WO2007125723A1

WO2007125723A1 - Glomerular slit protein inducer

Info

Publication number: WO2007125723A1
Application number: PCT/JP2007/057121
Authority: WO
Inventors: Toshikazu Nakamura; Shinya Mizuno; Kunio Iwatani
Original assignee: Osaka University; Kringle Pharma Inc.
Priority date: 2006-04-28
Filing date: 2007-03-30
Publication date: 2007-11-08

Abstract

Disclosed is an agent for inducing or maintaining a glomerular slit protein, which comprises, as an active ingredient, the following component (1) or (2): (1) (i) an HGF protein or (ii) a peptide which is a partial peptide of the HGF protein and which has substantially the same activities as those of the HGF protein, or a salt thereof; or (2) DNA comprising (i) DNA encoding the HGF protein, (ii) DNA encoding a peptide which is a partial peptide of the HGF protein and which has substantially the same activities as those of the HGF protein, or (iii) DNA which can hybridize with DNA having a nucleotide sequence complementary to that of the DNA (i) or (ii) under stringent conditions and which encodes a protein or peptide having substantially the same activities as those of the HGF protein.

Description

Specification

Glomerular slit protein inducer

Technical field

[oooi] The present invention relates to glomerular slit protein inducers and Z or maintenance agents, and more specifically, hepatocyte growth factor (hereinafter abbreviated as HGF) or DNA containing the DNA encoding it as an active ingredient. The present invention relates to glomerular slit protein inducers and Z or maintenance agents.

Background art

[0002] Proteinuria is not only an important symptom of kidney damage, but is itself considered to be an aggravating factor of progression of kidney damage. However, little has been clarified about the onset mechanism of proteinuria. In recent years, it has been clarified that there is a slit membrane between glomerular epithelial cells of the kidney, and this site has a barrier function to prevent permeation of proteins such as albumin, immunoglobulin or myoglobin. The slit membrane is composed of a slit protein such as nephrin (indicated by hrin) or podocin, and nephrin has been shown to form a filter structure of the slit membrane. For this reason, proteinuria is caused when the expression of the protein constituting the slit membrane is suppressed or the barrier function of the slit membrane is damaged or destroyed. Proteinuria usually accompanies renal disorders and renal diseases. The treatment of renal disorders is corticosteroids, immunosuppressants, antiplatelet drugs, or anticoagulants that treat inflammation in renal disorders. It is done by drug administration or protein-restricted diet therapy. However, these treatments are not currently aimed at inducing the expression of the protein constituting the slit membrane or suppressing or maintaining the destruction of the proteinuria barrier mechanism. HGF is known as one of the drugs for treating renal diseases (see Patent Documents 1 and 2 and Non-Patent Documents:! To 4). HGF was first identified as a potent mitogen for mature hepatocytes, and its gene cloning was performed in 1989 (see Non-Patent Documents 5 and 6). However, neither of these prior documents mentions the relationship between the HGF and the glial slit membrane's noria mechanism, and the relationship with the slit protein that constitutes the slit membrane.

Patent Document 1: JP-A-9 087199

Patent Document 2: Special Table 2001-516358 Non-Patent Literature 1: Mizuno et al., The 'Journal' of 'Tarinical' Investigation (J. Clin. Invest.), 1998, No. 101, pl827-1834

Non-Patent Document 2: Mizuno et al., American 'Journal' Ob Physiology '' Lena Nore 'Physology (Am. J. Physiol Renal Physiol), 2004, No. 286, pl 34— 1 43

Non-Patent Document 3: Liu Y., American 'Journal' of Physiol, 2004, No. 287, pF7—F16.

Non-Patent Document 4: Matsumoto K et al., Kidney y Int. 2001, 59th, p2023-2038

Non-Patent Document 5: Nakamura et al., Biochemical 'and' Biophysical 'and' Research 'Communications (Biochem. Biophys. Res. Commun.), 1984, pp. 12 2-14, p. 1450-1459

Non-Patent Document 6: Nakamura et al., Nature, 1989, No. 342, p. 44 0-443

Disclosure of the invention

Problems to be solved by the invention

[0003] An object of the present invention is to provide a drug effective for inducing and / or maintaining glomerular slit protein.

Means for solving the problem

[0004] As a result of various studies to solve the above problems, the present inventors have found that the HGF protein or a partial peptide of the HGF protein and a peptide having substantially the same activity as the HGF protein ( In the following, it may also be referred to as HGF protein, etc.), or DNA that encodes them or DNA that consists of a base sequence complementary to the DNA, and that hybridizes under stringent conditions, and is substantially the same as HGF protein. In particular, DNA containing a DNA encoding a protein having the same activity (hereinafter also referred to as HGF gene) induces and Z or maintains glomerular slit protein and suppresses proteinuria. I put it out. The inventors have further studied and completed the present invention.

That is, the present invention provides: [1] (1) (ii) HGF protein or (oral) Peptide of HGF protein and peptide having substantially the same activity as HGF protein or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA A glomerular slit protein inducer comprising, as an active ingredient, DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein Or maintenance agent,

[2] The active ingredient contains (i) an HGF protein or (oral) HGF protein partial peptide having substantially the same activity as the HGF protein, or a salt thereof. [1] glomerular slit protein inducer or maintainer according to [1],

[3] HGF protein strength (a) a protein comprising the same amino acid sequence as that represented by SEQ ID NO: 1, 2 or 5, or (b) a protein comprising substantially the same amino acid sequence as the amino acid sequence of (a) The glomerular slit protein inducer or maintainer according to [2] above,

[4] As an active ingredient, (i) DNA encoding HGF protein or (mouth) DNA encoding a peptide that is a partial peptide of HGF protein and having substantially the same activity as HGF protein or (c) those DNAs Which comprises a DNA encoding a protein or peptide that hybridizes with a DNA comprising a complementary base sequence under stringent conditions and has substantially the same activity as the HGF protein [1 ] Glomerular slit protein inducer or maintainer,

[5] DNA force encoding HGF protein (a) DNA consisting of the base sequence represented by SEQ ID NO: 3, 4 or 6 or (b) DNA consisting of a base sequence complementary to the base sequence of (a) above The glomerular slit protein inducer according to [4] above, which is a DNA comprising a DNA that hybridizes under stringent conditions and encodes a protein having substantially the same activity as the HGF protein. Or maintenance agent,

[6] The above [4] or [5], wherein the DNA is incorporated into a naked plasmid, an adenovirus vector, an adeno-associated virus vector, or a type I herpes simplex virus (HSV_1) vector. ] Glomerular slit protein inducer or maintainer, [7] Glomerular slit protein is on the membrane surface and / or in the cell surface of glomerular epithelial cells such as nephrin, podocine, and CD2-related protein, or the protein constituting the slit itself, or associated with this, stabilizes the slit. (1) to (6), characterized in that the glomerular slit protein inducer or maintainer according to any one of the above,

[8] (1) (i) HGF protein or (oral) Peptide or partial peptide of HGF protein having substantially the same activity as HGF protein, or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Related to the decrease in glomerular slit protein, characterized by containing as an active ingredient DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same quality of activity as HGF protein Preventive or therapeutic agent for proteinuria,

[9] (1) (i) HGF protein or (oral) Peptide or partial salt of HGF protein having substantially the same activity as HGF protein, or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Induction of glomerular slit protein characterized by administering to a mammal DNA comprising DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein Maintenance method,

[10] (1) (i) HGF protein or (oral) Peptide or partial salt of HGF protein having substantially the same activity as HGF protein, or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Use for the production of a glomerular slit protein inducer or maintainer of DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as the HGF protein; [11] (1) (i) HGF protein or (oral) Peptide or partial peptide of HGF protein having substantially the same activity as HGF protein, or a salt thereof, or (2) (i) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Use as a glomerular slit protein inducer or maintainer of DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as the HGF protein;

[12] (1) (i) HGF protein or (oral) Peptide of HGF protein and peptide having substantially the same activity as HGF protein or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Related to reduction of glomerular slit protein, characterized by administering to mammals DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein To prevent or treat proteinuria,

[13] (1) (i) HGF protein or (oral) Peptide having partial activity of HGF protein and having substantially the same activity as HGF protein, or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Production of a prophylactic or therapeutic agent for proteinuria associated with a decrease in glomerular slit protein of DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein Use for, and

[14] (1) (i) HGF protein or (mouth) Peptide of HGF protein and peptide having substantially the same activity as HGF protein or a salt thereof, or (2) (ii) HG F DNA encoding a protein or (mouth) a partial peptide of an HGF protein and encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to the DNA Hybridize under stringent conditions The present invention also relates to the use of a DNA containing a protein or peptide encoding a protein or peptide having substantially the same activity as the HGF protein as a prophylactic or therapeutic agent for proteinuria associated with glomerular slit protein reduction.

The invention's effect

[0006] A protein (slit protein) constituting a slit membrane having a function in the glomerular epithelial cells by administering the glomerular slit protein inducer and / or maintenance agent according to the present invention to a mammal or the like. Can be induced and maintained. The slit membrane has a barrier function that prevents permeation of proteins such as albumin, immunoglobulin, or myoglobin, so that the protein can be prevented from leaking into urine. Therefore, the glomerular slit protein inducer and / or maintenance agent according to the present invention exhibits an excellent preventive or therapeutic effect against proteinuria related to glomerular slit protein reduction and the like.

Brief Description of Drawings

[0007] FIG. 1 is a graph showing a nephrin score in ICGN mice. In the figure, normal indicates a healthy control group, 14 weeks indicates a pre-administration control group, 18 weeks (saline) indicates a control group, and 18 weeks (HGF) indicates an HGF administration group.

FIG. 2 is a diagram showing nephrin expression in glomeruli of ICGN mice. In the figure, raw food (18 weeks) represents the control group, and HGF (18 weeks) represents the HGF administration group.

FIG. 3 is a graph showing the CD2AP score in ICGN mice. In the figure, normal indicates a healthy control group, 14 weeks indicates a pre-administration control group, 18 weeks (saline) indicates a control group, and 18 weeks (HGF) indicates an HGF administration group.

FIG. 4 is a diagram showing the expression of CD2AP in the glomeruli of ICGN mice. In the figure, raw food (18 weeks) represents the control group, and HGF (18 weeks) represents the HGF administration group.

FIG. 5 is a graph showing a correlation between glomerular nephrin score and urinary protein concentration in ICGN mice. In the figure, ○ indicates a 14-week-old mouse (before administration of the test solution), the mouth indicates a control group, and the country indicates an HGF administration group.

FIG. 6 is a graph showing the correlation between glomerular CD2AP score 1 and urinary protein concentration in ICGN mice. In the figure, ○ indicates a 14-week-old mouse (before test solution administration), mouth indicates a control group, and country indicates an HGF administration group. FIG. 7 is a graph showing the nephrin score in STZ-administered mice. In the figure, normal indicates a healthy control group, STZ (saline) indicates a control group, and STZ (HGF) indicates an HGF administration group.

FIG. 8 is a graph showing nephrin expression in glomeruli of STZ-administered mice. In the figure

The 18th week (raw food) shows the control group, and the 18th week (HGF) shows the HGF administration group.

FIG. 9 is a graph showing a CD2AP score in STZ-administered mice. In the figure, normal indicates a healthy control group, STZ (saline) indicates a control group, and STZ (HGF) indicates an HGF administration group.

FIG. 10 is a graph showing the correlation between glomerular nephrin score and urinary protein concentration in diabetic mice. In the figure, the mouth indicates the control group, and the country indicates the HGF administration group.

FIG. 11 is a graph showing the correlation between glomerular CD2AP score 1 and urinary protein concentration in diabetic mice. In the figure, the mouth indicates the control group, and the country indicates the HGF administration group.

BEST MODE FOR CARRYING OUT THE INVENTION

The HGF protein used in the present invention is a known substance, and any protein prepared by various methods can be used as long as it is purified to the extent that it can be used as a medicine.

The HGF protein can be obtained, for example, by culturing primary cultured cells or cell lines that produce HGF protein, separating and purifying the culture supernatant, and separating the HGF protein. Alternatively, the gene encoding the HGF protein is incorporated into an appropriate vector by genetic engineering techniques, inserted into an appropriate host cell, and transformed into the host cell, and the target from the culture supernatant of this transformant. Recombinant HGF protein can also be obtained. (For example, see JP-A-5-111382, Biochem. Biophys. Res. Commun. 1989, No. 163, p. 967). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as E. coli, yeast or animal cells can be used. As long as the HGF protein thus obtained has substantially the same action as the natural HGF protein, one or more of its amino acid sequences (for example, 2-20, preferably 2 To: 10, more preferably 2 to 5; the same shall apply hereinafter.) The amino acid may be substituted, deleted or added, and the sugar chain may be substituted, deleted or added. You may have been. Examples of such HGF proteins include the following 5-amino acid deficient HGF proteins. Here, for amino acid sequences, “one or more amino acids are deleted, substituted or added” means genetic engineering techniques, site-specific mutations. It means that one or more amino acid residues have been deleted, substituted, or added by well-known technical means such as induction methods or by naturally occurring mutations. An HGF protein in which a sugar chain is substituted, deleted or added is, for example, an HGF protein in which a sugar chain added to a natural HGF protein is treated with an enzyme or the like to delete the sugar chain, or a sugar chain is not added. In this way, the amino acid sequence at the sugar chain addition site is mutated, or the amino acid sequence is mutated so that the sugar chain is added to a site different from the natural sugar chain addition site. Specifically, for example, with respect to the human HGF of Accession No. NP_001010932 registered in the NCBI database, the glycosylation site at position 289 Asn is Gin, position 397 A sn is Gin, position 471 Thr is Gly In addition, HGF [Fukuta K et al., Biochemical Journal, 388, 555-562 (2005), in which glycan was not added by replacing Asn at position 561 with Gin and Asn at position 648 with Gin, respectively. ] Etc. can be mentioned.

And a protein having at least about 80% homology with the amino acid sequence of the HGF protein, preferably a protein having about 90% or more homology, more preferably a protein having about 95% or more homology. A protein having substantially the same activity as the HGF protein is also included in the HGF protein used in the present invention. The term “homologous” in the above amino acid sequences means the degree of coincidence of amino acid residues constituting the respective sequences by comparing the primary structures of the proteins.

Examples of the HGF protein include those derived from humans represented by the amino acid sequence represented by Accession No. P14210 (SEQ ID NO: 1) or Accession No. NP-001010932 (SEQ ID NO: 2) registered in the NCBI database, for example. And the like are preferably mentioned. The HGF protein represented by SEQ ID NO: 2 is a 5-amino acid deficient HGF protein from which the five amino acid residues 161 to 165 of the amino acid sequence represented by SEQ ID NO: 1 have been deleted. Both of the proteins having the amino acid sequence represented by SEQ ID NO: 1 or 2 are human-derived natural HGF proteins, and have mitogenic activity, motogenic activity, etc. as HGF.

The protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or 2 is at least about 80% or more, preferably about 90% or more, with the amino acid sequence represented by SEQ ID NO: 1 or 2. More preferably about 95. Amino acid sequence with ₀ or more identity A protein having substantially the same activity as the HGF protein, for example, an amino acid sequence in which one or more amino acid residues are inserted or deleted from the amino acid sequence represented by SEQ ID NO: 1 or 2 A protein comprising an amino acid sequence in which one to a plurality of amino acid residues are substituted with another amino acid residue or an amino acid sequence in which one to a plurality of amino acid residues are modified, which is substantially the same as an HGF protein It is preferable that the protein has the same activity. The amino acid to be inserted or substituted may be an unnatural amino acid other than the 20 amino acids encoded by the gene. The unnatural amino acid may be any compound as long as it has an amino group and a carboxyl group, and examples thereof include γ-aminobutyric acid.

These proteins may be single or mixed proteins. Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or 2 include human-derived HGF such as Accession No. BAA14348 or AAC71655 registered in the NCBI database. Power is not limited to these.

In the HGF protein used in the present invention, the C-terminus may be any of a carboxyl group (one COOH), a carboxylate (one COO—), an amide (one CONH), or an ester (one COOR). Here, R in the ester is, for example, C16 alkyl group such as methinole, ethyl, n-propinole, isopropyl or n-butyl, for example, C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc., for example, phenyl. C6-12 aryl group such as naphthyl, for example, phenyl C1-2 alkyl group such as benzyl, phenethyl or C7-14 aralkyl group such as α-naphthyl C1-2 alkyl group such as naphthylmethyl Of these, C2-6 alkanolmethyl groups such as acetyloxymethyl and bivalyloxymethyl are used. When the HGF protein used in the present invention has a carboxyl group or carboxylate other than the C-terminus, the HGF protein in the present invention also includes an amidated or esterified carboxyl group or force lpoxylate. . As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Further, in the HGF protein used in the present invention, in the above-described protein, the amino group of the methionine residue at the terminus is a protecting group (for example, C 1-6 such as C2-6 alkyl group such as formyl group, acetyl, etc.). -6 sacyl group, etc.) Gnoretamyl group cleaved in the body is pyroglutamine oxidized, reactive group on the side chain of amino acid in the molecule (for example, —OH, —SH, amino group, imidazolyl group, indolinole group, guanidino group, etc.) Is protected with a suitable protecting group (for example, Cl_6 asinole group such as C2-6 alkanoyl group such as formyl group, acetyl and the like), or a sugar chain bound, loose glycoprotein, etc. These complex proteins are also included.

[0011] As a partial peptide of the HGF protein used in the present invention and having substantially the same activity as the HGF protein (hereinafter sometimes referred to as HGF partial peptide), the above-mentioned HGF protein partial Any peptide can be used as long as it has substantially the same activity as the HGF protein. In the present invention, the HGF partial peptide includes at least about 20 or more, preferably about 50 or more, more preferably about 100 or more amino acid sequences of the above-described amino acid sequences of the HGF protein. preferable. Specifically, for example, the amino acid sequence from the 32nd amino acid to the 210th amino acid from the N-terminal side of the human HGF amino acid sequence represented by SEQ ID NO: 1 (the first kringle from the N-terminal hairpin loop of HGF) Amino acid sequence from the N-terminal side to the 288th amino acid from the N-terminal side of the human HGF amino acid sequence represented by SEQ ID NO: 1 (the N-terminal of HGF) Preferable examples include peptides represented by the coordination IJ) from the hairpin loop to the second cling nore domain.

In the HGF partial peptide of the present invention, the C-terminus is any of carboxynole group (—COOH), carboxylate (one COO—), amide (one CONH) or ester (one COOR; R is as defined above). It may be. Furthermore, in the HGF partial peptide, the amino acid of the N-terminal methionine residue is protected with a protecting group, as in the HGF protein described above, and the Gin produced by cleavage of the N-terminal side in vivo is pyroglutamine oxidized. And those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.

[0012] Examples of the salt of HGF protein or a partial peptide thereof used in the present invention include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, And salts with maleic acid, succinic acid, tartaric acid, succinic acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).

[0013] The partial peptide of HGF protein or a salt thereof used in the present invention can be produced according to a known peptide synthesis method or by cleaving HGF protein with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, it may have a protecting group capable of constituting an HGF protein, or it may have a partial peptide or amino acid and a protecting group. If so, the ability to produce the desired peptide can be achieved by removing the protecting group. Known methods of condensation and elimination of protecting groups include, for example, M. Bodanszky and MA Ondetti, Peptide 'Synthesis (P-mark tide Synthesis), Interscience Publ ishers, New York (1966), Schroeder and Luebke, The' Examples include the methods described in Peptide, Academic Press, New York (1965) and the like. After the reaction, it is possible to purify and isolate a partial peptide of HGF protein by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, crystallization or recrystallization. If the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method. Conversely, if it is obtained by a salt, it is converted to a free form by a known method. can do.

In the present invention, “DNA encoding HGF protein” refers to DNA capable of expressing HGF protein. Examples of DNA containing DNA encoding HGF protein include, for example, Nature, 342, 440 (1989); Patent No. 2777678; Biochem. Biophys, Res. Commun., 198, 163, p. 967- 973; Pro Natl. Acad. Sci. USA, 1991, No. 88 (No. 16), p. 7001-7005 etc., for example, Accession No. M60718, M73240, AC004960, AY246560 in GeneBank / EMBL / DDBJ. , M29145 or M73240 and the like, DNA encoding human-derived HGF protein and the like are preferred. In addition, the DNA encoding the HGF protein of the present invention has a nucleotide sequence complementary to the DNA, DNA that hybridizes with the DNA under stringent conditions, and has substantially the same activity as the HGF protein. For example, DNA encoding a protein having mitogenic activity, motogenic activity, etc. is included. [0015] Specific examples of DNA encoding the HGF protein include, for example, DNA having the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or DNA having the base sequence represented by SEQ ID NO: 3 or 4 A DNA that hybridizes under stringent conditions with a DNA consisting of a complementary base sequence, preferably a DNA encoding a protein having substantially the same activity as the HGF protein, such as a mitogenic activity or a motogenic activity. Can be mentioned. Here, the nucleotide sequence represented by SEQ ID NO: 3 corresponds to nucleotide sequence 73 to 2259 of Accession No. M60718, and the DNA encodes an HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 1. It corresponds to. The nucleotide sequence represented by SEQ ID NO: 4 corresponds to the nucleotide sequence of Accession No. M73240 [corresponding to J Nos. 66 to 2237, and the DNA encodes an HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 2. It corresponds to.

[0016] A DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 4 and a DNA that hybridizes under stringent conditions are, for example, a colony hybridizer using the partial sequence of the DNA as a probe. It means DNA obtained by using the Chillon method, plaque hybridization method or Southern blot hybridization method. Specifically, using a filter to which DNA derived from colonies or plaques is immobilized, hybridization is performed at about 65 ° C. in the presence of about 0.7 to 1.0 M sodium chloride, and then about 0 to .:! ~ 2 times the concentration of the SSC solution (The composition of the 1 time concentration SSC solution consists of 150 mM sodium chloride, 15 mM sodium citrate) and wash the filter under conditions of about 65 ° C DNA that can be identified by. The stringent conditions are the same in the following.

[0017] Specifically, as a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 4, the base represented by SEQ ID NO: 3 or 4 Examples thereof include DNA having a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the sequence. Hybridization can be performed by known methods such as Molecular Cloning, A laboratory Manual, Third Edition (J. ^ ambrook et al., Old Spring Harbor

Lab. Press, 2001: The following is abbreviated as Molecular 'Cloning 3rd Edition'. ) And the like. When using a commercially available library, use the attached library. It can be performed according to the method described in the instructions.

[0018] Further, the DNA encoding the HGF protein of the present invention is not limited to the above, and the DNA encoding the HGF protein of the present invention is not limited as long as the expressed protein is a DNA having substantially the same action as the HGF protein. Can be used as For example, DNA that codes for a partial peptide of HGF protein and that codes for a peptide having substantially the same activity as HGF protein can be preferably used.

[0019] The DNA encoding the partial peptide of the HGF protein may be DNA encoding a peptide having the base sequence 1J encoding the partial peptide and having substantially the same activity as the HGF protein. It can be anything. Specific examples of the DNA encoding the partial peptide of the present invention include (a) a DNA having a partial base sequence of a DNA having the base sequence represented by SEQ ID NO: 3 or 4, and an HGF protein and a substance. (B) DNA encoding a peptide having a base sequence complementary to DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 3 or 4; Examples include DNA that hybridizes under stringent conditions and that encodes a protein having substantially the same activity as the HGF protein. More specifically, such DNA includes, for example, the 94th to 630th base sequences of the human HGF base sequence represented by SEQ ID NO: 3 (from the N-terminal hairpin loop of HGF). DNA having a DNA of a peptide up to 1 kringle domain) or the 94th base of the human HGF base sequence represented by SEQ ID NO: 3 up to the 864th base sequence (the second from the N-terminal hairpin loop of HGF) Preferred examples include DNA having a DNA that encodes a peptide up to the kringle domain.

[0020] DNA encoding HGF protein or a partial peptide of HGF protein and encoding a peptide having substantially the same quality of activity as HGF protein can be obtained by, for example, a conventional hybridization method or PCR method. For example, the DNA can be obtained with reference to a basic document such as Molecular 'Cloning 3rd Edition'.

[0021] DNA encoding HGF protein used in the present invention or a partial peptide of HGF protein, which encodes a peptide having substantially the same activity as HGF protein. Preferred examples of DNA containing DNA include genomic DNA, genomic DNA library, cDNA derived from cell 'tissue, cDNA library derived from cell or tissue, or synthetic DNA. Examples of the vector into which the genomic DNA fragment is cloned into the library include butteriophage, plasmid, cosmid, or phagemid.

In addition, RNA encoding the HGF protein used in the present invention or RNA encoding a partial peptide of the HGF protein, and an RNA encoding a peptide having substantially the same activity as the HGF protein, is also reverse transcriptase. Any HGF protein or partial peptide can be used in the present invention. Examples of the RNA include RNA obtained by preparing an mRNA fraction from cells or tissues and amplified by RT-PCR, and are within the scope of the present invention. The RNA can also be obtained by known means.

[0022] The HGF protein used in the present invention or the DNA encoding the same is preferably the above-mentioned human-derived one when applied to humans, but mammals other than humans (for example, monkeys, utensils, etc.). HGF protein derived from Shi, Horse, Pig, Hedge, Inu, Cat, Rat, Mouse, Chimpanzee, etc.) or DNA encoding it. Examples of such HGF include mouse-derived HGF (eg, Accession No. AAB31855, NP-034557, BAA01065, BAA01064, etc.) and rat-derived HGF [eg, Accession No. NP] registered in the NCBI database. — 58713 (protein consisting of amino acid sequence represented by SEQ ID NO: 5), etc.] Hushi-derived HGF (eg Accession No. NP — 00102692 1, XP874086, BAD02475 etc.), cat-derived HGF (eg Accession No. NP — 100 1009830, BAC10545, BAB21499, etc.), Inu-derived HGF (eg, Accession No. NP — 001002964, BAC57560, etc.) or chimpanzee-derived HGF (eg, Accession No. XP519174, etc.), etc.

[0023] The "glomerular slit protein" in the present invention refers to a protein constituting a slit membrane having a barrier function in glomerular epithelial cells. For example, nephrin, podosin or CD (cluster of differentiation) 2 related protein ( CD2AP; CD2-associated protein) or hi-kinin. The function of the slit membrane is to prevent permeation of proteins such as serum proteins such as albumin, immunoglobulin or myoglobin in glomerular epithelial cells. And a barrier function. The slit membrane can be observed as a linear material structure having a high electron density in a gap of about 5 to 60 nm in width existing between the foot processes of glomerular epithelial cells with an electron microscope or the like. “Induction of glomerular slit protein” means that the synthesis of the protein constituting the slit membrane is promoted, and the synthesis of the reduced protein in the slit membrane is promoted, or the formation of a new slit membrane. This includes the case where protein synthesis is promoted. Protein synthesis involves the process by which the slit protein gene is expressed and translated. “Maintenance of glomerular slit protein” means that the proteins constituting the slit membrane are kept in the same state and maintain the above-mentioned noria function in the slit membrane. Including suppression. “Proteinuria” refers to a state in which protein usually appears in a certain amount or more (usually about 15 OmgZ days) in urine. In the present invention, the above state is associated with a decrease in glomerular slit protein. Is mentioned. Proteinuria in the present invention includes all proteinuria related to glomerular slit protein reduction, such as proteinuria, orthostatic proteinuria, etc. found in kidney diseases associated with glomerular slit protein reduction. Asymptomatic proteinuria, physiological proteinuria, etc. found after intense exercise are included. To reduce glomerular slit protein, for example, when the slit film is quantitatively reduced due to damage, destruction, or decomposition of the slit film composed of the slit protein, the slit film is not quantitatively changed, but the slit film The case where the protein which comprises is reduced quantitatively is included. It also includes the case where the total amount of slit protein decreases as a result of the loss of survival and function of glomerular epithelial cells, which are slit-producing cells.

Diseases caused by mutations or decreases in slit protein include congenital childhood nephrotic syndrome, focal glomerulosclerosis, and the like. On the other hand, diseases in which a decrease in slit protein is a risk factor for progression of renal failure include diabetic nephropathy, acquired nephrotic syndrome (including minute changes or steroid resistance), renal diseases such as alport nephropathy, etc. Is included. The glomerular slit protein inducer and / or maintenance agent according to the present invention exerts an extremely excellent preventive or therapeutic effect on proteinuria associated with glomerular slit protein reduction and the like. The glomerular slit protein inducer and Z or maintenance agent of the present invention can be used for human forces \ non-human mammals (eg, monkeys, horses, horses, pigs, hidges, dogs, cats, rats, mice). , Usagi, Hamster, Guinea pig, Chimpanzee, etc.). When the glomerular slit protein inducer and / or maintenance agent of the present invention is administered to a patient, the administration form, administration method, dosage, etc. are the same when the active ingredient is HGF protein and DNA encoding HGF protein. The case may be slightly different.

For example, when the active ingredient is HGF protein, the preparation of the present invention can take various preparation forms, for example, liquids, solids, etc., but in general, only HGF protein is injected together with a conventional carrier. It is preferable to be formulated into an agent, propellant, sustained-release preparation (eg, depot). The injection may be either an aqueous injection or an oily injection. In the case of an aqueous injection, according to a known method, for example, an aqueous solvent (water for injection, purified water, etc.) is added to a pharmaceutically acceptable additive, for example, an isotonic agent (sodium sodium chloride, potassium chloride, glycerin, Mannitol, sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, tris buffer, glutamate) Buffer, epsilon aminocaproic acid buffer, etc.), preservatives (methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl parabenzoate, butyl parabenzoate, chlorobutanol, benzyl alcohol, benzoleocolium chloride, sodium dehydroacetate) , Sodium edetate, boric acid, borax, etc.), thickener (hydroxetyl se (Lulose, hydroxypropyl cellulose, polybulal alcohol, polyethylene glycol, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium quenate, ascorbic acid, dibutylhydroxytoluene, etc.) Prepared by dissolving HGF protein in a solution with appropriate addition of hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc., and then sterilizing by filtration with a filter etc., and then filling in an aseptic container. The power to do S. Further, an appropriate solubilizer such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, polyethylene glycol, etc.) or nonionic surfactant (polysorbate 80, polyoxyethylene hydrogenated castor oil 50, etc.) is further added. Also good. In the case of an oily injection, for example, sesame oil or soybean oil may be used as the oily solvent, and benzyl benzoate or benzyl alcohol may be added as a solubilizer. The prepared injection solution is usually filled in an appropriate ampoule or vial. The HGF protein content in the injection can be adjusted to generally about 0.0002-0. 2 w / v%, preferably about 0.001 to 0.1 lwZv%. In addition, liquid preparations such as injections It is desirable to store it after removing moisture by freeze storage or freeze drying. The freeze-dried product is used by re-dissolving and adding distilled water for injection at the time of use.

[0026] The HGF protein used in the present invention is a sustained-release preparation together with a biodegradable polymer.

(For example, a depot). By using HGF protein as a depot, effects such as reduction in the number of doses, sustained action and reduction of side effects can be expected. The sustained-release preparation can be produced according to a known method. The biodegradable polymer used in the sustained-release preparation can be appropriately selected from known biodegradable polymers. For example, polysaccharides such as starch, dextran, and chitosan; collagen, gelatin, etc. Proteins: Polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine; polylactic acid, polydaricholic acid, lactic acid-glycolic acid copolymer; poly force prolacton, poly-β-hydroxybutyric acid, polymalic acid, Polyanhydride or fumaric acid 'polyethylene glycol. Polyester such as butylpyrrolidone copolymer; Polyorthoester or polyalkyl cyanoacrylic acid such as polymethyl-α-cyanoacrylic acid; Polycarbonate such as polyethylene carbonate or polypropylene carbonate It is done. Preferred is polyester, and more preferred is polylactic acid or lactic acid darico-colic acid copolymer. When a lactic acid-glycolic acid copolymer is used, the composition ratio (lactic acid / daricholic acid) (mol%) varies depending on the sustained release period. For example, the sustained release period is about 2 weeks to 3 power months, preferably about In the case of 2 weeks to 1 power month, about 100/0 to 50/50 is preferred. The polylactic acid or lactic acid-glycolic acid copolymer generally has a weight average molecular weight of about 5,000 to 20,000 force S. Polylactic acid or lactic acid-glycolic acid copolymer can be produced according to a known production method, for example, the production method described in JP-A-61-28521. The mixing ratio of the biodegradable polymer and the HGF protein is not particularly limited. For example, the HGF protein is preferably about 0.01 to 30 wZw% with respect to the biodegradable polymer.

[0027] As an administration method, an injection is subcutaneously, intramuscularly, intravenously, or locally injected into the renal artery, or directly injected into a site close to the glomerulus of the kidney, or a sustained-release preparation (depot) is applied to the kidney. It is preferable to carry in the part close to the glomerulus. The dosage is appropriately selected according to the dosage form, the degree of disease, age, etc., but is usually about 1 μg to 500 mg, preferably about 10 xg to 100 mg, more preferably about 1 to 50 mg. In addition, the number of administration is the dosage form, It is appropriately selected depending on the degree of disease or age, etc., and can be administered once or continuously over a certain interval. In the case of continuous administration, the administration interval may be once a day to once every several months. For example, in the case of a sustained-release preparation (depot), it may be once every several months.

[0028] On the other hand, in order to administer the HGF gene to a patient, conventional methods such as separate experimental medicine, basic technology of gene therapy, Yodosha, 1996, separate experimental medicine, gene transfer & expression analysis experiment method, Yodosha 1997, The Gene Therapy Society of Japan, Gene Therapy Development Research Handbook, NTS, _{1999, and the} like.

Specific administration methods include, for example, a method in which an expression vector incorporating an HGF gene is locally injected into the kidney or nearby organ, or a naked HGF cDNA (plasmid) is directly injected from the renal artery, renal vein or ureter. For example, a method of performing the electoral position method on renal cells such as glomerular cells can be mentioned. Further, after removing cells from the patient's kidney tissue and the like, introducing the recombinant expression vector incorporating the HGF gene into the cells and transforming the cells, the transformed cells are transferred to the patient's kidney ( A method of transplanting to a glomerulus is preferable.

[0029] Expression vectors include naked plasmids, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses (type I herpes simplex viruses, etc.), vaccinia viruses, box viruses, polioviruses, syn By introducing the gene of interest into a DNA virus or RNA virus such as Vis virus, Sendai virus, SV40 or immunodeficiency virus (HIV) and infecting the cell with a recombinant virus, the HGF protein is encoded in the cell. It is possible to introduce DNA. Among them, type I herpes simplex virus (HSV_1) vector, adenovirus vector, adeno-associated virus (AAV) vector and the like are preferable.

[0030] As the preparation form of the HGF gene, various known preparation forms [for example, injections, sprays, sustained-release preparations (depots), microcapsules, etc.] suitable for each of the above administration forms are taken. You can get power S. Injections, sprays and sustained-release preparations (depots) can be prepared in the same way as for HGF protein. In addition, microcapsules are prepared by a known method (for example, core cell base) using, as a core substance, host cells into which an expression plasmid containing an HGF gene has been introduced. The particles can be produced as fine particles having a diameter of about:! To 500, preferably about 100 to 400 / im by covering with a coating material according to a method such as a suspension method, an interfacial polymerization method or a double nozzle method. Examples of the film material include carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, alginic acid or a salt thereof, gelatin, gelatin 'gum arabic, nitrosenolose, polybulal alcohol or hydroxypropyl cellulose, polylactic acid, polyglycolic acid, lactic acid. —Glycolic acid copolymer, chitosan-alginate, cellulose sulfate—metatalate, chitosan monocarboxymethyl cellulose, alginate monopolylysine monoalginate, and other film-forming polymers.

The content and dose of DNA in the preparation can be adjusted as appropriate depending on the disease to be treated, the age and weight of the patient. The dose varies depending on the type of HGF gene transfer vector, but it is usually 1 X 10 ⁶ pfu to 1 X 10 ¹² pfu, preferably 1 X 10 pfi! ~ 2 X 10 "pfu, more preferably 1.5 X 10 ⁷ pfu ~: · 5 X 10 ⁿ pfu¾r Preferably administered once every few days to several months.

[0031] The use of the present invention includes (1) (i) HGF protein or (oral) HGF protein partial peptide having substantially the same quality of activity as HGF protein, or a salt thereof, or ( 2) (i) DNA encoding HGF protein or (mouth) HGF protein partial peptide that encodes a peptide having substantially the same activity as HGF protein or (c) Complementary to these DNAs As an inducer or maintainer of glomerular slit protein of DNA that hybridizes with DNA consisting of a base sequence under stringent conditions and contains DNA encoding a protein or peptide having substantially the same activity as HGF protein Is the use of. The component (1) or (2) is used for producing a glomerular slit protein inducer or maintainer.

[0032] The method for inducing or maintaining glomerular slit protein of the present invention is a method of administering the above-mentioned component (1) or (2) to a mammal.

[0033] Furthermore, the use of the present invention is the use of the component (1) or (2) as a prophylactic or therapeutic agent for proteinuria associated with glomerular slit protein reduction. In addition, the production of a prophylactic or therapeutic agent for proteinuria associated with a decrease in glomerular slit protein of the above component (1) or (2). It is for use.

[0034] The method for preventing or treating proteinuria associated with the decrease in glomerular slit protein of the present invention is a method of administering the above-mentioned component (1) or (2) to a mammal.

[0035] The medicament and method of the present invention are suitable for use in humans having a disease caused by symptoms of proteinuria or mutation or decrease in slit protein. Proteinuria includes all proteinuria related to glomerular slit protein reduction, for example, asymptomatic such as proteinuria and orthostatic proteinuria seen in renal diseases associated with glomerular slit protein reduction. Proteinuria, physiological proteinuria, etc. found after intense exercise are included. In addition, diseases caused by mutations or decreases in slit protein include congenital childhood nephrotic syndrome, focal glomerulosclerosis, etc., and diabetic nephropathy, acquired nephrotic syndrome (microvariable or steroid). (Including resistance) and renal diseases such as Alport nephropathy.

Example

[0036] The present invention will be described below with reference to examples, but the present invention is not limited thereto. In the following examples, HGF is recombinant HGF (5-amino acid deficient HGF protein; HGF protein represented by SEQ ID NO: 2).

Example 1

Induction of glomerular slit protein expression in nephrotic syndrome model (ICGN mouse) by HGF administration

(Test solution)

It was prepared with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) so that the HGF concentration was 130 μg / mL.

(Method of administration)

Twelve 18-week-old ICGN mice that spontaneously developed nephrotic symptoms similar to Alport syndrome were used in the experiment. Six of them received HGF 500 gZkg / day subcutaneously every day for 4 weeks (up to 18 weeks of age) (HGF administration group). The remaining 6 mice were given subcutaneous saline alone for 4 weeks (up to 18 weeks of age) every day and used as a control group. In addition, 14-week-old ICGN mice (6 mice) before administration of HGF were used as a pre-dose control group, and 18-week-old ICR mice (6 mice) were used as a healthy control group (normal).

[0037] (Pathological specimen preparation) All mice were anesthetized after completion of dosing and necropsied. The removed kidney was immediately fixed in 70 (V / V)% ethanol, and paraffin sections were prepared according to a standard method. For the purpose of deparaffinizing the section and examining the expression of the slit protein, the primary antibody that is anti-nephrin monoremot IgG (PROGEN) or anti-CD2-related protein (CD2AP) Usagi IgG (Santa Cruz) is used at room temperature. The reaction was performed for 4 hours. Next, the reacted section was reacted at room temperature for 1 hour with a secondary antibody which was a piotine-labeled anti-guinea pig IgG (manufactured by CHEMICON) or a piotine-labeled anti-usagi IgG (manufactured by VectOT). This was thoroughly washed with phosphate buffered saline, and phosphate buffered saline containing avidin / peroxidase complex (Vector, Elite kit) was added onto the sections and reacted at room temperature for 1 hour. . Finally, the section thus treated was treated with phosphate buffered saline containing 3, 3'-daminobenzidine (Nacalai Testa Co., Ltd.) and hydrogen peroxide (Wako Pure Chemical Industries, Ltd.) and room temperature. For 4 to 8 minutes to visualize nephrin or CD2AP.

[0038] (Score unification)

Next, the kidney tissue of each mouse was observed with a microscope, and the expression intensity of nephrin or CD2AP was quantified according to the following score 1.

Score 1; no nephrin or CD2AP found in the entire glomerulus.

Score 1 0.5; Nephrin or CD2AP is found in less than one quarter of the glomeruli. Score 1; Nephrin or CD2AP is found in about one quarter to two quarters of the glomeruli.

Score 1 2: Nephrin or CD2AP is found in about 2/4 to 3/4 of all glomeruli.

Score 1 3: Nephrin or CD2AP is found in more than 3/4 of all glomeruli.

In the following, the nephrin expression intensity is unified as a nephrin score, and the CD2AP expression intensity is unified as a CD2AP score.

[0039] It should be noted that the numerical value is calculated by calculating the average score one after summing up the nephrin or CD2 AP scores for 20 or more glomeruli per mouse, and calculating the average nephrin score or the CD2AP score of the mouse. It was. Each group's score is shown as the average value (soil standard deviation) of each of the 6 mice. The student t_test is used to test the significance of differences between groups. went. Nephrin (Figs. 1 and 2) or CD2AP (Figs. 3 and 4), which decreases with the onset of nephrosis, was significantly increased by HGF administration. This indicates that administration of HGF to ICGN mice induces the slit proteins nephrin and CD2AP.

In the urine of 18-week-old ICGN mice (6 mice; control group) administered with physiological saline alone for 4 weeks daily and 18-week-old ICGN mice (6 mice; HGF-administered group) administered HGF for 4 weeks daily The protein concentration was measured. Urine was collected by bladder massage every 2 hours, and 12 doses were collected for 24 hours of urine. The protein in the urine for 24 hours was measured using A / GB test KOKO (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown as a scatter plot of urinary protein concentration and nephrin or CD2AP score (Figures 5 and 6). There was a negative correlation between urinary protein levels and nephrin or CD2AP scores. This indicates that the increase in urinary protein concentration is the same as the increase in nephrin or CD2AP score. In the HGF administration group, a decrease in urinary protein was observed with an increase in nephrin or CD2AP score as compared to the control group.

Example 2

Induction of glomerular slit protein expression in diabetic nephropathy (streptozotocin-treated mice) by HGF administration

(Test solution)

It was prepared with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) so that the HGF concentration was 80 μg / mL.

(Test method)

Streptozotocin (STZ) is administered intraperitoneally to 120 mg / kg, once daily for 2 days, and 80 mg / kg, once daily for 2 days, to 8 week old C57 / B6 female mice. More induced diabetes. The mice (12 mice) that induced diabetes were bred for 6 weeks (up to 14 weeks of age), and 6 mice were subcutaneously administered HGF 300 μg / kg / 12 hours for 4 weeks (up to 18 weeks of age) from the 7th week. (HGF administration group). The remaining 6 mice received subcutaneous saline alone for 4 weeks (up to 18 weeks of age) instead of HGF and used as a control group. Furthermore, diabetic mice (6 mice) 6 weeks after S TZ administration before HGF administration were used as a pre-administration control group and age-matched normal C57ZB6 mice (6 mice) as a healthy control group.

Next, pathological specimens were prepared in the same manner as described above, and scores were unified. Diabetes Decreased nephrin (Figs. 7 and 8) or CD2AP (Fig. 9) force was significantly increased by HGF administration. From this, it was found that administration of HGF induces the slit proteins nephrin and CD 2AP.

In addition, the urinary albumin concentration of 18-week-old STZ-administered mice (6 mice) administered with saline alone and 18-week-old STZ-administered mice (6 mice) administered HGF for 4 weeks daily was measured daily. Set. Urine was collected every 2 hours by bladder massage, and 6 doses were collected for 12 hours of urine. The albumin in the urine for 12 hours was measured using AZGB test KOKO (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown as a scatter plot of urinary albumin concentration and nephrin or CD2AP score (Figure 10 or 11). A negative correlation was observed between urinary albumin concentration and nephrin or CD2AP scores. This indicates that an increase in nephrin or CD2AP score results in a decrease in urinary albumin concentration. In the HGF group, a decrease in urinary albumin was observed with an increase in nephrin or CD2AP score compared to the control group.

Industrial applicability

The glomerular slit protein inducer and / or maintenance agent of the present invention can be used as a medicament for the prevention or treatment of proteinuria.

Claims

The scope of the claims

[1] (1) (i) HGF protein or (oral) peptide that is a partial peptide of HGF protein and has substantially the same activity as HGF protein, or a salt thereof, or (2) (i) HGF protein DNA encoding (or) a partial peptide of the (oral) HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to these DNAs Induction of glomerular slit protein characterized in that it contains DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein. Agent or maintenance agent.

[2] The active ingredient contains (i) an HGF protein or a (mouth) HGF protein partial peptide having substantially the same activity as the HGF protein or a salt thereof. The glomerular slit protein inducer or maintainer according to claim 1, wherein

[3] HGF protein strength (a) A protein comprising an amino acid sequence identical to the amino acid sequence represented by SEQ ID NO: 1, 2, or 5 or (b) substantially the same amino acid sequence as (a) above 3. The glomerular slit protein inducer or maintainer according to claim 2 characterized by the above.

[4] As an active ingredient, (i) DNA encoding HGF protein or (mouth) DNA encoding partial peptide of HGF protein which has substantially the same activity as HGF protein or (c) A DNA encoding a protein or peptide that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA and has substantially the same activity as the HGF protein. 2. An inducer or maintainer for glomerular slit protein according to item 1.

[5] DNA force encoding HGF protein (a) DNA consisting of the base sequence represented by SEQ ID NO: 3, 4 or 6 or (b) DNA consisting of a base sequence complementary to the base sequence of (a) above The glomerular slit protein according to claim 4, wherein the glomerular slit protein comprises a DNA that encodes a protein that hybridizes under stringent conditions to the protein and has substantially the same activity as the HGF protein. Inducing agent or maintenance agent.

[6] The DNA is incorporated into a naked plasmid, adenovirus vector, adeno-associated virus vector, or type I herpes simplex virus (HSV-1) vector. The inducer or maintainer of glomerular slit protein according to claim 4.

[7] Glomerular slit protein strength Nephrin, podocine, CD2-related protein, etc. on the membrane surface of the glomerular epithelial cell and / or in the cell, and the protein constituting the slit itself or associated with this stabilizes the slit. 2. The glomerular slit protein inducer or maintainer according to claim 1, wherein the glomerular slit protein inducer or maintainer is a protein to be produced.

[8] (1) (i) HGF protein or (oral) Peptide having partial activity of HGF protein and having substantially the same activity as HGF protein, or a salt thereof, or (2) (i) HGF protein DNA encoding (or) a partial peptide of the (oral) HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to these DNAs Reduced glomerular slit protein characterized by containing DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein A prophylactic or therapeutic agent for proteinuria related to.

[9] (1) (i) HGF protein or (oral) peptide that is a partial peptide of HGF protein and having substantially the same activity as HGF protein, or a salt thereof, or (2) (i) HGF protein DNA encoding (or) a partial peptide of the (oral) HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to these DNAs Induction of glomerular slit protein characterized by administering DNA containing a DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein. Introduction method or maintenance method.

[10] (1) (i) HGF protein or (oral) peptide that is a partial peptide of HGF protein and having substantially the same activity as HGF protein, or a salt thereof, or (2) (i) HGF protein DNA encoding (or) a partial peptide of the (oral) HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to these DNAs For the production of a glomerular slit protein inducer or maintainer of DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein. use.

[11] (1) (i) HGF protein or (oral) peptide that is a partial peptide of HGF protein and having substantially the same activity as HGF protein, or a salt thereof, or (2) (ii) HGF protein DNA encoding (or) a partial peptide of the (oral) HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to these DNAs Glomerular slit protein reduction characterized by administering to mammals DNA containing DNA encoding a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein A method for the prevention or treatment of proteinuria related to the disease

[12] (1) (i) HGF protein or (oral) peptide that is a partial peptide of HGF protein and having substantially the same activity as HGF protein, or a salt thereof, or (2) (i) HGF protein DNA encoding (or) a partial peptide of the (oral) HGF protein and a DNA encoding a peptide having substantially the same activity as the HGF protein, or (c) DNA comprising a nucleotide sequence complementary to these DNAs A prophylactic or therapeutic agent for proteinuria associated with a decrease in glomerular slit protein of DNA containing DNA that encodes a protein or peptide that hybridizes under stringent conditions and has substantially the same activity as HGF protein Use for manufacturing.