JP2009201364A - Diabetic nephropathy-sensitivity gene, and method for screening active ingredient of agent for preventing or treating diabetic nephropathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for searching a material capable of being an active ingredient for preventing or treating the onset or progress of diabetic nephropathy; to provide a method for selecting a patient having high latent risk of developing the diabetic nephropathy; and to provide a reagent kit capable of being effectively utilized for the method. <P>SOLUTION: The method for screening the active ingredient of an anti-diabetic nephropathy agent includes the following steps: (1) a step for bringing a test material into contact with a cell capable of expressing an ACC-β gene; (2) a step for measuring the expression level of the ACC-β gene of the cell brought into contact with the test material; and (3) a step for selecting the test material having the measured value smaller than the expression level of the ACC-β gene of a control cell not contacted with the test material. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention provides new knowledge regarding genes associated with the onset and progression of diabetic nephropathy. Based on this new knowledge, the present invention specifically relates to a method of screening for components useful for preventing or treating the onset and progression of diabetic nephropathy. Furthermore, the present invention provides a method for determining whether a subject has a high probability of developing diabetic nephropathy and selecting a patient with a high potential risk of developing diabetic nephropathy, and effectively uses the method. The present invention relates to a reagent kit that can be used.

  Diabetic nephropathy is the leading cause of end stage renal failure worldwide. Even in Japan, since 1998, diabetic nephropathy has overtaken chronic glomerulonephritis and has become the leading cause of new dialysis introduction, and in 2003 it has increased to over 40% of dialysis introduction patients. According to the results of the United Kingdom Prospective Diabetes Study (UKPDS), diabetic nephropathy patients progress to the next stage at a rate of 2-3% per year, and the annual mortality rate after the renal failure stage is about 20 %, And the prognosis is extremely poor. Therefore, it is very important from the viewpoint of both patient prognosis and medical economy to suppress the development of nephropathy and the introduction of dialysis by appropriate therapeutic intervention from the early stage (see Non-Patent Document 1). Based on the results of recent epidemiological studies and family surveys on diabetic nephropathy, nephropathy is restricted to some diabetic patients, and nephropathy is found in families with nephropathy. The existence of some genetic factor is suggested for the onset and progress of the disease, and many genes have been examined as a candidate gene for susceptibility to diabetic nephropathy. By elucidating the genetic factors of such diabetic nephropathy, it becomes possible to predict and select treatment methods for individual cases in advance, so-called customized medicine can be realized. However, it has not yet been clearly identified as a gene from which a consensus as a candidate gene for susceptibility to diabetic nephropathy can be obtained.

  The most well analyzed gene as the candidate gene is the 287 base pair insertion (I) / deletion (D) gene polymorphism present in the 16th intron of the angiotensin converting enzyme (ACE) gene. In normal case-control studies, there were many results showing a correlation between the D allele and nephropathy, and a meta-analysis of 18 case-control studies and a prospective follow-up study of type 1 diabetes cases revealed that the D allele was Although it is considered to be a risk gene for symptom, it is considered difficult to consider a so-called major gene (see Non-Patent Document 2).

  In recent years, the NCALD gene has been found as the candidate gene. Specifically, the gene is a diabetic nephropathy susceptibility gene that is closely related to the onset and progression of diabetic nephropathy, and a method for preventing or treating the onset and progression of diabetic nephropathy is developed. It has been reported that 3SNPs (SNP999, SNP1307, SNP1340) located within the NCALD gene have a significant relationship with the potential risk of developing diabetic nephropathy. (See Patent Document 1).

By the way, acetyl-CoA carboxylase (also referred to as “ACC” in the present invention) is an important intermediate in fatty acid synthesis by binding CO ₂ to the acetyl group of acetyl-CoA. It is known as an enzyme that catalyzes the synthesis of CoA (Human ACC-β: GenBank Accession No. NM — 001093, AC007637). This enzyme is considered to be a rate-determining enzyme for fatty acid biosynthesis and regulates the fatty acid biosynthesis rate. Its activity is promoted by citric acid.

  There are two types of ACCs: ACC-α (also referred to as “ACC-1”) and ACC-β (also referred to as “ACC-2” or “ACACB”). The former ACC-α is highly expressed in the liver, adipose tissue, and lactating mammary gland. On the other hand, the latter ACC-β is mainly expressed in skeletal muscle and myocardium and regulates β-oxidation of fatty acid in mitochondria by producing malonyl-CoA as an enzyme involved in fatty acid synthesis. It has been known.

  Recent studies have reported that the ACC-β is closely related to obesity and diabetes. Specifically, mice lacking the ACC-β gene (ACC-β knockout mice) do not get fat even when fed a high-fat, high-carbohydrate diet, so ACC-β can be a target for anti-obesity drugs (See Non-Patent Document 3). Moreover, since the inhibitor of ACC-β suppresses an increase in fat and improves insulin sensitivity, ACC-β is considered to be a target for diabetes drugs (see Non-Patent Document 4).

In subsequent studies, the correlation between the ACC-β gene (single nucleotide polymorphism) and the risk of myocardial infarction has been reported (see Patent Document 2), but the relationship with diabetic nephropathy has been reported. Absent.
"Diabetic complications" latest medicine separate volume ABC of new diagnosis and treatment (30), published by the latest medicine company, page 101 "Diabetic complications" latest medicine separate volume ABC of new diagnosis and treatment (30), published by the latest medicine company, page 93 Abu-Elheiga et al., 2001, Science, 291: 2613-2616 Harwood et al. Biol.Chem. 278: 37099-37111 JP 2007-129905 A International Publication (WO2004 / 058052)

  As mentioned above, elucidating the genetic factors of diabetic nephropathy and clarifying the relationship between the onset of diabetic nephropathy and its progression is to select the most appropriate treatment for each individual case (customized medicine) At the same time as providing a means to search for active ingredients in prophylactic and therapeutic agents against the onset and progression of diabetic nephropathy, and to enable the development of specific and highly safe pharmaceuticals It is thought to do.

  An object of the present invention is to provide a gene related to the onset and progression of diabetic nephropathy (hereinafter also referred to as “diabetic nephropathy susceptibility gene”), and the gene and diabetic nephropathy The purpose is to clarify the relationship between the onset and its progression. Another object of the present invention is to provide a method (screening method) for searching for a substance that can be an active ingredient for preventing or treating the onset and progression of diabetic nephropathy.

  Furthermore, the present invention provides a method for determining whether a subject has a high probability of developing diabetic nephropathy and selecting a patient with a high potential risk of developing diabetic nephropathy, and effectively uses the method. It aims at providing the reagent kit which can be utilized.

  In order to achieve the above object, the present inventors conducted genome-wide correlation analysis on diabetic nephropathy. As a result, SNP (single nucleotide polymorphism) that is strongly associated with diabetic nephropathy is human acetyl- 1 position [+4139 position G / A (SNP4139)] in intron 18 region (SEQ ID NO: 1) of CoA carboxylase gene (human acetyl-Coenzyme A carboxylase-β) (also referred to as “ACC-β gene” in the present invention) However, the A allele carrier having adenine (A) at the SNP locus (SNP4139) has a significantly higher risk of developing diabetic nephropathy than the non-A allele carrier (G allele carrier). Found it expensive. In addition, according to the haplotype of the ACC-β gene having adenine (A) at the SNP locus (SNP4139), the present inventors significantly increased the expression level of ACC-β, and excessively increased ACC-β. Among the expressed cells, osteopontin (Kidney International, Vol.63 (2003), p.454-463), a human monocyte chemotactic factor known as a marker gene (protein) of diabetic nephropathy The expression levels of MCP-1 (Kidney International, Vol.58 (2000), p.1492-1499) and IL-6 (Am J Nephrol 2006, 26, p.562-570) are also increasing. It was confirmed. This suggests that the increase (increase) in the expression of the ACC-β gene caused by the A allele of SNP (SNP4139) is related to the onset and progression of diabetic nephropathy.

  Based on these findings, the present inventors have proposed that the ACC-β gene is a diabetic nephropathy susceptibility gene that is closely related to the onset and progression of diabetic nephropathy, and prevents or treats the onset and progression of diabetic nephropathy. I was convinced that it would be a useful target gene for developing a method to do so. Furthermore, the present inventors have found a relationship between SNP (SNP4139) having a significant association with diabetic nephropathy located within the ACC-β gene and the potential risk of developing diabetic nephropathy, and from now on the subject It was convinced that the potential risk of developing diabetic nephropathy in the subject can be evaluated by measuring the base (G / A) of the SNP.

  The present invention has been completed based on such findings, and has the following specific embodiments.

I. Method for screening an effective component for prevention or treatment of diabetic nephropathy <1> A method for screening a preventive or therapeutic agent for diabetic nephropathy having the following steps:
(1) contacting the test substance with a cell capable of expressing the ACC-β gene;
(2) a step of measuring the expression level of the ACC-β gene in the cell contacted with the test substance; and (3) the measurement amount is higher than the expression level of the ACC-β gene in the control cell not contacted with the test substance. Selecting a small test substance.

Item 2. A method for screening a preventive or therapeutic agent for diabetic nephropathy having the following steps:
(1 ′) contacting a test substance with a cell capable of producing ACC-β or a cell fraction prepared from the cell;
(2 ′) a step of measuring the amount of ACC-β produced in the cell contacted with the test substance or the cell fraction thereof, and (3 ′) a control cell in which the test substance is not contacted with the test substance or a cell fraction thereof A step of selecting a test substance smaller than the amount of ACC-β produced per minute.

Item 3. A method for screening a preventive or therapeutic agent for diabetic nephropathy having the following steps:
(1 ″) contacting a test substance with a cell capable of producing ACC-β or a cell fraction prepared from the cell,
(2 ″) a step of detecting the ACC-β action of the cell contacted with the test substance or the cell fraction thereof; and (3 ″) a control cell or a cell fraction thereof wherein the detected action does not contact the test substance. A step of selecting a test substance lower than the action of ACC-β in minutes.

  Item 4. The action of ACC-β increases the expression level of any of the diabetic nephropathy-related factors selected from the group consisting of (1) osteopontin, MCP-1 and IL-6, (2) malonyl CoA Item 4. The screening method according to Item 3, wherein the method comprises: (3) inhibiting β-oxidation of fatty acids.

  Item 5. Item 4. The screening method according to any one of Items 1 to 3, wherein the cell capable of expressing the ACC-β gene or the cell capable of producing ACC-β is a tubular epithelial cell.

  Item 6. Item 5. The screening method according to any one of Items 1 to 4, which is a method for searching for an active ingredient of a prophylactic or therapeutic agent for diabetic nephropathy.

II. Item 7. Selection Method for Subjects with High Risk of Diabetic Nephropathy A step of measuring the mRNA expression level of the ACC-β gene in a biological sample derived from a subject (test sample), wherein the expression level in the test sample is ACC-β in a biological sample derived from a normal person (control sample) A method for selecting a subject who is highly likely to suffer from diabetic nephropathy, wherein an index that indicates that the subject is more likely to suffer from diabetic nephropathy is greater than the mRNA expression level of the gene.

  Item 8. A step of measuring a production amount of ACC-β in a biological sample derived from a subject (test sample), wherein the production amount in the test sample is a production of ACC-β in a biological sample derived from a normal person (control sample) A method for selecting subjects who are highly likely to suffer from diabetic nephropathy, wherein an amount greater than the amount is an indicator that the subject is likely to suffer from diabetic nephropathy.

  Item 9. Including a step of detecting the 4139th base present in intron 18 of the ACC-β gene in a biological sample (test sample) derived from a subject, and the subject is suffering from diabetic nephropathy that the base is adenine A method for selecting subjects who are highly likely to suffer from diabetic nephropathy, which is an indicator of high possibility.

III. 10. Diagnosis risk diagnosis kit for diabetic nephropathy A diagnostic kit for susceptibility to diabetic nephropathy, comprising a reagent for detecting the base at position 4139 present in intron 18 of the ACC-β gene.

  According to the screening method of the present invention, it is possible to obtain an effective component for preventing or treating the onset and progression of diabetic nephropathy. In addition, this method can be effectively used for the development of new drugs effective for the prevention or treatment of diabetic nephropathy.

  Furthermore, according to the diabetic nephropathy susceptibility gene and the diabetic nephropathy susceptibility SNP provided by the present invention, it is possible to easily detect the relative risk of developing nephropathy in a subject (particularly a diabetic patient). The detection method of the present invention is a method that can be easily carried out in vitro and without requiring specialized knowledge of a doctor or the like. For a subject (diabetic patient) who has been found to have a relatively high potential risk of developing nephropathy by the method of the present invention, the fact is notified and the onset of nephropathy is prevented in advance, or Appropriate measures can be taken to delay even a little. Therefore, the present invention is extremely useful as a test method for preventing the onset of diabetic nephropathy or for delaying the onset of diabetic nephropathy. Furthermore, this invention provides the reagent used in the said method, and it becomes possible to implement the said method simply by this.

1. Definition of terms used in the present invention Indications by abbreviations such as nucleotide sequences (nucleotide sequences) and nucleic acids in the present specification are defined by IUPAC-IUB [IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and conventional symbols in the field.

  In the present specification, unless otherwise specified, “gene” refers to double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand), and single-stranded DNA having a sequence complementary to the sense strand ( Antisense strands) and fragments thereof are included, and the term “gene” in the present specification indicates the regulatory region, coding region, exon, and intron without distinction unless otherwise specified. And

  Further, in this specification, “nucleotide”, “oligonucleotide” and “polynucleotide” are synonymous with nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.

  As used herein, “gene polymorphism” refers to alleles when there are two or more genetically determined alleles. Specifically, in a human population, one or more nucleotide substitutions, deletions, insertions, translocations, reverses at specific sites in one or more other individual genomes based on the genome sequence of one individual. When there is a mutation such as a position, it is statistically certain that the mutation is not a mutation that has occurred in the individual or individuals, or within the population at a frequency of 1% or more, not a specific mutation within the individual. If it is pedigree that it is present in the family, the mutation is referred to as a “gene polymorphism”. As used herein, the term “gene polymorphism” includes a so-called single nucleotide polymorphism (SNP), which is a polymorphism caused by a single nucleotide change. A “single nucleotide polymorphism” is a polymorphism caused by a single nucleic acid change. Polymorphisms are present at a frequency greater than 1%, preferably greater than 10% of the selected population. The “haplotype” refers to a combination of gene mutations existing in one allele (haploid).

  As used herein, “diabetic nephropathy susceptibility gene” refers to a gene that determines a predisposition to develop nephropathy or a predisposition to progress to nephropathy among diabetic patients.

  As used herein, “diabetic nephropathy” refers to a condition in which diabetes progresses and affects the kidneys, resulting in renal damage accompanied by proteinuria. Patients with diabetic nephropathy suffer from impaired renal function due to hyperglycemia, which impairs the ability to make raw urine, eventually resulting in renal failure. According to statistics, the number of patients who begin dialysis treatment for end-stage renal failure due to diabetic nephropathy is increasing year by year, and currently, diabetic nephropathy is ranked first as a causative disease for dialysis patients. More specifically, the “diabetic nephropathy” targeted by the present invention includes diabetic retinopathy and a urinary albumin excretion rate of 200 μg / min or higher, or a urinary albumin / creatinine ratio. Cases that are greater than or equal to 300 mg / gCr and cases that develop diabetic retinopathy and are receiving dialysis therapy are included.

  Each base number described in this specification is based on positional information of The National Center for Biotechnology Information data base. In this specification, position 4139, which is the position number of each SNP, means position information when the first base of intron 18 of the ACC-β gene is 1. The base sequence of intron 18 of the ACC-β gene is shown in SEQ ID NO: 1.

2. Diabetic nephropathy susceptibility gene and diabetic nephropathy susceptibility SNP
The present inventors conducted case-control correlation analysis and linkage disequilibrium (LD) mapping using gene-based SNPs in many Japanese patients with type 2 diabetes to search for diabetic nephropathy susceptibility genes. By doing so, intron 18 of human acetyl-Coenzyme A carboxylase-β gene (ACC-β gene) present in the chromosome 12 q24.11 (12q24.11) region A patient who has found one SNP (SNP4139) present on the base sequence and develops diabetic nephropathy has a significantly high frequency that the SNP is adenine (A). Is significantly related to the onset and progression of diabetic nephropathy (diabetic nephropathy susceptibility SNP). In addition, the present inventors also show that in the system overexpressing ACC-β, the expression of osteopontin, MCP-1 and IL-6, which are known as related factors of diabetic nephropathy, are also increased. From the above, it was found that the increase (increase) in the expression of the ACC-β gene is deeply related to the onset of diabetic nephropathy, and the ACC-β gene is closely related to the onset and progression of diabetic nephropathy. It was concluded that it is a disease susceptibility gene.

  The human ACC-β gene is a 128821 bp gene located at nucleotide numbers 108061594 to 108190414 in the genome sequence of human chromosome 12 (Genbank Sequence Accession IDs: NC-000012.10).

  Regarding the association between the ACC-β gene and the disease, conventionally, the association with obesity and diabetes (see Non-Patent Documents 3 and 4) and myocardial infarction (Patent Document 2) have been reported. No association has been reported with the disease.

  Examples of factors associated with increased (increased) expression of the ACC-β gene include the above-described diabetic nephropathy-susceptible SNPs. The SNP is located at position 4139 (G / A) (SNP4139) of intron 18 of the ACC-β gene.

  As shown in Example 1, type 2 diabetic patients in which SNP4139 has an adenine (A) haplotype have increased (increased) expression of the ACC-β gene and tend to develop nephropathy. Accordingly, the diabetic nephropathy-susceptible SNP is a genetic predisposing marker for diabetic nephropathy that regulates the ease of onset or progression of diabetic nephropathy, and is diabetic for human subjects, particularly type 2 diabetic patients. It is an index for judging the onset and progression of nephropathy.

  That is, when the base of the diabetic nephropathy-susceptible SNP (SNP4139) is adenine (A), the subject having the SNP is genetically prone to develop diabetic nephropathy or develops from diabetes to nephropathy. It can be judged that it has a constitution that is easy to progress (sensitive to diabetic nephropathy).

  One mechanism that the SNP is involved in the susceptibility to diabetic nephropathy is that rs2268388, a region containing SNP (SNP4139), has enhancer activity (see Example 7). It is speculated that it has some influence on the regulation of expression.

3. Method for Screening Components Effective for Prevention or Treatment of Diabetic Nephropathy As described above, the present inventors have shown that the increase (increase) in the expression of ACC-β gene is related to the onset and progression of diabetic nephropathy. I found that I was deeply involved. Therefore, ACC-β is involved in the suppression of the onset or progression of diabetic nephropathy, and has the effect of suppressing the production of ACC-β gene (mRNA expression) and reducing the production of ACC-β. A substance or a substance having an action of reducing the action of ACC-β is considered to be useful as an effective component for the prevention or treatment of diabetic nephropathy.

  The screening method of the present invention described below includes (3-1) a substance having an action of suppressing the expression (mRNA expression) of ACC-β gene, and (3-2) production of ACC-β among test substances. By searching for substances that have the effect of reducing the amount, or (3-3) substances that have the action of reducing the action of ACC-β, prophylactic or therapeutic agents for diabetic nephropathy (hereinafter collectively referred to as these) Thus, an active ingredient of an “anti-diabetic nephropathy agent”) is to be obtained.

  Examples of candidate substances that can be active ingredients of antidiabetic nephropathy agents include nucleic acids, peptides, proteins, organic compounds (including low molecular compounds and high molecular compounds), inorganic compounds, and the like. The screening method of the present invention can be carried out on samples containing these candidate substances (collectively referred to as “test substances”). Here, the sample containing the candidate substance includes a cell extract, an expression product of a gene library, a microorganism culture supernatant, a cell component, and the like.

(3-1) Method for screening substance having action of suppressing expression of ACC-β gene In the screening, a substance having an action of suppressing the expression of ACC-β gene is selected from the test substances as ACC-β mRNA. Is used as an index, and is obtained as an active ingredient of an antidiabetic nephropathy agent.

Specifically, the method can be carried out by performing the following steps (1) to (3).
(1) contacting the test substance with a cell capable of expressing the ACC-β gene;
(2) a step of measuring the expression level of the ACC-β gene in the cell contacted with the test substance; and (3) the measurement amount is higher than the expression level of the ACC-β gene in the control cell not contacted with the test substance. Selecting a small test substance.

  The cells used for such screening may be cells that can express the ACC-β gene, regardless of whether they are natural or recombinant. The origin of the ACC-β gene is not particularly limited, and it may be derived from humans, or may be derived from mammals such as mice other than humans or other biological species, but preferably human-derived ACCs. -β gene. Specific examples of such cells include tubular epithelial cells (including those derived from humans and other species) having the ACC-β gene, and primary cultured cells of isolated tubular epithelial cells. Can do.

  In addition, a transformed cell prepared by introducing an expression vector having the cDNA of the ACC-β gene and expressing ACC-β mRNA in accordance with a standard method can also be used. The category of cells used for screening includes tissues that are aggregates of cells.

  In the step (1) of the screening method (3-1) of the present invention, the condition for bringing the test substance into contact with the cell capable of expressing the ACC-β gene is not particularly limited, but the cell does not die and ACC-β It is preferable to select culture conditions (temperature, pH, medium composition, etc.) in which the gene can be expressed.

  The candidate substance is selected by, for example, contacting a test substance with a cell capable of expressing the ACC-β gene under the above conditions, and searching for a substance that suppresses the expression of the ACC-β gene and decreases the expression level of the mRNA. It can be carried out. Specifically, when ACC-β gene-expressable cells are cultured in the presence of a test substance, the ACC-β gene-expressing cells corresponding to the above are cultured in the absence of the test substance. The test substance that has been brought into contact with the cells can be selected as a candidate substance, using as an index the expression level (control expression level) of ACC-β mRNA obtained in this case.

  The measurement (detection and quantification) of the expression level of ACC-β mRNA is performed by measuring the expression level of ACC-β mRNA in an ACC-β gene-expressible cell with an oligonucleotide complementary to the base sequence of the ACC-β mRNA. It can be carried out by carrying out a known method such as Northern blot method using RT and the like, RT-PCR method, real-time quantitative PCR method, or measurement method using DNA array.

(3-2) Screening method for substances having an action to reduce ACC-β production The screening is carried out by selecting a substance having an action to reduce the production of ACC-β from the test substances, and determining the amount of ACC-β produced. Is used as an index, and is obtained as an active ingredient of an antidiabetic nephropathy agent.

The said method can be specifically implemented by performing the following process (1 ')-(3').
(1 ′) contacting a test substance with a cell capable of producing ACC-β or a cell fraction prepared from the cell,
(2 ′) a step of measuring the amount of ACC-β produced in the cell or cell fraction contacted with the test substance, and (3 ′) a control cell (ACC-β is not contacted with the test substance). A step of selecting a test substance that is smaller than the amount of ACC-β produced in a cell fraction (prepared from cells capable of producing ACC-β) that is not brought into contact with the test substance.

  The cells (including control cells) used for such screening may be cells that can express ACC-β and produce ACC-β regardless of whether it is natural or recombinant. The origin of the ACC-β gene is not particularly limited, and it may be derived from humans, or may be derived from mammals such as mice other than humans or other biological species, but preferably human-derived ACCs. -β gene. Specific examples of such cells include tubular epithelial cells (including those derived from humans and other species) having the ACC-β gene, and primary cultured cells of isolated tubular epithelial cells. Can do.

  In addition, a transformed cell prepared by introducing an expression vector having the cDNA of the ACC-β gene and capable of producing ACC-β according to a standard method can be used. The category of cells used for screening includes tissues that are aggregates of cells.

  In the step (1 ′) of the screening method (3-2) of the present invention, the condition for bringing the test substance into contact with the ACC-β-producing cell is not particularly limited, but the cell does not die and ACC-β It is preferable to select culture conditions (temperature, pH, medium composition, etc.) in which the gene is expressed and ACC-β can be produced.

  Selection of a candidate substance can be performed by, for example, contacting a test substance with a cell capable of producing ACC-β or a cell fraction thereof under the above conditions to search for a substance that reduces the amount of ACC-β produced. Specifically, the amount of ACC-β produced when ACC-β-producing cells or cell fractions thereof are cultured in the presence of the test substance is the ACC-β-producing cell corresponding to the above in the absence of the test substance or Using the ACC-β production amount (control production amount) obtained when the cell fraction is cultured as an index, the cell or the test substance brought into contact with the cell fraction can be selected as a candidate substance. it can.

  For the measurement (detection, quantification) of the ACC-β production amount, the amount of ACC-β obtained from the ACC-β-producing cell or its cell fraction is determined using an antibody against the ACC-β (anti-ACC-β antibody). It can be carried out by performing a known method such as Western blotting, immunoprecipitation or ELISA.

(3-3) Method for screening substance having action of reducing action of ACC-β In the screening, a substance having action of reducing the action of ACC-β is selected from the test substances, and the action of ACC-β is changed. It is a method of searching as an index and obtaining it as an active ingredient of an antidiabetic nephropathy agent.

Specifically, this method can be carried out by performing the following steps (1 ″) to (3 ″).
(1 ″) contacting a test substance with a cell capable of producing ACC-β or a cell fraction prepared from the cell,
(2 ″) a step of detecting the action of ACC-β of the cell or cell fraction contacted with the test substance, and (3 ″) a control cell that does not contact the test substance (ACC-β A test substance having a lower action than ACC-β in a cell capable of producing) or a cell fraction (prepared from a cell capable of producing ACC-β).

  As the cells used in the screening, and the method and conditions for contacting with the test substance in the step (2 ″), those described in the screening method of (3-2) described above can be used similarly.

  Selection of a candidate substance can be performed by contacting a test substance with a cell capable of producing ACC-β or a cell fraction thereof under the above conditions to search for a substance that reduces the action of ACC-β. Specifically, the ACC-β-producing cell corresponding to the above in the absence of the test substance is the action of ACC-β that occurs when the ACC-β-producing cell or a cell fraction thereof is cultured in the presence of the test substance. Alternatively, the test substance can be selected as a candidate substance using as an index the lower than the action (control action) of ACC-β obtained when the cell fraction is cultured.

  Here, as the action of ACC-β, in addition to increasing the expression level of diabetic nephropathy-related factors such as osteopontin, MCP-1 and IL-6, it has been conventionally known as the action of ACC-β. For example, an enzymatic action such as the production of malonyl-CoA, and an action of inhibiting fatty acid β-oxidation (Advanced Drug Delivery Reviews, Vol. 54 (2002), 1199-1212, etc.). In addition, the measurement of the expression level of the said protein or gene which is a related factor of diabetic nephropathy is possible according to the well-known method of those skilled in the art. The production of malonyl CoA and the inhibition of fatty acid β oxidation can also be measured by known methods.

The substance selected by the screening method of (3-1) to (3-3) described above reduces the production of ACC-β by suppressing the expression of the ACC-β gene in tubular epithelial cells, or reduces the production of ACC- It has an action of reducing the action of β, and can be used as an active ingredient of a composition for preventing the onset and progression of diabetic nephropathy or a composition for improving diabetic nephropathy The candidate substance selected by the screening method can be further screened using a non-human animal having a diabetic nephropathy. The candidate substance thus selected is a drug efficacy test using a non-human animal having a diabetic nephropathy, a safety test, a patient having a diabetic nephropathy (human), or a patient in a previous state (human) The clinical components may be subjected to clinical trials, and by carrying out these tests, it is possible to select and obtain active ingredients of a more practical composition for preventing or treating diabetic nephropathy.

  The material selected in this way is subjected to structural analysis as necessary, and then, depending on the type of the material, chemical synthesis, biological synthesis (including fermentation), or genetic engineering, And can be used to prepare a composition for the prevention and treatment of diabetic nephropathy.

4). Method for selecting test subject (diabetic nephropathy susceptible person) having high risk of suffering from diabetic nephropathy The present invention also relates to the subject's risk of developing / progressing diabetic nephropathy (prone to or easily develop diabetic nephropathy) A method of selecting a subject having a high risk from the subject. In the present invention, these high-risk subjects are referred to as “diabetic nephropathy susceptible persons”.

  As a screening method, (4-1) ACC-β gene expression level (mRNA expression level) is used as an index for test samples of subjects, and (4-2) ACC-β production level is used as an index. And (4-3) an ACC-β gene diabetic nephropathy susceptibility SNP as an index.

(4-1) Method for selecting a person susceptible to diabetic nephropathy using the expression level of ACC-β gene (mRNA expression level) as an index The method comprises the expression level of ACC-β gene in a biological sample derived from a subject, This can be done by measuring the expression level of ACC-β mRNA. Here, the biological sample may be a sample containing cells capable of expressing the ACC-β gene, and specific examples thereof include blood.

  The method for measuring the expression level of ACC-β mRNA can be performed according to a conventional method as described in the method (3-1) above.

  As a result of such measurement, when the expression level of ACC-β mRNA in the biological sample (test sample) of the subject is greater than the expression level (control expression level) of ACC-β mRNA in the biological sample (control sample) derived from a normal person In addition, it can be determined that the subject is likely to suffer from diabetic nephropathy (susceptibility to diabetic nephropathy).

  Here, “normal” is used in the sense that at least nephropathy has not occurred or is not a pre-stage of nephropathy. Specifically, when the urinary albumin excretion rate is less than 20 μg / min or the urinary albumin / creatinine ratio is less than 30 mg / gCr, it can be determined as “normal” with respect to nephropathy.

(4-2) Method for selecting a person susceptible to diabetic nephropathy using ACC-β production as an index The method can be performed by measuring the production of ACC-β in a biological sample derived from a subject. . Here, the biological sample may be a sample containing cells that express the ACC-β gene and can produce ACC-β, and specific examples thereof include blood.

  The method for measuring the amount of ACC-β production can be performed according to a standard method as described in the method (3-2) above.

  As a result of such measurement, when the production amount of ACC-β in the biological sample (test sample) of the subject is larger than the production amount (control production amount) of ACC-β in the biological sample (control sample) derived from a normal person, It can be determined that the subject is highly likely to suffer from diabetic nephropathy (susceptibility to diabetic nephropathy).

(4-3) Selection method of diabetic nephropathy susceptible person using diabetic nephropathy susceptibility SNP of ACC-β gene as an index The method is based on genomic DNA obtained from a subject, and the method of human ACC-β gene It has the step of detecting and identifying the base at position 4139 (SNP4139) of intron 18:
Preferably, the detection method of the present invention comprises a step of detecting and identifying a base at position 4139 (SNP4139) of intron 18 of the human ACC-β gene for genomic DNA obtained from a human subject. When the identified base is adenine (A), the subject can be determined and selected as a person susceptible to diabetic nephropathy (a person susceptible to diabetic nephropathy).

  According to this method, it is possible to determine and diagnose the risk of developing diabetic nephropathy and the risk of developing diabetic nephropathy, that is, the risk of developing diabetic nephropathy. The determination (diagnosis) of the risk of suffering from diabetic nephropathy can be performed mechanically without requiring the judgment of a person having specialized knowledge such as a doctor using the base of the SNP as a judgment criterion (determination index). . For this reason, it can be said that the method of the present invention is a method for detecting the risk of suffering from diabetic nephropathy.

  The genomic DNA is extracted and the target base is detected by a known method [for example, Bruce, et al., Geneme Analysis / A laboratory Manual (vol. 4), Cold Spring Harbor Laboratory, NY., (1999)]. Can be used.

  The sample from which genomic DNA is extracted is any cell (including cultured cells, excluding germ cells), tissue (including cultured tissue), organ, or body fluid (eg, saliva) isolated from subjects and clinical samples. Lymphatic fluid, airway mucosa, semen, sweat, urine, etc.). As the material, leukocytes or mononuclear cells separated from peripheral blood are preferable, and leukocytes are particularly preferable. These materials can be isolated according to methods commonly used in clinical laboratory tests.

For example, when using leukocytes as a material, leukocytes are first separated from peripheral blood isolated from a subject according to a conventional method. Subsequently, proteinase K and sodium dodecyl sulfate (SDS) are added to the obtained leukocytes to degrade and denature proteins, and then phenol / chloroform extraction is performed to obtain genomic DNA (including RNA). RNA can be removed by RNase as necessary. Incidentally, extraction of the genomic DNA is not limited to the above method, methods well known in the art (e.g., Sambrook J. et al,.. "Molecular Cloning:. A Laboratry Manual (2 nd Ed)" Cold Spring Harbor Laboratory, NY) and commercially available DNA extraction kits can be used. Further, if necessary, DNA containing the ACC-β gene on human chromosome 12 (12q24.11) or its intron 18 may be isolated. The DNA can be isolated by PCR or the like using genomic DNA as a template, using a primer that hybridizes to the ACC-β gene or intron 18 thereof.

  In the step of detecting the target base, a diabetic nephropathy-susceptible SNP that is extremely related to diabetic nephropathy is detected from the extract containing human genomic DNA obtained as described above. The base is detected by directly determining the base sequence in the LD block containing the ACC-β gene on human chromosome 12, preferably the intron 18, further isolated from a sample containing human genomic DNA. It may be by a method for examining the mutation of the base of the SNP located at.

  For example, as a method for detecting the target base, in addition to the method for directly determining the gene sequence of the corresponding region as described above, when the polymorphic sequence is a restriction enzyme recognition site, the difference in restriction enzyme cleavage pattern is used. Genotype determination method (hereinafter referred to as RFLP), polymorphism-specific probe-based hybridization method (for example, sticking a specific probe on a chip, glass slide, nylon membrane, etc. Determine the type of polymorphism by detecting the difference in hybridization intensity for different probes, or the efficiency of specific probe hybridization, and the amount of probe that polymerase degrades during template duplex amplification To identify the genotype, and to change the fluorescence emitted by certain double-strand-specific fluorescent dyes By detecting the temperature difference of double-stranded melting, a method for identifying the polymorphism by this, a complementary sequence is attached to both ends of the polymorphic site-specific oligo probe, and the probe in the self molecule depends on the temperature. And a method for specifying a genotype by using a difference between whether a secondary structure is formed or hybridized to a target region. In addition, a method in which a base-extension reaction is performed by polymerase from a template-specific primer and a base incorporated into a polymorphic site at that time is identified (by using dideoxynucleotide, each is fluorescently labeled, and each fluorescence is Detection method, method of detecting incorporated dideoxynucleotide by mass spectrometry), and further using template-specific primers followed by the presence or absence of base pairs complementary to the mutation site or non-complementary base pairs. There is a method to make it recognize.

  Hereinafter, conventionally known representative methods for detecting gene polymorphisms are listed, but the present invention is not limited to these.

  (a) RFLP (restriction fragment length polymorphism) method, (b) PCR-SSCP method (single-stranded DNA higher-order structure polymorphism analysis) [Biotechniques, 16, 296-297 (1994), and Biotechniques, 21 , 510-514 (1996)], (c) ASO (Allele Specific Oligonucleotide) hybridization method [Clin. Chim. Acta, 189, 153-157 (1990)], (d) Direct sequencing method [Biotechniques, 11, 246 -249 (1991)], (e) ARMS (Amplification Refracting Mutation System) method (Nuc. Acids. Res., 19, 3561-3567 (1991); Nuc. Acids. Res., 20, 4831-4837 (1992) (F) Denaturing Gradient Gel Electrophoresis (DGGE) method [Biotechniques, 27, 1016-1018 (1999)], (g) RNase A cleavage method [DNA Cell. Biol., 14, 87 -94 (1995)], (h) chemical cleavage method [Biotechniques, 21, 216-218 (1996)], (i) DOL (Dye-labeled Oligonucleotide Ligation) method [Genome Res., 8, 549-556 (1998) )], (J) T qMan-PCR method [Genet. Anal., 14, 143-149 (1999); J. Clin. Microbiol., 34, 2933-2936 (1996)], (k) Invader method [Science, 5109, 778-783 ( 1993); J. Biol. Chem., 30, 21387-21394 (1999); Nat. Biotechnol., 17, 292-296 (1999)], (l) MALDI-TOF / MS method (Matrix Assisted Laser Desorption-time of Flight / Mass Spectrometry) [Genome Res., 7, 378-388 (1997); Eur. J. Clin. Chem. Clin. Biochem., 35, 545-548 (1997)], (m) TDI (Template -directed Dye-terminator Incorporation method [Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)], (n) Molecular Beacons method [Nat. Biotechnol., 1, p49- 53 (1998); gene medicine, 4, p46-48 (2000)], (o) Dynamic Allele-Specific Hybridization (DASH) method [Nat. Biotechnol., 1.p.87 -88 (1999); gene medicine, 4, 47-48 (2000)], (p) padlock Probe (Padlock Probe) method [Nat. Genet., 3, p225-232 (1998); Gene medicine, 4, p50-51 (2000)], (q) UCAN method [Takara Shuzo Co., Ltd. home page (http: http://www.takara.co.jp)], (r) DNA chip or DNA microarray ("SNP gene polymorphism strategy" Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p128-135), (s) ECA method [ Anal. Chem., 72, 1334-1341, (2000)].

  The above are typical gene polymorphism detection methods. However, the present invention is not limited to the determination of the risk of developing diabetic nephropathy, and other known or future developed gene polymorphism detection methods are widely used. be able to. Moreover, when detecting the gene polymorphism of the present invention, these gene polymorphism detection methods may be used singly or in combination of two or more.

  As described above, the subject of the present invention ((4-1) to (4-3)) has been found to have a relatively high potential risk of developing or progressing to diabetic nephropathy. On the other hand, it is possible to notify that fact and to take appropriate measures in advance to prevent the onset or progression of diabetic nephropathy. Therefore, the present invention is extremely useful as a test method for preventing the onset and progression of diabetic nephropathy, and further as a test method for preventing the occurrence of other diseases and symptoms associated with diabetic nephropathy. is there.

5). Diabetes nephropathy morbidity risk diagnostic kit The present invention also provides a reagent kit (diagnostic kit) for diagnosing the morbidity risk of diabetic nephropathy. In particular, the present invention provides a diagnostic kit used in the method for selecting a person susceptible to diabetic nephropathy described in (4-3) above.

(5-1) Probe Diabetes on the ACC-β gene of human chromosome 12 (12q24.11) is used for the detection of the diabetic nephropathy-susceptible SNP described in (4-3) above and nucleotides containing the base. Oligos or polynucleotides that specifically hybridize to oligone or polynucleotides that contain SNP-sensitive SNPs and that can detect the SNPs are used. Such an oligo or polynucleotide specifically hybridizes to a continuous gene region having a length of 16 to 500 bases, preferably 20 to 200 bases, more preferably 20 to 50 bases including SNP4139 on the ACC-β gene. Thus, it is designed as an oligo or polynucleotide having the above base length.

  Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the second edition, conditions described in 1989), it means that cross-hybridization with other DNA does not occur significantly. Preferably, the oligo or polynucleotide preferably has a base sequence complementary to the base sequence of the gene region containing the SNP to be detected, but if such specific hybridization is possible, the oligo or polynucleotide is completely There is no need to be complementary.

  As such oligo or polynucleotide, the oligo or polynucleotide shown in the following (a) (however, when the oligo or polynucleotide is RNA, the base symbol “t” in the sequence listing should be read as “u”) And a continuous oligo or polynucleotide having a length of 16 to 500 bases.

  (a) A continuous oligo or polynucleotide having a length of 16 to 500 bases comprising a nucleotide located at position 4139 (SNP4139) of intron 18 in the base sequence of human ACC-β gene.

  The oligo or polynucleotide is a diabetic nephropathy susceptibility SNP (SNP4139) located on the ACC-β gene on human chromosome 12 in order to determine the susceptibility and progression of diabetic nephropathy for a subject. Designed as an oligo or polynucleotide “probe” that specifically hybridizes to the containing oligo or polynucleotide. These oligos or polynucleotides can be prepared based on the base sequence of the ACC-β gene, for example, using a commercially available nucleotide synthesizer according to a conventional method.

  More preferably, the probe is labeled with a radioactive substance, a fluorescent substance, a chemiluminescent substance, an enzyme, or the like (described later) so that the oligonucleotide containing each of the SNPs can be detected.

  The probe (oligo or polynucleotide) can be used by immobilizing on an arbitrary solid phase. Therefore, the present invention also provides the probe (oligo or polynucleotide) as an immobilized probe (for example, a gene chip, a cDNA microarray, an oligo DNA array, a membrane filter, etc. on which the probe is immobilized). The probe can be preferably used as a DNA chip for detecting a diabetic nephropathy susceptibility gene.

  The solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to. The immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).

  For example, in the TaqMan PCR method [Livak KJ. Gene Anal 14, 143 (1999), Morris T et al., J Clin Microbiol 34, 2933 (1996)], it is complementary to a region containing a diabetic nephropathy-susceptible SNP. An oligonucleotide having a length of about 20 bases is designed as a probe. The probe is labeled with a fluorescent dye at the 5 'end and a quenching substance at the 3' end and specifically hybridizes with the sample DNA, but does not emit light as it is, but is an extension reaction from the upstream of a separately added PCR primer. By this, the 5 'fluorescent dye bond is cleaved and detected by the released fluorescent dye.

  In the Invader method [Lyamichev V. et al., Nat Biotechnol 17, 292 (1999)], oligonucleotides complementary to the sequences adjacent to the polymorphic site (2 'on the 3' side and 5 'side) are designed as probes. Is done. Detection is achieved by these two probes and a third probe that is independent of the analyte.

(5-2) Primer The present invention also provides an oligonucleotide used as a primer for specifically amplifying a sequence region containing a diabetic nephropathy-susceptible SNP on the ACC-β gene of human chromosome 12.

  As such oligo or polynucleotide, the oligo or polynucleotide shown in the following (a) (however, when the oligo or polynucleotide is RNA, the base symbol “t” in the sequence listing should be read as “u”) And oligo- or polynucleotides having a length of 15 to 30 bases.

  (a) A continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 4139 (SNP4139) of intron 18 in the base sequence of human ACC-β gene.

  Such an oligonucleotide specifically hybridizes to a part of a continuous oligo or polynucleotide containing a base (nucleotide) of a diabetic nephropathy-susceptible SNP in the ACC-β gene, and specifically identifies the oligo or polynucleotide. It is designed as an oligonucleotide having a length of 15 to 30 bases, preferably about 18 to 25 bases for amplification. The length of the oligo or polynucleotide to be amplified is appropriately set depending on the detection method used, but is generally 15 to 1000 bases, preferably 20 to 500 bases, more preferably 20 to 200 bases. It is.

  The above-mentioned various oligonucleotides having a base sequence that specifically hybridizes to a base sequence of 15 bases or more in the vicinity of the diabetic nephropathy-susceptible SNP (SNP4139) on the ACC-β gene of human chromosome 12 are as follows: It can be used as a primer in the invention. In addition, these oligonucleotides can be prepared in accordance with a conventional method using, for example, a commercially available nucleotide synthesizer based on the base sequence of the human ACC-β gene.

  A method in which MALDI-TOF / MS (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry) is applied to the Mass Array method [Haff LA et al. Genome Res 7, 378 (1997), Little DP et al., Nature Medicine vol.3, No.12, 1413-1416, (1997)] will be used as an example to explain the specific usage of primers. In this case, the primer is hybridized to the sample DNA fixed on the silicon chip, and ddNTP is added to extend only one base. The single base extension product is then separated and the polymorphism is detected by mass spectrometry. In this method, it is desirable that the primer is designed to be as short as possible, usually 15 bases or more in length.

(5-3) Labeled substance The probe or primer of the present invention is added with an appropriate labeling substance for detecting a gene polymorphism, such as a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin and the like. Is included.

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′ , 7'-hexachloro-6-carboxylfluorescein, green fluorescent dye, fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems) , Yellow-green fluorescent dye), rhodamine or a derivative thereof [eg, tetramethylrhodamine (TMR)], but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, Amersham Pharmacia, oligonucleotide ECL 3'-oligo labeling system, etc.).

  In addition, the primer of the present invention includes those to which a linker sequence for detecting a polymorphism is added at the end. Examples of such a linker sequence include a flap (a sequence completely unrelated to the sequence near the polymorphism) added to the 5 ′ end of the oligonucleotide used in the above-described invader method.

  The above probes or primers (which may be labeled) can be used as a diagnostic reagent for the risk of developing diabetic nephropathy.

(5-4) Diabetes nephropathy risk diagnostic reagent kit (diagnostic kit)
The diagnostic kit of the present invention is an oligo or polynucleotide used as a probe or primer as a reagent for diagnosing the risk of developing diabetic nephropathy (note that these may be labeled or immobilized on a solid phase). At least one). In addition to the probe or primer, the diagnostic kit of the present invention may include other reagents necessary for carrying out the method of the present invention, such as a hybridization reagent, a probe label, a label detection agent, and a buffer as necessary. Instruments and the like may be included as appropriate.

  The present invention will be described with reference to the following examples, but the present invention is not limited to such examples. In the following examples, unless otherwise specified, genetic manipulation, cell culture, etc., Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience) and the like.

Example 1
Under Informed Consent, Shiga Medical University Hospital, Tokyo Women's Medical University Diabetes Center, Juntendo University School of Medicine Juntendo Hospital, Kawasaki Medical University Hospital, Iwate Medical University Hospital, Toride Cooperative Hospital, Kawai Clinic, Osaka City General Medical Center, Experiments were conducted on patients with type 2 diabetes who regularly visit outpatient clinics at Chiba Tokushukai Hospital or Osaka Industrial Hospital.

The target type 2 diabetic patients were classified into two groups according to the following diagnostic criteria:
1) Diabetic nephropathy: Diabetic nephropathy (urine albumin excretion rate of 200 μg / min or more, urinary albumin / creatinine ratio of 300 mg / gCr or more, or chronic dialysis therapy) and diabetic retinopathy A case with both,
2) Control group: Cases with diabetic retinopathy but no signs of renal dysfunction (patients with a urinary albumin excretion rate of less than 20 μg / min or a urinary albumin / creatinine ratio of less than 30 mg / gCr).

  The peripheral blood of the type 2 diabetic patient was collected, and 10 ml of the peripheral blood obtained was centrifuged at 3000 rpm for 5 minutes to remove serum. Subsequently, erythrocyte lysate was added to the obtained product and incubated at room temperature for 20 minutes. Thereafter, the incubated mixture was centrifuged at 3000 rpm for 5 minutes to remove the supernatant. Proteinase K was added to the resulting precipitate and incubated at 37 ° C. for 4 hours or longer. The obtained product was treated with phenol-chloroform, and isopropanol was added to the obtained upper layer (aqueous layer) to cause precipitation of DNA. Subsequently, centrifugation (12,000 rpm, 10 minutes) was performed, DNA was collected, 1 ml of 70% by volume ethanol was added, and the resulting DNA pellet was added to TE buffer [composition: 10 mM Tris-HCl, 1 mM EDTA ( pH 8.0)] to obtain a DNA sample.

  Using the DNA sample, genotype was determined by the invader method. SNPs to be analyzed were randomly selected from the Japanese SNP database (IMS-JST SNPs database: http://snp.ims.u-tokyo.ac.jp). The genotype of each SNP locus was analyzed by an invader method combined with Multiplex PCR (see, for example, Ohnishi Y. et al., J. Human. Genet., 46, 471-477, 2001). .

The reaction in Multiplex PCR includes a reaction solution containing a primer (SEQ ID NO: 2 and 3) that amplifies the region to be analyzed [composition: 16.6 mM (NH ₄ ) ₂ SO ₄ , 67 mM Tris (pH 8.8), 6. 7 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 6.7 μM EDTA, 1.5 mM dNTPs, 10 × Taq mix (2.5 U / μl Taq DNA polymerase, 31.25 U / μl Taq Antibody), 0 or 10% DMSO, each 0.3% 25 μM primer] was used for 5 minutes at 95 ° C., followed by 40 cycles of reaction at 95 ° C. for 15 seconds, 60 ° C. for 45 seconds and 72 ° C. for 3 minutes. .

The amplified product was diluted 1:11 with dH ₂ O and dispensed into each well of a 384 plate. Next, the plate was air-dried, and 3 μl of Invader reaction solution (composition: 10 × buffer, 10 × FRET probe, 10 × clevase, allele probe) was added to each well on the plate. The plate was then incubated at 63 ° C. for 20 minutes, and then fluorescence (520 nm, 546 nm) was measured for each well of the plate. The allele probe is one type of invader probe (for example, when the amplification region is a region containing SNP4139, and a probe having the base sequence described in SEQ ID NO: 4) and two types of probes corresponding to the respective alleles (for example, the amplification region is In the case of the region containing SNP4139, the G allele contains a probe having the base sequence shown in SEQ ID NO: 5, and the A allele has the probe having the base sequence shown in SEQ ID NO: 6).

  For 94 cases of diabetic nephropathy and 94 cases of the control group, 81315 SNP loci were genotyped. The SNPs that showed significant differences in genotype or allele frequency between the diabetic nephropathy group and the control group by evaluating statistical data using a 2 × 3 or 2 × 2 contingency table Selected.

  It should be noted that correlation analysis, statistical analysis of haplotype frequency and Hardy-Weinberg equilibrium and calculation of linkage disequilibrium coefficient (D ′) were carried out by Devlin B. et al. [Genomics, 29, 311-322, 1995] and Nielsen. DM.) Et al. [Am. J. Hum. Genet., 63, 1531-1540, 1998].

  In the above screening, SNP loci that showed a p-value of less than 0.01 between the diabetic nephropathy group and the control group were analyzed genome-wide for a larger number of patients.

  As a result, a strong correlation with diabetic nephropathy was observed at a specific SNP locus (SNP4139). The SNP is present in intron 18 (SEQ ID NO: 1) of the acetyl-CoA carboxylase-β (ACC-β) gene on chromosome 12, and the A allele frequency of the 4138th SNP is diabetic. It was significantly higher in the nephropathy group than in the control group (A allele frequency: 25% in diabetic nephropathy group, 17% in control group, p = 0.000001). To further verify the association between this SNP and diabetic nephropathy, an 8-year prospective cohort at Shiga Medical University (SUMS cohort) and a 10-year retrospective cohort study at Juntendo University (SA cohort) We examined the relationship with the progression of nephropathy. Table 1 shows the results of comparing the A allele frequency at the SNP locus (rs2268388; SNP4139, ACC-β intron 18, +4139 G / A) between the diabetic nephropathy group and the control group. As can be seen from this result, the same tendency was observed in all analyzes (genome-wide, SUMS cohort, SA cohort).

  Based on the results of these studies, the risk of developing diabetic nephropathy in the A allele holder at this SNP locus (rs2268388; SNP4139, ACC-β intron 18, +4139 G / A) It became clear that it was significantly higher than that.

Example 2 Stable introduction of ACC-β into NRK-52E cells and establishment of cell lines As shown in the examples described later, the ACC-β gene in which SNPs were found nearby was related to diabetic nephropathy. In order to investigate this, an in vitro functional analysis test was conducted. Prior to this, a stable cell line that stably overexpresses ACC-β in rat tubular epithelial cells (NRK-52E) was first established.

NRK-52E (5 × 10 ⁴ cells / 0.5 ml / well) was plated on a 24-well plate using serum-containing D-MEM medium, cultured in a 37 ° C., 5% CO ₂ environment, and the next day, human ACC After addition of retrovirus containing -β or GFP gene for 4 hours of infection, the medium was removed and replaced with serum-containing medium. Six days after the infection, limiting dilution (0.5 cells / 0.1 ml / well) was carried out and the cells were plated on a 96-well plate, and the culture was continued. Four ACC-β-introduced cell lines and three GFP-introduced cell lines were isolated.

  For stable cell lines, after culturing each plate, a lysis agent was added, and total RNA was column extracted according to a conventional method. Next, total RNA corresponding to 1 μg was reverse transcribed as a template for PCR, and the amount of ACC-β mRNA was measured by quantitative real-time PCR using synthetic primers. At this time, it was corrected based on the amount of GAPDH mRNA measured at the same time to obtain a relative value.

In addition, after culturing a stable cell line, protein was extracted by adding a lysing agent according to a conventional method. Next, 10 μg of an equivalent amount of protein was converted to SDS, electrophoresed, transferred to a PVDF membrane, and then subjected to Western blotting using an anti-ACC-β antibody.
Rat tubule epithelial cell NRK-52E was transfected with a retrovirus containing human ACC-β or GFP gene, and 4 clones of ACC-β cell line (A1-1, A1-3, The results of confirming the expression of each of A4-3, A7-2) and 3 clones of GFP cell line (# G2, # G3, # G7) by quantitative real-time PCR are shown in FIG. 1a. As shown in FIG. 1a, all 4 clones of the ACC-β cell line (A1-1, A1-3, A4-3, A7-2) showed the expression of human ACC-β, particularly the A4-3 clone and The expression of A7-2 clone was high.

  The result of confirming expression by Western blotting using an anti-ACC-β antibody is shown in FIG. 1b. As shown in FIG. 1b, a band of human ACC-β having a size of 280 kDa is observed in the A4-3 clone and the A7-2 clone, and the clone of the ACC-β cell line is also highly expressed at the protein level. found.

  The above facts indicate that an ACC-β overexpressing (ACC-β-introduced) stable cell line effective for examining the relationship between the ACC-β gene and the function in diabetic nephropathy has been established.

Example 3 Analysis of high sugar stimulation and expression level fluctuation of ACC-β stable cell line Using the stable cell line established in Example 2, the relationship between ACC-β gene and action in diabetic nephropathy was examined. .

First, a clone of an ACC-β-introduced stable cell line (1 × 10 ⁵ cells / 1 ml / well) was cultured in a 12-well plate containing a serum-containing medium containing 5.5 mM glucose, and the cells adhered to the next day. The medium was removed from the plate, washed once with PBS (−), and then replaced with serum-free medium containing insulin, transferrin, and selenium. After culturing for 24 hours, glucose or mannitol was added to a final sugar concentration of 25 mM, and the culture was further continued for 2 days. Then, total RNA was extracted, and osteopontin (Osteopontin) was obtained by quantitative real-time PCR. ) Or MCP-1 mRNA level was measured. At this time, it was corrected by the amount of GAPDH mRNA measured at the same time, and used as a relative value.

  Osteopontin is a factor associated with inflammation, and it has been reported that its expression is increased in kidneys with nephropathy in type 2 diabetes model rats (Kidney International, Vol. 63 (2003), p. .454-463). MCP-1 is also a factor associated with inflammation, and its concentration has been reported to increase in patients with diabetic nephropathy (Kidney International, Vol.58 (2000), p.1492-1499).

[result]
FIG. 2 shows the results of measuring the amount of osteopontin or MCP-1 mRNA by quantitative real-time PCR after ACC-β-introduced stable cell line A4-3 clone was stimulated with high glucose (black bar graph). . As can be seen from this result, both high expression levels of osteopontin and MCP-1 were observed by stimulation with high glucose. On the other hand, neither the increase in the expression of osteopontin nor MCP-1 was observed in the system in which mannitol was added in order to match the osmotic pressure when glucose was added. In addition, when the GFP-introduced stable cell line clone was used, the expression of osteopontin and MCP-1 did not increase even when stimulated with high glucose.

  As described above, since osteopontin and MCP-1 are both factors that are known to increase in expression in pathological conditions (diabetic nephropathy), the above ACC-β-introduced stable cell line is treated under conditions of high sugar concentration. When placed underneath (ie in vitro diabetic state), increased expression of any of these factors suggests that ACC-β is involved in diabetic nephropathy.

Example 4 Introduction of infection of ACC-β into human tubular epithelial cells RPTEC and analysis of fluctuation in expression level Using human-derived tubular epithelial cells RPTEC as cells capable of stably overexpressing human ACC-β, ACC- We investigated the relationship between β and its effect in diabetic nephropathy.

First, RPTEC (2 × 10 ⁵ cells / 2 ml / well) was plated on a 6-well plate containing serum-containing REGM medium and cultured in a 37 ° C., 5% CO ₂ environment. On the next day, adenovirus (50moi) containing human ACC-β gene or LacZ gene was added to infect for 4 hours, and then the medium was removed and replaced with serum-containing medium. Culture supernatants were collected 1, 2, 3 and 4 days after infection, then total RNA was extracted, and the amounts of ACC-β and IL-6 mRNA were measured by quantitative real-time PCR. At this time, it was corrected by the amount of GAPDH mRNA measured at the same time, and used as a relative value. In addition, the amount of IL-6 protein in the culture supernatant was quantified using an ELISA kit according to a conventional method.

  IL-6 is a cytokine related to inflammation, and it is known that the amount of IL-6 in urine and blood increases when diabetic nephropathy develops (Am. J. Nephrol. 2006, 26, p.562-570).

[result]
Human tubular epithelial cells RPTEC were introduced by infection with adenoviruses containing human ACC-β gene or LacZ gene, respectively, and expression of ACC-β gene was performed by quantitative real-time PCR method 1, 2, 3 and 4 days after infection ( The results of confirming (amount of mRNA) are shown in FIG. As shown in FIG. 3, overexpression of human ACC-β was observed 1 day after infection (24 hours), and peaked on the 2nd infection day (48 hours) until at least the 4th infection day (96 hours). Expression was observed.

  Moreover, the result of having measured the expression level (mRNA amount) of IL-6 by quantitative real-time PCR method about these cells is shown in FIG. As shown in FIG. 4a, an increase in the expression level of IL-6 was observed in the cells into which the ACC-β gene was introduced 2 days after infection (48 hours later) (indicated by “A” in the figure). Further increase on eyes (72 hours) and 4 days (96 hours). Moreover, the result of measuring the amount of IL-6 protein in the culture supernatant by ELISA is shown in FIG. 4b. As shown here, as shown here, the second day (24-48 hours) or the third day ( 48-72 hours), a marked increase in protein amount was observed, consistent with fluctuations in the amount of mRNA. On the other hand, such variation in IL-6 expression level was not observed in cells transfected with the LacZ gene (indicated by “L” in the figure).

  As described above, it was confirmed that IL-6 was elevated at both mRNA and protein levels in cells capable of stably over-expressing human ACC-β (human tubular epithelial cells). As described above, the expression of IL-6 is known to increase in the pathological condition (diabetic nephropathy), and the pathological condition is known to be alleviated by the neutralizing antibody. From these results, the above experimental results suggest that ACC-β is involved in human diabetic nephropathy.

Example 5 Analysis of mechanism of action of increase in IL-6 expression by introduction of ACC-β infection into human tubular epithelial cells RPTEC Based on the results of Example 4 above, increase in IL-6 production by ACC-β overexpression The mechanism of action of was investigated.

  First, in the same manner as in Example 4 above, human tubular epithelial cells RPTEC were infected with adenovirus containing human ACC-β gene or LacZ gene for 4 hours, and then rotenone, an inhibitor of mitochondrial complex I ( Rotenone) (5 μM) or p38 MAPK inhibitor SB203580 (10 μM) was added, and the culture was continued. The amount of IL-6 mRNA was measured by quantitative real-time PCR 3 days after infection. In addition, as a comparative control test, instead of rotenone and SB203580, the amount of IL-6 mRNA was similarly measured after adding DMSO and culturing in the same manner.

[result]
Results of measurement of IL-6 expression level (mRNA level) when rotenone (5 μM) was added to human tubular epithelial cell RPTEC overexpressed by introducing human ACC-β gene or LacZ gene FIG. 5a shows the results of measuring the expression level (mRNA amount) of IL-6 when SB203580 (10 μM) was added, respectively.

  As shown in FIGS. 5a and 5b, overexpression of ACC-β increased IL-6 expression (mRNA amount) (comparison control test: “DMSO” data in each figure). It was significantly suppressed by the addition of the inhibitor. This suggests that the increase in the amount of IL-6 mRNA due to ACC-β overexpression is partially mediated by mitochondrial complex I or p38 MAPK. That is, it is highly possible that activation of mitochondrial complex I or p38 MAPK is involved in the increase in IL-6 production due to ACC-β overexpression. In addition, IL-6 production was similarly reduced in the cells transfected with the LacZ gene by the addition of the above-described inhibitor. Therefore, even in a normal state in which ACC-β is not overexpressed, IL-6 production is also suppressed. It is suggested that these factors (mitochondrial complex I or p38 MAPK) are involved. That is, from these, it is considered that the expression of IL-6 is via the mitochondrial pathway.

Example 6 Examination of IL-6 mRNA Stability In order to investigate the action mechanism of IL-6 production increase by ACC-β overexpression, the involvement of ACC-β in the stability of IL-6 mRNA was examined.

  Specifically, in the same manner as in Example 4 above, human tubular epithelial cells RPTEC were infected with an adenovirus containing a human ACC-β gene or LacZ gene. Three days later, actinomycin D (10 μg / ml), a transcription inhibitor (RNA synthetase inhibitor), was added and culture was continued, and quantitative real-time PCR was performed at 30, 60, and 120 minutes after infection. The amount of IL-6 mRNA was measured.

[result]
The results are shown in FIG. As shown in FIG. 6, in human tubular epithelial cells overexpressing ACC-β, even when a transcription inhibitor (RNA synthetase inhibitor) is added, human tubular epithelial cells that do not express ACC-β In comparison, the decrease in IL-6 mRNA was significantly suppressed (degradation suppression), particularly after 60 minutes after the addition of the transcription inhibitor, significant decrease suppression of IL-6 mRNA (degradation suppression), In other words, increased stability was observed.

  From this, it is considered that the increase in IL-6 production when ACC-β is overexpressed is due to the increased stability of IL-6 mRNA. In other words, the increase in the stability of IL-6 mRNA due to overexpression of ACC-β is considered to be one of the mechanisms of action that the degradation rate of IL-6 mRNA is slowed down by ACC-β.

Example 7 Examination of enhancer activity In order to examine the function of the SNP present in rs2268388 present in intron 18 (SEQ ID NO: 1) of the ACC-β gene, the disease-susceptible allele (T) and insensitive allele (C) of rs2268388 A 28-bp oligoDNA sequence containing each of them was connected in tandem to create a sequence that was subcloned upstream of the SV40promoter of the pGL3 promoter vector to create a luciferase reporter construct. Normal human proximal tubular epithelial cells RPTEC were co-transfected with the luciferase reporter prepared above and the pRL-TK vector for correction, and after 4 hours, 7.2 mM glucose, 25 mM glucose, and 7.2 mM glucose + 17.8 mM mannitol After replacing with each medium, cell extracts were prepared 20 hours later, and luciferase activity was measured.

[result]
As shown in FIG. 7, the 28 bp oligoDNA sequence containing rs2268388 (SNP4139) had significant enhancer activity compared to the pGL3 promoter vector alone, regardless of genotype. From the above, it was suggested that there is a region capable of regulating the expression of ACC-β around rs2268388 (SNP4139) as a function of SNP.

Fig. A shows that rat tubule epithelial cell NRK-52E was transfected with a retrovirus containing human ACC-β or GFP gene, and 4 clones (A1-1, ACC-β cell line obtained by limiting dilution method). A1-3, A4-3, A7-2) and three clones of GFP cell line (# G2, # G3, # G7) were confirmed by quantitative real-time PCR (Example 2). The mRNA expression on the vertical axis means a numerical value corrected with the expression level of GAPDH. FIG. B shows the results of confirming the expression of the ACC-β cell line 4 clone and the GFP cell line 3 clone by Western blotting using an anti-ACC-β antibody (Example 2). ACC-β-introduced stable cell line A4-3 clone was stimulated with glucose (black bar) or mannitol (gray bar), and then the mRNA level of osteopontin or MCP-1 was measured by quantitative real-time PCR. Indicates. The white bar indicates the amount of osteopontin or MCP-1 mRNA in the ACC-β-introduced stable cell line A4-3 clone (Example 3). “Ratio” on the vertical axis means the relative ratio of the expression level when the expression level of untreated cells (Intact) that are not stimulated by glucose or mannitol is 1. Human tubular epithelial cells RPTEC were introduced by infection with adenovirus containing human ACC-β gene or LacZ gene, respectively, and quantitative real-time at 1, 2, 3 and 4 days after infection (24, 48, 72 and 96 hours) (Example 4) which shows the result of having confirmed the expression level (mRNA amount) of the ACC-β gene by the PCR method. In the figure, “N” represents the human tubular epithelial cell itself, “L” represents the human tubular epithelial cell introduced with the LacZ gene, and “A” represents the human tubular epithelial cell introduced with the ACC-β gene. “Fold increase” on the vertical axis indicates the expression level when the value obtained after 24 hours using cells in which neither the LacZ gene nor the ACC-b gene is introduced (No infection) is 1. It means relative ratio. FIG. A shows that human tubular epithelial cells RPTEC were introduced by infection with adenoviruses containing human ACC-β gene or LacZ gene, respectively, 1, 2, 3 and 4 days after infection (24, 48, 72 and 96 hours later). ) Shows the results of confirming the expression level (mRNA amount) of IL-6 by quantitative real-time PCR (Example 4). FIG. B shows the results of confirming the amount of IL-6 protein on infection 1-2 days (24-48 hours), 2-3 days (48-72 hours) and 3-4 days (72-96 hours). (Example 4). In the figure, “N” represents the human tubular epithelial cell itself, “L” represents the human tubular epithelial cell introduced with the LacZ gene, and “A” represents the human tubular epithelial cell introduced with the ACC-β gene. The “Fold increase” on the vertical axis shows the expression level when the value obtained 24 hours later using cells in which neither the LacZ gene nor the ACC-b gene is introduced (No infection) is 1. It means relative ratio. Fig. A shows IL-6 expression level (mRNA level) when rotenone (5 μM) is added to human tubular epithelial cell RPTEC overexpressed by introducing human ACC-β gene or LacZ gene FIG. B shows the results of measuring the expression level (mRNA amount) of IL-6 when SB203580 (10 μM) was added. In each figure, “DMSO” indicates the result of a comparative control test in which DMSO was added in place of rotenone and SB203580 and cultured in the same manner to measure the amount of IL-6 mRNA (Example 5). In the figure, “N” represents the human tubular epithelial cell itself, “L” represents the human tubular epithelial cell introduced with the LacZ gene, and “A” represents the human tubular epithelial cell introduced with the ACC-β gene. The “Fold increase” on the vertical axis shows the expression level when the value obtained 24 hours later using cells in which neither the LacZ gene nor the ACC-b gene is introduced (No infection) is 1. It means relative ratio. Human tubular epithelial cells RPTEC were infected with an adenovirus containing human ACC-β gene or LacZ gene, and then cultured with addition of a transcription inhibitor (RNA synthetase inhibitor) 30, 60, and 120 minutes The amount of IL-6 mRNA later is shown (Example 6). The results are shown as relative values (%) when the amount of IL-6 mRNA at 0 minutes in culture is 100%. 28 bp oligoDNA sequence containing the disease-susceptible allele (T) of rs2268388 (indicated by “T” in the figure) and 28 bp oligoDNA sequence containing the insensitive allele (C) of rs2268388 (in the figure, A luciferase reporter construct (in the figure, pGL3-promoter T3 (T3), and pGL3-) is inserted upstream of the SV40promoter of the pGL3 promoter vector, each of which has a tandem 3 sequence. promoter C3 (shown as C3)] and a pGL3 promoter vector (shown as pGL3-promoter (pro) in the figure) were prepared as comparative controls. The pRL-TK vector was introduced into RPTEC as a construct and correction, and cultured (7.2 mM glucose, 25 mM glucose, or 7.2 mM glucose + 17.8 mM mannitol), and then the luciferase activity of the cell extract was measured. Show. The results are shown as 1, where pGL3-promoter (pro), a comparative control, was cultured in 7.2 mM glucose and luciferase activity was measured (measured value).

SEQ ID NOs: 2 and 3 show the base sequences of primers for amplifying the rs2268388 region.
SEQ ID NO: 4 is the base sequence of the invader probe for detecting the amplified region containing SNP4139, SEQ ID NO: 5 is the base sequence of the allele probe for detecting the G allele of the amplified region containing SNP4139, and SEQ ID NO: 6 Indicates the nucleotide sequence of the allele probe for detecting the A allele in the amplified region containing SNP4139, respectively.

Claims (9)

A method for screening a preventive or therapeutic agent for diabetic nephropathy having the following steps:
(1) contacting the test substance with a cell capable of expressing the ACC-β gene;
(2) a step of measuring the expression level of the ACC-β gene in the cell contacted with the test substance; and (3) the measurement amount is higher than the expression level of the ACC-β gene in the control cell not contacted with the test substance. Selecting a small test substance. A method for screening a preventive or therapeutic agent for diabetic nephropathy having the following steps:
(1 ′) contacting a test substance with a cell capable of producing ACC-β or a cell fraction prepared from the cell;
(2 ′) a step of measuring the amount of ACC-β produced in the cell contacted with the test substance or the cell fraction thereof, and (3 ′) a control cell in which the test substance is not contacted with the test substance or a cell fraction thereof A step of selecting a test substance smaller than the amount of ACC-β produced per minute. A method for screening a preventive or therapeutic agent for diabetic nephropathy having the following steps:
(1 ″) contacting a test substance with a cell capable of producing ACC-β or a cell fraction prepared from the cell,
(2 ″) a step of detecting the action of ACC-β of the cell contacted with the test substance or the cell fraction thereof; and (3 ″) a control cell or a cell fraction thereof in which the action detected above does not contact the test substance. Selecting a test substance that is smaller than the ACC-β action of the minute.   The screening method according to any one of claims 1 to 3, wherein the cell capable of expressing the ACC-β gene or the cell capable of producing ACC-β is a tubular epithelial cell.   The screening method according to any one of claims 1 to 4, which is a method for searching for an active ingredient of a preventive or therapeutic agent for diabetic nephropathy.   Measuring the expression level of ACC-β mRNA in a test sample derived from a subject, wherein the expression level in the test sample is greater than the expression level of ACC-β mRNA in a control sample derived from a normal person. A method for selecting subjects who are highly likely to suffer from diabetic nephropathy, using the subject as an indicator that the subject is likely to suffer from diabetic nephropathy.   Measuring the production amount of ACC-β in a test sample derived from a subject, wherein the production amount in the test sample is larger than the production amount of ACC-β in a control sample derived from a normal person, A method for selecting subjects who are highly likely to suffer from diabetic nephropathy, which is an indicator that the subject is likely to suffer from diabetic nephropathy.   Including the step of detecting the 4139th base present in intron 18 of the ACC-β gene in a test sample derived from a test subject, and confirming that these bases are adenine, the test subject may suffer from diabetic nephropathy A method for selecting subjects who are highly likely to have diabetic nephropathy, which is an index of highness.   A diagnostic kit for susceptibility to diabetic nephropathy, comprising a reagent for detecting the base at position 4139 present in intron 18 of the ACC-β gene.

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