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WO2008001840A1 - Method for diagnosis of kidney/urological disease - Google Patents

Method for diagnosis of kidney/urological disease Download PDF

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Publication number
WO2008001840A1
WO2008001840A1 PCT/JP2007/062982 JP2007062982W WO2008001840A1 WO 2008001840 A1 WO2008001840 A1 WO 2008001840A1 JP 2007062982 W JP2007062982 W JP 2007062982W WO 2008001840 A1 WO2008001840 A1 WO 2008001840A1
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WIPO (PCT)
Prior art keywords
mca
acetyl
phe
protease activity
arg
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French (fr)
Japanese (ja)
Inventor
Takahisa Imamura
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Kumamoto University NUC
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Kumamoto University NUC
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Priority to JP2008522619A priority Critical patent/JP5481662B2/en
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • 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

Definitions

  • the present invention relates to a diagnostic method and a diagnostic kit for a kidney'urological disease. More specifically, the present invention relates to a method for diagnosing a renal urinary disease by measuring protease activity in urine, and a diagnostic kit for performing the above method.
  • the kidney filters about 1700 liters of blood per day to produce 1 liter of urine.
  • waste is excreted, water and salt concentration are adjusted, plasma is maintained at an appropriate pH, and physiologically necessary substances such as erythrocytes are produced and supplied to maintain the body.
  • the kidney is said to be a silent organ.Similar to the liver, the disease develops and progresses beyond the reserve capacity. ing. Since kidney status is significantly reflected in urinary components, tests for urine protein content, urine creatine concentration, hematuria, etc. have been conducted as screening for kidney disease, but all of these have low specificity with the disease. It is not enough to detect the initial stage.
  • the present invention aims to provide a diagnostic method and a diagnostic kit for a kidney 'urological disease, characterized by high specificity with the kidney' urological disease and less invasiveness to the patient. It was.
  • a real-time machine that is directly linked to the etiology of kidney and urinary disease and is detected even in the early stages of the disease. If it is possible to discover everything, it is possible to provide a diagnostic method and a diagnostic kit for kidney's urinary disease characterized by high specificity with kidney's urinary disease and less invasion to patients. Become. Proteases, which are proteolytic enzymes, are involved in protein metabolism in cells and secreted to perform various functions outside the cells.
  • protease activity in urine may change depending on the protease.
  • Protease can measure various activities by changing the amino acid sequence used as a substrate, and can detect it with high sensitivity by incorporating a fluorescent substance into the substrate. Therefore, the present inventors paid attention to various proteolytic enzyme (protease) activities appearing in urine and V that do not invade the patient at the time of specimen collection, and comprehensively detect them using various substrates.
  • proteolytic enzyme proteolytic enzyme
  • a method for diagnosing renal urological disease comprising measuring urinary protease activity using a plurality of substrates and analyzing a pattern of protease activity for the substrates.
  • Kidney 'The urological disease is rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer (1) to (4 ).
  • Urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and urine protease activity against acetyl-Phe-Lys-MCA in healthy subjects And the urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-His-Ala-MCA is equivalent to that in healthy subjects
  • MCA represents 4-methylcoumaryl-7-amide group
  • Protease activity in urine against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and in urine against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Indices that the protease activity of urine is higher than that of healthy subjects and that urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-His-Ala-MCA is equivalent to that of healthy subjects
  • MCA represents 4-methylcoumaryl-7-amide group
  • Urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and acetyl-Tyr-Arg-MCA and The urinary protease activity for chill-Phe-Lys-MCA is higher than that for healthy subjects, and the urinary protease activity for acetyl-His-Ala-MCA is equivalent to that for healthy subjects.
  • Protease activity in urine against acetyl-Asn-Phe-MCA is lower than that in healthy subjects, and in urine against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Protease activity is higher than that in healthy subjects, and acetyl-Phe-Arg- Chronic glomerulonephritis is determined based on the fact that the urinary protease activity against MCA and acetyl-His-Ala-MCA is the same as that in healthy subjects (where MCA is 4-methylcoumaryl-7 A method for diagnosing chronic glomerulonephritis.
  • the urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and the urinary protease activity against acetyl-His-Ala-MCA in healthy subjects Indices that urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA is equivalent to that in healthy subjects
  • MCA represents 4-methylcoumaryl-7-amide group
  • the protease activity in urine against acetyl-Tyr-Arg-MCA is higher than that in healthy subjects, and acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, It is determined that the cancer is bladder cancer using as an index the protease activity in urine against chill-Phe-Lys-MCA and acetyl-His-Ala-MCA (in the formula, MCA is 4- A methylcoumaryl-7-amide group), a method for diagnosing bladder cancer.
  • the diagnostic method of the present invention can diagnose a kidney's urinary disease quickly without any invasiveness to a patient.
  • the method for diagnosing renal urinary disease is a method characterized by measuring urinary protease activity. Specifically, urinary protease activity is measured by measuring the protease activity in urine using a plurality of substrates, and analyzing the pattern of protease activity for the substrates. Protease activity can be measured by measuring protease activity (hydrolysis activity) on a peptide substrate composed of two amino acids.
  • acetylyl X 1 — X 2 — Y (wherein X 1 and X 2 each independently represent an amino acid residue, and Y emits fluorescence when the bond with X 2 is cleaved)
  • the protease activity in urine can be measured by measuring the protease activity against the substrate indicated by
  • the amino acid residue X 1 shows, can be mentioned 19 kinds of amino acid residues ( ⁇ amino acid residue except Cys) of Trp from Gly.
  • Specific examples of the amino acid residue represented by X 2 include five types of amino acid residues, Lys, Phe, Val, Ala, and Arg.
  • Y represents a functional group that becomes a fluorescent compound when the bond with X 2 is cleaved.
  • functional groups include MCA (4-methylcoumaryum 7-amide) group, CMCA (4-chloromethyl coumaryi-7-amide), FMCA (4-trifluoromethylcoumary 7-amide), 110 ⁇ gun 110 groups. Or a FITC group.
  • acetyl-Asn-Phe-MCA acetyl-Phe-Arg-MCA
  • acetyl-Tyr-Arg-MCA acetyl-Phe-Lys-MCA
  • acetyl-His-Ala The urinary protease activity is measured by measuring the protease activity against MCA (wherein MCA represents 4-methylcoumaryl-7-amide group).
  • kidney and urinary diseases such as
  • Measurement of urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, or acetyl-His-Ala-MCA For example, it can be performed as follows. Dissolve the above five kinds of substrates in dimethyl sulfoxide, and add a solution (1 mM) diluted with 0.1M Tris-HCl, pH7.6, 150 mM NaCl to a 96-well plate.
  • the kidney's urinary disease was found to be rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, Kidney'urological diseases such as chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer can be diagnosed.
  • the diagnostic indicators are as described above in this specification and as shown in FIG.
  • urinary proteases for acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, and acetyl-His-Ala-MCA For example, when Asn-Phe is 86 or less, Phe-Arg is 103 or less, Ph e-Lys is 12 or more, Ding 1- ⁇ is 8.6 or more, and His-Ala is 7.4 or more (unit is cpu). / mg creatinine) can be determined to be an abnormal value.
  • acetyl-Asn-Phe-MCA acetyl-Phe-Arg-MCA
  • acetyl-Tyr-Arg-MCA acetyl-Phe-Lys-MCA
  • acetyl-His-Ala A diagnostic kit for kidney 'urinary diseases is provided, which contains -MCA (wherein MCA represents 4-methylcoumaryl-7-amide group).
  • the diagnostic kit of the present invention may further contain a solvent, a buffer solution, a measurement plate and the like as appropriate.
  • Urine was obtained from healthy individuals, nephritis patients, and urinary cancer patients, and the urine was used approximately 2 hours after the previous urination. 9 rapidly progressive glomerulonephritis, nephrotic syndrome 1
  • Urine was measured in 0 patients, IgA nephropathy 26 patients, chronic glomerulonephritis 12 patients, chronic renal failure 22 patients, prostate cancer 21 patients, bladder cancer 8 patients, and healthy volunteers 10 patients.
  • MCA is converted to AMC (7-amino-4-methylco) by protease hydrolysis between CAs. This was measured at an excitation wavelength of 355 nm and a detection wavelength of 450 nm. Five amino acids, Lys, Phe, Val, Ala, Arg, are selected for X, and Gly to Trp
  • a total of 95 types of substrates in which 19 types of amino acids were arranged were dissolved in dimethylsulfoxide, and 50 1 (1 mM) diluted with 0.1 M Tris-HCl, pH 7.6, 150 mM NaCl was added to a 96-well plate.
  • Urine 50 1 was added thereto and reacted at 37 ° C, and an increase in fluorescence intensity was detected over time with a fluorescence measuring device Arvo about every minute.
  • the activity with respect to each substrate was defined as the value with the highest rate of increase of 5 points out of 10 points. Since activity is affected by urine concentration, it was expressed as activity against Creatun 100 mg / dl.
  • the protease activity of 10 healthy subjects was measured.
  • -X-Arg-MCA system showed the most diverse activity, and activity against AR, LR, MR, HR, FR, YR, WR, PR, etc. was detected. In particular, FR activity was high.
  • activity against HK, FK, WK, and YK was observed. The highest FK activity was observed.
  • LF and NF were active in the X-Phe-MCA system.
  • Cathebsin B showed the strongest activity, and the highest activity among all substrates. Although the remaining -X-Va ⁇ MCA and -X-Ala-MCA substrates did not detect any activity at all, VV , IV, GA, HA weak activity was observed.
  • Acety ⁇ Asn-Phe-MCA was the substrate in which cathebsin B was most hydrolyzed and had the highest activity in the urine of healthy subjects. This activity was significantly decreased in IgA nephropathy (P 0.034) and chronic glomerulonephritis (P 0.034). In these diseases, there is a possibility that the production of protease related to this activity is decreased or the degradation of consumption is increased.
  • This activity is associated with rapidly progressive glomerulonephritis (P ⁇ 0.008), nephrotic syndrome (P ⁇ 0.023), IgA nephropathy (P ⁇ 0.045), chronic kidney disease other than chronic glomerulonephritis (P 0.08).
  • Significantly low strength in renal failure (P ⁇ 0.005) Cancer showed no significant difference. In these diseases, there is a possibility that the production of protease related to this activity may be reduced or consumption / degradation may be increased.
  • AcetyKTyr-Arg-MCA activity increases in disease as opposed to the activity that decreases in disease compared to healthy subjects, such as Acety ⁇ Asn- Phe-MCA and Acety ⁇ Phe-Arg-MCA described above It is active, has the ability to increase in both power and cancer, and has a very high diagnostic value. It is also noteworthy that this activity shows contrasting variation in the disease despite its very similar structure to acety ⁇ Phe-Arg-MCA.
  • Rapid progressive glomerulonephritis (P 0.019), nephrotic syndrome (P ⁇ 0.013), IgA nephropathy (P ⁇ 0.044), chronic glomerulonephritis (P ⁇ 0.023) Although it was significantly stronger, it was not recognized that there was a significant difference in cancer. The activity is high in various types of kidney diseases except chronic renal failure and does not increase in cancer. Therefore, it is specific for kidney disease, has a wide range of applications, and has high diagnostic utility. / ⁇ .
  • Protease activity for the five types of substrates used above is divided into activity that decreases and increases depending on the disease when compared between patients and healthy subjects.
  • the former is Acety ⁇ Asn- Phe- MCA and Acety ⁇ Phe- Arg- MCA hydrolysis activity, the latter being Acety ⁇ Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, and Acety ⁇ His-Ala-MCA hydrolysis activity.
  • Fig. 7 when these activities were compared for each disease, which were low, no difference, and high, compared with those in healthy subjects, different patterns were observed in seven diseases including nephritis and two cancers. It became clear to take This result shows that differential diagnosis of these diseases may be possible by comparing the activity against the above five kinds of substrates in urine between healthy subjects and patients.
  • the five renal diseases of interest this time can be differentiated by activity against four substrates except AcetyH "Iis-Ala-MCA, and the above two cancers can also be excluded. Cancers were identified by their activity against three different substrates: Acety ⁇ Phe- Arg- MCA ⁇ Acety ⁇ Phe- Lys- MCA ⁇ Acety ⁇ His- Ala- MCA, and the possibility of excluding kidney disease was also shown. Therefore, by measuring the activity in urine using the activity against these five kinds of substrates as a reporter and performing pattern analysis by comparison with healthy subjects, non-invasive and rapid treatment of nephritis, urinary tract cancer, etc. It becomes possible to diagnose kidney 'urological diseases.
  • FIG. 1 shows the activity patterns of human thrombin, human mouth kinase and human cathebsin B.
  • FIG. 2 shows protease activity against acetyl-Asn-Phe-MCA in the urine of patients with renal / urological diseases and healthy subjects.
  • FIG. 3 shows protease activity against acetyl-Phe-Arg-MCA in the urine of renal and urological disease patients and healthy subjects.
  • FIG. 4 shows protease activity against acetyl-Tyr-Arg-MCA in the urine of patients with renal / urological diseases and healthy subjects.
  • FIG. 5 shows protease activity against acetyl-Phe-Lys-MCA in the urine of patients with renal / urological diseases and healthy subjects.
  • FIG. 6 shows protease activity against acetyl-His-Ala-MCA in the urine of renal and urological disease patients and healthy subjects.
  • FIG. 7 shows the results of a comparison of protease activity against 5 kinds of substrates between renal and urological disease patients and normal subjects.

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Abstract

Disclosed are a method and a kit for the diagnosis of a kidney/urological disease, which are highly specific to a kidney/urological disease and are less invasive to a patient. The method for the diagnosis of a kidney/urological disease comprises measuring a protease activity in urine by using two or more substrates and analyzing the patterns of the protease activity against the substrates.

Description

明 細 書  Specification

腎臓 ·泌尿器疾患の診断方法  Diagnosis of kidney and urological diseases

技術分野  Technical field

[0001] 本発明は、腎臓'泌尿器疾患の診断方法及び診断キットに関する。より詳細には、 本発明は、尿中のプロテアーゼ活性を測定することによって腎臓'泌尿器疾患を診 断する方法、及び上記方法を行うための診断キットに関する。  [0001] The present invention relates to a diagnostic method and a diagnostic kit for a kidney'urological disease. More specifically, the present invention relates to a method for diagnosing a renal urinary disease by measuring protease activity in urine, and a diagnostic kit for performing the above method.

背景技術  Background art

[0002] 腎臓は一日に約 1700リットルの血液を濾過し尿 1リットルを産生する。これにより老 廃物排泄、水と塩分濃度調節、血漿の適切な pHの維持行い、さらにエリス口ポェチ ンなどの生理的に必要な物質を産生して生体維持のために供給している。腎臓は沈 黙の臓器といわれ、肝臓と同様に病気が力なり進行し予備能力を越えて機能低下が 起こって初めて症状が出現するので、症状が出現した時点では腎臓の状態は相当 重篤化している。腎臓の状態は尿成分にかなり反映されるので、腎疾患のスクリー二 ングとして尿蛋白質量、尿クレアチュン濃度、血尿などの検査が施行されているが、 いずれも疾患との特異性は低く病気の初期段階を検出するには充分ではない。糸球 体腎炎の最終診断は腎生検による腎臓組織の形態変化によって行われるが、これに よる診断は原因と必ずしも一致したものではなく初期では確定診断が困難であり、し 力も患者に与える侵襲が大きい。また、尿路系癌 (特に膀胱癌)は尿路に露出してい る場合が多ぐ尿にはこれらの癌情報も含んでいることが充分期待されるが、尿による これらの癌診断の試みは未だなされて ヽな 、。  [0002] The kidney filters about 1700 liters of blood per day to produce 1 liter of urine. As a result, waste is excreted, water and salt concentration are adjusted, plasma is maintained at an appropriate pH, and physiologically necessary substances such as erythrocytes are produced and supplied to maintain the body. The kidney is said to be a silent organ.Similar to the liver, the disease develops and progresses beyond the reserve capacity. ing. Since kidney status is significantly reflected in urinary components, tests for urine protein content, urine creatine concentration, hematuria, etc. have been conducted as screening for kidney disease, but all of these have low specificity with the disease. It is not enough to detect the initial stage. Although the final diagnosis of glomerulonephritis is made by morphological changes in kidney tissue by renal biopsy, this diagnosis is not always consistent with the cause and is difficult to make a definitive diagnosis at an early stage. Is big. In addition, urinary tract cancers (especially bladder cancer) are often exposed in the urinary tract, and it is highly expected that urine contains these cancer information. Hasn't been done yet.

発明の開示  Disclosure of the invention

発明が解決しょうとする課題  Problems to be solved by the invention

[0003] 本発明は、腎臓'泌尿器疾患との特異性が高ぐかつ患者に与える侵襲が少ないこ とを特徴とする腎臓'泌尿器疾患の診断方法及び診断キットを提供することを解決す べき課題とした。 [0003] The present invention aims to provide a diagnostic method and a diagnostic kit for a kidney 'urological disease, characterized by high specificity with the kidney' urological disease and less invasiveness to the patient. It was.

課題を解決するための手段  Means for solving the problem

[0004] 腎臓'泌尿器疾患の病因に直結し、かつ病気の初期でも検出されるリアルタイムマ 一力一を発見することができれば、腎臓'泌尿器疾患との特異性が高ぐかつ患者に 与える侵襲が少ないことを特徴とする腎臓'泌尿器疾患の診断方法及び診断キットを 提供することが可能になる。蛋白質分解酵素であるプロテアーゼは、細胞内で蛋白 質代謝等に関わるとともに分泌されて細胞外で様々な機能を果たしている。本発明 者らは、腎臓や尿路に炎症や腫瘍が発生すると、刺激を受けた臓器細胞からのプロ テアーゼ分泌変化や白血球、さらには腫瘍細胞力も分泌、また自壊あるいは傷害さ れた細胞力 漏出するプロテアーゼによって、尿中のプロテアーゼ活性は変化する 可能性があることを推定した。プロテア一ゼは基質となるアミノ酸配列を変えることに より種々の活性が測定でき、さらに基質に蛍光物質を組み入れることにより高感度検 出が可能である。そこで、本発明者らは、検体採取において患者への侵襲が全くな V、尿に出現する種々の蛋白質分解酵素 (プロテアーゼ)活性に注目し、多種の基質を 使って網羅的に検出し、これらの活性と、各種の腎臓'泌尿器疾患との関係を調べた 。その結果、本発明者らは、尿中のプロテアーゼ活性を測定することによって腎臓' 泌尿器疾患を診断できることを見出した。本発明はこれらの知見に基づいて完成した ものである。 [0004] A real-time machine that is directly linked to the etiology of kidney and urinary disease and is detected even in the early stages of the disease. If it is possible to discover everything, it is possible to provide a diagnostic method and a diagnostic kit for kidney's urinary disease characterized by high specificity with kidney's urinary disease and less invasion to patients. Become. Proteases, which are proteolytic enzymes, are involved in protein metabolism in cells and secreted to perform various functions outside the cells. When inflammation or tumor occurs in the kidney or urinary tract, the present inventors also secrete changes in protease secretion from stimulated organ cells, leukocytes, and also secrete tumor cell force, and self-destructed or damaged cell force leakage It was presumed that the protease activity in urine may change depending on the protease. Protease can measure various activities by changing the amino acid sequence used as a substrate, and can detect it with high sensitivity by incorporating a fluorescent substance into the substrate. Therefore, the present inventors paid attention to various proteolytic enzyme (protease) activities appearing in urine and V that do not invade the patient at the time of specimen collection, and comprehensively detect them using various substrates. The relationship between the activity of various kidneys and urinary diseases was investigated. As a result, the present inventors have found that renal and urological diseases can be diagnosed by measuring protease activity in urine. The present invention has been completed based on these findings.

[0005] 即ち、本発明によれば、以下の発明が提供される。  That is, according to the present invention, the following inventions are provided.

(1) 尿中のプロテアーゼ活性を複数の基質を用いて測定し、当該基質に対するプ 口テアーゼ活性のパターンを分析することを含む、腎臓'泌尿器疾患の診断方法。  (1) A method for diagnosing renal urological disease, comprising measuring urinary protease activity using a plurality of substrates and analyzing a pattern of protease activity for the substrates.

[0006] (2) ァセチル— X1— X2— Y (式中、 X1及び X2は、各々独立にアミノ酸残基を示し、 Y は、 X2との結合が切断されると蛍光を発する化合物になる官能基を示す)で示される 基質に対するプロテアーゼ活性を測定することにより、尿中のプロテアーゼ活性を測 定する、（1)に記載の方法。 [0006] (2) Acetyl — X 1 — X 2 — Y (wherein X 1 and X 2 each independently represent an amino acid residue, Y represents fluorescence when the bond with X 2 is cleaved) The method according to (1), wherein the protease activity in urine is measured by measuring the protease activity against a substrate represented by

[0007] (3) ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- MC A、ァセチル- Phe- Lys- MCA、又はァセチル- His- Ala- MCA (式中、 MCAは、 4-メチ ルクマリル- 7-アミド基を示す）に対するプロテアーゼ活性を測定することにより、尿中 のプロテアーゼ活性を測定する、（1)又は（2)に記載の方法。 [0007] (3) Acetyl-Asn-Phe-MCA, Acetyl-Phe-Arg-MCA, Acetyl-Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, or Acetyl-His-Ala-MCA (wherein The method according to (1) or (2), wherein the urinary protease activity is measured by measuring the protease activity against MCA (which represents 4-methylcoumaryl-7-amide group).

(4) ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- MC A、ァセチル- Phe- Lys- MCA、及びァセチル- His- Ala- MCA (式中、 MCAは、 4-メチ ルクマリル- 7-アミド基を示す）に対する尿プロテアーゼ活性の測定において、 Asn-P heでは 86以下、 Phe-Argでは 103以下、 Phe-Lysでは 12以上、丁 1- §では8.6以上、 His- Alaでは 7.4以上の場合（単位は cpu/mg creatinine)は異常値であると判断する、 (1)から（3)の何れかに記載の方法。 (4) Acetyl-Asn-Phe-MCA, Acetyl-Phe-Arg-MCA, Acetyl-Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, and Acetyl-His-Ala-MCA (where MCA is 4-methyl In the measurement of urinary protease activity against lucumaryl-7-amide group), Asn-Phe is 86 or less, Phe-Arg is 103 or less, Phe-Lys is 12 or more, Ding 1- § is 8.6 or more, His-Ala Then, if it is 7.4 or more (unit is cpu / mg creatinine), it is judged as an abnormal value. The method according to any one of (1) to (3).

[0008] (5) 腎臓 '泌尿器疾患が、急速進行性糸球体腎炎、ネフローゼ症候群、 IgA腎症、 慢性糸球体腎炎、慢性腎不全、前立腺癌、又は膀胱癌である、（1)から (4)の何れ かに記載の方法。 [0008] (5) Kidney 'The urological disease is rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer (1) to (4 ).

[0009] (6) ァセチル- Phe-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合よ りも低下しており、ァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健常 者の場合よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Tyr-Arg-M CA及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場合 と同等であることを指標として急速進行性糸球体腎炎であると判定する (式中、 MCA は、 4-メチルクマリル- 7-アミド基を示す)、急速進行性糸球体腎炎の診断方法。  [0009] (6) Urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and urine protease activity against acetyl-Phe-Lys-MCA in healthy subjects And the urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-His-Ala-MCA is equivalent to that in healthy subjects As a method of diagnosing rapidly progressive glomerulonephritis (where MCA represents 4-methylcoumaryl-7-amide group).

[0010] (7) ァセチル- Phe-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合よ りも低下しており、ァセチル- Tyr-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿 中のプロテアーゼ活性が健常者の場合よりも上昇しており、かつァセチル -Asn- Phe- MCA及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場 合と同等であることを指標としてネフローゼ症候群であると判定する（式中、 MCAは、 4-メチルクマリル- 7-アミド基を示す）、ネフローゼ症候群の診断方法。 [0010] (7) Protease activity in urine against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and in urine against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Indices that the protease activity of urine is higher than that of healthy subjects and that urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-His-Ala-MCA is equivalent to that of healthy subjects As determined as nephrotic syndrome (where, MCA represents 4-methylcoumaryl-7-amide group), a diagnostic method for nephrotic syndrome.

[0011] (8) ァセチル -Asn- Phe-MCA及びァセチル- Phe-Arg-MCAに対する尿中のプロテ ァーゼ活性が健常者の場合よりも低下しており、ァセチル -Tyr-Arg-MCA及びァセ チル -Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも上昇し ており、かつァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の 場合と同等であることを指標として IgA腎症であると判定する（式中、 MCAは、 4-メチ ルクマリル- 7-アミド基を示す）、 IgA腎症の診断方法。 [0011] (8) Urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and acetyl-Tyr-Arg-MCA and The urinary protease activity for chill-Phe-Lys-MCA is higher than that for healthy subjects, and the urinary protease activity for acetyl-His-Ala-MCA is equivalent to that for healthy subjects. A diagnostic method for IgA nephropathy, in which IgA nephropathy is determined as an index (wherein MCA represents 4-methylcoumaryl-7-amide group).

[0012] (9) ァセチル -Asn- Phe-MCAに対する尿中のプロテアーゼ活性が健常者の場合よ りも低下しており、ァセチル- Tyr-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿 中のプロテアーゼ活性が健常者の場合よりも上昇しており、かつァセチル -Phe-Arg- MCA及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場 合と同等であることを指標として慢性糸球体腎炎であると判定する (式中、 MCAは、 4 -メチルクマリル- 7-アミド基を示す)、慢性糸球体腎炎の診断方法。 [0012] (9) Protease activity in urine against acetyl-Asn-Phe-MCA is lower than that in healthy subjects, and in urine against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Protease activity is higher than that in healthy subjects, and acetyl-Phe-Arg- Chronic glomerulonephritis is determined based on the fact that the urinary protease activity against MCA and acetyl-His-Ala-MCA is the same as that in healthy subjects (where MCA is 4-methylcoumaryl-7 A method for diagnosing chronic glomerulonephritis.

[0013] (10) ァセチル- Phe-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合 よりも低下しており、ァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健 常者の場合よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Tyr-Arg- MCA及びァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健常者の場 合と同等であることを指標として慢性腎不全であると判定する (式中、 MCAは、 4-メ チルクマリル- 7-アミド基を示す)、慢性腎不全の診断方法。  [0013] (10) The urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and the urinary protease activity against acetyl-His-Ala-MCA in healthy subjects Indices that urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA is equivalent to that in healthy subjects As a method for diagnosing chronic renal failure (wherein, MCA represents 4-methylcoumaryl-7-amide group).

[0014] (11) ァセチル- Tyr-Arg-MCA及びァセチル- His-Ala-MCAに対する尿中のプロテ ァーゼ活性が健常者の場合よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセ チル- Phe-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活 性が健常者の場合と同等であることを指標として前立腺癌であると判定する (式中、 MCAは、 4-メチルクマリル- 7-アミド基を示す）、前立腺癌の診断方法。  [11] (11) The urinary protease activity against acetyl-Tyr-Arg-MCA and acetyl-His-Ala-MCA is higher than that in healthy subjects, and acetyl-Asn-Phe-MCA, Prostate cancer is determined by using the urinary protease activity against cetyl-Phe-Arg-MCA and acetyl-Phe-Lys-MCA as an index for healthy subjects (where MCA is 4-methylcoumaryl-7-amide group)), a method for diagnosing prostate cancer.

[0015] (12) ァセチル- Tyr-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合 よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Phe-Arg-MCA、ァセ チル- Phe-Lys-MCA及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性 が健常者の場合と同等であることを指標として膀胱癌であると判定する (式中、 MCA は、 4-メチルクマリル- 7-アミド基を示す)、膀胱癌の診断方法。  [12] (12) The protease activity in urine against acetyl-Tyr-Arg-MCA is higher than that in healthy subjects, and acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, It is determined that the cancer is bladder cancer using as an index the protease activity in urine against chill-Phe-Lys-MCA and acetyl-His-Ala-MCA (in the formula, MCA is 4- A methylcoumaryl-7-amide group), a method for diagnosing bladder cancer.

[0016] (13) ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- M CA、ァセチル- Phe- Lys- MCA、及びァセチル- His- Ala- MCA (式中、 MCAは、 4-メ チルクマリル- 7-アミド基を示す)を含む、腎臓'泌尿器疾患の診断キット。  [0016] (13) Acetyl-Asn-Phe-MCA, Acetyl-Phe-Arg-MCA, Acetyl-Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, and Acetyl-His-Ala-MCA (wherein , MCA includes 4-methylcoumaryl-7-amide group), a diagnostic kit for kidney 'urological diseases.

発明の効果  The invention's effect

[0017] 本発明によれば、腎臓'泌尿器疾患を高い特異性で診断することが可能である。ま た、本発明の診断方法は、患者に対する侵襲が全くなぐ迅速に腎臓'泌尿器疾患 を診断することができる。  [0017] According to the present invention, it is possible to diagnose kidney 'urinary disease with high specificity. In addition, the diagnostic method of the present invention can diagnose a kidney's urinary disease quickly without any invasiveness to a patient.

発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION

[0018] 以下、本発明の実施の形態についてさらに詳細に説明する。 本発明による腎臓'泌尿器疾患の診断方法は、尿中のプロテアーゼ活性を測定す ることを特徴とする方法である。具体的には、尿中のプロテアーゼ活性を複数の基質 を用いて測定し、当該基質に対するプロテアーゼ活性のパターンを分析することによ つて、腎臓'泌尿器疾患の診断を行う。プロテアーゼ活性は、 2アミノ酸カゝら構成され るペプチド基質に対するプロテアーゼ活性 (加水分解活性)を測定することによって 測定することができる。本発明では、例えば、ァセチルー X1— X2— Y (式中、 X1及び X2は、各々独立にアミノ酸残基を示し、 Yは、 X2との結合が切断されると蛍光を発す る化合物になる官能基を示す)で示される基質に対するプロテアーゼ活性を測定す ることにより、尿中のプロテアーゼ活性を測定することができる。 [0018] Hereinafter, embodiments of the present invention will be described in more detail. The method for diagnosing renal urinary disease according to the present invention is a method characterized by measuring urinary protease activity. Specifically, urinary protease activity is measured by measuring the protease activity in urine using a plurality of substrates, and analyzing the pattern of protease activity for the substrates. Protease activity can be measured by measuring protease activity (hydrolysis activity) on a peptide substrate composed of two amino acids. In the present invention, for example, acetylyl X 1 — X 2 — Y (wherein X 1 and X 2 each independently represent an amino acid residue, and Y emits fluorescence when the bond with X 2 is cleaved) The protease activity in urine can be measured by measuring the protease activity against the substrate indicated by

[0019] X1が示すアミノ酸残基としては、 Glyから Trpの 19種類のアミノ酸残基（Cys以外のァ ミノ酸残基)を挙げることができる。また、 X2が示すアミノ酸残基の具体例としては、 Ly s、 Phe、 Val、 Ala又は Argの 5種類のアミノ酸残基を挙げることができる。 [0019] The amino acid residue X 1 shows, can be mentioned 19 kinds of amino acid residues (§ amino acid residue except Cys) of Trp from Gly. Specific examples of the amino acid residue represented by X 2 include five types of amino acid residues, Lys, Phe, Val, Ala, and Arg.

[0020] Yは、 X2との結合が切断されると蛍光を発する化合物になる官能基を示す。このよう な官能基としては、 MCA (4- methylcoumary卜 7- amide)基， CMCA (4- chloromethyl coumaryi- 7- amide) 、 FMCA (4- trifluoromethylcoumaryト 7- amide) 、口 ~~グ ン 110基、又は FITC基などを挙げることができる。 [0020] Y represents a functional group that becomes a fluorescent compound when the bond with X 2 is cleaved. Such functional groups include MCA (4-methylcoumaryum 7-amide) group, CMCA (4-chloromethyl coumaryi-7-amide), FMCA (4-trifluoromethylcoumary 7-amide), 110 ~~ gun 110 groups. Or a FITC group.

[0021] 本発明にお 、て好ましくは、ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、 ァセチル- Tyr- Arg- MCA、ァセチル- Phe- Lys- MCA、又はァセチル- His- Ala- MCA ( 式中、 MCAは、 4-メチルクマリル- 7-アミド基を示す）に対するプロテアーゼ活性を測 定することにより、尿中のプロテアーゼ活性を測定する。上記した 5種類の基質に対 する尿中のプロテアーゼ活性を測定することによって、急速進行性糸球体腎炎、ネフ ローゼ症候群、 IgA腎症、慢性糸球体腎炎、慢性腎不全、前立腺癌、又は膀胱癌な どの腎臓'泌尿器疾患を特異的に診断することができる。  In the present invention, preferably, acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, or acetyl-His-Ala -The urinary protease activity is measured by measuring the protease activity against MCA (wherein MCA represents 4-methylcoumaryl-7-amide group). By measuring urinary protease activity for the above five substrates, rapid progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer It can specifically diagnose kidney and urinary diseases such as

[0022] ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- MCA、ァ セチル - Phe-Lys-MCA、又はァセチル- His-Ala-MCAに対する尿プロテアーゼ活性 の測定は、例えば、以下の通り行うことができる。上記 5種類の基質をジメチルスルホ キシドに溶解し、さらに 0.1M Tris-HCl, pH7.6, 150 mM NaClで希釈した溶液 (1 mM) を 96穴プレートに入れる。これに尿をカ卩えて 37°Cで反応させ、蛍光強度の増加を約 1 分毎に蛍光測定器で経時的に検出する。例えば、 10点のうち連続 5点の増加率が最 も高 、値を各基質に対するプロテアーゼ活性とすることができる。プロテアーゼ活性 は尿の濃度に影響されるので、クレアチュン 100mg/dlに対する活性として表すること ができる。被験者の尿中の各基質に対するプロテアーゼ活性を、コントロール値 (即 ち、健常者におけるプロテアーゼ活性)と比較することにより、腎臓'泌尿器疾患が、 急速進行性糸球体腎炎、ネフローゼ症候群、 IgA腎症、慢性糸球体腎炎、慢性腎不 全、前立腺癌、又は膀胱癌などの腎臓'泌尿器疾患を診断することができる。診断の 指標は、本明細書中に上記した記載した通りであり、また図 7にも示す通りである。具 体的には、ァセチル -Asn- Phe-MCA、ァセチル- Phe-Arg-MCA、ァセチル- Tyr-Arg -MCA,ァセチル- Phe-Lys-MCA、及びァセチル- His-Ala-MCAに対する尿プロテア ーゼ活性の測定において、例えば、 Asn-Pheでは 86以下、 Phe-Argでは 103以下、 Ph e-Lysでは 12以上、丁 1- §では8.6以上、 His-Alaでは 7.4以上の場合（単位は cpu/m g creatinine)は異常値であると判断することができる。 [0022] Measurement of urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, or acetyl-His-Ala-MCA For example, it can be performed as follows. Dissolve the above five kinds of substrates in dimethyl sulfoxide, and add a solution (1 mM) diluted with 0.1M Tris-HCl, pH7.6, 150 mM NaCl to a 96-well plate. Add urine to this and react at 37 ° C to increase fluorescence intensity by approximately 1 Detect with time with a fluorometer every minute. For example, the rate of increase of 5 consecutive points out of 10 is the highest, and the value can be the protease activity for each substrate. Since protease activity is affected by urine concentration, it can be expressed as activity against Creatun 100 mg / dl. By comparing the protease activity for each substrate in the urine of the subject with the control value (i.e., protease activity in healthy subjects), the kidney's urinary disease was found to be rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, Kidney'urological diseases such as chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer can be diagnosed. The diagnostic indicators are as described above in this specification and as shown in FIG. Specifically, urinary proteases for acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, and acetyl-His-Ala-MCA. For example, when Asn-Phe is 86 or less, Phe-Arg is 103 or less, Ph e-Lys is 12 or more, Ding 1- § is 8.6 or more, and His-Ala is 7.4 or more (unit is cpu). / mg creatinine) can be determined to be an abnormal value.

[0023] さらに本発明によれば、ァセチル -Asn- Phe-MCA、ァセチル- Phe-Arg-MCA、ァセ チル- Tyr-Arg-MCA、ァセチル- Phe-Lys-MCA、及びァセチル- His-Ala-MCA (式 中、 MCAは、 4-メチルクマリル- 7-アミド基を示す)を含む、腎臓'泌尿器疾患の診断 キットが提供される。なお、本発明の診断キットには、上記 5種類の基質に加えて、さ らに溶媒、緩衝液、測定プレートなどを適宜含めてもよい。  [0023] Further according to the present invention, acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Tyr-Arg-MCA, acetyl-Phe-Lys-MCA, and acetyl-His-Ala A diagnostic kit for kidney 'urinary diseases is provided, which contains -MCA (wherein MCA represents 4-methylcoumaryl-7-amide group). In addition to the above five types of substrates, the diagnostic kit of the present invention may further contain a solvent, a buffer solution, a measurement plate and the like as appropriate.

[0024] 以下の実施例により本発明を更に詳しく説明するが、本発明はこれらの実施例によ つて限定されるものではな 、。  [0024] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

実施例  Example

[0025] (1)実験方法 [0025] (1) Experimental method

尿は健常者、腎炎患者および泌尿器系癌患者のものを本人の承諾を得て、前回の 排尿力も約 2時間後の尿を用いた。急速進行性糸球体腎炎 9名、ネフローゼ症候群 1 Urine was obtained from healthy individuals, nephritis patients, and urinary cancer patients, and the urine was used approximately 2 hours after the previous urination. 9 rapidly progressive glomerulonephritis, nephrotic syndrome 1

0名、 IgA腎症 26名、慢性糸球体腎炎 12名、慢性腎不全 22名、前立腺癌 21名、膀胱 癌 8名、健常者 10名の尿を測定した。 Urine was measured in 0 patients, IgA nephropathy 26 patients, chronic glomerulonephritis 12 patients, chronic renal failure 22 patients, prostate cancer 21 patients, bladder cancer 8 patients, and healthy volunteers 10 patients.

[0026] Acetyl (ある ヽは succinyl)- X -X - MCA(4- methylcoumaryト 7- amide)を合成し、 X - M [0026] Acetyl (some succinyl) -X-X-MCA (4-methylcoumary 7-amide) was synthesized and X-M

1 2 2 1 2 2

CA間をプロテアーゼが加水分解することによって MCAが AMC(7-amino-4-methylco umarin)になり蛍光を発するので、これを励起波長 355nm、検出波長 450nmで測定し た。 Xには Lys、 Phe、 Val、 Ala、 Argの 5種類のアミノ酸を選定し、 Xには Glyから TrpのMCA is converted to AMC (7-amino-4-methylco) by protease hydrolysis between CAs. This was measured at an excitation wavelength of 355 nm and a detection wavelength of 450 nm. Five amino acids, Lys, Phe, Val, Ala, Arg, are selected for X, and Gly to Trp

2 1 twenty one

19種類のアミノ酸を配置した計 95種類の基質を dimethylsulfoxideに溶解し、さらに 0.1 M Tris-HCl, pH7.6, 150 mM NaClで希釈した 50 1(1 mM)を 96穴プレートに入れた。 これに尿 50 1を加えて 37°Cで反応させ、蛍光強度の増加を約 1分毎に蛍光測定器 A rvoで経時的に検出した。 10点のうち連続 5点の増加率が最も高い値を各基質に対す る活性とした。活性は尿の濃度に影響されるので、クレアチュン 100mg/dlに対する活 性として表した。検出された MCA基質加水分解活性の中から、充分かつ高頻度で加 水分解がみられる数種の基質を選び、患者と健常者との比較を行った。 Unpaired t-t estを行い、 Pく 0.05を有意とした。また、これらの活性をパターン解析し、これによる 分類と臨床診断との関連性を検討した。  A total of 95 types of substrates in which 19 types of amino acids were arranged were dissolved in dimethylsulfoxide, and 50 1 (1 mM) diluted with 0.1 M Tris-HCl, pH 7.6, 150 mM NaCl was added to a 96-well plate. Urine 50 1 was added thereto and reacted at 37 ° C, and an increase in fluorescence intensity was detected over time with a fluorescence measuring device Arvo about every minute. The activity with respect to each substrate was defined as the value with the highest rate of increase of 5 points out of 10 points. Since activity is affected by urine concentration, it was expressed as activity against Creatun 100 mg / dl. From the detected MCA substrate hydrolyzing activity, several types of substrates that were sufficiently and frequently hydrolyzed were selected and compared with patients. Unpaired t-test was performed, and P 0.05 was considered significant. We also analyzed the pattern of these activities and examined the relationship between classification and clinical diagnosis.

[0027] (2)結果 [0027] (2) Results

(2—1)尿中に存在すると推定される代表的なプロテアーゼの活性パターン ヒトトロンビン (1 IU/ml)は PR > AR > NF = PK = GAの順に活性を示し、従来より B oc-V-P-R-MCAが特異的な基質として使われて 、る事実によく一致して 、た。ヒトウ 口キナーゼ (10 nM)は AR > GK >AR = HR = PR = MR = QRの順に活性があり、 Arg や Lysを好むその性質に合致していた。ヒトカテブシン B(20 nM)活性は NF > GAが主 であった。し力し、 NFと GAは 3者共に、 PK、 ARと PRはトロンビンとゥロキナーゼとでカロ 水分解されていることから、 1つの基質に対する活性は必ずしも 1つのプロテアーゼに 由来しないものと認められる。  (2-1) Typical protease activity pattern presumed to be present in urine Human thrombin (1 IU / ml) is active in the order of PR> AR> NF = PK = GA, and B oc-VPR -MCA was used as a specific substrate and was in good agreement with the fact. Human mouth kinase (10 nM) was active in the order of AR> GK> AR = HR = PR = MR = QR, consistent with its preference for Arg and Lys. Human cathepsin B (20 nM) activity was mainly NF> GA. However, since NF and GA are all hydrolyzed by thrombin and urokinase for NF and GA, the activity against one substrate is not necessarily derived from one protease.

[0028] (2- 2)健常者尿中に検出される MCA基質加水分解活性の概要 [0028] (2-2) Overview of MCA substrate hydrolysis activity detected in urine of healthy volunteers

異常を知るためには健常者の活性が標準となるので、健常者 10名のプロテアーゼ 活性を測定した。 -X-Arg-MCA系に最も多種の活性が認められ、 AR、 LR、 MR、 HR、 FR、 YR、 WR、 PR等への活性が検出された。なかでも、 FR活性が高かった。次は、 -X - Lys-MCA系で HK、 FK、 WK、 YKへの活性がみられた力 FK活性が最も高かった。 あとは- X- Phe-MCA系で LF、 NFに活性があり、特にカテブシン Bが最も強い活性を 示した NFに対しては全基質のなかで最も高い活性があった。残りの- X-Va卜 MCA、 - X-Ala-MC A系の基質に対しては全く活性の検出されないものが多かったものの、 VV 、 IV、 GA、 HAへの弱い活性が認められた。 Since the normal activity is the standard for knowing the abnormality, the protease activity of 10 healthy subjects was measured. -X-Arg-MCA system showed the most diverse activity, and activity against AR, LR, MR, HR, FR, YR, WR, PR, etc. was detected. In particular, FR activity was high. Next, in the -X-Lys-MCA system, activity against HK, FK, WK, and YK was observed. The highest FK activity was observed. After that, LF and NF were active in the X-Phe-MCA system. Cathebsin B showed the strongest activity, and the highest activity among all substrates. Although the remaining -X-Va 卜 MCA and -X-Ala-MCA substrates did not detect any activity at all, VV , IV, GA, HA weak activity was observed.

[0029] (2- 3)腎臓'泌尿器疾患患者と健常者における尿プロテアーゼ活性の比較 [0029] (2-3) Comparison of urinary protease activity in renal and urological patients and healthy subjects

(2- 3 - 1) Acety卜 Asn- Phe- MCA加水分解活性（図 2)  (2- 3-1) Acety 卜 Asn- Phe-MCA hydrolysis activity (Fig. 2)

Acety卜 Asn- Phe-MCAは、図 1で示した様に、カテブシン Bが最もよく加水分解した 基質であり、健常者尿中の活性で最も高力つた。この活性は、 IgA腎症 (Pく 0.034)と 慢性糸球体腎炎 (Pく 0.034)で有意の低下が認められた。これらの疾患では、この活 性に関わるプロテア一ゼの産生低下あるいは消費 '分解が亢進している可能性が考 えられる。  As shown in Fig. 1, Acety 卜 Asn-Phe-MCA was the substrate in which cathebsin B was most hydrolyzed and had the highest activity in the urine of healthy subjects. This activity was significantly decreased in IgA nephropathy (P 0.034) and chronic glomerulonephritis (P 0.034). In these diseases, there is a possibility that the production of protease related to this activity is decreased or the degradation of consumption is increased.

[0030] (2- 3 - 2) Acety卜 Phe- Arg- MCA加水分解活性（図 3)  [0030] (2-3-2) Acety 卜 Phe- Arg-MCA hydrolysis activity (Fig. 3)

この活性は、慢性糸球体腎炎 (Pく 0.08)以外の腎疾患である急速進行性糸球体腎 炎 (P < 0.008)、ネフローゼ症候群 (P < 0.023)、 IgA腎症 (P < 0.045)、慢性腎不全 (P < 0.005)では有意に低力つた力 癌では有意な差はみとめられな力つた。これらの疾患 では、本活性に関わるプロテア一ゼの産生低下ある 、は消費 ·分解が亢進して 、る 可能性が考えられる。  This activity is associated with rapidly progressive glomerulonephritis (P <0.008), nephrotic syndrome (P <0.023), IgA nephropathy (P <0.045), chronic kidney disease other than chronic glomerulonephritis (P 0.08). Significantly low strength in renal failure (P <0.005) Cancer showed no significant difference. In these diseases, there is a possibility that the production of protease related to this activity may be reduced or consumption / degradation may be increased.

[0031] (2- 3 - 3) AcetyKTyr-Arg-MCA加水分解活性（図 4)  [0031] (2- 3-3) AcetyKTyr-Arg-MCA hydrolysis activity (Figure 4)

急速進行性糸球体腎炎 (Pく 0.15)と慢性腎不全 (Pく 0.18)以外である、ネフローゼ 症候群 く 0.034)、 IgA腎症 (Pく 0.0011)、慢性糸球体腎炎 (Pく 0.007)、前立腺癌 (P く 0.042)、膀胱癌 (Pく 0.047)といった癌を含めたすべての疾患においてこの活性は 有意に高力つた。これらの疾患では、この活性に関わるプロテア一ゼの産生亢進ある いは消費 ·分解が遅延して ヽる可能性が考えられる。 AcetyKTyr-Arg-MCA活性は、 前述の Acety卜 Asn- Phe- MCAや Acety卜 Phe- Arg- MCAの様に健常者に比較して疾 患では低下する活性とは対照的に、疾患で増加する活性であり、し力も癌でも増加 すること力 、極めて診断的価値が高い。また、 acety卜 Phe-Arg-MCAと構造的に極 めて類似しているにもかかわらず、疾患では対照的な変動を示すこともこの活性の特 記すべき点である。  Other than rapid progressive glomerulonephritis (P + 0.15) and chronic renal failure (P + 0.18), nephrotic syndrome (0.034), IgA nephropathy (P + 0.0011), chronic glomerulonephritis (P + 0.007), prostate This activity was significantly higher in all diseases including cancer (P + 0.042) and bladder cancer (P + 0.047). In these diseases, there is a possibility that the production of protease involved in this activity may be increased or consumption / degradation may be delayed. AcetyKTyr-Arg-MCA activity increases in disease as opposed to the activity that decreases in disease compared to healthy subjects, such as Acety 卜 Asn- Phe-MCA and Acety 卜 Phe-Arg-MCA described above It is active, has the ability to increase in both power and cancer, and has a very high diagnostic value. It is also noteworthy that this activity shows contrasting variation in the disease despite its very similar structure to acety 卜 Phe-Arg-MCA.

[0032] (2- 3 -4) Acety Phe- Lys- MCA加水分解活性（図 5)  [0032] (2 3 -4) Acety Phe- Lys-MCA hydrolysis activity (Figure 5)

慢性腎不全 (Pく 0.055)以外の腎疾患である急速進行性糸球体腎炎 (Pく 0.019)、 ネフローゼ症候群 (P < 0.013)、 IgA腎症 (P < 0.044)、慢性糸球体腎炎 (P < 0.023)にお いて有意に高力つたが、癌での有意差は認められな力つた。慢性腎不全を除く多種 の腎疾患で高値を示し、癌では上昇しない活性であることから、腎疾患に特異的で かつ応用も範囲も広く、診断的有用性の高! ヽ活性として興味深!/ヽ。 Rapid progressive glomerulonephritis (P 0.019), nephrotic syndrome (P <0.013), IgA nephropathy (P <0.044), chronic glomerulonephritis (P < 0.023) Although it was significantly stronger, it was not recognized that there was a significant difference in cancer. The activity is high in various types of kidney diseases except chronic renal failure and does not increase in cancer. Therefore, it is specific for kidney disease, has a wide range of applications, and has high diagnostic utility. / ヽ.

[0033] (2- 3 - 5) Acety卜 His- Ala- MCA加水分解活性（図 6)  [0033] (2- 3-5) Acety 卜 His- Ala- MCA hydrolysis activity (Fig. 6)

慢性腎不全 (Pく 0.014)と前立腺癌 (Pく 0.022)で有意に高力つた。疾患で高値を示 す活性として興味深!/、だけでなぐ Acety卜 Phe-Lys- MCAとは対照的に腎不全のみ で上昇し、かつ前立腺癌でのみ有意に上昇することから、この活性は Acety卜 Phe-Ly s-MCA活性との組み合わせにより、さらに高い診断的有用性が期待できる。  Chronic renal failure (P 0.014) and prostate cancer (P 0.022) were significantly higher. Interesting as an activity that shows a high value in the disease! /, In contrast to Acety 卜 Phe-Lys-MCA, this activity increases only in renal failure and significantly increases only in prostate cancer. Higher diagnostic utility can be expected by combining with Acety 卜 Phe-Ly s-MCA activity.

[0034] (3)まとめ  [0034] (3) Summary

上記で使用した 5種類の基質に対するプロテアーゼ活性は患者と健常者とで比較 すると、疾患によって低下する活性と上昇する活性に分けられ、前者は Acety卜 Asn- Phe- MCAと Acety卜 Phe- Arg- MCA加水分解活性であり、後者は Acety卜 Tyr- Arg- M CA、 Acetyl-Phe-Lys-MCA,および Acety卜 His- Ala- MCA加水分解活性である。これ らの活性を図 7に示す通り、健常者活性に対して低い、差なし、高いに分け各疾患で 比較したところ、腎炎と 2つの癌を含めた 7種疾患において、お互いに異なったパター ンをとることが明らかとなった。この結果は、尿中の上記 5種類の基質に対する活性を 健常者と患者で比較することにより、これらの疾患の鑑別診断が可能となりうることを 示している。  Protease activity for the five types of substrates used above is divided into activity that decreases and increases depending on the disease when compared between patients and healthy subjects. The former is Acety 卜 Asn- Phe- MCA and Acety 卜 Phe- Arg- MCA hydrolysis activity, the latter being Acety 卜 Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, and Acety 卜 His-Ala-MCA hydrolysis activity. As shown in Fig. 7, when these activities were compared for each disease, which were low, no difference, and high, compared with those in healthy subjects, different patterns were observed in seven diseases including nephritis and two cancers. It became clear to take This result shows that differential diagnosis of these diseases may be possible by comparing the activity against the above five kinds of substrates in urine between healthy subjects and patients.

[0035] 特に、今回注目した 5つの腎疾患では、 AcetyH"Iis-Ala-MCAを除く 4種の基質に対 する活性で鑑別でき、かつ上記の 2つの癌も除外できる。また、上記 2つの癌に関して は、 Acety卜 Phe- Arg- MCAゝ Acety卜 Phe- Lys- MCAゝ Acety卜 His- Ala- MCAの 3種の 基質に対する活性によって鑑別され、かつ腎疾患も除外できる可能性が示された。 従って、これらの 5種類の基質に対する活性をレポーターとして尿中活性を測定し健 常者との比較によるパターン解析を行うことにより、無侵襲性でかつ迅速に、腎炎や 尿路系癌などの腎臓'泌尿器疾患を診断することが可能になる。  [0035] In particular, the five renal diseases of interest this time can be differentiated by activity against four substrates except AcetyH "Iis-Ala-MCA, and the above two cancers can also be excluded. Cancers were identified by their activity against three different substrates: Acety 卜 Phe- Arg- MCA ゝ Acety 卜 Phe- Lys- MCA ゝ Acety 卜 His- Ala- MCA, and the possibility of excluding kidney disease was also shown. Therefore, by measuring the activity in urine using the activity against these five kinds of substrates as a reporter and performing pattern analysis by comparison with healthy subjects, non-invasive and rapid treatment of nephritis, urinary tract cancer, etc. It becomes possible to diagnose kidney 'urological diseases.

図面の簡単な説明  Brief Description of Drawings

[0036] [図 1]図 1は、ヒトトロンビン、ヒトウ口キナーゼ及びヒトカテブシン Bの活性パターンを示 す。 [図 2]図 2は、腎臓 ·泌尿器疾患患者及び健常者の尿におけるァセチル -Asn-Phe-M CAに対するプロテアーゼ活性を示す。 [0036] FIG. 1 shows the activity patterns of human thrombin, human mouth kinase and human cathebsin B. FIG. 2 shows protease activity against acetyl-Asn-Phe-MCA in the urine of patients with renal / urological diseases and healthy subjects.

[図 3]図 3は、腎臓 '泌尿器疾患患者及び健常者の尿におけるァセチル -Phe-Arg-M CAに対するプロテアーゼ活性を示す。  [FIG. 3] FIG. 3 shows protease activity against acetyl-Phe-Arg-MCA in the urine of renal and urological disease patients and healthy subjects.

[図 4]図 4は、腎臓 ·泌尿器疾患患者及び健常者の尿におけるァセチル -Tyr-Arg-M CAに対するプロテアーゼ活性を示す。  FIG. 4 shows protease activity against acetyl-Tyr-Arg-MCA in the urine of patients with renal / urological diseases and healthy subjects.

[図 5]図 5は、腎臓 ·泌尿器疾患患者及び健常者の尿におけるァセチル -Phe-Lys-M CAに対するプロテアーゼ活性を示す。  FIG. 5 shows protease activity against acetyl-Phe-Lys-MCA in the urine of patients with renal / urological diseases and healthy subjects.

[図 6]図 6は、腎臓 '泌尿器疾患患者及び健常者の尿におけるァセチル -His-Ala-M CAに対するプロテアーゼ活性を示す。  [FIG. 6] FIG. 6 shows protease activity against acetyl-His-Ala-MCA in the urine of renal and urological disease patients and healthy subjects.

[図 7]図 7は、 5種類の基質に対するプロテアーゼ活性を腎臓 '泌尿器疾患患者と健 常者とで比較した結果を示す。  [FIG. 7] FIG. 7 shows the results of a comparison of protease activity against 5 kinds of substrates between renal and urological disease patients and normal subjects.

Claims

請求の範囲 The scope of the claims [1] 尿中のプロテアーゼ活性を複数の基質を用いて測定し、当該基質に対するプロテア ーゼ活性のパターンを分析することを含む、腎臓'泌尿器疾患の診断方法。  [1] A method for diagnosing renal urological disease, comprising measuring protease activity in urine using a plurality of substrates and analyzing a pattern of protease activity for the substrates. [2] ァセチルー X1— X2— Y (式中、 X1及び X2は、各々独立にアミノ酸残基を示し、 Yは、 X2との結合が切断されると蛍光を発する化合物になる官能基を示す)で示される基 質に対するプロテアーゼ活性を測定することにより、尿中のプロテアーゼ活性を測定 する、請求項 1に記載の方法。 [2] Acetyl X 1 — X 2 — Y (wherein X 1 and X 2 each independently represents an amino acid residue, and Y becomes a compound that emits fluorescence when the bond to X 2 is cleaved) 2. The method according to claim 1, wherein the protease activity in urine is measured by measuring the protease activity with respect to the substrate represented by (showing a functional group). [3] ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- MCA、ァ セチル - Phe- Lys- MCA、又はァセチル- His- Ala- MCA (式中、 MCAは、 4-メチルクマ リル- 7-アミド基を示す）に対するプロテアーゼ活性を測定することにより、尿中のプロ テアーゼ活性を測定する、請求項 1又は 2に記載の方法。  [3] Acetyl-Asn-Phe-MCA, Acetyl-Phe-Arg-MCA, Acetyl-Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, or Acetyl-His-Ala-MCA (where MCA is The method according to claim 1 or 2, wherein the protease activity in urine is measured by measuring the protease activity against 4-methylcoumaryl-7-amide group. [4] ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- MCA、ァ セチル - Phe- Lys- MCA、及びァセチル- His- Ala- MCA (式中、 MCAは、 4-メチルクマ リル- 7-アミド基を示す）に対する尿プロテアーゼ活性の測定において、 Asn-Pheでは 86以下、 Phe-Argでは 103以下、 Phe-Lysでは 12以上、丁 1- §では8.6以上、 His-Ala では 7.4以上の場合 (単位は cpu/mg creatinine)は異常値であると判断する、請求項 1から 3の何れかに記載の方法。 [4] Acetyl-Asn-Phe-MCA, Acetyl-Phe-Arg-MCA, Acetyl-Tyr-Arg-MCA, Acetyl-Phe-Lys-MCA, and Acetyl-His-Ala-MCA (where MCA is Urinary protease activity for Asn-Phe, 86 or less for Phe-Arg, 103 or less for Phe-Lys, 12 or more for Phe-Lys, 8.6 or more for Ding 1- § , The method according to any one of claims 1 to 3, wherein in the case of His-Ala, when it is 7.4 or more (the unit is cpu / mg creatinine), it is judged as an abnormal value. [5] 腎臓，泌尿器疾患が、急速進行性糸球体腎炎、ネフローゼ症候群、 IgA腎症、慢性 糸球体腎炎、慢性腎不全、前立腺癌、又は膀胱癌である、請求項 1から 4の何れカゝ に記載の方法。  [5] The kidney or urinary disease is rapidly progressive glomerulonephritis, nephrotic syndrome, IgA nephropathy, chronic glomerulonephritis, chronic renal failure, prostate cancer, or bladder cancer. The method described in 1. [6] ァセチル -Phe-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも低 下しており、ァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健常者の 場合よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Tyr-Arg-MCA及 びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場合と同等 であることを指標として急速進行性糸球体腎炎であると判定する（式中、 MCAは、 4- メチルクマリル- 7-アミド基を示す)、急速進行性糸球体腎炎の診断方法。  [6] Urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and urinary protease activity against acetyl-Phe-Lys-MCA is higher than in healthy subjects And the rapid progress of the urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-His-Ala-MCA as an index A method for diagnosing rapidly progressive glomerulonephritis, wherein MCA represents 4-methylcoumaryl-7-amide group. [7] ァセチル -Phe-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも低 下しており、ァセチル- Tyr-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿中の プロテアーゼ活性が健常者の場合よりも上昇しており、かつァセチル -Asn- Phe-MC A及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場合と 同等であることを指標としてネフローゼ症候群であると判定する（式中、 MCAは、 4-メ チルクマリル- 7-アミド基を示す）、ネフローゼ症候群の診断方法。 [7] Protease activity in urine against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and urine against cetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Protease activity is higher than that in healthy subjects, and the urinary protease activity for acetyl-Asn-Phe-MCA and acetyl-His-Ala-MCA is the same as that in healthy subjects. A method for diagnosing nephrotic syndrome, which is determined to be nephrotic syndrome (wherein MCA represents 4-methylcoumaryl-7-amide group). [8] ァセチル -Asn- Phe-MCA及びァセチル- Phe-Arg-MCAに対する尿中のプロテア一 ゼ活性が健常者の場合よりも低下しており、ァセチル -Tyr-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも上昇しており 、かつァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場合と 同等であることを指標として IgA腎症であると判定する（式中、 MCAは、 4-メチルクマ リル- 7-アミド基を示す)、 IgA腎症の診断方法。  [8] Urinary protease activity against acetyl-Asn-Phe-MCA and acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and acetyl-Tyr-Arg-MCA and acetyl-Phe- The urinary protease activity against Lys-MCA is higher than that in healthy subjects, and the urinary protease activity against acetyl-His-Ala-MCA is equivalent to that in healthy subjects. A method for diagnosing IgA nephropathy, wherein MCA represents 4-methylcoumaryl-7-amide group. [9] ァセチル -Asn-Phe-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも低 下しており、ァセチル- Tyr-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿中の プロテアーゼ活性が健常者の場合よりも上昇しており、かつァセチル- Phe-Arg-MCA 及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の場合と同 等であることを指標として慢性糸球体腎炎であると判定する（式中、 MCAは、 4-メチ ルクマリル- 7-アミド基を示す)、慢性糸球体腎炎の診断方法。  [9] Urinary protease activity against acetyl-Asn-Phe-MCA is lower than that in healthy subjects, and urinary protease activity against acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA Chronic thread using as an index that the urinary protease activity for acetyl-Phe-Arg-MCA and acetyl-His-Ala-MCA is the same as that for healthy subjects. A method for diagnosing chronic glomerulonephritis that is determined to be glomerulonephritis (where MCA represents 4-methylcoumaryl-7-amide group). [10] ァセチル -Phe-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも低 下しており、ァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常者の 場合よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Tyr-Arg-MCA及 びァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健常者の場合と同 等であることを指標として慢性腎不全であると判定する（式中、 MCAは、 4-メチルク マリル- 7-アミド基を示す)、慢性腎不全の診断方法。  [10] Urinary protease activity against acetyl-Phe-Arg-MCA is lower than that in healthy subjects, and urinary protease activity against acetyl-His-Ala-MCA is higher than in healthy subjects And its urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Tyr-Arg-MCA and acetyl-Phe-Lys-MCA is chronic as an index. A method for diagnosing chronic renal failure, wherein renal failure is determined (wherein MCA represents 4-methylcomaryl-7-amide group). [11] ァセチル- Tyr-Arg-MCA及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ 活性が健常者の場合よりも上昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Phe-Arg-MCA及びァセチル- Phe-Lys-MCAに対する尿中のプロテアーゼ活性が健 常者の場合と同等であることを指標として前立腺癌であると判定する (式中、 MCAは 、 4-メチルクマリル- 7-アミド基を示す)、前立腺癌の診断方法。  [11] Urinary protease activity against acetyl-Tyr-Arg-MCA and acetyl-His-Ala-MCA is higher than that in healthy subjects, and acetyl-Asn-Phe-MCA, acetyl-Phe-Arg -Prostate cancer is determined based on the fact that protease activity in urine against MCA and acetyl-Phe-Lys-MCA is equivalent to that in healthy subjects (where MCA is 4-methylcoumaryl-7- A method for diagnosing prostate cancer. [12] ァセチル -Tyr-Arg-MCAに対する尿中のプロテアーゼ活性が健常者の場合よりも上 昇しており、かつァセチル -Asn- Phe-MCA、ァセチル- Phe-Arg-MCA、ァセチル- Ph e-Lys-MCA及びァセチル- His-Ala-MCAに対する尿中のプロテアーゼ活性が健常 者の場合と同等であることを指標として膀胱癌であると判定する (式中、 MCAは、 4- メチルクマリル- 7-アミド基を示す)、膀胱癌の診断方法。 [12] Urinary protease activity against acetyl-Tyr-Arg-MCA is higher than that in healthy subjects In healthy subjects with urinary protease activity against acetyl-Asn-Phe-MCA, acetyl-Phe-Arg-MCA, acetyl-Phe-Lys-MCA and acetyl-His-Ala-MCA A method for diagnosing bladder cancer, wherein it is determined that the cancer is bladder cancer using the equivalent as an index (wherein MCA represents 4-methylcoumaryl-7-amide group). ァセチル- Asn- Phe- MCA、ァセチル- Phe- Arg- MCA、ァセチル- Tyr- Arg- MCA、ァ セチル - Phe- Lys- MCA、及びァセチル- His- Ala- MCA (式中、 MCAは、 4-メチルクマ リル- 7-アミド基を示す)を含む、腎臓'泌尿器疾患の診断キット。 Acetyl- Asn- Phe- MCA, Acetyl- Phe- Arg- MCA, Acetyl- Tyr- Arg- MCA, Acetyl- Phe- Lys- MCA, and Acetyl- His- Ala- MCA (where MCA is 4- Diagnostic kit for kidney 'urinary diseases, comprising methylcoumaryl-7-amide group).
PCT/JP2007/062982 2006-06-28 2007-06-28 Method for diagnosis of kidney/urological disease Ceased WO2008001840A1 (en)

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JPH05168497A (en) * 1991-12-25 1993-07-02 Wako Pure Chem Ind Ltd Reagent for measurement of esterase or protease
JP2004528814A (en) * 2000-10-13 2004-09-24 チルドレンズ メディカル センター コーポレーション Non-invasive enzyme screening for tissue remodeling-related conditions

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JPH09281110A (en) * 1996-04-12 1997-10-31 Nippon Kayaku Co Ltd Inspecting method for renal function

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JPS6117956A (en) * 1984-07-04 1986-01-25 Nitto Boseki Co Ltd Novel substrate for measuring urinary kallikrein
JPH05168497A (en) * 1991-12-25 1993-07-02 Wako Pure Chem Ind Ltd Reagent for measurement of esterase or protease
JP2004528814A (en) * 2000-10-13 2004-09-24 チルドレンズ メディカル センター コーポレーション Non-invasive enzyme screening for tissue remodeling-related conditions

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