JP7055429B2 - Prophylactic or therapeutic agent for chronic kidney disease - Google Patents

Description

The present invention relates to a prophylactic or therapeutic agent for chronic kidney disease. More specifically, for the prophylactic or therapeutic agent for chronic kidney disease, the pharmaceutical composition for the prevention or treatment of chronic kidney disease, the screening method for the prophylactic or therapeutic agent for chronic kidney disease, and the prophylactic or therapeutic agent for chronic kidney disease. Regarding screening kits. This application claims priority under Japanese Patent Application No. 2017-208301 filed in Japan on October 27, 2017 and Japanese Patent Application No. 2018-072747 filed in Japan on April 4, 2018. And I will use those contents here.

Disorders of glomerular epithelial cells are a common condition in chronic kidney disease. Dialysis is required as chronic kidney disease progresses. Currently, the number of patients requiring dialysis is increasing, especially in Asia, which has become a major social problem (see, for example, Non-Patent Document 1).

Treatment to reduce proteinuria is needed to control the progression of chronic kidney disease. However, despite the low patient satisfaction with this treatment, no new drug has been developed for a long time.

Liyanage T., et al., Worldwide access to treatment for end-stage kidney disease: a systematic review., Lancet, 385 (9981), 1975-1982, 2015.

Against this background, it is an object of the present invention to provide a technique for preventing or treating chronic kidney disease.

［式（１）中、Ｒ^１は置換されていてもよい炭素数２～１６の直鎖状又は分岐鎖状のアルキル基を表す。］
［３］［１］又は［２］に記載の予防又は治療剤と、薬学的に許容される担体とを含有する、慢性腎臓病の予防又は治療用医薬組成物。
［４］慢性腎臓病の予防又は治療剤のスクリーニング方法であって、被験物質の存在下で、ネフリンタンパク質発現細胞を培養する工程と、前記細胞に抗ネフリン抗体を反応させる工程と、前記細胞のＦ－アクチンを検出する工程と、を備え、検出された前記Ｆ－アクチンの量が、前記被験物質の非存在下における前記Ｆ－アクチンの量と比較して増加することが、前記被験物質が慢性腎臓病の予防又は治療剤であることを示す、スクリーニング方法。
［５］慢性腎臓病の予防又は治療剤のスクリーニング方法であって、ネフリンタンパク質発現細胞に、抗ネフリン抗体を反応させる工程と、抗ネフリン抗体を反応させた前記細胞の培地に被験物質を添加する工程と、前記細胞のＦ－アクチンを検出する工程と、を備え、検出された前記Ｆ－アクチンの量が、前記被験物質の非存在下における前記Ｆ－アクチンの量と比較して増加することが、前記被験物質が慢性腎臓病の予防又は治療剤であることを示す、スクリーニング方法。
［６］慢性腎臓病の予防又は治療剤のスクリーニング方法であって、被験物質の存在下で、ネフリンタンパク質発現細胞を培養する工程と、前記細胞に抗ネフリン抗体を反応させる工程と、前記細胞におけるネフリンタンパク質のリン酸化を検出する工程であって、前記リン酸化が、マウスネフリンタンパク質の第１２３２番目のチロシン残基に相当するチロシン残基のリン酸化である工程と、を備え、検出された前記リン酸化が、前記被験物質の非存在下における前記リン酸化と比較して低下することが、前記被験物質が慢性腎臓病の予防又は治療剤であることを示す、スクリーニング方法。
［７］慢性腎臓病の予防又は治療剤のスクリーニング方法であって、ネフリンタンパク質発現細胞に、抗ネフリン抗体を反応させる工程と、抗ネフリン抗体を反応させた前記細胞の培地に被験物質を添加する工程と、前記細胞におけるネフリンタンパク質のリン酸化を検出する工程であって、前記リン酸化が、マウスネフリンタンパク質の第１２３２番目のチロシン残基に相当するチロシン残基のリン酸化である工程と、を備え、検出された前記リン酸化が、前記被験物質の非存在下における前記リン酸化と比較して低下することが、前記被験物質が慢性腎臓病の予防又は治療剤であることを示す、スクリーニング方法。
［８］慢性腎臓病の予防又は治療剤のスクリーニング方法であって、被験物質の存在下で細胞を培養する工程と、前記細胞におけるガングリオシドＧＭ３又はガングリオシドＧＭ３合成酵素遺伝子の発現量を測定する工程と、を備え、ガングリオシドＧＭ３又はガングリオシドＧＭ３合成酵素遺伝子の発現量が、前記被験物質の非存在下における発現量と比較して増加することが、前記被験物質が慢性腎臓病の予防又は治療剤であることを示す、スクリーニング方法。
［９］慢性腎臓病の予防又は治療剤のスクリーニング方法であって、被験物質の投与下で慢性腎臓病を発症する非ヒト動物モデルを飼育する工程と、前記非ヒト動物モデルの腎機能を評価する工程と、を備え、被験物質の投与下の前記非ヒト動物モデルの腎機能が、前記被験物質の非投与下の前記非ヒト動物モデルの腎機能と比較して向上したことが、前記被験物質が慢性腎臓病の予防又は治療剤であることを示す、スクリーニング方法。
［１０］ネフリンタンパク質発現細胞と、抗ネフリン抗体とを備える、慢性腎臓病の予防又は治療剤のスクリーニングキット。
［１１］ファロイジンを更に備える、［１０］に記載のスクリーニングキット。
［１２］抗リン酸化ネフリンタンパク質抗体を更に備え、前記抗体が、マウスネフリンタンパク質の第１２３２番目のチロシン残基に相当するチロシン残基のリン酸化を検出する抗体である、［１０］又は［１１］に記載のスクリーニングキット。
［１３］ガングリオシドＧＭ３に対する特異的結合物質、ガングリオシドＧＭ３合成酵素に対する特異的結合物質、ガングリオシドＧＭ３合成酵素遺伝子のｃＤＮＡを特異的に増幅するプライマーセット、又はガングリオシドＧＭ３合成酵素遺伝子のｍＲＮＡに特異的にハイブリダイズするプローブ、を備える、慢性腎臓病の予防又は治療剤のスクリーニングキット。The present invention includes the following aspects.
[1] A prophylactic or therapeutic agent for chronic kidney disease containing a substance that increases the expression level of ganglioside GM3 as an active ingredient.
[2] The prophylactic or therapeutic agent according to [1], wherein the substance that increases the expression level of ganglioside GM3 is a compound represented by the following formula (1) or a derivative thereof.

[In formula (1), R ¹ represents a linear or branched alkyl group having 2 to 16 carbon atoms which may be substituted. ]
[3] A pharmaceutical composition for preventing or treating chronic kidney disease, which comprises the prophylactic or therapeutic agent according to [1] or [2] and a pharmaceutically acceptable carrier.
[4] A method for screening a preventive or therapeutic agent for chronic kidney disease, which comprises a step of culturing nephrin protein-expressing cells in the presence of a test substance, a step of reacting the cells with an anti-nephrin antibody, and a step of reacting the cells with an anti-nephrin antibody. The test substance comprises a step of detecting F-actin, wherein the detected amount of the F-actin is increased as compared with the amount of the F-actin in the absence of the test substance. A screening method that indicates that it is a prophylactic or therapeutic agent for chronic kidney disease.
[5] A method for screening a preventive or therapeutic agent for chronic kidney disease, in which a step of reacting a nephrin protein-expressing cell with an anti-nephrin antibody and a test substance are added to the medium of the cell reacted with the anti-nephrin antibody. The step comprises a step of detecting F-actin in the cells, and the amount of the detected F-actin is increased as compared with the amount of the F-actin in the absence of the test substance. However, a screening method showing that the test substance is a prophylactic or therapeutic agent for chronic kidney disease.
[6] A method for screening a prophylactic or therapeutic agent for chronic kidney disease, which comprises a step of culturing a nephrin protein-expressing cell in the presence of a test substance, a step of reacting the cell with an anti-nephrin antibody, and a step of reacting the cell with an anti-nephrin antibody. The step of detecting the phosphorylation of a nephrin protein, wherein the phosphorylation is a phosphorylation of a tyrosine residue corresponding to the 1232nd tyrosine residue of the mouse nephrin protein. A screening method in which a decrease in phosphorylation as compared to the phosphorylation in the absence of the test substance indicates that the test substance is a prophylactic or therapeutic agent for chronic kidney disease.
[7] A method for screening a prophylactic or therapeutic agent for chronic kidney disease, in which a step of reacting a nephrin protein-expressing cell with an anti-nephrin antibody and a test substance are added to the medium of the cell reacted with the anti-nephrin antibody. The step of detecting the phosphorylation of the nephrin protein in the cell, wherein the phosphorylation is the phosphorylation of the tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein. A screening method, wherein the detected phosphorylation is reduced as compared with the phosphorylation in the absence of the test substance, indicating that the test substance is a prophylactic or therapeutic agent for chronic kidney disease. ..
[8] A method for screening a prophylactic or therapeutic agent for chronic kidney disease, which comprises a step of culturing cells in the presence of a test substance and a step of measuring the expression level of a ganglioside GM3 or ganglioside GM3 synthase gene in the cells. The test substance is a prophylactic or therapeutic agent for chronic kidney disease, wherein the expression level of the ganglioside GM3 or the ganglioside GM3 synthase gene is increased as compared with the expression level in the absence of the test substance. A screening method that shows that.
[9] A method for screening a preventive or therapeutic agent for chronic kidney disease, in which a step of breeding a non-human animal model that develops chronic kidney disease under administration of a test substance and evaluation of the renal function of the non-human animal model are evaluated. The test is that the renal function of the non-human animal model under the administration of the test substance is improved as compared with the renal function of the non-human animal model under the non-administration of the test substance. A screening method that indicates that the substance is a prophylactic or therapeutic agent for chronic kidney disease.
[10] A screening kit for a preventive or therapeutic agent for chronic kidney disease, comprising nephrin protein-expressing cells and an anti-nephrin antibody.
[11] The screening kit according to [10], further comprising phalloidin.
[12] Further comprising an anti-phosphorylated nephrin protein antibody, wherein the antibody is an antibody that detects phosphorylation of a tyrosine residue corresponding to the 1232nd tyrosine residue of mouse nephrin protein, [10] or [11]. ] The screening kit described in.
[13] A specific binding substance for ganglioside GM3, a specific binding substance for ganglioside GM3 synthase, a primer set that specifically amplifies the cDNA of the ganglioside GM3 synthase gene, or a specific hybrid to the mRNA of the ganglioside GM3 synthase gene. A screening kit for the prevention or treatment of chronic kidney disease, comprising a probe for soybean.

According to the present invention, it is possible to provide a technique for preventing or treating chronic kidney disease.

6 is a fluorescence micrograph in which a nephrin protein and a ganglioside GM3 (hereinafter, may be referred to as “GM3”) are detected in Experimental Example 1. It is a fluorescence micrograph in which nephrin protein and GM3 were detected in Experimental Example 2. It is a photograph which shows the result of the immunothin layer chromatography in Experimental Example 3. 6 is a fluorescence micrograph showing the results of detecting GM3 and F-actin in Experimental Example 4. 6 is a fluorescence micrograph showing the results of detecting GM3 and F-actin in Experimental Example 5. It is a photograph which shows the analysis result by the immunoprecipitation-Western blotting method in Experimental Example 6. It is a photograph which shows the analysis result by the immunoprecipitation-Western blotting method in Experimental Example 7. It is a photograph which shows the analysis result by the Western blotting method in Experimental Example 8. 6 is a fluorescence micrograph showing the result of detecting F-actin in Experimental Example 9. 6 is a fluorescence micrograph showing the result of detecting GM3 in Experimental Example 9. It is a graph which shows the change of the urinary albumin (mg / g, creatinine) of a mouse calculated in Experimental Example 10. (A) is a graph showing the measurement results of the serum albumin amount of mice in Experimental Example 10. (B) is a graph showing the measurement results of the serum creatinine amount of mice in Experimental Example 10. (A) to (g) are micrographs showing the results of PAS staining of mouse kidney tissue sections in Experimental Example 10. It is a graph which shows the result of quantifying the obstacle induction amount based on the result of FIGS. 13 (a) to 13 (g). (A) to (f) are micrographs showing the results of PAS staining of mouse kidney tissue sections and immunostaining of p57 in Experimental Example 10. 3 is a graph showing the results of measuring the number of p57-positive cells per unit area based on the results of FIGS. 15 (a) to 15 (f). It is a typical fluorescence micrograph in which GM3 and nephrin protein were detected in Experimental Example 11. It is a typical electron micrograph in Experimental Example 11. It is a typical fluorescence micrograph in which GM3 was detected in Experimental Example 12. It is a typical fluorescence micrograph in which GM3 was detected in Experimental Example 13.

[Preventive or therapeutic agent for chronic kidney disease]
In one embodiment, the present invention provides a prophylactic or therapeutic agent for chronic kidney disease, which comprises a substance that increases the expression level of ganglioside GM3 (the abundance of ganglioside GM3) as an active ingredient.

As will be described later in Examples, the inventors increase the expression level of ganglioside GM3 in cells from the results of studies using model mice that develop chronic kidney disease and the results of studies using a chronic kidney disease cell model. It has been clarified that the substance can be used as a preventive or therapeutic agent for chronic kidney disease. Here, as the cells, nephrin protein-expressing cells are suitable.

Chronic kidney disease targeted by the prophylactic or therapeutic agent of the present embodiment includes chronic kidney disease due to damage to podocytes of glomerules, and more specifically, microvariant nephrotic syndrome (MCNS). , Nest-like segmental glomerular sclerosis (FSGS), diabetic nephropathy and the like.

The substance that increases the expression level of ganglioside GM3 may be a substance that increases the expression level of the ganglioside GM3 synthase gene in cells.

As the prophylactic or therapeutic agent of the present embodiment, any substance that increases the expression level of ganglioside GM3 in cells can be used without particular limitation, but a more specific example is represented by the following formula (1). Compounds or derivatives thereof.

［式（１）中、Ｒ^１は置換されていてもよい炭素数２～１６の直鎖状又は分岐鎖状のアルキル基を表す。］

[In formula (1), R ¹ represents a linear or branched alkyl group having 2 to 16 carbon atoms which may be substituted. ]

As will be described later in the examples, the inventors have clarified that the compound represented by the above formula (1) can be used as a prophylactic or therapeutic agent for chronic kidney disease. From the results of the examples, it is considered that the prophylactic or therapeutic agent for chronic kidney disease of the present embodiment enhances the expression of ganglioside GM3.

In the formula (1), the carbon number of the linear alkyl group which may be substituted, which is represented by ^R1 , is 2 to 16 as long as it exerts a function as a preventive or therapeutic agent for chronic kidney disease. It may be 2 to 10, or it may be 2 to 5. More specific linear alkyl groups include, but are not limited to, ethyl group, propyl group, butyl group, pentyl group, heptyl group, octyl group, nonyl group, decyl group and the like.

The carbon number of the substituted chain-like alkyl group represented by ^R1 may be 2 to 16 as long as it exerts a function as a preventive or therapeutic agent for chronic kidney disease. It may be up to 10 or 2 to 5. More specific branched alkyl groups include, for example, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a 2-methylheptyl group, a 3-methylheptyl group, a 4-methylheptyl group and the like. However, it is not limited to these.

Examples of the substituent of the group represented by R ¹ include an amino group and a halogen atom. Examples of the halogen atom include fluorine, chlorine, bromine and iodine.

Examples of the derivative of the compound represented by the above formula (1) include an ester of the compound represented by the above formula (1), an amide of the compound represented by the above formula (1), and the like.

Examples of the more specific compound represented by the above formula (1) include valproic acid, butyric acid, propionic acid and the like. The following formula (2) shows the chemical formula of valproic acid, the following formula (3) shows the chemical formula of butyric acid, and the following formula (4) shows the chemical formula of propionic acid.

[Pharmaceutical composition]
The above-mentioned preventive or therapeutic agent for chronic kidney disease is preferably formulated as a pharmaceutical composition. That is, in one embodiment, the present invention provides a pharmaceutical composition for preventing or treating chronic kidney disease, which comprises the above-mentioned preventive or therapeutic agent for chronic kidney disease and a pharmaceutically acceptable carrier.

The pharmaceutical composition may be in a dosage form used orally or in a dosage form used parenterally. Dosage forms used orally include, for example, tablets, capsules, elixirs, microcapsules and the like. Dosage forms used parenterally include, for example, injections, inhalants, suppositories, patches and the like.

Examples of the pharmaceutically acceptable carrier include solvents such as sterile water and physiological saline; binders such as gelatin, cornstarch, tragant gum and arabic rubber, excipients such as crystalline cellulose; and swelling agents such as alginic acid. Can be mentioned.

The pharmaceutical composition may further contain additives. As additives, lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; flavoring agents such as peppermint and red mono oil; stabilizers of benzyl alcohol and phenol; buffers such as phosphates and sodium acetate. Agents; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.

The pharmaceutical composition can be formulated by appropriately combining the above carriers and additives and mixing them in a generally accepted unit dose form required for pharmaceutical practice.

Administration to patients is performed, for example, by intranasal, transbronchial, intramuscular, percutaneous, or oral methods known to those skilled in the art, in addition to intraarterial injection, intravenous injection, subcutaneous injection, and the like. sell. The dose varies depending on the weight and age of the patient, the symptoms of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.

The dose of the pharmaceutical composition for the prevention or treatment of chronic kidney disease varies depending on the type of active ingredient, the patient's symptoms, etc., but in the case of oral administration, it is generally per day for adults (assuming a weight of 60 kg). It is considered appropriate to administer about 0.1 to 5000 mg of the active ingredient (preventive or therapeutic agent for chronic kidney disease) once a day or in several divided doses.

When administered parenterally, the dose varies depending on the type of active ingredient, patient's symptoms, administration method, etc., but when systemic administration is performed, it is generally for adults (assuming a body weight of 60 kg) per day. It is considered appropriate to administer about 0.1 to 500 mg of the active ingredient (preventive or therapeutic agent for chronic kidney disease) once a day or in several divided doses. In the case of local administration, generally, in adults (assuming a body weight of 60 kg), about 0.001 to 10 mg of the active ingredient (preventive or therapeutic agent for chronic kidney disease) per day is applied once a day or in a number. It may be appropriate to administer in divided doses.

[Screening method for preventive or therapeutic agents for chronic kidney disease]
(First Embodiment)
In one embodiment, the present invention is a method for screening a prophylactic or therapeutic agent for chronic kidney disease, in which a step of culturing nephrin protein-expressing cells in the presence of a test substance and reacting the cells with an anti-nephrin antibody are allowed. The step comprises a step of detecting F-actin in the cells, and the amount of the detected F-actin is increased as compared with the amount of the F-actin in the absence of the test substance. Provided a screening method showing that the test substance is a prophylactic or therapeutic agent for chronic kidney disease.

In the screening method of the present embodiment, the test substance is not particularly limited, and examples thereof include a natural compound library, a synthetic compound library, an existing drug library, and a metabolite library.

As the nephrin protein-expressing cell, a cell that originally expresses nephrin protein, such as a podocyte (podocyte) existing in the glomerular region of the kidney, may be used. A cell expressing a nephrin protein by introducing an expression vector of the nephrin protein into a cell in which the amount of the nephrin protein is small may be used. Here, the expression vector is not particularly limited, and a plasmid vector, a virus vector, or the like can be used.

When a cell originally expressing nephrin protein is used as the nephrin protein-expressing cell, the origin of the nephrin protein-expressing cell is not particularly limited, but among them, a cell derived from a species to which a prophylactic or therapeutic agent for chronic kidney disease is administered. It is preferable that the cells are derived from human or mouse.

When an expression vector for a nephrin protein is introduced into a cell, the nephrin protein can be used without particular limitation as long as it is a mammalian-derived nephrin protein. Among them, the nephrin protein derived from the species to which the prophylactic or therapeutic agent for chronic kidney disease is to be administered is preferable, and the nephrin protein derived from human or mouse is preferable.

The accession number of the human nephrin protein UniProtKD is O60500, and the accession number of the mouse nephrin protein UniProtKD is Q9QZS7.

The nephrin protein-expressing cells are preferably kidney-derived cells. Among them, cells derived from the species to which the prophylactic or therapeutic agent for chronic kidney disease is to be administered are preferable, and cells derived from humans or mice are preferable. Specific examples of the cells include cells expressing nephrin protein in HEK293 cells, which is the human fetal kidney cell line used in the examples, and podocytes originally expressing nephrin protein. Not limited.

As the anti-nephrin antibody that reacts with the nephrin protein-expressing cells, an antibody that specifically reacts with the nephrin protein expressed by the nephrin protein-expressing cells can be used, and a polyclonal antibody is preferable.

For example, when a nephrin protein-expressing cell expresses a mouse nephrin protein, it is preferable to use an anti-mouse nephrin antibody. Among them, a polyclonal antibody produced by immunizing a rabbit with a DNA encoding a mouse nephrin protein by a gene immunization method is preferable.

The antibody may be an antibody fragment. Examples of the antibody fragment include F (ab') ₂ , Fab', Fab, Fv, scFv and the like.

The anti-nephrin antibody may be a specific binding substance other than the antibody as long as it has specific binding property to the nephrin protein. Examples of the specific binding substance other than the antibody include nucleic acid aptamers and peptide aptamers.

As described below in the Examples, the inventors have developed a chronic kidney disease cell model in which an anti-nephrin antibody is reacted with a nephrin protein-expressing cell. Then, it was clarified that when the anti-nephrin antibody was reacted with the nephrin protein-expressing cells, the F-actin of the cells decreased. Therefore, by using this chronic kidney disease cell model, a prophylactic or therapeutic agent for chronic kidney disease can be screened.

Here, the nephrin protein-expressing cells used in the chronic kidney disease cell model are the same as those described above, and may be cells that originally express nephrin protein, and may or may not originally express nephrin protein. The cell may be a cell in which the expression vector of the nephrin protein is introduced into a cell in which the amount is small to express the nephrin protein.

More specifically, first, a step of culturing nephrin protein-expressing cells in the presence of a test substance is carried out, and then a step of reacting the above cells with an anti-nephrin antibody is carried out. Then, a step of detecting F-actin in the above cells is carried out. F-actin is a polymer in which a large number of G-actin are polymerized.

The method for detecting F-actin is not particularly limited, and examples thereof include a method for detecting the label after reacting with the labeled phalloidin. Examples of the label include a fluorescent label. In this case, the detection of F-actin can be carried out by observation with a fluorescent microscope.

Then, when the detected amount of F-actin is increased as compared with the amount of F-actin in the absence of the test substance, it is determined that the test substance is a preventive or therapeutic agent for chronic kidney disease. be able to.

(Second Embodiment)
In one embodiment, the present invention is a method for screening a prophylactic or therapeutic agent for chronic kidney disease, in which a step of reacting a nephrin protein-expressing cell with an anti-nephrin antibody and a medium of the cell reacted with the anti-nephrin antibody. The step of adding the test substance to the cell and the step of detecting the F-actin of the cell are provided, and the amount of the detected F-actin is the amount of the F-actin under non-administration of the test substance. A comparative increase provides a screening method indicating that the test substance is a prophylactic or therapeutic agent for chronic kidney disease.

The screening method of the present embodiment is mainly different from the screening method of the first embodiment in the timing of administering the test substance. In the screening method of the present embodiment, the steps of detecting the test substance, nephrin protein-expressing cells, anti-nephrin antibody, and F-actin are the same as those of the screening method of the first embodiment.

According to the screening method of the present embodiment, it is possible to efficiently screen a drug that is administered after the onset of chronic kidney disease and has a therapeutic effect.

(Third Embodiment)
In one embodiment, the present invention is a method for screening a prophylactic or therapeutic agent for chronic kidney disease, in which a step of culturing a nephrin protein-expressing cell in the presence of a test substance and the reaction of the anti-nephrin antibody with the cell. The step of detecting the phosphorylation of the nephrin protein in the cell, wherein the phosphorylation is the phosphorylation of the tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein. A screening method, wherein the detected phosphorylation is reduced as compared with the phosphorylation in the absence of the test substance, indicating that the test substance is a prophylactic or therapeutic agent for chronic kidney disease. I will provide a.

The screening method of the present embodiment is different from the screening method of the first embodiment and the screening method of the second embodiment mainly in the method of determining whether or not the agent is a preventive or therapeutic agent for chronic kidney disease.

In the screening method of the present embodiment, the steps of detecting the test substance, nephrin protein-expressing cells, anti-nephrin antibody, and F-actin are the same as those of the screening method of the first embodiment.

As will be described later in the examples, the inventors have shown that when an anti-nephrin antibody is reacted with a nephrin protein-expressing cell, the nephrin protein is phosphorylated. Therefore, a prophylactic or therapeutic agent for chronic kidney disease can be screened using the phosphorylation of the nephrin protein as an index.

The amino acid residue phosphorylated by reacting with the anti-nephrine antibody was the tyrosine residue at position 1232 in the case of mouse nephrin protein. Therefore, when a human nephrin protein or a nephrin protein of a species other than human or mouse is used as the nephrin protein expressed in cells, phosphorylation of the tyrosine residue corresponding to the tyrosine residue at position 1232 of the mouse nephrin protein is performed. It should be detected.

The tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein is, for example, the 1217th tyrosine residue in the case of the human nephrin protein.

The tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein can also be specified as follows. First, a step of culturing a nephrin protein-expressing cell in the absence of a test substance and a step of reacting the cell with an anti-nephrin antibody are carried out. Subsequently, the phosphorylated tyrosine residue of the above nephrin protein is identified. Phosphorylated tyrosine residues can be detected by Western blotting or the like using a phosphorylated nephrin protein-specific antibody. The phosphorylated tyrosine residue identified as a result is a tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein.

Phosphorylation of the tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein detected in the presence of the test substance is the phosphorylation (the 1232th mouse nephrin protein) in the absence of the test substance. If it is reduced as compared with the phosphorylation of the tyrosine residue corresponding to the tyrosine residue in the above, it can be determined that the test substance is a prophylactic or therapeutic agent for chronic kidney disease.

(Fourth Embodiment)
In one embodiment, the present invention is a method for screening a prophylactic or therapeutic agent for chronic kidney disease, in which a step of reacting a nephrin protein-expressing cell with an anti-nephrin antibody and a medium of the cells reacted with the anti-nephrin antibody are performed. In the step of adding the test substance to the cell and the step of detecting the phosphorylation of the nephrin protein in the cells, the phosphorylation corresponds to the phosphorylation of the tyrosine residue corresponding to the 1232nd tyrosine residue of the mouse nephrin protein. The said test substance is a prophylactic or therapeutic agent for chronic kidney disease, wherein the detected phosphorylation is reduced as compared with the phosphorylation in the absence of the test substance. A screening method is provided to show that.

The screening method of the present embodiment is mainly different from the screening method of the third embodiment in the timing of administering the test substance. In the screening method of the present embodiment, the steps of detecting the phosphorylation of the test substance, the nephrin protein-expressing cell, the anti-nephrin antibody, and the nephrin protein are the same as those of the screening method of the third embodiment.

According to the screening method of the present embodiment, it is possible to efficiently screen a drug that is administered after the onset of chronic kidney disease and has a therapeutic effect.

(Fifth Embodiment)
In one embodiment, the invention is a method of screening for a prophylactic or therapeutic agent for chronic kidney disease, the step of culturing cells in the presence of a test substance and the expression of a ganglioside GM3 or ganglioside GM3 synthase gene in the cells. A step of measuring the amount is provided, and the expression level of the ganglioside GM3 or the ganglioside GM3 synthase gene is increased as compared with the expression level in the absence of the test substance. Provided is a screening method showing that it is a prophylactic or therapeutic agent.

The screening method of the present embodiment is the screening method of the first embodiment, the screening method of the second embodiment, the screening method of the third embodiment, and the screening method of the fourth embodiment is a preventive or therapeutic agent for chronic kidney disease. The method of determining whether or not it is is mainly different.

In the screening method of the present embodiment, the test substance is the same as the screening method of the first embodiment.

Further, the cell is not particularly limited, and is preferably, for example, a cell derived from a kidney, but the cell is not limited thereto. As the cells, nephrin protein-expressing cells similar to those in the screening method of the first embodiment, the screening method of the second embodiment, the screening method of the third embodiment, and the screening method of the fourth embodiment may be used. ..

The expression level of GM3 can be measured by, for example, a thin layer chromatography method or the like. The expression level of the GM3 synthase gene may be measured at the protein level by, for example, Western blotting, or may be measured at the mRNA level by, for example, a quantitative RT-PCR method, a microarray, or the like.

The GM3 synthase is also called lactosylceramide α2,3 sialic acid transferase. The accession number of the human GM3 synthase UniProtKD is Q9UNP4, and the accession number of the mouse GM3 synthase UniProtKD is O88829.

As will be described later in Examples, the inventors have shown that increasing the expression level of GM3 in a chronic kidney disease cell model can prevent or treat chronic kidney disease. Therefore, it can be said that the test substance that increases the expression level of GM3 is a prophylactic or therapeutic agent for chronic kidney disease.

That is, when the expression level of the GM3 or GM3 synthase gene detected in the presence of the test substance in the screening method of the present embodiment is increased as compared with the expression level in the absence of the test substance, the test is concerned. It can be determined that the substance is a prophylactic or therapeutic agent for chronic kidney disease.

(Sixth Embodiment)
In one embodiment, the present invention is a method for screening a prophylactic or therapeutic agent for chronic kidney disease, wherein a non-human animal model that develops chronic kidney disease under the administration of a test substance is bred, and the non-human animal. It comprises a step of evaluating the renal function of the model, and the renal function of the non-human animal model under the administration of the test substance is improved as compared with the renal function of the non-human animal model under the non-administration of the test substance. Provided is a screening method showing that the test substance is a preventive or therapeutic agent for chronic kidney disease.

The screening method of the present embodiment is the screening method of the first embodiment, the screening method of the second embodiment, the screening method of the third embodiment, and the screening method of the fourth embodiment are non-humans who develop chronic kidney disease. The main difference is that it uses an animal model.

In the screening method of the present embodiment, the test substance is the same as the screening method of the first embodiment.

As a non-human animal model that develops chronic kidney disease, for example, when an anti-mouse nephrin rabbit polyclonal antibody described later in an example is administered to a mouse, the mouse becomes chronic kidney disease (more accurately, focal segmental). A model or the like that develops glomerulosclerosis, hereinafter sometimes referred to as "FSGS") can be used. Here, the non-human animal is not limited to a mouse, and may be, for example, a rat, a pig, a goat, a sheep, a monkey, or the like.

In the screening method of the present embodiment, renal function includes, for example, measurement of blood albumin content, measurement of urinary albumin content, measurement of progression of glomerular sclerosing lesion by histological analysis of renal specimen, unit area of podocyte. It can be evaluated by measuring the number of hits, measuring the abundance of ganglioside GM3, measuring the abundance of nephrin protein, and the like.

In the screening method of the present embodiment, when the renal function of the non-human animal model under the administration of the test substance is improved as compared with the renal function of the non-human animal model under the non-administration of the test substance, the test substance is released. It can be determined that it is a preventive or therapeutic agent for chronic kidney disease.

[Screening kit for preventive or therapeutic agents for chronic kidney disease]
(First Embodiment)
In one embodiment, the present invention provides a screening kit for a prophylactic or therapeutic agent for chronic kidney disease, which comprises nephrin protein-expressing cells and an anti-nephrin antibody.

By using the kit of this embodiment, the above-mentioned method for screening a preventive or therapeutic agent for chronic kidney disease can be carried out.

In the kit of the present embodiment, as the nephrin protein-expressing cell and the anti-nephrin antibody, the same method as the screening method for a prophylactic or therapeutic agent for chronic kidney disease according to the first embodiment described above can be used.

The kit of this embodiment may further include phalloidin. Phalloidin is a reagent that detects F-actin. By providing phalloidin, the kit of this embodiment can be suitably used for the method for screening a prophylactic or therapeutic agent for chronic kidney disease according to the above-mentioned first embodiment and second embodiment.

The kit of the present embodiment further comprises an anti-phosphorylated nephrin protein antibody, which antibody may be an antibody that detects phosphorylation of a tyrosine residue corresponding to the 1232nd tyrosine residue of mouse nephrin protein. ..

Here, the antibody for detecting the phosphorylation of the tyrosine residue corresponding to the 1232th tyrosine residue of the mouse nephrin protein is the same as the method for screening a preventive or therapeutic agent for chronic kidney disease according to the third embodiment described above. Can be used.

By comprising an antibody that detects phosphorylation of the tyrosine residue corresponding to the tyrosine residue at the 1232th tyrosine residue of the mouse nephrin protein, the kit of the present embodiment can be used in the chronic form according to the third embodiment and the fourth embodiment described above. It can be suitably used as a method for screening a prophylactic or therapeutic agent for kidney disease.

The antibody that detects phosphorylation of the tyrosine residue corresponding to the tyrosine residue at position 1232 of the mouse nephrin protein may be a specific binding substance other than the antibody. Examples of the specific binding substance other than the antibody include nucleic acid aptamers and peptide aptamers.

(Second Embodiment)
In one embodiment, the invention is specific for a specific binding agent for GM3, a specific binding agent for GM3 synthase, a primer set that specifically amplifies the cDNA of the GM3 synthase gene, or the mRNA of the GM3 synthase gene. Provided is a screening kit for a prophylactic or therapeutic agent for chronic kidney disease, which comprises a probe that hybridizes to.

By using the kit of the present embodiment, it is possible to carry out the method for screening a preventive or therapeutic agent for chronic kidney disease according to the above-mentioned fifth embodiment.

<< Specific binding substance >>
Specific binding substances include, for example, antibodies, antibody fragments, aptamers and the like. Examples of the aptamer include nucleic acid aptamers and peptide aptamers.

The specific binding substance is not particularly limited as long as it can specifically bind to the target protein or target glycolipid, and may be commercially available. Further, the specific binding substance may be fixed on the carrier to form a protein chip or the like.

《Primer set》
The primer set is not particularly limited as long as it can amplify the cDNA of the GM3 synthase gene.

"probe"
The probe is not particularly limited as long as it specifically hybridizes to the mRNA of the GM3 synthase gene. The probe may be immobilized on a carrier to form a DNA microarray or the like.

[Other embodiments]
In one embodiment, the present invention provides a method for preventing or treating chronic kidney disease, which comprises a step of administering an effective amount of a compound represented by the following formula (1) to a patient or a patient in need of treatment. ..

［式（１）中、Ｒ^１は置換されていてもよい炭素数２～１６の直鎖状又は分岐鎖状のアルキル基を表す。］

[In formula (1), R ¹ represents a linear or branched alkyl group having 2 to 16 carbon atoms which may be substituted. ]

In one embodiment, the present invention provides a compound represented by the above formula (1) for the prevention or treatment of chronic kidney disease.

In one embodiment, the present invention provides the use of a compound represented by the above formula (1) for producing a prophylactic or therapeutic agent for chronic kidney disease.

In these embodiments, more limited examples of the compound represented by the above formula (1) are the same as those described above for the embodiments of the preventive or therapeutic agent for chronic kidney disease.

[Experimental Example 1]
(Study using model mice that develop chronic kidney disease)
The inventors have previously developed a model mouse that develops chronic kidney disease. In this model mouse, when an anti-mouse nephrin rabbit polyclonal antibody is administered to a mouse, the mouse develops chronic kidney disease (more accurately, focal segmental glomerulosclerosis, hereinafter may be referred to as "FSGS"). It is a thing. As the above-mentioned anti-mouse nephrin rabbit polyclonal antibody, one prepared by immunizing a rabbit by a gene immunization method with DNA encoding a mouse nephrin protein is used.

This model mouse exhibits all the phenomena such as proteinuria, edema, and hypercholesterolemia observed in human chronic kidney disease and nephrotic syndrome, and is a model mouse that accurately reflects human chronic kidney disease. Is considered to be.

Therefore, we conducted a study using this mouse model. Specifically, first, 0.5 to 5 mg of anti-mouse nephrin rabbit polyclonal antibody was intravenously administered to mice. Subsequently, before, 90 minutes, 3 hours, and 7 days after the administration of the antibody, the kidneys of the mice were excised to prepare thin film tissue sections, and the nephrin protein and ganglioside GM3 in the glomerulus were described by the inventor, respectively. It was detected by fluorescent immunostaining using an anti-nephrin antibody and an anti-GM3 antibody (model "A2582", Tokyo Kasei Kogyo Co., Ltd.) prepared by the above.

FIG. 1 is a fluorescence micrograph in which a nephrin protein and GM3 are detected. The magnification is 400 times. In FIG. 1, "nephrin" means the result of detecting nephrin protein, "GM3" means the result of detecting GM3, and "Overlay" means the result of detecting nephrin protein and GM3. It means that it is the result of synthesizing the detected result.

As a result, it was clarified that the expression levels of the nephrin protein and GM3 decreased when the anti-nephrin antibody was administered to the mice. A decrease in the expression levels of nephrin protein and GM3 was observed 90 minutes after administration of the anti-mouse nephrin rabbit polyclonal antibody.

[Experimental Example 2]
(Examination using a chronic kidney disease cell model)
Based on the results of the mouse model used in Experimental Example 1, it was examined whether or not a model of chronic kidney disease could be constructed at the cultured cell level.

Specifically, first, an expression vector of mouse nephrin protein was introduced into HEK293 cells, which is a human fetal kidney cell line. Subsequently, anti-mouse nephrin rabbit polyclonal antibody was added to the medium of the cells at a final concentration of 5 to 30 μg / mL.

Subsequently, immediately after administration of the antibody, 6 hours later, and 48 hours later, each cell was fixed with paraformaldehyde, and the nephrin protein, F-actin, and ganglioside GM3 in each cell were prepared by the inventors. , Phalloidin (model "A34055", Invitrogen) and anti-GM3 antibody (type "A2582", Tokyo Kasei Kogyo Co., Ltd.).

FIG. 2 is a fluorescence micrograph in which nephrin protein and GM3 are detected. The magnification is 400 times. In FIG. 2, "nephrin" means the result of detecting the nephrin protein, "F-actin" means the result of detecting F-actin, and "GM3" means the result of detecting GM3. Means that

As a result, it was clarified that when the anti-mouse Neflin rabbit polyclonal antibody was reacted with HEK293 cells into which the mouse Neflin protein expression vector was introduced, Neflin protein decreased, F-actin decreased, and GM3 decreased. This result indicates that a system in which an anti-nephrin antibody is reacted with HEK293 cells into which an expression vector for mouse nephrin protein has been introduced can be used as a model for chronic kidney disease cells.

[Experimental Example 3]
(Examination of GM3 expression level in chronic kidney disease cell model)
The expression level of ganglioside GM3 was examined using the same chronic kidney disease cell model as in Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody was reacted with HEK293 cells into which an expression vector of mouse nephrin protein was introduced.

Specifically, anti-nephrin rabbit polyclonal antibody was added to the medium of HEK293 cells into which the expression vector of mouse nephrin protein was introduced at a final concentration of 5 to 30 μg / mL, and the expression level of GM3 was examined after 6 hours and 48 hours. bottom. In addition, as a control, the expression level of GM3 in cells to which no anti-nephrine antibody was added to the medium was examined. In addition, a standard product of GM3 was used as a positive control. GM3 was detected by immunothin layer chromatography using an anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.).

FIG. 3 is a photograph showing the results of immunothin layer chromatography. In FIG. 3, lane 1 is the analysis result of the standard product of GM3, lane 2 is the analysis result of HEK293 cells to which the anti-nephrine antibody was not added to the medium, and lane 3 is the analysis result of the anti-nephrin antibody added to the medium. It is an analysis result of HEK293 cells. In FIG. 3, "*" and "**" indicate the band of GM3.

As a result, it was clarified that the expression level of GM3 was decreased by the addition of the anti-mouse nephrin rabbit polyclonal antibody. In particular, the decrease in the band indicated by "*" in FIG. 3 was remarkable.

From the above results, it was confirmed again that the addition of the anti-mouse nephrin rabbit polyclonal antibody reduced the expression level of GM3 in HEK293 cells into which the mouse nephrin protein expression vector was introduced.

[Experimental Example 4]
(Examination of addition of GM3 to chronic kidney disease cell model)
Using the same chronic kidney disease cell model as in Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody is reacted with HEK293 cells introduced with a mouse nephrin protein expression vector, the effect of addition of GM3 to the medium is investigated. bottom.

Specifically, ganglioside GM3 was added to the medium of HEK293 cells into which an expression vector of mouse nephrin protein was introduced in the presence and absence of anti-mouse nephrin rabbit polyclonal antibody, and its effect was examined.

The anti-mouse nephrin rabbit polyclonal antibody was added to the medium at a final concentration of 5-30 μg / mL. GM3 was also added to the medium at a final concentration of 0, 125 or 250 μM. Subsequently, 48 hours after the start of the experiment, each cell was fixed with paraformaldehyde, and GM3 and F-actin in each cell were treated with anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.) and phalloidin (type "A34055", respectively). , Invitrogen) was detected by fluorescent staining.

FIG. 4 is a fluorescence micrograph showing the results of detecting GM3 and F-actin. The magnification is 400 times. In FIG. 4, "Nep Ab" means an anti-mouse nephrin rabbit polyclonal antibody, "+" means that it was added, and "-" means that it was not added. Further, "GM3" means the result of detecting GM3, "F-actin" means the result of detecting F-actin, and "DAPI" means 4', 6-diamidino-2. -It means that it is the result of staining the nucleus with phenylindole (DAPI).

As a result, it was confirmed again that when the anti-mouse nephrin rabbit polyclonal antibody was reacted with HEK293 cells into which the mouse nephrin protein expression vector was introduced, F-actin decreased and GM3 decreased. It was also clarified that the decrease of F-actin can be suppressed by adding GM3 to the medium of the chronic kidney disease cell model.

[Experimental Example 5]
(Examination of addition of valproic acid to chronic kidney disease cell model)
Using the same chronic kidney disease cell model as in Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody is reacted with HEK293 cells into which an expression vector of mouse nephrin protein has been introduced, the effect of addition of valproic acid on the medium was used. investigated.

Specifically, an anti-mouse nephrin rabbit polyclonal antibody was reacted with a medium of HEK293 cells into which an expression vector of mouse nephrin protein was introduced in the presence of valproic acid, and its effect was examined.

The anti-mouse nephrin rabbit polyclonal antibody was added to the medium at a final concentration of 5-30 μg / mL. Valproic acid was also added to the medium at a final concentration of 0, 1, 2 or 3 mM. Subsequently, 48 hours after the start of the experiment, each cell was fixed with paraformaldehyde, and GM3 and F-actin in each cell were treated with anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.) and phalloidin (type "A34055", respectively). , Invitrogen) was detected by fluorescent staining.

FIG. 5 is a fluorescence micrograph showing the results of detecting GM3 and F-actin. The magnification is 400 times. In FIG. 5, "Nep Ab" means an anti-mouse nephrin rabbit polyclonal antibody, and "+" means that it was added. Further, "VPA" means valproic acid, "GM3" means the result of detecting GM3, "F-actin" means the result of detecting F-actin, and "Overlay". "" Means that it is the result of synthesizing the result of detecting GM3 and the result of detecting F-actin, and "DAPI" means the result of staining the nucleus with DAPI.

As a result, it was clarified that the decrease of F-actin due to the addition of the anti-nephrin antibody was suppressed in the presence of valproic acid. This result means that valproic acid can be used as a prophylactic or therapeutic agent for chronic kidney disease.

[Experimental Example 6]
(Examination of interaction between GM3 and nephrin protein)
Using the same chronic kidney disease cell model as in Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody is reacted with HEK293 cells introduced with a mouse nephrin protein expression vector, the interaction between ganglioside GM3 and nephrin protein is performed. investigated.

Specifically, first, all proteins were extracted from HEK293 cells into which an expression vector of mouse nephrin protein was introduced, and the interaction between GM3 and nephrin protein was detected by immunoprecipitation-Western blotting.

For comparison, an anti-mouse nephrin rabbit polyclonal antibody having a final concentration of 5 to 30 μg / mL was added to the medium of HEK293 cells into which an expression vector of mouse nephrin protein was introduced, and 24 hours later, all proteins were extracted and immunoprecipitation was performed. -The interaction between GM3 and nephrin protein was detected by Western blotting.

The anti-nephrin antibody prepared by the inventors was used for immunoprecipitation of mouse nephrin protein. An anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.) was used for immunoprecipitation of GM3. In addition, the anti-nephrin antibody prepared by the inventors was used for the detection of the mouse nephrin protein by the Western blotting method.

FIG. 6 is a photograph showing the analysis results by the immunoprecipitation-Western blotting method. In FIG. 6, "IP" means immunoprecipitation, "IB" means that it was detected by Western blotting, "GM3" means that an anti-GM3 antibody was used, and "Nephrin" means anti-nephrin. "Untreat" means that the antibody was used, "untreat" means that the anti-mouse nephrin rabbit polyclonal antibody was not reacted, and "Ab treat" means that the anti-mouse nephrin rabbit polyclonal antibody was reacted.

As a result, it was clarified that GM3 and nephrin protein interacted in HEK293 cells into which the mouse nephrin protein expression vector was introduced. It was also clarified that when the anti-mouse Neflin rabbit polyclonal antibody was reacted with HEK293 cells into which the mouse Neflin protein expression vector was introduced, the interaction between GM3 and Neflin protein was reduced.

[Experimental Example 7]
(Examination of the effect of valproic acid on the interaction between GM3 and nephrin protein)
Using the same chronic kidney disease cell model as in Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody is reacted with HEK293 cells introduced with a mouse nephrin protein expression vector, for the interaction between ganglioside GM3 and nephrin protein. The effect of valproic acid was investigated.

Specifically, an anti-mouse nephrin rabbit having a final concentration of 5 to 30 μg / mL in the medium of HEK293 cells into which an expression vector of mouse nephrin protein was introduced, in the presence of valproic acid having a final concentration of 3 mM or in the absence of valproic acid. Polyclonal antibody was added, total protein was extracted 24 hours later, and the interaction between GM3 and nephrin protein was detected by immunoprecipitation-Western blotting.

The anti-nephrin antibody prepared by the inventors was used for immunoprecipitation of mouse nephrin protein. An anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.) was used for immunoprecipitation of GM3. In addition, the anti-nephrin antibody prepared by the inventors was used for the detection of the mouse nephrin protein by the Western blotting method.

FIG. 7 is a photograph showing the analysis results by the immunoprecipitation-Western blotting method. In FIG. 7, "Input" means that the sample is the same as that used for the immunoprecipitation-Western blotting method, "IP" means immunoprecipitation, and "IB" is detected by the Western blotting method. "GM3" means that an anti-GM3 antibody was used, "Nephrin" means that an anti-nephrin antibody was used, and "Ab treat" means that an anti-mouse nephrin rabbit was used in the absence of valproic acid. It means that the polyclonal antibody was reacted, and "Ab + VPA treat" means that the anti-mouse nephrin rabbit polyclonal antibody was reacted in the presence of valproic acid.

As a result, in the presence of valproic acid, the decrease in the interaction between GM3 and nephrin protein was suppressed by reacting the anti-mouse nephrin rabbit polyclonal antibody with HEK293 cells into which the expression vector of mouse nephrin protein was introduced, and GM3 and GM3 It was revealed that the interaction with the nephrin protein was maintained.

[Experimental Example 8]
(Examination of phosphorylation of nephrin protein)
The phosphorylation of nephrin protein was examined using a chronic kidney disease cell model similar to that of Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody was reacted with HEK293 cells into which an expression vector of mouse nephrin protein was introduced.

Specifically, an anti-mouse nephrin rabbit having a final concentration of 5 to 30 μg / mL in the medium of HEK293 cells into which an expression vector of mouse nephrin protein was introduced, in the presence of valproic acid having a final concentration of 3 mM or in the absence of valproic acid. Polyclonal antibody was added, total protein was extracted after 0, 30, 60 and 120 minutes, and phosphorylation of nephrin protein was examined by Western blotting. For comparison, a sample to which anti-mouse nephrin rabbit polyclonal antibody was not added was also prepared.

Examples of the antibody that detects phosphorylated nephrin protein include an antibody that recognizes phosphorylation of the 1232th tyrosine residue of mouse nephrin protein (model "ab80298", Abcum), and the 1191th tyrosine residue of mouse nephrin protein. An antibody (type "ab80299", Abcum) that simultaneously recognizes the phosphorylation of the tyrosine residue at position 1208 and the phosphorylation of the 1208th tyrosine residue was used. In addition, the anti-nephrin antibody prepared by the inventors was used for the detection of the mouse nephrin protein by the Western blotting method. In addition, as a loading control, GAPDH protein was detected using an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (model "2118", CellSignalingTechnology Japan Co., Ltd.).

FIG. 8 is a photograph showing the analysis results by the Western blotting method. In FIG. 8, "Untreat" means that the anti-mouse nephrin rabbit polyclonal antibody was not reacted, "Ab treat" means that the anti-mouse nephrin rabbit polyclonal antibody was reacted, and "Ab + VPA treat" means that the anti-mouse nephrin rabbit polyclonal antibody was reacted. It means that the anti-mouse nephrin rabbit polyclonal antibody was reacted in the presence of acid.

Further, "PY1232" means that an antibody recognizing the phosphorylation of the 1232th tyrosine residue of the mouse nephrin protein was used, and "PY1191 + 1208" means the phosphorylation of the 1191th tyrosine residue of the mouse nephrin protein. And 1208th tyrosine residue phosphorylation was used at the same time, "Nephrin" means that an anti-nephrin antibody was used, and "GAPDH" means that an anti-GAPDH antibody was used. Means.

As a result, it was clarified that when the anti-mouse nephrin rabbit polyclonal antibody was added to the medium of HEK293 cells into which the expression vector of mouse nephrin protein was introduced, the tyrosine residue at position 1232 of the mouse nephrin protein was strongly phosphorylated. rice field. In addition, it was clarified that the phosphorylation of mouse nephrin protein was suppressed by the addition of anti-mouse nephrin rabbit polyclonal antibody in the presence of valproic acid.

[Experimental Example 9]
(Examination of compounds other than valproic acid)
The effects of compounds other than valproic acid were investigated using a chronic kidney disease cell model similar to Experimental Example 2, that is, a model in which an anti-mouse nephrin rabbit polyclonal antibody was reacted with HEK293 cells into which an expression vector for mouse nephrin protein was introduced. ..

Specifically, anti-mouse in the presence of valproic acid, butyric acid, propionic acid or γ-aminobutyric acid (hereinafter, may be referred to as “GABA”) in the medium of HEK293 cells into which an expression vector of mouse nephrin protein has been introduced. Neflin rabbit polyclonal antibody was reacted and its effect was investigated. The following formula (2) shows the chemical formula of valproic acid, the following formula (3) shows the chemical formula of butyric acid, the following formula (4) shows the chemical formula of propionic acid, and the following formula (5) shows the chemical formula of GABA.

The anti-mouse nephrin rabbit polyclonal antibody was added to the medium at a final concentration of 5-30 μg / mL. In addition, valproic acid, butyric acid, propionic acid and γ-aminobutyric acid were each added to the medium at a final concentration of 3 mM. Subsequently, 48 hours after the start of the experiment, each cell was fixed with paraformaldehyde, and F-actin and GM3 in each cell were treated with phalloidin (type "A34055", Invitrogen) and anti-GM3 antibody (type "A2582", Tokyo, respectively). It was detected by fluorescent staining using Kasei Kogyo Co., Ltd.).

FIG. 9 is a fluorescence micrograph showing the result of detecting F-actin, and FIG. 10 is a fluorescence micrograph showing the result of detecting GM3. The magnification is 400 times. In FIGS. 9 and 10, "Nep Ab" means an anti-mouse nephrin rabbit polyclonal antibody, "-" means that it was not added, and "+" means that it was added. Further, "VPA" means valproic acid, "BTA" means butyric acid, "PPA" means propionic acid, and "GABA" means γ-aminobutyric acid.

Further, "F-actin" means that it is the result of detecting F-actin, "GM3" means that it is the result of detecting GM3, and "DAPI" is the result of staining the nucleus with DAPI. It means that there is. Further, in FIG. 9, "Overlay" means the result of synthesizing the result of detecting F-actin and the result of staining the nucleus with DAPI, and in FIG. 10, "Overlay" is the result of detecting GM3. It means that it is the result of synthesizing the result of staining the nucleus with DAPI.

As a result, as shown in FIG. 9, it was clarified that the addition of valproic acid, butyric acid, and propionic acid to the medium can suppress the decrease of F-actin due to the addition of the anti-mouse nephrin rabbit polyclonal antibody. .. On the other hand, it was clarified that the degree of suppression of the decrease in F-actin was low when GABA was added to the medium.

Further, as shown in FIG. 10, it was clarified that by adding valproic acid, butyric acid, and propionic acid to the medium, the decrease of GM3 due to the addition of the anti-mouse nephrin rabbit polyclonal antibody can be suppressed. On the other hand, it was clarified that the degree of suppression of the decrease in GM3 was low when GABA was added to the medium.

[Experimental Example 10]
(Administration of valproic acid to model mice that develop chronic kidney disease 1)
Using model mice that develop chronic kidney disease similar to Experimental Example 1 (more accurately, focal segmental glomerulosclerosis, hereinafter sometimes referred to as "FSGS"), the effects of in vivo valproic acid administration investigated.

C57BL / 6N mice (7 weeks old) were intraperitoneally administered with valproic acid once daily at a dose of 100 mg / kg for 28 days. Then, on the 14th day from the start of administration of valproic acid, anti-mouse nephrin rabbit polyclonal antibody was administered once in the tail vein at a dose of 0.5 mg / animal, 1 mg / animal or 2 mg / animal, and the onset of chronic kidney disease occurred. Was induced.

For comparison, anti-mouse nephrin rabbit polyclonal antibody was administered once in the tail vein at a dose of 2 mg / animal without administration of valproic acid, and only valproic acid was administered in the group that induced the onset of chronic kidney disease. A group that did not induce the onset of chronic kidney disease was also prepared.

Subsequently, on days 1, 2, 3, 5, and 14 days after administration of the anti-mouse nephrin rabbit polyclonal antibody, urine was collected from each mouse and subjected to urine biochemical examination.

In addition, on the 28th day from the start of administration of valproic acid (14th day from the administration of anti-mouse nephrin rabbit polyclonal antibody), kidneys were excised from each mouse to prepare tissue sections. At this time, blood was also collected and a serum biochemical test was performed.

<< Results of urine biochemical test >>
FIG. 11 is a graph showing changes in urinary albumin (mg / g / creatinine) of each mouse calculated based on the results of urinary biochemical tests. In FIG. 11, "FSGS" shows the results of a group in which 2 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered without administration of valproic acid to induce the onset of chronic kidney disease, and "VPA only" was valproic acid only. The results of the group that did not induce the onset of chronic kidney disease were shown, and "Ab 0.5 mg + VPA" was the result of the group that received 0.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody and daily administration of valproic acid. "Ab 1 mg + VPA" shows the result of the group in which 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily, and "Ab 2 mg + VPA" showed the result of administration of 2 mg / animal of anti-mouse nephrin rabbit polyclonal antibody. The results of the group receiving daily administration of valproic acid are shown, and "ARB" is the range of the maximum amount of urinary albumin calculated from the suppression rate of urinary albumin by administration of ARB, which has been reported in humans and rats. Is shown.

Specifically, for humans, for example, Iino Y., et al., Renoprotective effect of losartan in comparison to amlodipine in patients with chronic kidney disease and hypertension --a report of the Japanese Losartan Therapy Intended for the Global Renal Protection in In Hypertensive Patients (JLIGHT) study., Hypertens Res., 27 (1), 21-30, 2004., ARB (losartan) -administered group showed a 47.9% decrease in albumin urine 12 months after the start of treatment. Have been described.

In addition, 34 non-diabetic patients were found in Ono M., et al., Predictors of proteinuria reduction by monotherapy with an angiotensin receptor blocker, olmesartan., J Renin Angiotensin Aldosterone Syst., 13 (2), 239-243, 2012. It has been described that as a result of administration of ARB (olmesartan) to patients with sexual nephropathy, albuminuria decreased by 44% after 8 weeks.

In addition, Usta M., et al., Efficacy of losartan in patients with primary focal segmental glomerulosclerosis resistant to immunosuppressive treatment., J Intern Med., 253 (3), 329-334, 2003. It is described that as a result of administration of ARB (losartan), albumin urine decreased by 48% after 12 months.

In addition, on page 435 of Schleier's "Kidney Disease and Pathophysiology" (Medical Science International, published in May 2011), the optimal level of antiproteinuria effect obtained by treatment is unknown, but before treatment. It is stated that it is recommended to aim for a reduction of at least 30-40% from urinary protein levels in the kidney.

For rats, Takahashi A., et al., Angiotensin II type 1 receptor blockade ameliorates proteinuria in puromycin aminonucleoside nephropathy by inhibiting the reduction of NEPH1 and nephrin., J Nephrol. 27 (6), 627-634, 2014. Has described that administration of ARB (ilvesartane) to a rat model of PAN nephropathy (MCNS) resulted in a 34% reduction in albuminuria after 8 days.

As a result, it was clarified that the administration of valproic acid significantly reduced the amount of urinary protein in mice modeled for chronic kidney disease. It was also clarified that the degree of decrease in urinary protein amount by administration of valproic acid was significantly higher than the degree of decrease in urinary protein amount by administration of the existing drug ARB.

<< Results of serum biochemical test >>
FIG. 12A is a graph showing the measurement results of the serum albumin amount of each mouse. Further, FIG. 12B is a graph showing the measurement results of the serum creatinine amount of each mouse. In FIGS. 12 (a) and 12 (b), "Normal" shows the measurement results in normal mice, and "VPA" shows the results of the group in which only valproic acid was administered and did not induce the onset of chronic kidney disease. "FSGS" shows the results of the group in which 2 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and no valproic acid was administered, and "Ab 0.5 mg + VPA" was administered in 0.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody. "Ab 1 mg + VPA" shows the results of the group receiving daily administration of valproic acid, "Ab 2 mg + VPA" shows the results of the group receiving 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody, and "Ab 2 mg + VPA" shows the results of the group receiving daily administration of valproic acid. The results of the group receiving 2 mg / animal of Neflin rabbit polyclonal antibody and daily administration of valproic acid are shown, and "#" indicates that there is a significant difference in P <0.05 with respect to the results of the "Normal" group. ..

From the results of FIG. 12 (a), it was clarified that administration of valproic acid normalizes hypoalbuminemia in chronic kidney disease model mice. Moreover, from the result of FIG. 12 (b), it was clarified that the administration of valproic acid normalizes the renal dysfunction of the chronic kidney disease model mouse.

<< PAS staining of kidney tissue sections >>
Kidney tissue sections of each mouse were stained with PAS and observed under a microscope to observe sclerosing lesions. 13 (a) to 13 (g) are micrographs showing the results of PAS staining of kidney tissue sections of mice in each group. The scale bar indicates 20 μm. FIG. 13 (a) is a representative result of the group in which only valproic acid was administered and did not induce the onset of chronic kidney disease, and FIG. 13 (b) was administered with 0.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody. FIG. 13 (c) shows typical results of the group in which 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily. 13 (d) is a representative result of the group in which 2 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily, and FIG. 13 (e) shows 0.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody. It is a typical result of the group to be administered and not to receive valproic acid, and FIG. 13 (f) is a representative of the group to which 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and no valproic acid was administered. As a result, FIG. 13 (g) is a representative result of the group in which 1.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was not administered. Further, in FIGS. 13 (d) to 13 (g), the region surrounded by the line indicates a cured region, and the arrowhead indicates an adhesion.

Further, FIG. 14 is a graph showing the results of quantifying the amount of injury induction based on the results of FIGS. 13 (a) to 13 (g). In FIG. 14, "VPA only" shows the results of the group in which only valproic acid was administered and did not induce the onset of chronic kidney disease, and "Ab 0.5 mg" was 0.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody. "Ab 0.5 mg + VPA" shows the results of the group that received 0.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody and received valproic acid daily, showing the results of the group that received and did not receive valproic acid. "Ab 1 mg" shows the results of the group in which 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and no valproic acid was administered, and "Ab 1 mg + VPA" was administered with 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody and valproic acid. "Ab 1.5 mg" shows the result of the group administered daily, "Ab 2 mg + VPA" shows the result of the group administered 1.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody and did not administer valproic acid, and "Ab 2 mg + VPA" is anti. The results of the group in which 2 mg / animal of mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily are shown.

As a result, it was clarified that the administration of valproic acid can significantly suppress the progression of glomerular sclerosing lesions in the kidney of a mouse model of chronic kidney disease. More specifically, when the dose of anti-mouse nephrin rabbit polyclonal antibody was 0.5 mg, administration of valproic acid was able to suppress the amount of injury induction by 74%. Further, when the dose of the anti-mouse nephrin rabbit polyclonal antibody was 1 mg, the amount of injury induction could be suppressed by 65% by the administration of valproic acid. In addition, when the dose of anti-mouse nephrin rabbit polyclonal antibody was 2 mg and valproic acid was administered daily, the dose of anti-mouse nephrin rabbit polyclonal antibody was 1.5 mg and compared with the case where valproic acid was not administered. , The amount of injury induction could be suppressed by 58%. Here, when the dose of the anti-mouse nephrin rabbit polyclonal antibody was 1.5 mg and valproic acid was administered daily, the amount of injury induction was expected to be suppressed by 58% or more.

<< PAS staining of kidney tissue sections and immunostaining of p57 >>
Kidney tissue sections of each mouse were PAS stained and further immunostained with p57. p57 is a podocyte marker. 15 (a) to 15 (f) are micrographs showing the results of PAS staining and immunostaining of p57 of kidney tissue sections of mice in each group. The scale bar indicates 20 μm. FIG. 15 (a) is a representative result of the group that did not induce the onset of chronic kidney disease, and FIG. 15 (b) shows that 2 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and no valproic acid was administered. FIG. 15 (c) is a representative result of the group in which only valproic acid was administered and did not induce the onset of chronic kidney disease, and FIG. 15 (d) is an anti-mouse nephrin. It is a typical result of the group in which 0.5 mg / animal of rabbit polyclonal antibody was administered and valproic acid was administered daily. FIG. 15 (e) shows that 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily. Representative results of the group, FIG. 15 (f) is a representative result of the group in which 2 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily.

Further, FIG. 16 is a graph showing the results of measuring the number of p57-positive cells per unit area based on the results of FIGS. 15 (a) to 15 (f). In FIG. 16, “Vehicle” showed the results of the group that did not induce the onset of chronic kidney disease, and “FSGS” was administered with 1.5 mg / animal of anti-mouse nephrin rabbit polyclonal antibody and was not administered with valproic acid. The results of the group were shown, "VPA" showed the results of the group in which only valproic acid was administered and did not induce the onset of chronic kidney disease, and "Ab 0.5 mg + VPA" showed the anti-mouse nephrin rabbit polyclonal antibody 0.5 mg / The results of the group in which animals were administered and valproic acid was administered daily were shown, "Ab 1 mg + VPA" showed the results of the group in which 1 mg / animal of anti-mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily, and "Ab 2 mg + VPA" was anti The results of the group in which 2 mg / animal of mouse nephrin rabbit polyclonal antibody was administered and valproic acid was administered daily are shown.

As a result, it was clarified that the administration of valproic acid suppressed the decrease in the number of podocytes in the kidney of a mouse model of chronic kidney disease. More specifically, when the dose of anti-mouse nephrin rabbit polyclonal antibody was 1.5 mg and no valproic acid was administered, the number of podocytes did not induce the onset of chronic kidney disease (hereinafter, "" It decreased to 66% of the "control group"). In contrast, when the dose of anti-mouse nephrin rabbit polyclonal antibody was 0.5 mg, administration of valproic acid was able to retain 92% of the number of podocytes in the control group. In addition, when the dose of anti-mouse nephrin rabbit polyclonal antibody was 1 mg, administration of valproic acid was able to retain 87% of the number of podocytes in the control group. In addition, when the dose of anti-mouse nephrin rabbit polyclonal antibody was 2 mg, administration of valproic acid was able to retain 74% of the number of podocytes in the control group.

[Experimental Example 11]
(Administration of valproic acid to model mice that develop chronic kidney disease 2)
The effect of valproic acid administration in vivo was investigated using model mice developing chronic kidney disease (FSGS) similar to Experimental Example 1.

C57BL / 6N mice (7 weeks old) were intraperitoneally administered with valproic acid once daily at a dose of 100 mg / kg for 28 days. Then, on the 14th day from the start of administration of valproic acid, anti-mouse nephrin rabbit polyclonal antibody was administered once in the tail vein at a dose of 2 mg / animal to induce the onset of chronic kidney disease.

For comparison, no valproic acid was administered, and only valproic acid, a group in which the anti-mouse nephrin rabbit polyclonal antibody was administered once in the tail vein at a dose of 2 mg / animal to induce the onset of chronic kidney disease, was administered. We also prepared a group that did not induce the onset of chronic kidney disease and a group that did not treat anything.

Subsequently, on the 14th day after administration of the anti-mouse nephrin rabbit polyclonal antibody, a kidney was excised from each mouse to prepare a tissue section. Subsequently, ganglioside GM3 and nephrin protein in the glomerulus were detected by fluorescent immunostaining using an anti-GM3 antibody (model "A2582", Tokyo Kasei Kogyo Co., Ltd.) and an anti-nephrin antibody prepared by the inventors, respectively. ..

FIG. 17 is a typical fluorescence micrograph in which GM3 and nephrin protein were detected. The scale bar is 20 μm. In FIG. 17, "GM3" means the result of detecting GM3, and "Nephrin" means the result of detecting the nephrin protein. In addition, "Control" means the result of the group of mice treated with nothing, and "VPA" means the result of the group in which only valproic acid was administered and the onset of chronic kidney disease was not induced. "FSGS" means that it is the result of a group in which anti-mouse nephrin rabbit polyclonal antibody was administered without administration of valproic acid to induce the onset of chronic kidney disease, and "VPA + FSGS" means valproic acid. It means that it is the result of the group to which the anti-mouse nephrin rabbit polyclonal antibody was administered on the 14th day from the start of administration of.

As a result, it was clarified that administration of valproic acid to mice increased the amount of GM3 accumulated in the glomerulus. In addition, it was revealed that the expression levels of nephrin protein and GM3 were decreased in the chronic kidney disease (FSGS) model in which the anti-nephrin antibody was administered to mice. On the other hand, it was clarified that the decrease of GM3 was remarkably suppressed when the anti-nephrin antibody was administered to the mice after pre-administration of valproic acid.

In addition, on the 14th day after administration of the anti-mouse nephrin rabbit polyclonal antibody, an electron microscope specimen was prepared from the kidney excised from each mouse, and the shape of the podocyte foot process in the glomerulus was observed with an electron microscope.

FIG. 18 is a typical electron micrograph. The scale bar is 1 μm. In FIG. 18, "Control" means the result of the untreated group of mice, and "VPA" is the result of the group that received only valproic acid and did not induce the onset of chronic kidney disease. Meaning that, "FSGS" means that it is the result of a group in which anti-mouse nephrin rabbit polyclonal antibody was administered without administration of valproic acid to induce the onset of chronic kidney disease, and "VPA + FSGS" is It means that it is the result of the group to which the anti-mouse nephrin rabbit polyclonal antibody was administered 14 days after the start of administration of valproic acid.

As a result, in a chronic kidney disease (FSGS) model in which an anti-nephrin antibody was administered to mice, fusion and involution of the podocyte foot process were observed. On the other hand, when the anti-nephrin antibody was administered to the mice after pre-administration of valproic acid, almost no fusion and regression of podocytes were observed, and it was clarified that the normal shape was maintained.

The above results further support that administration of valproic acid can prevent the onset of chronic kidney disease.

[Experimental Example 12]
(Administration of valproic acid to model mice that develop chronic kidney disease 3)
The effect of valproic acid administration in vivo was investigated using model mice developing chronic kidney disease (FSGS) similar to Experimental Example 1. In this experimental example, valproic acid was orally administered instead of intraperitoneally.

C57BL / 6N mice (7 weeks old) were treated with valproic acid by drinking water for 14 days. The daily intake of valproic acid was estimated to be 145.2 mg / kg. Then, on the 15th day from the start of drinking water administration of valproic acid, anti-mouse nephrin rabbit polyclonal antibody was administered once in the tail vein at a dose of 2 mg / animal to induce the onset of chronic kidney disease. Then, valproic acid was further administered by drinking water for 14 days.

For comparison, no valproic acid was administered, and only valproic acid, a group in which the anti-mouse nephrin rabbit polyclonal antibody was administered once in the tail vein at a dose of 2 mg / animal to induce the onset of chronic kidney disease, was administered. We also prepared a group that did not induce the onset of chronic kidney disease and a group that did not treat anything.

Subsequently, on the 28th day from the start of drinking water administration of valproic acid, kidneys were excised from each mouse to prepare tissue sections. Subsequently, ganglioside GM3 in the glomerulus was detected by fluorescent immunostaining using an anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.).

FIG. 19 is a typical fluorescence micrograph in which GM3 is detected. The scale bar is 20 μm. In FIG. 19, "GM3" means the result of detecting GM3, and "GMR6" means the clone name of the anti-GM3 antibody. In addition, "Normal" means the result of the group of mice treated with nothing, and "VPA" is the result of the group in which only valproic acid was administered by drinking water and did not induce the onset of chronic kidney disease. That is, "FSGS" means that it is the result of the group in which anti-mouse nephrin rabbit polyclonal antibody was administered without administration of valproic acid to induce the onset of chronic kidney disease, and "VPA + FSGS" means valpro. It means that it is the result of the group in which the anti-mouse nephrin rabbit polyclonal antibody was administered on the 15th day from the start of the drinking water administration of the acid, and the valproic acid was further administered by drinking water for 14 days.

As a result, it was revealed that the expression level of GM3 was decreased in the chronic kidney disease (FSGS) model in which the anti-nephrin antibody was administered to the mice. On the other hand, it was clarified that when valproic acid was administered to mice by drinking water, the decrease in GM3 due to the administration of the anti-nephrin antibody was remarkably suppressed.

This result indicates that the onset of chronic kidney disease can be prevented even when valproic acid is orally administered.

[Experimental Example 13]
(Examination of GM3 expression level in glomeruli of human chronic kidney disease patients)
Tissue sections were prepared from human renal biopsy tissue. Subsequently, ganglioside GM3 in the glomerulus was detected by fluorescent immunostaining using an anti-GM3 antibody (model "A2582", Tokyo Chemical Industry Co., Ltd.).

FIG. 20 is a typical fluorescence micrograph in which GM3 is detected. The scale bar is 40 μm. In FIG. 20, "GM3" means the result of detecting GM3, "GMR6" means the clone name of the anti-GM3 antibody, and "DAPI" means 4', 6-diamidino-2-phenylindole ( It means that it is the result of staining the nucleus with DAPI). In addition, "Normal" means that it is the result of normal human glomerulus, and "MCNS" means that it is the result of the glomerulus of a patient with microvariant nephrotic syndrome, which is a kind of chronic kidney disease. "FSGS" means that it is the result of the glomerulus of a patient with focal segmental glomerulosclerosis, a type of chronic kidney disease.

As a result, it was clarified that GM3 is abundantly expressed in normal glomeruli even in humans. On the other hand, it was revealed that the expression level of GM3 was significantly reduced in the glomerulus of patients with diseases caused by podocyte injury such as MCNS and FSGS.

This result indicates that the phenomenon confirmed in the model mouse can be applied to chronic kidney disease in humans.

According to the present invention, it is possible to provide a technique for preventing or treating chronic kidney disease.

Claims (2)

A prophylactic or therapeutic agent for microvariant nephrotic syndrome, focal segmental glomerulosclerosis or diabetic nephropathy, which contains valproic acid, butyric acid or propionic acid as an active ingredient. A pharmaceutical composition for the prevention or treatment of microvariant nephrotic syndrome, focal segmental glomerulosclerosis or diabetic nephropathy, which comprises the prophylactic or therapeutic agent according to claim 1 and a pharmaceutically acceptable carrier. thing.

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