. are known. Protein can be measured.
  • a preferable measurement method from the viewpoint of required time, good handleability, measurement accuracy, and the like is an immunological measurement method.
  • immunological measurement methods include Western blotting, immunohistochemical staining, immunoelectron microscopic observation, EIA, ELISA, RIA, FIA, FLISA, ELISPOT, SPR, QCM, DPI and the like.
  • the preferred immunoassay is the western blotting method.
  • Antibodies for measuring Tpbg protein by immunoassay and kits using the same are commercially available. Therefore, they can be purchased as appropriate to measure Tpbg protein. It is also possible to prepare an antibody that specifically recognizes the Tpbg protein according to a conventional method, and to measure the Tpbg protein by combining commercially available secondary antibodies.
  • the antibody that specifically recognizes the Tpbg protein may be a polyclonal antibody or a monoclonal antibody.
  • Tpbg protein When Tpbg protein is detected in urine collected from a subject, it can be predicted that the subject has reactivity with a therapeutic agent for nephritis.
  • the amount of the Tpbg protein for determining that the Tpbg protein has been detected is not particularly limited, and it is preferable to understand that the Tpbg protein exists if it is detected even in a very small amount.
  • the nephritis therapeutic agent is not particularly limited as long as it is used for the treatment of nephritis.
  • immunosuppressive agents such as pergarin, rapamycin, and leflunomide
  • antibody drugs such as rituximab, ofatumumab, alemtuzumab, gemtuzumab ozogamicin, 131-I tocitumomab, and daclizumab. These may use only 1 type, but can also combine 2 or more types.
  • Preferred immunosuppressants are cyclosporine, tacrolimus, mycophenolate mofetil and mizoribine.
  • a preferred antibody drug is rituximab.
  • the subject to be predicted for reactivity to the nephritis therapeutic agent based on the presence of Tpbg protein in urine is preferably a subject from which WT1 protein is detected in urine.
  • the presence of WT1 protein in urine means that the renal function of the subject is reduced. A method for measuring WT1 protein will be described later.
  • the subject for which the reactivity to the nephritis therapeutic drug is predicted based on the presence of the Tpbg protein in urine is preferably a subject diagnosed as having chronic kidney disease.
  • chronic kidney disease means that (1) renal disorder is obvious by urinalysis, blood test, image diagnosis, etc. (for example, proteinuria has appeared), or (2) glomerular filtration rate (GFR) ) Is less than 60 ml / min / 1.73 m 2 , meaning that one or both have been sustained for more than 3 months.
  • kidney disease There are various primary diseases diagnosed as chronic kidney disease, such as diabetic nephropathy, chronic glomerulonephritis, nephrosclerosis, polycystic kidney disease, rapidly progressive glomerulonephritis, chronic pyelonephritis, hypertension , SLE nephritis, amyloid kidney, renal urinary tract disorder, gout kidney, renal urinary calculus, renal dysplasia, pregnancy kidney / pregnancy toxemia, renal / urinary tract tuberculosis and the like.
  • a reagent for measuring Tpbg protein in urine (for example, Tpbg protein is specific) It is also possible to prepare a test kit in order to predict the reactivity to a nephritis therapeutic drug using an antibody that recognizes.
  • a test kit includes an antibody that specifically recognizes the Tpbg protein, an antibody that specifically recognizes the antibody (secondary antibody), a buffer solution, an enzyme, an enzyme reaction stop solution, a solid phase carrier, and the like. Can be included. These can be appropriately selected and designed according to the method for detecting the Tpbg protein.
  • the kit may further contain a reagent (for example, an antibody) that specifically recognizes the WT1 protein described below.
  • a reagent for example, an antibody
  • an antibody that specifically recognizes the WT1 protein and an antibody that specifically recognizes the Tpbg protein it is possible to predict the responsiveness to a therapeutic agent for nephritis in a patient suffering from progressive renal disease.
  • the decrease in renal function can be predicted using the presence of WT1 protein in the urine of a subject as an index. Similar to the measurement of Tpbg protein described above, measurement of WT1 protein in urine can be performed using any method. The immunoassay described for measuring Tpbg protein can be suitably used. Antibodies for measuring WT1 protein and kits using the same are also commercially available. Therefore, they can be purchased as appropriate to measure WT1 protein. It is also possible to prepare an antibody specifically recognizing the WT1 protein according to a conventional method and measure the WT1 protein by combining commercially available secondary antibodies. The antibody that specifically recognizes the WT1 protein may be a polyclonal antibody or a monoclonal antibody.
  • the renal function of the subject is predicted to decrease.
  • the amount of WT1 protein in urine for determining that WT1 protein has been detected is not particularly limited, and it is preferable to understand that WT1 protein is present if it is detected even in a very small amount.
  • Renal function can be evaluated by, for example, glomerular filtration rate.
  • the decrease in kidney function here means a decrease in kidney function that exceeds the decrease in kidney function that normally occurs based on aging.
  • the subject to be predicted to have a decreased renal function based on the presence of WT1 protein in urine is preferably a subject diagnosed as having chronic kidney disease.
  • a reagent for measuring the WT1 protein in the urine for example, WT1 protein specifically It is also possible to prepare a test kit for predicting a decrease in renal function using a recognizing antibody.
  • a test kit includes, in addition to an antibody that specifically recognizes the WT1 protein, an antibody that specifically recognizes the antibody (secondary antibody), a buffer solution, an enzyme, an enzyme reaction stop solution, a solid phase carrier, and the like. Can be included. These can be appropriately selected and designed according to the method for detecting the Tpbg protein.
  • a subject whose renal function is predicted to be reduced due to the presence of WT1 protein in urine is appropriately treated in consideration of the primary disease and the like in order to delay the rate of decrease in renal function.
  • the presence of Tpbg protein in the urine described above can be further measured, and further treatment content and schedule can be determined according to the result. By doing so, it becomes possible to provide an appropriate (therapeutically responsive) treatment for a subject whose renal function is expected to be reduced.
  • a bank of urine samples from patients with chronic kidney disease who had a definitive diagnosis was obtained by renal biopsy. These samples were approved by the Ethics Committee and consent forms were obtained. The background data was anonymized so that it could be linked, and collected so that individual identification was not possible. Similarly, the kidney tissue subjected to biopsy was anonymized and banked. In addition, clinical data such as albuminuria, renal function, and drugs used were collected from patients over time.
  • Example 1 Usefulness of WT1 protein as CKD prognostic prediction marker Urine was collected from CKD patients to examine its components, and WT1 protein was detected in urine and WT1 protein was detected in urine. There were two urine WT1-negative groups (20 each). For both groups, no significant difference was found as shown in Table 1 below when comparing sex, age, serum Cr level, eGFR, and urine protein / uCr ratio at the start of follow-up (at the time of renal biopsy). The patient was followed up for 3 years. Measurement of WT1 was performed by Western blotting.
  • Albumin amount and WT1 protein amount were measured using urine samples of 4 patients arbitrarily selected from 2 groups and urine samples of 4 normal subjects as comparison targets. Each sample was subjected to SDS-polyacrylamide electrophoresis and transferred to a nitrocellulose membrane. Western blotting was performed using an ECL-advance kit (GE Healthcare Japan, Tokyo, Japan) with anti-WT1 antibody (1: 400 dilution, rabbit polyclonal, Santa Cruz, Santa Cruz, CA) as the primary antibody. The nitrocellulose membrane after transfer was stained with urine protein by CBB staining. As a control for urinary albumin, bovine serum albumin was used. The measurement results are shown in FIG.
  • EGFR was calculated based on the patient's age and serum Cr level 3 years after the start of follow-up, and compared with the value at the start of follow-up, changes in eGFR (Fig. 2) and changes in eGFR ( ⁇ eGFR ) (FIG. 3).
  • Fig. 2 changes in eGFR
  • ⁇ eGFR changes in eGFR
  • FIG. 3 changes in eGFR
  • the average amount of change in eGFR over 3 years in Japanese was used.
  • Example 2 Usefulness of Tpbg protein as CKD therapeutic response marker 1
  • the presence of urinary Tpbg protein in 20 urinary WT1-positive group patients was measured by Western blotting, and was classified into two groups (10 each): a urinary Tpbg positive group and a urinary Tpbg negative group.
  • Table 2 there was no significant difference in sex, age, serum Cr level, eGFR, and urine protein / uCr ratio in both groups.
  • Tpbg protein appears in the urine of a patient suffering from progressive nephritis. Although no influence on podocytes could be confirmed by the current histopathological analysis, steroid treatment was effective for all patients who were positive for urinary Tpbg, and renal function was improved. As an example, urine was collected over time during hospitalization for patient c: (lupus nephritis). In accordance with the progress of steroid treatment, SDS-polyacrylamide electrophoresis was performed using urine from before treatment to 5 weeks after the start of treatment, and transferred to a nitrocellulose membrane.
  • Example 3 Usefulness of Tpbg protein as CKD therapeutic response marker 2
  • the presence of urinary Tpbg protein was measured by Western blotting in another urinary WT1-positive group patient.
  • the measurement results are shown in FIG.
  • the findings of the subjects indicated by (A) to (F) in FIG. 6 are as follows.
  • D IgA nephropathy
  • E Lupus nephritis
  • F ANCA-related nephritis
  • the sex and urine protein / creatinine ratio of patients (1-11) shown in FIG. 6 were as follows. 1: Male, 0.01 (urine Tp / Cr ratio) 2: Male, 6.31 (urine Tp / Cr ratio) 3: Female, 3.64 (urine Tp / Cr ratio) 4: Female, 5.48 (urine Tp / Cr ratio) 5: Male, 3.02 (urine Tp / Cr ratio) 6: Female, 1.03 (urine Tp / Cr ratio) 7: Female, 1.71 (urine Tp / Cr ratio) 8: Female, 0.44 (urine Tp / Cr ratio) 9: Female, 3.09 (urine Tp / Cr ratio) 10: Male, 0.90 (urine Tp / Cr ratio) 11: Female, 0.50 (urine Tp / Cr ratio)
  • B and C are patients with no inflammation in the kidney.
  • IgA nephropathy patient of D in the patient who lost the inflammation activity (“6” in FIG. 6), the detection level of Tpbg was low, but the patient with the inflammation activity (“7” in FIG. 6).
  • E and F which are renal diseases belonging to nephritis, high levels of Tpbg expression were observed as in the results of Example 2.
  • E and F which are renal diseases belonging to nephritis
  • E and F which are renal diseases belonging to nephritis
  • high levels of Tpbg expression were observed as in the results of Example 2.
  • Prognosis prediction and treatment response prediction are possible. Therefore, as a WT1 protein and / or Tpbg protein biomarker, it is possible to establish a tailor-made treatment method for improving prognosis by accurately elucidating the pathological condition and establishing a diagnostic method that accurately reflects the pathological condition. .

Abstract

Provided is a means or the like for more accurately predicting the prognosis of kidney disease, or the responsiveness of kidney disease to a treatment. In this method, Tpbg protein in urine collected from a subject is measured in order to predict the responsiveness to a therapeutic agent for nephritis.

Description

腎臓疾患に関するマーカー及びその利用Markers for kidney disease and use thereof
　本発明は、広く腎臓疾患の治療に関する。 The present invention relates generally to the treatment of kidney disease.
　進行性の慢性腎臓病（Chronic Kidney Disease、ＣＫＤ）は、不可逆的な糸球体硬化に陥り、遂には末期腎不全、透析導入へと進行するだけでなく、心筋梗塞及び脳卒中といった心血管イベント、更には死亡の重要なリスクとなることが明らかになってきた。本邦でも、末期腎不全から透析に至る患者は増加を続け、2011年末でついに30万人を超えた。慢性維持透析患者の年間医療費だけでも、一兆五千億円にのぼっており、医療費に削減という観点からもＣＫＤの効果的な治療手段が求められている。 Progressive chronic kidney disease (Chronic Kidney Disease, CKD) falls into irreversible glomerulosclerosis and eventually progresses to end stage renal failure, dialysis induction, as well as cardiovascular events such as myocardial infarction and stroke, Has become a significant risk of death. In Japan, the number of patients from end-stage renal failure to dialysis continued to increase, and finally exceeded 300,000 at the end of 2011. The annual medical cost of chronic maintenance dialysis patients alone is 1.5 trillion yen, and effective treatment means for CKD is required from the viewpoint of reducing medical costs.
　ＣＫＤは、糸球体硬化という共通のプロセスを経て、慢性腎不全・透析に至るが、ＣＫＤには、治療によって腎機能低下が食い止められるもの（微小変化型ネフローゼ症候群、及び一部の腎炎・膜性腎症など）と、治療を施しても腎機能低下が進行し、糸球体硬化、及び腎不全に至るものの２種類が存在する。しかし、殆ど全てのＣＫＤにおいて、アルブミン尿が出現し、血清クレアチニン値が上昇するため、現行の腎機能検査及び尿蛋白定量による評価では、予めそのような病態の違い（即ち、腎機能低下を伴うか否か）を把握し得ず、結果として闇雲な治療が行われている重大な問題がある。また、ＣＫＤと判断された患者の腎機能の予後を予測することも困難である。 CKD goes through a common process of glomerulosclerosis, leading to chronic renal failure and dialysis. However, CKD can prevent a decrease in renal function by treatment (microvariable nephrotic syndrome, and some nephritis and membranous). There are two types, such as nephropathy), and even if treatment is performed, the kidney function declines and leads to glomerulosclerosis and renal failure. However, in almost all CKD, albuminuria appears and the serum creatinine level rises. Therefore, the current renal function test and urinary protein quantification are evaluated in advance in such pathological differences (ie, accompanied by decreased renal function). There is a serious problem that cannot be grasped as a result, and as a result, dark cloud treatment is performed. It is also difficult to predict the prognosis of renal function in patients who are determined to be CKD.
　腎臓病の確定診断は、現在のところ腎生検によるしかない。しかし、腎生検は、侵襲的であり、リスクを伴うため、繰り返し施行することは困難である。よって、長期に渡る腎臓病の経過は主に腎機能と尿蛋白定量による評価しかなされていないため、腎臓内の病態の変化を正確にとらえることはできない。 The definitive diagnosis of kidney disease is currently only by renal biopsy. However, renal biopsies are invasive and risky and are difficult to perform repeatedly. Therefore, since the long-term progress of kidney disease is mainly evaluated by renal function and urine protein quantification, changes in the pathological condition in the kidney cannot be accurately captured.
　また、各種腎炎においても、病理診断は同様であっても、その治療に対する反応と腎機能の予後はさまざまであり、現状では、何らかの治療をしながら、その経過を見守るしかない。現在行われている治療の中では、ステロイド剤、又は免疫抑制薬等を用いた腎炎の治療の成績は比較的良い。しかしながら、糖尿病、高血圧、又は高齢者に腎炎が発症した場合、そのポドサイトの障害が炎症による障害なのか、代謝異常、又は血行力学的異常、或いは加齢などの影響なのかを判別することはできない。従って、ステロイド剤等を用いた治療が奏効するかどうかを予測することは困難である。 Also, even in various types of nephritis, even if the pathological diagnosis is the same, the response to the treatment and the prognosis of the renal function are various, and at present, there is no choice but to observe the progress of some treatment. Among the currently performed treatments, the results of the treatment of nephritis using steroids or immunosuppressants are relatively good. However, when diabetes, hypertension, or nephritis develops in the elderly, it is not possible to determine whether the podocyte disorder is due to inflammation, metabolic abnormalities, hemodynamic abnormalities, or effects such as aging . Therefore, it is difficult to predict whether a treatment using a steroid or the like will be effective.
　このような現状の下、本発明は、腎臓疾患の予後又は治療に対する反応性等をより正確に予測するための手段等を提供すること等を課題とする。 Under such circumstances, it is an object of the present invention to provide means for more accurately predicting prognosis or responsiveness to treatment of kidney diseases.
　斯かる課題を解決するために実施された研究により次のような知見が得られた。即ち、腎生検によって確定診断が得られた多数のＣＫＤ症例について、全尿中のポドサイト由来タンパク質の測定及びポドサイト障害の評価を行い、検出されたタンパク質と患者の腎予後との関連性を調べた。その結果、全尿中にWilms Tumor 1 protein（ＷＴ１タンパク質）が検出された場合には、腎機能が低下し、ＷＴ１タンパク質が検出されない場合には、腎機能の低下はみられないという傾向を見出した。また、ポドサイトに炎症が生じることで出現するTrophoblast glycoprotein（Ｔｐｂｇタンパク質）が検出された場合は、ステロイド剤等を用いた現行の治療が有効であり、ＷＴ１タンパク質が検出された場合であっても腎機能が改善するが、Ｔｐｂｇタンパク質が検出されない場合には、そのような治療は有効ではないことが確認された。斯かる知見及び更なる検討に基づき、以下に代表される発明が提供される。 The following findings were obtained from research conducted to solve such problems. That is, for a large number of CKD cases with a definitive diagnosis by renal biopsy, podocyte-derived protein in whole urine is measured and podocyte damage is evaluated, and the relationship between the detected protein and the renal prognosis of the patient is examined. It was. As a result, when Wilms Tumor 1 protein (WT1 protein) is detected in the whole urine, the kidney function decreases, and when WT1 protein is not detected, the kidney function does not decrease. It was. In addition, when Trophoblast glycoprotein (Tpbg protein) that appears due to inflammation in podocytes is detected, the current treatment using a steroid or the like is effective, and even when WT1 protein is detected, kidney If the function improves, but no Tpbg protein is detected, it was confirmed that such treatment is not effective. Based on such knowledge and further studies, the following inventions are provided.
項１．
腎炎治療薬に対する反応性を予測するために、被検体から採取した尿中のＴｐｂｇタンパク質を測定する方法。
項２．
該尿中のＴｐｂｇタンパク質の存在が治療反応性を有することを示す、項１に記載の方法。
項３．
該被検体が、その尿中にＷＴ１タンパク質の存在が予め確認された被検体である、請求項１又は２に記載の方法。
項４．
該被検体が慢性腎臓病を患った被検体である、項１～３のいずれかに記載の方法。
項５．
尿中のＴｐｂｇタンパク質を検出するための試薬を含む、腎炎治療薬に対する反応性を予測するためのキット。
項６．
更に、ＷＴ１タンパク質を検出するための試薬を含む、項５に記載のキット。
項７．
該Ｔｐｂｇタンパク質を検出するための試薬が、該Ｔｐｂｇタンパク質を特異的に認識する抗体である、項５に記載のキット。
項８．
該ＷＴ１タンパク質を検出するための試薬が、該ＷＴ１タンパク質を特異的に認識する抗体である、項６に記載のキット。
項９．
（１）被検体から尿を採取する工程、
（２）該尿中のＴｐｂｇタンパク質を測定する工程、及び
（３）前記工程（２）において、尿中にＴｐｂｇタンパク質の存在が確認された被検体に腎炎治療薬を投与する工程
を含む、腎炎を患う被検体の治療方法。
項１０．
工程（２）の前に、（４）該尿中のＷＴ１タンパク質を測定する工程を含み、
該尿中にＷＴ１タンパク質の存在が確認された被検体の尿について、前記工程（２）を行う、項９記載の治療方法。
項１１．
該被検体が慢性腎臓病を患った被検体である、項９に記載の方法。
項１２．
Ｔｐｂｇタンパク質の測定を被検体の全尿に対して直接免疫学的方法で行う、項９に記載の方法。
項１３．
ＷＴ１タンパク質の測定を被検体の全尿に対して直接免疫学的方法で行う、項１０に記載の方法。 Item 1.
A method of measuring Tpbg protein in urine collected from a subject in order to predict reactivity to a therapeutic agent for nephritis.
Item 2.
Item 2. The method according to Item 1, wherein the presence of the Tpbg protein in the urine is indicative of therapeutic response.
Item 3.
The method according to claim 1 or 2, wherein the subject is a subject whose presence of WT1 protein is confirmed in advance in the urine.
Item 4.
Item 4. The method according to any one of Items 1 to 3, wherein the subject is a subject suffering from chronic kidney disease.
Item 5.
A kit for predicting responsiveness to a therapeutic agent for nephritis, comprising a reagent for detecting Tpbg protein in urine.
Item 6.
Item 6. The kit according to Item 5, further comprising a reagent for detecting WT1 protein.
Item 7.
Item 6. The kit according to Item 5, wherein the reagent for detecting the Tpbg protein is an antibody that specifically recognizes the Tpbg protein.
Item 8.
Item 7. The kit according to Item 6, wherein the reagent for detecting the WT1 protein is an antibody that specifically recognizes the WT1 protein.
Item 9.
(1) collecting urine from a subject,
(2) a step of measuring Tpbg protein in the urine, and (3) a step of administering a nephritis therapeutic agent to the subject in which presence of the Tpbg protein is confirmed in the urine in the step (2). For the treatment of a subject suffering from the disease.
Item 10.
Before step (2), (4) measuring WT1 protein in the urine,
Item 10. The treatment method according to Item 9, wherein the step (2) is performed on the urine of a subject in which the presence of the WT1 protein is confirmed in the urine.
Item 11.
Item 10. The method according to Item 9, wherein the subject is a subject suffering from chronic kidney disease.
Item 12.
Item 10. The method according to Item 9, wherein the measurement of Tpbg protein is performed directly on the whole urine of a subject by an immunological method.
Item 13.
Item 11. The method according to Item 10, wherein the measurement of WT1 protein is performed directly on the whole urine of a subject by an immunological method.
　本発明によれば、腎疾患（特に、進行性慢性腎臓病）の罹患の有無、罹患の可能性、その進展、予後、及び／又は治療反応性の正確な予測を簡便且つ非侵襲的に行うことが可能である。よって、本発明により、腎疾患が進展しない（又は腎機能が低下しない）と予測される被検体に対しては、むやみな薬の処方を制御することが可能となり、医療費の抑制及び患者負担の軽減を図ることが可能となる。一方、腎症が、第2期、第3期、第4期、又は第5期に進展しうると予測される患者に対しては、積極的に腎症進展予防薬を使うことにより、進行時期の遅延、或いは進行防止が可能となる。 According to the present invention, it is possible to simply and non-invasively accurately predict the presence or absence of kidney disease (particularly, progressive chronic kidney disease), the possibility of morbidity, its progress, prognosis, and / or therapeutic response. It is possible. Therefore, according to the present invention, it is possible to control the prescription of irrelevant drugs for a subject whose kidney disease is predicted not to progress (or renal function does not decrease), thereby suppressing medical costs and patient burden. Can be reduced. On the other hand, for patients whose nephropathy is predicted to progress to the 2nd, 3rd, 4th, or 5th stage, the use of a prophylactic agent for the progression of nephropathy It becomes possible to delay the timing or prevent progress.
健常者及びＣＫＤ患者の全尿を試料として、ウェスタンブロット法でＷＴ１タンパク質を測定した結果を示す。The result of having measured the WT1 protein by the western blotting method using the whole urine of a healthy person and a CKD patient as a sample is shown. ＷＴ１タンパク質陰性ＣＫＤ患者及びＷＴ１タンパク質陽性ＣＫＤ患者についての、経過観察開始から3年間での腎機能（ｅＧＦＲ）の変化を示す。縦軸の単位は、ml/min/1.73m2である。The change of a renal function (eGFR) in a WT1 protein negative CKD patient and a WT1 protein positive CKD patient in 3 years from follow-up start is shown. The unit of the vertical axis is ml / min / 1.73m 2 . ＷＴ１タンパク質陰性ＣＫＤ患者、ＣＫＤ患者全般（日本人全体）、及びＷＴ１タンパク質陽性ＣＫＤ患者についての、経過観察開始から3年間での腎機能の変化量（ΔｅＧＦＲ）を示す。縦軸の単位は、ΔｅＧＦＲである。The amount of change in renal function (ΔeGFR) for 3 years from the start of follow-up for WT1 protein negative CKD patients, CKD patients in general (all Japanese), and WT1 protein positive CKD patients is shown. The unit of the vertical axis is ΔeGFR. 健常者及びＣＫＤ患者の全尿を試料として、ウェスタンブロット法でTpbgタンパク質を測定した結果を示す。The result of having measured Tpbg protein by the western blotting method using the whole urine of a healthy person and a CKD patient as a sample is shown. 治療前から治療開始後５週までの間に、患者ｃから採取した尿について、Ｔｐｂｇタンパク質の量を測定した結果を示す。The results of measuring the amount of Tpbg protein in urine collected from patient c during the period from before treatment to 5 weeks after the start of treatment are shown. 健常者及びＣＫＤ患者の全尿を試料として、ウェスタンブロット法でTpbgタンパク質を測定した結果を示す。The result of having measured Tpbg protein by the western blotting method using the whole urine of a healthy person and a CKD patient as a sample is shown.
　本書において「被検体」には、ヒト及び他の哺乳動物（例えば、マウス、ラット、ウサギ、イヌ、ネコ、ウシ、ウマ、ブタ、サル等）が含まれる。好ましい被検体はヒトである。また、被検体は、腎機能の低下又は障害が生じている（例えば、慢性腎臓病と診断されている）か、又はその可能性が疑われるヒト又は他の哺乳動物であることが好ましい。 As used herein, “subject” includes humans and other mammals (eg, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.). A preferred subject is a human. In addition, the subject is preferably a human or other mammal that has a decreased or impaired renal function (eg, has been diagnosed with chronic kidney disease) or is suspected.
　本書において、「Ｔｐｂｇタンパク質」とは、Trophoblast glycoproteinの略称であり、72ｋＤａの糖タンパクである。Ｔｐｂｇタンパク質は、従前より上皮細胞の表現系の制御に関与することが知られており、近年、糸球体腎炎におけるポドサイト損傷に関与することが報告されている（非特許文献１）。 In this document, “Tpbg protein” is an abbreviation for Trophoblast® glycoprotein and is a 72 kDa glycoprotein. Tpbg protein has been known to be involved in the regulation of epithelial cell phenotype, and has recently been reported to be involved in podocyte damage in glomerulonephritis (Non-patent Document 1).
　本書において、タンパク質の測定の対象となる尿は、医療機関等において通常採取されるもの（例えば、蓄尿、随時尿、早朝第一尿等）であれば特に限定されないが、被検体からの随時尿が好ましい。また、尿は、濃縮や各主成分の分離が行われていても良いが、採取した尿は 1500rpmで、5 分間遠心し、その上清を使用することが望ましい。より簡便な予測方法を提供するという観点から、全尿をそのまま（特定の画分のみを抽出することなく）、目的タンパク質の測定に用いることが好ましい。 In this document, the urine subject to protein measurement is not particularly limited as long as it is normally collected in a medical institution or the like (for example, urine collection, occasional urine, early morning urine, etc.), but anytime urine from the subject. Is preferred. In addition, urine may be concentrated or separated from each main component, but it is desirable that the collected urine is centrifuged at 1500 rpm for 5 minutes and the supernatant is used. From the viewpoint of providing a simpler prediction method, it is preferable to use whole urine as it is (without extracting only a specific fraction) for measurement of the target protein.
１．尿中Ｔｐｂｇを指標とした腎炎治療薬に対する反応性の予測
　被検体の尿中のＴｐｂｇタンパク質を指標として腎炎治療薬に対する反応性を予測することができる。尿中のＴｐｂｇタンパク質は、任意の方法で測定することが出来る。液体中の特定のタンパク質を検出する種々の方法（例えば、凝集法、比濁法、表面プラズモン共鳴（SPR）法、免疫学的方法等）が知れられているため、これらを適宜選択してＴｐｂｇタンパク質を測定することが出来る。所要時間、取扱い性の良さ、及び測定精度等の観点から好ましい測定方法は、免疫学的測定法である。免疫学的測定法としては、例えば、ウェスタンブロッティング法、免疫組織染色、免疫電子顕微鏡観察、ＥＩＡ、ＥＬＩＳＡ、ＲＩＡ、ＦＩＡ、ＦＬＩＳＡ、ＥＬＩＳＰＯＴ、ＳＰＲ、ＱＣＭ、ＤＰＩ等を挙げることが出来る。一実施形態において、好ましい免疫学的測定法は、ウェスタンブロッティング法である。 1. Prediction of reactivity to therapeutic drug for nephritis using urinary Tpbg as an index The reactivity to therapeutic drug for nephritis can be predicted using Tpbg protein in the urine of a subject as an index. Tpbg protein in urine can be measured by any method. Various methods for detecting a specific protein in a liquid (for example, aggregation method, turbidimetric method, surface plasmon resonance (SPR) method, immunological method, etc.) are known. Protein can be measured. A preferable measurement method from the viewpoint of required time, good handleability, measurement accuracy, and the like is an immunological measurement method. Examples of immunological measurement methods include Western blotting, immunohistochemical staining, immunoelectron microscopic observation, EIA, ELISA, RIA, FIA, FLISA, ELISPOT, SPR, QCM, DPI and the like. In one embodiment, the preferred immunoassay is the western blotting method.
　免疫学的測定法でＴｐｂｇタンパク質を測定するための抗体及びそれを用いたキットは商業的に入手可能である。よって、それらを適宜購入してＴｐｂｇタンパク質を測定することが出来る。また、Ｔｐｂｇタンパク質を特異的に認識する抗体を常法に従って作成し、商業的に入手可能な二次抗体を組み合わせて、Ｔｐｂｇタンパク質を測定することも可能である。Ｔｐｂｇタンパク質を特異的に認識する抗体は、ポリクローナル抗体であっても、モノクローナル抗体であっても良い。 Antibodies for measuring Tpbg protein by immunoassay and kits using the same are commercially available. Therefore, they can be purchased as appropriate to measure Tpbg protein. It is also possible to prepare an antibody that specifically recognizes the Tpbg protein according to a conventional method, and to measure the Tpbg protein by combining commercially available secondary antibodies. The antibody that specifically recognizes the Tpbg protein may be a polyclonal antibody or a monoclonal antibody.
　被検体から採取した尿に、Ｔｐｂｇタンパク質が検出された場合、当該被検体は、腎炎治療薬に対する反応性を有すると予測することが出来る。Ｔｐｂｇタンパク質が検出されたと判定するためのＴｐｂｇタンパク質の量は、特に制限されず、極微量でも検出されれば、存在すると解することが好ましい。 When Tpbg protein is detected in urine collected from a subject, it can be predicted that the subject has reactivity with a therapeutic agent for nephritis. The amount of the Tpbg protein for determining that the Tpbg protein has been detected is not particularly limited, and it is preferable to understand that the Tpbg protein exists if it is detected even in a very small amount.
　腎炎治療薬は、腎炎の治療に用いられるものであれば特に制限されないが、例えば、シクロスポリン、タクロリムス、ミゾリビン、ミコフェノール酸モフェチル、アザチオプリン、メチルプレドニゾロン、シクロフォスファミド、メトトレキセート、ブレキナールナトリウム、デオキシスパーガリン、ラパマイシン、及びレフルノマイド等の免疫抑制剤、並びに、リツキシマブ、オファツムマブ、アレムツズマブ、ゲムツズマブ・オゾガマイシン、１３１－Ｉトシツモマブ、及びダクリズマブ等の抗体医薬を挙げることが出来る。これらは、一種のみを用いても良いが２種以上を組み合わせることも出来る。好ましい免疫抑制剤は、シクロスポリン、タクロリムス、ミコフェノール酸モフェチル及びミゾリビンである。好ましい抗体医薬は、リツキシマブである。 The nephritis therapeutic agent is not particularly limited as long as it is used for the treatment of nephritis. Examples include immunosuppressive agents such as pergarin, rapamycin, and leflunomide, and antibody drugs such as rituximab, ofatumumab, alemtuzumab, gemtuzumab ozogamicin, 131-I tocitumomab, and daclizumab. These may use only 1 type, but can also combine 2 or more types. Preferred immunosuppressants are cyclosporine, tacrolimus, mycophenolate mofetil and mizoribine. A preferred antibody drug is rituximab.
　尿中のＴｐｂｇタンパク質の存在に基づいて腎炎治療薬に対する反応性を予測する対象となる被検体は、好ましくは尿中にＷＴ１タンパク質が検出される被検体であることが好ましい。尿中のＷＴ１タンパク質の存在は、被検体の腎機能が低下することを意味する。ＷＴ１タンパク質の測定方法等は後述する。 The subject to be predicted for reactivity to the nephritis therapeutic agent based on the presence of Tpbg protein in urine is preferably a subject from which WT1 protein is detected in urine. The presence of WT1 protein in urine means that the renal function of the subject is reduced. A method for measuring WT1 protein will be described later.
　尿中のＴｐｂｇタンパク質の存在に基づいて腎炎治療薬に対する反応性を予測する対象となる被検体は、好ましくは慢性腎臓病を患っていると診断される被検体であることが好ましい。ここで慢性腎臓病とは、（１）尿検査、血液検査、画像診断等で腎障害が明らかであること（例えば、タンパク尿が出ていること）、又は（２）糸球体濾過量（ＧＦＲ）が60ml/分/1.73m2未満であることの、いずれか又は両方が、３ヶ月以上持続している状態を意味する。 The subject for which the reactivity to the nephritis therapeutic drug is predicted based on the presence of the Tpbg protein in urine is preferably a subject diagnosed as having chronic kidney disease. Here, chronic kidney disease means that (1) renal disorder is obvious by urinalysis, blood test, image diagnosis, etc. (for example, proteinuria has appeared), or (2) glomerular filtration rate (GFR) ) Is less than 60 ml / min / 1.73 m 2 , meaning that one or both have been sustained for more than 3 months.
　慢性腎臓病と診断される原疾患は種々であり、例えば、糖尿病性腎症、慢性糸球体腎炎、腎硬化症、多発性嚢胞腎、急速進行性糸球体腎炎、慢性腎孟腎炎、高血症、ＳＬＥ腎炎、アミロイド腎、腎尿路障害、痛風腎、腎尿路結石、腎形成不全、妊娠腎／妊娠中毒症、腎・尿路結核等を挙げることが出来る。これらの中でも慢性糸球体腎炎、急速進行性糸球体腎炎、又はＳＬＥ腎炎が原疾患で慢性腎臓病と診断された場合は、尿中Ｔｐｂｇタンパク質が陽性である場合の治療反応性がより高いため好ましい。 There are various primary diseases diagnosed as chronic kidney disease, such as diabetic nephropathy, chronic glomerulonephritis, nephrosclerosis, polycystic kidney disease, rapidly progressive glomerulonephritis, chronic pyelonephritis, hypertension , SLE nephritis, amyloid kidney, renal urinary tract disorder, gout kidney, renal urinary calculus, renal dysplasia, pregnancy kidney / pregnancy toxemia, renal / urinary tract tuberculosis and the like. Among these, when chronic glomerulonephritis, rapid progressive glomerulonephritis, or SLE nephritis is diagnosed as chronic kidney disease as the primary disease, it is preferable because treatment reactivity when urinary Tpbg protein is positive is higher. .
　尿中のＴｐｂｇタンパク質の存在によって、被検体が腎炎治療薬に反応性があるか否かを判断することが出来るため、尿中のＴｐｂｇタンパク質を測定するための試薬（例えば、Ｔｐｂｇタンパク質を特異的に認識する抗体）を用いて、腎炎治療薬に対する反応性を予測するために検査キットを作製することも可能である。そのような検査キットには、Ｔｐｂｇタンパク質を特異的に認識する抗体の他に、当該抗体を特異的に認識する抗体（二次抗体）、緩衝液、酵素、酵素反応停止液、固相担体等を含めることが出来る。これらは、Ｔｐｂｇタンパク質を検出する手法に応じて適宜選択し、設計することが出来る。また、当該キットは、後述するＷＴ１タンパク質を特異的に認識する試薬（例えば、抗体）を更に含んでいても良い。ＷＴ１タンパク質を特異的に認識する抗体とＴｐｂｇタンパク質を特異的に認識する抗体とを含むことにより、進行性の腎疾患を患う患者において、腎炎の治療薬に対する反応性を予測することが出来る。 Since the presence of Tpbg protein in urine can determine whether or not a subject is responsive to a nephritis therapeutic agent, a reagent for measuring Tpbg protein in urine (for example, Tpbg protein is specific) It is also possible to prepare a test kit in order to predict the reactivity to a nephritis therapeutic drug using an antibody that recognizes. Such a test kit includes an antibody that specifically recognizes the Tpbg protein, an antibody that specifically recognizes the antibody (secondary antibody), a buffer solution, an enzyme, an enzyme reaction stop solution, a solid phase carrier, and the like. Can be included. These can be appropriately selected and designed according to the method for detecting the Tpbg protein. The kit may further contain a reagent (for example, an antibody) that specifically recognizes the WT1 protein described below. By including an antibody that specifically recognizes the WT1 protein and an antibody that specifically recognizes the Tpbg protein, it is possible to predict the responsiveness to a therapeutic agent for nephritis in a patient suffering from progressive renal disease.
２．尿中ＷＴ１を指標とした腎機能低下の予測
　被検体の尿中のＷＴ１タンパク質の存在を指標として腎機能の低下を予測することが出来る。上述するＴｐｂｇタンパク質の測定と同様に、尿中のＷＴ１タンパク質の測定は、任意の手法を用いて行うことができる。Ｔｐｂｇタンパク質の測定について記載した、免疫学的測定法が好適に使用され得る。ＷＴ１タンパク質を測定するための抗体及びそれを用いたキットも商業的に入手可能である。よって、それらを適宜購入してＷＴ１タンパク質を測定することが出来る。また、ＷＴ１タンパク質を特異的に認識する抗体を常法に従って作成し、商業的に入手可能な二次抗体を組み合わせて、ＷＴ１タンパク質を測定することも可能である。ＷＴ１タンパク質を特異的に認識する抗体は、ポリクローナル抗体であっても、モノクローナル抗体であっても良い。 2. Prediction of decrease in renal function using urine WT1 as an index The decrease in renal function can be predicted using the presence of WT1 protein in the urine of a subject as an index. Similar to the measurement of Tpbg protein described above, measurement of WT1 protein in urine can be performed using any method. The immunoassay described for measuring Tpbg protein can be suitably used. Antibodies for measuring WT1 protein and kits using the same are also commercially available. Therefore, they can be purchased as appropriate to measure WT1 protein. It is also possible to prepare an antibody specifically recognizing the WT1 protein according to a conventional method and measure the WT1 protein by combining commercially available secondary antibodies. The antibody that specifically recognizes the WT1 protein may be a polyclonal antibody or a monoclonal antibody.
　被検体から採取した尿に、ＷＴ１タンパク質が検出された場合、当該被検体の腎機能は低下すると予測される。ＷＴ１タンパク質が検出されたと判定するための尿中のＷＴ１タンパク質の量は、特に制限されず、極微量でも検出されれば、存在すると解することが好ましい。腎機能は、例えば、糸球体濾過量によって評価することができる。ここでの腎機能の低下とは、通常、加齢に基なって生じる腎機能の低下を超える腎機能の低下を意味する。 When WT1 protein is detected in urine collected from a subject, the renal function of the subject is predicted to decrease. The amount of WT1 protein in urine for determining that WT1 protein has been detected is not particularly limited, and it is preferable to understand that WT1 protein is present if it is detected even in a very small amount. Renal function can be evaluated by, for example, glomerular filtration rate. The decrease in kidney function here means a decrease in kidney function that exceeds the decrease in kidney function that normally occurs based on aging.
　尿中のＷＴ１タンパク質の存在に基づいて腎機能の低下を予測する対象となる被検体は、好ましくは慢性腎臓病を患っていると診断される被検体であることが好ましい。 The subject to be predicted to have a decreased renal function based on the presence of WT1 protein in urine is preferably a subject diagnosed as having chronic kidney disease.
　尿中のＷＴ１タンパク質の存在に基づいて、被検体の腎機能が低下するか否かを判断することが出来るため、尿中のＷＴ１タンパク質を測定するための試薬（例えば、ＷＴ１タンパク質を特異的に認識する抗体）を用いて、腎機能の低下を予測するために検査キットを作製することも可能である。そのような検査キットには、ＷＴ１タンパク質を特定的に認識する抗体の他に、当該抗体を特異的に認識する抗体（二次抗体）、緩衝液、酵素、酵素反応停止液、固相担体等を含めることが出来る。これらは、Ｔｐｂｇタンパク質を検出する手法に応じて適宜選択し、設計することが出来る。 Based on the presence of WT1 protein in the urine, it can be determined whether or not the renal function of the subject is reduced. Therefore, a reagent for measuring the WT1 protein in the urine (for example, WT1 protein specifically It is also possible to prepare a test kit for predicting a decrease in renal function using a recognizing antibody. Such a test kit includes, in addition to an antibody that specifically recognizes the WT1 protein, an antibody that specifically recognizes the antibody (secondary antibody), a buffer solution, an enzyme, an enzyme reaction stop solution, a solid phase carrier, and the like. Can be included. These can be appropriately selected and designed according to the method for detecting the Tpbg protein.
　尿中のＷＴ１タンパク質の存在により腎機能の低下が予測される被検体には、腎機能の低下速度を遅らせるために、原疾患等を考慮して、適切な治療を行うことが好ましい。例えば、上述する尿中のＴｐｂｇタンパク質の存在を更に測定し、その結果に応じて、更なる治療内容及びスケジュールを決定することが出来る。そうすることにより、腎機能の低下が予測される被検体により適切な（治療反応性のある）治療を手供することが可能になる。 It is preferable that a subject whose renal function is predicted to be reduced due to the presence of WT1 protein in urine is appropriately treated in consideration of the primary disease and the like in order to delay the rate of decrease in renal function. For example, the presence of Tpbg protein in the urine described above can be further measured, and further treatment content and schedule can be determined according to the result. By doing so, it becomes possible to provide an appropriate (therapeutically responsive) treatment for a subject whose renal function is expected to be reduced.
　以下、実施例により本発明についてさらに詳細に説明するが、本発明はこれらに制限されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
　腎生検により、確定診断の得られた慢性腎臓病患者の尿サンプルのバンク化を行った。これらサンプルは倫理委員会で承認され、同意書が得られたものであり、その背景データを連結可能なように匿名化を行い、個人同定ができないようにして収集した。生検を施行した腎組織も、同様に匿名化し、バンク化を行った。また、アルブミン尿、腎機能、使用薬剤等の臨床データも患者から経時的に集積した。 A bank of urine samples from patients with chronic kidney disease who had a definitive diagnosis was obtained by renal biopsy. These samples were approved by the Ethics Committee and consent forms were obtained. The background data was anonymized so that it could be linked, and collected so that individual identification was not possible. Similarly, the kidney tissue subjected to biopsy was anonymized and banked. In addition, clinical data such as albuminuria, renal function, and drugs used were collected from patients over time.
実施例１：ＷＴ１タンパク質のＣＫＤ予後予測マーカーとしての有用性
　ＣＫＤ患者から尿を採取して成分を調べ、尿中にＷＴ１タンパク質が検出された尿中ＷＴ１陽性群と尿中にＷＴ１タンパク質が検出されなかった尿中ＷＴ１陰性群の2群に分けた（各20名）。両群について、経過観察開始時（腎生検時）の性別、年齢、血清Cr値、eGFR、及び尿蛋白/uCr比を比較すると下記表１の通り有意差は認められなかった。その後3年間、腎機能等を経過観察した。ＷＴ１の測定は、ウェスタンブロット法で行った。 Example 1: Usefulness of WT1 protein as CKD prognostic prediction marker Urine was collected from CKD patients to examine its components, and WT1 protein was detected in urine and WT1 protein was detected in urine. There were two urine WT1-negative groups (20 each). For both groups, no significant difference was found as shown in Table 1 below when comparing sex, age, serum Cr level, eGFR, and urine protein / uCr ratio at the start of follow-up (at the time of renal biopsy). The patient was followed up for 3 years. Measurement of WT1 was performed by Western blotting.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　2群より任意に選択した4名の患者の尿サンプル、及び比較対象として正常者4名の尿サンプルを用いて、アルブミン量及びWT1タンパク質量を測定した。各サンプルについてSDS－ポリアクリルアミド電気泳動を行い、ニトロセルロース膜にトランスファーした。抗ＷＴ１抗体(1:400希釈、ラビットポリクローナル、Santa Cruz社, Santa Cruz, CA）を一次抗体として、ECL advanceキット（GEヘルスケア・ジャパン、東京、日本）を用いてウェスタンブロット法を行なった。トランスファー後のニトロセルロース膜は、CBB染色にて尿中タンパク質を青染した。尿中アルブミンのコントロールとしては、bovine serum albuminを用いた。測定結果を図１に示す。 Albumin amount and WT1 protein amount were measured using urine samples of 4 patients arbitrarily selected from 2 groups and urine samples of 4 normal subjects as comparison targets. Each sample was subjected to SDS-polyacrylamide electrophoresis and transferred to a nitrocellulose membrane. Western blotting was performed using an ECL-advance kit (GE Healthcare Japan, Tokyo, Japan) with anti-WT1 antibody (1: 400 dilution, rabbit polyclonal, Santa Cruz, Santa Cruz, CA) as the primary antibody. The nitrocellulose membrane after transfer was stained with urine protein by CBB staining. As a control for urinary albumin, bovine serum albumin was used. The measurement results are shown in FIG.
　経過観察開始時から3年後の患者の年齢、血清Cr値を元に、eGFRを算出し、経過観察開始時の値と比較して、eGFRの変化（図２）及びeGFRの変化量（ΔeGFR）（図３）を測定した。変化量の測定には、参考値として、検討した40名の患者のeGFRを元に、日本人における3年間のeGFRの変化量平均を利用した。 EGFR was calculated based on the patient's age and serum Cr level 3 years after the start of follow-up, and compared with the value at the start of follow-up, changes in eGFR (Fig. 2) and changes in eGFR (ΔeGFR ) (FIG. 3). For the measurement of the amount of change, as a reference value, based on the eGFR of 40 patients studied, the average amount of change in eGFR over 3 years in Japanese was used.
　図２及び３に示されるように、ＷＴ１陽性患者群では、同年齢・同腎機能の日本人の平均的な腎機能の推移と比較して、重度の腎機能低下が見られた。尚、ＷＴ１陽性とは、0.00005μｇ/μｌ以上の濃度でＷＴ１タンパク質が検出されたことを意味する。一方、尿中ＷＴ１陰性の患者群では、腎機能の保持／改善がみられ、腎機能の低下は見られなかった。この結果から、尿中ＷＴ１陽性のＣＫＤ患者では、患者の年齢、腎機能、及び尿タンパク量等に関係なく、腎機能が低下する傾向があることが確認された。一方、尿中ＷＴ１陰性のＣＫＤ患者の場合は、腎機能は低下しない傾向があることが判明した。即ち、ＣＫＤ被検体の尿サンプル中のＷＴ１タンパク質の有無を調べることにより、予後の予測が可能であることが示された。 As shown in FIGS. 2 and 3, in the WT1-positive patient group, a severe decrease in renal function was observed as compared with the transition of average renal function in Japanese of the same age and renal function. Note that WT1 positive means that WT1 protein was detected at a concentration of 0.00005 μg / μl or more. On the other hand, in the urine WT1-negative patient group, retention / improvement of renal function was observed, and no decrease in renal function was observed. From this result, it was confirmed that in urinary WT1-positive CKD patients, the renal function tends to decrease regardless of the patient's age, renal function, urine protein amount, and the like. On the other hand, in the case of CKD patients who are negative for urine WT1, it has been found that renal function tends not to decrease. That is, it was shown that prognosis can be predicted by examining the presence or absence of WT1 protein in the urine sample of the CKD subject.
実施例２：Ｔｐｂｇタンパク質のＣＫＤ治療反応性マーカーとしての有用性１
　尿中ＷＴ１陽性群患者20名について、尿中Ｔｐｂｇタンパク質の存在をウェスタンブロット法で測定し、尿中Ｔｐｂｇ陽性群と尿中Ｔｐｂｇ陰性群の2群（各10名）に分類した。下記の表２に示すとおり、両群において、性別、年齢、血清Cr値、eGFR、及び尿蛋白/uCr比には有意差は認められなかった。 Example 2: Usefulness of Tpbg protein as CKD therapeutic response marker 1
The presence of urinary Tpbg protein in 20 urinary WT1-positive group patients was measured by Western blotting, and was classified into two groups (10 each): a urinary Tpbg positive group and a urinary Tpbg negative group. As shown in Table 2 below, there was no significant difference in sex, age, serum Cr level, eGFR, and urine protein / uCr ratio in both groups.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　上記2群の患者から任意に選択した8名の尿サンプル及び健常者4名の尿サンプルについて、SDS－ポリアクリルアミド電気泳動を行い、ニトロセルロース膜にトランスファーした。抗Ｔｐｂｇ抗体(1:500希釈、ラビットポリクローナル抗体、MBLにて受託作成）を一次抗体として、ECL advanceキット（GEヘルスケア・ジャパン、東京、日本）を用いてウェスタンブロット法を行なった（図４）。図４において(a)～(l)で示される被験者の所見は下記の通りである。
(a):健常者（尿潜血プラス、尿蛋白－）
(b):糖尿病
(c):ループス腎炎
(d):巣状糸球体硬化症（腎炎）
(e):糖尿病（尿潜血＋、尿蛋白－）
(f):健常者
(g):健常者
(h):健常者
(i):糖尿病＋膜性腎症（腎炎）（尿潜血＋、尿蛋白±）
(j):IgA腎症（腎炎）
(k):糖尿病＋急速進行性糸球体腎炎
(l):IgA腎症（腎炎） Eight urine samples arbitrarily selected from the two groups of patients and urine samples of four healthy subjects were subjected to SDS-polyacrylamide electrophoresis and transferred to a nitrocellulose membrane. Western blotting was performed using an ECL advance kit (GE Healthcare Japan, Tokyo, Japan) with an anti-Tpbg antibody (diluted 1: 500, rabbit polyclonal antibody, prepared by MBL) as the primary antibody (FIG. 4). ). The findings of the test subjects indicated by (a) to (l) in FIG. 4 are as follows.
(a): Healthy subject (urinary occult blood plus, urine protein-)
(b): Diabetes
(c): Lupus nephritis
(d): Focal glomerulosclerosis (nephritis)
(e): Diabetes (urine occult blood +, urine protein-)
(f): Healthy person
(g): Healthy subject
(h): Healthy subject
(i): Diabetes + membranous nephropathy (nephritis) (urinary occult blood +, urine protein ±)
(j): IgA nephropathy (nephritis)
(k): Diabetes mellitus + rapidly progressive glomerulonephritis
(l): IgA nephropathy (nephritis)
　図４の結果から、Ｔｐｂｇタンパク質は進行性の腎炎を患う患者の尿中に現れることが確認された。ポドサイトへの影響は、現状の病理組織学的解析ではいずれも確認できなかったが、尿中Ｔｐｂｇ陽性である全ての患者に対するステロイド治療は奏効し、腎機能の改善を得ることができた。一例として、患者c : （ループス腎炎）に対して、入院中に経時的に尿を採取した。ステロイド治療の経過にあわせて、治療前から治療開始後5週までの尿を用いて、ＳＤＳ－ポリアクリルアミド電気泳動を行い、ニトロセルロース膜にトランスファーした。抗Ｔｐｂｇ抗体(1:500希釈、ラビットポリクローナル抗体、MBLにて受託作成）を一次抗体として、ECL advanceキット（GEヘルスケア・ジャパン、東京、日本）を用いてウェスタンブロット法を行なった。図５に示される結果のように、Ｔｐｂｇの検出レベルが低下したことから、患者ｃの腎機能は治療によって回復することが確認された。 4, it was confirmed that the Tpbg protein appears in the urine of a patient suffering from progressive nephritis. Although no influence on podocytes could be confirmed by the current histopathological analysis, steroid treatment was effective for all patients who were positive for urinary Tpbg, and renal function was improved. As an example, urine was collected over time during hospitalization for patient c: (lupus nephritis). In accordance with the progress of steroid treatment, SDS-polyacrylamide electrophoresis was performed using urine from before treatment to 5 weeks after the start of treatment, and transferred to a nitrocellulose membrane. Western blotting was performed using an ECL advance kit (GE Healthcare Japan, Tokyo, Japan) using an anti-Tpbg antibody (diluted 1: 500, rabbit polyclonal antibody, commissioned with MBL) as a primary antibody. As the result shown in FIG. 5, since the detection level of Tpbg was lowered, it was confirmed that the renal function of patient c was recovered by treatment.
実施例３：Ｔｐｂｇタンパク質のＣＫＤ治療反応性マーカーとしての有用性２
　実施例２と同様にして、別の尿中ＷＴ１陽性群患者について、尿中Ｔｐｂｇタンパク質の存在をウェスタンブロット法で測定した。測定結果を図（６）に示す。図６において(A)～(F)で示される被験者の所見は下記の通りである。
A:健常者
B:微小変化型ネフローゼ症候群
C:膜性腎症
D:IgA腎症
E:ループス腎炎
F:ANCA関連腎炎 Example 3: Usefulness of Tpbg protein as CKD therapeutic response marker 2
In the same manner as in Example 2, the presence of urinary Tpbg protein was measured by Western blotting in another urinary WT1-positive group patient. The measurement results are shown in FIG. The findings of the subjects indicated by (A) to (F) in FIG. 6 are as follows.
A: Healthy people
B: Minimal change nephrotic syndrome
C: membranous nephropathy
D: IgA nephropathy
E: Lupus nephritis
F: ANCA-related nephritis
　図６に示される患者（１～１１）の性別及び尿タンパク質／クレアチニン比は下記の通りであった。
１：男性、０．０１（尿Tp/Cr比）
２：男性、６．３１（尿Tp/Cr比）　
３：女性、３．６４（尿Tp/Cr比）
４：女性、５．４８（尿Tp/Cr比）
５：男性、３．０２（尿Tp/Cr比）
６：女性、１．０３（尿Tp/Cr比）
７：女性、１．７１（尿Tp/Cr比）
８：女性、０．４４（尿Tp/Cr比）
９：女性、３．０９（尿Tp/Cr比）
１０：男性、０．９０（尿Tp/Cr比）
１１：女性、０．５０（尿Tp/Cr比） The sex and urine protein / creatinine ratio of patients (1-11) shown in FIG. 6 were as follows.
1: Male, 0.01 (urine Tp / Cr ratio)
2: Male, 6.31 (urine Tp / Cr ratio)
3: Female, 3.64 (urine Tp / Cr ratio)
4: Female, 5.48 (urine Tp / Cr ratio)
5: Male, 3.02 (urine Tp / Cr ratio)
6: Female, 1.03 (urine Tp / Cr ratio)
7: Female, 1.71 (urine Tp / Cr ratio)
8: Female, 0.44 (urine Tp / Cr ratio)
9: Female, 3.09 (urine Tp / Cr ratio)
10: Male, 0.90 (urine Tp / Cr ratio)
11: Female, 0.50 (urine Tp / Cr ratio)
　B及びCは、腎に炎症を伴わない患者である。DのIgA腎症患者については、炎症の活動性がなくなった患者（図６における「６」）では、Tpbgの検出レベルは低かったが、炎症の活動性がある患者（図６における「７」）では、高いレベルのTpbgの発現がみられた。また、腎炎に属する腎疾患であるE及びFでは、実施例２の結果と同様に高いレベルのTpbgの発現が見られた。尚、尿中Ｔｐｂｇ陽性群と尿中Ｔｐｂｇ陽性群との間に、性別、年齢、血清Cr値、eGFR、及び尿蛋白/uCr比に関する有意差は認められなかった。 B and C are patients with no inflammation in the kidney. As for the IgA nephropathy patient of D, in the patient who lost the inflammation activity (“6” in FIG. 6), the detection level of Tpbg was low, but the patient with the inflammation activity (“7” in FIG. 6). ) Showed high levels of Tpbg expression. In addition, in E and F, which are renal diseases belonging to nephritis, high levels of Tpbg expression were observed as in the results of Example 2. There were no significant differences in sex, age, serum Cr level, eGFR, and urinary protein / uCr ratio between the urinary Tpbg positive group and the urinary Tpbg positive group.
　慢性腎臓病は病状が進行するまで症状が現れず、発見又は治療が遅れることが多く、根治が困難なケースが非常に多い。そのため末期腎不全・透析患者が増加し続けている要因ともなっている。アルブミン尿及びタンパク尿の評価は重要であるが、微小変化型ネフローゼ症候群に代表される、アルブミン尿は大量にみられるものの腎機能低下を来さない疾患と、糖尿病性腎症のようにタンパク尿の増加とともに腎機能低下が進行する疾患は、従来の検査法では早期には識別ができない。しかしながら、上記試験結果で示されたように、ＷＴ１タンパク質及び／又はＴｐｂｇタンパク質の尿中での存在を検出し、指標とすることにより、非侵襲的かつ従来の病理組織学的解析では得られなかった予後予測・治療奏効性予測が可能となる。従って、ＷＴ１タンパク質及び／又はＴｐｂｇタンパク質バイオマーカーとして、精確な病態解明と、その病態を忠実に反映する診断方法を樹立することで、予後改善のためのテーラーメード治療法を確立することが可能になる。 Chronic kidney disease does not show symptoms until the disease progresses, and is often delayed in discovery or treatment, and is very difficult to cure. For this reason, end-stage renal failure / dialysis patients continue to increase. Evaluation of albuminuria and proteinuria is important, but there is a large amount of albuminuria, such as minimal change nephrotic syndrome, but it does not cause a decrease in renal function, and proteinuria such as diabetic nephropathy. Diseases with progressively decreased renal function with increasing levels cannot be identified early by conventional testing methods. However, as shown in the above test results, the presence of WT1 protein and / or Tpbg protein in urine is detected and used as an index, which cannot be obtained by noninvasive and conventional histopathological analysis. Prognosis prediction and treatment response prediction are possible. Therefore, as a WT1 protein and / or Tpbg protein biomarker, it is possible to establish a tailor-made treatment method for improving prognosis by accurately elucidating the pathological condition and establishing a diagnostic method that accurately reflects the pathological condition. .

Claims (8)

  1. 腎炎治療薬に対する反応性を予測するために、被検体から採取した尿中のＴｐｂｇタンパク質を測定する方法。  A method of measuring Tpbg protein in urine collected from a subject in order to predict reactivity to a therapeutic agent for nephritis.
  2. 該尿中のＴｐｂｇタンパク質の存在が治療反応性を有することを示す、請求項１に記載の方法。  2. The method of claim 1, wherein the presence of Tpbg protein in the urine is indicative of therapeutic response.
  3. 該被検体が、その尿中にＷＴ１タンパク質の存在が予め確認された被検体である、請求項１又は２に記載の方法。  The method according to claim 1 or 2, wherein the subject is a subject whose presence of WT1 protein is confirmed in advance in the urine.
  4. 該被検体が慢性腎臓病を患った被検体である、請求項１～３のいずれかに記載の方法。  The method according to any one of claims 1 to 3, wherein the subject is a subject suffering from chronic kidney disease.
  5. 尿中のＴｐｂｇタンパク質を検出するための試薬を含む、腎炎治療薬に対する反応性を予測するためのキット。  A kit for predicting responsiveness to a therapeutic agent for nephritis, comprising a reagent for detecting Tpbg protein in urine.
  6. 更に、ＷＴ１タンパク質を検出するための試薬を含む、請求項５に記載のキット。 The kit according to claim 5, further comprising a reagent for detecting WT1 protein.
  7. 該Ｔｐｂｇタンパク質を検出するための試薬が、該Ｔｐｂｇタンパク質を特異的に認識する抗体である、請求項５に記載のキット。 The kit according to claim 5, wherein the reagent for detecting the Tpbg protein is an antibody that specifically recognizes the Tpbg protein.
  8. 該ＷＴ１タンパク質を検出するための試薬が、該ＷＴ１タンパク質を特異的に認識する抗体である、請求項６に記載のキット。 The kit according to claim 6, wherein the reagent for detecting the WT1 protein is an antibody that specifically recognizes the WT1 protein.
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CN110606890A (en) * 2015-12-24 2019-12-24 凯惠科技发展(上海)有限公司 TPBG antibody, preparation method thereof, conjugate thereof and application thereof
CN110698558A (en) * 2015-12-24 2020-01-17 凯惠科技发展(上海)有限公司 TPBG antibody, preparation method thereof, conjugate thereof and application thereof
CN110698558B (en) * 2015-12-24 2021-09-10 凯惠科技发展(上海)有限公司 TPBG antibody, preparation method thereof, conjugate thereof and application thereof
CN110606890B (en) * 2015-12-24 2021-11-26 凯惠科技发展(上海)有限公司 TPBG antibody, preparation method thereof, conjugate thereof and application thereof
JP2020502507A (en) * 2016-12-16 2020-01-23 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングＭｅｒｃｋ Ｐａｔｅｎｔ Ｇｅｓｅｌｌｓｃｈａｆｔ ｍｉｔ ｂｅｓｃｈｒａｅｎｋｔｅｒ Ｈａｆｔｕｎｇ Use of galectin-3 binding protein detected in urine to monitor the severity and progression of lupus nephritis
JP7271421B2 (en) 2016-12-16 2023-05-11 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング Methods of using galectin-3 binding protein detected in urine to monitor the severity and progression of lupus nephritis

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