JP4845486B2 - Diabetes nephropathy susceptibility gene and method for screening active ingredient of preventive or therapeutic agent for diabetic nephropathy - Google Patents

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. Further, the present invention relates to a method for determining whether or not a subject has a high probability of developing diabetic nephropathy, and selecting a patient having a high risk of developing diabetic nephropathy, as well as the method. The present invention relates to a reagent kit that can be used effectively.

  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 diabetic nephropathy, it is 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 a 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).

  By the way, the human neurocalcin delta gene (NCALD gene) was cloned from the human fetal brain in 2001 by Wang et al. (See Non-Patent Document 3). NCALD, which is an expression product of this NCALD gene, is a protein belonging to the nerve calcium sensor (NCS) family, and is known to be expressed mainly in the photoreceptors and nerves of the retina (Non-Patent Documents 3 to 6). reference). NCALD is known to have a structure having two pairs of EF-hand calcium binding loop sequences and myristoylated signal sites in the N-terminal region (Non-patent Documents 3, 7 to 10). . However, the clear function is unknown at present.

In subsequent studies, the relationship between the NCALD gene and breast cancer (see Patent Document 1), the relationship with the treatment effect of psoriasis (see Patent Document 2), and the relationship with the precursor of human β-amyloid (Patent Document 3). Etc.) have been reported, but no association with diabetic nephropathy has been reported.
"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 Wang W, et al., Biochim Biophys Acta. 2001 Mar 19; 1518 (1-2): 162-7 Polans A, et al., Trends Neurosci 19: 547-554, 1996 Hideka H, et al., Neurosci Res 16: 73-77, 1993 Palczewski K, et al., Bioessays 22: 337-350, 2000 Terasawa M, et al., J Biol Chem 267: 29596-19599, 1992 Zozulya S, et al., Proc Natl Acad Sci USA 89: 11569-11573, 1992 Zozulya S, et al., Method Enzymol 250: 383-393, 1995 Vijay-Kumar S, et al., Nat Struct Biol 6: 80-88, 1999 International Publication (WO03 / 004989) International Publication (WO04 / 028479) International Publication (WO05 / 023858)

  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.

  Further, the present invention relates to a method for determining whether or not a subject has a high probability of developing diabetic nephropathy, and selecting a patient having a high risk of developing diabetic nephropathy, as well as the method. It is an object to provide a reagent kit that can be used effectively.

  In order to achieve the above object, the present inventors have conducted a large-scale correlation analysis of diabetic nephropathy and genome. As a result, SNP (single nucleotide polymorphism) that is strongly associated with diabetic nephropathy is 3 locations within exon 4 region of human neurocalcin delta gene (hereinafter referred to as “NCALD gene”) [+999 position A / T (SNP999), +1307 position T / C (SNP1307), +1340 position G / A (SNP1340)] and the expression level of NCALD mRNA according to the haplotype of the NCALD gene which is present and has thymine (T), cytosine (C) and adenine (A) in the SNP sites (SNP999, SNP1307, SNP1340), respectively Was found to decrease significantly. This suggests that the decrease in NCALD gene expression caused by these SNPs is related to the onset and progression of diabetic nephropathy.

  Moreover, when the present inventors suppressed the expression of the NCALD gene in the proximal tubule epithelial cell using the siRNA technique, the expression of E-cadherin, which is a cell adhesion factor, was suppressed in the epithelial cell. It was confirmed that the expression of a smooth muscle actin (α-SMA) was induced in the epithelial cells and that the cell migration ability was enhanced. These phenomena are observed in the process of conversion of tubular epithelial cells to mesenchymal cells (Tubular Epithelial-Mesencymal Transition, TEMT) (Liu Y, et al., J Am Soc Nephrol 15: 1-12, 2004; Savagner P, et al., Bio Essays 23: 912-923, 2001; Okada H, et al., Am J Physiol 273: F563-F574, 1997; Kalluri R, et al., J Clin Invest 112: 1776-1784, 2003; Li Y, et al., J Clin Invest 112: 503-516, 2003), and recently model mice and humans with obstructive kidney disease In chronic kidney disease, TEMT has been reported to be deeply involved in the progression of the disease (Iwano M, et al., J Clin Invest 110: 341-350, 2002; Oldfield MD, et al., J Clin Invest 108: 1853-1863, 2001; Li JH, et al., Am J pathol 164: 1384-1397, 2004; Ina K, et al., J Med Election Microsc 35: 87-95, 2002), NCALD gene Decreased expression of TEMT, which leads to renal interstitial lines It was thought that it was brought to the development of diabetic nephropathy. Moreover, it has been conventionally known that TEMT is induced by TGF-β (Okada H, et al., Am J Physiol 273: F563-F574, 1997; Bottinger EP, et al., J Am Soc Nephrol 13: 2600-2610, 2002), the study by the present inventors has confirmed that the treatment of tubular epithelial cells with TGF-β reduces the expression of NCALD in a dose-dependent manner. This suggested that the NCALD gene is deeply involved in the induction of TEMT by TGF-β.

  Based on these findings, the inventors of the present invention have disclosed that the NCALD gene is a diabetic nephropathy susceptibility gene that is closely related to the onset and progression of diabetic nephropathy, and prevents the onset and progression of diabetic nephropathy or We were convinced that it would be a useful target gene for developing methods to treat. Furthermore, the present inventors have found a relationship between 3SNPs (SNP999, SNP1307, SNP1340) having a significant association with diabetic nephropathy located within the NCALD gene and the potential risk of developing diabetic nephropathy, It was convinced that the potential risk of developing diabetic nephropathy in the subject can be evaluated by measuring the base of the 3SNPs for the subject.

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

I. Item 1. Method for screening effective components for prevention or treatment of diabetic nephropathy A screening method for a substance that enhances the expression of the NCALD gene, comprising the following steps:
(1) A step of contacting a test substance with a cell capable of expressing the NCALD gene,
(2) a step of measuring the expression level of the NCALD gene in the cell contacted with the test substance, and (3) a test substance in which the measurement amount is larger than the expression level of the NCALD gene in the control cell not contacted with the test substance Process to select.
Item 2. A screening method for a substance that increases the amount of NCALD produced, comprising the following steps:
(1 ′) contacting a test substance with a cell capable of producing NCALD or a cell fraction prepared from the cell;
(2 ′) a step of measuring the amount of NCALD produced in the cell contacted with the test substance or the cell fraction thereof, and (3 ′) the above measured amount being a control cell or a fraction of the cell that is not contacted with the test substance A step of selecting a test substance larger than the production amount of NCALD.

Item 3. Screening method for substances that enhance NCALD function:
(1 ") a step of contacting a test substance with a cell capable of producing NCALD or a cell fraction prepared from this cell,
(2 ″) a step of detecting the NCALD function of the cell contacted with the test substance or a fraction of the cell, and (3 ″) a control cell or a fraction of the cell in which the detected function is not contacted with the test substance. A step of selecting a test substance having a function larger than that of NCALD.
Item 4. Item 4. The screening method according to Item 3, wherein the function of NCALD is inhibition of cell mesenchyme.
Item 5. Item 4. The screening method according to Item 3, wherein the function of NCALD is induction of adhesion molecule expression.
Item 6. Item 6. The screening method according to Item 5, wherein the adhesion molecule is E-cadherin.
Item 7. Item 4. The screening method according to Item 3, wherein the function of NCALD is suppression of actin expression.
Item 8. Item 8. The screening method according to Item 7, wherein the actin is α-smooth muscle actin.
Item 9. Item 4. The screening method according to Item 3, wherein the function of NCALD is suppression of extracellular matrix production.
Item 10. Item 4. The screening method according to Item 3, wherein the function of NCALD is suppression of cell migration ability or invasion.

Item 11. Item 11. The screening method according to any one of Items 1 to 10, further comprising contacting TGF-β with a cell capable of expressing the NCALD gene or a cell capable of producing NCALD.
Item 12. Item 12. The screening method according to any one of Items 1 to 11, wherein the cell capable of expressing the NCALD gene or the cell capable of producing NCALD is a proximal tubular epithelial cell.
Item 13. Item 13. The screening method according to any one of Items 1 to 12, which is a method for searching for an active ingredient of a prophylactic or therapeutic agent for diabetic nephropathy.

II. Item 14. Selection Method for Subjects with High Risk of Diabetic Nephropathy A step of measuring the mRNA expression level of the NCALD gene in a test sample derived from a subject, wherein the expression level in the test sample is smaller than the mRNA expression level of the NCALD gene in a control sample derived from a normal person This is an indicator that the subject is highly likely to suffer from diabetic nephropathy, and a method for selecting subjects who are likely to suffer from diabetic nephropathy.
Item 15. Measuring the production amount of NCALD in a test sample derived from a subject, wherein the production amount in the test sample is smaller than the production amount of NCALD in a control sample derived from a normal person, A method for selecting a subject who is highly likely to suffer from diabetic nephropathy, which is an indicator that the subject is likely to suffer from diabetic nephropathy.
Item 16. A step of detecting at least one of the base at 999, the base at 1307, and the base at 1340 in the fourth exon of the NCALD gene in a test sample derived from a subject, At least one of the bases of the following conditions:
999th base: thymine 1307th base: cytosine 1340th base: satisfying adenine is an indicator that the subject is likely to suffer from diabetic nephropathy. A method for selecting subjects who are likely to be affected.

III. Diagnosis risk diagnosis kit for diabetic nephropathy Item 17. Diabetes nephropathy susceptibility diagnosis comprising a reagent for detecting at least one of the base at 999, the base at 1307, and the base at 1340 present in the fourth exon of the NCALD gene kit.

  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 SNPs 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 subjects (diabetic patients) whose potential risk of developing nephropathy has been found to be relatively high by the method of the present invention, is this notified and whether to prevent the onset of nephropathy in advance? Or, you can take appropriate measures 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 “gene polymorphism”. As used herein, the term “gene polymorphism” refers to a single nucleotide polymorphism (SNP (or SNPs)), which is a polymorphism caused by a single nucleotide change, and extends over a plurality of nucleotides continuous. Both polymorphisms are included. 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 starting dialysis treatment due to end stage renal failure due to diabetic nephropathy is increasing year by year, and at present, diabetic nephropathy is No. 1 as the causative disease of dialysis patients. More specifically, the “diabetic nephropathy” targeted by the present invention includes diabetic retinopathy and the urinary albumin excretion rate exceeds 200 μg / min or the urinary albumin / creatinine ratio is 300 mg. Cases exceeding / gCr, as well as cases that develop diabetic retinopathy and are undergoing artificial 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 the present specification, the position numbers 999, 1307, and 1340, which are position numbers of each SNP, mean position information when the first base of the fourth exon of the NCALD gene is 1.

2. Diabetic nephropathy susceptibility gene and diabetic nephropathy susceptibility SNP
In order to search for diabetic nephropathy susceptibility genes, the present inventors conducted case-control-related analysis and linkage disequilibrium (LD) mapping using gene-based SNPs in many Japanese patients with type 2 diabetes. By doing so, a linkage disequilibrium state is present on the base sequence of the fourth exon of the human neurocalcin delta gene (NCALD gene) present in the chromosome 8 q22-23 (8q22-23) region. 3 SNPs (SNP999, SNP1307, SNP1340) were found, and these SNPs were found to be significantly related to the onset and progression of diabetic nephropathy (diabetic nephropathy sensitive SNPs). Furthermore, the present inventors have shown that mRNA derived from the NCALD gene having a specific haplotype of the SNPs has a significantly reduced stability, that the neurocalcin delta is expressed in E-cadherin and α-SMA, The present inventors have found that the cell migration ability is affected, and concluded that the NCALD gene is a diabetic nephropathy susceptibility gene that is deeply involved in the onset and progression of diabetic nephropathy.

  The human NCALD gene is a 43972 bp gene (Gene Name: human neurocalcin delta gene) located at base numbers 102767947 to 103205665 in the genome sequence of human chromosome 8 (Genbank Sequence Accession IDs: NC 000008).

  Regarding the relationship between the NCALD gene and the disease, the relationship with breast cancer (see Patent Document 1), the relationship with the therapeutic effect of psoriasis (see Patent Document 2), and the precursor of human β-amyloid Although relatedness (refer patent document 3) etc. are reported, the relationship with diabetic nephropathy has not been reported.

  As described in detail above and in the Examples, when the expression of the NCALD gene in the proximal tubular epithelial cells was suppressed using the siRNA technique, expression of E-cadherin, which is a cell adhesion factor, in the epithelial cells. Suppression, induction of α-SMA expression, and enhancement of cell migration were observed. Here, E-cadherin is a protein abundantly contained in epithelial cells, and plays a role of adhering cells to each other and fixing the cells on the tissue membrane. Since mesenchymal cells do not express E-cadherin, the cells can move freely. On the other hand, α-SMA is a protein specifically found in mesenchymal cells (mesenchymal cell protein). In general, when tubular epithelial cell-stromal cell transformation (TEMT) occurs, the expression of E-cadherin is suppressed and the expression of mesenchymal cell proteins begins, resulting in disruption of intercellular junctions (basal It is known that cell morphology changes and cells acquire high motility (cell migration, invasion). As a result, extracellular matrix is produced, resulting in fibrosis of the kidney stroma leading to the development of nephropathy (Liu Y, et al., J Am Soc Nephrol 15: 1-12, 2004; Savagner P, et al., Bio Essays 23: 912-923, 2001; Okada H, et al., Am J Physiol 273: F563-F574, 1997; Kalluri R, et al., J Clin Invest 112: 1776-1784, 2003; Li Y, et al., J Clin Invest 112: 503-516, 2003; Iwano M, et al., J Clin Invest 110: 341-350, 2002; Oldfield MD, et al., J Clin Invest 108: 1853- 1863, 2001; Li JH, et al., Am J pathol 164: 1384-1397, 2004; Ina K, et al., J Med Election Microsc 35: 87-95, 2002).

  From the above, the above-mentioned phenomenon observed from suppressing the expression of the NCALD gene is that the mesenchymal mechanism of the proximal tubular epithelial cells proceeds due to the decrease in the expression of the NCALD gene in diabetic patients, and the renal It shows that the stroma becomes fibrotic, that is, nephropathy develops or progresses.

  By the way, the above-mentioned three diabetic nephropathy susceptibility SNPs can be mentioned as factors related to the decrease in expression of the NCALD gene. These SNPs are all present in the fourth exon of the NCALD gene. One is at position 999 (A / T) (SNP999) of the fourth exon of the NCALD gene, and the other is position 1307 (T / C) (SNP1307) at the fourth exon of the NCALD gene. Is located at position 1340 (G / A) (SN1340) of the fourth exon of the NCALD gene.

  As shown in the Examples, patients with type 2 diabetes who have haplotypes of SNP999 as thymine (T), SNP1307 as cytosine (C), and SNP1340 as adenine (A) have decreased NCALD gene expression and have nephropathy. There was a tendency to develop symptoms. Accordingly, these diabetic nephropathy-susceptible SNPs are genetic predisposing markers for diabetic nephropathy that regulate the ease of onset or progression of diabetic nephropathy. It becomes an index to judge onset and easy progression.

That is, at least one, preferably two, and more preferably all of these diabetic nephropathy-susceptible SNPs have the following conditions:
SNP 999 base: Thymine (T)
SNP 1307 base: cytosine (C)
SNP 1340 base: adenine (A)
If the condition is true, the subject having the SNPs is genetically susceptible to developing diabetic nephropathy or having a constitution that is likely to develop and progress from diabetes to nephropathy (sensitive to diabetic nephropathy). Judgment can be made.

  Although these SNPs are not restricted as a mechanism related to the susceptibility of diabetic nephropathy, the production of NCALD is reduced by destabilizing the mRNA expressed by NCALD from the results of Example 1. it is conceivable that.

3. Method for Screening Effective Components for Prevention or Treatment of Diabetic Nephropathy As described above, the present inventors show that the decrease in the expression of NCALD gene is deeply related to the onset and progression of diabetic nephropathy. I found. From this fact, NCALD is involved in the suppression of the onset or progression of diabetic nephropathy, and enhances the expression of NCALD gene (mRNA expression) to increase the amount of NCALD produced, or NCALD A substance having an action of enhancing the function 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 enhancing NCALD gene expression (mRNA expression) among test substances, and (3-2) increasing the production amount of NCALD. By searching for a substance having an action, or (3-3) a substance having an action of enhancing the function of NCALD, a prophylactic or therapeutic agent for diabetic nephropathy (hereinafter collectively referred to as “antidiabetic” The active ingredient of “nephropathy agent”).

  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) Screening method for substances having the action of enhancing the expression of NCALD gene The screening is performed by selecting substances having the action of enhancing the expression of NCALD gene from the test substances using the expression level of NCALD mRNA as an index. It is a method of searching and obtaining 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) A step of contacting a test substance with a cell capable of expressing the NCALD gene,
(2) a step of measuring the expression level of the NCALD gene in the cell contacted with the test substance, and (3) a test substance in which the measurement amount is larger than the expression level of the NCALD gene in the control cell not contacted with the test substance Process to select.

  The cells used for such screening may be cells that can express the NCALD gene, regardless of whether they are natural or recombinant. The origin of the NCALD gene is not particularly limited, and may be derived from a human, or from a mammal other than a human, such as a mouse, or other biological species, but preferably a human-derived NCALD gene. is there. Specific examples of such cells include proximal tubular epithelial cells having NCALD gene (including those derived from humans and other species), and primary cultured cells of isolated proximal tubular epithelial cells. Can be mentioned.

  In addition, a transformed cell prepared by introducing an expression vector having the cDNA of the NCALD gene and expressing NCALD mRNA in accordance with a conventional 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 NCALD gene-expressable cell is not particularly limited, but the cell does not die and the NCALD gene is expressed. It is preferable to select the culture conditions (temperature, pH, medium composition, etc.) to be obtained.

  Selection of candidate substances can be performed, for example, by contacting a test substance with a cell capable of expressing the NCALD gene under the above conditions, and searching for a substance that enhances the expression of the NCALD gene and increases its mRNA expression level. . Specifically, the expression level of NCALD mRNA when cells capable of expressing NCALD gene are cultured in the presence of a test substance is NCALD obtained when cells corresponding to NCALD gene are cultured in the absence of the test substance. The test substance that has been brought into contact with the cell can be selected as a candidate substance using as an index an expression level greater than the expression level of mRNA (control expression level).

  The measurement (detection and quantification) of the expression level of NCALD mRNA is carried out using the Northern blot method using an NCALD mRNA expression level of a cell capable of expressing NCALD gene using an oligonucleotide having a sequence complementary to the nucleotide sequence of the NCALD mRNA. It can be carried out by carrying out a known method such as RT-PCR method or real-time quantitative PCR method or a measuring method using a DNA array.

  TGF-β is known as a cytokine that plays a central role in tissue fibrosis and is known to induce epithelial-mesenchymal transition (EMT) in vivo. Therefore, when such a substance is allowed to coexist in the above screening system (for example, added to the medium), screening that reflects the in vivo environment becomes possible. Therefore, the NCALD gene-expressable cell may be contacted with TGF-β in the step (1) of the screening method or in the previous step. In addition, by doing this, it may be easier to catch the fluctuations in NCALD by inducing in vitro the factor groups that are induced in vivo in nephropathy.

(3-2) Method for screening a substance having an action to increase NCALD production The screening searches for a substance having an action to increase NCALD production from test substances using the production amount of NCALD as an index, It is a method of obtaining 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 NCALD or a cell fraction prepared from the cell,
(2 ′) a step of measuring the amount of NCALD produced in the cell or cell fraction contacted with the test substance, and (3 ′) a control cell in which the above test amount is not contacted with the test substance (cells capable of producing NCALD) Or a step of selecting a test substance larger than the amount of NCALD produced in a cell fraction (prepared from a cell capable of producing NCALD) which is not contacted with the test substance.

  The cells (including control cells) used for such screening may be any cells that can produce NCALD by expressing the NCALD gene, regardless of whether the cells are natural or recombinant. The origin of the NCALD gene is not particularly limited, and may be derived from a human, or from a mammal other than a human, such as a mouse, or other biological species, but preferably a human-derived NCALD gene. is there. Specific examples of such cells include proximal tubular epithelial cells having NCALD gene (including those derived from humans and other species), and primary cultured cells of isolated proximal tubular epithelial cells. Can be mentioned.

  In addition, a transformed cell prepared in a state capable of producing NCALD by introducing an expression vector having the cDNA of the NCALD gene according to a conventional 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 (3-2) of the present invention, the condition for bringing the test substance into contact with a cell capable of producing NCALD is not particularly limited, but the cell does not die and the NCALD gene is expressed. In addition, it is preferable to select culture conditions (temperature, pH, medium composition, etc.) that NCALD can produce.

  Selection of candidate substances can be performed, for example, by contacting a test substance with a cell capable of producing NCALD or a cell fraction thereof under the above conditions to search for a substance that increases the production amount of NCALD. Specifically, the amount of NCALD produced when culturing NCALD-producing cells or cell fractions thereof in the presence of a test substance is the same as the above-described NCALD-producing cells or cell fractions in the absence of the test substance. The test substance brought into contact with the cell or the cell fraction can be selected as a candidate substance, using as an index the production amount of NCALD (control production amount) obtained in this case.

  The measurement (detection and quantification) of the NCALD production amount is carried out by measuring the amount of NCALD obtained from NCALD-producing cells or cell fractions thereof by Western blotting, immunoprecipitation, ELISA using an antibody against the NCALD (anti-NCALD antibody). It can implement by performing well-known methods, such as.

  For the same reason as described above, a cell capable of producing NCALD or a cell fraction thereof may be contacted with TGF-β during the step (1 ′) of the screening method or in the previous step.

(3-3) Method of screening for substance having action of enhancing NCALD function The screening is carried out by searching for a substance having an action of enhancing the function of NCALD from test substances using the function of NCALD as an index. It is a method of acquiring as an active ingredient of a diabetic nephropathy agent.

Specifically, this method can be carried out by performing the following steps (1 ″) to (3 ″).
(1 ") a step of contacting a test substance with a cell capable of producing NCALD or a cell fraction prepared from this cell,
(2 ″) a step of detecting the NCALD function of the cell or cell fraction contacted with the test substance, and (3 ″) a control cell (cell capable of producing NCALD) in which the detected function is not contacted with the test substance. Or a test substance having a function larger than the NCALD function of the cell fraction (prepared from a cell capable of producing NCALD).

  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 candidate substances can be performed by contacting a test substance with a cell capable of producing NCALD or a cell fraction thereof under the above conditions to search for a substance that increases the function of NCALD. Specifically, the NCALD-producing cell or cell fraction corresponding to the above in the absence of the test substance is cultured when the NCALD-producing cell or cell fraction thereof is cultured in the presence of the test substance. The test substance can be selected as a candidate substance using as an index the function larger than the NCALD function (control function) obtained in this case.

  Here, as a function of NCALD, a function of inhibiting the conversion from epithelial cells to mesenchymal cells (function of inhibiting cell mesenchyme) can be mentioned. Specifically, (i) function to induce adhesion molecule expression (adhesion molecule expression induction function), (ii) function to suppress expression of mesenchymal cell specific protein (function to suppress expression of mesenchymal cell specific protein) ), (Iii) functions to suppress extracellular matrix production (extracellular matrix production suppression function), and (iv) functions to suppress cell migration or invasion (cell migration ability / invasion suppression function). be able to.

  As shown in Examples described later, by reducing the expression of the NCALD gene in the proximal tubular epithelial cells, (i) the expression of E-cadherin, an adhesion molecule, is suppressed, and (ii) actin (α-smooth expression of muscle actin; α-SMA) was induced. It was also observed that (iv) cell migration was enhanced by reducing the expression of the NCALD gene in proximal tubular epithelial cells. From these, it is considered that NCALD is involved in the conversion (mesenchymalization) into mesenchymal cells in the proximal tubular epithelial cells. In other words, due to the decrease in NCALD gene expression, loss of adhesion molecules → mesenchymal cell specific protein expression → basement membrane breakdown → cell migration / wetting → extracellular matrix production → tissue fibrosis → It is thought that nephropathy, such as nephropathy, has progressed to nephropathy. Therefore, NCALD directly or indirectly inhibits cell mesenchyme, specifically (i) adhesion molecule expression inducing function, (ii) mesenchymal cell specific protein expression suppressing function, (iii) It is considered to exert a function of suppressing production of extracellular matrix and (iv) a function of suppressing cell migration and invasion. Here, specifically, E-cadherin is specifically used as an adhesion molecule, and actin, preferably α-smooth muscle actin (α-SMA), is specifically used as an extracellular matrix. Can include fibronectin, type I collagen, type III collagen, laminin, and the like.

  These NCALD functions include, for example, adhesion molecules (eg, E-cadherin), mesenchymal cell specific proteins (eg, α-SMA), or extracellular matrix (eg, fibronectin, type I collagen, type III collagen). , Laminin) can be measured by measuring and comparing by a known method such as RT-PCR or ELISA. The function of NCALD can also be measured by measuring and comparing fibroblast-like changes in epithelial cells and tissue wettability by observation using an electron microscope.

  In addition, for the same reason as described above, a cell capable of producing NCALD or a cell fraction thereof may be contacted with TGF-β in the step (1 ″) of the above screening method or a previous stage thereof.

The substance selected by the screening method of (3-1) to (3-3) increases the expression of NCALD gene in the proximal tubular epithelial cell to increase the production of NCALD, or has a function of NCALD. It has an enhancing action, and can be used as an active ingredient in a composition for preventing the onset or progression of diabetic nephropathy or a composition for improving diabetic nephropathy. The candidate substance thus obtained 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 a subject who has a high risk of suffering from diabetic nephropathy (a person who is susceptible to diabetic nephropathy) The present invention also relates to the risk of suffering from or progression of diabetic nephropathy (from diabetic nephropathy). The present invention provides a method for selecting a subject having a high risk by measuring whether it is easy to develop and easy to progress / whether it is difficult to develop and difficult to progress. In the present invention, these high-risk subjects are referred to as “diabetic nephropathy susceptible persons”.

  As a screening method, (4-1) a method using the expression level of the NCALD gene (mRNA expression level) as an index, and (4-2) a method using the NCALD production level as an index for the test sample of the subject And (4-3) a method using NCALD gene-sensitive diabetic nephropathy-sensitive SNPs as an index.

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

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

  As a result of the measurement, when the expression level of NCALD mRNA in the test sample of the subject is smaller than the expression level (control expression level) of NCALD mRNA in the control sample derived from a normal person, the subject is diabetic. It can be judged that there is a high possibility of suffering from nephropathy (susceptibility to diabetic nephropathy). Here, “the expression level of NCALD mRNA is small” does not need to be compared with the control expression level, and includes the case where NCALD mRNA is not expressed at all, that is, the expression level of NCALD mRNA is substantially zero. It is.

  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 diabetic nephropathy susceptible person using NCALD production as an index This method can be performed by measuring the production of NCALD in a test sample derived from a subject. Here, the test sample may be a biological sample containing cells that express the NCALD gene and can produce NCALD, and specific examples thereof include blood.

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

  As a result of such measurement, when the NCALD production amount in the subject test sample is smaller than the NCALD production amount (control production amount) in the control sample derived from a normal person, the subject is diabetic nephropathy. It is possible to determine that there is a high possibility of suffering from diabetes (susceptibility to diabetic nephropathy). Here, “the production amount of NCALD is small” does not need to be compared with the control production amount, and includes the case where NCALD is not produced at all, that is, the production amount of NCALD is substantially zero.

(4-3) Selection method for diabetic nephropathy susceptible patients using NCALD gene-sensitive diabetic nephropathy susceptibility SNPs as an index This method is based on the following three SNPs for genomic DNA obtained from a subject. Detecting and identifying a base for at least one of the SNPs:
(1) Base 999 (SNP999) of the fourth exon of human NCALD gene
(2) Base at position 1307 (SNP1307) of exon 4 of human NCALD gene
(3) Base at position 1340 (SNP1340) of the fourth exon of the human NCALD gene.

  Preferably, it is a method having a step of detecting and identifying a base for two or more SNPs in these SNPs, and more preferably a method having a step of detecting and identifying a base for all three SNPs in these SNPs. .

Preferably, the detection method of the present invention comprises a step of detecting and identifying one, preferably at least two, more preferably three bases selected from the above SNPs for genomic DNA obtained from a human subject. Furthermore, at least the base detected and identified has the following conditions:
(1) Base at position 999 (SNP999) of the fourth exon of human NCALD gene: thymine
(2) Base at position 1307 (SNP1307) of exon 4 of human NCALD gene: cytosine
(3) Base of position 1340 (SNP1340) of the 4th exon of human NCALD gene: When the adenine is satisfied, the subject is susceptible to diabetic nephropathy (susceptible to diabetic nephropathy) Can be determined and selected.

  According to this method, it is possible to determine and diagnose the risk of diabetic nephropathy susceptibility and diabetic nephropathy susceptibility, 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 bases of the SNPs as judgment criteria (judgment indicators). . 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 specimen from which genomic DNA is extracted is any cell (including cultured cells, excluding germ cells), tissue (including cultured tissue), organ, or body fluid (for example, isolated from subjects and clinical specimens). , Saliva, lymph, 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 leukocytes are used 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. Furthermore, if necessary, DNA containing the NCALD gene on human chromosome 8 (8q22-23) or its fourth exon 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 NCALD gene or its fourth exon.

  In the step of detecting the target base, diabetic nephropathy-sensitive SNPs that are highly related to diabetic nephropathy are detected from the extract containing human genomic DNA obtained as described above. The base is detected by directly determining the base sequence in the NCALD gene on human chromosome 8, preferably the LD block containing the fourth exon, further isolated from a sample containing human genomic DNA, A method for examining the mutation of the bases of various SNPs located may be used.

  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 test 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 The person can be informed of this, and appropriate measures can be taken 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) Probes Diabetic nephropathy-sensitive SNPs described in (4-3) above and nucleotides containing the base are detected by detecting diabetic kidneys on the NCALD gene of human chromosome 8 (8q22-23). Oligos or polynucleotides that can specifically hybridize to oligos or polynucleotides containing disease-susceptible SNPs and detect the SNPs are used. Such oligos or polynucleotides are 16 to 500 bases long, preferably 20 to 200 bases long, more preferably 20 to 50 bases long each containing individual SNPs (SNP999, SNP1307, SNP1340) on the NCALD gene. It is designed as an oligo or polynucleotide having the above base length so as to specifically hybridize with the gene region.

  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.

  Such an oligo or polynucleotide is at least one oligo or polynucleotide selected from the group consisting of the following (a) to (c) (provided that when the oligo or polynucleotide is RNA, the base symbol in the sequence listing) “T” shall be read as “u”), and may be a continuous oligo or polynucleotide having a length of 16 to 500 bases.

(a) In the nucleotide sequence of the human NCALD gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases comprising a nucleotide located at position 999 (SNP999) of the fourth exon,
(b) in the base sequence of the human NCALD gene, a continuous oligo or polynucleotide having a length of 16 to 500 bases containing a nucleotide located at position 1307 (SNP1307) of the fourth exon;
(c) A continuous oligo or polynucleotide having a length of 16 to 500 bases comprising a nucleotide located at position 1340 (SNP1340) of the fourth exon in the base sequence of human NCALD gene.

  In order to determine the susceptibility to or progression of diabetic nephropathy in a subject, the oligo or polynucleotide is a diabetic nephropathy sensitive SNPs (SNP999, SNP1307) located on the NCALD gene on human chromosome 8. Or an oligo or polynucleotide “probe” that specifically hybridizes to an oligo or polynucleotide comprising SNP1340). In addition, these oligos or polynucleotides can be prepared based on the nucleotide sequence of the NCALD 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 case of the TaqMan PCR method (Livak KJ. Gene Anal 14, 143 (1999), Morris T et al., J Clin Microbiol 34, 2933 (1996)), which is an example of the ASO method, is susceptible to various types of diabetic nephropathy. An oligonucleotide having a length of about 20 bases complementary to the region containing SNPs 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 Invade method [Lyamichev V. et al., Nat Biotechnol 17, 292 (1999)], which is another example of the ASO method, the sequences adjacent to the polymorphic site (2 'on the 3' side and 2 'on the 5' side) Complementary oligonucleotides are designed as probes. 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 diabetic nephropathy-susceptible SNPs on the NCALD gene of human chromosome 8.

  Such an oligo or polynucleotide includes at least one oligo or polynucleotide selected from the group consisting of the following (a) to (c) (provided that when the oligo or polynucleotide is RNA, a base symbol in the sequence listing) “T” can be read as “u”), and can be exemplified by oligos or polynucleotides having a length of 15 to 30 bases.

(a) a continuous oligo or polynucleotide having a length of 16 bases or more, comprising a nucleotide located at position 999 (SNP999) of the fourth exon in the base sequence of the human NCALD gene;
(b) In the nucleotide sequence of the human NCALD gene, a continuous oligo or polynucleotide having a length of 16 bases or more including a nucleotide located at position 1307 (SNP1307) of the fourth exon,
(c) In the nucleotide sequence of the human NCALD gene, a continuous oligo or polynucleotide having a length of 16 bases or more, comprising a nucleotide located at position 1340 (SNP1340) of the fourth exon.

  Such an oligonucleotide specifically hybridizes to a part of a continuous oligo or polynucleotide containing a base (nucleotide) of diabetic nephropathy-susceptible SNPs in the NCALD gene, and the oligo or polynucleotide specifically 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 or more bases in the vicinity of various diabetic nephropathy-susceptible SNPs (SNP999, SNP1307, SNP1340) on the NCALD gene of human chromosome 8. In the present invention, it can be used as a primer. In addition, these oligonucleotides can be prepared according to a conventional method using, for example, a commercially available nucleotide synthesizer based on the base sequence of the human NCALD 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. Further, 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 Protocol Genetics (Current Protocol) The method described in Publishing Associates and Wiley-Interscience) was used.

Example 1
Under informed consent, Shiga Medical University, Tokyo Women's Medical University, Juntendo University, Kawasaki Medical University, Iwate Medical University, Toride Kyodo Hospital, Kawai Clinic, Osaka City General Medical Center, Chiba Tokushukai Hospital, or Osaka Labor Hospital An experiment was conducted on type 2 diabetic patients who visited regularly.

The target type 2 diabetic patients were classified into two groups according to the following diagnostic criteria:
1) Diabetic nephropathy cases: patients with diabetic retinopathy and obvious diabetic nephropathy (urinary albumin excretion rate exceeding 200 μg / min or urinary albumin / creatinine ratio exceeding 300 mg / g Cr) or chronic Patients undergoing dialysis therapy,
2) Control group: patients with no signs of renal dysfunction but with diabetic retinopathy [urinary albumin excretion rate less than 20 μg / min or urinary albumin / creatinine ratio less than 30 mg / g Cr].

  The peripheral blood of the type 2 diabetic patient was collected, and 7 ml of the peripheral blood obtained was centrifuged at 3000 rpm for 10 minutes to remove plasma. 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 70% ethanol was added, and the resulting DNA pellet was mixed with 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 determined using an invader method combined with Multiplex PCR [see, for example, Ohnishi et al. Human. Genet. , 46, 471-477, 2001, etc.].

  The gene polymorphism was searched in more detail by the direct sequence method. The target site was amplified using PCR primers designed based on GenBank information [accession number: NM_032041] related to the genome sequence containing NCALD. In designing PCR primers, as described in Bedell et al. [Bioinformatics, 16, 1040-1041, 2000], the trade name: REPEAT MASKER program [supplied by the University of Washington, web page address: http: // Available at repeatmasker.genome.washington.edu] to eliminate repeated elements from the search.

The reaction conditions in Multiplex PCR are as follows: reaction solution [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 dNTP, 10 × Taq mix (2.5 U / μl Taq DNA polymerase, 31.25 U / μl Taq Antibody), 0 or 10% DMSO, each 0.25 μM primer], reaction at 95 ° C. for 5 minutes, then 95 ° C. for 15 minutes Forty cycles of reaction were carried out, with one second consisting of 45 seconds at 60 ° C. and 3 minutes at 72 ° C.

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.

  For 94 patients with diabetic nephropathy and 94 individuals in the control group, 81315 SNP loci were genotyped. Then, by evaluating statistical data using a 2 × 3 or 2 × 2 contingency table, a significant difference in genotype or allelic frequency between patients with diabetic nephropathy and control individuals The indicated SNP was 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 performed by Devlin B. et al. [Genomics, 29, 311-322, 1995] and Nielsen. DM.) Et al. [Am. J. et al. Hum. Genet. , 63, 1531-1540, 1998].

  In the above screening, SNP loci that showed a p value of less than 0.01 between patients with nephropathy cases and individuals in the control group were analyzed for a larger number of patients.

  As a result, as shown in Table 1, the genotype distribution of one SNP locus (1340 G / A, hereinafter also referred to as “SNP1340”) in the fourth exon of the NCALD gene is strongly related to diabetic nephropathy. [A vs. G, χ2 = 16.9, p = 0.004, odds ratio 1.59, 95% CI 1.27 to 1.98]. In the table, “HWE Test” means Hardy-Weinberg equilibrium test.

また、ＮＣＡＬＤ遺伝子におけるランドマークＳＮＰ（SNP1340）周辺の領域を連鎖不均衡マッピングすることにより、図１に示すように、ランドマーク部位（SNP1340）（図中、＊で示す）の約３０ｋｂ上流及び２０ｋｂ下流に、この領域における連鎖不均衡が広がっていることが示された。

Further, by performing linkage disequilibrium mapping of the region around the landmark SNP (SNP1340) in the NCALD gene, as shown in FIG. 1, about 30 kb upstream and 20 kb of the landmark site (SNP1340) (indicated by * in the figure) Downstream, it was shown that linkage disequilibrium in this region is widening.

  This suggests that a region important for susceptibility to diabetic nephropathy probably exists within this 50 kb high linkage disequilibrium block. Within this linkage disequilibrium block, there are two genes, the NCALD gene and the TFCP2L3 gene. However, since the expression of the TFCP2L3 gene was not observed in the kidney in both the quantitative PCR method and the in situ hybridization method, the NCALD gene was considered to be a candidate for susceptibility to diabetic nephropathy.

  Therefore, other polymorphisms in the NCALD gene were then examined.

  As a result, 115 polymorphisms, specifically 108 SNPs and 7 insertion / deletion polymorphisms were identified on the NCALD gene separately from SNP1340. Of these, when linkage disequilibrium mapping (LD mapping) was performed on a polymorphism of 16 SNPs (see FIG. 2) presumed to be accompanied by a functional mutation, two SNPs of exon 4 (+999 A / T (hereinafter, referred to as “the SNPs”) Linkage disequilibrium was observed at +1307 T / C (hereinafter also referred to as “SNP1307”)). In addition, the position number of SNP shown in a figure is a number when the first base of the exon or intron which each positions is set to 1.

Example 2
In order to examine the influence of the NCALD gene by the three SNPs (SNP999, SNP1307, SNP1340) identified in Example 1, the stability of the expression product mRNA of the NCALD gene into which each SNP was introduced was examined.

  First, the full length cDNA of the NCALD gene was PCR cloned and inserted into a pCRII-TOPO vector (Invitrogen). Thereafter, thymine (T), cytosine (C), and adenine (A) were introduced into each of the three SNP sites (SNP999, SNP1307, SNP1340) of exon 4 using a mutagenesis kit (Stratagene). From the resulting vector, RNA was synthesized and purified using Ribomax Large Scale RNA Production System-T7 (Promega).

  On the other hand, normal human proximal tubule epithelial cells (manufactured by Sanko Junyaku Co., Ltd.) were washed with PBS, and then eluted buffer (0.5% Nonidet P-40; 20 mM HEPES buffer, pH 8.0; 20% glycerol (v / v); The suspension was suspended in 400 mM NaCl, 0.5 mM dithiothreitol; 0.2 mM EDTA, 0.1% protease inhibitor cocktail (manufactured by Nacalai) and left on ice for 30 minutes. Thereafter, the mixture was centrifuged at 15,000 G for 30 minutes, and the supernatant was collected and used as a cell extract.

  25 μg of the above RNA and the above cell extract diluted 1000 times were mixed and allowed to stand at room temperature for 0 to 15 minutes. This reaction solution was incubated at 65 ° C. for 15 minutes and electrophoresed on a formamide agarose gel (Susceptible). As a control, RNA prepared from a vector prepared so that the three SNPs (SNP999, SNP1307, SNP1340) of exon 4 of the NCALD gene were adenine (A), thymine (T), and guanine (G), respectively. Similarly, it was reacted with the cell extract and subjected to electrophoresis (Non-susceptible).

  After electrophoresis, it was stained with ethidium bromide, scanned with LAS-3000 (Fuji Film), and the fluorescence intensity of each band corresponding to NCALD mRNA was measured and quantified.

  FIG. 3 shows the fluorescence intensity of NCALD mRNA of haplotype (Non-susceptible) in which the three SNPs of exon 4 (SNP999, SNP1307, SNP1340) are adenine (A), thymine (T), and guanine (G), respectively. And SNP999, SNP1307 and SNP1340 show changes over time in the fluorescence intensity of the haplotype (Susceptible) NCALD mRNA, which are thymine (T), cytosine (C), and adenine (A), respectively.

  As can be seen from the results, the fluorescence intensity half-life of NCALD mRNA of major haplotypes (Non-susceptible) whose three SNPs of exon 4 (SNP999, SNP1307, SNP1340) are A, T, and G was 10 minutes. On the other hand, the fluorescence intensity half-life of NCALD mRNA of haplotype (susceptible) in which T, C, and A are respectively introduced into these SNPs positions is as short as 6.7 minutes, and the stability is clearly lowered. It was recognized that

Example 3
The siRNA was used to suppress NCALD gene expression, and the effect on diabetic nephropathy-related factors was examined.

As siRNA sequences specific to the NCALD gene, siRNA having a sequence specific to the nucleotide sequence 373-391 of the NCALD gene (siRNA # 1) and siRNA having a sequence specific to the 597-615 position (siRNA # 2) Selected:
siRNA # 1 (nt373-391): 5'-TTC ATC ATC GCC TTG AGT GdTdT-3 '(SEQ ID NO: 1)
siRNA # 2 (nt597-615): 5'-TAG AGA CGG AAA ACT CTC CdTdT-3 '(SEQ ID NO: 2).

  Double strands of these siRNAs (siRNA # 1, siRNA # 2) are prepared and transferred to normal human proximal tubular epithelial cells. Control siRNA (5'-AATTCTCCGAACGTGTCACGT-3 (SEQ ID NO: 3), manufactured by Qiagen) Transfected with. As a transfection reagent, Gene Silencer siRNA Transfection Reagent (manufactured by Gene Therapy System) was used. After 48 hours, cells were lysed and quantitative PCR and Western blotting were performed.

  From the obtained cells, RNA was collected using a nucleospin column (manufactured by Makerei Nagel), and cDNA was synthesized using the Superscript III first strand system (Invitrogen). Using this cDNA, the expression level (mRNA level) of E-cadherin gene and α-SMA gene was measured by quantitative PCR.

In the quantitative PCR (thermal profile), after incubation at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, 40 cycles of reactions with 95 ° C. for 30 seconds, 63 ° C. for 30 seconds and 72 ° C. for 30 seconds are performed. Was carried out. For quantitative PCR, 1 × TaKaRa Ex Taq ™ buffer (Takara Bio), 200 nM dNTP mixture, 1/20000 SYBR Green, each 800 nM primer, 0.05 U Ex Taq ^™ DNA polymerase (Takara Bio) A reaction solution having 2.75 ng TaqStart Antibody (trade name) (manufactured by Clontech) and 5 ng template was used.

Quantitative PCR was also performed using the following primers:
About NCALD gene (cDNA)
Sense primer: 5'-AGC ATG TAC GAC CTG GAC GG-3 '(SEQ ID NO: 4),
Antisense primer: 5-TTT GGC TCC TCG GAT GAA CT-3 ′ (SEQ ID NO: 5),
About the E-cadherin gene (cDNA)
Sense primer: 5'-GCC CAT TTC CTA AAA ACC TGG-3 '(SEQ ID NO: 6),
Antisense primer: 5'-TTG GAT GAC ACA GCG TGA GAG-3 '(SEQ ID NO: 7),
About GAPDH gene (cDNA)
Sense primer: 5′-AGG TGA AGG TCG GAG TCA ACG-3 ′ (SEQ ID NO: 8),
Antisense primer: 5′-GCT CCT GGA AGA TGG TGA TGG-3 ′ (SEQ ID NO: 9),
About 36B4 gene (cDNA)
Sense primer: 5'-AAG AAC ACC ATG ATG CGC AAG-3 '(SEQ ID NO: 10),
Antisense primer: 5′-CCT TAT TGG CCA GCA ACA TGT C-3 ′ (SEQ ID NO: 11).

  The GAPDH gene and 36B4 gene were used as controls (internal standard genes).

  The results are shown in panels a) and c) of FIG. Panel a) shows the result of measuring the expression level (mRNA amount) of the E-cadherin gene by quantitative PCR, and panel c) shows the result of measuring the expression level (mRNA amount) of the α-SMA gene by quantitative PCR. In addition, all have shown with the ratio with respect to the expression level of 36E4 gene which is an internal standard gene.

  As shown in FIG. 4, the siRNA (siRNA # 1 or siRNA # 2) for the NCALD gene decreases the expression level (mRNA amount) of the E-cadherin gene, while the expression level (mRNA) of the α-SMA gene decreases. Increased.

  Normal human proximal tubular epithelial cells transfected with siRNA (siRNA # 1 or siRNA # 2) were treated with 1% NP40, 1% Triton X-100, 0.35 mg / ml PMSF, 9.5 μg / ml leupeptin, 13. It was dissolved in a PBS solution containing 75 μg / ml pepstatin A. Subsequently, the supernatant was collected by centrifugation at 14,000 G, and this was used as a cell extract. The amount of protein in the cell extract was quantified using a Bradford protein assay reagent (manufactured by Bio-Rad).

  The cell extract thus obtained was electrophoresed by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was reacted with anti-E-cadherin antibody overnight after blocking. After washing, it was reacted with an HRP-labeled anti-mouse antibody and detected with an ECL plus western blotting detection system (Amersham). The result is shown in panel b) of FIG.

  As can be seen, siRNA (siRNA # 1 or siRNA # 2) against the NCALD gene decreased the amount of E-cadherin protein.

Example 4
Using an improved Boyden chamber, the influence of the NCALD gene on cell migration ability was examined.

  Normal human proximal tubular epithelial cells transfected with the aforementioned siRNA (siRNA # 1 or siRNA # 2) were seeded above a 24-well Boyden chamber (manufactured by Coaster) and cultured for 24 hours. Thereafter, the filter was taken out, fixed with ethanol, and the cells remaining above the Boyden chamber were removed with cotton. Only cells that migrated to the lower side of the filter were stained with 0.2% crystal violet, and the number of cells present in the same area was measured. The result is shown in FIG. Panel a) shows a microscopic image, and b) shows the number of cells measured.

  As shown in FIG. 5, migration ability was increased in normal human proximal tubular epithelial cells transfected with siRNA (siRNA # 1 or siRNA # 2) against the NCALD gene as compared to the control.

  From the above, the stability of NCALD mRNA was reduced by introducing three SNPs susceptible to diabetic nephropathy into the NCALD gene. Moreover, the expression of NCALD gene was suppressed and the production amount of NCALD was reduced, resulting in a decrease in the expression level of E-cadherin, an increase in α-SMA expression, and an increase in cell migration ability. From these facts, it is considered that suppression of NCALD gene expression and suppression of NCALD production are probably involved in the onset and progression of diabetic nephropathy.

It is a figure which shows the linkage disequilibrium mapping around a NCALD gene. In the figure, * on the NCALD gene indicates the position of the landmark SNP (SNP1340). Of the polymorphisms in the NCALD gene, 16SNPs presumed to be accompanied by functional mutation are shown. Among them, the SNP (SNP 1340) at position +1340 marked with * is a landmark SNP. The position number of each SNPs indicates the number when the first base of the intron or exon in which each SNP exists is +1. It is a figure which shows the influence with respect to stability of the expression product (mRNA) of a NCALD gene of SNPs (SNP1340, SNP1307, SNP999). Non-susceptible (Haplotype) indicates a haplotype that is insensitive to diabetic nephropathy, and Susceptible (Haplotype) indicates a haplotype that is susceptible to diabetic nephropathy. It is a figure which shows the influence of siRNA with respect to the expression of NCALD gene in a normal human proximal tubule epithelial cell. Panel a) shows the result of measuring the expression level of the E-cadherin gene by quantitative PCR, b) shows the result of Western blotting, and c) shows the result of measuring the expression level of the α-SMA gene by quantitative PCR. Both the results of panels a) and c) are shown as a ratio to the expression level of 36E4 gene, which is an internal standard gene. The influence on the cell migration ability of siRNA with respect to the expression of the NCALD gene in a normal human proximal tubule epithelial cell is shown. Panel a) shows a microscopic image, and b) shows the number of cells measured.

Claims (11)

A method for screening a substance that enhances the expression of an NCALD gene, comprising the following steps:
(1) A step of contacting a test substance with a cell capable of expressing the NCALD gene,
(2) a step of measuring the expression level of the NCALD gene in the cell contacted with the test substance, and (3) a test substance in which the measurement amount is larger than the expression level of the NCALD gene in the control cell not contacted with the test substance Process to select,
Furthermore, the said screening method including the process which makes TGF- (beta) contact the cell which can express a NCALD gene in a process (1) or its previous step. A method for screening for a substance that increases the amount of NCALD produced, comprising the following steps:
(1 ′) contacting a test substance with a cell capable of producing NCALD or a cell fraction prepared from the cell;
(2 ′) a step of measuring the amount of NCALD produced in the cell contacted with the test substance or the cell fraction thereof, and (3 ′) the above measured amount being a control cell or a fraction of the cell that is not contacted with the test substance Selecting a test substance larger than the amount of NCALD produced,
Furthermore, in the step (1 ′) or a previous step thereof, the screening method comprising a step of contacting TGF-β with a cell capable of producing NCALD or a cell fraction prepared from the cell. A method for screening for a substance that enhances the function of NCALD, comprising:
(1 ") a step of contacting a test substance with a cell capable of producing NCALD or a cell fraction prepared from this cell,
(2 ″) a step of detecting the NCALD function of the cell contacted with the test substance or a fraction of the cell, and (3 ″) a control cell or a fraction of the cell in which the detected function is not contacted with the test substance. Selecting a test substance larger than the function of NCALD,
Furthermore, in the step (1 ″) or a previous step thereof, the screening method comprising a step of contacting TGF-β with a cell capable of producing NCALD or a cell fraction prepared from the cell. The screening method according to claim 3, wherein the function of NCALD is inhibition of cell mesenchyme. The screening method according to claim 3, wherein the function of NCALD is induction of adhesion molecule expression. The screening method according to claim 5, wherein the adhesion molecule is E-cadherin. The screening method according to claim 3, wherein the function of NCALD is suppression of actin expression. The screening method according to claim 7, wherein the actin is α-smooth muscle actin. The screening method according to claim 3, wherein the function of NCALD is suppression of extracellular matrix production. The screening method according to claim 3, wherein the function of NCALD is suppression of cell migration ability or invasion. The screening method according to any one of claims 1 to 10, wherein the cell capable of expressing the NCALD gene or the cell capable of producing NCALD is a proximal tubular epithelial cell.

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