JPWO2003057874A1 - Disease marker for kidney disease and use thereof - Google Patents

Abstract

The present invention provides a disease marker reflecting a renal disease associated with glomerular fibrosis, a method for detecting a renal disease associated with glomerular fibrosis using the disease marker, and a screening method for a drug useful for improving the disease. . The present invention uses a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of the Ceramide Glucosyltransferase (CGT) gene and / or a polynucleotide complementary thereto as a disease marker for renal disease associated with glomerular fibrosis, It can also be carried out by contacting a test substance with CGT gene-expressing cells and screening for an agent for improving renal disease associated with glomerular fibrosis by examining the presence or absence or increase / suppression of CGT gene expression.

Description

（式中、Ｒ１は炭素数５〜１５のアルキル基またはベンジルオキシ基、Ｒ２は―（ＣＨ_２）_４―基または―（ＣＨ_２）_２−Ｏ−（ＣＨ_２）_２―基を意味する。）
で示される化合物またはその薬学上許容される塩である、項２３に記載の糸球体線維化を伴う腎疾患の改善または治療剤。
項２６． Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの活性を抑制する物質が、Ｄ−スレオ−１−フェニル−２−デカノイルアミノ−３−モルフォリノ−１−プロパノール（Ｄ−ＰＤＭＰ）、（１Ｒ，２Ｒ）−１−フェニル−２−ベンジルオキシカルボニルアミノ−３−ピロリジノ−１−プロパノール（ＰＢＰＰ）、（１Ｒ，２Ｒ）−２−ヘキサノイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＨＰＰ）、（１Ｒ，２Ｒ）−２−オクタノイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＯＰＰ）、（１Ｒ，２Ｒ）−２−デカノイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＤＰＰ）、（１Ｒ，２Ｒ）−２−パルミトイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＰＰＰ）、及びこれらの薬学上許容される塩よりなる群から選択される少なくとも１種である、項２３に記載の糸球体線維化を伴う腎疾患の改善または治療剤。
項２７． Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質が、一般式：

（式中、Ｒ３は任意の官能基、またＲ４は環中の窒素原子とともに複素環を形成する任意の官能基を意味する。）
で示される化合物またはその薬学上許容される塩を被験物質として、項１８または１９のいずれかに記載のスクリーニング方法を行うことによって得られるものである、項２３に記載の糸球体線維化を伴う腎疾患の改善または治療剤。
項２８． Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質が、一般式：

（式中、Ｒ５は炭素数１〜２０の直鎖状または分岐状のアルキル基を意味する。）
で示される化合物、一般式：

（式中、Ｒ６は炭素数１〜２０の直鎖状または分岐状のアルキル基を意味する。）
で示される化合物、またはこれらの薬学上許容される塩である、項２３に記載の糸球体線維化を伴う腎疾患の改善または治療剤。
項２９． Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの活性を抑制する物質が、Ｎ−ブチル−デオキシノジリマイシン、Ｎ−ノニル−デオキシノジリマイシン、Ｎ−デシル−デオキシノジリマイシン、Ｎ−ブチル−デオキシガラクトノジリマイシン、およびこれらの薬学上許容される塩よりなる群から選択される少なくとも１種である、項２３に記載の糸球体線維化を伴う腎疾患の改善または治療剤。
項３０． Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質が、一般式：

（式中、Ｒ７は任意の官能基を意味する。）
で示される化合物またはその薬学上許容される塩を被験物質として、項１８または１９のいずれかに記載のスクリーニング方法を行うことによって得られるものである、項２３に記載の糸球体線維化を伴う腎疾患の改善または治療剤。
項３１． 配列番号１若しくは２に記載のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ遺伝子の塩基配列中の連続する少なくとも１５塩基を有するポリヌクレオチドおよび／またはそれに相補的なポリヌクレオチドの、糸球体線維化を伴う腎疾患の疾患マーカーとしての使用。
項３２． 配列番号３に記載のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅのアミノ酸配列からなるタンパク質を認識する抗体の、糸球体線維化を伴う腎疾患の疾患マーカーとしての使用。
項３３． 疾患マーカーが糸球体線維化を伴う腎疾患の疾患の検出においてプローブまたはプライマーとして用いられるものである、項３１または３２記載の使用。
項３４． 糸球体線維化を伴う腎疾患の検出方法における下記の工程（ａ）及び（ｂ）の使用：
（ａ）被験者の生体試料から調製されたＲＮＡまたは該ＲＮＡから転写された相補的ポリヌクレオチドと請求項１または２に記載の疾患マーカーとを結合させる工程、
（ｂ）該疾患マーカーに結合した生体試料由来のＲＮＡまたは該ＲＮＡから転写された相補的ポリヌクレオチドを、上記疾患マーカーを指標として測定する工程。
項３５． 糸球体線維化を伴う腎疾患の検出方法における下記の工程（ａ）及び（ｂ）の使用：
（ａ）被験者の腎組織抽出物と項３または４に記載の疾患マーカーとを接触させる工程、
（ｂ）該疾患マーカーに結合した腎組織抽出物中のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅまたはその部分ペプチドを、上記疾患マーカーを指標として測定する工程。
項３６． 糸球体線維化を伴う腎疾患の検出方法における下記の工程（ｉ）または工程（ｉｉ−ａ）および（ｉｉ−ｂ）の使用：
（ｉ）被験者の腎組織中に内在するグルコシルセラミドの量を測定する工程、または
（ｉｉ−ａ）被験者の腎組織抽出物とＵＤＰ−グルコース及びセラミドとを接触させる工程、および
（ｉｉ−ｂ）上記（ｉｉ−ａ）の工程に基づいて生じるグルコシルセラミドの生成量を測定する工程。
項３７．標識化されていてもよいＵＤＰ−グルコースまたはセラミドの、糸球体線維化を伴う腎疾患の検査試薬としての使用。
項３８．糸球体線維化を伴う腎疾患の改善または治療剤の有効成分を取得するための、項１６乃至１９のいずれかに記載するスクリーニング方法の使用。
項３９．糸球体線維化を伴う腎疾患の改善または治療剤を製造するための、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ遺伝子の発現を抑制する物質の使用。
項４０．糸球体線維化を伴う腎疾患の改善または治療剤を製造するための、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの生成、機能または活性を抑制する物質の使用。
項４１．Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質が、一般式：

（式中、Ｒ１は炭素数５〜１５のアルキル基またはベンジルオキシ基、Ｒ２は―（ＣＨ_２）_４―基または―（ＣＨ_２）_２−Ｏ−（ＣＨ_２）_２―基を意味する。）
で示される化合物またはその薬学上許容される塩である項４０記載の使用。
項４２．Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質が、一般式：

（式中、Ｒ５は炭素数１〜２０の直鎖状または分岐状のアルキル基を意味する。）
で示される化合物、一般式：

（式中、Ｒ６は炭素数１〜２０の直鎖状または分岐状のアルキル基を意味する。）
で示される化合物、またはこれらの薬学上許容される塩である項４０記載の使用。
発明を実施するための最良の形態
以下、本明細書において、アミノ酸、（ポリ）ペプチド、（ポリ）ヌクレオチドなどの略号による表示は、ＩＵＰＡＣ−ＩＵＢの規定〔ＩＵＰＡＣ−ＩＵＢ Ｃｏｍｍｕｎｉｃａｔｉｏｎ ｏｎ Ｂｉｏｌｏｇｉｃａｌ Ｎｏｍｅｎｃｌａｔｕｒｅ，Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．，１３８：９（１９８４）〕、「塩基配列又はアミノ酸配列を含む明細書等の作成のためのガイドライン」（日本国特許庁編）、および当該分野における慣用記号に従う。
本明細書において「遺伝子」または「ＤＮＡ」とは、２本鎖ＤＮＡのみならず、それを構成するセンス鎖およびアンチセンス鎖といった各１本鎖ＤＮＡを包含する趣旨で用いられる。またその長さによって特に制限されるものではない。従って、本明細書において遺伝子（ＤＮＡ）とは、特に言及しない限り、ヒトゲノムＤＮＡを含む２本鎖ＤＮＡおよびｃＤＮＡを含む１本鎖ＤＮＡ（正鎖）並びに該正鎖と相補的な配列を有する１本鎖ＤＮＡ（相補鎖）、およびこれらの断片のいずれもが含まれる。なお、遺伝子またはＤＮＡは、機能領域の別を問うものではなく、例えば発現制御領域、コード領域、エキソン、またはイントロンを含むことができる。
本明細書において「ポリヌクレオチド」とは、ＲＮＡおよびＤＮＡのいずれをも包含する趣旨で用いられる。なお、上記ＤＮＡには、ｃＤＮＡ、ゲノムＤＮＡ、及び合成ＤＮＡのいずれもが含まれる。また上記ＲＮＡには、ｔｏｔａｌ ＲＮＡ、ｍＲＮＡ、ｒＲＮＡ、及び合成のＲＮＡのいずれもが含まれる。
本明細書において「タンパク質」または「（ポリ）ペプチド」には、特定の塩基配列で示される「タンパク質」または「（ポリ）ペプチド」だけでなく、これらと生物学的機能が同等であることを限度として、その同族体（ホモログ）、誘導体、および変異体が包含される。なお、上記変異体には、天然に存在するアレル変異体、天然に存在しない変異体、及び人為的に欠失、置換、付加および挿入されることによって改変されたアミノ酸配列を有する変異体が包含される。なお、上記変異体としては、変異のないタンパク質または（ポリ）ペプチドと、少なくとも７０％、好ましくは８０％、より好ましくは９５％、さらにより好ましくは９７％相同なものを挙げることができる。
本明細書でいう「抗体」には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、またはＦａｂフラグメントやＦａｂ発現ライブラリーによって生成されるフラグメントなどのように抗原結合性を有する上記抗体の一部が包含される。
さらに本明細書において「疾患マーカー」とは、腎疾患、特に糸球体線維化を伴う腎疾患の罹患の有無若しくは罹患の程度を診断するために、直接または間接的に利用されるものである。具体的には、糸球体線維化を伴う腎疾患患者の腎組織において特異的に発現上昇しているＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ（ＣＧＴ）遺伝子またはその発現産物（タンパク質、ＣＧＴ）を特異的に認識し、また結合することのできる（ポリ）（オリゴ）ヌクレオチドまたは抗体が包含される。これらの（ポリ）（オリゴ）ヌクレオチドおよび抗体は、上記性質に基づいて、生体内で発現した上記遺伝子（ＣＧＴ遺伝子）及びタンパク質（ＣＧＴ）を検出し、定量するためのプローブとして、また（オリゴ）ヌクレオチドは生体内で発現した上記遺伝子（ＣＧＴ遺伝子）を増幅するためのプライマーとして有効に利用することができる。
また本明細書において、「正常者」とは、腎疾患、具体的には糸球体の線維化を伴う腎疾患に罹患していない者を意味する。より具体的には、血清クレアチニン値検査、または尿中タンパク質や糸球体濾過率等の測定の結果から、腎疾患に罹患していないと診断された者を意味する。また本明細書において、「正常な腎組織」とは、当該正常者の腎組織を意味するものである。
本発明は、前述するように、ＣＧＴ遺伝子の発現が糸球体線維化を伴う腎疾患患者の腎組織で特異的に増大しており、且つｉｎ ｖｉｔｒｏ及びｉｎ ｖｉｖｏにおいて、ＣＧＴの阻害剤の投与によって糸球体硬化をもたらす糸球体の線維化が抑制もしくは改善されるという知見に基づくものである。従って、当該ＣＧＴ遺伝子及びその発現産物〔タンパク質、（ポリ）（オリゴ）ペプチド〕は、腎疾患、特に糸球体線維化を伴う腎疾患の解明、並びに診断に有効に利用することができる。そして、かかる利用によって医学並びに臨床学上、有用な情報や手段を得ることができる。また、これらＣＧＴ遺伝子及びその発現産物（ＣＧＴ）並びにそれらからの派生物（例えば、ＣＧＴに対する抗体など）は、上記腎疾患の治療、並びに該治療に有効に用いられる薬剤の開発に好適に利用することができる。さらに、個体または腎組織における、上記ＣＧＴ遺伝子の発現またはその発現産物（ＣＧＴ）の有無またはその程度の測定（定性、定量）、または該遺伝子の変異またはその発現不全の検出は、糸球体線維化を伴う腎疾患の解明や診断に有効に利用することができる。
本発明でいう腎疾患には、具体的には糸球体線維化を伴う慢性腎炎、糸球体線維化を伴う糖尿病性腎症、糸球体線維化を伴うＩｇＡ腎症、糸球体線維化を伴う遺伝性腎疾患、糸球体線維化を伴う腎不全、及び糸球体線維化を伴う糸球体硬化症などが包含される。なお、本明細書において「糸球体線維化を伴う腎疾患」は、単に「糸球体の線維化」と言い換えることができる。
以下、ＣＧＴ遺伝子、並びに当該遺伝子の発現産物（ＣＧＴ）やそれらの派生物について、具体的な用途を説明する。
（１）糸球体線維化を伴う腎疾患の疾患マーカー及びその応用
（１−１）ポリヌクレオチド
本発明の配列番号１および２の塩基配列で示すポリヌクレオチドは、それぞれヒト由来のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ（ＣＧＴ）の遺伝子としていずれも公知の遺伝子である（ＧｅｎＢａｎｋ Ａｃｃ．Ｎｏ．ＸＭ＿００５５５５とＤ５０８４０）。なお、これらの遺伝子は２１位と６３８位の２カ所の塩基配列を異にするものの、ともに同一のアミノ酸配列を有するＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ（ＣＧＴ）をコードする遺伝子である。
本発明は、前述するように、ＣＧＴ遺伝子の発現上昇の有無やその程度を検出（定性・定量的測定）することによって糸球体線維化を伴う腎疾患の罹患の有無や罹患の程度（進行度・進展度）を特異的に検出することができ、該疾患の診断を正確に行うことができるという発想に基づくものである。すなわち、本発明は、被験者における上記遺伝子の発現上昇の有無またはその程度を測定することによって、被験者について糸球体線維化を伴う腎疾患の罹患の有無またはその程度を診断することのできるツールとして有用な疾患マーカーを提供するものである。
かかる本発明の疾患マーカーは、配列番号１または２に記載のＣＧＴ遺伝子の塩基配列中の連続する少なくとも１５塩基を有するポリヌクレオチド及び／またはそれに相補的なポリヌクレオチドからなることを特徴とする。
ここで相補的なポリヌクレオチド（相補鎖、逆鎖）とは、配列番号１または２に示される塩基配列（ＣＧＴ遺伝子の全長配列）、または該塩基配列において連続した少なくとも１５塩基長の塩基配列を有する配列（部分配列）（ここでは便宜上、これら全長配列及び部分配列を「正鎖」ともいう）に対して、Ａ：ＴおよびＧ：Ｃといった塩基対関係に基づいて、塩基的に相補的な関係にあるポリヌクレオチドを意味するものである。ただし、かかる相補鎖は、対象とする正鎖の塩基配列に対して完全な相補配列である場合に限らず、対象とする正鎖とストリンジェントな条件でハイブリダイズすることができる程度の相補関係を有するものであってもよい。なお、ここでストリンジェントな条件は、Ｂｅｒｇｅｒ ａｎｄ Ｋｉｍｍｅｌ（１９８７，Ｇｕｉｄｅ ｔｏ Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ Ｔｅｃｈｎｉｑｕｅｓ Ｍｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ，Ｖｏｌ．１５２，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｓａｎ Ｄｉｅｇｏ ＣＡ）に教示されるように、複合体或いはプローブを結合する核酸の融解温度（Ｔｍ）に基づいて決定することができる。例えばハイブリダイズ後の洗浄条件として、通常「１×ＳＳＣ、０．１％ＳＤＳ、３７℃」程度の条件を挙げることができる。相補鎖はかかる条件で洗浄しても対象とする正鎖とハイブリダイズ状態を維持するものであることが好ましい。特に制限されないが、より厳しいハイブリダイズ条件として「０．５×ＳＳＣ、０．１％ＳＤＳ、４２℃」程度の洗浄条件、さらに厳しくは「０．１×ＳＳＣ、０．１％ＳＤＳ、６５℃」程度の洗浄条件で洗浄しても正鎖と相補鎖とがハイブリダイス状態を維持する条件を挙げることができる。具体的には、このような相補鎖として、対象の正鎖の塩基配列と完全に相補的な関係にある塩基配列からなる鎖、並びに該鎖と少なくとも９０％、好ましくは９５％の相同性を有する塩基配列からなる鎖を例示することができる。
また、正鎖側のポリヌクレオチドには、配列番号１または２に記載の塩基配列（全長配列）またはその部分配列を有するものだけでなく、上記相補鎖の塩基配列に対してさらに相補的な関係にある塩基配列からなる鎖を含めることができる。
さらに上記正鎖のポリヌクレオチド及び相補鎖（逆鎖）のポリヌクレオチドは、各々一本鎖の形態で疾患マーカーとして使用されても、また二本鎖の形態で疾患マーカーとして使用されてもよい。
本発明の糸球体線維化を伴う腎疾患の疾患マーカーは、具体的にはＣＧＴ遺伝子の塩基配列に関する配列番号１または２に記載される塩基配列（全長配列）からなるポリヌクレオチドであってもよいし、その相補配列からなるポリヌクレオチドであってもよい。また、配列番号１または２で示されるＣＧＴ遺伝子もしくは該遺伝子に由来するポリヌクレオチドを選択的に（特異的に）認識するものであれば、上記全長配列またはその相補配列の各々部分配列からなるポリヌクレオチドであってもよい。この場合、上記全長配列またはその相補配列の塩基配列から任意に選択される少なくとも１５個の連続した塩基長を有するポリヌクレオチドを挙げることができる。
なお、ここで「選択的に（特異的に）認識する」とは、例えばノーザンブロット法を用いる場合には、ＣＧＴ遺伝子またはこれらに由来するポリヌクレオチドが特異的に検出できること、またＲＴ−ＰＣＲ法を用いる場合にはＣＧＴ遺伝子またはこれらに由来するポリヌクレオチドが特異的に生成されることを意味するが、これらに限定されることなく、当業者が上記ノーザンブロット法における検出物または上記ＲＴ−ＰＣＲ法における生成物が、ＣＧＴ遺伝子またはこれに由来するポリヌクレオチドに由来すると判断できるものであればよい。
そのような本発明の疾患マーカーは、配列番号１または２で示されるＣＧＴ遺伝子の塩基配列をもとに、例えばｐｒｉｍｅｒ ３（ｈｔｔｐ：／／ｗｗｗ．ｇｅｎｏｍｅ．ｗｉ．ｍｉｔ．ｅｄｕ／ｃｇｉ−ｂｉｎ／ｐｒｉｍｅｒ／ｐｒｉｍｅｒ３．ｃｇｉ）あるいはベクターＮＴＩ（Ｉｎｆｏｍａｘ社製）を利用して設計することができる。
具体的にはＣＧＴ遺伝子の塩基配列をｐｒｉｍｅｒ３またはベクターＮＴＩのソフトウエアにかけて得られる、プライマーまたはプローブの候補配列、若しくは少なくとも該配列を一部に含む配列をプライマーまたはプローブとして使用することができる。
本発明で用いる疾患マーカーは、上述するように連続する少なくとも１５塩基の長さを有するものであればよいが、具体的にはマーカーの用途に応じて、長さを適宜選択し設定することができる。
本発明において糸球体線維化を伴う腎疾患の検出（診断）は、被験者の生体組織、特に腎組織におけるＣＧＴ遺伝子の発現上昇の有無またはそのレベル（発現上昇程度）を評価することによって行われる。この場合、上記本発明の疾患マーカーは、上記遺伝子の発現によって生じたＲＮＡまたはそれに由来するポリヌクレオチドを特異的に認識し増幅するためのプライマーとして、または該ＲＮＡまたはそれに由来するポリヌクレオチドを特異的に検出するためのプローブとして利用することができる。
上記疾患マーカーを、糸球体線維化を伴う腎疾患の検出においてプライマーとして用いる場合には、通常１５ｂｐ〜１００ｂｐ、好ましくは１５ｂｐ〜５０ｂｐ、より好ましくは１５ｂｐ〜３５ｂｐの塩基長を有するものが例示できる。また検出プローブとして用いる場合には、通常１５ｂｐ〜全配列の塩基数、好ましくは１５ｂｐ〜１ｋｂ、より好ましくは１００ｂｐ〜１ｋｂの塩基長を有するものが例示できる。
本発明の疾患マーカーをプライマーとして用いる場合、好ましいプライマーとしては、センス鎖については、ＣＧＴ遺伝子の塩基配列７８０−７９９位に位置する配列を有するセンス鎖（２０ｂｐ）：５’−ＡＡＡＧＴＡＧＧＣＴＴＧＧＴＴＣＡＣＧＧ−３’（配列番号４）、アンチセンス鎖については、ＣＧＴ遺伝子の塩基配列（配列番号１）の１６０８−１６２７位に位置する配列（２０ｂｐ）に対するアンチセンス鎖（２０ｂｐ）：５’−ＣＡＧＡＴＧＣＡＡＧＴＧＣＣＡＴＧＣＡＡ−３’（配列番号５）を例示することができる。
また本発明の疾患マーカーをプローブとして用いる場合、好ましいプローブとしては、上記プライマーを用いて増幅される配列、すなわちＣＧＴ遺伝子の塩基配列７８０−１６２７位に位置する配列（配列番号６）またはその相補配列を例示することができる。なお、本発明の疾患マーカー（ＲＮＡに対しては相補鎖）は、放射性同位元素（^３２Ｐ、^３３Ｐなど：ＲＩ）、蛍光物質、化学発光物質、または酵素などで標識されていてもよく、かかる標識疾患マーカーはプローブ（検出マーカー）として好適に利用することができる。
本発明の疾患マーカーは、ノーザンブロット法、ＲＴ−ＰＣＲ法、ｉｎ ｓｉｔｕハイブリダーゼーション法などといった、特定遺伝子を特異的に認識し、また検出する公知の方法において、常法に従ってプライマーまたはプローブとして利用することができ、これによって糸球体線維化を伴う腎疾患に関連するＣＧＴ遺伝子の発現上昇の有無またはそのレベル（発現上昇程度）を評価することができる。なお、測定対象試料としては、使用する検出方法の種類に応じて、被験者の腎組織の一部をバイオプシ等で採取し、そこから常法に従って調製したｔｏｔａｌ ＲＮＡを用いてもよいし、さらに該ＲＮＡをもとにして調製される各種のポリヌクレオチドを用いてもよい。なお、採取する腎組織は、腎皮質及び腎髄質の別を問わないが、好ましくは腎皮質である。
また、生体組織におけるＣＧＴ遺伝子の遺伝子発現レベルは、ＤＮＡチップを利用して検出あるいは定量することもできる。この場合、本発明の疾患マーカーは当該ＤＮＡチップのプローブとして使用することができる。かかるＤＮＡチップを、生体組織から採取したＲＮＡをもとに調製される標識ＤＮＡまたは標識ＲＮＡとハイブリダイズさせて、ハイブリダイズによって形成された上記プローブ（疾患マーカー）と標識ＤＮＡまたはＲＮＡとの複合体を該標識ＤＮＡまたはＲＮＡの標識を指標として検出することにより生体組織中でのＣＧＴ遺伝子の発現量が測定でき、これによって該遺伝子の発現上昇の有無またはそのレベル（発現上昇程度）を評価することができる。
本発明の疾患マーカーは、糸球体線維化を伴う腎疾患の検出（罹患の有無や罹患の程度の診断）に有用である。当該検出は被験者の腎組織におけるＣＧＴ遺伝子の発現量を上記疾患マーカーを用いて測定することにより実施することができる。具体的には、糸球体線維化を伴う腎疾患でＣＧＴ遺伝子が特異的に発現誘導されていることから（発現上昇を示す）、当該検出は被験者の腎組織におけるＣＧＴ遺伝子の遺伝子発現量を上記疾患マーカーを用いて測定し、被験者における該遺伝子発現量の上昇の有無を、正常者の腎組織におけるＣＧＴ遺伝子の遺伝子発現量との対比から判定することによって行うことができる。この場合、被験者の腎組織での該遺伝子の発現量が正常者の腎組織の発現量と比べて２倍以上、好ましくは３倍以上多ければ、当該被験者について糸球体線維化を伴う腎疾患の罹患が疑われる。
（１−２）抗体
また本発明は、糸球体線維化を伴う腎疾患の疾患マーカーとしてＣＧＴ遺伝子の発現産物（タンパク質、ＣＧＴ）を特異的に認識することのできる抗体を提供する。
ＣＧＴとしては配列番号１または２に示されるポリヌクレオチドによってコードされるタンパク質を挙げることができる。なお、配列番号１または２に示されるポリヌクレオチドによってコードされるタンパク質の具体的態様としては配列番号３で示されるアミノ酸配列を有するタンパク質を例示することができる。
なお、ここで配列番号３に示すタンパク質は、スフィンゴリピッド合成系の律速酵素であるヒト由来のＣＧＴ（Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ）として公知のタンパク質であり、その酵素学的性質や取得方法についてもＳＨＡＹＭＡＮ ＪＡ，ＡＢＥ Ａ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ ２０００；３１１：４２−９“ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ：ＡＳＳＡＹ ＡＮＤ ＰＲＯＰＥＲＴＩＥＳ”及びＭＡＲＫＳ ＤＬ，ＰＡＵＬ Ｐ，ＫＡＭＩＳＡＫＡ Ｙ，ＰＡＧＡＮＯ ＲＥ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ．２０００；３１１：５０−９．“ＭＥＴＨＯＤＳ ＦＯＲ ＳＴＵＤＹＩＮＧ ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ”に記載されるように公知である。ただし、当該ＣＧＴ酵素が糸球体線維化を伴う腎疾患に何らかの形で関与していることの認識は、本発明で初めて見いだされたものである。
本発明の抗体は、その形態に特に制限はなく、ＣＧＴを免疫抗原とするポリクローナル抗体であっても、またそのモノクローナル抗体であってもよく、さらには当該ＣＧＴのアミノ酸配列のうち、連続する少なくとも８アミノ酸からなるポリペプチドに対して抗原結合性を有する抗体も、本発明の抗体に含まれる。
これらの抗体の製造方法は、すでに周知であり、本発明の抗体もこれらの常法に従って製造することができる（Ｃｕｒｒｅｎｔ ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ ｅｄｉｔ．Ａｕｓｕｂｅｌ ｅｔ ａｌ．（１９８７）Ｐｕｂｌｉｓｈ．Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ．Ｓｅｃｔｉｏｎ １１．１２−１１．１３）。具体的には、本発明の抗体がポリクローナル抗体の場合には、常法に従って大腸菌等で発現し精製したＣＧＴを用いて、あるいは常法に従って当該ＣＧＴの部分アミノ酸配列を有するオリゴペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、常法に従って大腸菌等で発現し精製したＣＧＴ、あるいはこれらタンパク質の部分アミノ酸配列を有するオリゴペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞の中から得ることができる（Ｃｕｒｒｅｎｔ ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ ｅｄｉｔ．Ａｕｓｕｂｅｌ ｅｔ ａｌ．（１９８７）Ｐｕｂｌｉｓｈ．Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ．Ｓｅｃｔｉｏｎ １１．４−１１．１１）。
また、抗体の作製に使用されるタンパク質は、本発明により提供される遺伝子の配列情報（配列番号１または２）に基づいて、ＤＮＡクローニング、各プラスミドの構築、宿主へのトランスフェクション、形質転換体の培養および培養物からのタンパク質の回収の操作により得ることができる。これらの操作は、当業者に既知の方法、あるいは文献記載の方法（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ，Ｔ．Ｍａｎｉａｔｉｓ ｅｔ ａｌ．，ＣＳＨ Ｌａｂｏｒａｔｏｒｙ（１９８３），ＤＮＡ Ｃｌｏｎｉｎｇ，ＤＭ．Ｇｌｏｖｅｒ，ＩＲＬ ＰＲＥＳＳ（１９８５））などに準じて行うことができる。具体的にはＣＧＴをコードする遺伝子が所望の宿主細胞中で発現できる組み換えＤＮＡ（発現ベクター）を作成し、これを宿主細胞に導入して形質転換し、該形質転換体を培養して、得られる培養物から、目的タンパク質を回収することによって実施することができる。また、これらＣＧＴは、本発明により提供されるアミノ酸配列の情報（配列番号３）に従って、一般的な化学合成法（ペプチド合成）によって製造することもできる。
なお、本発明のＣＧＴには、配列番号３に示すアミノ酸配列を有するタンパク質のみならず、その相同物も包含される。該相同物としては、上記配列番号３で示されるアミノ酸配列において、１若しくは複数のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、且つ配列番号３で示されるアミノ酸配列を有するタンパク質の公知の機能と同等の生物学的機能を有するか、及び／または免疫学的活性において同等の活性を有するタンパク質を挙げることができる。
なお、ここで配列番号３で示されるアミノ酸配列を有するタンパク質の公知の機能と同等の生物学的機能を有するタンパク質としてはＣＧＴと生化学的または薬理学的機能において同等の機能を有するタンパク質を挙げることができる。また、上記タンパク質と免疫学的活性において同等の活性を有するタンパク質としては、適当な動物あるいはその細胞において特定の免疫反応を誘発し、かつＣＧＴに対する抗体と特異的に結合する能力を有するタンパク質を挙げることができる。
なお、タンパク質におけるアミノ酸の変異数や変異部位は、その生物学的機能及び／または免疫学的活性が保持される限り制限はない。生物学的機能や免疫学的活性を喪失することなくアミノ酸残基が、どのように、何個置換、挿入あるいは欠失されればよいかを決定する指標は、当業者に周知のコンピュータプログラム、例えばＤＮＡ Ｓｔａｒ ｓｏｆｔｗａｒｅを用いて見出すことができる。例えば変異数は、典型的には、全アミノ酸のうち１０％以内であり、好ましくは全アミノ酸のうち５％以内であり、さらに好ましくは全アミノ酸のうち１％以内である。また置換されるアミノ酸は、当該アミノ酸で置換した後に得られるタンパク質がＣＧＴの生物学的機能及び／または免疫学的活性を保持している限り、特に制限されない。好ましくは、タンパク質の構造保持の観点から、残基の極性、電荷、可溶性、疎水性、親水性並びに両親媒性など、置換前のアミノ酸と似た性質を有するアミノ酸である。例えば、Ａｌａ、Ｖａｌ、Ｌｅｕ、Ｉｌｅ、Ｐｒｏ、Ｍｅｔ、Ｐｈｅ及びＴｒｐは互いに非極性アミノ酸に分類されるアミノ酸であり、Ｇｌｙ、Ｓｅｒ、Ｔｈｒ、Ｃｙｓ、Ｔｙｒ、Ａｓｎ及びＧｌｎは互いに非荷電性アミノ酸に分類されるアミノ酸であり、Ａｓｐ及びＧｌｕは互いに酸性アミノ酸に分類されるアミノ酸であり、またＬｙｓ、Ａｒｇ及びＨｉｓは互いに塩基性アミノ酸に分類されるアミノ酸である。ゆえに、これらのアミノ酸の性質を指標として同群に属するアミノ酸の中からを適宜選択することができる。
また本発明の抗体は、ＣＧＴの部分アミノ酸配列を有するオリゴペプチドを用いて調製されるものであってよい。かかる抗体誘導のために用いられオリゴペプチドは、機能的な生物活性を有することは要しないが、ＣＧＴと同様な免疫原特性を有するものであることが望ましい。好ましくはかかる免疫原特性を有し、ＣＧＴのアミノ酸配列において連続する少なくとも８つのアミノ酸からなるポリペプチドを例示することができる。
かかるポリペプチドに対する抗体の生成は、宿主に応じて種々のアジュバントを用いて免疫学的反応を高めることによって行うこともできる。限定はされないが、そのようなアジュバントには、フロイントアジュバント；水酸化アルミニウムのようなミネラルゲル；並びにリゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油乳剤、キーホールリンペットヘモシアニン及びジニトロフェノールのような表面活性物質；ＢＣＧ（カルメット−ゲラン桿菌）やコリネバクテリウム−パルヴムなどのヒトアジュバントなどがある。
本発明の抗体はＣＧＴに特異的に結合する性質を有することから、該抗体を利用することによって、被験者の腎組織内に発現した上記タンパク質（ＣＧＴ）を特異的に検出し、また定量することができる。すなわち、当該抗体は被験者の腎組織内におけるＣＧＴ生成の有無を検出するためのプローブとして有用である。
具体的には、患者の腎組織の一部をバイオプシ等で採取し、そこから常法に従って上記のタンパク質（ＣＧＴ）を含む腎組織抽出物（タンパク質（ＣＧＴ）含有画分）を調製して、例えばウェスタンブロット法、ＥＬＩＳＡ法など公知の検出方法において、上記抗体を常法に従ってプローブとして使用することによって、上記腎組織抽出物中（すなわち腎組織）に存在するＣＧＴを検出することができる。
前述するように、糸球体線維化を伴う腎疾患患者の腎組織ではＣＧＴ遺伝子が発現上昇を示していることから、被験者の腎組織におけるＣＧＴの量が正常な腎組織におけるＣＧＴの量に比して上昇しているか否かを判定することによって、糸球体線維化を伴う腎疾患の罹患の有無を診断することができる。この場合、被験者の腎組織におけるＣＧＴ遺伝子の発現産物（ＣＧＴ）の量が正常な腎組織における発現産物量と比べて２倍以上、好ましくは３倍以上多いことが判定されれば、当該被験者において糸球体線維化を伴う腎疾患の罹患が疑われる。
（２）糸球体線維化を伴う腎疾患の検出方法
本発明は、糸球体線維化を伴う腎疾患の検出方法を提供する。
（２−１）当該検出方法としては、第１に前述する本発明の糸球体線維化を伴う腎疾患の疾患マーカーを利用する方法を挙げることができる。
具体的には、当該検出方法は、被験者の腎組織の一部をバイオプシなどで採取し、そこに含まれるＣＧＴ遺伝子の遺伝子発現レベルまたはこれらの遺伝子に由来するタンパク質（ＣＧＴ）を検出し、または定量する工程を有する。ここで得られた結果に基づいて該被験者が糸球体線維化を伴う腎疾患に罹患しているか否か、または罹患している場合にはその程度を判断することができる。
本発明の腎疾患の検出方法は次の（ａ）および（ｂ）の工程を含むものである：
（ａ）被験者の生体試料と本発明の疾患マーカーを接触させる工程、
（ｂ）生体試料中のＣＧＴ遺伝子の発現レベル、またはその遺伝子産物（タンパク質、ＣＧＴ）の量を、上記疾患マーカーを指標として測定する工程。
さらに、本発明の腎疾患の検出方法は上記工程（ａ）および（ｂ）に加えて、さらに下記の工程（ｃ）を含むことができる：
（ｃ）上記（ｂ）の測定結果に基づいて、被験者について糸球体線維化を伴う腎疾患の罹患を判断する工程。
当該（ｃ）工程は、例えば、工程（ｂ）において被験者について得られる測定結果を、正常者について同様にして得られる測定結果と対比して、得られる疾患マーカーへのポリヌクレオチドの結合量の異同に基づいて行うことができる。
ここで用いられる生体試料は、被験者の腎組織から調製されるＲＮＡ若しくはそれからさらに調製されるポリヌクレオチド、被験者の腎組織から調製されるタンパク質、またはタンパク質含有画分を含む被験者の腎組織抽出物、または腎組織などである。かかるＲＮＡ、ポリヌクレオチド、タンパク質または腎組織抽出物（タンパク質含有画分）は、被験者の腎組織の一部をバイオプシ等で採取し、そこから常法に従って調製することができる。
本発明の診断方法は、測定対象として用いる生体試料の種類に応じて、具体的には下記のようにして実施される。
（ｉ） 測定対象の生体試料としてＲＮＡまたはそれから調製されるポリヌクレオチドを利用する場合
この場合、上記工程（ａ）および（ｂ）として下記の工程を用いることができる。
（ａ）被験者の生体試料から調製されたＲＮＡまたは該ＲＮＡから転写された相補的ポリヌクレオチドと請求項１または２に記載の疾患マーカーとを結合させる工程、
（ｂ）該疾患マーカーに結合した生体試料由来のＲＮＡまたは該ＲＮＡから転写された相補的ポリヌクレオチドを、上記疾患マーカーを指標として測定する工程。
検出に用いる生体試料としてＲＮＡまたはそれから調製されるポリペプチド（以下、ＲＮＡ等という）を利用する場合は、本発明の腎疾患の検出方法は該ＲＮＡ等中のＣＧＴ遺伝子の発現を検出し、またその程度を測定することによって実施される。具体的には、前述する本発明のポリヌクレオチドからなる疾患マーカー（ＣＧＴ遺伝子の塩基配列において連続する少なくとも１５塩基を有するポリヌクレオチド及び／又はその相補的なポリヌクレオチド）をプライマーまたはプローブとして用いて、上記ＲＮＡ等を対象にしてノーザンブロット法、ＲＴ−ＰＣＲ法、ＤＮＡチップ解析法、ｉｎ ｓｉｔｕハイブリダイゼーション解析法などの公知の方法を行うことにより実施できる。
ノーザンブロット法を利用する場合は、本発明の上記疾患マーカーをプローブとして用いることによって、ＲＮＡ等中のＣＧＴ遺伝子の発現の有無やその発現の程度を検出、測定することができる。具体的には、本発明の疾患マーカー（ＲＮＡに対しては相補鎖）を放射性同位元素（^３２Ｐ、^３３Ｐなど：ＲＩ）や蛍光物質などで標識し、それを、常法に従ってナイロンメンブレン等にトランスファーした被験者の生体組織由来のＲＮＡ等とハイブリダイズさせた後、形成された疾患マーカー（ポリヌクレオチド）とＲＮＡ等との二重鎖を、疾患マーカーの標識物（ＲＩ若しくは蛍光物質等）に由来するシグナルを放射線検出器（ＢＡＳ−１８００ＩＩ、富士フィルム社製）または蛍光検出器等で検出、測定する方法を例示することができる。また、ＡｌｋＰｈｏｓ Ｄｉｒｅｃｔ Ｌａｂｅｌｉｎｇ ａｎｄ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ａｍｅｒｓｈａｍ Ｐｈａｒａｍｃｉａ Ｂｉｏｔｅｃｈ社製）を用いて、該プロトコールに従って疾患マーカー（ポリヌクレオチド）を標識し、被験者の生体組織由来のＲＮＡ等とハイブリダイズさせた後、疾患マーカーの標識物に由来するシグナルをマルチバイオイメージャーＳＴＯＲＭ８６０（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）で検出、測定する方法を使用することもできる。
ＲＴ−ＰＣＲ法を利用する場合は、本発明の上記疾患マーカー（ポリヌクレオチド）をプライマーとして用いることによって、ＲＮＡ等中のＣＧＴ遺伝子の発現の有無やその程度を検出、測定することができる。具体的には、被験者の生体組織由来のＲＮＡから常法に従ってｃＤＮＡを調製して、これを鋳型として標的のＣＧＴ遺伝子領域が増幅できるように、本発明の疾患マーカーから調製した一対のプライマー〔上記ｃＤＮＡ（−鎖）に結合する本発明の疾患マーカー（正鎖）、上記ｃＤＮＡの逆鎖（＋鎖）に結合する本発明の疾患マーカー（相補鎖）〕をこれとハイブリダイズさせて、常法に従ってＰＣＲ法を行い、得られた増幅二本鎖ＤＮＡを検出する方法を例示することができる。なお、増幅された二本鎖ＤＮＡの検出は、上記ＰＣＲを予めＲＩや蛍光物質で標識しておいたプライマーを用いて行うことによって産生される標識二本鎖ＤＮＡを検出する方法、産生された二本鎖ＤＮＡを常法に従ってナイロンメンブレン等にトランスファーさせて、標識した疾患マーカーをプローブとして使用してこれとハイブリダイズさせて検出する方法などを用いることができる。なお、生成された標識二本鎖ＤＮＡ産物はアジレント２１００バイオアナライザ（横河アナリティカルシステムズ社製）などで測定することができる。また、ＳＹＢＲ Ｇｒｅｅｎ ＲＴ−ＰＣＲ Ｒｅａｇｅｎｔｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）で該プロトコールに従ってＲＴ−ＰＣＲ反応液を調製し、ＡＢＩ ＰＲＩＭＥ ７７００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）で反応させて、該反応物を検出することもできる。
また、ＤＮＡチップ解析を利用する場合は、本発明の上記疾患マーカーをＤＮＡプローブ（１本鎖または２本鎖ポリヌクレオチド）として貼り付けたＤＮＡチップを用意し、これに被験者の生体組織由来のＲＮＡから常法によって調製されたｃＲＮＡとハイブリダイズさせて、形成されたＤＮＡとｃＲＮＡとの二本鎖に、別途ＲＩや蛍光物質等で標識してなる本発明の疾患マーカー（ポリヌクレオチド）を標識プローブとして結合させて検出する方法を挙げることができる。また、上記ＤＮＡチップとして、ＣＧＴ遺伝子の発現レベルの検出、測定が可能なＤＮＡチップを用いることもできる。かかる遺伝子の発現レベルを検出、測定することができるＤＮＡチップとしては、Ａｆｆｙｍｅｔｒｉｘ社のＧｅｎｅ Ｃｈｉｐ Ｈｕｍａｎ Ｇｅｎｏｍｅ Ｕ９５ Ａ，Ｂ，Ｃ，Ｄ，Ｅを挙げることができる。かかるＤＮＡチップを用いた、被験者ＲＮＡ中のＣＧＴ遺伝子の発現レベルの検出、測定については、実施例において詳細に説明する。
糸球体線維化に伴う腎疾患の診断は、好適には、上記の如くして測定される本発明の疾患マーカーに対するポリヌクレオチドの結合量に反映される、被験者の腎組織ＲＮＡ等中におけるＣＧＴ遺伝子の発現量を、同様にして測定される正常者の腎組織ＲＮＡ等中におけるＣＧＴ遺伝子の発現量と対比して、両者の違いに基づいて行うことができる。具体的には、被験者の当該発現量が正常者の発現量よりも多い場合には、該被験者について糸球体線維化を伴う腎疾患の罹患を疑うことができる、この場合、被験者のＣＧＴ遺伝子の発現量が正常者のそれよりも２倍以上、好ましくは３倍以上高ければ、より高い信頼性をもって、該被験者について腎疾患の罹患を認定することができる。
（ｉｉ） 測定対象物としてタンパク質を用いる場合
測定対象物としてタンパク質を用いる場合は、本発明の検出方法は生体試料中のＣＧＴを検出し、その量を測定することによって実施される。具体的には、被験者の腎組織抽出物（タンパク質含有画分）を、抗体に関する本発明の疾患マーカー（配列番号３に示すアミノ酸配列からなるタンパク質を認識する抗体）と接触させて、該疾患マーカーに結合したＣＧＴ又はその部分ペプチドを、ウエスタンブロット法などの方法で、本発明の疾患マーカーを指標として検出し、また定量する方法を挙げることができる。
この場合、前述する工程（ａ）および（ｂ）として下記の工程を用いることができる。
（ａ）被験者の腎組織抽出物と請求項３または４に記載の疾患マーカーとを接触させる工程、
（ｂ）該疾患マーカーに結合した腎組織抽出物中のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅまたはその部分ペプチドを、上記疾患マーカーを指標として測定する工程。
ウエスタンブロット法は、一次抗体として本発明の上記疾患マーカーを用いた後、二次抗体として上記一次抗体に結合性を有する^１２５Ｉなどの放射性同位元素や蛍光物質で標識してなる抗体を用いて、ＣＧＴ又はその部分ペプチドと疾患マーカー（一次抗体）との複合体を標識し、次いで該放射性同位元素若しくは蛍光物質由来のシグナルを放射線測定器（ＢＡＳ−１８００ＩＩ：富士フィルム社製など）若しくは蛍光検出器で検出し、測定することによって実施できる。また、一次抗体として本発明の上記疾患マーカーを用いた後、ＥＣＬ Ｐｌｕｓ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｃｔｉｏｎ Ｓｙｓｔｅｍ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）を用いて、該プロトコールに従って検出し、マルチバイオイメージャーＳＴＯＲＭ８６０（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製）で測定することもできる。
糸球体線維化を伴う腎疾患の検出は、好適には上記の如くして測定される本発明の疾患マーカーに対するＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅまたはその部分ペプチドの結合量に反映される、被験者の腎組織におけるＣＧＴ遺伝子の発現産物（ＣＧＴ）の量を、同様にして測定される正常な腎組織における当該ＣＧＴの量と比較し、両者の違いに基づいて行うことができる。具体的には、被験者の腎組織中のＣＧＴ量が正常者のＣＧＴ量よりも多い場合には、該被験者について糸球体線維化を伴う腎疾患の罹患を疑うことができる、この場合、被験者の腎組織中のＣＧＴ量が正常者のそれよりも２倍以上、好ましくは３倍以上高ければ、より高い信頼性をもって、該被験者について腎疾患の罹患を認定することができる。
なお、被験者の腎組織と正常な腎組織とのＣＧＴ遺伝子の発現の程度またはＣＧＴ量の比較は、被験者の生体試料と正常者の生体試料を対象とした測定を並行して行うことで実施できる。並行して行なわない場合は、複数（少なくとも２つ、好ましくは３以上、より好ましくは５以上）の正常な腎組織を用いて均一な測定条件で測定して得られたＣＧＴ遺伝子の発現の程度、若しくはその遺伝子発現産物であるＣＧＴの量の平均値または統計的中間値を、正常者のＣＧＴ遺伝子発現レベル若しくはＣＧＴ量として、比較に用いることができる。
ところで、ＣＧＴは前述するようにスフィンゴリピッド合成系の酵素であり、ＵＤＰ−グルコースとセラミドからグルコシルセラミドを生成する反応を触媒することが知られている（ＳＨＡＹＭＡＮ ＪＡ，ＡＢＥ Ａ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ ２０００；３１１：４２−９“ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ：ＡＳＳＡＹ ＡＮＤ ＰＲＯＰＥＲＴＩＥＳ．及びＭＡＲＫＳ ＤＬ，ＰＡＵＬ Ｐ，ＫＡＭＩＳＡＫＡ Ｙ，ＰＡＧＡＮＯ ＲＥ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ．２０００；３１１：５０−９．”ＭＥＴＨＯＤＳ ＦＯＲ ＳＴＵＤＹＩＮＧ ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ”）。従って、腎組織中のＣＧＴ量は、該腎組織中の当該ＣＧＴ酵素活性を測定することによっても、評価することができる。
（２−２）従って、本発明は糸球体線維化を伴う腎疾患の検出方法として、第２に、被験者の腎組織中のＣＧＴ酵素活性に基づいて該疾患の罹患の有無を検出する方法を提供する。
具体的には、本発明の検出方法は、（ａ）被験者の腎組織中のＣＧＴ酵素活性を測定する工程を実施することで行うことができる。
この場合、具体的には腎組織中のＣＧＴ酵素活性は、例えば、（ｉ）腎組織中に内在するグルコシルセラミド（内因性グルコシルセラミド）の量を測定するか、または（ｉｉ）腎組織抽出物にＵＤＰ−グルコースとセラミドを配合して、当該外来的に添加したＵＤＰ−グルコースとセラミドに基づいて生成するグルコシルセラミド（外因性グルコシルセラミド）の量を測定することによって、測定し評価することができる。当該（ｉ）の方法は、腎組織中に本来的に存在するＵＤＰ−グルコースとセラミドに対して腎組織中のＣＧＴの作用によって生成する内在性のグルコシルセラミドの量に基づいてＣＧＴ活性を評価する方法である。また（ｉｉ）の方法は腎組織抽出物に外来的に配合したＵＤＰ−グルコースとセラミドに対して腎組織中のＣＧＴの作用によって生成する外来性のグルコシルセラミドの量に基づいてＣＧＴ活性を評価する方法である。当該（ｉｉ）の方法は、具体的には上記（ａ）の工程として下記工程を実施することによって行うことができる：
（ａ−１）被験者の腎組織抽出物とＵＤＰ−グルコース及びセラミドとを接触させる工程、
（ａ−２）上記（ａ）の工程に基づいて生じるグルコシルセラミドの生成量を測定する工程。
腎組織抽出物は、腎組織中に存在するＣＧＴの量や活性、ＵＤＰ−グルコース、セラミド及びグルコシルセラミドの量を低減させない方法を用いて調製することが好ましい。かかる方法としてＳＨＡＹＭＡＮ ＪＡ，ＡＢＥ Ａ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ ２０００；３１１：４２−９“ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ：ＡＳＳＡＹ ＡＮＤ ＰＲＯＰＥＲＴＩＥＳ”、またはＭＡＲＫＳ ＤＬ，ＰＡＵＬ Ｐ，ＫＡＭＩＳＡＫＡ Ｙ，ＰＡＧＡＮＯ ＲＥ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ．２０００；３１１：５０−９．“ＭＥＴＨＯＤＳ ＦＯＲ ＳＴＵＤＹＩＮＧ ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ”に記載される方法を挙げることができる。
（ｉ）の方法において、腎組織中に内在するグルコシルセラミドの量は、具体的には上記方法に従って得られる腎組織抽出物から脂質画分を抽出し、これをＨＰＣＬに供することによって測定することができる。
（ｉｉ）の方法は、具体的には上記の腎組織抽出物０．１μｇ〜０．１ｇあたりに、ＵＤＰ−グルコース及びセラミドをそれぞれ０．１ｐｇ〜０．１ｍｇ程度の割合で加え、得られる混合物を３７℃で１時間程度反応させて、イソプロパノールと硫酸ナトリウムの混合物等の反応停止剤を添加後、得られた反応物について、上記方法に従ってグルコシルセラミドの量を測定することによって実施することができる。この時に、コントロールとしてＵＤＰ−グルコース及びセラミドを添加しない腎組織抽出物についても同様に実験を行い、得られるグルコシルセラミドの内在量（内因性グルコシルセラミドの量）を差し引くことによって、外因性グルコシルセラミドの量を評価することができる。
上記において用いられるセラミドとは、ＣＧＴの基質となるものであれば、天然由来／非天然由来のいずれであってもよく、セラミドの誘導体も本発明でいうセラミドの範疇に含まれる。セラミドの誘導体としては、具体的にはＮ−アシルスフィンゴシンを挙げることができる。ここでアシルとは、炭素数２〜２０程度のアシル基を指し、好ましくは炭素数８のアシル基を有するオクタノイルスフィンゴシン（Ｏｃｔａｎｏｙｌｓｐｈｉｎｇｏｓｉｎｅ）を挙げることができる。
また、上記（ｉｉ）の方法において腎組織抽出物と反応させるＵＤＰ−グルコース及びセラミドのいずれか少なくとも１方を、放射性同位体、蛍光試薬、化学発光試薬または酵素試薬などで標識したものを使用することによって、外因性グルコシルセラミドの量を選択的に測定し評価することができる。具体的には、一例として、腎組織抽出物とセラミド及びＵＤＰ−^３Ｈ−グルコースを混合して、３７℃で１時間程度反応させた後、イソプロパノールと硫酸ナトリウムの混合物等の反応停止剤を添加して反応を停止し、得られた反応溶液からｔ−ブチルメチルエーテルで^３Ｈ−グルコシルセラミドを抽出して、該標識グルコシルセラミドの放射活性を液体シンチレーションカウンターで測定する方法を例示することができる。
このように本発明の糸球体線維化を伴う腎疾患の診断は、いずれか少なくとも１方が標識化されていてもよいＵＤＰ−グルコース及びセラミドを検査試薬として用いて実施することができる。よって本発明は、いずれか少なくとも１方が標識化されていてもよいＵＤＰ−グルコース及びセラミドの糸球体線維化を伴う腎疾患の検査試薬としての使用（用途）をも提供するものである。当該検査試薬は、少なくともＵＤＰ−グルコースとセラミドとが予め混合された状態で包装されてなるものであってもよいし、また用時に混合できるように、別個に包装されてなるものであってもよい。なお、ＵＤＰ−グルコース及び／またはセラミドを標識する場合、使用される標識剤としては特に制限されず、従来公知のアッセイ系で使用される標識剤、例えば放射性同位体、蛍光試薬、化学発光試薬または酵素試薬などを用いることができる。また、本発明の検査試薬の中には、同一包装物中または別個の包装物として、他の成分（例えば、反応緩衝液、反応停止剤、酵素反応基質、ｐＨ調整剤）を含めることもできる。
本発明の糸球体線維化を伴う腎疾患の検出は、好適にはさらに前述する方法によって得られる被験者の腎組織に内在するグルコシルセラミドまたは外来的に生じるグルコシルセラミドの量を、正常者のそれらと対比して、両者の異同に基づいて行うことができる。
具体的には、（ｉ）の場合、上記（ａ）の工程に加えてさらに下記の工程を行うことが好ましい：
（ｂ）上記（ａ）の結果を正常者の腎組織中に内在するグルコシルセラミドの量と対比して、その異同に基づいて、被験者について糸球体線維化を伴う腎疾患の罹患を判断する工程。
（ｉｉ）の場合は、上記（ａ−１）および（ａ−２）の工程に加えてさらに下記の工程を行うことが好ましい：
（ｂ）上記（ａ−２）の測定結果を、同様にして得られる正常者のグルコシルセラミド生成量と対比して、その異同に基づいて、被験者について糸球体線維化を伴う腎疾患の罹患を判断する工程。
上記（ｂ）の工程において、被験者に内在するグルコシルセラミドの量または外来的に生じるグルコシルセラミドの量が正常者のそれよりも多い場合には、該被験者について糸球体線維化を伴う腎疾患を疑うことができる。この場合、被験者についてグルコシルセラミドの量が正常者のそれよりも２倍以上、好ましくは３倍以上高ければ、より高い信頼性をもって、該被験者について腎疾患の罹患を認定することができる。
（３）候補薬のスクリーニング方法
（３−１）遺伝子発現レベルを指標とするスクリーニング方法
本発明は、配列番号１または２に記載の塩基配列を有するＣＧＴ遺伝子の発現を抑制する物質をスクリーニングする方法を提供する。
本発明のスクリーニング方法は次の工程（ａ）、（ｂ）及び（ｃ）を含む：
（ａ）被験物質とＣＧＴ遺伝子を発現可能な細胞とを接触させる工程、
（ｂ）被験物質を接触させた細胞のＣＧＴ遺伝子の発現量を測定し、該発現量を被験物質を接触させない対照細胞の上記遺伝子の発現量と比較する工程、および（ｃ）上記の比較結果に基づいて、ＣＧＴ遺伝子の発現量を減少させる被験物質を選択する工程。
かかるスクリーニングに用いられる細胞としてはＣＧＴ遺伝子を発現する細胞であって、かつ腎臓由来の細胞を挙げることができる。腎臓由来の細胞としては、具体的には、抗Ｔｈｙ−１抗体を投与したＷｉｓｔａｒラット（チャールズリバー社製）、もしくは６分の５腎臓摘出Ｗｉｓｔａｒラット（チャールズリバー社製）から単離・調製した初代腎臓培養細胞などを挙げることができる。またＮＲＫ−４９Ｆ細胞（Ｎｏｒｍａｌ Ｒａｔ Ｋｉｄｎｅｙ Ｃｅｌｌ：ＡＴＣＣ Ｎｕｍｂｅｒ ＣＲＬ−１５７０）のような株化細胞も用いることができる。その際、糸球体線維化を伴う腎炎またはこれに近い状態の細胞を用いることもでき、かかる細胞は、上記ＣＧＴ遺伝子を発現する腎臓由来の細胞をＴＧＦ−β（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ−β）あるいはＰＤＧＦ（ｐｌａｔｅｌｅｄ−ｄｅｒｉｖｅｄ ｇｒｏｗｔｈ ｆａｃｔｏｒ）等の線維化原因因子で処理することにより調製することができる。さらに本発明のスクリーニングに用いられる細胞の範疇には、細胞の集合体である組織も含まれる。
実施例に示すように、糸球体線維化を伴う腎疾患（糸球体硬化症）に罹患した患者の腎組織には、特異的にＣＧＴ遺伝子が高く発現している。また既存のＣＧＴ阻害剤の投与実験により、糸球体線維化抑制効果が認められた。これらの知見から、ＣＧＴ遺伝子の発現の増加（発現誘導）が糸球体線維化を伴う腎疾患の原因になっていると考えられる。本発明の上記スクリーニング方法は、当該知見に基づいて、糸球体線維化を伴う腎疾患の緩和／抑制作用を有する（糸球体線維化を伴う腎疾患に対して改善／治療効果を発揮する）物質（以下、「候補物質」ともいう）を、上記ＣＧＴ遺伝子の発現レベルの減少、あるいは発現誘導の抑制を指標として探索しようとするものである。
ここで候補物質となりえるものとしては、制限されないが、核酸（ＣＧＴ遺伝子のアンチセンス分子を含む）、ペプチド、タンパク質、有機化合物、無機化合物などであり、本発明のスクリーニング方法は、具体的にはこれらの候補物質となり得る被験物質を上記細胞と接触させることにより行うことができる。また細胞との接触は、かかる被験物質を含む試料（被験試料）を用いて行うこともでき、かかる被験試料としては細胞抽出液、遺伝子ライブラリーの発現産物、または合成低分子化合物、合成ペプチド、若しくは天然化合物などを含む試料を例示することができる。
また、スクリーニングに際して、被験物質と細胞とを接触させる条件は、特に制限されないが、該細胞が死なないように、その培養条件（温度、ｐＨ、培地組成など）を大きく変化させない条件を採用することが好ましい。
本発明のスクリーニング方法によれば、ＣＧＴ遺伝子の発現を抑制する物質を探索することによって、糸球体線維化を伴う腎疾患の改善薬または治療薬の有効成分となる候補物質を取得することができる。この意味で、本発明のスクリーニング方法は被験物質の中から糸球体線維化を伴う腎疾患の改善薬または治療薬の有効成分を探索し、選別し、取得する方法として位置づけることができる。
候補物質の探索（選別）は、具体的には被験物質と接触させる細胞としてＣＧＴ遺伝子を発現する細胞を用いる場合は、被験物質を添加した細胞におけるＣＧＴ遺伝子の発現レベルが被験物質を添加しない細胞のそのレベルに比して低くなることをもって行うことができる。
より具体的には、例えば、上記細胞として抗Ｔｈｙ−１抗体を投与したＷｉｓｔａｒラットや６分の５腎臓摘出Ｗｉｓｔａｒラットから単離・調製した初代腎臓培養細胞を用いる場合、被験物質を加えた初代腎臓培養細胞のＣＧＴ遺伝子の発現量を、被験物質を加えず溶媒のみを加えた初代腎臓培養細胞（対照細胞）のＣＧＴ遺伝子の発現量と比較し、添加によりその発現量の低下をもたらす被験物質を候補物質として選別することができる。
このようなＣＧＴ遺伝子の発現の検出や定量は、前記細胞から調製したＲＮＡ又はそれから転写された相補的なポリヌクレオチドを用いて、ノーザンブロット法、ＲＴ−ＰＣＲ法など公知の方法の他、ＤＮＡチップなどを利用して実施できる。これら公知の方法については、前記本発明の腎疾患の検出方法に関する（２−１）の項を参照されたい。指標とするＣＧＴ遺伝子発現の低下（減少）の程度は、被験物質を添加した細胞におけるＣＧＴ遺伝子の発現量が被験物質を添加しない対照細胞での発現量と比較して１０％以上、好ましくは３０％以上、特に好ましくは５０％以上の低下（減少）を例示することができる。
またＣＧＴ遺伝子発現の検出及び定量は、ＣＧＴ遺伝子の発現を制御する遺伝子領域（発現制御領域）に、例えばルシフェラーゼ遺伝子などのマーカー遺伝子をつないだ融合遺伝子を導入した細胞株を用いて、マーカー遺伝子由来のタンパク質の活性を測定することで実施することもできる。本発明のＣＧＴ遺伝子の発現抑制物質のスクリーニング方法には、かかるマーカー遺伝子の発現量を指標として標的物質を探索する方法も包含される。この意味において請求項１６に記載する「Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ遺伝子」の概念には、ＣＧＴ遺伝子の発現制御領域とマーカー遺伝子との融合遺伝子が含まれる。
なお、上記マーカー遺伝子としては、発光反応や抵触反応を触媒する酵素の構造遺伝子が好ましく、具体的には上記のルシフェラーゼ遺伝子のほか、クロラムフェニコール・アセチルトランスフェラーゼ遺伝子、βグルクロニダーゼ遺伝子、βガラクトシダーゼ遺伝子、及びエクオリン遺伝子などのレポーター遺伝子もまた使用することができる。ここでＣＧＴ遺伝子の発現制御領域としては、例えば該遺伝子の転写開始部位上流約１ｋｂ、好ましくは約２ｋｂを用いることができる。融合遺伝子の作成、およびマーカー遺伝子由来の活性測定は公知の方法で行うことができる。
以上のスクリーニング方法により、被験物質から選別される物質はＣＧＴ遺伝子の発現抑制剤として位置づけることができる。これらの物質は、ＣＧＴ遺伝子の発現を抑制することによって、糸球体線維化を伴う腎疾患の発症や進展、ならびに該疾患の進展による糸球体硬化症への移行を抑制し得ると考えられる。よって、これらの物質は、糸球体線維化を伴う腎疾患を緩和、抑制（改善、治療）する薬物または糸球体硬化症への進展及び発症を予防する薬物の有力な候補物質となる。
（３−２）ＣＧＴ産生量を指標とするスクリーニング方法
本発明は、配列番号３に記載のアミノ酸配列を有するＣＧＴの産生を抑制する物質をスクリーニングする方法を提供する。
本発明のスクリーニング方法は次の工程（ａ）、（ｂ）及び（ｃ）を含む：
（ａ）被験物質とＣＧＴを産生可能な細胞とを接触させる工程、
（ｂ）被験物質を接触させた細胞におけるＣＧＴの産生量を測定し、該産生量を被験物質を接触させない対照細胞の上記ＣＧＴの産生量と比較する工程、および
（ｃ）上記の比較結果に基づいて、ＣＧＴの産生量を減少させる被験物質を選択する工程。
かかるスクリーニングに用いられる細胞は、内在性および外来性を問わず、ＣＧＴ遺伝子を発現し、ＣＧＴを産生する細胞を挙げることができる。具体的には、抗Ｔｈｙ−１抗体を投与したＷｉｓｔａｒラット（チャールズリバー社製）、もしくは６分の５腎臓摘出Ｗｉｓｔａｒラット（チャールズリバー社製）から単離・調製した初代腎臓培養細胞、ＮＲＫ−４９Ｆ細胞（Ｎｏｒｍａｌ Ｒａｔ Ｋｉｄｎｅｙ Ｃｅｌｌ：ＡＴＣＣ Ｎｕｍｂｅｒ ＣＲＬ−１５７０）などの株化細胞を挙げることができる。また前述するように当該スクリーニングに用いられる細胞の範疇には、細胞の集合体である組織も含まれる。
実施例に示すように、糸球体線維化を伴う腎疾患（糸球体硬化症）に罹患した患者の腎組織には、特異的にＣＧＴ遺伝子が高く発現している。また既存のＣＧＴ阻害剤の投与実験により、糸球体線維化抑制効果が認められた。これらの知見から、ＣＧＴ遺伝子の発現の増加（発現誘導）が糸球体線維化を伴う腎疾患の原因になっていると考えられる。本発明のスクリーニング方法は、ＣＧＴ遺伝子の発現に対応して増減するＣＧＴの産生量を指標として（具体的にはＣＧＴの産生量の減少を指標として）、糸球体線維化を伴う腎疾患の緩和／抑制作用を有する（糸球体線維化を伴う腎疾患に対して改善／治療効果を発揮する）物質（候補物質）を探索しようとするものである。
ここで候補物質となりえるものとしては、制限されないが、核酸（ＣＧＴ遺伝子のアンチセンス分子を含む）、ペプチド、タンパク質、有機化合物、無機化合物などであり、本発明のスクリーニング方法は、具体的にはこれらの候補物質となり得る被験物質を上記細胞と接触させることにより行うことができる。また細胞との接触は、かかる被験物質を含む試料（被験試料）を用いて行うこともでき、かかる被験試料としては細胞抽出液、遺伝子ライブラリーの発現産物、または合成低分子化合物、合成ペプチド、若しくは天然化合物などを含む試料を例示することができる。
また、スクリーニングに際して、被験物質と細胞とを接触させる条件は、特に制限されないが、該細胞が死なないように、その培養条件（温度、ｐＨ、培地組成など）を大きく変化させない条件を採用することが好ましい。
本発明のスクリーニング方法によれば、ＣＧＴの産生を抑制する物質を探索することによって、糸球体線維化を伴う腎疾患の改善薬または治療薬の有効成分となる候補物質を取得することができる。この意味で、本発明のスクリーニング方法は被験物質の中から糸球体線維化を伴う腎疾患の改善薬または治療薬の有効成分を探索し、選別し、取得する方法として位置づけることができる。
候補物質の探索（選別）は、具体的には被験物質を添加した細胞におけるＣＧＴの産生量が被験物質を添加しない細胞のＣＧＴ産生量に比して低くなることをもって行うことができる。
より具体的には、例えば、上記細胞として抗Ｔｈｙ−１抗体を投与したＷｉｓｔａｒラットや６分の５腎臓摘出Ｗｉｓｔａｒラットから単離・調製した初代腎臓培養細胞を用いる場合、被験物質を加えた初代腎臓培養細胞のＣＧＴの産生量を、被験物質を加えず溶媒のみを加えた初代腎臓培養細胞（対照細胞）のＣＧＴの産生量と比較し、添加によりその産生量の低下をもたらす被験物質を候補物質として選別することができる。
このようなＣＧＴ産生量は、抗体に関する本発明の疾患マーカー（配列番号３に示すアミノ酸配列からなるタンパク質を認識する抗体）を利用したウエスタンブロット法などの方法に従って測定することができる。これらの方法については、前記本発明の腎疾患の検出方法に関する（２−２）の項を参照されたい。指標とするＣＧＴ産生量の低下（減少）の程度は、被験物質を添加した細胞におけるＣＧＴの産生量が被験物質を添加しない対照細胞での産生量と比較して１０％以上、好ましくは３０％以上、特に好ましくは５０％以上の低下（減少）を例示することができる。
以上のスクリーニング方法により、被験物質から選別される物質はＣＧＴの産生抑制剤として位置づけることができる。これらの物質は、ＣＧＴの産生を抑制することによって、糸球体線維化を伴う腎疾患の発症や進展、ならびに該疾患の進展による糸球体硬化症への移行を抑制し得ると考えられる。よって、これらの物質は、糸球体線維化を伴う腎疾患を緩和、抑制（改善、治療）する薬物または糸球体硬化症への進展及び発症を予防する薬物の有力な候補物質となる。
ＣＧＴは前述するように生体内に存在するスフィンゴリピッド合成系の酵素であり（ＳＨＡＹＭＡＮ ＪＡ，ＡＢＥ Ａ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ ２０００；３１１：４２−９“ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ：ＡＳＳＡＹ ＡＮＤ ＰＲＯＰＥＲＴＩＥＳ．及びＭＡＲＫＳ ＤＬ，ＰＡＵＬ Ｐ，ＫＡＭＩＳＡＫＡ Ｙ，ＰＡＧＡＮＯ ＲＥ．ＭＥＴＨＯＤＳ ＥＮＺＹＭＯＬ．２０００；３１１：５０−９．”ＭＥＴＨＯＤＳ ＦＯＲ ＳＴＵＤＹＩＮＧ ＧＬＵＣＯＳＹＬＣＥＲＡＭＩＤＥ ＳＹＮＴＨＡＳＥ”）、それ自体が特定の機能や活性を有している。前述する本発明のスクリーニング方法は、ＣＧＴ遺伝子の発現量またはＣＧＴ産生量を指標とするものであるが、上記ＣＧＴの機能（活性）はこれらのＣＧＴ遺伝子発現量およびＣＧＴ産生量に応じて変動しえるものである。よって、同様のスクリーニングは、ＣＧＴの機能（活性）を指標として行うこともできる。
（３−３）ＣＧＴ（タンパク質）の機能または活性を指標とするスクリーニング方法
本発明は、ＣＧＴの機能または活性を抑制する物質をスクリーニングする方法を提供する。
本発明のスクリーニング方法は次の工程（ａ）、（ｂ）及び（ｃ）を含む：
（ａ）被験物質をＣＧＴに接触させる工程、
（ｂ）上記工程に起因して生じるＣＧＴの機能または活性を測定し、該活性を被験物質を接触させない場合のＣＧＴの機能または活性と比較する工程、
（ｃ）（ｂ）の比較結果に基づいて、ＣＧＴの機能または活性の低下をもたらす被験物質を選択する工程。
実施例に示すように、糸球体線維化を伴う腎疾患（糸球体硬化症）に罹患した患者の腎組織には、特異的にＣＧＴ遺伝子の発現が増大している。また既存のＣＧＴ阻害剤の投与実験により、糸球体線維化抑制効果が認められた。これらの知見から、ＣＧＴ遺伝子でコードされるタンパク質（ＣＧＴ）の機能または活性の増大が糸球体線維化を伴う腎疾患の原因となっていると考えられる。本発明のスクリーニング方法は、かかる知見に基づいて、当該腎疾患の緩和／抑制作用を有する（糸球体線維化を伴う腎疾患に対して改善／治療効果を発揮する）物質（候補物質）を、ＣＧＴの機能または活性の低下もしくは抑制を指標として探索しようとするものである。
すなわち、本発明のスクリーニング方法によれば、ＣＧＴの機能または活性を抑制する物質を探索することによって、糸球体線維化を伴う腎疾患の改善薬または治療薬の有効成分となる候補物質を取得することができる。その意味で本発明のスクリーニング方法は、被験物質の中から糸球体線維化を伴う腎疾患の改善薬または治療薬の有効成分を探索し、選別し、また取得するための方法として位置づけることができる。
具体的には、当該スクリーニング方法は、ＵＤＰ−グルコースとセラミドとの反応を触媒してグルコシドセラミドを生成するというＣＧＴの酵素学的作用を利用して下記の工程（ａ）、（ｂ）及び（ｃ）に従って実施することができる。
（ａ）被験物質の存在下で、ＣＧＴ、ＵＤＰ−グルコース、及びセラミドを反応させる工程、
（ｂ）上記反応の結果生じたグルコシルセラミドの生成量を測定し、該生成量を、被験物質の非存在下で上記（ａ）の反応を行って生じるグルコシルセラミドの生成量と比較する工程、
（ｃ）上記（ｂ）の比較結果に基づいて、グルコシルセラミドの生成量の減少をもたらす被験物質を選択する工程。
ここで用いられるセラミドとは、ＣＧＴの基質となるものであれば、天然由来／非天然由来のいずれであってもよく、セラミドの誘導体も本発明でいうセラミドの範疇に含まれる。セラミドの誘導体として、具体的にはＮ−アシルスフィンゴシンを挙げることができる。ここでアシルとは、炭素数２〜２０程度のアシル基を指し、好ましくは炭素数８のアシル基を有するオクタノイルスフィンゴシン（Ｏｃｔａｎｏｙｌｓｐｈｉｎｇｏｓｉｎｅ）を挙げることができる。
また被験物質としては、核酸、ペプチド、タンパク質（ＣＧＴに対する抗体を含む）、有機化合物または無機化合物などの任意のものを用いることができる。ＣＧＴの取得の由来も特に制限されず、天然由来のＣＧＴ酵素であっても、また遺伝子組み換え技術によって、例えば非細胞系もしくは各種細胞系（バクテリア、酵母、昆虫細胞、動物細胞）にて生産、精製して取得されるヒト組み換え型ＣＧＴ酵素であってもよい。また、当該ＣＧＴ、並びにＵＤＰ−グルコース及びセラミドは商業的に入手可能であり、簡便には市販のものを使用することができる。なお、ＵＤＰ−グルコースまたはセラミドは、少なくともいずれか１方が放射性同位体、蛍光試薬、化学発光試薬などの任意のラベルで標識されていてもよく、こうすることで反応によって生じるグルコシルセラミドを選択的に検出し、定量することができる。この場合、反応系に配合する各成分の割合は特に制限されないが、ＣＧＴ、並びにＵＤＰ−グルコース及びセラミド等のＣＧＴの基質の配合量として、それぞれ０．１ｐｇ〜０．１ｍｇ程度の割合を例示することができる。
具体的なスクリーニング方法としては、一例としてＣＧＴをセラミド、ＵＤＰ−^３Ｈ−グルコース、及び被験物質と混合し、ｔ−ブチルメチルエーテルで^３Ｈ−グルコシルセラミドを抽出して該標識グルコシドセラミドの放射活性を液体シンチレーションカウンターで測定することにより、生成したグルコシドセラミドを定量する方法を挙げることができる。当該方法は、具体的には、下記の操作によって行うことができる；
▲１▼上記混合物を３７℃にて１時間インキュベートし、１ｍｌのｉｓｏｐｒｏｐａｎｏｌ、０．８ｍｌの５０ｍｇ／ｍｌ硫酸ナトリウムを添加し反応を止める。
▲２▼混合後６ｍｌのｔ−ｂｕｔｙｌ ｍｅｔｈｙｌ ｅｔｈｅｒを添加しさらに混合する。
▲３▼１５００ｘｇで１０分間遠心分離した後、上清６ｍｌを４５℃のドライブロックヒーターで乾燥させ、０．５ｍｌの蒸留水に溶解して、５ｍｌのＡＣＳＩＩを添加して、液体シンチレーションカウンターで放射活性を測定する。
斯くして選抜取得される被験物質は、糸球体線維化を伴う腎疾患を緩和、抑制（改善、治療）する薬物の有力な候補物質となる。
上記本発明のスクリーニング方法によって選別された候補物質は、さらに糸球体線維化を伴う腎疾患モデル動物を用いた薬効試験、安全性試験、さらに糸球体線維化を伴う腎疾患患者への臨床試験に供してもよく、これらの試験を実施することによって、より実用的な腎疾患改善または治療薬を取得することができる。このようにして選別された物質は、さらにその構造解析結果に基づいて、化学的合成、生物学的合成（発酵）または遺伝子学的操作によって、工業的に製造することができる。
（４）腎疾患の改善・治療剤
本発明は、糸球体線維化を伴う腎疾患の改善・治療剤を提供するものである。
本発明はＣＧＴ遺伝子の発現が糸球体線維化を伴う腎疾患と関連しており、またＣＧＴに対する既存の阻害剤が糸球体の繊維化抑制効果を有しているという新たな知見からＣＧＴ遺伝子の発現を抑制する物質、ＣＧＴの産生を抑制する物質、あるいはＣＧＴの機能または活性を抑制する物質が、上記疾患の改善または治療に有効であるという考えに基づくものである。すなわち、本発明の腎疾患の改善・治療剤はＣＧＴ遺伝子の発現を抑制する物質、ＣＧＴの産生を抑制する物質、あるいはＣＧＴの機能又は活性を抑制する物質を有効成分とするものである。
本発明で用いる有効成分、すなわちＣＧＴ遺伝子の発現抑制物質、ＣＧＴの産生抑制物質、あるいはＣＧＴの機能または活性抑制物質には、それぞれＣＧＴ遺伝子の発現抑制作用、ＣＧＴの産生抑制作用、あるいはＣＧＴの機能または活性抑制作用を有するものであればよく、従来公知のもののほか、従来公知でないものも含まれる。従来公知でないものは、被験物質の中から上記のスクリーニング方法を利用して選別し、取得することができる。また、有効物質は上記スクリーニング方法を利用して選別された物質に関する情報に基づいてさらに常法に従って製造されたものであってもよい。
ここで腎疾患の改善・治療剤の有効成分となり得るＣＧＴの機能または活性抑制物質としては、下記の一般式

（式中、Ｒ１は炭素数５〜１５のアルキル基またはベンジルオキシ基を、Ｒ２は―（ＣＨ_２）_４―基または―（ＣＨ_２）_２−Ｏ−（ＣＨ_２）_２―基を意味する。）
で示される化合物（１）またはその薬学的に許容される塩を挙げることができる。
具体的には１−フェニル−２−アルカノイルアミノ−３−モルフォリノ−１−プロパノール、あるいは１−フェニル−２−アルカノイルアミノ−３−ピロリジノ−１−プロパノールおよびその構造的アナログを挙げることができる（ＷＯ００／６２７７９；Ｃｈｅｍｉｃａｌ Ｐｈｙｓ．Ｌｉｐｉｄｓ（１９８０），２６（３），２６５−７８；Ｊ．Ｂｉｏｃｈｅｍ．１２７，４８５−４９１（２０００）等参照）。ここで、前者１−フェニル−２−アルカノイルアミノ−３−モルフォリノ−１−プロパノールの構造的アナログとして、特に好ましくはＤ−スレオ−１−フェニル−２−デカノイルアミノ−３−モルフォリノ−１−プロパノール（Ｄ−ＰＤＭＰ）を挙げることができる。また、後者１−フェニル−２−アルカノイルアミノ−３−ピロリジノ−１−プロパノールの構造的アナログとして、（１Ｒ，２Ｒ）−２−ヘキサノイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＨＰＰ）、（１Ｒ，２Ｒ）−２−オクタノイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＯＰＰ）、（１Ｒ，２Ｒ）−２−デカノイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＤＰＰ）、（１Ｒ，２Ｒ）−２−パルミトイルアミノ−１−フェニル−３−ピロリジノ−１−プロパノール（ＰＰＰＰ）および（１Ｒ，２Ｒ）−１−フェニル−２−ベンジルオキシカルボニルアミノ−３−ピロリジノ−１−プロパノール（ＰＢＰＰ）を挙げることができる。なお、これらの物質は、いずれも例えば前記文献等に基づいて製造することができる。
また、これらに近縁するＣＧＴの機能または活性抑制物質は、下記の一般式：

（式中、Ｒ３は任意の官能基、またＲ４は環中の窒素原子とともに複素環を形成する任意の官能基を意味する。）
で示される化合物（２）またはその薬学上許容される塩を被験物質として、前述の（３−３）の項で詳述する本発明のスクリーニング方法を行うことにより、取得することができる。上記複素環としては、各々置換基を有していても良いピロリジン、イミダゾリジン、ピラゾリジン、ピペリジン、ピペラジン、及びモルフォリンを例示することができる。なお、上記化合物には立体異性体をはじめとする各種の異性体が含まれる。
ゆえに、本発明には、上記化合物（２）またはその薬学上許容される塩を被験物質として下記１）の工程（ａ）、（ｂ）および（ｃ）を実施するか、または下記２）の工程（ｄ）、（ｅ）および（ｆ）を実施する、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質のスクリーニング方法が含まれる：
１）（ａ）被験物質をＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅに接触させる工程、
（ｂ）上記工程に起因して生じるＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を測定し、該機能または活性を被験物質を接触させない場合のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性と比較する工程、および
（ｃ）（ｂ）の比較結果に基づいて、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性の低下をもたらす被験物質を選択する工程；
２）（ｄ）被験物質の存在下で、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ、ＵＤＰ−グルコース、及びセラミドを反応させる工程、
（ｅ）上記反応の結果生じたグルコシルセラミドの生成量を測定し、該生成量を、被験物質の非存在下で上記（ａ）の反応を行って得られるグルコシルセラミドの生成量と比較する工程、および
（ｆ）上記（ｂ）の比較結果に基づいて、グルコシルセラミドの生成量の減少をもたらす被験物質を選択する工程。
また本発明には、上記スクリーニング方法で被験物質の中から選別され取得される候補物質を有効成分とする、糸球体線維化を伴う腎疾患の改善または治療剤が含まれる。
さらにＣＧＴの機能または活性抑制物質の別の具体例として、一般式（３）：

（式中、Ｒ５は炭素数１〜２０の直鎖状または分岐状のアルキル基を意味する。）
で示されるＮ−アルキル−デオキシノジリマイシン（ＮＡ−ＤＮＪ）、一般式（４）：

（式中、Ｒ６は炭素数１〜２０の直鎖状または分岐状のアルキル基を意味する。）
で示されるＮ−アルキル−デオキシガラクトノジリマイシン（ＮＡ−ＧＮＪ）、またはこれらの構造的アナログを挙げることができる（ＷＯ００／６２７７９、ＷＯ００／６２７８０、特許第２８３１０６８号公報、ＷＯ００／３３８４３、ＷＯ０１／０７０７８、Ｂｉｏｃｈｅｍｉｃａｌ Ｐｈａｒｍａｃｏｌｏｇｙ，Ｖｏｌ．５６，ｐｐ．４２１−４３０（１９９８）、Ｂｉｏｃｈｅｍｉｃａｌ Ｐｈａｒｍａｃｏｌｏｇｙ，Ｖｏｌ．５９，ｐｐ．８２１−８２９（２０００）等参照）。なお、当該化合物は薬学的に許容される塩の態様を有していてもよい。
ここでＲ３で示されるアルキル基としては、炭素数１〜２０の直鎖又は分枝のアルキル基を挙げることができるが、好ましくは炭素数１〜１０のアルキル基であり、より炭素数４のブチル基である。Ｎ−アルキル−デオキシノジリマイシンとして具体的には、Ｎ−ブチル−デオキシノジリマイシン（ＮＢ−ＤＮＪ）、Ｎ−ノニル−デオキシノジリマイシン、およびＮ−デシル−デオキシノジリマイシンが、またＮ−アルキル−デオキシガラクトノジリマイシンとして具体的にはＮ−ブチル−デオキシガラクトノジリマイシン（ＮＢ−ＤＧＪ）などを挙げることができる。中でも特に好ましくはＮ−ブチル−デオキシノジリマイシン（ＮＢ−ＤＮＪ）である。これらの物質は、例えば前記文献やＵＳ４１８２７６７、ＵＳ４６３９４３６、ＥＰ００１２２７８、ＥＰ０６２４６５２、ＵＳ４２６６０２５，ＵＳ４４０５７１４，ＵＳ５１５１５１９等に基づいて製造することができる。
また、これらＮＡ−ＤＮＪおよびＮＡ−ＧＮＪに近縁するＣＧＴの機能または活性抑制物質は、下記の一般式：

（式中、Ｒ７は任意の官能基を意味する。）
で示される化合物（５）またはその薬学上許容される塩を被験物質として、前述の（３−３）の項で詳述する本発明のスクリーニング方法を行うことにより、取得することができる。なお、上記化合物には立体異性体をはじめとする各種の異性体が含まれる。
ゆえに、本発明には、上記化合物（５）またはその薬学上許容される塩を被験物質として下記１）の工程（ａ）、（ｂ）および（ｃ）を実施するか、または下記２）の工程（ｄ）、（ｅ）および（ｆ）を実施する、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を抑制する物質のスクリーニング方法が含まれる：
１）（ａ）被験物質をＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅに接触させる工程、
（ｂ）上記工程に起因して生じるＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性を測定し、該機能または活性を被験物質を接触させない場合のＣｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性と比較する工程、および
（ｃ）（ｂ）の比較結果に基づいて、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅの機能または活性の低下をもたらす被験物質を選択する工程；
２）（ｄ）被験物質の存在下で、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ、ＵＤＰ−グルコース、及びセラミドを反応させる工程、
（ｅ）上記反応の結果生じたグルコシルセラミドの生成量を測定し、該生成量を、被験物質の非存在下で上記（ａ）の反応を行って得られるグルコシルセラミドの生成量と比較する工程、および
（ｆ）上記（ｂ）の比較結果に基づいて、グルコシルセラミドの生成量の減少をもたらす被験物質を選択する工程。
また本発明には、上記スクリーニング方法で被験物質の中から選別され取得される候補物質を有効成分とする、糸球体線維化を伴う腎疾患の改善または治療剤が含まれる。
以上詳述するＣＧＴの機能または活性抑制物質、ならびにＣＧＴ遺伝子の発現抑制物質、またはＣＧＴの産生抑制物質は、そのままもしくは自体公知の薬学的に許容される担体（賦形剤、増量剤、結合剤、滑沢剤などが含まれる）や慣用の添加剤などと混合して医薬組成物として、特に腎疾患の改善または治療を目的とした医薬組成物として調製することができる。当該医薬組成物は、調製する形態（錠剤、丸剤、カプセル剤、散剤、顆粒剤、シロップ剤などの経口投与剤；注射剤、点滴剤、外用剤、坐剤などの非経口投与剤）等に応じて経口投与または非経口投与することができる。また投与量は、有効成分の種類、投与経路、投与対象または患者の年齢、体重、症状などによって異なり一概に規定できないが、１日投与用量として、数百ｍｇ〜２ｇ、好ましくは数十ｍｇ程度を、１日１〜数回にわけて投与することができる。
なお、上記の物質がＤＮＡによりコードされるものであれば、該ＤＮＡを遺伝子治療用ベクターに組み込み、遺伝子治療を行うことも考えられる。さらに、上記の物質がＣＧＴ遺伝子に対するアンチセンスヌクレオチドの場合は、そのまま、若しくは遺伝子治療用ベクターにこれを組み込むことにより遺伝子治療を行うこともできる。これらの場合も、投与量、投与方法は患者の体重や年齢、症状などにより変動するが、当業者であれば適宜選択することが可能である。
実施例
次に、本発明を実施例によりさらに具体的に説明する。しかし、本発明はこれらの実施例になんら限定されるものではない。
実施例１： 正常および糸球体硬化（線維化）状態の腎臓での遺伝子発現の変動解析
ヒト糸球体硬化症患者の腎臓組織６サンプルとヒトの正常な腎臓組織５５サンプルからそれぞれ調製したｔｏｔａｌ ＲＮＡを用いて、ＤＮＡチップ解析を行った。ＤＮＡチップ解析は、Ａｆｆｙｍｅｔｒｉｘ社Ｇｅｎｅ Ｃｈｉｐ Ｈｕｍａｎ Ｇｅｎｏｍｅ Ｕ９５Ａ，Ｂ，Ｃ，Ｄ，Ｅを用いて行った。具体的には、解析は、（１）ｔｏｔａｌ ＲＮＡからのｃＤＮＡの調製、（２）該ｃＤＮＡからラベル化ｃＲＮＡの調製、（３）ラベル化ｃＲＮＡのフラグメント化、（４）フラグメント化ｃＲＮＡとプローブアレイとのハイブリダイズ、（５）プローブアレイの染色、（６）プローブアレイのスキャン、及び（７）遺伝子発現解析、の手順で行った。
（１）ｔｏｔａｌ ＲＮＡからのｃＤＮＡの調製
ヒト糸球体硬化症患者の腎臓組織６サンプル、ヒトの正常な腎臓組織５５サンプルから常法に従って調製した各ｔｏｔａｌ ＲＮＡ １０μｇ、Ｔ７−（ｄＴ）２４プライマー（Ａｍｅｒｓｈａｍ社製）１００ｐｍｏｌを含む１１μＬの混合液を、７０℃、１０分間加熱後、氷上で冷却した。冷却後、ＳｕｐｅｒＳｃｒｉｐｔ Ｃｈｏｉｃｅ Ｓｙｓｔｅｍ ｆｏｒ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ（Ｇｉｂｃｏ−ＢＲＬ社製）に含まれる５×Ｆｉｒｓｔ Ｓｔｒａｎｄ ｃＤＮＡ Ｂｕｆｆｅｒ ４μＬ、該キットに含まれる０．１Ｍ ＤＴＴ ２μＬ、該キットに含まれる１０ｍＭ ｄＮＴＰ Ｍｉｘ １μＬを添加し、４２℃で２分間加熱した。更に、該キットに含まれるＳｕｐｅｒ ＳｃｒｉｐｔＩＩ ＲＴ ２μＬ（４００Ｕ）を添加し、４２℃で１時間加熱した後、氷上で冷却した。冷却後、ＤＥＰＣ処理水（ナカライテスク社製）９１μＬ、該キットに含まれる５×Ｓｅｃｏｎｄ Ｓｔｒａｎｄ Ｒｅａｃｔｉｏｎ Ｂｕｆｆｅｒ ３０μＬ、１０ｍＭ ｄＮＴＰ Ｍｉｘ ３μＬ、該キットに含まれるＥ．ｃｏｌｉ ＤＮＡ Ｌｉｇａｓｅ １μＬ（１０Ｕ）、該キットに含まれるＥ．ｃｏｌｉ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ Ｉ ４μＬ（４０Ｕ）、該キットに含まれるＥ．ｃｏｌｉ ＲＮａｓｅＨ １μＬ（２Ｕ）を添加し、１６℃で２時間反応させた。次いで、該キットに含まれるＴ４ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ ２μＬ（１０Ｕ）を加え、１６℃で５分間反応させた後、０．５Ｍ ＥＤＴＡ １０μＬを添加した。次いで、フェノール／クロロホルム／イソアミルアルコール溶液（ニッポンジーン社製）１６２μＬを添加し、混合した。該混合液を、予め室温、１４，０００ｒｐｍ、３０秒間遠心分離しておいたＰｈａｓｅ Ｌｏｃｋ Ｇｅｌ Ｌｉｇｈｔ（エッペンドルフ社製）に移し、室温下１４，０００ｒｐｍで２分間遠心分離した後、１４５μＬの水層をエッペンドルフチューブに移した。得られた溶液に、７．５Ｍ酢酸アンモニウム溶液７２．５μＬ、エタノール３６２．５μＬを加え混合した後、４℃で１４，０００ｒｐｍ、２０分間遠心分離した。遠心分離後、上清を捨て、作製したｃＤＮＡを含むＤＮＡペレットを得た。その後、該ペレットに８０容量％エタノール０．５ｍＬを添加し、４℃で１４，０００ｒｐｍ、５分間遠心分離した後、上清を捨てた。再度上記と同様の操作を行った後、該ペレットを乾燥させて、ＤＥＰＣ処理水（ナカライテスク社製）１２μＬに溶解した。
以上の操作によって、ヒト糸球体硬化症患者の腎臓組織６サンプル、ヒトの正常な腎臓組織５５サンプルからそれぞれ調製したｔｏｔａｌＲＮＡからｃＤＮＡを取得した。
（２）ｃＤＮＡからラベル化ｃＲＮＡの調製
（１）で得られた各ｃＤＮＡ溶液５μＬに、ＤＥＰＣ処理水（ナカライテスク社製）１７μＬ、ＢｉｏＡｒｒａｙ Ｈｉｇｈ Ｙｉｅｌｄ ＲＮＡ Ｔｒａｎｓｃｒｉｐｔ Ｌａｂｅｌｉｎｇ Ｋｉｔ（ＥＮＺＯ社製）に含まれる１０×ＨＹ Ｒｅａｃｔｉｏｎ Ｂｕｆｆｅｒ ４μＬ、該キットに含まれる１０×Ｂｉｏｔｉｎ Ｌａｂｅｌｅｄ Ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ ４μＬ、該キットに含まれる１０×ＤＴＴ ４μＬ、該キットに含まれる１０×ＲＮａｓｅ Ｉｎｈｉｂｉｔｏｒ Ｍｉｘ ４μＬ、該キットに含まれる２０×Ｔ７ ＲＮＡ Ｐｏｌｙｍｅｒａｓｅ ２μＬを混合し、３７℃で５時間反応させて、ラベルｃＲＤＮＡを調製した。反応後、該反応液にＤＥＰＣ処理水６０μＬを加え、次いでＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔを用いて添付プロトコールに従い、調製したラベル化ｃＲＮＡを精製した。
（３）ラベル化ｃＲＮＡのフラグメント化
（２）で調製した各ラベル化ｃＲＮＡ ２０μｇを含む溶液に、５×Ｆｒａｇｍｅｎｔａｔｉｏｎ Ｂｕｆｆｅｒ（２００ｍＭ トリス−酢酸 ｐＨ８．１（Ｓｉｇｍａ社製）、５００ｍＭ酢酸カリウム（Ｓｉｇｍａ社製）、１５０ｍＭ酢酸マグネシウム（Ｓｉｇｍａ社製））８μＬを加えて調製した反応液４０μＬを９４℃で３５分間加熱した後、氷中に置いた。これによって、各ラベル化ｃＲＮＡをフラグメント化した。
（４）フラグメント化ｃＲＮＡとプローブアレイとのハイブリダイズ
（３）で得られた各フラグメント化ｃＲＮＡ ４０μＬに、５ｎＭ Ｃｏｎｔｒｏｌ Ｏｌｉｇｏ Ｂ２（Ａｍｅｒｓｈａｍ社製）４μＬ、１００×Ｃｏｎｔｒｏｌ ｃＲＮＡ Ｃｏｃｋｔａｉｌ ４μＬ、Ｈｅｒｒｉｎｇ ｓｐｅｒｍ ＤＮＡ（Ｐｒｏｍｅｇａ社製）４０μｇ、Ａｃｅｔｙｌａｔｅｄ ＢＳＡ（Ｇｉｂｃｏ−ＢＲＬ社製）２００μｇ、２×ＭＥＳ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｂｕｆｆｅｒ（２００ｍＭ ＭＥＳ、２Ｍ［Ｎａ^＋］、４０ｍＭ ＥＤＴＡ、０．０２％ Ｔｗｅｅｎ２０、ｐＨ６．５〜６．７：Ｐｉｅｒｃｅ社製）２００μＬ、ＤＥＰＣ処理水１４４μＬを混合し、４００μＬのハイブリカクテルを得た。得られた各ハイブリカクテルを９９℃で５分間加熱し、更に４５℃で５分間加熱した。加熱後、室温で１４，０００ｒｐｍ、５分間遠心分離し、ハイブリカクテル上清を得た。
一方、１×ＭＥＳ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｂｕｆｆｅｒで満たしたＨｕｍａｎ ｇｅｎｏｍｅ Ｕ９５プローブアレイ（Ａｆｆｙｍｅｔｒｉｘ社製）を、ハイブリオーブン内で、４５℃、６０ｒｐｍで１０分間回転させた後、１×ＭＥＳ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｂｕｆｆｅｒを除去してプローブアレイを調製した。上記で得られたハイブリカクテル上清２００μＬを該プローブアレイにそれぞれ添加し、ハイブリオーブン内で４５℃、６０ｒｐｍで１６時間回転させ、フラグメント化ｃＲＮＡとハイブリダイズしたプローブアレイを得た。
（５）プローブアレイの染色
（４）で得られたハイブリダイズ済みプローブアレイのそれぞれからハイブリカクテルを回収除去した後、Ｎｏｎ−Ｓｔｒｉｎｇｅｎｔ Ｗａｓｈ Ｂｕｆｆｅｒ（６×ＳＳＰＥＺ［２０×ＳＳＰＥ（ナカライテスク社製）を希釈］、０．０１％Ｔｗｅｅｎ２０、０．００５％Ａｎｔｉｆｏａｍ０−３０［Ｓｉｇｍａ社製］）を添加して、該溶液でプローブアレイを満たした。次にＮｏｎ−Ｓｔｒｉｎｇｅｎｔ Ｗａｓｈ ＢｕｆｆｅｒおよびＳｔｒｉｎｇｅｎｔ Ｗａｓｈ Ｂｕｆｆｅｒ（１００ｍＭ ＭＥＳ、０．１Ｍ ＮａＣｌ、０．０１％Ｔｗｅｅｎ２０）をセットしたＧｅｎｅＣｈｉｐ Ｆｌｕｉｄｉｃｓ Ｓｔａｔｉｏｎ ４００（Ａｆｆｙｍｅｔｒｉｘ社製）の所定の位置にフラグメント化ｃＲＮＡとハイブリダイズしたプローブアレイを装着した。その後、染色プロトコールＥｕＫＧＥ−ＷＳ２に従って、１次染色液〔１０μｇ／ｍＬ Ｓｔｒｅｐｔａｖｉｄｉｎ Ｐｈｙｃｏｅｒｙｔｈｒｉｎ（ＳＡＰＥ）［Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅ社製］、２ｍｇ／ｍＬ Ａｃｅｔｙｌａｔｅｄ ＢＳＡ、１００ｍＭ ＭＥＳ、１Ｍ ＮａＣｌ（Ａｍｂｉｏｎ社製）、０．０５％Ｔｗｅｅｎ２０、０．００５％Ａｎｔｉｆｏａｍ０−３０〕、２次染色液〔１００μｇ／ｍＬ Ｇｏａｔ ＩｇＧ［Ｓｉｇｍａ社製］、３μｇ／ｍＬ Ｂｉｏｔｉｎｙｌａｔｅｄ Ａｎｔｉ−Ｓｔｒｅｐｔａｖｉｄｉｎ ａｎｔｉｂｏｄｙ［Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ社製］、２ｍｇ／ｍＬ Ａｃｅｔｙｌａｔｅｄ ＢＳＡ、１００ｍＭ ＭＥＳ、１Ｍ ＮａＣｌ、０．０５％Ｔｗｅｅｎ２０、０．００５％Ａｎｔｉｆｏａｍ０−３０〕により、プローブアレイを染色した。
（６）プローブアレイのスキャン、及び（７）遺伝子発現解析
（５）で染色した各プローブアレイをＨＰ ＧｅｎｅＡｒｒａｙ Ｓｃａｎｎｅｒ（Ａｆｆｙｍｅｔｒｉｘ社製）に供し、染色パターンを読み取った。染色パターンをもとにＧｅｎｅＣｈｉｐ Ｗｏｒｋｓｔａｔｉｏｎ Ｓｙｓｔｅｍ（Ａｆｆｙｍｅｔｒｉｘ社製）によってプローブアレイ上の遺伝子の発現を解析した。次に、解析プロトコールに従ってＮｏｒｍａｌｉｚａｔｉｏｎおよび遺伝子発現の比較解析を行った。
以上のＤＮＡチップ解析による遺伝子発現の比較解析結果から、正常腎臓組織に比べて糸球体硬化症患者の腎臓組織で３倍以上の発現増を示したプローブを選抜した。
選抜したプローブの中から、さらに糸球体硬化症患者の腎組織で高頻度で発現上昇しているプローブを選抜した。具体的には、ＤＮＡチップ解析結果から得られるＡｂｓ Ｃａｌｌを指標にして、糸球体硬化症患者の腎組織でのＡｂｓ ＣａｌｌがＰｒｅｓｅｎｔである例数が４例以上であるプローブを選抜した。
その結果、Ｃｅｒａｍｉｄｅ Ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ（ＣＧＴ）遺伝子（プローブ名：６１３１５＿ａｔ）が選抜された。ＣＧＴ遺伝子は正常の腎臓と比較して糸球体硬化症の腎臓では３．１倍の発現上昇が観察された。
実施例２： ＣＧＴ阻害剤による繊維芽細胞のプロテオグリカン合成阻害
ＣＧＴ遺伝子と糸球体硬化症または糸球体線維化を伴う腎疾患との関連性を確認するために、腎炎等の組織線維化の原因因子として知られるＴＧＦ−β（Ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ−ｂｅｔａ）［Ｎａｔｕｒｅ，３４６（６２８２），ｐｐ．３７１−３７４，１９９０，Ｊｕｌｙ ２６］あるいはＰＤＧＦ（Ｐｌａｔｅｌｅｔ−ｄｅｒｉｖｅｄ ｇｒｏｗｔｈ ｆａｃｔｏｒ）［Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ，８８（１５），ｐｐ．６５６０−６５６４，１９９１ Ａｕｇ．１；Ｊ Ｃｌｉｎ Ｉｎｖｅｓｔ，９２（６），ｐｐ．２５９７−２６０１，１９９３ Ｄｅｃ；Ａｍ Ｊ Ｐａｔｈｏｌ，１５４（１），ｐｐ．１６９−１７９，１９９９ Ｊａｎ．；Ｎｅｐｈｒｏｎ，８１（４），ｐｐ．４２８−４３３，１９９９；Ｋｉｄｎｅｙ Ｉｎｔ，５９（４），ｐｐ．１３２４−１３３２，２００１ Ａｐｒ．］に依存して産生されるプロテオグリカンの合成を、ＣＧＴの阻害剤が阻害するか否かを検討した。
株化細胞であるＮＲＫ−４９Ｆ細胞（Ｎｏｒｍａｌ Ｒａｔ Ｋｉｄｎｅｙ Ｃｅｌｌ：ＡＴＣＣ Ｎｕｍｂｅｒ ＣＲＬ−１５７０）を１．３×１０^４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌの割合で９６穴プレートにまき込み、１０％ＦＢＳ含有ＤＭＥＭ培地中、ＣＯ_２インキュベーターで一晩培養した。翌日、培養上清を吸引除去し、５％ＦＢＳ含有ＲＰＭＩ培地に交換し、ＣＧＴの既存の阻害剤、ＴＧＦ−β（終濃度３ｎｇ／ｍｌ）またはＰＤＧＦ（終濃度１０ｎｇ／ｍｌ）、^３５Ｓ−Ｎａ_２ＳＯ_４（終濃度１０μＣｉ／ｍｌ）を、計１００μｌ／ｗｅｌｌの割合で添加した。
なお、ここで組織線維化の原因因子としてＴＧＦ−βを配合した系（ＴＧＦ−β依存的プロテオグリカン産生系）にはＣＧＴの既存の阻害剤として、Ｄ−ＰＤＭＰ（Ｄ−ｔｈｒｅｏ−１−ｐｈｅｎｙｌ−２−ｄｅｃａｎｏｌｙａｍｉｎｏ−３−ｍｏｒｐｈｏｌｉｎｏ−１−ｐｒｏｐａｎｏｌ：和光純薬工業）（終濃度３μＭ、３０μＭ）およびＮＢ−ＤＮＪ（Ｎ−Ｂｕｔｙｌｄｅｏｘｙｎｏｊｉｒｉｍｙｃｉｎ：和光純薬工業）（終濃度３μＭ、３０μＭ）の２種類を各々別個に、また組織線維化の原因因子としてＰＤＧＦを配合した系（ＰＤＧＦ依存的プロテオグリカン産生系）にはＣＧＴの既存の阻害剤として、Ｄ−ＰＤＭＰ（終濃度１μＭ、１０μＭ）を用いた。更に培養を約２４時間継続した後に培養上清を回収し、以下の条件にてＳＤＳ−ＰＡＧＥを行った。
すなわち、前記培養上清２０μｌとサンプルバッファー（×５：６２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８）、５０％グリセロール、５％ＳＤＳ、２５％ ２−メルカプトエタノール、０．０５％ブロムフェノールブルー）５μｌを混和し、１００℃で５分処理した後、１０μｌ／ｗｅｌｌの割合でゲル（４％ポリアクリルアミドゲル：テフコ社）にアプライし、１８ｍＡ／枚の定圧電流で３時間泳動を行った。ゲルをゲルドライヤーにて乾燥させた後、イメージングプレートに２４時間露光した。その後、電気泳動されたプロテオグリカンの放射活性をＢＡＳ２０００により定量し、以下の計算式によりＴＧＦ−β依存的プロテオグリカン産生阻害率、並びにＰＤＧＦ依存的プロテオグリカン産生阻害率を計算した。

▲１▼ＴＧＦ−β（又はＰＤＧＦ）添加、ＣＧＴ阻害剤非添加時の放射活性
▲２▼ＴＧＦ−β（又はＰＤＧＦ）添加、ＣＧＴ阻害剤添加時の放射活性
▲３▼ＴＧＦ−β（又はＰＤＧＦ）非添加、ＣＧＴ阻害剤非添加時の放射活性
▲４▼ＴＧＦ−β（又はＰＤＧＦ）非添加、ＣＧＴ阻害剤添加時の放射活性
その結果得られたＴＧＦ−β依存的なプロテオグリカン産生阻害率を図１に、またＰＤＧＦ依存的なプロテオグリカン産生阻害率を図２に、それぞれ示す。この結果からわかるようにＣＧＴ阻害剤であるＤ−ＰＤＭＰおよびＮＢ−ＤＮＪのいずれもが組織線維化の原因因子であるＴＧＦ−βに依存するプロテオグリカン産生阻害活性を示し、またＰＤＭＰが同様に組織線維化の原因因子であるＰＤＧＦに依存するプロテオグリカン産生阻害活性を示したことから、ＣＧＴ阻害剤は、これら原因因子による細胞外基質（プロテオグリカン）の産生亢進を抑制すること、すなわち線維化抑制作用を有することが示された。
実施例３： ノーザンブロット法によるＣＧＴの発現変化の評価
正常ラット（ウイスターラット：日本チャールズリバー（株））および、ウイスターラット（日本チャールズリバー（株））に抗Ｔｈｙ−１モノクローナル抗体（ＯＸ−７：Ｂｉｏｓｏｕｒｃｅｉｎｔｅｒｎａｔｉｏｎａｌ）を投与して作成したＴｈｙ−１腎炎モデルラットから採取した腎皮質から、Ｔｒｉｚｏｌ（ＧｉｂｃｏＢＲＬ）を用いてｔｏｔａｌ ＲＮＡを単離した。ＲＮＡ １０μｇを電気泳導した後にナイロンメンブレンに転写し、ＣＧＴに特異的なｃＤＮＡをプローブとしてノーザンブロット法を実施し、転写レベルをＢＡＳ−２０００（フジフィルム）を用いて定量的に解析することによって、正常ラットの腎皮質およびＴｈｙ−１腎炎モデルラットの腎皮質それぞれにおけるＣＧＴ遺伝子の発現量を調べた。その結果を、図３に、正常ラットの腎皮質中の発現量に対するＴｈｙ−１腎炎モデルラットの腎皮質中の発現量を「ＣＧＴ相対発現量」として示す。これからわかるように、正常ラットの腎皮質中のＣＧＴ遺伝子の発現量と比較して、Ｔｈｙ−１腎炎モデルラットの腎皮質中のＣＧＴ遺伝子の発現量が上昇しているのが観察された。
実施例４： 抗Ｔｈｙ−１腎炎モデルに対するＣＧＴ阻害剤の効果
ウイスターラット（日本チャールズリバー（株））（８匹）に生理食塩水（大塚製薬（株）製）で希釈した抗 Ｔｈｙ−１モノクローナル抗体（ＯＸ−７：Ｂｉｏｓｏｕｒｃｅｉｎｔｅｒｎａｔｉｏｎａｌ）を０．４ｍｇ／ｋｇ（体重）の割合で尾静脈より投与した（ｄａｙ０）。その後、各ラットの体重を測定し、この測定結果に基づいて無作為に無処置群（４匹）、ＮＢ−ＤＮＪ投与群（４匹）に群分けを行った。また抗Ｔｈｙ−１モノクローナル抗体投与を行わない正常対照群（４匹）も設定した。
ＯＸ−７投与約６０分後よりＮＢ−ＤＮＪ投与群（４匹）に対してＮＢ−ＤＮＪの投与を開始し、ｄａｙ６まで１日１回５ｍｇ／ｈｅａｄ（約２０ｍｇ／ｋｇ）腹腔内投与を行った。ｄａｙ７に当該ＮＢ−ＤＮＪ投与群（ＯＸ−７＋ＮＢ−ＤＮＪ投与群）、並びに無処置群（ＯＸ−７投与群）及び正常対照群のそれぞれのラットから腎臓を摘出し、各左腎を１０％中性緩衝ホルマリン溶液を用いて還流し、２μｍの組織切片を作成し、ＰＡＳ染色を施した。ＮＢ−ＤＮＪ投与群（ＯＸ−７＋ＮＢ−ＤＮＪ投与群）、無処置群（ＯＸ−７投与群）及び正常対照群のそれぞれラットの腎臓の糸球体組織（任意箇所）について、１００倍の視野倍率で撮影した像を図４に示す。図４の▲１▼と▲２▼との対比からわかるように、Ｔｈｙ−１腎炎モデルラットでは糸球体硬化の病態を示す糸球体基底膜の肥厚（線維化）が認められたが、ＮＢ−ＤＮＪ投与により当該糸球体の線維化の病態（病変）が改善することが確認された（▲３▼）。
このことから、ＣＧＴ阻害剤が糸球体線維化の予防または／および改善に有効であること、すなわちＣＧＴ阻害剤が糸球体線維化に伴う腎疾患の発症の予防、または／および当該腎疾患の改善または治療に有効であると考えられた。
実施例５： ＣＧＴタンパクの生産、精製
全長ヒトＣＧＴをコードする遺伝子（配列番号１または２）を、ＳＵＰＥＲ ＳＣＲＩＰＴ^ＴＭｃＤＮＡ ｌｉｂｒａｒｙ（ＧＩＢＣＯ ＢＲＬ）から常法によりクローニングし、これをｐＩＶＥＸ２．４ａ（Ｒｏｃｈｅ社）にサブクローニングしてＣＧＴ発現ベクターを調製する。当該ＣＧＴ発現ベクターを鋳型としてＲａｐｉｄ Ｔｒａｎｓｌａｔｉｏｎ Ｓｙｓｔｅｍ（Ｒｏｃｈｅ社）にてＣＧＴ（タンパク質）を合成する。次いで、得られたＣＧＴをベクターに付加されたヒスチジンタグを用いて、ニッケルカラム（アマシャムファルマシア）にて精製する。
実施例６： ＣＧＴ阻害剤のスクリーニング
以下の反応混合液を調製する。

まず上記ＣＧＴ以外の各成分を混合し、これにＣＧＴを加えて反応を開始し、３７℃にて１時間インキュベートする。コントロールとして、被験物質を含まないＤＭＳＯ溶液のみを含む反応混合物についても、同様の反応を行う。１ｍｌのｉｓｏｐｒｏｐａｎｏｌ、０．８ｍｌの５０ｍｇ／ｍｌ硫酸ナトリウムを添加して反応を止め、さらに６ｍｌのｔ−ｂｕｔｙｌ ｍｅｔｈｙｌ ｅｔｈｅｒを添加して混合する。１５００ｘｇで１０分遠心分離した後、上清６ｍｌを４５℃のドライブロックヒーターで乾燥させて、これを０．５ｍｌの蒸留水に溶解して、５ｍｌのＡＣＳＩＩを添加し液体シンチレーションカウンターで反応生成物（^３Ｈ−標識Ｇｌｕｃｏｓｙｌｃｅｒａｍｉｄ）の放射活性を特定して^３Ｈ−標識Ｇｌｕｃｏｓｙｌｃｅｒａｍｉｄの生成量を測定する。被験物質存在下での当該放射活性が、コントロールである被験物質非存在下の放射活性と比較して低下している場合に、その被験物質を、糸球体線維化を伴う腎疾患を緩和または抑制（改善、治療）する候補化合物として選択する。
産業上の利用可能性
本発明によって、糸球体線維化を伴う腎疾患、例えば慢性腎炎、糖尿病性腎症、ＩｇＡ腎症、遺伝性腎疾患などの腎疾患で発現が特異的に増大している遺伝子（ＣＧＴ遺伝子）が明らかとなり、当該遺伝子発現産物の生成またはその活性の増大が糸球体線維化を伴う腎疾患の発症並びに進行（進展）に関与していることが示唆された。従って。かかるＣＧＴ遺伝子は糸球体線維化を伴う腎疾患の遺伝子診断に用いられるマーカー遺伝子（プローブ、プライマー）として有用である。かかるマーカー遺伝子によれば糸球体線維化を伴う腎疾患であるかどうか、その原因を明らかにすることができ（診断精度が向上）、これによってより適切な治療を施すことが可能となる。すなわち、糸球体線維化を伴う腎疾患の適切な治療のためのツールとして利用することができる。
また、本発明において、上記ＣＧＴを阻害することによって糸球体線維化の病態が改善し、またその進行を予防することが確認されたことから、該ＣＧＴの産生を抑制する化合物またはＣＧＴの機能または活性を抑制する化合物は、上記糸球体線維化を伴う腎疾患の治療薬または／および予防剤として有用であると思われる。従って、本発明のＣＧＴ遺伝子によれば、その発現の抑制若しくは減少を指標にすることによって、糸球体線維化を伴う腎疾患の治療薬または予防薬（糸球体線維化の進行抑制剤）の有効成分となり得る候補化合物をスクリーニング選別することが可能である。また本発明が提案するＣＧＴ活性の抑制作用を指標としたスクリーニング法によれば、糸球体線維化を伴う腎疾患の治療薬または予防薬（糸球体線維化の進行抑制剤）の有効成分となり得る候補化合物を簡便に、探索し取得することができる。すなわち、本発明は、糸球体線維化を伴う腎疾患の治療薬または予防薬（糸球体線維化の進行抑制剤）の開発に有用である。
【配列表】

【図面の簡単な説明】
図１は、実施例２において、既存のＣＧＴ阻害剤（Ｄ−ＰＤＭＰ［Ｄ−ｔｈｒｅｏ−１−ｐｈｅｎｙｌ−２−ｄｅｃａｎｏｙｌａｍｉｎｏ−３−ｍｏｒｐｈｏｌｉｎｏ−１−ｐｒｏｐａｎｏｌ］、ＮＢ−ＤＮＪ［Ｎ−ｂｕｔｙｌｄｅｏｘｙｎｏｊｉｒｉｍｙｃｉｎ］：各々３μＭ及び３０μＭ使用）について、ＴＧＦ−β（Ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ−ｂｅｔａ）依存的プロテオグリカン産生阻害率を調べた結果を示す図である。
図２は、実施例２において、既存のＣＧＴ阻害剤（Ｄ−ＰＤＭＰ：１μＭ及び１０μＭ）についてＰＤＧＦ（Ｐｌａｔｅｌｅｔ−ｄｅｒｉｖｅｄ ｇｒｏｗｔｈ ｆａｃｔｏｒ）依存的プロテオグリカン産生阻害率を調べた結果を示す図である。
図３は、実施例３において、正常ラットとＴｈｙ−１腎炎モデルラットについて腎皮質中でのＣＧＴ発現量を比較した結果を示す図である。
図４は、実施例４において、▲１▼正常対照群、▲２▼無処置群（ＯＸ−７投与群）及び▲３▼ＮＢ−ＤＮＪ投与群（ＯＸ−７＋ＮＢ−ＤＮＪ投与群）のそれぞれのラットの腎臓の糸球体組織切片についてＰＡＳ染色を行い、該組織の任意箇所を１００倍の視野倍率で撮影した像を示す図である。 Technical field
The present invention relates to a disease marker useful for diagnosis of renal diseases associated with glomerular fibrosis such as chronic nephritis, diabetic nephropathy, IgA nephropathy, renal failure or glomerulosclerosis. More specifically, the present invention relates to a disease marker that can be effectively used as a primer or a detection probe in the diagnosis of renal diseases associated with glomerular fibrosis. The present invention also relates to a method (diagnostic method) for detecting a renal disease associated with glomerular fibrosis using such a disease marker.
Furthermore, the present invention relates to a method for screening a substance useful as an active ingredient of an agent for ameliorating or treating renal disease associated with glomerular fibrosis, and glomerular fibrosis using the substance prepared by the method as an active ingredient. The present invention relates to a drug for ameliorating or treating accompanying kidney disease.
Background art
In recent years, patients with chronic nephritis such as IgA nephropathy and hereditary kidney disease, or renal diseases such as diabetic nephropathy progress to end-stage renal disease and dialysis therapy is introduced year by year. For this reason, the medical expenses spent on dialysis treatment are becoming enormous.
When glomerulonephritis such as chronic nephritis or diabetic nephropathy develops, mesangial enlargement and glomerular fibrosis (or glomerulosclerosis) are observed as pathological findings, and if this progresses, it may lead to glomerulosclerosis. Are known.
For the treatment of such renal diseases associated with glomerulosclerosis, pharmacotherapy using blood pressure regulators, steroids, or immunosuppressants has been conventionally performed, but it is still impossible to improve the underlying pathology. However, the present situation is that only the effect of the time delay until the end stage renal disease is obtained.
Both mesangial enlargement and glomerular fibrosis (or glomerulosclerosis) seen as pathological findings of glomerulonephritis mentioned above are tissue fibers in the renal tissue mainly due to increased production of extracellular matrix centering on the mesangial region It is thought that the cause is. For this reason, it is possible to effectively improve and treat renal diseases such as chronic nephritis by inhibiting the process leading to tissue fibrosis or restoring the fibrotic tissue. Longed for. However, there is no therapeutic agent for chronic nephritis that has been marketed so far based on such a mechanism. Conventionally, renal function diagnosis is performed by urine protein testing by collecting urine, and there is no simple method for determining the presence or absence of renal disease associated with glomerular fibrosis (fibrosis of kidney tissue).
On the other hand, in the recent medical field, it is desired to select an appropriate treatment method according to the symptoms of individual patients as well as kidney diseases. In recent years when the need for improvement in QOL (Quality of life) in an aging society has been recognized, it is not a treatment that is common to all people, but that appropriate treatment is given according to the symptoms of individual patients. Is strongly demanded. In order to carry out such so-called tailor-made treatments, disease markers that accurately reflect the patient's symptoms and their causes (genetic background) for each disease are useful, and therefore research aimed at exploration and development thereof is necessary. It is the current situation that is being performed vigorously.
The CGT targeted by the present invention is an enzyme of a sphingolipid synthesis system and is known to catalyze a reaction for producing glucosylceramide from UDP-glucose and ceramide (SHAYMAN JA, ABE A. METHODS ENZYMOL 2000). 311: 42-9 “GLUCOSYLCERAMIDE SYNTHASE: ASSAY AND PROPERITES. And MARKS DL, PAUL P, KAMISAKA Y, PAGANO RE.
Disclosure of the invention
An object of this invention is to provide the disease marker useful for the diagnosis of the renal disease accompanying glomerular fibrosis. More specifically, an object of the present invention is to provide a disease marker that specifically reflects renal tissue fibrosis, specifically, renal disease associated with glomerular fibrosis. Another object of the present invention is to provide a method for detecting a renal disease associated with glomerular fibrosis using the disease marker. A further object of the present invention is to provide a method for screening a drug useful for improving or treating a disease associated with glomerular fibrosis using the disease marker, and a drug useful for improving or treating the disease. To do.
The present inventors have conducted intensive research to solve the above-mentioned problems. As a result, the gene of Ceramide Glucosyltransferase (hereinafter also referred to as “CGT”), which is the rate-limiting enzyme of the sphingolipid synthesis system (hereinafter referred to as “CGT gene”). However, it was found to be expressed specifically higher in the kidney tissue of glomerulosclerosis patients with advanced glomerular fibrosis compared to normal subjects. The present inventors have further conducted research based on this finding, thereby suppressing the production of extracellular matrix that causes glomerular sclerosis by the inhibitor of CGT, thereby suppressing fibrosis of renal tissue. (In vitro), the expression of the gene is similarly increased in the renal cortex of a kidney disease model animal (Thy-1 model) with glomerular fibrosis, and the model animal is treated with the CGT inhibitor. It was confirmed that the fibrosis condition (lesion) of the glomerulus was improved (in vivo) by administration. Based on these various findings, the present inventors have shown that CGT is involved in the development of renal diseases accompanied by glomerular fibrosis (fibrosis of kidney tissue, glomerular sclerosis), and the expression level of CGT gene ( Or the amount of CGT produced) or the enzyme activity of CGT becomes an index (marker) for the diagnosis of renal disease accompanied by glomerular fibrosis, and the progression of the renal disease by inhibiting the production of CGT or its action (function, activity) It was convinced that the progression (onset) to glomerulosclerosis could be prevented, and that renal disease with glomerular fibrosis could be improved / treated. The present invention has been developed based on such knowledge.
That is, the present invention is as follows.
Item 1. A polynucleotide having at least 15 contiguous bases in the nucleotide sequence of the Ceramide Glucosyltransferase gene described in SEQ ID NO: 1 or 2 and / or a polynucleotide complementary thereto, which may be labeled with glomerular fibrosis Disease marker for kidney disease.
Item 2. Item 2. The disease marker according to Item 1, which is used as a probe or a primer in detection of renal disease associated with glomerular fibrosis.
Item 3. A disease marker for renal disease associated with glomerular fibrosis, comprising an antibody that recognizes a protein comprising the amino acid sequence of Ceramide Glucosyltransferase described in SEQ ID NO: 3.
Item 4. Item 4. The disease marker according to Item 3, which is used as a probe in detection of renal disease associated with glomerular fibrosis.
Item 5. A method for detecting renal disease associated with glomerular fibrosis, comprising the following steps (a) and (b):
(A) binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA to the disease marker according to Item 1 or 2,
(B) A step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index.
Item 6. The method for detecting a renal disease associated with glomerular fibrosis according to Item 5, further comprising the following step (c) following the steps (a) and (b):
(C) A step of determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the measurement result of (b).
Item 7. The determination of the presence of kidney disease associated with glomerular fibrosis in step (c) is performed by comparing the measurement result obtained for the subject in step (b) with the measurement result obtained in the same manner for a normal subject. Item 7. A step of determining the morbidity of a renal disease associated with glomerular fibrosis according to Item 6, which is performed based on the difference in the binding amount of the polynucleotide.
Item 8. A method for detecting renal disease associated with glomerular fibrosis comprising the following steps (a) and (b):
(A) contacting the renal tissue extract of the subject with the disease marker according to Item 3 or 4,
(B) A step of measuring Ceramide Glucosyltransferase or a partial peptide thereof in a renal tissue extract bound to the disease marker using the disease marker as an index.
Item 9. Item 8. The method for detecting renal disease associated with glomerular fibrosis according to Item 8, further comprising the following step (c) following steps (a) and (b):
(C) A step of determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the measurement result of (b).
Item 10. The determination of the presence of kidney disease associated with glomerular fibrosis in step (c) is performed by comparing the measurement result obtained for the subject in step (b) with the measurement result obtained in the same manner for a normal subject. Item 10. The step of determining the onset of renal disease associated with glomerular fibrosis according to Item 9, which is performed based on the difference in binding amount of Ceramide Glucosyltransferase or a partial peptide thereof.
Item 11. A method for detecting renal disease associated with glomerular fibrosis comprising the following step (a):
(A) A step of measuring the amount of glucosylceramide present in the renal tissue of the subject.
Item 12. The method for detecting a renal disease associated with glomerular fibrosis according to Item 11, comprising the following step (b) following the step (a):
(B) A step of comparing the result of (a) above with the amount of glucosylceramide inherent in the kidney tissue of a normal person, and determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the difference .
Item 13. A method for detecting renal disease associated with glomerular fibrosis comprising the following steps (a) and (b):
(A) a step of contacting a renal tissue extract of a subject with UDP-glucose and ceramide;
(B) The process of measuring the production amount of the glucosylceramide produced based on the process of said (a).
Item 14. Item 14. The method for detecting renal disease associated with glomerular fibrosis according to Item 13, further comprising the following step (c) following the steps (a) and (b):
(C) A step of comparing the measurement result of (b) above with the amount of glucosylceramide produced by a normal person and determining whether the subject is suffering from renal disease with glomerular fibrosis based on the difference.
Item 15. A test agent for renal disease associated with glomerular fibrosis, having UDP-glucose and ceramide, at least one of which is labeled, in the same package or as a separate package.
Item 16. A screening method for a substance that suppresses the expression of Ceramide Glucosyltransferase gene comprising the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of expressing the Ceramide Glucosyltransferase gene;
(B) measuring the expression level of the Ceramide Glucosyltransferase gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that decreases the expression level of the Ceramide Glucosyltransferase gene based on the comparison result of (b).
Item 17. A screening method for a substance that suppresses the production of Ceramide Glucosyltransferase comprising the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of producing Ceramide Glucosyltransferase;
(B) measuring the production amount of Ceramide Glucosyltransferase in a cell contacted with a test substance, and comparing the production amount with the production amount of Ceramide Glucosyltransferase in the control cell not contacted with the test substance;
(C) A step of selecting a test substance that reduces the production amount of Ceramide Glucosyltransferase based on the comparison result of (b).
Item 18. A screening method for a substance that suppresses the function or activity of Ceramide Glucosyltransferase comprising the following steps (a), (b) and (c):
(A) contacting the test substance with Ceramide Glucosyltransferase;
(B) measuring the function or activity of Ceramide Glucosyltransferase resulting from the above step and comparing the function or activity with the function or activity of Ceramide Glucosyltransferase in the case where the test substance is not contacted,
(C) A step of selecting a test substance that causes a decrease in the function or activity of Ceramide Glucosyltransferase based on the comparison result of (b).
Item 19. A screening method for a substance that suppresses the function or activity of Ceramide Glucosyltransferase comprising the following steps (a), (b) and (c):
(A) reacting Ceramide Glucosyltransferase, UDP-glucose, and ceramide in the presence of a test substance;
(B) A step of measuring the amount of glucosylceramide produced as a result of the reaction and comparing the amount of production with the amount of glucosylceramide obtained by performing the reaction (a) in the absence of the test substance. ,
(C) A step of selecting a test substance that causes a decrease in the production amount of glucosylceramide based on the comparison result of (b).
Item 20. Item 20. The screening method according to any one of Items 16 to 19, which is a method for searching for an active ingredient of an agent for improving or treating a renal disease associated with glomerular fibrosis.
Item 21. An agent for improving or treating renal diseases associated with glomerular fibrosis, comprising as an active ingredient a substance that suppresses expression of the Ceramide Glucosyltransferase gene.
Item 22. Item 22. The agent for improving or treating renal disease associated with glomerular fibrosis according to Item 21, wherein the substance that suppresses expression of the Ceramide Glucosyltransferase gene is obtained by the screening method according to Item 16.
Item 23. An agent for improving or treating renal diseases associated with glomerular fibrosis, comprising as an active ingredient a substance that suppresses the generation, function, or activity of Ceramide Glucosyltransferase.
Item 24. Item 20. Improvement or treatment of renal disease associated with glomerular fibrosis according to Item 23, wherein the substance that suppresses production, function, or activity of Ceramide Glucosyltransferase is obtained by the screening method according to any one of Items 17 to 19. Agent.
Item 25. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R1 is an alkyl group having 5 to 15 carbon atoms or a benzyloxy group, and R2 is — (CH₂)₄-Group or-(CH₂)₂-O- (CH₂)₂-Means a group. )
Item 24. An agent for ameliorating or treating a renal disease associated with glomerular fibrosis according to Item 23, which is a compound represented by:
Item 26. Substances that suppress the activity of Ceramide Glucosyltransferase are D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R, 2R) -1-phenyl-2-benzyl Oxycarbonylamino-3-pyrrolidino-1-propanol (PBPP), (1R, 2R) -2-hexanoylamino-1-phenyl-3-pyrrolidino-1-propanol (PHPP), (1R, 2R) -2- Octanoylamino-1-phenyl-3-pyrrolidino-1-propanol (POPP), (1R, 2R) -2-decanoylamino-1-phenyl-3-pyrrolidino-1-propanol (PDPP), (1R, 2R ) -2-palmitoylamino-1-phenyl-3-pyrrolidino-1-propyl Propanol (PPPP), and is at least one selected from the group consisting acceptable salts their pharmaceutically, improving or treating agent for renal diseases accompanied by glomerular fibrosis according to claim 23.
Item 27. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R3 represents an arbitrary functional group, and R4 represents an arbitrary functional group that forms a heterocyclic ring with a nitrogen atom in the ring.)
Item 20. Accompanied by glomerular fibrosis according to Item 23, obtained by performing the screening method according to Item 18 or 19, using the compound represented by the following or a pharmaceutically acceptable salt thereof as a test substance: An agent for improving or treating renal diseases.
Item 28. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R5 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
A compound represented by the general formula:

(In the formula, R6 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
Item 24. An agent for ameliorating or treating a renal disease associated with glomerular fibrosis according to Item 23, which is a compound represented by the formula: or a pharmaceutically acceptable salt thereof.
Item 29. Substances that suppress the activity of Ceramide Glucosyltransferase are N-butyl-deoxynojirimycin, N-nonyl-deoxynojirimycin, N-decyl-deoxynojirimycin, N-butyl-deoxygalactonojirimycin, and pharmaceutically acceptable thereof. Item 24. The agent for improving or treating renal disease associated with glomerular fibrosis according to Item 23, which is at least one selected from the group consisting of:
Item 30. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R7 means any functional group.)
Item 20. Accompanied by glomerular fibrosis according to Item 23, obtained by performing the screening method according to Item 18 or 19, using the compound represented by the following or a pharmaceutically acceptable salt thereof as a test substance: An agent for improving or treating renal diseases.
Item 31. Use of a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of the Ceramide Glucosyltransferase gene described in SEQ ID NO: 1 or 2 and / or a polynucleotide complementary thereto as a disease marker for renal diseases associated with glomerular fibrosis .
Item 32. Use of an antibody recognizing a protein comprising the amino acid sequence of Ceramide Glucosyltransferase described in SEQ ID NO: 3 as a disease marker for renal disease accompanied by glomerular fibrosis.
Item 33. Item 33. The use according to Item 31 or 32, wherein the disease marker is used as a probe or a primer in detecting a disease of renal disease accompanied by glomerular fibrosis.
Item 34. Use of the following steps (a) and (b) in a method for detecting renal disease associated with glomerular fibrosis:
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker according to claim 1 or 2;
(B) A step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index.
Item 35. Use of the following steps (a) and (b) in a method for detecting renal disease associated with glomerular fibrosis:
(A) contacting the renal tissue extract of the subject with the disease marker according to Item 3 or 4,
(B) A step of measuring Ceramide Glucosyltransferase or a partial peptide thereof in a renal tissue extract bound to the disease marker using the disease marker as an index.
Item 36. Use of the following step (i) or steps (ii-a) and (ii-b) in a method for detecting renal disease associated with glomerular fibrosis:
(I) measuring the amount of glucosylceramide present in the renal tissue of the subject, or
(Ii-a) contacting the subject's renal tissue extract with UDP-glucose and ceramide; and
(Ii-b) The process of measuring the production amount of the glucosylceramide produced based on the process of said (ii-a).
Item 37. Use of optionally labeled UDP-glucose or ceramide as a test reagent for renal diseases associated with glomerular fibrosis.
Item 38. Item 20. Use of the screening method according to any one of Items 16 to 19 for obtaining an active ingredient of an agent for improving or treating a renal disease associated with glomerular fibrosis.
Item 39. Use of a substance that suppresses the expression of the Ceramide Glucosyltransferase gene for producing an agent for improving or treating a renal disease associated with glomerular fibrosis.
Item 40. Use of a substance that suppresses the generation, function, or activity of Ceramide Glucosyltransferase for producing an agent for ameliorating or treating a renal disease associated with glomerular fibrosis.
Item 41. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R1 is an alkyl group having 5 to 15 carbon atoms or a benzyloxy group, and R2 is — (CH₂)₄-Group or-(CH₂)₂-O- (CH₂)₂-Means a group. )
Item 41. The use according to Item 40, which is a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof:
Item 42. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R5 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
A compound represented by the general formula:

(In the formula, R6 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
Item 41. The use according to Item 40, which is a compound represented by the formula: or a pharmaceutically acceptable salt thereof.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, in this specification, indications with abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc. are defined in IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. et al. Biochem. , 138: 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.
In this specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.
In the present specification, “polynucleotide” is used to include both RNA and DNA. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes any of total RNA, mRNA, rRNA, and synthetic RNA.
In this specification, “protein” or “(poly) peptide” means not only “protein” or “(poly) peptide” represented by a specific base sequence, but also biological functions equivalent to these. Limits include its homologues, derivatives, and variants. The mutants include naturally occurring allelic mutants, non-naturally occurring mutants, and mutants having amino acid sequences modified by artificial deletion, substitution, addition and insertion. Is done. Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein or (poly) peptide without mutation.
As used herein, the term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or an antibody having an antigen binding property such as a Fab fragment or a fragment generated by a Fab expression library. Some are included.
Further, in the present specification, the “disease marker” is used directly or indirectly for diagnosing the presence / absence of morbidity or the degree of morbidity of renal diseases, particularly renal diseases associated with glomerular fibrosis. Specifically, it specifically recognizes and binds to the Ceramide Glucosyltransferase (CGT) gene or its expression product (protein, CGT) whose expression is specifically increased in the kidney tissue of a renal disease patient with glomerular fibrosis Included are (poly) (oligo) nucleotides or antibodies that can be made. These (poly) (oligo) nucleotides and antibodies are used as probes for detecting and quantifying the gene (CGT gene) and protein (CGT) expressed in vivo based on the above properties, and (oligo) The nucleotide can be effectively used as a primer for amplifying the gene (CGT gene) expressed in vivo.
In the present specification, the term “normal person” means a person who does not suffer from kidney disease, specifically, kidney disease accompanied by glomerular fibrosis. More specifically, it means a person who has been diagnosed as not suffering from renal disease based on the results of a serum creatinine test or measurement of urinary protein, glomerular filtration rate, and the like. In this specification, “normal kidney tissue” means the kidney tissue of the normal person.
In the present invention, as described above, the expression of CGT gene is specifically increased in the renal tissue of a renal disease patient with glomerular fibrosis, and administration of an inhibitor of CGT in vitro and in vivo. This is based on the knowledge that glomerular fibrosis that causes glomerulosclerosis is suppressed or improved. Therefore, the CGT gene and its expression product [protein, (poly) (oligo) peptide] can be effectively used for elucidation and diagnosis of renal diseases, particularly renal diseases associated with glomerular fibrosis. And by such use, useful information and means in medicine and clinical science can be obtained. Further, these CGT genes and their expression products (CGT) and derivatives thereof (for example, antibodies to CGT, etc.) are suitably used for the treatment of the above-mentioned renal diseases and the development of drugs that are effectively used for the treatment. be able to. Furthermore, the presence or absence (qualitative, quantitative) of the expression of the above-mentioned CGT gene or its expression product (CGT) in an individual or kidney tissue, or the detection of a mutation of the gene or its expression failure is detected by glomerular fibrosis. It can be effectively used for elucidation and diagnosis of renal diseases associated with.
Specific examples of renal diseases in the present invention include chronic nephritis with glomerular fibrosis, diabetic nephropathy with glomerular fibrosis, IgA nephropathy with glomerular fibrosis, and genetics with glomerular fibrosis. Renal disease, renal failure with glomerular fibrosis, glomerulosclerosis with glomerular fibrosis, and the like are included. In the present specification, “renal disease accompanied by glomerular fibrosis” can be simply referred to as “glomerular fibrosis”.
Hereinafter, specific uses of the CGT gene, the expression product (CGT) of the gene and their derivatives will be described.
(1) Disease marker for renal disease associated with glomerular fibrosis and its application
(1-1) Polynucleotide
The polynucleotides represented by the nucleotide sequences of SEQ ID NOs: 1 and 2 of the present invention are genes known as human ceramide glucosyltransferase (CGT) genes, respectively (GenBank Acc. No. XM_005555 and D50840). These genes encode Ceramide Glucosyltransferase (CGT), which have the same amino acid sequence, although the nucleotide sequences at 21 and 638 are different.
As described above, the present invention detects whether or not CGT gene expression is increased or not (qualitative / quantitative measurement) to determine whether or not a kidney disease is associated with glomerular fibrosis, and the degree of progression (degree of progression). The degree of progress) can be specifically detected, and is based on the idea that the disease can be diagnosed accurately. That is, the present invention is useful as a tool capable of diagnosing the presence or degree of renal disease associated with glomerular fibrosis in a subject by measuring the presence or degree of the increase in the expression of the gene in the subject. Provide a disease marker.
The disease marker of the present invention is characterized by comprising a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of the CGT gene described in SEQ ID NO: 1 or 2 and / or a polynucleotide complementary thereto.
Here, the complementary polynucleotide (complementary strand, reverse strand) is the base sequence shown in SEQ ID NO: 1 or 2 (full-length sequence of CGT gene), or a base sequence having a length of at least 15 bases continuous in the base sequence. Complementary to the base sequence (partial sequence) (for convenience, these full-length sequences and partial sequences are also referred to as “positive strands”) based on base pair relationships such as A: T and G: C It is intended to mean a related polynucleotide. However, such a complementary strand is not limited to a complete complementary sequence to the target positive strand base sequence, but is complementary to the extent that it can hybridize with the target positive strand under stringent conditions. It may have. It should be noted that the stringent conditions here are taught by a combination of Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although not particularly limited, as stricter hybridization conditions, washing conditions of about “0.5 × SSC, 0.1% SDS, 42 ° C.”, more strictly “0.1 × SSC, 0.1% SDS, 65 ° C.” The conditions under which the normal strand and the complementary strand maintain a hybrid state even when washed under such washing conditions can be mentioned. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the target base strand, and at least 90%, preferably 95% homology with the strand. An example is a chain composed of a base sequence having the same.
In addition, the polynucleotide on the positive strand side has not only a nucleotide sequence (full length sequence) described in SEQ ID NO: 1 or 2 or a partial sequence thereof, but also a complementary relationship to the nucleotide sequence of the complementary strand. The chain | strand which consists of a base sequence in can be included.
Furthermore, the above-mentioned normal-strand polynucleotide and complementary-strand (reverse strand) polynucleotide may each be used as a disease marker in a single-stranded form, or may be used as a disease marker in a double-stranded form.
The disease marker for renal disease associated with glomerular fibrosis according to the present invention may be specifically a polynucleotide comprising the base sequence (full-length sequence) described in SEQ ID NO: 1 or 2 relating to the base sequence of the CGT gene. Further, it may be a polynucleotide consisting of its complementary sequence. In addition, as long as it selectively recognizes (specifically) the CGT gene represented by SEQ ID NO: 1 or 2 or a polynucleotide derived from the gene, a polynucleotide comprising each partial sequence of the full-length sequence or its complementary sequence It may be a nucleotide. In this case, a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence can be mentioned.
Here, “selectively (specifically)” means that, for example, when Northern blotting is used, the CGT gene or a polynucleotide derived therefrom can be specifically detected, and RT-PCR method. Means that the CGT gene or a polynucleotide derived therefrom is specifically produced, but the present invention is not limited thereto, and those skilled in the art will be able to detect the detection product in the Northern blot method or the RT-PCR. Any product that can be judged to be derived from the CGT gene or a polynucleotide derived from the CGT gene may be used.
Such a disease marker of the present invention is based on the base sequence of the CGT gene represented by SEQ ID NO: 1 or 2, for example, primer 3 (http: // www. genome. wi. mit. edu / cgi-bin / primer / primer3. cgi) Or a vector NTI (manufactured by Infomax).
Specifically, a primer or probe candidate sequence obtained by applying the base sequence of the CGT gene to the software of primer3 or vector NTI, or a sequence containing at least the sequence as a part can be used as a primer or probe.
The disease marker used in the present invention is not particularly limited as long as it has a length of at least 15 consecutive bases as described above. Specifically, the length can be appropriately selected and set according to the use of the marker. it can.
In the present invention, detection (diagnosis) of a renal disease associated with glomerular fibrosis is performed by evaluating the presence or level of CGT gene expression in a living tissue of a subject, particularly kidney tissue, or its level (degree of expression increase). In this case, the disease marker of the present invention is used as a primer for specifically recognizing and amplifying the RNA produced by the expression of the gene or a polynucleotide derived therefrom, or specifically the polynucleotide derived therefrom. It can be used as a probe for detection.
When the disease marker is used as a primer in the detection of a renal disease associated with glomerular fibrosis, those having a base length of usually 15 bp to 100 bp, preferably 15 bp to 50 bp, more preferably 15 bp to 35 bp can be exemplified. When used as a detection probe, those having a base length of usually 15 bp to the total number of sequences, preferably 15 bp to 1 kb, more preferably 100 bp to 1 kb can be exemplified.
When the disease marker of the present invention is used as a primer, a preferable primer is a sense strand having a sequence located at positions 780 to 799 of the base sequence of the CGT gene (20 bp): 5′-AAAGTAGGCCTTGGTTCACGG-3 ′ ( SEQ ID NO: 4), for the antisense strand, the antisense strand (20 bp) for the sequence (20 bp) located at positions 1608-1627 of the base sequence of the CGT gene (SEQ ID NO: 1): 5′-CAGATCGCAGTGCCCATGCAA-3 ′ (sequence Number 5) can be exemplified.
When the disease marker of the present invention is used as a probe, a preferred probe is a sequence amplified using the above primer, that is, a sequence (SEQ ID NO: 6) located at positions 780-1627 of the CGT gene or its complementary sequence. Can be illustrated. The disease marker of the present invention (complementary strand for RNA) is a radioisotope (³²P,³³P or the like: RI), a fluorescent substance, a chemiluminescent substance, or an enzyme may be labeled, and such a labeled disease marker can be suitably used as a probe (detection marker).
The disease marker of the present invention is used as a primer or probe according to a conventional method in a known method for specifically recognizing and detecting a specific gene, such as Northern blotting, RT-PCR, in situ hybridization, etc. Thus, it is possible to evaluate the presence or level of CGT gene expression associated with renal disease associated with glomerular fibrosis or the level thereof (degree of increased expression). In addition, according to the kind of detection method to be used, a sample of a subject's kidney tissue may be collected with a biopsy or the like, and a total RNA prepared according to a conventional method may be used as the measurement target sample. Various polynucleotides prepared based on RNA may be used. The kidney tissue to be collected is not limited to the renal cortex and the renal medulla, but is preferably the renal cortex.
The gene expression level of the CGT gene in living tissue can also be detected or quantified using a DNA chip. In this case, the disease marker of the present invention can be used as a probe for the DNA chip. A complex of the above-mentioned probe (disease marker) and labeled DNA or RNA formed by hybridizing such a DNA chip with labeled DNA or labeled RNA prepared based on RNA collected from living tissue Can be measured using the label of the labeled DNA or RNA as an indicator to measure the expression level of the CGT gene in living tissue, thereby evaluating the presence or level of the gene expression (degree of increase in expression). Can do.
The disease marker of the present invention is useful for the detection of renal diseases associated with glomerular fibrosis (diagnosis of presence or absence and degree of morbidity). The detection can be performed by measuring the expression level of the CGT gene in the renal tissue of the subject using the disease marker. Specifically, since the CGT gene expression is specifically induced in renal diseases associated with glomerular fibrosis (indicating increased expression), the detection is based on the expression level of the CGT gene in the renal tissue of the subject. The measurement can be performed using a disease marker, and the presence or absence of an increase in the gene expression level in the subject can be determined from a comparison with the gene expression level of the CGT gene in the normal human kidney tissue. In this case, if the expression level of the gene in the kidney tissue of the subject is 2 times or more, preferably 3 times or more compared to the expression level of the kidney tissue of a normal subject, the subject has renal disease associated with glomerular fibrosis. Suspected of being affected.
(1-2) Antibody
The present invention also provides an antibody capable of specifically recognizing an expression product (protein, CGT) of a CGT gene as a disease marker for renal disease associated with glomerular fibrosis.
CGT can include a protein encoded by the polynucleotide shown in SEQ ID NO: 1 or 2. A specific example of the protein encoded by the polynucleotide shown in SEQ ID NO: 1 or 2 is a protein having the amino acid sequence shown in SEQ ID NO: 3.
Here, the protein shown in SEQ ID NO: 3 is a protein known as human-derived CGT (Ceramide Glucosyltransferase), which is the rate-limiting enzyme of the sphingolipid synthesis system, and its enzymatic properties and acquisition method are also SHAYMAN JA, ABE. A. METHODS ENZYMOL 2000; 311: 42-9 “GLUCOSYLCERAMIDE SYNTHASE: ASSAY AND PROPERITES” and MARKS DL, PAUL P, KAMISAKAY, PAGANO RE. METHODS ENZYMOL. 2000; 311: 50-9. Known as described in "METHODS FOR STUDYING GLUCOSYLCERAMIDE SYNTHESE". However, the recognition that the CGT enzyme is involved in some form in renal diseases associated with glomerular fibrosis was found for the first time in the present invention.
The form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody having CGT as an immunoantigen or a monoclonal antibody thereof, and further, at least a continuous amino acid sequence of the CGT. Antibodies having an antigen binding property to a polypeptide consisting of 8 amino acids are also included in the antibody of the present invention.
Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley and Sons. Section 11.12-11.13). Specifically, when the antibody of the present invention is a polyclonal antibody, CGT expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of the CGT is synthesized according to a conventional method. It is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of monoclonal antibodies, spleen cells and bone marrow obtained by immunizing non-human animals such as mice with CGT expressed and purified in Escherichia coli or the like according to conventional methods or oligopeptides having partial amino acid sequences of these proteins are obtained. It can be obtained from hybridoma cells prepared by cell fusion with tumor cells (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley and Sons. Section 11.11-11.11). .
The protein used for the production of the antibody is DNA cloning, construction of each plasmid, transfection into a host, transformant based on the sequence information (SEQ ID NO: 1 or 2) of the gene provided by the present invention. And the recovery of the protein from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)) and the like. Can be done. Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding CGT in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. The target protein can be recovered from the culture obtained. These CGTs can also be produced by a general chemical synthesis method (peptide synthesis) in accordance with the amino acid sequence information (SEQ ID NO: 3) provided by the present invention.
The CGT of the present invention includes not only a protein having the amino acid sequence shown in SEQ ID NO: 3, but also homologues thereof. As the homologue, a known protein having an amino acid sequence represented by SEQ ID NO: 3 consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3 Mention may be made of proteins having a biological function equivalent to that of and / or having an equivalent activity in immunological activity.
Here, as a protein having a biological function equivalent to the known function of the protein having the amino acid sequence represented by SEQ ID NO: 3, a protein having a function equivalent to CGT in biochemical or pharmacological functions is exemplified. be able to. Examples of a protein having an immunological activity equivalent to that of the above protein include a protein capable of inducing a specific immune reaction in an appropriate animal or its cells and specifically binding to an antibody against CGT. be able to.
The number of amino acid mutations and mutation sites in the protein are not limited as long as the biological function and / or immunological activity is maintained. Indicators that determine how many amino acid residues need to be substituted, inserted or deleted without loss of biological function or immunological activity are computer programs well known to those skilled in the art, For example, it can be found using DNA Star software. For example, the number of mutations is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids. The amino acid to be substituted is not particularly limited as long as the protein obtained after substitution with the amino acid retains the biological function and / or immunological activity of CGT. From the viewpoint of maintaining the structure of the protein, an amino acid having properties similar to the amino acid before substitution, such as residue polarity, charge, solubility, hydrophobicity, hydrophilicity and amphiphilicity, is preferable. For example, Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are amino acids classified as nonpolar amino acids, and Gly, Ser, Thr, Cys, Tyr, Asn and Gln are mutually non-charged amino acids. Asp and Glu are amino acids that are classified as acidic amino acids, and Lys, Arg, and His are amino acids that are classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected from the properties of these amino acids as an index.
The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of CGT. The oligopeptide used for antibody induction does not need to have functional biological activity, but desirably has immunogenic properties similar to CGT. Preferably, a polypeptide having such immunogenic properties and consisting of at least 8 consecutive amino acids in the amino acid sequence of CGT can be exemplified.
Generation of antibodies against such polypeptides can also be performed by enhancing the immunological response using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant; mineral gels such as aluminum hydroxide; and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances; human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
Since the antibody of the present invention has a property of specifically binding to CGT, the protein (CGT) expressed in the renal tissue of the subject is specifically detected and quantified by using the antibody. Can do. That is, the antibody is useful as a probe for detecting the presence or absence of CGT generation in the renal tissue of the subject.
Specifically, a part of a patient's kidney tissue is collected with a biopsy or the like, and a kidney tissue extract (protein (CGT) -containing fraction) containing the above protein (CGT) is prepared therefrom according to a conventional method. For example, in known detection methods such as Western blotting and ELISA, CGT present in the renal tissue extract (ie, renal tissue) can be detected by using the antibody as a probe according to a conventional method.
As described above, since the expression of CGT gene is increased in the kidney tissue of a renal disease patient with glomerular fibrosis, the amount of CGT in the kidney tissue of the subject is larger than the amount of CGT in the normal kidney tissue. It is possible to diagnose the presence or absence of kidney disease associated with glomerular fibrosis. In this case, if it is determined that the amount of the expression product (CGT) of the CGT gene in the kidney tissue of the subject is 2 times or more, preferably 3 times or more compared to the amount of the expression product in the normal kidney tissue, Suspected of having kidney disease with glomerular fibrosis.
(2) Method for detecting renal disease associated with glomerular fibrosis
The present invention provides a method for detecting renal disease associated with glomerular fibrosis.
(2-1) Examples of the detection method include a method of using a disease marker for renal disease accompanied by glomerular fibrosis according to the present invention described above.
Specifically, in the detection method, a part of the kidney tissue of the subject is collected with a biopsy or the like, and the gene expression level of the CGT gene contained therein or a protein (CGT) derived from these genes is detected, or A step of quantifying. Based on the results obtained here, it is possible to determine whether or not the subject suffers from kidney disease accompanied by glomerular fibrosis, and if so, the degree thereof.
The method for detecting renal disease of the present invention comprises the following steps (a) and (b):
(A) contacting the biological sample of the subject with the disease marker of the present invention,
(B) A step of measuring the expression level of the CGT gene in the biological sample or the amount of the gene product (protein, CGT) using the disease marker as an index.
Furthermore, in addition to the above steps (a) and (b), the method for detecting a renal disease of the present invention can further include the following step (c):
(C) A step of determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the measurement result of (b).
In the step (c), for example, the measurement result obtained for the subject in the step (b) is compared with the measurement result obtained in the same manner for a normal person, and the amount of binding of the polynucleotide to the disease marker obtained is different. Can be done based on.
The biological sample used herein is an RNA prepared from a subject's kidney tissue or a polynucleotide further prepared therefrom, a protein prepared from the subject's kidney tissue, or a subject's kidney tissue extract containing a protein-containing fraction, Or kidney tissue. Such RNA, polynucleotide, protein, or kidney tissue extract (protein-containing fraction) can be prepared according to a conventional method from a part of a subject's kidney tissue collected by biopsy or the like.
The diagnostic method of the present invention is specifically performed as follows according to the type of biological sample used as a measurement target.
(I) When using RNA or a polynucleotide prepared therefrom as a biological sample to be measured
In this case, the following steps can be used as the steps (a) and (b).
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker according to claim 1 or 2;
(B) A step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index.
When RNA or a polypeptide prepared therefrom (hereinafter referred to as RNA or the like) is used as the biological sample used for detection, the renal disease detection method of the present invention detects the expression of the CGT gene in the RNA or the like. This is done by measuring the degree. Specifically, using a disease marker (polynucleotide having at least 15 bases continuous in the base sequence of the CGT gene and / or its complementary polynucleotide) comprising the above-described polynucleotide of the present invention as a primer or probe, It can be carried out by performing known methods such as the Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization analysis method on the above RNA and the like.
When using the Northern blot method, the presence or absence of the expression of the CGT gene in RNA or the like and the degree of expression thereof can be detected and measured by using the disease marker of the present invention as a probe. Specifically, the disease marker of the present invention (complementary strand for RNA) is defined as a radioisotope (³²P,³³P, etc .: labeled with RI) or a fluorescent substance, and hybridized with RNA derived from the body tissue of a subject transferred to a nylon membrane or the like according to a conventional method, and then a disease marker (polynucleotide) formed A method of detecting and measuring a double-stranded RNA or the like with a radiation detector (BAS-1800II, manufactured by Fuji Film Co., Ltd.) or a fluorescence detector from a signal derived from a marker (RI or fluorescent substance) of a disease marker Can be illustrated. In addition, using AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharmacia Biotech), a disease marker (polynucleotide) is labeled according to the protocol, and hybridized with RNA derived from the biological tissue of the subject, and then the disease marker A method of detecting and measuring a signal derived from a label with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.
When the RT-PCR method is used, the presence or absence of CGT gene expression in RNA or the like can be detected and measured by using the disease marker (polynucleotide) of the present invention as a primer. Specifically, a pair of primers prepared from the disease marker of the present invention is prepared so that a cDNA can be prepared from RNA derived from a biological tissue of a subject according to a conventional method, and a target CGT gene region can be amplified using this as a template. The disease marker (forward strand) of the present invention that binds to cDNA (-strand), the disease marker of the present invention (complementary strand) that binds to the reverse strand (+ strand) of the above cDNA] and the like are hybridized with conventional methods. A method of detecting the amplified double-stranded DNA obtained by performing the PCR method in accordance with can be exemplified. In addition, the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and a labeled disease marker is used as a probe and hybridized with the marker to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems), and reacted with ABI PRIME 7700 Sequence Detection System (manufactured by Applied Biosystems). You can also.
When DNA chip analysis is used, a DNA chip on which the above-mentioned disease marker of the present invention is attached as a DNA probe (single-stranded or double-stranded polynucleotide) is prepared, and RNA derived from the biological tissue of the subject is prepared on this. The disease marker (polynucleotide) of the present invention, which is hybridized with a cRNA prepared by a conventional method from the above, and separately labeled with a RI or a fluorescent substance on the formed double strand of DNA and cRNA, is labeled probe And a method of detecting by binding. Further, as the DNA chip, a DNA chip capable of detecting and measuring the expression level of the CGT gene can also be used. Examples of DNA chips that can detect and measure the expression level of such genes include Affymetrix's Gene Chip Human Genome U95 A, B, C, D, and E. The detection and measurement of the expression level of the CGT gene in the subject RNA using such a DNA chip will be described in detail in Examples.
Diagnosis of renal disease associated with glomerular fibrosis is preferably performed by the CGT gene in the renal tissue RNA of the subject, which is reflected in the amount of polynucleotide bound to the disease marker of the present invention measured as described above. Can be performed based on the difference between the expression level of CGT gene and the expression level of CGT gene in renal tissue RNA or the like of normal subjects measured in the same manner. Specifically, when the subject's expression level is higher than that of normal subjects, the subject can be suspected of suffering from renal disease associated with glomerular fibrosis. In this case, the subject's CGT gene If the expression level is 2 times or more, preferably 3 times or more higher than that of a normal subject, the subject can be confirmed to have renal disease with higher reliability.
(Ii) When protein is used as a measurement object
When using protein as a measurement object, the detection method of the present invention is carried out by detecting CGT in a biological sample and measuring the amount thereof. Specifically, a renal tissue extract (protein-containing fraction) of a subject is brought into contact with the disease marker of the present invention relating to an antibody (an antibody that recognizes a protein comprising the amino acid sequence shown in SEQ ID NO: 3), and the disease marker Examples include a method of detecting and quantifying CGT or a partial peptide thereof bound to γ by a method such as Western blotting using the disease marker of the present invention as an index.
In this case, the following steps can be used as the steps (a) and (b) described above.
(A) contacting the subject's renal tissue extract with the disease marker according to claim 3 or 4,
(B) A step of measuring Ceramide Glucosyltransferase or a partial peptide thereof in a renal tissue extract bound to the disease marker using the disease marker as an index.
Western blotting uses the disease marker of the present invention as a primary antibody, and then has a binding property to the primary antibody as a secondary antibody.¹²⁵Using an antibody labeled with a radioisotope such as I or a fluorescent substance, a complex of CGT or a partial peptide thereof and a disease marker (primary antibody) is labeled, and then a signal derived from the radioisotope or fluorescent substance Is detected and measured with a radiation measuring instrument (BAS-1800II: manufactured by Fuji Film Co., Ltd.) or a fluorescence detector. In addition, after using the disease marker of the present invention as a primary antibody, ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech) is used for detection according to the protocol, and multibioimager STORM 860 (manufactured by Amersham Pharma Bio). Can also be measured.
Detection of renal disease associated with glomerular fibrosis is preferably performed by the CGT gene in the renal tissue of the subject reflected in the binding amount of Ceramide Glucosyltransferase or a partial peptide thereof to the disease marker of the present invention measured as described above. The amount of the expression product (CGT) can be compared with the amount of the CGT in normal kidney tissue measured in the same manner, and can be performed based on the difference between the two. Specifically, when the amount of CGT in the subject's renal tissue is greater than the amount of CGT in the normal subject, the subject can be suspected of suffering from renal disease associated with glomerular fibrosis. If the amount of CGT in the renal tissue is 2 times or more, preferably 3 times or more higher than that of a normal person, the subject can be confirmed to have kidney disease with higher reliability.
In addition, the comparison of the expression level or the amount of CGT of the CGT gene between the kidney tissue of the subject and the normal kidney tissue can be performed by performing measurements on the subject biological sample and the normal subject biological sample in parallel. . When not performed in parallel, the degree of expression of CGT gene obtained by measurement under uniform measurement conditions using a plurality of (at least 2, preferably 3 or more, more preferably 5 or more) normal kidney tissues Alternatively, the average value or statistical intermediate value of the amount of CGT that is a gene expression product thereof can be used as a CGT gene expression level or CGT amount of a normal person for comparison.
By the way, as described above, CGT is an enzyme of a sphingolipid synthesis system and is known to catalyze a reaction for producing glucosylceramide from UDP-glucose and ceramide (SHAYMAN JA, ABE A. METHODS ENZYMOL 2000; 311). : 42-9 “GLUCOSYLCERAMIDE SYNTHASE: ASSAY AND PROPERITES. And MARKS DL, PAUL P, KAMISAKA Y, PAGANO RE. METHODS ENZYMOL. 2000; The amount of CGT of can also be determined by measuring the CGT enzyme activity in the kidney tissue. Worth can be.
(2-2) Accordingly, the present invention provides a method for detecting the presence or absence of the disease based on the CGT enzyme activity in the kidney tissue of the subject as a second method for detecting a renal disease associated with glomerular fibrosis. provide.
Specifically, the detection method of the present invention can be performed by performing (a) a step of measuring CGT enzyme activity in the renal tissue of a subject.
In this case, specifically, the CGT enzyme activity in the kidney tissue is measured by, for example, (i) measuring the amount of glucosylceramide (endogenous glucosylceramide) inherent in the kidney tissue, or (ii) the kidney tissue extract. Can be measured and evaluated by blending UDP-glucose and ceramide and measuring the amount of glucosylceramide (exogenous glucosylceramide) produced based on the exogenously added UDP-glucose and ceramide. . The method (i) evaluates CGT activity based on the amount of endogenous glucosylceramide produced by the action of CGT in renal tissue with respect to UDP-glucose and ceramide originally present in renal tissue. Is the method. In the method (ii), CGT activity is evaluated based on the amount of exogenous glucosylceramide produced by the action of CGT in renal tissue against UDP-glucose and ceramide exogenously mixed in the renal tissue extract. Is the method. Specifically, the method (ii) can be performed by carrying out the following steps as the step (a):
(A-1) A step of bringing a subject's renal tissue extract into contact with UDP-glucose and ceramide;
(A-2) The process of measuring the production amount of the glucosylceramide produced based on the process of said (a).
The kidney tissue extract is preferably prepared using a method that does not reduce the amount or activity of CGT present in the kidney tissue, the amount of UDP-glucose, ceramide, or glucosylceramide. As such a method, SHAYMAN JA, ABE A.I. METHODS ENZYMOL 2000; 311: 42-9 “GLUCOSYLCERAMIDE SYNTHASE: ASSAY AND PROPERITES”, or MARKS DL, PAUL P, KAMISAKA Y, PAGANO RE. METHODS ENZYMOL. 2000; 311: 50-9. The method described in "METHODS FOR STUDYING GLUCOSYLCERAMIDE SYNTHESE" can be mentioned.
In the method (i), the amount of glucosylceramide present in the kidney tissue is specifically measured by extracting a lipid fraction from the kidney tissue extract obtained according to the above method and subjecting it to HPCL. Can do.
Specifically, the method of (ii) is a mixture obtained by adding UDP-glucose and ceramide at a ratio of about 0.1 pg to 0.1 mg per 0.1 μg to 0.1 g of the above-mentioned kidney tissue extract, respectively. Can be carried out at 37 ° C. for about 1 hour, and after adding a reaction terminator such as a mixture of isopropanol and sodium sulfate, the obtained reaction product can be measured by measuring the amount of glucosylceramide according to the above method. . At this time, a kidney tissue extract to which UDP-glucose and ceramide were not added as a control was also subjected to the same experiment, and the endogenous amount of glucosylceramide obtained (the amount of endogenous glucosylceramide) was subtracted. The amount can be evaluated.
The ceramide used in the above may be any of natural or non-natural origin as long as it is a substrate for CGT, and ceramide derivatives are also included in the category of ceramide in the present invention. Specific examples of ceramide derivatives include N-acyl sphingosine. Here, acyl refers to an acyl group having about 2 to 20 carbon atoms, and preferably includes octanoyl sphingosine having an acyl group having 8 carbon atoms.
In addition, a method in which at least one of UDP-glucose and ceramide to be reacted with a renal tissue extract in the method (ii) is labeled with a radioisotope, a fluorescent reagent, a chemiluminescent reagent, an enzyme reagent, or the like is used. Thus, the amount of exogenous glucosylceramide can be selectively measured and evaluated. Specifically, as an example, renal tissue extract and ceramide and UDP-³After mixing H-glucose and reacting at 37 ° C. for about 1 hour, the reaction was stopped by adding a reaction terminator such as a mixture of isopropanol and sodium sulfate, and t-butyl methyl ether was obtained from the resulting reaction solution. so³A method of extracting H-glucosylceramide and measuring the radioactivity of the labeled glucosylceramide with a liquid scintillation counter can be exemplified.
Thus, the diagnosis of renal diseases associated with glomerular fibrosis according to the present invention can be carried out using UDP-glucose and ceramide, which may be labeled at least one of them, as a test reagent. Therefore, the present invention also provides use (use) of a reagent for examination of renal diseases associated with glomerular fibrosis of UDP-glucose and ceramide, at least one of which may be labeled. The test reagent may be packaged in a state where at least UDP-glucose and ceramide are premixed, or may be packaged separately so that they can be mixed at the time of use. Good. In addition, when labeling UDP-glucose and / or ceramide, the labeling agent to be used is not particularly limited, and a labeling agent used in a conventionally known assay system, for example, a radioisotope, a fluorescent reagent, a chemiluminescent reagent or An enzyme reagent or the like can be used. Further, in the test reagent of the present invention, other components (for example, reaction buffer, reaction terminator, enzyme reaction substrate, pH adjusting agent) can be contained in the same package or as separate packages. .
The detection of a renal disease associated with glomerular fibrosis according to the present invention is preferably performed by further measuring the amount of glucosylceramide inherent in the kidney tissue of a subject obtained by the above-described method or exogenously generated glucosylceramide with those of a normal subject. In contrast, it can be performed based on the difference between the two.
Specifically, in the case of (i), it is preferable to perform the following steps in addition to the step (a):
(B) A step of comparing the result of (a) above with the amount of glucosylceramide inherent in the kidney tissue of a normal person, and determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the difference .
In the case of (ii), it is preferable to perform the following steps in addition to the steps (a-1) and (a-2):
(B) The measurement result of (a-2) is compared with the amount of glucosylceramide produced by a normal person obtained in the same manner, and based on the difference, the subject is affected with renal disease associated with glomerular fibrosis. Judgment process.
In the step (b), when the amount of glucosylceramide inherent in the subject or the amount of exogenously generated glucosylceramide is greater than that of a normal subject, the subject is suspected of having renal disease associated with glomerular fibrosis. be able to. In this case, if the amount of glucosylceramide is about 2 times or more, preferably 3 times or more higher than that of a normal subject, the morbidity of the kidney disease can be recognized for the subject with higher reliability.
(3) Candidate drug screening method
(3-1) Screening method using gene expression level as an index
The present invention provides a method of screening for a substance that suppresses the expression of the CGT gene having the base sequence set forth in SEQ ID NO: 1 or 2.
The screening method of the present invention comprises the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of expressing the CGT gene;
(B) measuring the expression level of the CGT gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance, and (c) the comparison result described above A step of selecting a test substance that reduces the expression level of the CGT gene based on the above.
Examples of cells used for such screening include cells that express the CGT gene and cells derived from the kidney. Specifically, the kidney-derived cells were isolated and prepared from Wistar rats (Charles River) administered with anti-Thy-1 antibody or 5/6 nephrectomized Wistar rats (Charles River). Examples include primary kidney cultured cells. Cell lines such as NRK-49F cells (Normal Rat Kidney Cell: ATCC Number CRL-1570) can also be used. At that time, nephritis accompanied by glomerular fibrosis or cells in a state close thereto can be used, and such cells are expressed as TGF-β (transforming growth factor-β) or PDGF. It can be prepared by treating with a fibrosis-causing factor such as (plated-derived growth factor). Furthermore, the category of cells used in the screening of the present invention includes tissues that are aggregates of cells.
As shown in the Examples, the CGT gene is specifically highly expressed in the renal tissue of a patient suffering from renal disease (glomerulosclerosis) associated with glomerular fibrosis. Moreover, the glomerular fibrosis inhibitory effect was recognized by the administration experiment of the existing CGT inhibitor. From these findings, it is considered that an increase in expression of CGT gene (expression induction) is responsible for kidney disease accompanied by glomerular fibrosis. The screening method of the present invention is based on the above findings, and has an action of alleviating / suppressing renal diseases associated with glomerular fibrosis (exhibiting improvement / therapeutic effect on renal diseases associated with glomerular fibrosis). (Hereinafter also referred to as “candidate substance”) is to be searched using as an index the decrease in the expression level of the CGT gene or the suppression of expression induction.
Examples of candidate substances used here include, but are not limited to, nucleic acids (including CGT gene antisense molecules), peptides, proteins, organic compounds, inorganic compounds, and the like. It can be performed by contacting a test substance that can be a candidate substance with the cells. Contact with cells can also be performed using a sample containing the test substance (test sample). Examples of the test sample include cell extracts, gene library expression products, synthetic low molecular compounds, synthetic peptides, Or the sample containing a natural compound etc. can be illustrated.
In screening, the conditions for bringing the test substance into contact with the cells are not particularly limited, but conditions for not greatly changing the culture conditions (temperature, pH, medium composition, etc.) should be adopted so that the cells do not die. Is preferred.
According to the screening method of the present invention, by searching for a substance that suppresses the expression of the CGT gene, a candidate substance that becomes an active ingredient of a drug for improving or treating a renal disease associated with glomerular fibrosis can be obtained. . In this sense, the screening method of the present invention can be positioned as a method for searching for, selecting and acquiring an active ingredient of a remedy or therapeutic agent for renal diseases associated with glomerular fibrosis from test substances.
In the search (selection) of the candidate substance, specifically, when a cell expressing the CGT gene is used as a cell to be contacted with the test substance, the expression level of the CGT gene in the cell to which the test substance is added is a cell to which the test substance is not added. It can be done with a lower than that level.
More specifically, for example, when using primary kidney cultured cells isolated and prepared from Wistar rats administered with anti-Thy-1 antibody or 5/6 nephrectomized Wistar rats as the above-mentioned cells, the primary to which a test substance is added Comparison of the expression level of the CGT gene in the kidney cultured cell with the expression level of the CGT gene in the primary kidney cultured cell (control cell) to which only the solvent is added without adding the test substance, Can be selected as candidate substances.
Such detection and quantification of the expression of the CGT gene may be performed by using a RNA chip prepared from the cells or a complementary polynucleotide transcribed therefrom, as well as known methods such as Northern blotting and RT-PCR, as well as DNA chips. Etc. can be implemented. For these known methods, see the section (2-1) regarding the method for detecting a renal disease of the present invention. The degree of decrease (decrease) in the expression of CGT gene as an index is 10% or more, preferably 30 compared with the control cell in which the expression level of CGT gene in the cell to which the test substance is added is not compared with that in the control cell to which the test substance is not added. % (Particularly preferably 50% or more) can be exemplified.
The detection and quantification of CGT gene expression is derived from a marker gene using a cell line in which a fusion gene in which a marker gene such as a luciferase gene is connected to a gene region (expression control region) that controls CGT gene expression. It can also be carried out by measuring the activity of the protein. The screening method for a CGT gene expression-suppressing substance of the present invention includes a method for searching for a target substance using the expression level of the marker gene as an index. In this sense, the concept of “Ceramide Glucosyltransferase gene” described in claim 16 includes a fusion gene of an expression control region of a CGT gene and a marker gene.
The marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a conflict reaction. Specifically, in addition to the luciferase gene described above, a chloramphenicol / acetyltransferase gene, a β-glucuronidase gene, a β-galactosidase gene , And reporter genes such as the aequorin gene can also be used. Here, as the expression control region of the CGT gene, for example, about 1 kb upstream of the transcription start site of the gene, preferably about 2 kb can be used. Creation of a fusion gene and measurement of activity derived from a marker gene can be performed by known methods.
By the above screening method, a substance selected from the test substance can be positioned as a CGT gene expression inhibitor. These substances are considered to be able to suppress the onset and progression of renal diseases associated with glomerular fibrosis and the transition to glomerulosclerosis due to the progression of the diseases by suppressing the expression of the CGT gene. Therefore, these substances are potential candidates for drugs that alleviate or suppress (improve or treat) renal diseases associated with glomerular fibrosis or drugs that prevent progression to and onset of glomerulosclerosis.
(3-2) Screening method using CGT production as an index
The present invention provides a method of screening for a substance that suppresses the production of CGT having the amino acid sequence set forth in SEQ ID NO: 3.
The screening method of the present invention comprises the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of producing CGT;
(B) measuring the amount of CGT produced in the cells contacted with the test substance, and comparing the amount of production with the amount of CGT produced in the control cells not contacted with the test substance;
(C) A step of selecting a test substance that reduces the production amount of CGT based on the comparison result.
Regardless of whether endogenous or exogenous, cells used for such screening include cells that express the CGT gene and produce CGT. Specifically, primary kidney cultured cells isolated and prepared from Wistar rats (Charles River) administered with anti-Thy-1 antibody or 5/6 nephrectomized Wistar rats (Charles River), NRK- Examples include cell lines such as 49F cells (Normal Rat Kidney Cell: ATCC Number CRL-1570). As described above, the category of cells used in the screening includes tissues that are aggregates of cells.
As shown in the Examples, the CGT gene is specifically highly expressed in the renal tissue of a patient suffering from renal disease (glomerulosclerosis) associated with glomerular fibrosis. Moreover, the glomerular fibrosis inhibitory effect was recognized by the administration experiment of the existing CGT inhibitor. From these findings, it is considered that an increase in expression of CGT gene (expression induction) is responsible for kidney disease accompanied by glomerular fibrosis. The screening method of the present invention uses the production amount of CGT that increases or decreases in accordance with the expression of the CGT gene as an index (specifically, uses the decrease in the production amount of CGT as an index) to alleviate renal disease associated with glomerular fibrosis. It is intended to search for a substance (candidate substance) having a suppressive action (providing an improvement / therapeutic effect on a renal disease accompanied by glomerular fibrosis).
Examples of candidate substances used here include, but are not limited to, nucleic acids (including CGT gene antisense molecules), peptides, proteins, organic compounds, inorganic compounds, and the like. It can be performed by contacting a test substance that can be a candidate substance with the cells. Contact with cells can also be performed using a sample containing the test substance (test sample). Examples of the test sample include cell extracts, gene library expression products, synthetic low molecular compounds, synthetic peptides, Or the sample containing a natural compound etc. can be illustrated.
In screening, the conditions for bringing the test substance into contact with the cells are not particularly limited, but conditions for not greatly changing the culture conditions (temperature, pH, medium composition, etc.) should be adopted so that the cells do not die. Is preferred.
According to the screening method of the present invention, by searching for a substance that suppresses the production of CGT, a candidate substance that becomes an active ingredient of a drug for improving or treating a renal disease associated with glomerular fibrosis can be obtained. In this sense, the screening method of the present invention can be positioned as a method for searching, selecting, and obtaining an active ingredient of a remedy or therapeutic agent for renal diseases associated with glomerular fibrosis from test substances.
Specifically, the search (selection) of the candidate substance can be performed when the CGT production amount in the cell to which the test substance is added is lower than the CGT production amount in the cell to which the test substance is not added.
More specifically, for example, when using primary kidney cultured cells isolated and prepared from Wistar rats administered with anti-Thy-1 antibody or 5/6 nephrectomized Wistar rats as the above-mentioned cells, the primary to which a test substance is added Compare the amount of CGT produced by cultured kidney cells with the amount of CGT produced by primary kidney cultured cells (control cells) in which only the solvent is added without adding the test substance, and candidates for test substances that cause a decrease in the amount of production when added are candidates Can be sorted as a substance.
Such an amount of CGT production can be measured according to a method such as Western blotting using the disease marker of the present invention relating to an antibody (an antibody recognizing a protein comprising the amino acid sequence shown in SEQ ID NO: 3). For these methods, refer to the section (2-2) regarding the method for detecting a renal disease of the present invention. The degree of reduction (decrease) in the CGT production amount as an index is 10% or more, preferably 30%, in comparison with the production amount in the control cell to which the test substance is not added. As described above, a decrease (decrease) of 50% or more is particularly preferable.
By the above screening method, a substance selected from the test substance can be positioned as a CGT production inhibitor. It is considered that these substances can suppress the onset and progress of renal diseases accompanied by glomerular fibrosis and the transition to glomerulosclerosis due to the progress of the diseases by suppressing the production of CGT. Therefore, these substances are potential candidates for drugs that alleviate or suppress (improve or treat) renal diseases associated with glomerular fibrosis or drugs that prevent progression to and onset of glomerulosclerosis.
CGT is an enzyme of a sphingolipid synthesis system that exists in the living body as described above (SHAYMAN JA, ABE A. METHODS ENZYMOL 2000; 311: 42-9 “GLUCOSYLCERAMIDERAMESYNTHASE: ASSAY AND PROPARTIES., MARKS PAL. KAMISAKA Y, PAGANO RE.METHODS ENZYMOL.2000; 311: 50-9. "METHODS FOR STUDYING GLUCOSYLCERAMIDE SYNTHESE"), which itself has a specific function and activity. The expression level of the gene or the production amount of CGT is used as an index. In which may vary in accordance with the CGT gene expression and CGT production amount. Therefore, a similar screening can be performed function of the CGT (activity) as an indicator.
(3-3) Screening method using CGT (protein) function or activity as an index
The present invention provides a method for screening for a substance that suppresses the function or activity of CGT.
The screening method of the present invention comprises the following steps (a), (b) and (c):
(A) contacting the test substance with CGT;
(B) a step of measuring the function or activity of CGT caused by the above-mentioned step and comparing the activity with the function or activity of CGT when the test substance is not contacted;
(C) A step of selecting a test substance that causes a decrease in CGT function or activity based on the comparison result of (b).
As shown in the Examples, the expression of CGT gene is specifically increased in the renal tissue of a patient suffering from renal disease (glomerulosclerosis) accompanied by glomerular fibrosis. Moreover, the glomerular fibrosis inhibitory effect was recognized by the administration experiment of the existing CGT inhibitor. From these findings, it is considered that an increase in the function or activity of the protein (CGT) encoded by the CGT gene causes kidney disease accompanied by glomerular fibrosis. Based on such findings, the screening method of the present invention has a substance (candidate substance) having an action of alleviating / suppressing the kidney disease (providing an improvement / treatment effect on kidney disease accompanied by glomerular fibrosis), It is intended to search for the decrease or inhibition of CGT function or activity as an index.
That is, according to the screening method of the present invention, by searching for a substance that suppresses the function or activity of CGT, a candidate substance that becomes an active ingredient of an agent for improving or treating a renal disease associated with glomerular fibrosis is obtained. be able to. In that sense, the screening method of the present invention can be positioned as a method for searching, selecting, and acquiring an active ingredient of a remedy or therapeutic agent for renal diseases associated with glomerular fibrosis from test substances. .
Specifically, the screening method uses the enzymatic action of CGT to produce a glucoside ceramide by catalyzing the reaction between UDP-glucose and ceramide, and the following steps (a), (b) and ( c).
(A) reacting CGT, UDP-glucose, and ceramide in the presence of a test substance;
(B) a step of measuring the amount of glucosylceramide produced as a result of the reaction and comparing the amount of production with the amount of glucosylceramide produced by performing the reaction (a) in the absence of the test substance,
(C) A step of selecting a test substance that causes a decrease in the production amount of glucosylceramide based on the comparison result of (b).
The ceramide used here may be any of natural and non-natural origins as long as it is a substrate for CGT, and ceramide derivatives are also included in the category of ceramide in the present invention. Specific examples of ceramide derivatives include N-acyl sphingosine. Here, acyl refers to an acyl group having about 2 to 20 carbon atoms, and preferably includes octanoyl sphingosine having an acyl group having 8 carbon atoms.
Moreover, as a test substance, arbitrary things, such as a nucleic acid, a peptide, protein (including the antibody with respect to CGT), an organic compound, or an inorganic compound, can be used. The origin of acquisition of CGT is not particularly limited, and even a naturally-derived CGT enzyme can be produced in a non-cellular system or various cell systems (bacteria, yeast, insect cells, animal cells) by genetic recombination techniques. It may be a human recombinant CGT enzyme obtained by purification. Moreover, the said CGT, UDP-glucose, and ceramide are commercially available, and a commercially available thing can be used simply. In addition, at least one of UDP-glucose or ceramide may be labeled with an arbitrary label such as a radioisotope, a fluorescent reagent, a chemiluminescent reagent, and the like so that glucosylceramide generated by the reaction is selectively used. Can be detected and quantified. In this case, the proportion of each component to be blended in the reaction system is not particularly limited, but the proportion of about 0.1 pg to 0.1 mg is exemplified as the amount of CGT and the substrate of CGT such as UDP-glucose and ceramide, respectively. be able to.
As a specific screening method, for example, CGT is ceramide, UDP-³Mix with H-glucose and the test substance, and use t-butyl methyl ether.³A method of quantifying the produced glucoside ceramide by extracting H-glucosylceramide and measuring the radioactivity of the labeled glucoside ceramide with a liquid scintillation counter can be mentioned. Specifically, the method can be performed by the following operations;
(1) The above mixture is incubated at 37 ° C. for 1 hour, and 1 ml of isopropanol and 0.8 ml of 50 mg / ml sodium sulfate are added to stop the reaction.
(2) After mixing, 6 ml of t-butyl methyl ether is added and further mixed.
(3) After centrifugation at 1500 × g for 10 minutes, 6 ml of the supernatant is dried with a drive lock heater at 45 ° C., dissolved in 0.5 ml of distilled water, added with 5 ml of ACSII, and radiated with a liquid scintillation counter. Measure activity.
Thus, the test substance selected and acquired is a promising candidate substance for a drug that alleviates or suppresses (improves or treats) renal disease associated with glomerular fibrosis.
Candidate substances selected by the screening method of the present invention are further used in drug efficacy tests, safety tests, and clinical trials for patients with renal diseases associated with glomerular fibrosis. By carrying out these tests, a more practical renal disease ameliorating or therapeutic agent can be obtained. The substance thus selected can be industrially produced by chemical synthesis, biological synthesis (fermentation), or genetic manipulation based on the structural analysis result.
(4) Kidney disease improvement / treatment agent
The present invention provides an agent for improving / treating renal diseases associated with glomerular fibrosis.
The present invention is based on the new finding that CGT gene expression is associated with renal diseases associated with glomerular fibrosis, and that existing inhibitors of CGT have an effect of suppressing glomerular fibrosis. This is based on the idea that a substance that suppresses expression, a substance that suppresses the production of CGT, or a substance that suppresses the function or activity of CGT is effective for the improvement or treatment of the above-mentioned diseases. That is, the agent for improving / treating renal disease of the present invention comprises a substance that suppresses the expression of CGT gene, a substance that suppresses the production of CGT, or a substance that suppresses the function or activity of CGT as an active ingredient.
The active ingredient used in the present invention, that is, a CGT gene expression inhibitor, a CGT production inhibitor, or a CGT function or activity inhibitor, is a CGT gene expression inhibitor, a CGT production inhibitor, or a CGT function, respectively. Or what is necessary is just to have an activity inhibitory effect, and what was not conventionally known besides the conventionally well-known thing is also contained. Those not conventionally known can be selected and obtained from the test substances using the screening method described above. In addition, the effective substance may be produced according to a conventional method based on information on a substance selected using the above screening method.
Here, as a CGT function or activity inhibitor which can be an active ingredient of an agent for improving / treating renal disease, the following general formula

(Wherein R1 represents an alkyl group having 5 to 15 carbon atoms or a benzyloxy group, and R2 represents — (CH₂)₄-Group or-(CH₂)₂-O- (CH₂)₂-Means a group. )
Or a pharmaceutically acceptable salt thereof.
Specific examples include 1-phenyl-2-alkanoylamino-3-morpholino-1-propanol, 1-phenyl-2-alkanoylamino-3-pyrrolidino-1-propanol, and structural analogs thereof (WO00). Chemical Phys. Lipids (1980), 26 (3), 265-78; J. Biochem. 127, 485-491 (2000), etc.). Here, as the structural analog of the former 1-phenyl-2-alkanoylamino-3-morpholino-1-propanol, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol is particularly preferable. (D-PDMP). Further, as a structural analog of the latter 1-phenyl-2-alkanoylamino-3-pyrrolidino-1-propanol, (1R, 2R) -2-hexanoylamino-1-phenyl-3-pyrrolidino-1-propanol (PHPP) ), (1R, 2R) -2-octanoylamino-1-phenyl-3-pyrrolidino-1-propanol (POPP), (1R, 2R) -2-decanoylamino-1-phenyl-3-pyrrolidino-1 -Propanol (PDPP), (1R, 2R) -2-palmitoylamino-1-phenyl-3-pyrrolidino-1-propanol (PPPP) and (1R, 2R) -1-phenyl-2-benzyloxycarbonylamino-3 -Pyrrolidino-1-propanol (PBPP) can be mentioned. In addition, all of these substances can be manufactured based on the above-mentioned literatures, for example.
In addition, CGT functions or activity inhibitors related to these are represented by the following general formula:

(In the formula, R3 represents an arbitrary functional group, and R4 represents an arbitrary functional group that forms a heterocyclic ring with a nitrogen atom in the ring.)
The compound (2) represented by the above or a pharmaceutically acceptable salt thereof as a test substance can be obtained by performing the screening method of the present invention described in detail in the above section (3-3). Examples of the heterocyclic ring include pyrrolidine, imidazolidine, pyrazolidine, piperidine, piperazine, and morpholine, each of which may have a substituent. The above compound includes various isomers including stereoisomers.
Therefore, in the present invention, steps (a), (b) and (c) of the following 1) are carried out using the compound (2) or a pharmaceutically acceptable salt thereof as a test substance, or the following 2): A method of screening for a substance that inhibits the function or activity of Ceramide Glucosyltransferase that performs steps (d), (e), and (f) is included:
1) (a) contacting a test substance with Ceramide Glucosyltransferase;
(B) measuring the function or activity of Ceramide Glucosyltransferase resulting from the above step, and comparing the function or activity with the function or activity of Ceramide Glucosyltransferase when the test substance is not contacted; and
(C) selecting a test substance that causes a decrease in the function or activity of Ceramide Glucosyltransferase based on the comparison result of (b);
2) (d) reacting Ceramide Glucosyltransferase, UDP-glucose, and ceramide in the presence of a test substance;
(E) A step of measuring the amount of glucosylceramide produced as a result of the reaction and comparing the amount of production with the amount of glucosylceramide obtained by performing the reaction (a) in the absence of the test substance. ,and
(F) A step of selecting a test substance that causes a decrease in the production amount of glucosylceramide based on the comparison result of (b).
The present invention also includes an agent for improving or treating renal diseases associated with glomerular fibrosis, which comprises, as an active ingredient, a candidate substance that is selected and obtained from test substances by the screening method described above.
Furthermore, as another specific example of the CGT function or activity inhibitor, the general formula (3):

(In the formula, R5 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
N-alkyl-deoxynojirimycin (NA-DNJ) represented by the general formula (4):

(In the formula, R6 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
And N-alkyl-deoxygalactonojirimycin (NA-GNJ) represented by the following formulas, or structural analogs thereof (WO00 / 62779, WO00 / 62780, Japanese Patent No. 283068, WO00 / 33843, WO01 / 07078). , Biochemical Pharmacology, Vol. 56, pp. 421-430 (1998), Biochemical Pharmacology, Vol. 59, pp. 821-829 (2000), etc.). In addition, the said compound may have the aspect of a pharmaceutically acceptable salt.
Here, examples of the alkyl group represented by R3 include a linear or branched alkyl group having 1 to 20 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms, and more preferably 4 carbon atoms. It is a butyl group. Specific examples of N-alkyl-deoxynojirimycin include N-butyl-deoxynojirimycin (NB-DNJ), N-nonyl-deoxynojirimycin, and N-decyl-deoxynojirimycin, Specific examples of galactonojirimycin include N-butyl-deoxygalactonojirimycin (NB-DGJ). Of these, N-butyl-deoxynojirimycin (NB-DNJ) is particularly preferred. These substances can be produced based on, for example, the above-mentioned literature and US Pat. No. 4,182,767, US Pat. No. 4,639,436, EP0012278, EP0624652, US4262605, US4405714, US5151519, and the like.
In addition, the CGT function or activity inhibitor related to NA-DNJ and NA-GNJ is represented by the following general formula:

(In the formula, R7 means any functional group.)
The compound (5) represented by the formula (5) or a pharmaceutically acceptable salt thereof as a test substance can be obtained by performing the screening method of the present invention described in detail in the above section (3-3). The above compound includes various isomers including stereoisomers.
Therefore, in the present invention, steps (a), (b) and (c) of the following 1) are carried out using the compound (5) or a pharmaceutically acceptable salt thereof as a test substance, or the following 2): A method of screening for a substance that inhibits the function or activity of Ceramide Glucosyltransferase that performs steps (d), (e), and (f) is included:
1) (a) contacting a test substance with Ceramide Glucosyltransferase;
(B) measuring the function or activity of Ceramide Glucosyltransferase resulting from the above step, and comparing the function or activity with the function or activity of Ceramide Glucosyltransferase when the test substance is not contacted; and
(C) selecting a test substance that causes a decrease in the function or activity of Ceramide Glucosyltransferase based on the comparison result of (b);
2) (d) reacting Ceramide Glucosyltransferase, UDP-glucose, and ceramide in the presence of a test substance;
(E) A step of measuring the amount of glucosylceramide produced as a result of the reaction and comparing the amount of production with the amount of glucosylceramide obtained by performing the reaction (a) in the absence of the test substance. ,and
(F) A step of selecting a test substance that causes a decrease in the production amount of glucosylceramide based on the comparison result of (b).
The present invention also includes an agent for improving or treating renal diseases associated with glomerular fibrosis, which comprises, as an active ingredient, a candidate substance that is selected and obtained from test substances by the screening method described above.
The CGT function or activity inhibitory substance, CGT gene expression inhibitory substance, or CGT production inhibitory substance described in detail above is a pharmaceutically acceptable carrier (excipient, extender, binder) as it is or known per se. , And other additives or the like, and can be prepared as a pharmaceutical composition, particularly as a pharmaceutical composition for the purpose of improving or treating renal diseases. The pharmaceutical composition is prepared in a form (oral administration such as tablets, pills, capsules, powders, granules, syrups; parenteral administration such as injections, drops, external preparations, suppositories, etc.), etc. Depending on the dosage, it can be administered orally or parenterally. The dose varies depending on the type of active ingredient, administration route, administration subject or patient's age, weight, symptoms, etc., and cannot be defined unconditionally, but the daily dose is several hundred mg to 2 g, preferably about several tens mg. Can be administered in one to several times a day.
If the above substance is encoded by DNA, it may be considered that the DNA is incorporated into a gene therapy vector to perform gene therapy. Furthermore, when the above substance is an antisense nucleotide for the CGT gene, gene therapy can be performed as it is or by incorporating it into a gene therapy vector. In these cases, the dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but those skilled in the art can appropriately select them.
Example
Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
Example 1: Analysis of gene expression fluctuations in normal and glomerulosclerotic (fibrotic) kidneys
DNA chip analysis was performed using total RNA prepared from 6 kidney tissue samples of human glomerulosclerosis patients and 55 normal human kidney tissue samples, respectively. DNA chip analysis was performed using Affymetrix Gene Chip Human Genome U95A, B, C, D, E. Specifically, analysis includes (1) preparation of cDNA from total RNA, (2) preparation of labeled cRNA from the cDNA, (3) fragmentation of labeled cRNA, (4) fragmented cRNA and probe array And (5) staining of the probe array, (6) scanning of the probe array, and (7) gene expression analysis.
(1) Preparation of cDNA from total RNA
11 μL of a mixed solution containing 10 μg of total RNA prepared according to a conventional method from 6 samples of kidney tissue of human glomerulosclerosis patients and 55 samples of normal human kidney tissue, and 100 pmol of T7- (dT) 24 primer (manufactured by Amersham). The mixture was heated at 70 ° C. for 10 minutes and then cooled on ice. After cooling, 4 μL of 5 × First Strand cDNA Buffer included in SuperScript Choice for cDNA Synthesis (manufactured by Gibco-BRL), 2 μL of 0.1 M DTT included in the kit, and 1 mM of TP included in 1M of TP And heated at 42 ° C. for 2 minutes. Further, Super Script II RT 2 μL (400 U) included in the kit was added, heated at 42 ° C. for 1 hour, and then cooled on ice. After cooling, 91 μL of DEPC treated water (manufactured by Nacalai Tesque), 3 μL of 5 × Second Strand Reaction Buffer included in the kit, 3 μL of 10 mM dNTP Mix, E.E included in the kit. E. coli DNA Ligase 1 μL (10 U), E. coli included in the kit. coli DNA Polymerase I 4 μL (40 U), E. coli contained in the kit. 1 μL (2 U) of E. coli RNase H was added and reacted at 16 ° C. for 2 hours. Next, 2 μL (10 U) of T4 DNA Polymerase contained in the kit was added, and the mixture was reacted at 16 ° C. for 5 minutes, and then 10 μL of 0.5 M EDTA was added. Subsequently, 162 μL of a phenol / chloroform / isoamyl alcohol solution (Nippon Gene) was added and mixed. The mixture was transferred to Phase Lock Gel Light (Eppendorf), which had been centrifuged at 14,000 rpm for 30 seconds in advance, and centrifuged at 14,000 rpm for 2 minutes at room temperature, and then a 145 μL aqueous layer was added. Transferred to Eppendorf tube. The resulting solution was mixed with 72.5 μL of a 7.5 M ammonium acetate solution and 362.5 μL of ethanol, and then centrifuged at 14,000 rpm for 20 minutes at 4 ° C. After centrifugation, the supernatant was discarded, and a DNA pellet containing the prepared cDNA was obtained. Thereafter, 0.5 mL of 80% by volume ethanol was added to the pellet and centrifuged at 14,000 rpm for 5 minutes at 4 ° C., and then the supernatant was discarded. After the same operation as described above, the pellets were dried and dissolved in 12 μL of DEPC-treated water (Nacalai Tesque).
Through the above operation, cDNA was obtained from total RNA prepared from 6 samples of kidney tissue of human glomerulosclerosis patients and 55 samples of normal human kidney tissue.
(2) Preparation of labeled cRNA from cDNA
17 μL of DEPC-treated water (manufactured by Nacalai Tesque), 5 μL of each cDNA solution obtained in (1), 10 × HY Reaction Buffer 4 μL included in BioArray High Yield RNA Transscript Labeling Kit (manufactured by ENZO), included in the kit 10 × Biotin Labeled Ribonucleotides 4 μL, 10 × DTT 4 μL included in the kit, 10 × RNase Inhibitor Mix 4 μL included in the kit, and 20 × T7 RNA Polymerase 2 μL included in the kit were mixed at 37 ° C. for 5 hours. By reacting, labeled cRDNA was prepared. After the reaction, 60 μL of DEPC-treated water was added to the reaction solution, and then the prepared labeled cRNA was purified using RNeasy Mini Kit according to the attached protocol.
(3) Fragmentation of labeled cRNA
In a solution containing 20 μg of each labeled cRNA prepared in (2), 5 × Fragmentation Buffer (200 mM Tris-acetic acid pH 8.1 (manufactured by Sigma), 500 mM potassium acetate (manufactured by Sigma), 150 mM magnesium acetate (manufactured by Sigma) )) 40 μL of the reaction solution prepared by adding 8 μL was heated at 94 ° C. for 35 minutes and then placed on ice. This fragmented each labeled cRNA.
(4) Hybridization of fragmented cRNA and probe array
40 μL of each fragmented cRNA obtained in (3), 4 μL of 5 nM Control Oligo B2 (manufactured by Amersham), 4 μL of 100 × Control cRNA Cocktail, 40 μg of Herring super DNA (manufactured by Promega), manufactured by CoSAB ) 200 μg, 2 × MES Hybridization Buffer (200 mM MES, 2M [Na⁺], 40 mM EDTA, 0.02% Tween 20, pH 6.5 to 6.7: manufactured by Pierce) 200 μL and DEPC-treated water 144 μL were mixed to obtain 400 μL of hybrid cocktail. Each obtained hybrid cocktail was heated at 99 ° C. for 5 minutes, and further heated at 45 ° C. for 5 minutes. After heating, the mixture was centrifuged at 14,000 rpm for 5 minutes at room temperature to obtain a hybrid cocktail supernatant.
On the other hand, a human genome U95 probe array (manufactured by Affymetrix) filled with 1 × MES Hybridization Buffer was rotated at 45 ° C. and 60 rpm for 10 minutes in a hybrid oven, and then 1 × MES Hybridization Buffer was removed to remove the probe array. Was prepared. 200 μL of the hybrid cocktail supernatant obtained above was added to the probe array and rotated in a hybridization oven at 45 ° C. and 60 rpm for 16 hours to obtain a probe array hybridized with the fragmented cRNA.
(5) Staining the probe array
After collecting and removing the hybrid cocktail from each of the hybridized probe arrays obtained in (4), Non-Stringent Wash Buffer (6 × SSPEZ [20 × SSPE (manufactured by Nacalai Tesque)] was diluted, 0.01% Tween 20, 0.005% Antifoam 0-30 (manufactured by Sigma)) was added to fill the probe array with the solution. Next, a fragmented cRNA and a fragmented cRNA in a predetermined position of GeneChip Fluidics Station 400 (manufactured by Affymetrix) set with Non-Stringent Wash Buffer and Stringent Wash Buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) A probe array was attached. After that, according to the staining protocol EuKGE-WS2, the primary staining solution [10 μg / mL Streptavidin Phycoerythrin (SAPE) (Molecular Probe), 2 mg / mL Acetylated BSA, 100 mM MES, 1M NaCl (Ambion), 0.05% Tween20, 0.005% Antifoam 0-30], secondary staining solution [100 μg / mL Goat IgG [manufactured by Sigma], 3 μg / mL Biotinylated Anti-Streptavidin antibody [Vector Laboratories 100 mM / mL, BioSemiconductor AME, mLS / mL, BS / mL 1M NaCl, 0.05% Tween 20, 0.005% Ant Ifoam0-30], the probe array was stained.
(6) Probe array scan, and (7) Gene expression analysis
Each probe array stained in (5) was subjected to HP GeneArray Scanner (manufactured by Affymetrix), and the staining pattern was read. Based on the staining pattern, gene expression on the probe array was analyzed by GeneChip Workstation System (Affymetrix). Next, normalization and comparative analysis of gene expression were performed according to the analysis protocol.
From the above comparative analysis results of gene expression by DNA chip analysis, a probe was selected that showed an expression increase of 3 times or more in the kidney tissue of glomerulosclerosis patients compared to normal kidney tissue.
From the selected probes, probes that were highly expressed in the renal tissue of glomerulosclerosis patients were selected. Specifically, using Abs Call obtained from the results of DNA chip analysis as an index, a probe having 4 or more cases in which Abs Call in the renal tissue of glomerulosclerosis patients is Present was selected.
As a result, the Ceramide Glucosyltransferase (CGT) gene (probe name: 61315_at) was selected. A 3.1-fold increase in expression of the CGT gene was observed in glomerulosclerosis kidneys compared to normal kidneys.
Example 2: Inhibition of fibroblast proteoglycan synthesis by CGT inhibitors
In order to confirm the relationship between CGT gene and glomerulosclerosis or renal disease accompanied by glomerular fibrosis, TGF-β (Transforming growth factor-beta) known as a causative factor of tissue fibrosis such as nephritis [Nature] , 346 (6282), pp. 371-374, 1990, July 26] or PDGF (Platelet-derived growth factor) [Proc Natl Acad Sci USA, 88 (15), pp. 6560-6564, 1991 Aug. 1; J Clin Invest, 92 (6), pp. 2597-2601, 1993 Dec; Am J Pathol, 154 (1), pp. 169-179, 1999 Jan. Nephron, 81 (4), pp .; 428-433, 1999; Kidney Int, 59 (4), pp. 1324-1332, 2001 Apr. It was investigated whether CGT inhibitors inhibit the synthesis of proteoglycans produced depending on
NRK-49F cells (Normal Rat Kidney Cell: ATCC Number CRL-1570) which is a cell line, 1.3 × 10⁴cells / 100 μl / well in 96-well plates, 10% FBS-containing DMEM medium, CO₂The cells were cultured overnight in an incubator. The next day, the culture supernatant was removed by aspiration and replaced with 5% FBS-containing RPMI medium. Existing inhibitors of CGT, TGF-β (final concentration 3 ng / ml) or PDGF (final concentration 10 ng / ml),³⁵S-Na₂SO₄(Final concentration 10 μCi / ml) was added at a total rate of 100 μl / well.
In addition, in the system | strain (TGF-beta dependent proteoglycan production system) which mix | blended TGF-beta as a causative factor of a tissue here, as an existing inhibitor of CGT, D-PDMP (D-threo-1-phenyl- 2-decanolamino-3-morpholino-1-propanol: Wako Pure Chemical Industries (final concentration 3 μM, 30 μM) and NB-DNJ (N-Butyldeoxynojirimycin) (final concentration 3 μM, 30 μM), respectively. Separately and in a system (PDGF-dependent proteoglycan production system) containing PDGF as a causative factor of tissue fibrosis, D-PDMP (final concentrations 1 μM, 10 μM) was used as an existing inhibitor of CGT. Furthermore, after continuing culture | cultivation for about 24 hours, culture supernatants were collect | recovered and SDS-PAGE was performed on the following conditions.
That is, 20 μl of the culture supernatant and 5 μl of sample buffer (× 5: 625 mM Tris-HCl (pH 6.8), 50% glycerol, 5% SDS, 25% 2-mercaptoethanol, 0.05% bromphenol blue) are mixed. Then, after treatment at 100 ° C. for 5 minutes, it was applied to a gel (4% polyacrylamide gel: Tefco) at a rate of 10 μl / well, and electrophoresis was performed at a constant pressure of 18 mA / sheet for 3 hours. The gel was dried with a gel dryer and then exposed to an imaging plate for 24 hours. Thereafter, the radioactivity of the electrophoresed proteoglycan was quantified by BAS2000, and the inhibition rate of TGF-β-dependent proteoglycan production and the inhibition rate of PDGF-dependent proteoglycan production were calculated by the following formulas.

(1) Radioactivity when TGF-β (or PDGF) is added and no CGT inhibitor is added
(2) Radioactivity when TGF-β (or PDGF) is added or CGT inhibitor is added
(3) Radioactivity when TGF-β (or PDGF) is not added and CGT inhibitor is not added
(4) Radioactivity when TGF-β (or PDGF) is not added or CGT inhibitor is added
The resulting inhibition rate of TGF-β-dependent proteoglycan production is shown in FIG. 1, and the inhibition rate of PDGF-dependent proteoglycan production is shown in FIG. As can be seen from these results, both of the CGT inhibitors D-PDMP and NB-DNJ exhibit proteoglycan production inhibitory activity dependent on TGF-β, which is a causative factor of tissue fibrosis, and PDMP is similarly treated with tissue fiber. Since CGT inhibitors showed proteoglycan production inhibitory activity dependent on PDGF, which is the causative factor of glycation, CGT inhibitors have the effect of suppressing the production of extracellular matrix (proteoglycan) by these causative factors, that is, fibrosis inhibitory action It was shown that.
Example 3: Evaluation of changes in CGT expression by Northern blotting
Thy-1 nephritis model prepared by administering anti-Thy-1 monoclonal antibody (OX-7: Biosource international) to normal rats (Wistar rat: Nippon Charles River Co., Ltd.) and Wistar rats (Nihon Charles River Co., Ltd.) Total RNA was isolated from renal cortex collected from rats using Trizol (GibcoBRL). After 10 μg of RNA was electrophoresed and transferred to a nylon membrane, Northern blotting was performed using CGT-specific cDNA as a probe, and the transcription level was quantitatively analyzed using BAS-2000 (Fujifilm). The expression level of the CGT gene in the renal cortex of normal rats and the renal cortex of Thy-1 nephritis model rats was examined. The results are shown in FIG. 3 as “CGT relative expression level” as the expression level in the renal cortex of the Thy-1 nephritis model rat relative to the expression level in the renal cortex of normal rats. As can be seen, it was observed that the expression level of the CGT gene in the renal cortex of the Thy-1 nephritis model rat was increased compared to the expression level of the CGT gene in the renal cortex of the normal rat.
Example 4: Effect of CGT inhibitor on anti-Thy-1 nephritis model
0.4 mg / kg (weight) of an anti-Thy-1 monoclonal antibody (OX-7: Biosource international) diluted with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) in Wistar rats (Nippon Charles River Co., Ltd.) (8 animals) ) At a rate of () from the tail vein (day 0). Thereafter, the body weight of each rat was measured, and based on the measurement results, the rats were randomly divided into an untreated group (4 animals) and an NB-DNJ-administered group (4 animals). In addition, a normal control group (4 animals) not administered with anti-Thy-1 monoclonal antibody was also set.
About 60 minutes after OX-7 administration, administration of NB-DNJ to the NB-DNJ administration group (4 animals) was started, and intraperitoneal administration was performed once a day until day 6 at 5 mg / head (about 20 mg / kg). It was. On day 7, the kidneys were removed from the rats of the NB-DNJ administration group (OX-7 + NB-DNJ administration group), the non-treatment group (OX-7 administration group), and the normal control group, and each left kidney was in 10% The solution was refluxed with a neutral buffered formalin solution, a 2 μm tissue section was prepared, and PAS staining was performed. With respect to glomerular tissue (arbitrary location) of rat kidneys in the NB-DNJ administration group (OX-7 + NB-DNJ administration group), the non-treatment group (OX-7 administration group) and the normal control group, respectively, at a field magnification of 100 times The photographed image is shown in FIG. As can be seen from the comparison between (1) and (2) in FIG. 4, in the Thy-1 nephritis model rat, glomerular basement membrane thickening (fibrosis) indicating the pathogenesis of glomerulosclerosis was observed, but NB- It was confirmed that DNJ administration improved the fibrotic fibrosis condition (lesion) (3).
Therefore, the CGT inhibitor is effective in preventing or / and improving glomerular fibrosis, that is, the CGT inhibitor prevents the onset of renal disease associated with glomerular fibrosis, and / or improves the renal disease. Or it was considered effective for treatment.
Example 5: Production and purification of CGT protein
A gene (SEQ ID NO: 1 or 2) encoding full-length human CGT is expressed as SUPER SCRIPT.^TMA CGT expression vector is prepared by cloning from cDNA library (GIBCO BRL) by a conventional method and subcloning it into pIVEX 2.4a (Roche). CGT (protein) is synthesized by Rapid Translation System (Roche) using the CGT expression vector as a template. Next, the obtained CGT is purified with a nickel column (Amersham Pharmacia) using the histidine tag added to the vector.
Example 6: Screening for CGT inhibitors
Prepare the following reaction mixture.

First, components other than the above CGT are mixed, CGT is added thereto to start the reaction, and the mixture is incubated at 37 ° C. for 1 hour. As a control, the same reaction is performed for a reaction mixture containing only a DMSO solution containing no test substance. The reaction is stopped by adding 1 ml isopropanol, 0.8 ml 50 mg / ml sodium sulfate, and 6 ml t-butyl methyl ether is added and mixed. After centrifugation at 1500 × g for 10 minutes, 6 ml of the supernatant is dried with a drive lock heater at 45 ° C., dissolved in 0.5 ml of distilled water, 5 ml of ACSII is added, and the reaction product is obtained using a liquid scintillation counter. (³Identify the radioactivity of H-labeled Glucosylceramid)³The amount of H-labeled Glucosylceramid produced is measured. When the radioactivity in the presence of the test substance is reduced compared to the radioactivity in the absence of the control test substance, the test substance is used to alleviate or suppress renal disease associated with glomerular fibrosis. Select candidate compounds for (improve, treat).
Industrial applicability
According to the present invention, a gene (CGT gene) whose expression is specifically increased in renal diseases associated with glomerular fibrosis, for example, renal diseases such as chronic nephritis, diabetic nephropathy, IgA nephropathy, hereditary renal disease, etc. It was clarified, and it was suggested that the generation of the gene expression product or the increase in its activity is involved in the onset and progression (progression) of renal diseases associated with glomerular fibrosis. Therefore. Such a CGT gene is useful as a marker gene (probe, primer) used for genetic diagnosis of renal diseases associated with glomerular fibrosis. According to such a marker gene, it is possible to clarify whether or not the disease is a renal disease accompanied by glomerular fibrosis (improvement in diagnosis accuracy), thereby enabling more appropriate treatment. That is, it can be used as a tool for appropriate treatment of renal diseases associated with glomerular fibrosis.
In addition, in the present invention, it has been confirmed that inhibiting the above-mentioned CGT improves the pathology of glomerular fibrosis and prevents its progression, so that the compound that suppresses the production of CGT or the function of CGT or A compound that suppresses the activity seems to be useful as a therapeutic or / and prophylactic agent for the above-mentioned renal diseases associated with glomerular fibrosis. Therefore, according to the CGT gene of the present invention, by using the suppression or decrease in its expression as an index, the therapeutic or prophylactic agent (inhibitor of glomerular fibrosis progression) associated with glomerular fibrosis is effective. Candidate compounds that can be components can be screened and selected. Further, according to the screening method proposed by the present invention using the inhibitory action on CGT activity as an index, it can be an active ingredient of a therapeutic or prophylactic agent (inhibitor of glomerular fibrosis progression) associated with glomerular fibrosis. Candidate compounds can be easily searched and acquired. That is, the present invention is useful for the development of a therapeutic or prophylactic agent for renal diseases associated with glomerular fibrosis (an agent for suppressing the progression of glomerular fibrosis).
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the results of Example 2 in which an existing CGT inhibitor (D-PDMP [D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol]], NB-DNJ [N-butyldeoxyroxycin]: It is a figure which shows the result of having investigated the TGF- (beta) (Transforming growth factor-beta) dependence proteoglycan production inhibition rate about 3 micromol and 30 micromol use.
FIG. 2 is a graph showing the results of examining the PDGF (Platelet-derived growth factor) -dependent proteoglycan production inhibition rate of an existing CGT inhibitor (D-PDMP: 1 μM and 10 μM) in Example 2.
FIG. 3 is a diagram showing the results of comparison of CGT expression levels in the renal cortex for normal rats and Thy-1 nephritis model rats in Example 3.
FIG. 4 shows the results of Example 1 in each of (1) normal control group, (2) no treatment group (OX-7 administration group) and (3) NB-DNJ administration group (OX-7 + NB-DNJ administration group). It is a figure which shows the image which performed PAS dyeing | staining about the glomerular tissue section | slice of the rat kidney, and image | photographed the arbitrary locations of this structure | tissue by 100 times the visual field magnification.

Claims (42)

A polynucleotide having at least 15 contiguous bases in the nucleotide sequence of the Ceramide Glucosyltransferase gene described in SEQ ID NO: 1 or 2 and / or a polynucleotide complementary thereto, which may be labeled with glomerular fibrosis Disease marker for kidney disease. The disease marker according to claim 1, which is used as a probe or a primer in detection of a renal disease associated with glomerular fibrosis. A disease marker for renal disease associated with glomerular fibrosis, comprising an antibody that recognizes a protein comprising the amino acid sequence of Ceramide Glucosyltransferase described in SEQ ID NO: 3. The disease marker according to claim 3, which is used as a probe in the detection of a renal disease associated with glomerular fibrosis. A method for detecting renal disease associated with glomerular fibrosis, comprising the following steps (a) and (b):
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker according to claim 1 or 2;
(B) A step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index. The method for detecting renal disease associated with glomerular fibrosis according to claim 5, further comprising the following step (c) following steps (a) and (b):
(C) A step of determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the measurement result of (b). The determination of the presence of kidney disease associated with glomerular fibrosis in step (c) is performed by comparing the measurement result obtained for the subject in step (b) with the measurement result obtained in the same manner for a normal subject. The step of determining the morbidity of a renal disease associated with glomerular fibrosis according to claim 6, which is performed based on the difference in the amount of binding of the polynucleotide. A method for detecting renal disease associated with glomerular fibrosis comprising the following steps (a) and (b):
(A) contacting the subject's renal tissue extract with the disease marker according to claim 3 or 4,
(B) A step of measuring Ceramide Glucosyltransferase or a partial peptide thereof in a renal tissue extract bound to the disease marker using the disease marker as an index. The method for detecting renal disease associated with glomerular fibrosis according to claim 8, further comprising the following step (c) following steps (a) and (b):
(C) A step of determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the measurement result of (b). The determination of the presence of kidney disease associated with glomerular fibrosis in step (c) is performed by comparing the measurement result obtained for the subject in step (b) with the measurement result obtained in the same manner for a normal subject. The process of determining the morbidity of the renal disease accompanying glomerular fibrosis of Claim 9 performed based on difference in the binding amount of Ceramide Glucosyltransferase or its partial peptide. A method for detecting renal disease associated with glomerular fibrosis comprising the following step (a):
(C) A step of measuring the amount of glucosylceramide present in the renal tissue of the subject. The method for detecting a renal disease associated with glomerular fibrosis according to claim 11, comprising the following step (b) following the step (a):
(D) A step of comparing the result of (a) above with the amount of glucosylceramide inherent in the kidney tissue of a normal person, and determining whether the subject is suffering from renal disease associated with glomerular fibrosis based on the difference . A method for detecting renal disease associated with glomerular fibrosis comprising the following steps (a) and (b):
(A) a step of contacting a renal tissue extract of a subject with UDP-glucose and ceramide;
(B) a step of measuring the amount of glucosylceramide produced based on the step (a), The method for detecting renal disease associated with glomerular fibrosis according to claim 13, further comprising the following step (c) following the steps (a) and (b):
(C) A step of comparing the measurement result of (b) above with the amount of glucosylceramide produced by a normal person and determining whether the subject is suffering from renal disease with glomerular fibrosis based on the difference. A test agent for renal disease associated with glomerular fibrosis, having UDP-glucose and ceramide, at least one of which is labeled, in the same package or as a separate package. A screening method for a substance that suppresses the expression of Ceramide Glucosyltransferase gene comprising the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of expressing the Ceramide Glucosyltransferase gene;
(B) measuring the expression level of the Ceramide Glucosyltransferase gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that decreases the expression level of the Ceramide Glucosyltransferase gene based on the comparison result of (b). A screening method for a substance that suppresses the production of Ceramide Glucosyltransferase comprising the following steps (a), (b) and (c):
(A) contacting the test substance with a cell capable of producing Ceramide Glucosyltransferase;
(B) measuring the production amount of Ceramide Glucosyltransferase in a cell contacted with a test substance, and comparing the production amount with the production amount of Ceramide Glucosyltransferase in the control cell not contacted with the test substance;
(C) A step of selecting a test substance that reduces the production amount of Ceramide Glucosyltransferase based on the comparison result of (b). A screening method for a substance that suppresses the function or activity of Ceramide Glucosyltransferase comprising the following steps (a), (b) and (c):
(A) contacting the test substance with Ceramide Glucosyltransferase;
(B) measuring the function or activity of Ceramide Glucosyltransferase resulting from the above step and comparing the function or activity with the function or activity of Ceramide Glucosyltransferase in the case where the test substance is not contacted,
(C) A step of selecting a test substance that causes a decrease in the function or activity of Ceramide Glucosyltransferase based on the comparison result of (b). A screening method for a substance that suppresses the function or activity of Ceramide Glucosyltransferase comprising the following steps (a), (b) and (c):
(A) reacting Ceramide Glucosyltransferase, UDP-glucose, and ceramide in the presence of a test substance;
(B) A step of measuring the amount of glucosylceramide produced as a result of the reaction and comparing the amount of production with the amount of glucosylceramide obtained by performing the reaction (a) in the absence of the test substance. ,
(C) A step of selecting a test substance that causes a decrease in the production amount of glucosylceramide based on the comparison result of (b). The screening method according to any one of claims 16 to 19, which is a method for searching for an active ingredient of an agent for improving or treating a renal disease associated with glomerular fibrosis. An agent for improving or treating renal diseases associated with glomerular fibrosis, comprising as an active ingredient a substance that suppresses expression of the Ceramide Glucosyltransferase gene. The agent for improving or treating renal disease associated with glomerular fibrosis according to claim 21, wherein the substance that suppresses expression of the Ceramide Glucosyltransferase gene is obtained by the screening method according to claim 16. An agent for improving or treating renal diseases associated with glomerular fibrosis, comprising as an active ingredient a substance that suppresses the generation, function, or activity of Ceramide Glucosyltransferase. 24. Improvement of renal disease associated with glomerular fibrosis according to claim 23, wherein the substance that suppresses production, function or activity of Ceramide Glucosyltransferase is obtained by the screening method according to claim 17. Or therapeutic agent. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(Wherein R1 represents an alkyl group having 5 to 15 carbon atoms or a benzyloxy group, and R2 represents a — (CH ₂ ) ₄ — group or a — (CH ₂ ) ₂ —O— (CH ₂ ) ₂ — group. )
The improvement or therapeutic agent of the renal disease accompanying glomerular fibrosis of Claim 23 which is the compound shown by these, or its pharmaceutically acceptable salt. Substances that suppress the activity of Ceramide Glucosyltransferase are D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), (1R, 2R) -1-phenyl-2-benzyl Oxycarbonylamino-3-pyrrolidino-1-propanol (PBPP), (1R, 2R) -2-hexanoylamino-1-phenyl-3-pyrrolidino-1-propanol (PHPP), (1R, 2R) -2- Octanoylamino-1-phenyl-3-pyrrolidino-1-propanol (POPP), (1R, 2R) -2-decanoylamino-1-phenyl-3-pyrrolidino-1-propanol (PDPP), (1R, 2R ) -2-palmitoylamino-1-phenyl-3-pyrrolidino-1-propyl Propanol (PPPP), and is at least one selected from the group consisting acceptable salts their pharmaceutically, improving or treating agent for renal diseases accompanied by glomerular fibrosis of claim 23. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R3 represents an arbitrary functional group, and R4 represents an arbitrary functional group that forms a heterocyclic ring with a nitrogen atom in the ring.)
The glomerular fibrosis of Claim 23 obtained by performing the screening method in any one of Claim 18 or 19 by making into a test substance the compound shown by these, or its pharmacologically acceptable salt. An agent for improving or treating renal diseases. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R5 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
A compound represented by the general formula:

(In the formula, R6 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
The improvement or therapeutic agent of the renal disease accompanying glomerular fibrosis of Claim 23 which is a compound shown by these, or these pharmacologically acceptable salts. Substances that inhibit the activity of Ceramide Glucosyltransferase are N-butyl-deoxynojirimycin, N-nonyl-deoxynojirimycin, N-decyl-deoxynojirimycin, N-butyl-deoxygalactonojirimycin, and pharmaceutically acceptable thereof. The agent for improving or treating renal disease associated with glomerular fibrosis according to claim 23, wherein the agent is at least one selected from the group consisting of: A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R7 means any functional group.)
The glomerular fibrosis of Claim 23 obtained by performing the screening method in any one of Claim 18 or 19 by making into a test substance the compound shown by these, or its pharmacologically acceptable salt. An agent for improving or treating renal diseases. Use of a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of the Ceramide Glucosyltransferase gene described in SEQ ID NO: 1 or 2 and / or a polynucleotide complementary thereto as a disease marker for renal diseases associated with glomerular fibrosis . Use of an antibody recognizing a protein comprising the amino acid sequence of Ceramide Glucosyltransferase described in SEQ ID NO: 3 as a disease marker for renal disease accompanied by glomerular fibrosis. The use according to claim 31 or 32, wherein the disease marker is used as a probe or primer in the detection of a disease of renal disease accompanied by glomerular fibrosis. Use of the following steps (a) and (b) in a method for detecting renal disease associated with glomerular fibrosis:
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed from the RNA and the disease marker according to claim 1 or 2;
(B) A step of measuring RNA derived from a biological sample bound to the disease marker or a complementary polynucleotide transcribed from the RNA using the disease marker as an index. Use of the following steps (a) and (b) in a method for detecting renal disease associated with glomerular fibrosis:
(A) contacting the subject's renal tissue extract with the disease marker according to claim 3 or 4,
(B) A step of measuring Ceramide Glucosyltransferase or a partial peptide thereof in a renal tissue extract bound to the disease marker using the disease marker as an index. Use of the following step (i) or steps (ii-a) and (ii-b) in a method for detecting renal disease associated with glomerular fibrosis:
(I) a step of measuring the amount of glucosylceramide present in the kidney tissue of the subject, or (ii-a) a step of contacting the kidney tissue extract of the subject with UDP-glucose and ceramide, and (ii-b) The process of measuring the production amount of the glucosylceramide produced based on the process of said (ii-a). Use of optionally labeled UDP-glucose or ceramide as a test reagent for renal diseases associated with glomerular fibrosis. 20. Use of the screening method according to any one of claims 16 to 19 for obtaining an active ingredient of an agent for improving or treating a renal disease associated with glomerular fibrosis. Use of a substance that suppresses the expression of the Ceramide Glucosyltransferase gene for producing an agent for improving or treating a renal disease associated with glomerular fibrosis. Use of a substance that suppresses the generation, function, or activity of Ceramide Glucosyltransferase for producing an agent for ameliorating or treating a renal disease associated with glomerular fibrosis. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(Wherein R1 represents an alkyl group having 5 to 15 carbon atoms or a benzyloxy group, and R2 represents a — (CH ₂ ) ₄ — group or a — (CH ₂ ) ₂ —O— (CH ₂ ) ₂ — group. )
The use according to claim 40, which is a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof. A substance that suppresses the function or activity of Ceramide Glucosyltransferase is represented by the general formula:

(In the formula, R5 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
A compound represented by the general formula:

(In the formula, R6 means a linear or branched alkyl group having 1 to 20 carbon atoms.)
The use according to claim 40, which is a compound represented by the formula: or a pharmaceutically acceptable salt thereof.

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