JP5479480B2 - 長期透析を予測するための尿中バイオマーカー - Google Patents
長期透析を予測するための尿中バイオマーカー Download PDFInfo
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- JP5479480B2 JP5479480B2 JP2011529017A JP2011529017A JP5479480B2 JP 5479480 B2 JP5479480 B2 JP 5479480B2 JP 2011529017 A JP2011529017 A JP 2011529017A JP 2011529017 A JP2011529017 A JP 2011529017A JP 5479480 B2 JP5479480 B2 JP 5479480B2
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Description
本明細書中で用いる場合、「バイオマーカー」との用語は、慢性腎疾患の進行性発症に関連するいずれかの生物学的化合物を指す。例えば、バイオマーカーは、ヒアルロン酸、またはその代謝産物もしくは誘導体のいずれかを含む。
腎臓は、脇腹(脊柱のどちらかの側の上腹部の背面)に位置する。腎臓は、腹部の深部にあり、脊髄、下部胸郭および背部の強い筋肉により保護されている。この配置が、腎臓を多くの外力から保護する。腎臓が十分に守られている理由の1つは、腎臓が非常に血管に富んだ臓器であり、つまり、多量の血液供給を受けているからである。もし傷害されれば、深刻な出血が起こり得る。
急性(突発性)腎不全は、電解質を失うことなく、老廃物を除去し、尿を濃縮する腎臓の能力が突然失われることである。腎臓ダメージには多くの考えられる原因がある。そのようなものとしては、限定するものではないが、血流の低下(外傷、手術、重篤な疾病、敗血性ショック、大出血、熱傷、または脱水による極端な低血圧に伴って生じる)、急性尿細管壊死(ATN)、急性腎盂腎炎または敗血症などの腎臓を直接的に傷害する感染、尿路閉塞(閉塞性尿路疾患)、間質性腎炎または急性腎炎症候群などの自己免疫性腎疾患、腎臓の細血管内での凝固を引き起こす障害、特発性血栓性血小板減少性紫斑病(ITTP)、輸血反応、悪性高血圧、強皮症、溶血性尿毒症症候群、出血性胎盤早期剥離または前置胎盤などの出産の障害が挙げられる。
急性腎不全とは異なり、慢性腎不全はゆっくりと悪化する。最も多くは、腎機能を徐々に失わせるいずれかの疾患の結果として生じる。それは、軽度の機能不全から重篤な腎不全まで及び得る。疾患が終末期腎疾患(ESRD)をもたらし得る。
透析(すなわち、例えば、腎代替療法)は、腎臓がそれを行うことができない場合に、血液から毒性物質(不純物または老廃物)を除去する方法であり、数種類の異なる方法を用いて行うことができる。例えば、腹膜透析では、腹部の中の腹膜を用いることにより、老廃物をろ過することができる。腹部は、毒物の除去を助ける特別な溶液で満たされる。その溶液は一定時間腹部内に留まり、その後、排出される。この形態の透析は家庭で行うことができるが、毎日行わなければならない。あるいは、血液透析は、体外の特別なフィルターを通して血液を循環させることにより行うことができる。血液は、毒物の除去を助ける溶液と共に、フィルターを通って流れる。
一実施形態では、本発明は、急性腎傷害(AKI)後の腎機能の回復を予測するための、尿中ヒアルロン酸(HA)バイオマーカーを意図する。一実施形態では、このバイオマーカーの特定が、治療強度を合わせるための患者の層化をもたらし、それにより不要な長期の合併症を防ぐ。
ヒアルロン酸(当技術分野では、ヒアルロン酸塩およびヒアルロナンとしても知られ、HAと略す)は、N-アセチル-D-グルコサミンおよびD-グルクロン酸の交互する単位を有する、直鎖非分岐多糖鎖を含むグリコサミノグリカンである(Laurent TC, Fraser RE. Hyaluronan. FASEB J 1992;6:2397-2404; and Delpech B, Girard N, Bertrand P. Hyaluronan: fundamental principles and applications in cancer. J Intern Med 1997;242: 41-48)。HAは、細菌および動物の両方をはじめとする種々のタイプの生物学的材料中に遍在する。ヒトでは、HAは、臍帯、目の硝子体液、軟骨および滑液中に高濃度で見出される。少量のHAが、CSF、リンパ、血液、血清および尿中に存在する。HAレベルは、関節リウマチ、肝硬変、およびウィルムス腫瘍などの疾患と関連している。HAは、非特異的な腫瘍一般に関連しているが、その用途は従来、特定の臨床上の腫瘍の発見、治療および管理に適用されてこなかった。HAは、癌をはじめとするいくつかの病理生理学的状態で働いていることが知られている。
尿中ヒアルロン酸は、初めに、アッセイされるまで-20℃で保存された排泄された尿(中間尿)検体を採取することによって測定することができる。HAアッセイは、ビオチン化プロテオグリカンG1ドメイン(HA結合性)領域を用いるヒアルロナンに対するELISAプレートアッセイに基づくことができる(Fosang et al. Matrix, 10: 306-131 (1990))。一実施形態では、ヒト臍帯HA(25μg/ml)でコーティングした96ウェルマイクロタイタープレートを用いることによりアッセイを改変することができ、このプレートを、リン酸緩衝生理食塩水(PBS)+0.05%Tween 20(PBS+Tween)中の尿検体の段階希釈物およびビオチン化ウシ鼻軟骨HA結合性タンパク質(1μg/ml)と共にインキュベートする。室温で16時間のインキュベーションに続いて、ウェルをPBS+Tweenで洗浄した。これらのウェルに結合したHA結合性タンパク質を、アビジン・ビオチン検出系およびABTS(2,2’アジノ-ビス(3-エチル-ベンズチアゾリン-6-スルホン酸)基質(Vector Laboratories, Burlingame, Calif.)を用いて定量した。標準グラフは、吸光度(405nm)をヒト臍帯HA濃度(ng/ml)に対してプロットすることにより作成することができる。このグラフを用いて、尿検体の各希釈中のHA濃度を算出することができる。数回のそのような測定から、各サンプル中の平均HA濃度を決定し、続いて、尿サンプル中のクレアチニン濃度(mg/ml)に対して標準化した。
(a)固体支持体(好ましくは、マイクロタイターウェル)をHAでコーティングするステップ;
(b)HA結合性タンパク質(HABP)が固体支持体上にコーティングされたHAおよびサンプル中のHA(もし存在すれば)に結合することができる条件下で、腎傷害および/または腎疾患を有することが疑われる者から採取した生物学的流体(尿サンプルなど)の存在下で、HABPを、コーティングした固体支持体と接触させ、インキュベートするステップ;
(c)固体支持体上にコーティングされたHAに結合しているHABPの量を測定し、それによりサンプル中に存在するHAの量を測定するステップ
を含む方法を用いて予測することができる。
正常被験体および患者由来の尿検体は、10倍に濃縮して、PBSに対して十分に透析することができる。透析検体のそれぞれの約2ml(約20mgのタンパク質)を、PBSで平衡化したSepharose 6 CL-Bカラム(1.5×120cm)(Pharmacia, Piscataway, N.J.)にアプライした。カラムにPBSを7ml/hrで流し、3.5ml画分を回収した。画分は、HAについて、上述のようなELISA様アッセイによりアッセイした。標準的な球状のタンパク質マーカーと、HAおよびHA断片などの直鎖多糖とは異なる形状を有するので、カラムを、ヒト臍帯静脈HA(Mr約2×106D)ならびにHA断片であるF1、F2およびF3で較正した。
ヒアルロニダーゼ(HAアーゼ)は、HA中のN-アセチルグルコサミン結合を加水分解することによりHAを分解するエンドグリコシダーゼ酵素である。ヒアルロニダーゼによるHAの制限分解は、特定の長さ(約3〜25二糖単位)のHA断片の生成をもたらし、この断片は血管形成性である(West et al., Angiogenesis induced by degradation products of hyaluronic acid. Science, 228: 1324-1326, 1985)。脊椎動物では、ヒアルロニダーゼは、中性pHで活性なもの(至適pH 5.0)と、酸性pHで活性なもの(pH3.5〜4.0)の2つのクラスに分類することができる(Roden et al., Enzymatic pathways of hyaluronan catabolism. In: The Biology of hyaluronan, (J. Whelan, ed.), pp. 60-86, New York, Wiley Chichister (Ciba Foundation Symposium 143), 1989; West et al., ibid.; Gold, Purification and properties of hyaluronidase from human liver. Biochem. J., 205: 69-74, 1982; Fraser and Laurent, Turnover and metabolism of Hyaluronan. in: Biology of Hyaluronan, (J. Whelan, ed.), pp. 41-59, New York, Wiley Chichister (Ciba Foundation Symposium 143), 1989; Zhu et al., Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum heme-binding protein. J. Biol. Chem., 269: 32092-32097, 1994; Lin et al., A hyaluronidase a
ctivity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus layer surrounding the egg. J. Cell Biol., 125: 1157-1163, 1995)。例えば、精巣ヒアルロニダーゼは中性タイプであり、肝臓ヒアルロニダーゼは酸性の至適pHを有する。HAとヒアルロニダーゼの両者の協調的作用が、胎児発達、血管形成、血管リモデリング、免疫監視および腫瘍進行の間に重要な役割を果たしていることが知られている(McCormick and Zetter, Adhesive interactions in angiogenesis and metastasis. Pharmacol. Ther., 53: 239- 260, 1992; Hobarth et al., Topical chemo-prophylaxis of superficial bladder cancer by mitomycin C and adjuvant hyaluronidase, Eur. Urol., 21: 206-210, 1992; Knudson et al., The role and regulation of tumor-associated hyaluronan. In: The Biology of Hyaluronan (J. Whelan, ed.) pp. 150-169, New York, Wiley, Chichester (Ciba Foundation Symposium 143), 1989; Lin et al., Urinary hyaluronic acid is a Wilms' tumor marker. J. Ped. Surg., 30: 304-308, 1995; Stern et al., Hyaluronidase levels in urine from Wilms' tumor patients. J. Natl. Canc. Inst., 83: 1569-1574, 1991)。
一実施形態では、本発明は、長期透析を予測するための診断用キットを意図する。一実施形態では、キットは、HAおよび/またはHAアーゼ、HABPおよびマーカーまたはマーカーと結合したHABP、ならびに生物学的サンプル(すなわち、例えば、尿サンプル)中のHAおよび/またはHAアーゼの存在の検出における使用に好適な付属の試薬を含む。本発明によって意図される診断用キットの例は、慣用のディップスティックテストデバイスである。
ヒト尿サンプル中の標準化ヒアルロン酸
ヒアルロン酸を、上述のようにヒト尿中で測定した。2週間(すなわち、14日間;D1〜D14)尿中のHAを採取および分析することにより、経時変化を作成した。提示したデータは、腎傷害に罹患してから28日後に回復した患者と回復しなかった患者を示す(すなわち、例えば、R28=回復患者;およびNR28=非回復患者)。14日間の採取期間の間で、第1日(D1)、第7日(D7)および第14日(D14)にサンプルを分析した。図1を参照されたい。
ヒト尿サンプル中の標準化ヒアルロン酸レベルの絶対値
本実施例のデータは、腎傷害28日後(R28)と60日後(R60)の両方で回復を示した患者、および非回復患者(NR28およびNR60)について、実施例1に従い採取した腎傷害後のD1、D7および/またはD14で取得したサンプルにおける、標準化されたヒアルロン酸レベルの絶対値の間の差異を調べる。
ヒト尿サンプル中の標準化ヒアルロン酸レベルの相対値
本実施例では、回復患者と非回復患者との間の差異の大きさをさらに説明するために、実施例2に従いデータを再プロットする。特に、データをパーセンテージで表す(すなわち、D7÷D1、D14÷D1、D7÷D14、またはD14÷D7)。
腎傷害後D14での長期透析の予測
真陽性と偽陽性との間の関係を評価するために、実施例2に従い提示されたデータを解析し、再プロットした。特に、尿中HA/クレアチニンの持続的な上昇(すなわち、D1とD14の測定値の間の差異)があった条件下では、患者が腎傷害後D60に長期透析に付されている可能性を予測することに対して感度が高かった。図6を参照されたい。結論として、データは、腎傷害後D1〜D14で持続的に上昇した尿中HAを示す患者は、D60で(かつもっとも可能性があるのはその後に)透析に付されるであろうことを示唆している。
Claims (17)
- (a)(i)急性腎傷害を示す患者;
(ii)該患者由来の少なくとも2種類の尿サンプル
を準備するステップ;
(b)該尿サンプル中のヒアルロン酸の、該急性腎傷害後の第1日と第14日の間の持続的な上昇を検出するステップ;および
(c)該患者が長期透析を必要とすることを予測するステップ
を含む方法。 - 前記サンプルを、重症急性腎傷害に対する代替療法の開始後の第1日および第14日に採取する、請求項1に記載の方法。
- 慢性腎疾患発症を予測するために前記ヒアルロン酸をモニタリングするステップをさらに含む、請求項1に記載の方法。
- 前記予測を、腎傷害の少なくとも60日後に行う、請求項1に記載の方法。
- (a)(i)急性腎傷害を示し、慢性腎疾患発症のリスクがある患者;
(ii)該患者由来の少なくとも2種類の尿サンプル
を準備するステップ;
(b)該尿サンプル中のヒアルロン酸の、該急性腎傷害後の第1日と第14日の間の持続的な上昇を検出するステップ;および
(c)該患者が慢性腎疾患を予防するための治療を必要とすることを予測するステップ
を含む方法。 - 前記予測を腎疾患後の第14日に開始する、請求項5に記載の方法。
- (a)(i)急性腎傷害に罹患した患者;
(ii)該患者由来の複数の尿中ヒアルロン酸レベルおよび尿中クレアチニンレベルを、該急性腎傷害後の第1日と第14日の間に経時的に取得するステップ;
(b)尿中クレアチニンレベルに対して標準化した、尿中ヒアルロン酸レベルの経時変化を作成するステップ;および
(c)慢性腎疾患発症を予測するステップ
を含む方法。 - 前記患者が長期腎代替療法を必要とすることを予測するステップをさらに含む、請求項7に記載の方法。
- (a)急性腎傷害を示す患者由来の少なくとも2種類の尿サンプルを取得するステップ;
(b)該尿サンプル中のヒアルロン酸の、該急性腎傷害後の第1日と第14日の間の持続的な上昇を検出するステップ;および
(c)該患者が長期透析を必要とすることを予測するステップ
を含むインビトロの方法。 - 前記サンプルを、重症急性腎傷害に対する代替療法の開始後の第1日および第14日に採取する、請求項9に記載の方法。
- 慢性腎疾患発症を予測するために前記ヒアルロン酸をモニタリングするステップをさらに含む、請求項9に記載の方法。
- 前記予測を、腎傷害の少なくとも60日後に行う、請求項9に記載の方法。
- (a)(i)急性腎傷害を示し、慢性腎疾患発症のリスクがある患者由来の少なくとも2種類の尿サンプルを取得するステップ;
(b)該尿サンプル中のヒアルロン酸の、該急性腎傷害後の第1日と第14日の間の持続的な上昇を検出するステップ;および
(c)該患者が慢性腎疾患を予防するための治療を必要とすることを予測するステップ
を含む、インビトロの方法。 - 前記予測を腎疾患後の第14日に開始する、請求項13に記載の方法。
- (a)(i)急性腎傷害に罹患した患者由来の複数の尿中ヒアルロン酸レベルおよび尿中クレアチニンレベルを、該急性腎傷害後の第1日と第14日の間に経時的に取得するステップ;
(b)尿中クレアチニンレベルに対して標準化した、尿中ヒアルロン酸レベルの経時変化を作成するステップ;および
(c)慢性腎疾患発症を予測するステップ
を含む、インビトロの方法。 - 前記患者が長期腎代替療法を必要とすることを予測するステップをさらに含む、請求項15に記載の方法。
- 患者に対する長期透析の必要性の予測のための、該患者から取得される尿サンプルにおいてインビトロで測定されるヒアルロン酸濃度の、該患者が急性腎傷害に罹患した後の第1日と第14日の間の持続的な上昇の使用。
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BRPI0919296A2 (pt) | 2015-12-15 |
US9057722B2 (en) | 2015-06-16 |
CA2737779C (en) | 2018-05-22 |
EP2344882B1 (en) | 2017-05-10 |
CN102216776B (zh) | 2015-01-28 |
WO2010036342A2 (en) | 2010-04-01 |
US9389222B2 (en) | 2016-07-12 |
US8501489B2 (en) | 2013-08-06 |
US20140329256A1 (en) | 2014-11-06 |
US20150330975A1 (en) | 2015-11-19 |
KR20110097760A (ko) | 2011-08-31 |
AU2009297098B2 (en) | 2015-01-22 |
JP2012503772A (ja) | 2012-02-09 |
HK1160927A1 (en) | 2012-08-17 |
KR101680392B1 (ko) | 2016-11-28 |
EP2344882A4 (en) | 2013-01-23 |
MX2011003174A (es) | 2011-09-15 |
AU2009297098A1 (en) | 2010-04-01 |
AU2009297098C1 (en) | 2015-07-30 |
US20130299427A1 (en) | 2013-11-14 |
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