WO2015119253A1 - 腎疾患の予防または治療剤 - Google Patents
腎疾患の予防または治療剤 Download PDFInfo
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- WO2015119253A1 WO2015119253A1 PCT/JP2015/053415 JP2015053415W WO2015119253A1 WO 2015119253 A1 WO2015119253 A1 WO 2015119253A1 JP 2015053415 W JP2015053415 W JP 2015053415W WO 2015119253 A1 WO2015119253 A1 WO 2015119253A1
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- renal
- nephropathy
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- renal failure
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- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/775—Apolipopeptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a preventive or therapeutic agent for renal diseases.
- Acute renal failure is a disease caused by various causes such as renal ischemia, nephrotoxic toxins, and sepsis. The rate of long-term hospitalization and mortality is high. It has been increasing in recent years. It is known that there are not a few cases in which acute renal failure does not fully cure and becomes chronic, resulting in chronic renal failure.
- Pharmacological, dietary, and resting treatments are used as treatments for chronic kidney disease.
- antihypertensive drugs, diuretics, active vitamin D preparations, oral adsorbed carbon preparations, potassium adsorbents, phosphorus adsorbents, etc. are used as pharmacotherapy and are associated with slowing the progression of the disease or decreasing kidney function
- the aim is to improve symptoms.
- none of the treatment methods can sufficiently stop the progression of the disease, and a new treatment method is required.
- there is no reliable treatment for acute renal failure, and the development of an immediate treatment is desired.
- kidney disease is a major issue.
- Chronic renal failure is the most common disease in older cats, and kidney failure is said to be the leading cause of death in older cats.
- urinary tract stones and urinary tract infections caused kidney function to deteriorate and acute kidney failure (or acute kidney injury), and many became chronic kidney failure 15 Death by about age.
- there is no satisfactory treatment for kidney disease and a new treatment method is required.
- AIM apoptosis-inhibitor-of-macrophage
- Non-patent Document 1 AIM increases blood concentration with obesity, is taken up by adipocytes by CD36-mediated endocytosis, and induces degradation of accumulated triglycerides (lipolysis). Is suggested (Non-Patent Document 2). AIM also releases free fatty acids from adipocytes through the breakdown of neutral fat, and the released fatty acids induce and maintain chronic inflammation in adipose tissue through stimulation of toll-like receptors.
- Metabolic syndrome is based on the acquisition of insulin resistance associated with obesity, but since chronic inflammation in adipose tissue is important, AIM is considered to be related to metabolic syndrome (Non-patent Document 3). Furthermore, the present inventor has clarified that obese AIM ⁇ ⁇ ⁇ KO mice loaded with a high calorie diet show a pathological condition similar to human NASH pathological conditions such as obesity, fatty liver, fibrosis of the liver parenchyma, and carcinogenesis. Has reported the possibility of application to (patent document 1). However, the relationship between AIM and kidney disease has not been known so far.
- An object of the present invention is to provide a preventive or therapeutic agent for renal diseases. Another object of the present invention is to provide a method for evaluating or screening a preventive or therapeutic agent for renal disease using a new model mouse for renal disease. Yet another object of the present invention is to provide a method for predicting the prognosis of renal diseases.
- the present inventor for AIM knockout mice subjected to unilateral ureteral obstruction (UUO) or transient renal ischemia / reperfusion (IR; ischemia / reperfusion) after unilateral nephrectomy,
- UUO ureteral obstruction
- IR transient renal ischemia / reperfusion
- IR transient renal ischemia / reperfusion
- a prophylactic / therapeutic agent for kidney disease comprising a nucleic acid comprising AIM or a partial peptide thereof or a base sequence encoding them;
- a prophylactic / therapeutic agent for renal disease comprising a drug that induces the expression of AIM or a drug that stabilizes AIM;
- the renal disease is acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease or IgM nephropathy
- the present invention can provide a prophylactic / therapeutic agent for renal diseases containing AIM as an active ingredient. Moreover, according to the screening method using the kidney disease model mouse of the present invention, a substance effective for prevention / treatment of kidney disease can be searched. Moreover, the effect of a known preventive / therapeutic agent for renal disease can be evaluated using the renal disease model mouse of the present invention. Furthermore, the present invention can provide a prognostic method for predicting renal disease and a test method for renal disease by measuring the AIM concentration in the sample of the subject.
- A Azan and hematoxylin co-stained images of normal kidney tissue pieces and post-UUO kidney tissue pieces of AIM KO mice and WT mice subjected to unilateral ureteral ligation.
- B Azan, PAS and hematoxylin co-stained images of normal kidney tissue pieces and UUO kidney tissue pieces of AIM KO mice and WT mice subjected to unilateral ureteral ligation. It is a hematoxylin-eosin stained image of the kidney tissue pieces of AIM KO mice and WT mice subjected to transient renal ischemia reperfusion after unilateral nephrectomy.
- A Immunostained images of F4 / 80 of kidney tissue pieces of AIM KO mice and WT mice subjected to transient renal ischemia reperfusion after unilateral nephrectomy.
- B is a graph showing the expression level of MCP-1 in kidney tissue pieces of AIM KO mice and WT mice subjected to transient renal ischemia reperfusion after unilateral nephrectomy. *: P ⁇ 0.05, **: P ⁇ 0.01
- A A hematoxylin-eosin stained image of a kidney tissue piece of an AIM-KO mouse that had undergone transient renal ischemia / reperfusion after unilateral nephrectomy and was administered rAIM or PBS.
- B is a graph showing the BUN value of AIM ⁇ ⁇ ⁇ KO mice subjected to transient renal ischemia / reperfusion after unilateral nephrectomy and administered with rAIM or PBS. These are phase-contrast images of kidney tissue fragments and immunostained images for AIM of AIM KO mice that had undergone transient renal ischemia / reperfusion after unilateral nephrectomy and were administered rAIM.
- N Indicates a necrotic lesion. Arrow: indicates AIM. It is a graph which shows the urinary AIM value of the WT mouse
- a graph showing the area of dead cell mass in the proximal tubule of the kidney tissue pieces of AIM KO mice and WT mice subjected to bilateral transient renal ischemia reperfusion as a percentage of the total area per section is there.
- *: P ⁇ 0.05 It is a figure which shows the flow cytometer analysis result of the cell derived from the kidney of the 7th day after IR of the AIM KO mouse and WT mouse which performed bilateral transient renal ischemia reperfusion.
- FIG. 3 shows sequential PAS staining images and immunostaining images with anti-AIM antibodies of kidney tissue pieces of AIM KO mice subjected to bilateral transient renal ischemia reperfusion. It is a graph showing the phagocytic ability of AIM-KO mice after bilateral IR treatment, the kidney on the third day after IR was treated with collagenase, and F4 / 80 positive macrophages were isolated using a FACS sorter. . It is the graph which showed the phagocytic ability, after isolating the macrophage which differentiated the bone marrow cell derived from AIM-KO by M-CSF using a FACS sorter.
- A: A graph showing the correlation between eGFR (glomerular filtration rate; ml / min / 1.73m 2 ) or blood creatinine level (mg / dl) and blood AIM concentration in patients with chronic renal failure (n 55). is there.
- A Western blot images of AIM and IgM for each size fraction of cat-derived serum intravenously injected with recombinant cat AIM (1 mg).
- B Western blot images of AIM and IgM for each size fraction of mouse-derived serum. It is the western blot image of AIM and IgM with respect to each size fraction of the serum derived from the cat which intravenously injected recombinant mouse AIM (1 mg). It is a figure which shows a cat AIM cDNA sequence (SEQ ID NO: 5). The translation start point (atg) and translation stop point (tga) are shown in bold. Also, non-coding ⁇ sequence is shown in italics. The base sequence encoding the leader peptide is indicated by a solid underline.
- FIG. 3 is a view showing hydrophobicity of a leader peptide sequence of a feline AIM protein. It is a figure which shows the hydrophobicity of the leader peptide sequence of a human AIM protein. It is a figure which shows the hydrophobicity of the leader peptide sequence of a mouse
- the present invention provides a preventive / therapeutic agent for renal diseases, comprising AIM or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding them.
- AIM in the present invention is an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (amino acid sequence of human-derived AIM protein) or SEQ ID NO: 4 (amino acid sequence of cat-derived AIM protein). Contains protein.
- AIM is isolated and purified from macrophages that are immune cells of warm-blooded animals (eg, humans, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, birds, etc.). It may be a protein.
- it may be a protein synthesized chemically or by a cell-free translation system, or a recombinant produced from a transformant introduced with a nucleic acid containing a base sequence encoding the amino acid sequence. It may be a protein.
- amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is about 60% or more, preferably about Examples include amino acid sequences having homology of 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
- homology refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered).
- Similar amino acids means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution).
- amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is about 60% or more with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.
- Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 include the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.
- a protein having substantially the same amino acid sequence and having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is preferred.
- activity refers to, for example, atherosclerotic lesion macrophage apoptosis inhibitory activity, arteriosclerosis maintenance / promoting activity, adipocyte differentiation inhibiting activity, adipocyte lipid droplet thawing activity, adipocyte shrinking activity, CD36 binding activity, It refers to endocytic activity on fat cells, FAS binding activity, FAS function inhibitory activity, anti-obesity activity, and the like. “Substantially homogeneous” indicates that their activities are qualitatively (eg, physiologically or pharmacologically) the same.
- the activities are equivalent, but quantitative factors such as the degree of these activities (for example, about 0.1 to about 10 times, preferably about 0.5 to about 2 times) and the molecular weight of the protein. May be different.
- the activity can be measured according to a method known per se.
- the AIM of the present invention includes, for example, (1) one or more of the amino acid sequences represented by SEQ ID NO: 2 or SEQ ID NO: 4 (preferably about 1 to 100, preferably 1 to About 50 amino acids, more preferably about 1 to 10, particularly preferably 1 to several (2, 3, 4 or 5) amino acid sequences deleted.
- amino acid sequences represented by 4 preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, and particularly preferably 1 to number (2, 3 , 4 or 5) amino acid sequence added, (3) 1 or more (preferably about 1 to 50, preferably about the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 Is an amino acid in which about 1 to 10 amino acids, more preferably 1 to several (2, 3, 4 or 5) amino acids are inserted Sequence, (4) one or more of the amino acid sequences represented by SEQ ID NO: 2 or SEQ ID NO: 4 (preferably about 1 to 50, preferably about 1 to 10, more preferably 1 to A protein containing an amino acid sequence in which several (2, 3, 4 or 5) amino acids are substituted with other amino acids, or (5) an amino acid sequence combining them is also included.
- the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity
- the AIM of the present invention is preferably a human AIM protein having the amino acid sequence represented by SEQ ID NO: 2 (GenBank accession number: AAD01446), a cat AIM protein having the amino acid sequence represented by SEQ ID NO: 4, or A homologue [for example, a mouse homologue registered in GenBank as accession number: AAD01445, etc.] in other mammals, more preferably a human AIM protein or sequence comprising the amino acid sequence represented by SEQ ID NO: 2 It is a feline AIM protein consisting of the amino acid sequence represented by No. 4.
- proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation.
- the AIM of the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, has a C-terminal carboxyl group (—COOH), carboxylate (—COO ⁇ ), amide ( Either -CONH 2 ) or ester (-COOR) may be used.
- R in the ester for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C 6-12 aryl groups such as ⁇ -naphthyl; phenyl-C 1-2 alkyl groups such as benzyl and phenethyl; C 7- such as ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl; 14 aralkyl group; pivaloyloxymethyl group is used.
- a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl
- a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl
- the AIM of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention.
- the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
- the amino group of the N-terminal amino acid residue is protected with a protecting group (for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as formyl group, acetyl group, etc.).
- N-terminal glutamine residue that can be cleaved in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) a suitable protecting group (e.g., formyl group, those protected by C 1-6 an acyl group) such as C 1-6 alkanoyl group such as acetyl group, or a sugar chain-binding Also included are complex proteins such as so-called glycoproteins.
- a substituent on the side chain of an amino acid in the molecule eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.
- a suitable protecting group e.g., formyl group, those protected by C 1-6 an acyl group
- C 1-6 alkanoyl group such as acetyl
- a partial peptide of AIM is a peptide having the above-mentioned partial amino acid sequence of AIM and has substantially the same quality of activity as AIM. Any one may be used.
- substantially the same quality of activity has the same meaning as described above. Further, “substantially the same quality of activity” can be measured as in the case of AIM. Since AIM contains three SRCR (Scavenger-Receptor Cysteine-Rich) domains containing many cysteines, each SRCR domain can be used as a partial peptide of the present invention.
- SRCR1 domain amino acid numbers 24-125 of the amino acid sequence represented by SEQ ID NO: 2
- SRCR2 domain amino acid numbers 24-125 of the amino acid sequence represented by SEQ ID NO: 2
- SRCR3 domain amino acid numbers 244 to 346 of the amino acid sequence represented by SEQ ID NO: 2
- the SRCR1 domain (amino acids 24 to 125 of the amino acid sequence represented by SEQ ID NO: 4) and the SRCR2 domain (amino acids represented by SEQ ID NO: 4)
- Partial amino acid sequences including amino acid numbers 139 to 239), SRCR3 domain (amino acid numbers 244 to 346 of amino acid sequences represented by SEQ ID NO: 4), and any combination of SRCR domains Those having an array are also used.
- the size of the partial peptide of the present invention is not particularly limited as long as it includes the functional domain described above, but preferably includes 50 or more partial amino acid sequences, more preferably 100 or more partial amino acid sequences. More preferably, those containing 200 or more partial amino acid sequences are included.
- the partial amino acid sequence may be one continuous partial amino acid sequence, or may be a concatenation of a plurality of discontinuous partial amino acid sequences.
- the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁇ ), an amide (—CONH 2 ), or an ester (—COOR).
- R in the ester include the same as those described above for AIM.
- the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention.
- the ester in this case, for example, the same ester as the C-terminal ester is used.
- the amino group of the N-terminal amino acid residue is protected with a protecting group
- the N-terminal glutamine residue is pyroglutamine oxidized
- Examples include those in which a substituent on the side chain of the amino acid is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
- the AIM or its partial peptide used in the present invention may be in the form of a salt.
- a salt with a physiologically acceptable acid eg, inorganic acid, organic acid
- a base eg, alkali metal salt
- a physiologically acceptable acid addition salt is particularly preferable.
- Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
- inorganic acids eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
- organic acids eg acetic acid, formic acid, propionic acid, fumaric acid, maleic
- AIM can be produced from the above-mentioned mammalian macrophages by a known protein purification method. Specifically, after homogenizing mammalian macrophages and removing cell debris by low-speed centrifugation, the supernatant is centrifuged at high speed to precipitate a cell membrane-containing fraction, and the supernatant is subjected to reverse phase chromatography and ion exchange chromatography. AIM or a salt thereof can be prepared by subjecting to chromatography such as affinity chromatography.
- AIM or a partial peptide thereof can also be produced according to a known peptide synthesis method (hereinafter, in the description of these chemical synthesis, the full-length AIM and its partial peptide are simply referred to as AIM unless otherwise specified. ).
- the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
- the target protein can be produced by removing the protecting group.
- the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) and (2).
- the AIM thus obtained can be purified and isolated by a known purification method.
- the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
- AIM obtained by the above method is a free form
- the free form can be converted into an appropriate salt by a known method or a method according thereto
- AIM is obtained as a salt
- the salt can be converted into a free form or other salt by a known method or a method analogous thereto.
- AIM can also be produced by culturing a transformant containing a nucleic acid encoding it and separating and purifying AIM from the resulting culture.
- the nucleic acid encoding AIM or a partial peptide thereof may be DNA or RNA, or may be a DNA / RNA chimera.
- DNA is used.
- the nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.
- DNA encoding AIM or a partial peptide thereof include genomic DNA, warm-blooded animals (eg, humans, cows, monkeys, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, mice, rabbits, hamsters, birds). Macrophage-derived cDNA, synthetic DNA, and the like.
- Any genomic DNA encoding AIM or a partial peptide thereof may be any cell of the animal [for example, hepatocytes, spleen cells, neurons, glial cells, pancreatic ⁇ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, Epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, Basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, Stem cells or cancer cells] or any tissue in which those cells exist [eg, brain, brain parts (eg, olfactory bulb, amygdala
- Amplification can also be performed directly by the method and Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).
- genomic DNA and cDNA encoding AIM or a partial peptide thereof can be obtained from a colony DNA library and cDNA library prepared by inserting the above genomic DNA and a fragment of total RNA or mRNA into an appropriate vector.
- they can be cloned by plaque hybridization method or PCR method, respectively.
- the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
- DNA encoding AIM examples include DNA containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
- Examples of the DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 include, for example, about 60% or more, preferably approximately about the nucleotide sequence represented by SEQ ID NO: 1.
- DNA encoding a protein containing a base sequence having homology of 70% or more, more preferably about 80% or more, particularly preferably about 90% or more and having substantially the same activity as AIM is used. It is done.
- NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
- the DNA encoding AIM is preferably a DNA containing the base sequence encoding the human AIM protein represented by the base sequence represented by SEQ ID NO: 1 (GenBank accession number: AF011429), or represented by SEQ ID NO: 3. DNA containing the base sequence encoding the feline AIM protein represented by the base sequence shown in FIG. 6 or a homolog thereof in other mammals [eg, mouse homolog registered in GenBank as accession number: AF011428].
- the DNA encoding the partial peptide of the present invention includes a base sequence encoding a peptide comprising an amino acid sequence identical or substantially identical to a part of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Anything can be used.
- the DNA encoding the partial peptide of the present invention for example, (1) DNA containing a partial base sequence of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or (2) sequence About 60% or more, preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more of DNA containing a partial base sequence of the base sequence represented by No. 1 or SEQ ID NO: 3.
- DNA encoding a protein containing a homologous base sequence and having substantially the same activity as AIM described above can be used.
- a DNA encoding AIM or a partial peptide thereof is amplified by PCR using a synthetic DNA primer having a part of a base sequence encoding the AIM or a partial peptide thereof, or is incorporated into a suitable expression vector, It can be cloned by hybridization with a DNA fragment encoding a part or the entire region of AIM or a labeled synthetic DNA.
- Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). it can. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual.
- Hybridization can be preferably performed according to stringent conditions.
- High stringent conditions include, for example, a hybridization reaction at 45 ° C. in 6 ⁇ SSC (sodium chloride / sodium citrate), followed by one or more washings at 65 ° C. in 0.2 ⁇ SSC / 0.1% SDS.
- SSC sodium chloride / sodium citrate
- a person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc.
- the desired stringency can be easily adjusted.
- hybridization can be performed according to the method described in the instruction manual attached to the library.
- An expression vector containing DNA encoding AIM or a partial peptide thereof is produced, for example, by excising a target DNA fragment from DNA encoding AIM and ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
- Expression vectors include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); retroviruses, Animal virus vectors such as vaccinia virus and adenovirus are used.
- the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
- SR ⁇ promoter when the host is an animal cell, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex) Virus thymidine kinase) promoter and the like are used.
- CMV promoter, SR ⁇ promoter and the like are preferable.
- the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter and the like are preferable.
- an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired.
- Selectable markers include, for example, dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter abbreviated as amp r ), neomycin resistance gene ( hereinafter sometimes abbreviated as neo r, include G418 resistance) and the like.
- the target gene can also be selected using a medium that does not contain thymidine.
- a base sequence (signal codon) encoding a signal sequence suitable for the host is added (or replaced with a native signal codon) to the 5 ′ end of the DNA encoding AIM or a partial peptide thereof. May be.
- the host is Escherichia, PhoA signal sequence, OmpA signal sequence, etc .; when the host is an animal cell, insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc. Each is used.
- AIM or a partial peptide thereof can be produced by transforming a host with an expression vector containing DNA encoding the above-mentioned AIM or a partial peptide thereof and culturing the resulting transformant.
- the host for example, Escherichia bacteria, animal cells and the like are used.
- the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA).
- animal cells examples include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO (dhfr ⁇ ) cells), mouse L Cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells and the like are used.
- Transformation can be performed according to a known method depending on the type of host.
- Escherichia bacteria are, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene (Proceedings of the National Academy of Sciences of the USA) Gene), Volume 17, 107 (1982) and the like.
- Animal cells should be transformed according to the method described in, for example, Cell Engineering Annex 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do.
- the culture of the transformant can be performed according to a known method depending on the type of the host.
- a medium for culturing a transformant whose host is an Escherichia bacterium for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
- an agent such as 3 ⁇ -indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
- Culturing of a transformant whose host is an Escherichia bacterium is usually performed at about 15 to about 43 ° C. for about 3 to about 24 hours.
- aeration or agitation may be performed.
- a minimal essential medium containing about 5 to about 20% fetal bovine serum [Science, Vol. 122, 501 (1952) ], Dulbecco's Modified Eagle Medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI1640 Medium [The Journal of the American Medical Association, 199 Volume 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Volume 73, 1 (1950)] etc. It is done.
- the pH of the medium is preferably about 6 to about 8.
- the culture is usually performed at about 30 ° C. to about 40 ° C. for about 15 to about 60 hours. You may perform ventilation
- AIM can be produced inside or outside the transformant.
- AIM or a partial peptide thereof can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
- a method known per se For example, when AIM or a partial peptide thereof is extracted from cultured cells or cell cytoplasm, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or freezing.
- ultrasound, lysozyme and / or freezing For example, a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by thawing or the like is appropriately used.
- the buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 TM .
- AIM or a partial peptide thereof is secreted outside the cells (cells)
- a method of separating the culture supernatant from the culture by centrifugation or filtration is used. Isolation and purification of AIM or its partial peptide contained in the thus obtained soluble fraction and culture supernatant can be performed according to a method known per se. Such methods include the use of solubilities such as salting out and solvent precipitation; mainly using differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
- a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.
- AIM AIM or a partial peptide thereof thus obtained can be confirmed by enzyme immunoassay or Western blotting using an antibody against AIM.
- AIM or a partial peptide thereof or a salt thereof obtained as described above or a nucleic acid containing a base sequence encoding AIM or a partial peptide thereof (herein sometimes referred to as AIMs) is used to prevent the onset of renal disease.
- AIMs a nucleic acid containing a base sequence encoding AIM or a partial peptide thereof
- -It can be provided as a therapeutic agent.
- the present invention can also use an agent that induces the expression of AIM or an agent that stabilizes AIM, instead of AIMs.
- Examples of drugs that induce AIM expression include compounds having an AIM transcription activity, and examples of the compounds include transcription factors that can bind to the promoter region of the AIM gene.
- the inventors have also found that AIM is expressed in macrophages. Therefore, a macrophage differentiation inducer is mentioned as an agent which induces the expression of AIM.
- the macrophage differentiation inducer is not particularly limited as long as it can induce differentiation of macrophages from progenitor cells such as granulocyte macrophage colony forming cells (CFU-GM) and macrophage colony forming cells (CFU-M).
- a sphere macrophage colony-stimulating factor (GM-CSF), a macrophage colony-stimulating factor (M-CSF), or the like can be used.
- GM-CSF and M-CSF may be proteins isolated and purified from mammalian tissues / cells by the above-mentioned known means, or may be biochemically synthesized by chemical synthesis or cell-free translation systems. It may be a synthesized protein. Alternatively, it may be a recombinant protein produced from a transformant introduced with a nucleic acid containing a base sequence encoding the protein.
- Examples of drugs that stabilize AIM include compounds that inhibit AIM degradation, and compounds that inhibit urinary excretion.
- Examples of the compound that inhibits degradation include protease inhibitors and proteasome inhibitors.
- protease inhibitors include serine protease inhibitors (fluorinated 4- (2-aminoethyl) benzenesulfonyl hydrochloride (AEBSGF), aprotinin, trypsin inhibitor, etc.), cysteine protease inhibitors (E-64, leupeptin, etc.) Etc.
- Examples of proteasome inhibitors include lactacystin, MG-115, MG-132, proteasome inhibitor I and the like.
- Examples of compounds that inhibit urinary excretion include compounds that impart to AIM a molecular weight that cannot pass through the glomerular basement membrane. Since IgM binds to AIM (WO2013 / 162021; Arai et al., Cell Reports 3: 1187-1198, 2013), IgM can be mentioned as a compound that inhibits urinary excretion. However, there is concern about immune system side effects when administering IgM itself, so the Fc part of IgM, which is the binding site for AIM, was fused with a protein with a molecular weight that is filtered through tubules and not excreted in the urine. A fusion protein is preferably used.
- the protein to be fused is not limited, but is preferably a protein with less side effects, such as albumin.
- the connection may be directly connected, or may be connected using a hinge portion.
- the hinge portion include those in which FLAG tags are arranged in parallel.
- Such molecules can be produced in a conventional manner as a single recombinant protein by linking the genes encoding them.
- bonded with IgM may be AIM derived from arbitrary warm-blooded animals, AIM derived from a cat may be excluded.
- AIM knockout mice are subjected to unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy or bilateral transient renal ischemia reperfusion Showed symptoms of kidney disease.
- AIMs, drugs that induce the expression of AIM, drugs that stabilize AIM, or compounds that can replace the function of AIM that can be explored by the screening methods described below are the onset and progression of renal disease. It is suggested that can be prevented and treated.
- an agent that induces the expression of AIM or an agent that stabilizes AIM is human or other warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig) Cattle, cats, dogs, monkeys, birds, preferably cats).
- the renal diseases to which the pharmaceutical composition containing the AIMs of the present invention, the drug that induces the expression of AIM or the drug that stabilizes AIM include, for example, acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome Diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease or IgM nephropathy, preferably acute renal failure or chronic renal failure.
- a typical example of nephropathy associated with collagen disease is lupus nephritis.
- a pharmaceutical composition containing the AIMs of the present invention a drug that induces the expression of AIM or a drug that stabilizes AIM has low toxicity and is used as a liquid or as a pharmaceutical composition of an appropriate dosage form as human or other Of warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, birds, preferably cats, etc.) orally (eg, intravascular administration, subcutaneous administration) Etc.).
- warm-blooded animals eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, birds, preferably cats, etc.
- ally eg, intravascular administration, subcutaneous administration
- injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included.
- Such an injection can be prepared according to a known method.
- a method for preparing an injection for example, the AIMs of the present invention, a drug that induces the expression of AIM or a drug that stabilizes AIM is dissolved in a sterile aqueous liquid or oily liquid that is usually used for injection. It can be prepared by turbidity or emulsification.
- an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)), etc. may be used in combination.
- alcohol eg, ethanol
- polyalcohol eg, Propylene glycol, polyethylene glycol
- nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
- oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubil
- the prepared injection solution is preferably filled in a suitable ampoule.
- a suppository used for rectal administration may be prepared by mixing the above AIMs, an agent that induces expression of AIM, or an agent that stabilizes AIM into an ordinary suppository base.
- compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
- Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
- a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
- the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
- dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
- the AIMs of the present invention, an agent that induces the expression of AIM or an agent that stabilizes AIM is, for example, usually 5 to 500 mg per dosage unit form, especially 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms. It is preferably contained.
- the dose of the above-mentioned prophylactic / therapeutic agent containing the AIMs of the present invention, a drug that induces the expression of AIM or a drug that stabilizes AIM varies depending on the administration subject, target disease, symptom, administration route, etc.
- the AIM of the present invention is used as a single dose, usually about 0.01 to 20 mg / kg body weight, preferably 0.1 to 10 mg / kg body weight.
- compositions may contain other active ingredients as long as no unfavorable interaction is caused by the combination with the AIMs of the present invention, a drug that induces the expression of AIM, or a drug that stabilizes AIM. Good.
- agents that induce the expression of AIM are other agents useful for the treatment of renal diseases, such as antihypertensive agents (eg, angiotensin converting enzyme inhibitors, angiotensin II).
- antihypertensive agents eg, angiotensin converting enzyme inhibitors, angiotensin II.
- Receptor antagonists calcium antagonists, renin inhibitors, alpha blockers, beta blockers, etc.); diuretics (eg, carbonic anhydrase inhibitors, loop diuretics, thiazide diuretics, antialdosterone drugs, potassium retention) Diuretics, etc.); active vitamin D3 preparations (eg, calcitriol, alpha-cacildol, maxacalcitol, falecalcitriol, etc.); oral adsorbed carbon preparations (eg, activated carbon, etc.); potassium correctors (eg, polystyrene) Phosphorus adsorbent (eg, calcium carbonate, calcium acetate, sevelamer hydrochloride, lanthanum carbonate, etc.), manufactured by erythropoiesis stimulating factor It may be used in combination with an agent (erythropoiesis stimulating agent; ESA) (eg, erythropoietin preparation), amino acid infusion preparation, and the like.
- ESA
- the present invention also provides prevention of renal disease for administration to cats, comprising AIM characterized by binding to cat IgM or a partial peptide thereof, or a nucleic acid containing a base sequence encoding them.
- AIM that binds to cat IgM in the present invention is a protein that contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4 and that can bind to cat IgM.
- a protein may be, for example, a protein chemically synthesized or biochemically synthesized in a cell-free translation system, or from a transformant introduced with a nucleic acid containing a base sequence encoding the amino acid sequence. It may be a recombinant protein produced.
- the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 is about 60% or more, preferably about 70% or more, more preferably about 80%, with the amino acid sequence represented by SEQ ID NO: 4.
- the amino acid sequences having homology of about 90% or more, most preferably about 95% or more are particularly preferable.
- the “homology” may be the same as described above. More preferably, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 4 is about 60% or more, preferably about 70% or more, more preferably the amino acid sequence represented by SEQ ID NO: 4. Is an amino acid sequence having about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more identity.
- an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 is used as a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4.
- a protein having substantially the same activity as that of the protein containing the amino acid sequence represented by SEQ ID NO: 4 is preferred.
- substantially the same quality of activity has the same meaning as described above.
- the AIM protein that can bind to cat IgM may be any AIM protein as long as it can bind to cat IgM in cat blood.
- it must be a protein containing the SRCR3 domain of mouse-derived AIM. Is preferred.
- an AIM protein containing amino acid numbers 246 to 348 in the amino acid sequence represented by SEQ ID NO: 6 can be mentioned.
- the protein containing the SRCR3 domain of mouse-derived AIM may be a protein in which the SRCR3 domain originally possessed by AIM is replaced with the SRCR3 domain of mouse-derived AIM.
- the partial peptide of AIM that binds to cat IgM may be any peptide as long as it is a peptide having the partial amino acid sequence of AIM that binds to cat IgM and has substantially the same activity as AIM. .
- substantially the same quality of activity has the same meaning as described above.
- SRCR1 domain amino acid numbers 24-125 of the amino acid sequence represented by SEQ ID NO: 4
- SRCR2 domain amino acid numbers 139 to 239
- SRCR3 domain amino acid numbers 244 to 346 of amino acid sequences represented by SEQ ID NO: 4
- amino acid numbers 244 to 346 of amino acid sequences represented by SEQ ID NO: 4 amino acid numbers 244 to 346 of amino acid sequences represented by SEQ ID NO: 4
- amino acid numbers 244 to 346 of amino acid sequences represented by SEQ ID NO: 4 amino acid numbers 244 to 346 of amino acid sequences represented by SEQ ID NO: 4
- the partial peptide is not particularly limited in size as long as it includes the functional domain.
- DNA containing a base sequence encoding AIM that binds to cat IgM or a partial peptide thereof examples include DNA containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 3. It is done.
- the DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 3 is, for example, about 60% or more, preferably about 70% or more with the nucleotide sequence represented by SEQ ID NO: 3.
- DNA encoding a protein containing a base sequence having a homology of about 80% or more, particularly preferably about 90% or more and having substantially the same quality of activity as AIM is used.
- the “homology” may be the same as described above.
- the DNA is prepared in the same manner as described above.
- the cat can be given as an administration target of the pharmaceutical composition containing AIM binding to the cat IgM of the present invention or a partial peptide thereof, or a nucleic acid containing the base sequence encoding them.
- renal diseases to which the pharmaceutical composition containing AIM binding to the cat IgM of the present invention or a partial peptide thereof, or a nucleic acid containing a nucleotide sequence encoding them, include acute renal failure, chronic nephritis, chronic kidney Insufficiency, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease or IgM nephropathy, preferably acute renal failure or chronic renal failure.
- a typical example of nephropathy associated with collagen disease is lupus nephritis.
- a pharmaceutical composition containing AIM binding to the cat IgM of the present invention or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding them, has low toxicity and is administered to a subject orally or parenterally as described above. can do.
- the dosage form, dosage, etc. may be the same as described above.
- AIM knockout mice can be used for unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy, or bilateral transient renal ischemia reperfusion. Showed symptoms. This is because AIM knockout mice placed under conditions of unilateral ureteral ligation, transient renal ischemia reperfusion after bilateral nephrectomy or bilateral transient renal ischemia reperfusion It is suggested that it can be provided as a new model mouse. Therefore, the present invention is obtained by performing unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy, or bilateral transient renal ischemia reperfusion on non-human mammals deficient in AIM expression. Provided is a screening method for a prophylactic / therapeutic agent for renal diseases using animals.
- AIM-deficient non-human mammal means a non-human mammal in which the expression of endogenous AIM is inactivated, and in addition to AIM KO animals produced from ES cells in which AIM is knocked out (KO), Also included are knockdown (KD) animals in which AIM expression is inactivated by antisense or RNAi technology.
- knockout (KO) means that complete mRNA cannot be produced by destroying or removing the endogenous gene
- knockdown (KD)” By inhibiting the translation from mRNA to protein, it means inactivating the expression of the endogenous gene as a result.
- the AIM KO / KD animal of the present invention may be simply referred to as “KO / KD animal of the present invention”.
- the AIM KO animal of the present invention is disclosed in, for example, Miyazaki T. et al. (J. Exp. Med., 189, 413-422, 1999, or WO2013 / 162021).
- non-human mammal that can be the subject of the present invention is not particularly limited as long as it is a mammal other than a human in which a transgenic system has been established.
- a mouse, rat, cow, monkey, pig, sheep, goat Examples include rabbits, dogs, cats, guinea pigs, hamsters, rats, and mice.
- Rabbits, dogs, cats, guinea pigs, hamsters and the like are preferable, and rodents with relatively short ontogeny and biological cycle and easy reproduction are more preferable from the viewpoint of production of disease model animals, especially mice (for example, Preferred are C57BL / 6 strain, BALB / c strain, DBA2 strain, etc.
- AIM gene sequence derived from a target non-human mammal is isolated according to a conventional method, for example, (1) other DNA fragments (for example, drug resistance genes) in the exon part or promoter region Or the reporter gene, etc.), or the exon or promoter function is disrupted, or (2) the gene is deleted by excising all or part of the AIM using the Cre-loxP system or Flp-frt system Or (3) insert a stop codon into the protein coding region to disable complete protein translation, or (4) a DNA sequence that terminates gene transcription into the transcription region (eg, polyA addition signal, etc.) ) Insert and complete By disabling the synthesis of mRNA, a DNA strand having a DNA sequence constructed so as to inactivate the gene (hereinafter ab
- the homologous recombinant can be obtained, for example, by introducing the above targeting vector into embryonic stem cells (ES cells).
- ES cells are cells derived from the inner cell mass (ICM) of fertilized eggs at the blastocyst stage, and can be cultured and maintained in an undifferentiated state in vitro.
- ICM cells are the cells that will form the embryo body in the future, and are the stem cells that form the basis of all tissues including germ cells.
- ES cells may be established cell lines, or may be newly established according to the method of Evans and Kaufman (Nature 292, 154, 1981).
- ES cells derived from 129 mice are generally used, but since the immunological background is unclear, a purely alternative and immunologically genetic
- B57 1 mice C57BL / 6 and DBA / ES cells established from F 1
- BDF 1 mouse has C57BL / 6 mouse in the background in addition to the advantage that the number of eggs collected is large and the egg is strong, so the ES cell derived from this is C57BL / 6 mouse.
- ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions.
- MJ Evans and MH Kaufman Nature 292, 154, 1981; GR Martin, Proceedings of National Academy of Sciences USA (Proc. Natl Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985]
- AIM-deficient non-human mammalian cells obtained by differentiating ES cells into which a targeting vector has been introduced are useful for in vitro cell biology of AIM.
- the targeting vector when the targeting vector is designed to destroy the function of the exon or promoter by inserting another DNA fragment into the exon part or promoter region of AIM, the vector is, for example, Such a configuration can be adopted.
- the targeting vector is homologous to the target site 5 ′ upstream and 3 ′ downstream of the other DNA fragment, respectively. It is necessary to include the sequence (5 ′ arm and 3 ′ arm).
- DNA fragments to be inserted are not particularly limited, but when a drug resistance gene or a reporter gene is used, ES cells in which a targeting vector is integrated into the chromosome can be selected using drug resistance or reporter activity as an index.
- drug resistance genes include neomycin phosphotransferase II (nptII) gene and hygromycin phosphotransferase (hpt) gene.
- Reporter genes include, for example, ⁇ -galactosidase (lacZ) gene, chloramphenicol acetyl. Examples thereof include, but are not limited to, transferase (cat) genes.
- the drug resistance or reporter gene is preferably under the control of any promoter that can function in mammalian cells.
- viral promoters such as SV40-derived early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, murine leukemia virus (MoMuLV) LTR, adenovirus (AdV) derived early promoter
- CMV cytomegalovirus
- RSV Rous sarcoma virus
- MoMuLV murine leukemia virus
- AdV adenovirus
- promoters of mammalian constituent protein genes such as ⁇ -actin gene promoter, PGK gene promoter, and transferrin gene promoter.
- a promoter that controls transcription of the gene in the targeting vector is not required.
- the targeting vector preferably has a sequence (also called a polyadenylation (polyA) signal, terminator) that terminates transcription of mRNA from the drug resistance or reporter gene, for example, Terminator sequences derived from viral genes or from various mammalian or avian genes can be used.
- a sequence also called a polyadenylation (polyA) signal, terminator
- terminator terminates transcription of mRNA from the drug resistance or reporter gene
- Terminator sequences derived from viral genes or from various mammalian or avian genes can be used.
- SV40-derived terminator or the like is used.
- gene recombination in mammals is mostly non-homologous, and the introduced DNA is randomly inserted at any position on the chromosome. Therefore, depending on selection (positive selection) such as detection of drug resistance or reporter gene expression, only clones targeted to the endogenous AIM targeted by homologous recombination could not be selected efficiently. It is necessary to confirm the integration site by Southern hybridization or PCR for all clones.
- HSV-tk herpes simplex virus-derived thymidine kinase
- a diphtheria toxin gene is linked instead of the HSV-tk gene, cells into which the vector has been randomly inserted are killed by the toxin produced by the vector, so that homologous recombinants can be removed in the absence of a drug. You can also choose.
- the electroporation method is selected from the viewpoint that a large number of cells can be treated.
- the conditions used for gene transfer into normal animal cells may be used as they are.
- 10 6 After ES cells in the logarithmic growth phase are treated with trypsin and dispersed into single cells, 10 6 It can be carried out by suspending in a medium so as to be ⁇ 10 8 cells / ml, transferring to a cuvette, adding 10 to 100 ⁇ g of the targeting vector, and applying an electric pulse of 200 to 600 V / cm.
- ES cells incorporating a targeting vector can be assayed by screening chromosomal DNA isolated and extracted from colonies obtained by culturing single cells on feeder cells by Southern hybridization or PCR, When drug resistance genes or reporter genes are used as other DNA fragments, transformants can be selected at the cell stage using their expression as an index. For example, when a vector containing the nptII gene is used as a positive selection marker gene, the ES cells after gene transfer treatment are cultured in a medium containing a neomycin antibiotic such as G418, and the resulting resistant colonies are transformed. Select as a candidate.
- the vector When a vector containing the HSV-tk gene is used as a negative selection marker gene, the vector is cultured in a medium containing ganciclovir, and the resulting resistant colony is selected as a candidate for a homologous recombinant. Each of the obtained colonies is transferred to a culture plate, and after trypsin treatment and medium exchange are repeated, a part is left for culture, and the rest is subjected to PCR or Southern hybridization to confirm the presence of the introduced DNA.
- ES cells in which the introduced DNA has been confirmed to be integrated are returned into embryos derived from the same type of non-human mammal, they are integrated into the ICM of the host embryo to form a chimeric embryo.
- a chimeric KO animal can be obtained by transplanting this to a temporary parent (female for embryo transfer) and further allowing development.
- ES cells contribute to the formation of primordial germ cells that differentiate into eggs and sperm in the chimeric animal, germline chimeras will be obtained, and mating them will genetically fix AIM expression deficiency. KO animals can be made.
- the chimera embryos are prepared by adhering the early embryos up to the morula stage (aggregate chimera method) and by microinjecting cells into the blastocyst division (injection chimera method) There is.
- aggregate chimera method aggregate chimera method
- blastocyst division injection chimera method
- a method of creating an aggregate chimera by injecting ES cells into the zona pellucida of an 8-cell embryo or a micromanipulator As a method that is easy to operate, a method of producing an aggregate chimera by co-culturing and aggregating an ES cell mass and an 8-cell embryo from which the zona pellucida has been removed is also performed.
- the host embryo can be collected in the same manner from a non-human mammal that can be used as an egg-collecting female in gene transfer into a fertilized egg, which will be described later. It is preferable to collect a host embryo from a mouse having a different color from the strain from which the ES cells are derived so that the contribution rate of the ES cells can be determined by the coat color. For example, if ES cells are derived from 129 mice (hair color: Agouti), C57BL / 6 mice (hair color: black) and ICR mice (hair color: albino) are used as eggs for collection, and ES cells are C57BL / 6 or DBF.
- the germline chimera formation ability largely depends on the combination of the ES cell and the host embryo, it is more preferable to select a combination having a high germline chimera formation ability.
- a host embryo derived from the C57BL / 6 strain for ES cells derived from the 129 strain, and a host embryo derived from the BALB / c strain for ES cells derived from the C57BL / 6 strain. Etc. are preferred.
- the female mouse for egg collection is preferably about 4 to about 6 weeks of age, and the male mouse for mating is preferably of the same strain of about 2 to about 8 months of age.
- the mating may be natural mating, but is preferably performed after gonad stimulating hormone (follicle stimulating hormone and then luteinizing hormone) is administered to induce superovulation.
- blastocyst stage embryos (for example, in the case of mice, about 3.5 days after mating) are collected from the uterus of the female for egg collection (or early embryos before the morula stage are collected from the oviduct) Thereafter, ES cells (about 10 to about 15) into which the targeting vector has been introduced into the dividing space of the blastocyst using a micromanipulator may be cultured in a medium for embryo culture (described later).
- ES cells about 10 to about 15
- ES cells (about 10 to about 15) into which the targeting vector has been introduced into the dividing space of the blastocyst using a micromanipulator may be cultured in a medium for embryo culture (described later).
- a medium for embryo culture (described later).
- the female non-human mammal for embryo transfer a non-human mammal that can be used as a female for embryo transfer in gene transfer into a fertilized egg can be similarly used.
- 8-cell embryos and morulas are collected from the oviduct and uterus of the female egg collection (or early embryos before the 8-cell stage).
- the zona pellucida can be cultivated in an acidic Tyrode solution after being cultured in the embryo culture medium (described later) until the 8-cell stage or morula stage.
- the obtained morula or blastocyst is transplanted into the uterus of a female non-human mammal for embryo reception in the same manner as described above.
- a chimeric non-human mammal can be obtained by natural delivery or caesarean section. It is only necessary to continue suckling for naturally delivered embryos, and if a baby is delivered by caesarean section, the offspring are fed by a separately prepared nursing female (a female non-human mammal that is normally mated and delivered). be able to.
- chimeric mice of the same sex as the ES cells usually male ES cells are used, so male chimeric mice are selected.
- a chimeric mouse (for example, 50% or more) having a high ES cell contribution rate is selected from the phenotype such as hair color.
- a chimeric mouse obtained from a chimeric embryo of D3 cells which are male ES cells derived from 129 mice, and a host embryo derived from a C57BL / 6 mouse, it is preferable to select male mice with a high proportion of agouti's coat color. preferable.
- a germline chimeric non-human mammal (founder) into which the targeting vector obtained as described above is introduced is usually obtained as a heterozygote in which only one AIM of homologous chromosome is KO.
- Phase to AIM both homologous chromosomes obtain a homozygote is KO may be crossed siblings heterozygous out of the F 1 animal obtained as described above. Selection of heterozygotes can be assayed by screening for example F 1 animals separated extracted chromosomal DNA from the tail by Southern hybridization or PCR method. 1/4 of the F 2 animals obtained are homozygotes.
- a virus comprising a positive selection marker gene inserted between the 5 ′ and 3 ′ arms and a DNA containing a negative selection marker gene outside the arm
- a method for infecting non-human mammal ES cells for example, Proc. Natl. Acad. Sci. USA) Vol. 99, Proceedings of National Academy of Sciences USA No. 4, pages 2140-2145, 2002.
- seed cells in a suitable incubator such as a dish
- add a viral vector to the culture solution may coexist with polybrene if desired
- culture for 1-2 days As described above, the selective agent is added and the culture is continued to select cells into which the vector has been incorporated.
- DNA encoding AIM antisense RNA or siRNA is introduced using a known per se transgenic production technique, and is introduced into the target non-human mammalian cell.
- shRNA shRNA
- DNA containing a base sequence complementary to the target region of the target polynucleotide that is, DNA capable of hybridizing with the target polynucleotide is "antisense" to the target polynucleotide.
- Antisense DNA having a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide encoding AIM or a part thereof is complementary or substantially complementary to the base sequence of the polynucleotide encoding AIM. Any antisense DNA may be used as long as it contains a complementary base sequence or a part thereof and has an action capable of suppressing the expression of the polynucleotide.
- the nucleotide sequence substantially complementary to the polynucleotide encoding AIM is, for example, about 70% or more, preferably about 80% or more, with respect to the region overlapping with the nucleotide sequence of the complementary strand of the polynucleotide.
- the nucleotide sequence has a homology of about 90% or more, most preferably about 95% or more.
- an antisense DNA having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a nearby base sequence
- antisense DNA directed to RNA degradation by RNaseH it is about 70% or more, preferably about 80% or more, more preferably about 90% or more, with the complementary strand of the entire nucleotide sequence of AIM-encoding polynucleotide containing intron
- each of the antisense DNAs having a homology of about 95% or more is suitable.
- an antisense DNA comprising a base sequence complementary or substantially complementary to the base sequence registered as GenBank accession No.AF011428, or a part thereof
- Preferred examples include antisense DNA containing a base sequence complementary to the base sequence or a part thereof.
- an antisense DNA having a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding AIM or a part thereof was cloned, Alternatively, it can be designed and synthesized based on the determined base sequence information of DNA encoding AIM.
- Such antisense DNA can inhibit AIM replication or expression. That is, the antisense DNA of the present invention can hybridize with RNA (mRNA or initial transcription product) transcribed from AIM, and inhibits mRNA synthesis (processing) or function (translation into protein). it can.
- the target region of the antisense DNA of the present invention is not particularly limited in length as long as the antisense DNA hybridizes, and as a result, the translation into AIM is inhibited, and the mRNA encoding the protein is not limited.
- the short sequence may be about 10 bases, and the long sequence may be the entire mRNA or initial transcription product sequence.
- the 3 ′ end palindromic region or the 3 ′ end hairpin loop or the like can be selected as a preferred target region of the antisense DNA, but any region within the AIM can be selected as a target.
- the intron portion of the gene can be used as the target region.
- the antisense DNA of the present invention not only hybridizes with AIM mRNA or initial transcription product to inhibit translation into protein, but also binds to AIM, which is a double-stranded DNA, to form a triplex. May be formed so that RNA transcription can be inhibited.
- a DNA: RNA hybrid may be formed to induce degradation by RNaseH.
- the most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known.
- the hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
- ribozyme has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate.
- AIM mRNA itself has a double-stranded structure
- the target sequence can be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
- a hybrid ribozyme in which a sequence modified with tRNA is further linked can be used [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
- RNA interference (RNAi) phenomenon in which siRNA is introduced into cells and the mRNA homologous to the RNA is degraded, has been known for a long time in nematodes, insects and plants.
- siRNA can be designed as appropriate using commercially available software (eg, RNAi ⁇ ⁇ Designer; Invitrogen) based on the base sequence information of the target mRNA.
- the antisense oligo DNA and ribozyme of the present invention determine the target sequence of mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence of AIM, and are commercially available DNA / RNA automatic synthesizers (Applied Biosystems, Beckman). Etc.) can be prepared by synthesizing a complementary sequence thereto.
- the synthesized antisense oligo DNA or ribozyme is inserted downstream of the promoter of the expression vector through an appropriate linker (adapter) sequence as necessary, so that a DNA expression vector encoding the antisense oligo RNA or ribozyme can be obtained.
- Expression vectors that can be used here include plasmids derived from E.
- plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as ⁇ phage, retroviruses such as Moloney leukemia virus, lentiviruses, adeno-associated viruses, and vaccinia viruses. Or animals such as baculoviruses or insect viruses are used.
- plasmids preferably E. coli-derived, Bacillus subtilis-derived or yeast-derived, especially E. coli-derived plasmids
- animal viruses preferably retroviruses, lentiviruses
- promoters examples include SV40-derived early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, murine leukemia virus (MoMuLV) LTR, adenovirus (AdV) -derived early promoter.
- CMV cytomegalovirus
- RSV Rous sarcoma virus
- MoMuLV murine leukemia virus
- AdV adenovirus
- promoters of mammalian constituent protein genes such as ⁇ -actin gene promoter, PGK gene promoter, transferrin gene promoter, and the like.
- a DNA expression vector encoding a longer antisense RNA (for example, the full-length complementary strand of AIM mRNA) can be obtained by using an AIM cDNA cloned by a conventional method, if necessary, via an appropriate linker (adapter) sequence. It can be prepared by inserting in the reverse direction downstream of the promoter.
- DNA encoding siRNA can be prepared by separately synthesizing as DNA encoding sense strand or antisense strand and inserting each into an appropriate expression vector.
- siRNA expression vector one having a Pol III promoter such as U6 or H1 can be used.
- siRNA is formed by transcription and annealing of the sense strand and the antisense strand in the animal cell introduced with the vector.
- shRNA can be prepared by inserting a unit in which a length (for example, about 15 to 25 bases) between the sense strand and the antisense strand to form an appropriate loop structure is inserted into an appropriate expression vector.
- a Pol III promoter such as U6 or H1
- the shRNA transcribed in the animal cell into which the expression vector has been introduced forms a loop by itself, and then is processed by an endogenous enzyme dicer or the like to form a mature siRNA.
- knockdown can be achieved by RNAi by expressing microRNA (miRNA) containing a target siRNA sequence with a Pol II promoter.
- miRNA microRNA
- a tissue-specific knockdown is also possible by a promoter exhibiting tissue-specific expression.
- a method for introducing an expression vector containing DNA encoding AIM antisense RNA, siRNA, shRNA, or miRNA into a cell a method known per se is appropriately used depending on the target cell.
- a microinjection method is used for introduction into an early embryo such as a fertilized egg.
- calcium phosphate coprecipitation method, electroporation method, lipofection method, retrovirus infection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method and the like can be used.
- the gene when a retrovirus or a lentivirus is used as a vector, the gene can be introduced simply by adding the virus to an early embryo or ES cell, culturing for 1 to 2 days, and infecting the cell with the virus. May be achievable. Individual regeneration (establishment of a founder), passage (production of a homozygote), and the like from ES cells can be performed by the same method as described above in the KO animal of the present invention.
- an expression vector containing DNA encoding AIM antisense RNA, siRNA, shRNA, or miRNA is introduced into an early embryo (fertilized egg) of a target non-human mammal by a microinjection method.
- the microinjection of DNA into a fertilized egg can be performed according to a conventional method using a known apparatus such as a micromanipulator. Briefly, a fertilized egg placed in a microdrop of embryo culture medium is aspirated and fixed with a holding pipette and the DNA solution is directly injected into the male or female pronucleus, preferably into the male pronucleus, using an injection pipette. inject.
- the introduced DNA is preferably highly purified by CsCl density gradient ultracentrifugation or an anion exchange resin column.
- the introduced DNA is preferably linearized by cleaving the vector portion using a restriction enzyme.
- the fertilized egg after introduction of DNA is cultured from the 1st cell stage to the blastocyst stage in 5% carbon dioxide gas / 95% air in the embryo culture medium using the microdrop culture method, and then pseudopregnant female. Transplanted into the fallopian tube or uterus of a non-human mammal.
- the female non-human mammal for embryo transfer may be of the same kind as the animal from which the early embryo to be transplanted is derived. For example, when transplanting a mouse early embryo, an ICR female mouse (preferably about 8 to about 10 weeks old) is preferably used.
- Examples of a method for putting a female non-human mammal for embryo transfer into a pseudopregnant state include, for example, the same type of vasectomized (ligated) male non-human mammal (for example, in the case of a mouse, an ICR male mouse (preferably about 2 There is known a method of selecting those in which the presence of vaginal plugs has been confirmed by mating with those a month old or older)).
- Embryonic females may be naturally ovulated, or administered luteinizing hormone-releasing hormone (generally abbreviated as LHRH) or its analog prior to mating with a vasectomized (ligated) male. Alternatively, fertility induced may be used.
- LHRH analogs include [3, 5-DiI-Tyr 5 ] -LH-RH, [Gln 8 ] -LH-RH, [D-Ala 6 ] -LH-RH, [des-Gly 10 ]- Examples include LH-RH, [D-His (Bzl) 6 ] -LH-RH, and their ethylamide.
- the dose of LHRH or an analog thereof and the timing of mating with a male non-human mammal after the administration vary depending on the type of non-human mammal.
- the non-human mammal is a mouse (preferably an ICR mouse, etc.)
- the dose of the analog is usually about 10-60 ⁇ g / individual, preferably about 40 ⁇ g / individual.
- the embryo is transplanted to the uterus of the female for embryo reception, and if it is earlier (for example, the 1-cell to 8-cell embryo), the fallopian tube.
- a female for embryo transfer a female whose a certain number of days has passed since pseudopregnancy is appropriately used depending on the stage of development of the transplanted embryo. For example, in the case of mice, female mice about 0.5 days after pseudopregnancy are preferable for transplanting 2-cell stage embryos, and female mice about 2.5 days after pseudopregnancy are preferable for transplanting blastocyst stage embryos.
- anesthetizing females for embryo transfer preferably Avertin, Nembutal, etc. are used
- incisions are drawn to draw out ovaries, and initial embryos (about 5 to about 10) suspended in embryo culture medium are pipetted for embryo transfer Inject into the fallopian tube or near the fallopian junction at the uterine horn.
- the offspring is a separately provided female female (for example, in the case of a mouse, a female mouse normally mated and delivered) Can be fed into ICR female mice))).
- AIM antisense RNA, siRNA, shRNA, or miRNA at the fertilized egg cell stage is ensured so that the introduced DNA is present in all germ line cells and somatic cells of the subject non-human mammal. Whether or not the introduced DNA is incorporated into the chromosomal DNA can be assayed, for example, by screening the chromosomal DNA separated and extracted from the tail of the offspring by Southern hybridization or PCR.
- the presence of the expression vector in the germ line cells of the offspring non-human mammal (F 0 ) obtained as described above means that all of the progeny (F 1 ) animals have their germ line cells and somatic cells in all. It means that an expression vector exists.
- F 0 animals are obtained as heterozygotes having the introduced DNA only on one of the homologous chromosomes. Individual F 0 individuals are randomly inserted on different chromosomes unless homologous recombination is used. To obtain homozygotes with expression vectors on both homologous chromosomes, F 0 animals and non-transgenic animals are crossed to create F 1 animals and heterozygotes that have the introduced DNA only on one of the homologous chromosomes. You only have to cross your brothers and sisters. If only introduced DNA is integrated into a locus, 1/4 of the obtained F 2 animals would be homozygotes.
- a virus containing DNA encoding AIM antisense RNA, siRNA, shRNA, or miRNA is used.
- examples include a method of infecting an early embryo or ES cell.
- a fertilized egg it is preferable to remove the zona pellucida prior to infection.
- the selective agent is added as described above, and the culture is continued to select cells into which the vector has been incorporated.
- AIM-deficient non-human mammal of the present invention described in Miyazaki T. et al. (J. Exp. Med., 189, 413-422, 1999, or WO2013 / 162021) or obtainable by the above technique
- transient renal ischemia reperfusion after unilateral nephrectomy or bilateral transient renal ischemia reperfusion (1) Compared with the control kidney, necrotic tubular cells accumulate in the kidney that has undergone ureteral ligation or transient renal ischemia reperfusion, and the renal parenchyma becomes fibrotic.
- AIM administration improves the above (1) to (6).
- AIM KO mice chronic kidney damage associated with acute kidney injury (acute kidney failure) triggered by ureteral stones, ascending urinary tract infections, or ureteral compression / obstruction caused by tumors, etc., and tumors, thrombus, Or it is a new discovery that it is close to the pathology of chronic renal injury associated with ischemic renal injury due to renal stenosis / occlusion due to diabetes, hypertension and the like.
- necrotic tubular cell accumulation in kidneys undergoing ureteral ligation or transient renal ischemia reperfusion compared to control kidneys and It means that extensive fibrosis of the renal parenchyma is observed.
- Accumulation of necrotic tubular cells can be confirmed by, for example, renal tissue fragments by hematoxylin / eosin staining, and fibrosis of the renal parenchyma can be confirmed by simultaneous staining of Azan staining and hematoxylin staining.
- AIM knockout mice showed a significant difference from the control kidneys from the 14th day after ureter ligation.
- the collapse of the glomerular structure can be confirmed by, for example, renal tissue fragments by hematoxylin-eosin staining, and glomerular fibrosis can be confirmed by simultaneous staining of Azan staining and hematoxylin staining.
- AIM knockout mice showed a significant difference compared to normal kidneys from day 14 after ureter ligation. In AIM knockout mice, a significant difference was observed compared to normal kidneys from day 7 after transient ischemia-reperfusion. (3) Compared with the control kidney, the expression of inflammatory cytokines is enhanced in the kidney subjected to ureteral ligation or transient renal ischemia reperfusion.
- the survival rate of non-human mammals deficient in AIM expression is low after unilateral ureter ligation and unilateral nephrectomy in AIM deficient non-human mammals of the present invention.
- the survival rate is reduced as compared to a control non-human mammal.
- the above-mentioned (1) to (6) are improved by administration of AIM.
- AIM administration shows a significant decrease in BUN value, necrotic tubular cell accumulation and glomerular structure collapse, and associated kidneys It means that parenchymal and glomerular fibrosis is clearly improved, inflammatory cytokine expression is decreased, survival rate is improved, and macrophage infiltration is suppressed.
- non-human mammals with AIM expression deficiency are treated with unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy, or bilateral transient renal ischemia reperfusion. It is shown that it can be used as a model animal and can be used for screening for preventive / therapeutic drugs for renal diseases.
- the screening method of the present invention includes the following steps.
- a test substance is applied to a non-human mammal deficient in AIM expression under conditions of unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy or bilateral transient renal ischemia reperfusion Administering, (2) A step of observing one or more of the following characteristics of a non-human mammal deficient in AIM expression administered with a test substance: (I) accumulation of necrotic tubular cells and fibrosis of the renal parenchyma, (Ii) Collapse and fibrosis of the glomerular structure, (Iii) expression level of inflammatory cytokines in the kidney, (Iv) the proportion of macrophages in the kidney, (V) BUN value, (Vi) survival rate, (3) A step of selecting a test substance that improves at least one of the above characteristics as compared to the case of non-test substance administration.
- unilateral ureteral ligation refers to ligating the ureter of the unilateral kidney.
- Urinary ligation can induce necrosis of tubules and glomeruli in the renal parenchyma of the ligated kidney, which subsequently causes inflammation and fibrosis, and ultimately renders the kidney dysfunctional.
- Transient renal ischemia / reperfusion after unilateral nephrectomy means that one kidney is removed in advance and ischemia is induced by ligating the remaining renal artery after 2 weeks. This refers to releasing post-ligation and reperfusion of blood flow. Due to this transient ischemia, necrosis of the tubule proceeds with mild fibrosis for about 3 days, and renal function deteriorates accordingly.
- Bilateral transient renal ischemia / reperfusion refers to induction of ischemia by ligating the renal arteries of both kidneys without removing one kidney and releasing the ligation 30 minutes later. Reperfusion is said to be performed.
- Test substances to be administered to non-human mammals with AIM expression deficiency include proteins, peptides, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. May be used.
- the test substance is administered at the same time, even before unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy, or before bilateral transient renal ischemia reperfusion.
- a non-human mammal deficient in AIM expression may have undergone unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy, or bilateral transient renal ischemia reperfusion. It may be after the characteristics are observed.
- the administration method may be oral or parenteral. Oral administration can be mixed with feed or drinking water. Examples of parenteral administration include intraperitoneal administration, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc., rectal administration with a suppository, and the like.
- the administration may be a single administration or multiple administrations.
- the characteristics of a non-human mammal deficient in AIM expression administered with a test substance are usually observed after 3 or 4 days, preferably after 7 days, more preferably after 14 days after administration of the test substance.
- renal kidney tissue fragments removed from the mammal are stained with hematoxylin and eosin, or Azan and hematoxylin, and the degree of staining is quantified. Can be observed.
- the excised kidney kidney tissue pieces should be stained with hematoxylin / eosin staining or Azan staining and hematoxylin, and the degree of staining should be quantified. Can be observed.
- the expression level of inflammatory cytokines in the kidney can be measured by quantitative RT-PCR or the like.
- examples of the inflammatory cytokine to be measured include MCP-1, IL-1 ⁇ , and IL-6.
- the ratio of macrophages in the kidney can be confirmed by identifying Mac-1 positive cells with a flow cytometer or the like.
- the BUN value can be observed by measuring the blood urea nitrogen concentration.
- the observation results of the characteristics obtained as described above are compared with the case of non-test substance administration.
- a correlation diagram between the characteristics and the presence or absence of kidney disease may be prepared in advance, and the obtained observation result of the characteristics may be compared with the correlation diagram.
- the comparison is preferably performed based on the presence or absence of a significant difference.
- the test substance can be selected as a prophylactic / therapeutic agent for renal diseases.
- the improvement here is that (i) the degree of accumulation of necrotic tubular cells (hematoxylin and eosin staining, or Azan staining and hematoxylin staining) is significantly lower than that when no test substance is administered , (Ii) the degree of glomerular structure collapse (hematoxylin and eosin staining, or Azan staining and hematoxylin staining) is significantly lower than when no test substance is administered, (iii) inflammatory cytokines in the kidney (Iv) the ratio of macrophages in the kidney is significantly lower than when no test substance is administered, and (v) the BUN value is a test substance. It means that it is significantly lower than that in the case of non-administration, and (vi) that the survival rate is significantly higher than that in the case of no administration of
- the selected test substance When used as a prophylactic / therapeutic agent for renal diseases, it can be formulated in the same manner as the AIMs of the present invention and administered by the same administration route / dose. The same applies to the kidney disease that is the target of the prophylactic / therapeutic agent.
- AIM expression-deficient non-human mammals are unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy or bilateral transient renal ischemia reperfusion under the condition of a model animal for renal disease Therefore, the mammal can be used in a method for evaluating a prophylactic / therapeutic agent for renal diseases. Therefore, the present invention also provides a non-human mammal deficient in AIM expression by unilateral ureteral ligation, transient renal ischemia reperfusion after unilateral nephrectomy, or bilateral transient renal ischemia reperfusion. Provided is a method for evaluating the preventive / therapeutic effect of a preventive / therapeutic agent for renal diseases using the obtained animal.
- the evaluation method of the present invention includes the following steps. (1) Prevention of renal disease in non-human mammals deficient in AIM expression under conditions of unilateral ureter ligation, transient renal ischemia reperfusion after unilateral nephrectomy or bilateral transient renal ischemia reperfusion Administering a therapeutic agent, (2) A step of observing one or more of the following characteristics of a non-human mammal with AIM expression deficiency administered with an agent for preventing or treating renal disease: (I) accumulation of necrotic tubular cells and fibrosis of the renal parenchyma, (Ii) Collapse and fibrosis of the glomerular structure, (Iii) expression level of inflammatory cytokines in the kidney, (Iv) the proportion of macrophages in the kidney, (V) BUN value, (Vi) survival rate, (3) A step of evaluating the effect of the renal disease preventive / therapeutic agent by comparing one or more of the above characteristics with the case of no administration of the renal disease preventive / therapeutic agent
- the preventive / therapeutic agent for renal disease administered to a non-human mammal deficient in AIM expression may be a known preventive / therapeutic agent for renal disease.
- the method for observing the characteristics observed by the evaluation method of the present invention may be performed according to the description in the screening method.
- the improvement here may be the same as described above.
- the blood AIM concentration of patients with chronic kidney disease correlates with renal function (eGFR: glomerular filtration rate).
- eGFR glomerular filtration rate
- the prognosis of patients with chronic kidney disease can be predicted by measuring the blood AIM concentration of the subject.
- the present invention provides a method for predicting prognosis of patients with renal disease, comprising measuring AIM concentration in a sample of a subject.
- the subject to which the prediction method of the present invention can be applied is not particularly limited, and examples thereof include a subject who has or is suspected of developing acute renal failure or chronic kidney disease.
- chronic kidney disease include, but are not limited to, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease Or IgM nephropathy is included.
- a sample collected from the above-mentioned subject and particularly containing an AIM gene product eg, RNA, protein, its degradation product, etc.
- an AIM gene product eg, RNA, protein, its degradation product, etc.
- body fluids such as blood, plasma, serum, lymph, urine, sweat, saliva, joint fluid or fractions thereof, or cells contained therein, particularly macrophages, preferably blood, plasma, and serum.
- the measurement of AIM concentration in a sample collected from a subject can be performed by preparing a RNA (eg, total RNA, mRNA) fraction from the sample and measuring the transcript of the AIM gene contained in the fraction. it can.
- the RNA fraction can be prepared using a known method such as guanidine-CsCl ultracentrifugation or AGPC, but using a commercially available RNA extraction kit (eg, RNeasy Mini Kit; manufactured by QIAGEN, etc.) High-purity total RNA can be prepared quickly and easily from a small amount of macrophages.
- a means for detecting the transcription product of the AIM gene in the RNA fraction for example, a method using hybridization (Northern blot, dot blot, DNA chip analysis, etc.) or PCR (RT-PCR, competitive PCR, real-time PCR, etc.) ) And the like.
- Quantitative PCR methods such as competitive PCR and real-time PCR are preferred in that changes in the expression of the AIM gene can be detected quickly and easily in a quantitative manner from a small amount of macrophages.
- the measurement of the transcription product of the AIM gene can be performed using a nucleic acid (probe) that can hybridize with the transcription product of the gene.
- a nucleic acid include a nucleic acid capable of hybridizing under high stringency conditions with a nucleic acid containing the base sequence shown in the transcription product of the AIM gene (for example, the base sequence shown in SEQ ID NO: 1).
- the highly stringent conditions include the aforementioned conditions. More preferably, a nucleic acid containing a base sequence complementary to the base sequence shown in the transcription product of the AIM gene (for example, the base sequence shown in SEQ ID NO: 1) can be mentioned.
- the nucleic acid used as the probe may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid. In the case of a single strand, an antisense strand can be used.
- the length of the nucleic acid is not particularly limited as long as it can specifically hybridize with the target nucleic acid, and is, for example, about 15 bases or more, preferably about 30 bases or more.
- the nucleic acid is preferably labeled with a labeling agent in order to enable detection and quantification of the target nucleic acid.
- a radioisotope for example, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
- the radioisotope for example, [ 32 P], [ 3 H], [ 14 C] and the like are used.
- the enzyme those which are stable and have high specific activity are preferable.
- ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
- the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
- luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
- biotin- (strept) avidin can also be used for binding between the probe and the labeling agent.
- the RNA fraction prepared as described above is separated by gel electrophoresis, and then transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, and prepared as described above.
- the amount of AIM gene expressed is determined by measuring the amount of label bound to the membrane for each band using an appropriate method after hybridization under the above-mentioned stringent conditions. Can be measured.
- the expression level of the AIM gene can be measured by subjecting the membrane spotted with the RNA fraction to a hybridization reaction in the same manner and measuring the labeling amount of the spot.
- quantitative PCR is used as a method for measuring AIM concentration.
- Examples of quantitative PCR include competitive PCR and real-time PCR.
- the set of oligonucleotides used as primers in PCR is a DNA that can hybridize specifically with the sense strand (coding strand) and antisense strand (non-coding strand) of the AIM gene transcript, and is sandwiched between them.
- the set of oligonucleotides used as primers includes a nucleic acid (sense strand) containing the base sequence shown in SEQ ID NO: 1 and a nucleic acid that can hybridize under highly stringent conditions
- examples include a nucleic acid that can hybridize with a nucleic acid (antisense strand) containing a base sequence complementary to the base sequence under highly stringent conditions.
- highly stringent conditions have the same meaning as described above. More preferably, a nucleic acid containing a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid can be mentioned.
- RT-PCR is a method in which a known amount of another template nucleic acid that can be amplified by a set of primers that can amplify the target DNA is used as a competitor in the reaction solution to cause an amplification reaction competitively.
- a known amount of competitor nucleic acid is used (such as different sizes, different migration patterns of restriction enzyme-treated fragments).
- the competitor nucleic acid may be DNA or RNA.
- cDNA may be synthesized by reverse transcription reaction from the RNA fraction prepared as described above, and then PCR may be performed in the presence of the above primer set and competor.
- a competitor is added to the RNA fraction. Is added to carry out the reverse transcription reaction, and the primer set is further added to carry out PCR. In the latter case, the efficiency of the reverse transcription reaction is taken into consideration, so that the absolute amount of the original mRNA can be estimated.
- real-time PCR is a method for monitoring the amount of amplification in real time using a fluorescent reagent and requires an apparatus in which a thermal cycler and a spectrofluorometer are integrated.
- a thermal cycler and a spectrofluorometer are integrated.
- Such devices are commercially available.
- a reagent that emits fluorescence by binding to the above primer set and double-stranded DNA such as SYBR Green I and ethidium bromide ( Intercalator), a nucleic acid that can be used as the above probe (however, the nucleic acid hybridizes to the target nucleic acid in the amplification region) and a fluorescent substance (eg, FAM, HEX, TET, FITC, etc.) and a quencher (Example: TAMRA, DABCYL, etc.)
- a fluorescent reagent such as a modified one (TaqMan TM probe or Molecular Beacon probe) is added to the PCR reaction system.
- the intercalator binds to the synthesized double-stranded DNA and emits fluorescence when irradiated with excitation light, the amount of amplification product produced can be monitored by measuring the fluorescence intensity, thereby increasing the amount of original template cDNA. Can be estimated.
- TaqMan TM probe is an oligonucleotide that can be hybridized to the target nucleic acid amplification region, modified at both ends with a fluorescent substance and a quenching substance, and hybridizes to the target nucleic acid during annealing, but does not emit fluorescence due to the presence of the quenching substance, During the extension reaction, the fluorescent substance is released by being decomposed by the exonuclease activity of DNA polymerase, and emits fluorescence. Therefore, the amount of amplification product generated can be monitored by measuring the fluorescence intensity, thereby estimating the amount of the original template cDNA.
- the Molecular Beacon probe is an oligonucleotide that can be hybridized to the amplification region of the target nucleic acid and has a hairpin secondary structure with both ends modified with a fluorescent substance and a quencher, and is quenched when it takes a hairpin structure. Fluorescence is not emitted due to the presence of the substance, but is emitted when the distance between the fluorescent substance and the quenching substance is increased by hybridization to the target nucleic acid during annealing. Therefore, the amount of amplification product generated can be monitored by measuring the fluorescence intensity, thereby estimating the amount of the original template cDNA. Since real-time RT-PCR can monitor the amount of PCR amplification in real time, it does not require electrophoresis and can analyze the expression of the AIM gene more quickly.
- the measurement of AIM concentration in a sample collected from a subject can be examined by preparing a protein fraction from the sample and detecting AIM contained in the fraction. Detection of AIM can be performed by immunoassay (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody against AIM. Alternatively, AIM can also be detected using mass spectrometry such as MALDI-TOFMS.
- the antibody against AIM is a polyclonal antibody that is commonly used as a sensitizing antigen with a protein containing the same or substantially the same amino acid sequence or partial amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. It can be obtained according to antibody or monoclonal antibody production techniques.
- the prediction method of the present invention may be a method including the following steps. (1) A step of measuring AIM concentration in samples of healthy subjects and subjects, (2) A step of comparing the AIM concentration measured by a healthy person with the AIM concentration measured by a subject.
- the renal function of the AIM of the present invention deteriorates after 2 to 3 years in patients with chronic kidney disease whose blood concentration is lower than a certain level. Therefore, as a result of measuring the AIM concentration as described above, it can be determined that there is a high possibility that the subject's chronic kidney disease will worsen in the future if it is lower than that of a healthy person or a certain level. .
- a correlation diagram between the deterioration of chronic kidney disease and the AIM concentration may be prepared in advance, and the obtained measurement results may be compared with the correlation diagram. The comparison is preferably performed based on the presence or absence of a significant difference.
- the prediction method of the present invention (3) When the AIM concentration in the subject is significantly higher than that in healthy subjects, chronic kidney disease in the subject May include a step of determining that there is a high possibility of deterioration in the future.
- the present invention provides a test method for acute renal failure, which comprises measuring the AIM concentration in a sample of a subject.
- the subject to which the test method of the present invention can be applied is not particularly limited, and examples thereof include subjects who are likely to develop or suspected of developing acute renal failure.
- the sample used in the test method of the present invention is as described in the method for predicting prognosis of a renal disease patient of the present invention, and preferably includes urine.
- the measurement of the AIM concentration in the sample collected from the test subject is as described in the prognosis prediction method for a renal disease patient of the present invention.
- the inspection method of the present invention may be a method including the following steps. (1) A step of measuring AIM concentration in samples of healthy subjects and subjects, (2) A step of comparing the AIM concentration measured by a healthy person with the AIM concentration measured by a subject.
- the AIM of the present invention has a significantly higher urinary concentration in patients with acute renal failure than in healthy individuals. Therefore, as a result of measuring the AIM concentration as described above, when the AIM concentration is significantly higher than that of a healthy person, it can be determined that the subject suffers from acute renal failure.
- a correlation diagram between acute renal failure and AIM concentration may be prepared in advance, and the obtained measurement results may be compared with the correlation diagram. The comparison is preferably performed based on the presence or absence of a significant difference.
- test method of the present invention A step of determining that the patient is suffering from the disease may be included.
- kits for diagnosing or prognosing renal diseases are not particularly limited as long as it is a kit for simply carrying out the above-described inspection method or prediction method of the present invention.
- the kit (A) a nucleic acid probe or nucleic acid primer capable of hybridizing with a transcription product of the AIM gene, and / or (b) an antibody against AIM.
- each nucleic acid or antibody specifically recognizes a different part of the base sequence of the AIM gene, or specifically recognizes a different epitope of the translation product of the AIM gene. It can be recognized.
- kits of the present invention includes the reagent containing the nucleic acid (a) as a component
- examples of the nucleic acid include the probe nucleic acid or primer oligonucleotide described above in the test method or prediction method of the present invention.
- the nucleic acid capable of detecting the expression of the AIM gene can be provided as a solid in a dry state or in the state of alcohol precipitation, or dissolved in water or a suitable buffer (eg, TE buffer). It can also be provided.
- the nucleic acid can be provided in a state of being previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substance and can be used after labeling.
- the nucleic acid can be provided in a state immobilized on an appropriate solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like.
- functional groups such as amino groups, aldehyde groups, SH groups, and biotin are introduced into nucleic acids in advance, and functional groups capable of reacting with the nucleic acids on solid phases (eg, aldehydes).
- solid phases eg, aldehydes.
- Group, amino group, SH group, streptavidin, etc. and solid phase and nucleic acid are cross-linked by covalent bond between both functional groups, or polyanionic nucleic acid is coated with polycation and solid phase is coated.
- Examples of the method include immobilization of nucleic acid using electric coupling, but are not limited thereto.
- the nucleic acid contained in the kit is particularly preferably constructed so that the expression of the AIM gene can be detected by the same method (eg, Northern blot, dot blot, DNA array technology, quantitative RT-PCR, etc.). .
- kits of the present invention includes the reagent containing the antibody (b) as a component
- examples of the antibody include the antibodies described above in the examination method or the prediction method of the present invention.
- the reagent constituting the kit of the present invention is a substance other than the nucleic acid or antibody capable of detecting the expression of the AIM gene and other substances necessary for the reaction for detecting the expression of the gene, and is stored in a coexisting state Thus, a substance that does not adversely influence the reaction can be further contained.
- the reagent may be provided with a separate reagent containing other substances necessary in the reaction for detecting the expression of the AIM gene.
- the reaction for detecting the expression of the AIM gene is PCR
- examples of the other substance include a reaction buffer, dNTPs, and a heat-resistant DNA polymerase.
- the reaction for detecting the expression of the AIM gene is an antigen-antibody reaction
- examples of the other substance include a reaction buffer, a competitor antibody, and a labeled secondary antibody (for example, the primary antibody is a rabbit anti-human).
- an AIM antibody mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), blocking solution, and the like can be mentioned.
- sequence numbers in the sequence listing in the present specification indicate the following sequences.
- [SEQ ID NO: 1] The base sequence of human AIM is shown.
- [SEQ ID NO: 2] The amino acid sequence of human AIM is shown.
- [SEQ ID NO: 3] The base sequence of cat AIM is shown.
- [SEQ ID NO: 4] The amino acid sequence of cat AIM is shown.
- [SEQ ID NO: 5] The complementary sequence of the cat AIM transcript is shown.
- SEQ ID NO: 6 The amino acid sequence of mouse AIM is shown.
- Example 1 Inhibition of progression of chronic renal failure or renal fibrosis by AIM
- UUO ureteral ligation
- the structure of normal kidney was not different from WT or AIM-KO.
- Example 2 AIM-induced acute renal failure (until day 7) to chronic progression of renal damage (chronic renal failure) (day 14)
- Another renal disease model is the transient renal ischemia reperfusion (IR) model .
- transient renal ischemia reperfusion one kidney is removed in advance, and the remaining renal artery is ligated to make it ischemic. After 30 minutes, the ligation is released and the blood flow is reperfused.
- tubule necrosis progresses for about 3 days with mild fibrosis, and the renal function deteriorates accordingly.
- IR was performed on AIM-deficient mice (AIM-KO) and wild-type mice (WT), respectively, and the progress was observed (FIG. 2).
- Example 3 Inhibition of prolongation of inflammation after chronic renal failure by AIM (chronic renal injury) Transient renal ischemia / reperfusion (IR) performed in Example 2 was performed in wild-type mice (WT) and AIM-deficient mice ( AIM-KO) and postoperative inflammatory macrophage infiltration was observed by immunostaining of F4 / 80, a macrophage marker (FIG. 3A). On day 3 after surgery, infiltration of F4 / 80 positive macrophages was clearly enhanced in AIM KO mice compared to WT. On the 14th day after the operation, macrophage infiltration was reduced in WT, but worsened in AIM KO mice.
- AIM chronic renal injury
- IR transient renal ischemia / reperfusion
- MCP-1 one of the inflammatory cytokines
- Example 4 Recovery of acute renal failure by AIM AIM-KO mice were subjected to the IR performed in Example 2, and on the third, fourth, and seventh days, the renal function was most transiently deteriorated, respectively.
- rAIM Recombinant AIM
- the progression of necrotic cell accumulation and glomerular destruction progressed, and the renal function (BUN value) improved to some extent from the third day, but returned to normal. There was no recovery, and the second half gradually showed a trend of deterioration.
- the BUN value improved significantly after rAIM administration on the third day, and already returned to the normal range on the seventh day, and further decreased thereafter. Histologically, necrotic tubular cells were removed on day 7, and the structure of the renal parenchyma was also normalized. That is, it was revealed that AIM administration can enhance the removal of necrotic cells, promote the regeneration of tubules, and restore renal function.
- Example 5 Attachment of AIM to tubular epithelial cell mass necrotized due to acute renal failure and removal of necrotic lesion
- AIM-KO mice were subjected to IR performed in Example 2 and intravenously administered rAIM (100 ⁇ g).
- the kidney sections were stained with anti-AIM antibody after 3, 6 and 12 hours. 3 hours after administration of AIM, it was confirmed that AIM was strongly attached to the necrotic part of the tubule (the upper part of FIG. 5: The necrotic lesion was indicated by N in the photograph by phase-contrast.
- the lower part of FIG. 5: AIM signal (arrowhead) was observed overlapping the necrotic lesion).
- the necrotic area was reduced with time, and only traces were confirmed after 12 hours. That is, it is considered that the recovery of renal function after IR by AIM (Example 4) promoted the removal of AIM by adhering to the necrotic lesion, and accompanying this, suppression of inflammation and tissue regeneration proceeded accordingly.
- Example 6 Appearance of urinary AIM after acute renal failure Bilateral transient renal ischemia reperfusion (IR), in which one kidney is not removed and the renal arteries of both kidneys are ligated to become ischemic
- IR Bilateral transient renal ischemia reperfusion
- the urine of the applied wild type mouse (WT) was detected by ELISA on the first and seventh days after IR (FIG. 6). Almost no AIM was detected in the urine of normal mice (before IR), but a large amount of AIM was detected in the urine on the first day after the most severe IR.
- Urinary AIM decreased with recovery from renal injury (7 days after IR).
- urine excreted during IR-induced renal injury is diluted urine, the urinary AIM value was normalized with the urinary creatinine value.
- Example 8 Clinical score Bilateral IR was applied to WT and AIM-KO mice, and the clinical score was analyzed over time (FIG. 8). The clinical scores were scored for lack of movement agility, opacity of the eyes, decreased responsiveness to painful stimuli in the tail, and poor fur, respectively (0: no abnormalities, 1: mild symptoms, 2: moderate Symptoms, 3: severe symptoms), and the total was graphed. In WT, the score reached its maximum value on the first day after IR and gradually decreased, but in AIM-KO, the score remained high.
- Example 10 Renal dysfunction associated with acute renal failure (renal tissue: PAS staining) Bilateral IR was applied to WT and AIM-KO mice, and renal tissues were analyzed by PAS staining over time (FIG. 10). Similar to the clinical score in Example 8 and the BUN value in Example 9, renal dysfunction peaked on day 1 in WT and then recovered, and on day 7, normal tubular structure and brush border (booklet border) The proximal tubular epithelium with the arrowheads) regenerated. However, in AIM-KO, the disorder continued, and PAS-positive dead cell clusters accumulated in the proximal tubules and were not removed even after 7 days.
- AIM inflammatory cytokine
- Example 13 Prolonged inflammation after acute renal failure due to AIM (infiltrating macrophages) Bilateral IR was applied to WT and AIM-KO mice, and the kidneys on day 7 after IR were treated with collagenase and analyzed with a flow cytometer to examine the proportion of macrophages (Mac-1 positive cells) (Fig. 13). Similar to the results of Example 12, AIM-KO showed a higher percentage of macrophages in the kidney than WT. Three mice were analyzed for each, and similar results were obtained. FIG. 13 presents a representative result.
- Example 14 Accumulation of AIM in intraepithelial cell mass of mouse tubule necrotic due to acute renal failure About kidney serial sections of WT mice subjected to bilateral IR, immunostaining with PAS staining (left) and anti-AIM antibody ( (Right) was performed (FIG. 14). AIM (white) was found to accumulate in many dead cell masses.
- Example 15 Accumulation of AIM in necrotic intratubular epithelial cell mass of patients with acute renal failure Renal serial sections of human patients who died of acute renal failure due to renal infarction were immunized with PAS staining (left) and anti-AIM antibody Staining (right) was performed ( Figure 15). As in the IR mouse of Example 14, it can be seen that AIM (white) is accumulated in many dead cell clusters.
- Example 16 Detection of urinary AIM in acute renal failure patients and acute renal failure mice WT mice subjected to bilateral IR and 3 patients and 3 healthy individuals who were transported to hospital with acute renal failure (AKI) About 5 animals, AIM concentration was analyzed by ELISA for urine before IR, 1 day after IR, and 7 days after (FIG. 16). Almost no AIM was found in the urine of healthy people, but it was found in the urine of AKI patients. In mice, a significant amount of AIM was found in the urine after 1 day of IR, where renal injury was significant, but the amount of AIM decreased on the 7th day when renal injury improved, even though it was not observed before IR.
- Example 17 Reduction of tubule epithelial cell mass by AIM accumulation AID-KO mice were subjected to bilateral IR, and 200 ⁇ g of rAIM was administered 3 days after IR. Staining and immunostaining with anti-AIM antibody were performed (FIG. 17). The dead cell mass in which AIM accumulated was rapidly reduced.
- Example 18 In vitro phagocytosis assay (experiment using kidney-derived macrophages) AIM-KO mice were subjected to bilateral IR, the kidney on the third day after IR was treated with collagenase, and F4 / 80 positive macrophages were isolated using a FACS sorter, and then their phagocytic ability was analyzed (FIG. 18). ).
- rAIM recombinant AIM
- BSA bovine serum albumin
- HK2 dead cells that were not coated with rAIM or BSA were used. Macrophages and the above three types of HK2 dead cells were incubated, and FITC-positive HK2 dead cells incorporated into the macrophages were analyzed over time with a flow cytometer (FACS). It was shown that HK2 dead cells coated with rAIM versus HK2 dead cells coated with BSA or uncoated HK2 dead cells were phagocytosed by macrophages more efficiently.
- Example 19 In vitro phagocytosis assay (experiment using bone marrow-derived macrophages) An experiment similar to Example 18 was performed using macrophages obtained by differentiating AIM-KO-derived bone marrow cells with M-CSF as phagocytic cells (FIG. 19). In Example 18, since there was no difference in the way of phagocytosis between HK2 dead cells coated with BSA and HK2 dead cells not coated, HK2 dead cells not coated were not used in this example. Similar to the results of Example 18, phagocytosis of HK2 dead cells coated with rAIM increased.
- Example 21 Recovery of clinical score by AIM administration The clinical score of the mouse of Example 20 was examined in the same manner as in Example 8 (Fig. 21). A marked recovery of clinical score was observed after rAIM administration.
- Example 22 Renal Function Recovery by AIM Administration BUN was measured over time for the mice of Example 20 as in Example 9 (FIG. 22). A significant decrease in BUN levels was observed with rAIM administration.
- Example 23 Removal of necrotic intratubular epithelial cell mass by AIM administration After bilateral IR was applied to AIM-KO mice, rAIM 200 ⁇ g / mouse or an equal volume of PBS was applied from day 1 to day 3. Intravenous injection was performed, and the state of the kidney on day 3 and day 7 was analyzed by PAS staining (FIG. 23). PBS administration resulted in accumulation of dead cells in the proximal tubules, but significant removal was observed in rAIM-treated mice, and tubule cells with brush borders recovered on day 7.
- Example 24 Quantification of necrotic intratubular epithelial cell mass after AIM administration
- n 3 each
- Example 25 Reduction of inflammatory response by administration of AIM (inflammatory cytokine)
- AIM inflammatory cytokine
- RNA was extracted from the kidney on day 7 after IR, and the inflammatory cytokines IL-1 ⁇ and IL-6 were analyzed by quantitative RT-PCR (FIG. 25).
- both IL-1 ⁇ and IL-6 decreased compared to the PBS administration group, suggesting that the inflammatory reaction associated with acute renal failure was also suppressed by rAIM administration.
- Example 26 Effect of AIM on acute renal failure due to non-lethal (mild) IR
- ischemia was performed for 30 minutes, and the lethality of AIM-KO Induced a high degree of acute renal failure.
- the ischemia time was shortened (30 minutes ⁇ 25 minutes), and AIM-KO induced renal failure in AIM-KO mice with a survival rate of 100% on the 7th day.
- Example 28 Chronic renal failure due to AIM deficiency The same mild IR as in Example 26 was applied to WT and AIM-KO mice, and the state of the kidney on day 28 after IR was PAS stained (to see dead cell mass) ) And Azan staining (to see fibrosis) (FIG. 28). Even on the 28th day, in AIM-KO, some PAS-positive dead cell mass remained as compared with WT. AIM-KO showed marked fibrosis (Azan positive), and the tubule structure was more fragile than WT.
- Example 29 Enhancement of renal fibrosis due to AIM deficiency
- AIM-KO increased all fibrosis markers compared to WT.
- Example 30 Blood AIM value in human diabetic chronic renal failure patients Age is almost equal, (1) healthy subjects (male 142, women: 54), (2) no renal disorder (Cre ⁇ 1.0 mg / dl The blood AIM concentration was measured separately for men and women for diabetic patients (male: 70, females: 57) and (3) diabetic patients with chronic renal failure (male: 146, females: 54) (FIG. 30). For both men and women, there was no significant difference in AIM values between (1) and (2), but it was significantly lower in (3).
- Example 31 Correlation between Renal Function and Blood AIM in Human Chronic Kidney Disease Patients
- CKD-causing diseases include diabetic nephropathy, glomerulonephritis, hypertensive nephropathy, IgA nephropathy and the like.
- the age of patients is 60 years old or younger in both sexes. As shown in FIG. 31A, the blood AIM value and the renal function showed a significant correlation.
- AIM can be a useful marker for predicting not only current renal function but also prognosis in CKD patients.
- Example 32 Blood AIM deficiency (or marked reduction) in cats
- Anti-AIM antibody (Rab2: A polyclonal antibody produced by immunizing rabbits with mouse rAIM from each of the sera of dogs (3 animals), cats (3 animals), and mice. And immunoblotting was performed under reducing conditions (FIG. 32A). As a result, a signal was confirmed in the dog serum, but almost no signal was detected in all three cats. This is not an antibody problem, but feline AIM cDNA was cloned (see Example 36), incorporated into the pCAGGS expression vector with the HA tag attached to the C-terminus, and anti-HA antibody from the culture supernatant expressed in HEK293T cells.
- feline rAIM protein purified using the column could be detected by immunoblotting under reducing conditions using this antibody to the same extent as mouse rAIM (FIG. 32B). From these results, it was found that the cats examined in this example expressed functional AIM mRNA but hardly existed as AIM protein in the blood.
- Example 33 Binding ability of feline AIM and IgM in feline blood Recombinant feline AIM (transfected with HEK293T cells transfected with a plasmid in which feline AIM cDNA shown in Fig. 35 was inserted into an expression vector) was purified from the culture supernatant. 1 mg) was intravenously injected into a cat (mongrel, male 2 years and 3 months old), blood was collected after 1 hour, and serum was separated. Serum was subjected to gel size fractionation, and each fraction was analyzed for AIM and IgM by Western blotting. As shown in FIG. 33A, the fraction in which AIM is present and the fraction in which IgM are present are clearly different.
- Example 34 Binding ability of mouse AIM and IgM in feline blood
- the same test as in Example 33 was performed using mouse AIM.
- Recombinant mouse AIM (1 mg) was intravenously injected into a cat (mongrel, male 2 years and 3 months old), and blood was collected 1 hour later and serum separated. Serum was subjected to gel size fractionation, and each fraction was analyzed for AIM and IgM by Western blotting. As shown in FIG. 34, the fraction in which mouse AIM was present and the fraction in which cat IgM were present completely matched.
- mouse AIM binds to IgM and is thought to be stable in blood.
- Example 35 Mouse AIM binding site to IgM A plurality of recombinant modified mouse AIMs lacking the C-terminus were prepared. The binding of these modified mouse AIM and IgM was confirmed in vitro. As a result, the modified mouse AIM lacking a part of the SRCR3 domain significantly decreased the binding to IgM. Therefore, it was found that the SRCR3 domain of mouse AIM is important for binding to IgM.
- Example 36 Cat AIM cDNA sequence
- the full-length cat AIM cDNA (SEQ ID NO: 5) was isolated (FIG. 35). The sequence, especially the sequence encoding the leader peptide, was significantly different from the published sequence.
- Example 37 Hydrophobicity of leader peptide
- the hydrophobicity of the leader peptide sequence of each AIM protein of cat, human, mouse, and dog is shown (FIGS. 36 to 39). All had sufficient hydrophobicity and met the conditions as secreted proteins.
- the cat analyzed the leader peptide from the cDNA we isolated, and the canine AIM from the predicted sequence (GenBank Accession No .: XM_846487.2) published on NCBI Resources.
- Example 38 Comparison of AIM amino acid sequences of human, cat, and mouse Comparison of amino acid sequences of the leader peptide (LS), each SRCR, and the hinge part in three is shown (FIG. 40).
- the amino acids that are common to all three are indicated by “*”, those that are common to only humans and cats are “.”, And those that are common only to humans and mice are indicated by “:”. .
- Example 39 Analysis of feline blood AIM
- an HA tag was linked to the feline AIM cDNA.
- Recombinant feline AIM protein (rcAIM-HA) loaded with HA peptide at the C-terminus was produced by integrating DNA into an expression vector and introducing the gene into HEK293T cells. Mice were immunized to establish anti-cat AIM monoclonal antibodies. Using the antibody, the AIM concentration in blood of cats (48 individuals) of various strains was analyzed by the reduced Western blotting method (FIG. 41). RcAIM-HA was used as a concentration control.
- Example 40 Establishment of IR method for cats A method for inducing acute renal failure by IR was established in cats with high blood AIM concentrations. Under general anesthesia, the bilateral renal arteries were ligated with a clamp for 1 hour under a laparoscope and then released. Thereafter, blood and urine were collected over time to examine renal function.
- FIG. 42 shows changes in blood BUN value and Cre value. BUN and Cre values peaked at 12 hours after IR, but did not improve until the 7th day. That is, it was suggested that there is a high possibility that recovery from acute renal failure is impaired as in AIM-KO mice.
- Example 41 Analysis of AIM in blood and urine in cats after IR
- AIM is detected in urine after IR, which is thought to accumulate in dead cell mass clogged in tubules.
- FIG. 43 As a result of analyzing the serum and urine of cats after IR by the reduced Western blotting method using an anti-cat AIM antibody, AIM was not detected in the urine after IR (FIG. 43). There was also no obvious change in the amount of AIM in the blood.
- Example 42 Presence or absence of AIM accumulation in dead cell mass in the proximal tubule of a cat induced with acute renal failure
- a bilateral IR was applied to the cat and the kidney on the third day was treated with AIM in the necrotic cell mass in the tubule It was analyzed by immunostaining as to whether or not accumulated (FIG. 44).
- a necrotic cell mass was detected in the proximal tubule (white arrow), but AIM accumulation was not observed at the same location.
- Example 41 it was shown that AIM was not detected in the urine after IR. As a result, it was suggested that AIM did not reach the dead cell mass in the tubule.
- Example 43 Effect of AIM Administration on Cats Induced with Acute Renal Failure Similar to the method described in Example 40, the cat (female 5 years old) was given bilateral IR and after 24 hours under anesthesia, an arterial catheter Was inserted from the groin artery, the catheter tip was advanced to the renal artery, and 25 ml (rAIM: 25 mg) of each solution of rAIM 50 mg dissolved in PBS 50 ml was injected into the unilateral renal artery. In another cat (also a female 5 years old), IR and catheterization were similarly performed, and 25 ml each of PBS alone was injected into the unilateral renal artery. FIG.
- GFR Glomerular Filtration Rate
- Example 44 Effect of AIM administration on cats with induced acute renal failure Similar to the method described in Example 43, bilateral IR was given and kidney was 24 hours after rAIM or PBS injection (48 hours after IR). The tissue was analyzed by PAS staining ( Figure 46). Cats injected with PBS showed necrosis and loss of tubule epithelial cells, destruction of tubule structure, and proliferation of stroma, but tubule epithelial cells had already recovered in the cat injected with rAIM and the booklet edge was restored. And the structure was restored. In addition, the stroma was thinner than the cat injected with PBS, and no growth was observed. Histologically, the cure of renal failure by rAIM is clear.
- Example 45 Effect of AIM Administration on Cats AIM or vehicle is administered daily to cats 6-8 years old.
- the renal function (BUN value) is measured 2 to 4 weeks after administration.
- AIM is useful for the prevention of feline renal function deterioration and renal failure.
- Similar results can be obtained by using an agent capable of agonistically adjusting the function of AIM (including a partial peptide of AIM having AIM activity) or an agent that induces the expression of AIM instead of AIM.
- Example 46 Effect of administration of modified AIM to cats
- the SRCR3 domain of cat AIM is modified to the SRCR3 domain of mouse AIM to produce a modified cat AIM that binds to IgM.
- the renal function (BUN value) is measured 2 to 4 weeks after administration.
- BUN value The renal function
- deterioration of renal function observed in the vehicle administration group is suppressed. Therefore, it can be seen that AIM is useful for the prevention of feline renal function deterioration and renal failure.
- the modified cat AIM binds to and stabilizes IgM in the blood, the effectiveness can be obtained by administration of the modified cat AIM at a lower dose than administration of the cat AIM.
- the modified cat AIM may be bound to cat IgM and is not limited.
- the present invention can provide a prophylactic / therapeutic agent for renal diseases containing AIM as an active ingredient.
- the renal disease model mouse of the present invention contributes to elucidation of the onset mechanism of renal disease, and further, according to the screening method using the model mouse, it is possible to search for a substance effective for prevention / treatment of renal disease. it can.
- the effect of a known preventive / therapeutic agent for renal disease can be evaluated using the renal disease model mouse of the present invention.
- this invention can provide the diagnostic method of a renal disease.
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Abstract
Description
本発明者は、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。
[1]AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸を含有してなる、腎疾患の予防・治療剤;
[2]AIMの発現を誘導する薬剤またはAIMを安定化させる薬剤を含有してなる、腎疾患の予防・治療剤;
[3]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である前記[1]または[2]に記載の予防・治療剤;
[4]腎疾患が急性腎不全または慢性腎不全である前記[1]~[3]のいずれか1つに記載の予防・治療剤;
[5]予防・治療の対象がヒトである前記[1]~[4]のいずれか1つに記載の予防・治療剤;
[6]予防・治療の対象がネコである前記[1]~[4]のいずれか1つに記載の予防・治療剤;
[7]AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、前記[6]に記載の予防・治療剤;
[8]ネコIgMと結合するAIMもしくはその部分ペプチドが、マウス由来AIMのSRCR3ドメインを含む、前記[7]に記載の予防・治療剤;
[9]AIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって得られる動物を用いる、腎疾患の予防・治療剤のスクリーニング方法;
[10]以下の工程を含むことを特徴とする、前記[9]に記載のスクリーニング方法
(1)片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行う条件下、AIM発現不全非ヒト哺乳動物に被検物質を投与する工程、
(2)被検物質を投与されたAIM発現不全非ヒト哺乳動物の下記特性のいずれか一項目以上を観察する工程:
(i)壊死した尿細管細胞の蓄積と腎実質の線維化、
(ii)糸球体構造の崩壊と線維化、
(iii)腎臓における炎症性サイトカインの発現量、
(iv)腎臓におけるマクロファージの割合、
(v)BUN値、
(vi)生存率、
(3)被検物質非投与の場合と比較して、前記特性のいずれか一項目以上が改善される被検物質を選択する工程;
[11]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である前記[9]または[10]に記載のスクリーニング方法;
[12]AIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって得られる動物を用いる、腎疾患予防・治療剤の予防・治療効果の評価方法;
[13]以下の工程を含むことを特徴とする、前記[12]に記載の評価方法
(1)片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行う条件下、AIM発現不全非ヒト哺乳動物に腎疾患予防・治療剤を投与する工程、
(2)腎疾患予防・治療剤を投与されたAIM発現不全非ヒト哺乳動物の下記特性のいずれか一項目以上を観察する工程:
(i)壊死した尿細管細胞の蓄積と腎実質の線維化、
(ii)糸球体構造の崩壊と線維化、
(iii)腎臓における炎症性サイトカインの発現量、
(iv)腎臓におけるマクロファージの割合、
(v)BUN値、
(vi)生存率、
(3)前記特性のいずれか一項目以上を腎疾患予防・治療剤非投与の場合と比較して、腎疾患予防・治療剤の効果を評価する工程;
[14]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である前記[12]または[13]に記載の評価方法;
[15]被検者の試料中のAIM濃度を測定することを含む、腎疾患患者の予後の予測方法;
[16]試料が血液または血清である、前記[15]に記載の予測方法;
[17]AIM濃度の測定方法が抗AIM抗体を用いた免疫学的方法である、前記[15]または[16]に記載の予測方法;
[18]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である前記[15]~[17]のいずれか1つに記載の予測方法;
[19]被検者の試料中のAIM濃度を測定することを含む、急性腎不全の検査方法;
[20]試料が尿である、[19]に記載の検査方法;
[21]AIM濃度の測定方法が抗AIM抗体を用いた免疫学的方法である、[19]または[20]に記載の検査方法;
[22]以下の(a)または(b)を含む、腎疾患の診断または予後の予測キット:
(a)AIM遺伝子の転写産物とハイブリダイズし得る核酸プローブまたは核酸プライマー、および/または
(b)AIMに対する抗体;
[23]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である[22]に記載のキット;
[24]AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸を対象に有効量投与することを含む、腎疾患の予防・治療方法;
[25]AIMの発現を誘導する薬剤またはAIMを安定化させる薬剤を対象に有効量投与することを含む、腎疾患の予防・治療方法;
[26]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、前記[24]または[25]に記載の予防・治療方法;
[27]腎疾患が急性腎不全または慢性腎不全である、前記[24]~[26]のいずれか1つに記載の予防・治療方法;
[28]予防・治療の対象がヒトである、前記[24]~[27]のいずれか1つに記載の予防・治療方法;
[29]予防・治療の対象がネコである、前記[24]~[27]のいずれか1つに記載の予防・治療方法;
[30]AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、前記[29]に記載の予防・治療方法;
[31]腎疾患の予防・治療に用いるための、AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸;
[32]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、前記[31]に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸;
[33]腎疾患が急性腎不全または慢性腎不全である、前記[31]または[32]に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸;
[34]予防・治療の対象がヒトである、前記[31]~[33]のいずれか1つに記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸;
[35]予防・治療の対象がネコである、前記[31]~[33]のいずれか1つに記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸;
[36]AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、前記[35]に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸;
[37]腎疾患の予防・治療に用いるための、AIMの発現を誘導する薬剤またはAIMを安定化させる薬剤;
[38]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、前記[37]に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤;
[39]腎疾患が急性腎不全または慢性腎不全である、前記[37]または[38]に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤;
[40]予防・治療の対象がヒトである、前記[37]~[39]のいずれか1つに記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤;
[41]予防・治療の対象がネコである、前記[37]~[39]のいずれか1つに記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤;
[42]AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、前記[41]に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤;
[43]腎疾患の予防・治療剤を製造するための、AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸またはAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤の使用;
[44]腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、前記[43]に記載の使用。
[45]腎疾患が急性腎不全または慢性腎不全である、前記[43]または[44]に記載の使用;
[46]予防・治療の対象がヒトである、前記[43]~[45]のいずれか1つに記載の使用;
[47]予防・治療の対象がネコである、前記[43]~[45]のいずれか1つに記載の使用;
[48]AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、前記[47]に記載の使用;
を提供する。
本発明におけるAIMは、配列番号:2(ヒト由来AIM蛋白質のアミノ酸配列)または配列番号:4(ネコ由来AIM蛋白質のアミノ酸配列)で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質である。
AIMは、例えば、温血動物(例えば、ヒト、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジー、トリなど)の免疫細胞であるマクロファージから単離・精製される蛋白質であってよい。また、化学合成もしくは無細胞翻訳系で生化学的に合成された蛋白質であってもよいし、あるいは上記アミノ酸配列をコードする塩基配列を含む核酸を導入された形質転換体から産生される組換え蛋白質であってもよい。
本明細書におけるアミノ酸配列の相同性は、相同性計算アルゴリズムNCBI BLAST(National Center for Biotechnology Information Basic Local Alignment Search Tool)を用い、以下の条件(期待値=10;ギャップを許す;マトリクス=BLOSUM62;フィルタリング=OFF)にて計算することができる。アミノ酸配列の相同性を決定するための他のアルゴリズムとしては、例えば、Karlinら,Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993)に記載のアルゴリズム[該アルゴリズムはNBLASTおよびXBLASTプログラム(version 2.0)に組み込まれている(Altschulら,Nucleic Acids Res., 25: 3389-3402 (1997))]、Needlemanら, J .Mol. Biol., 48: 444-453 (1970)に記載のアルゴリズム[該アルゴリズムはGCGソフトウェアパッケージ中のGAPプログラムに組み込まれている]、MyersおよびMiller, CABIOS, 4: 11-17 (1988)に記載のアルゴリズム[該アルゴリズムはCGC配列アラインメントソフトウェアパッケージの一部であるALIGNプログラム(version 2.0)に組み込まれている]、Pearsonら, Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988)に記載のアルゴリズム[該アルゴリズムはGCGソフトウェアパッケージ中のFASTAプログラムに組み込まれている]等が挙げられ、それらも同様に好ましく用いられ得る。
より好ましくは、配列番号:2または配列番号:4で表されるアミノ酸配列と実質的に同一のアミノ酸配列とは、配列番号:2または配列番号:4で表されるアミノ酸配列と約60%以上、好ましくは約70%以上、さらに好ましくは約80%以上、特に好ましくは約90%以上、最も好ましくは約95%以上の同一性を有するアミノ酸配列である。
前記活性の測定は、自体公知の方法に準じて行なうことができる。
上記のようにアミノ酸配列が挿入、欠失または置換されている場合、その挿入、欠失または置換の位置は、蛋白質の活性が保持される限り特に限定されない。
ここでエステルにおけるRとしては、例えば、メチル、エチル、n-プロピル、イソプロピル、n-ブチルなどのC1-6アルキル基;例えば、シクロペンチル、シクロヘキシルなどのC3-8シクロアルキル基;例えば、フェニル、α-ナフチルなどのC6-12アリール基;例えば、ベンジル、フェネチルなどのフェニル-C1-2アルキル基;α-ナフチルメチルなどのα-ナフチル-C1-2アルキル基などのC7-14アラルキル基;ピバロイルオキシメチル基などが用いられる。
本発明のAIMがC末端以外にカルボキシル基(またはカルボキシレート)を有している場合、カルボキシル基がアミド化またはエステル化されているものも本発明の蛋白質に含まれる。この場合のエステルとしては、例えば上記したC末端のエステルなどが用いられる。
さらに、本発明のAIMには、N末端のアミノ酸残基のアミノ基が保護基(例えば、ホルミル基、アセチル基などのC1-6アルカノイルなどのC1-6アシル基など)で保護されているもの、生体内で切断されて生成し得るN末端のグルタミン残基がピログルタミン酸化したもの、分子内のアミノ酸の側鎖上の置換基(例えば-OH、-SH、アミノ基、イミダゾール基、インドール基、グアニジノ基など)が適当な保護基(例えば、ホルミル基、アセチル基などのC1-6アルカノイル基などのC1-6アシル基など)で保護されているもの、あるいは糖鎖が結合したいわゆる糖蛋白質などの複合蛋白質なども含まれる。
AIMは、システインを多く含む3つのSRCR(Scavenger-Receptor Cysteine-Rich)ドメイン含んでいることから、それぞれのSRCRドメインを本発明の部分ペプチドとして使用できる。具体的には、例えば、配列番号:2で表されるアミノ酸配列のうち、SRCR1ドメイン(配列番号:2で表されるアミノ酸配列のうち、アミノ酸番号24~125)、SRCR2ドメイン(配列番号:2で表されるアミノ酸配列のうち、アミノ酸番号138~239)、SRCR3ドメイン(配列番号:2で表されるアミノ酸配列のうち、アミノ酸番号244~346)をそれぞれ含む部分アミノ酸配列やSRCRドメインの任意の組合せを含む部分アミノ酸配列を有するものなどが用いられる。また、配列番号:4で表されるアミノ酸配列のうち、SRCR1ドメイン(配列番号:4で表されるアミノ酸配列のうち、アミノ酸番号24~125)、SRCR2ドメイン(配列番号:4で表されるアミノ酸配列のうち、アミノ酸番号139~239)、SRCR3ドメイン(配列番号:4で表されるアミノ酸配列のうち、アミノ酸番号244~346)をそれぞれ含む部分アミノ酸配列やSRCRドメインの任意の組合せを含む部分アミノ酸配列を有するものなども用いられる。本発明の部分ペプチドは、上記の機能ドメインを含む限りそのサイズに特に制限はないが、好ましくは50個以上の部分アミノ酸配列を含むもの、より好ましくは100個以上の部分アミノ酸配列を含むもの、さらに好ましくは200個以上の部分アミノ酸配列を含むものが挙げられる。該部分アミノ酸配列は一個の連続した部分アミノ酸配列であってもよく、あるいは不連続な複数の部分アミノ酸配列が連結されたものであってもよい。
さらに、本発明の部分ペプチドには、上記したAIMと同様に、N末端のアミノ酸残基のアミノ基が保護基で保護されているもの、N末端のグルタミン残基がピログルタミン酸化したもの、分子内のアミノ酸の側鎖上の置換基が適当な保護基で保護されているもの、あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合ペプチドなども含まれる。
ペプチド合成法は、例えば、固相合成法、液相合成法のいずれであってもよい。AIMを構成し得る部分ペプチドもしくはアミノ酸と残余部分とを縮合し、生成物が保護基を有する場合は保護基を脱離することにより目的とする蛋白質を製造することができる。
ここで、縮合や保護基の脱離は、自体公知の方法、例えば、以下の(1)および(2)に記載された方法に従って行われる。
(1)M. BodanszkyおよびM. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966年)
(2)SchroederおよびLuebke, The Peptide, Academic Press, New York(1965年)
上記方法で得られるAIMが遊離体である場合には、該遊離体を公知の方法あるいはそれに準じる方法によって適当な塩に変換することができるし、逆にAIMが塩として得られた場合には、該塩を公知の方法あるいはそれに準じる方法によって遊離体または他の塩に変換することができる。
AIMまたはその部分ペプチドをコードするDNAとしては、ゲノムDNA、温血動物(例えば、ヒト、ウシ、サル、ウマ、ブタ、ヒツジ、ヤギ、イヌ、ネコ、モルモット、ラット、マウス、ウサギ、ハムスター、トリなど)のマクロファージ由来のcDNA、合成DNAなどが挙げられる。AIMまたはその部分ペプチドをコードするゲノムDNAであれば、前記動物のあらゆる細胞[例えば、肝細胞、脾細胞、神経細胞、グリア細胞、膵β細胞、骨髄細胞、メサンギウム細胞、ランゲルハンス細胞、表皮細胞、上皮細胞、杯細胞、内皮細胞、平滑筋細胞、線維芽細胞、線維細胞、筋細胞、脂肪細胞、免疫細胞(例、マクロファージ、T細胞、B細胞、ナチュラルキラー細胞、肥満細胞、好中球、好塩基球、好酸球、単球)、巨核球、滑膜細胞、軟骨細胞、骨細胞、骨芽細胞、破骨細胞、乳腺細胞、肝細胞もしくは間質細胞、またはこれら細胞の前駆細胞、幹細胞もしくはガン細胞など]もしくはそれらの細胞が存在するあらゆる組織[例えば、脳、脳の各部位(例、嗅球、扁桃核、大脳基底球、海馬、視床、視床下部、大脳皮質、延髄、小脳)、脊髄、下垂体、胃、膵臓、腎臓、肝臓、生殖腺、甲状腺、胆嚢、骨髄、副腎、皮膚、肺、消化管(例、大腸、小腸)、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子宮、骨、関節、脂肪組織(例、褐色脂肪組織、白色脂肪組織)、骨格筋など]より調製したゲノムDNA画分を鋳型として用い、Polymerase Chain Reaction(以下、「PCR法」と略称する)によって直接増幅することができ、AIMまたはその部分ペプチドをコードするcDNAであれば、マクロファージより調製した全RNAもしくはmRNA画分をそれぞれ鋳型として用い、PCR法およびReverse Transcriptase-PCR(以下、「RT-PCR法」と略称する)によって直接増幅することもできる。あるいは、AIMまたはその部分ペプチドをコードするゲノムDNAおよびcDNAは、上記したゲノムDNAおよび全RNAもしくはmRNAの断片を適当なベクター中に挿入して調製されるゲノムDNAライブラリーおよびcDNAライブラリーから、コロニーもしくはプラークハイブリダイゼーション法またはPCR法などにより、それぞれクローニングすることもできる。ライブラリーに使用するベクターは、バクテリオファージ、プラスミド、コスミド、ファージミドなどいずれであってもよい。
配列番号:1または配列番号:3で表される塩基配列と実質的に同一な塩基配列を含むDNAとしては、例えば、配列番号:1で表される塩基配列と約60%以上、好ましくは約70%以上、さらに好ましくは約80%以上、特に好ましくは約90%以上の相同性を有する塩基配列を含有し、前記したAIMと実質的に同質の活性を有する蛋白質をコードするDNAなどが用いられる。
本明細書における塩基配列の相同性は、相同性計算アルゴリズムNCBI BLAST(National Center for Biotechnology Information Basic Local Alignment Search Tool)を用い、以下の条件(期待値=10;ギャップを許す;フィルタリング=ON;マッチスコア=1;ミスマッチスコア=-3)にて計算することができる。塩基配列の相同性を決定するための他のアルゴリズムとしては、上記したアミノ酸配列の相同性計算アルゴリズムが同様に好ましく例示される。
ハイストリンジェントな条件としては、例えば、6×SSC(sodium chloride/sodium citrate)中45℃でのハイブリダイゼーション反応の後、0.2×SSC/0.1%SDS中65℃での一回以上の洗浄などが挙げられる。当業者は、ハイブリダイゼーション溶液の塩濃度、ハイブリダゼーション反応の温度、プローブ濃度、プローブの長さ、ミスマッチの数、ハイブリダイゼーション反応の時間、洗浄液の塩濃度、洗浄の温度等を適宜変更することにより、所望のストリンジェンシーに容易に調節することができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、該ライブラリーに添付された使用説明書に記載の方法に従って行なうことができる。
発現ベクターとしては、大腸菌由来のプラスミド(例、pBR322,pBR325,pUC12,pUC13);動物細胞発現プラスミド(例:pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo);レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどが用いられる。
プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。
例えば、宿主が動物細胞である場合、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV(サイトメガロウイルス)プロモーター、RSV(ラウス肉腫ウイルス)プロモーター、MoMuLV(モロニーマウス白血病ウイルス)LTR、HSV-TK(単純ヘルペスウイルスチミジンキナーゼ)プロモーターなどが用いられる。なかでも、CMVプロモーター、SRαプロモーターなどが好ましい。
宿主がエシェリヒア属菌である場合、trpプロモーター、lacプロモーター、recAプロモーター、λPLプロモーター、lppプロモーター、T7プロモーターなどが好ましい。
また、必要に応じて、宿主に合ったシグナル配列をコードする塩基配列(シグナルコドン)を、AIMまたはその部分ペプチドをコードするDNAの5’末端側に付加(またはネイティブなシグナルコドンと置換)してもよい。例えば、宿主がエシェリヒア属菌である場合、PhoA・シグナル配列、OmpA・シグナル配列などが;宿主が動物細胞である場合、インスリン・シグナル配列、α-インターフェロン・シグナル配列、抗体分子・シグナル配列などがそれぞれ用いられる。
宿主としては、例えば、エシェリヒア属菌、動物細胞などが用いられる。
エシェリヒア属菌としては、例えば、エシェリヒア・コリ(Escherichia coli)K12・DH1〔プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンシイズ・オブ・ザ・ユーエスエー(Proc. Natl. Acad. Sci. USA), 60巻, 160(1968)〕,エシェリヒア・コリJM103〔ヌクレイック・アシッズ・リサーチ(Nucleic Acids Research),9巻,309(1981)〕,エシェリヒア・コリJA221〔ジャーナル・オブ・モレキュラー・バイオロジー(Journal of Molecular Biology),120巻,517(1978)〕,エシェリヒア・コリHB101〔ジャーナル・オブ・モレキュラー・バイオロジー, 41巻, 459(1969)〕,エシェリヒア・コリC600〔ジェネティックス(Genetics), 39巻, 440(1954)〕などが用いられる。
エシェリヒア属菌は、例えば、プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンジイズ・オブ・ザ・ユーエスエー(Proc. Natl. Acad. Sci. USA), 69巻, 2110(1972)やジーン(Gene), 17巻, 107(1982)などに記載の方法に従って形質転換することができる。
動物細胞は、例えば、細胞工学別冊8 新細胞工学実験プロトコール,263-267 (1995)(秀潤社発行)、ヴィロロジー(Virology),52巻,456(1973)に記載の方法に従って形質転換することができる。
宿主がエシェリヒア属菌である形質転換体を培養する場合の培地としては、例えば、グルコース、カザミノ酸を含むM9培地〔ミラー(Miller),ジャーナル・オブ・エクスペリメンツ・イン・モレキュラー・ジェネティックス(Journal of Experiments in Molecular Genetics),431-433,Cold Spring Harbor Laboratory,New York 1972〕が好ましい。必要により、プロモーターを効率よく働かせるために、例えば、3β-インドリルアクリル酸のような薬剤を培地に添加してもよい。
宿主がエシェリヒア属菌である形質転換体の培養は、通常約15~約43℃で、約3~約24時間行なわれる。必要により、通気や撹拌を行ってもよい。
宿主が動物細胞である形質転換体を培養する場合の培地としては、例えば、約5~約20%の胎児ウシ血清を含む最小必須培地(MEM)〔サイエンス(Science),122巻,501(1952)〕,ダルベッコ改変イーグル培地(DMEM)〔ヴィロロジー(Virology),8巻,396(1959)〕,RPMI1640培地〔ジャーナル・オブ・ザ・アメリカン・メディカル・アソシエーション(The Journal of the American Medical Association),199巻,519(1967)〕,199培地〔プロシージング・オブ・ザ・ソサイエティ・フォー・ザ・バイオロジカル・メディスン(Proceeding of the Society for the Biological Medicine),73巻,1(1950)〕などが用いられる。培地のpHは、好ましくは約6~約8である。培養は、通常約30℃~約40℃で、約15~約60時間行なわれる。必要に応じて通気や撹拌を行ってもよい。
以上のようにして、形質転換体の細胞内または細胞外にAIMを製造せしめることができる。
例えば、AIMまたはその部分ペプチドを培養菌体あるいは細胞の細胞質から抽出する場合、培養物から公知の方法で集めた菌体あるいは細胞を適当な緩衝液に懸濁し、超音波、リゾチームおよび/または凍結融解などによって菌体あるいは細胞を破壊した後、遠心分離やろ過により可溶性蛋白質の粗抽出液を得る方法などが適宜用いられる。該緩衝液は、尿素や塩酸グアニジンなどの蛋白質変性剤や、トリトンX-100TMなどの界面活性剤を含んでいてもよい。また、AIMまたはその部分ペプチドが菌体(細胞)外に分泌される場合には、培養物から遠心分離またはろ過等により培養上清を分取するなどの方法が用いられる。
このようにして得られた可溶性画分、培養上清中に含まれるAIMまたはその部分ペプチドの単離精製は、自体公知の方法に従って行うことができる。このような方法としては、塩析や溶媒沈澱法などの溶解度を利用する方法;透析法、限外ろ過法、ゲルろ過法、およびSDS-ポリアクリルアミドゲル電気泳動法などの主として分子量の差を利用する方法;イオン交換クロマトグラフィーなどの荷電の差を利用する方法;アフィニティークロマトグラフィーなどの特異的親和性を利用する方法;逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法;等電点電気泳動法などの等電点の差を利用する方法;などが用いられる。これらの方法は、適宜組み合わせることもできる。
本発明におけるネコIgMと結合するAIMは、配列番号:4で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含み、かつネコIgMと結合することができる蛋白質である。
そのような蛋白質は例えば、化学合成もしくは無細胞翻訳系で生化学的に合成された蛋白質であってもよいし、あるいは上記アミノ酸配列をコードする塩基配列を含む核酸を導入された形質転換体から産生される組換え蛋白質であってもよい。
具体的には、例えば、配列番号:4で表されるアミノ酸配列のうち、SRCR1ドメイン(配列番号:4で表されるアミノ酸配列のうち、アミノ酸番号24~125)、SRCR2ドメイン(配列番号:4で表されるアミノ酸配列のうち、アミノ酸番号139~239)、SRCR3ドメイン(配列番号:4で表されるアミノ酸配列のうち、アミノ酸番号244~346)をそれぞれ含む部分アミノ酸配列やSRCRドメインの任意の組合せを含む部分アミノ酸配列を有するものなども用いられる。上記の部分ペプチドは、上記の機能ドメインを含む限りそのサイズに特に制限はない。
配列番号:3で表される塩基配列と実質的に同一な塩基配列を含むDNAとしては、例えば、配列番号:3で表される塩基配列と約60%以上、好ましくは約70%以上、さらに好ましくは約80%以上、特に好ましくは約90%以上の相同性を有する塩基配列を含有し、前記したAIMと実質的に同質の活性を有する蛋白質をコードするDNAなどが用いられる。「相同性」については、上記と同様であってよい。該DNAは、上記した方法と同様に調製される。
また、哺乳動物以外にもニワトリなどの鳥類を本発明で対象とする「非ヒト哺乳動物」と同様の目的に用いることができる。
ES細胞は胚盤胞期の受精卵の内部細胞塊(ICM)に由来し、インビトロで未分化状態を保ったまま培養維持できる細胞をいう。ICMの細胞は将来、胚本体を形成する細胞であり、生殖細胞を含むすべての組織の基になる幹細胞である。ES細胞としては、既に樹立された細胞株を用いてもよく、また、EvansとKaufmanの方法(ネイチャー(Nature)第292巻、154頁、1981年)に準じて新しく樹立したものでもよい。例えば、マウスES細胞の場合、現在、一般的には129系マウス由来のES細胞が使用されているが、免疫学的背景がはっきりしていないので、これに代わる純系で免疫学的に遺伝的背景が明らかなES細胞を取得するなどの目的で、例えば、C57BL/6マウスやC57BL/6の採卵数の少なさをDBA/2との交雑により改善したBDF1マウス(C57BL/6とDBA/2とのF1)から樹立されるES細胞なども良好に用いることができる。BDF1マウスは、採卵数が多く、かつ卵が丈夫であるという利点に加えて、C57BL/6マウスを背景に持つので、これ由来のES細胞は疾患モデルマウスを作製したとき、C57BL/6マウスと戻し交雑することでその遺伝的背景をC57BL/6マウスに代えることが可能である点で有利に用い得る。ES細胞は、適当な条件により、高密度に至るまで単層培養するか、または細胞集塊を形成するまで浮遊培養することにより、頭頂筋、内臓筋、心筋などの種々のタイプの細胞に分化させることが可能であり〔M. J. Evans及びM. H. Kaufman,ネイチャー(Nature)第292巻、154頁、1981年;G. R. Martin, プロシーディングズ・オブ・ナショナル・アカデミー・オブ・サイエンシーズ・ユーエスエー(Proc. Natl. Acad. Sci. U.S.A.)第78巻、7634頁、1981年;T. C. Doetschmanら,ジャーナル・オブ・エンブリオロジー・アンド・エクスペリメンタル・モルフォロジー、第87巻、27頁、1985年〕、本発明のターゲッティングベクターを導入されたES細胞を分化させて得られるAIM発現不全非ヒト哺乳動物細胞は、インビトロにおけるAIMの細胞生物学的検討において有用である。
AIMをコードするポリヌクレオチドの塩基配列に、相補的もしくは実質的に相補的な塩基配列またはその一部を有するアンチセンスDNAとしては、AIMをコードするポリヌクレオチドの塩基配列に相補的もしくは実質的に相補的な塩基配列またはその一部を含有し、該ポリヌクレオチドの発現を抑制し得る作用を有するものであれば、いずれのアンチセンスDNAであってもよい。
特に、AIMをコードするポリヌクレオチドの相補鎖の全塩基配列のうち、(a)翻訳阻害を指向したアンチセンスDNAの場合は、AIMのN末端部位をコードする部分の塩基配列(例えば、開始コドン付近の塩基配列など)の相補鎖と約70%以上、好ましくは約80%以上、より好ましくは約90%以上、最も好ましくは約95%以上の相同性を有するアンチセンスDNAが、(b)RNaseHによるRNA分解を指向するアンチセンスDNAの場合は、イントロンを含むAIMをコードするポリヌクレオチドの全塩基配列の相補鎖と約70%以上、好ましくは約80%以上、より好ましくは約90%以上、最も好ましくは約95%以上の相同性を有するアンチセンスDNAがそれぞれ好適である。
さらに、本発明のアンチセンスDNAは、AIMのmRNAもしくは初期転写産物とハイブリダイズして蛋白質への翻訳を阻害するだけでなく、二本鎖DNAであるAIMと結合して三重鎖(トリプレックス)を形成し、RNAの転写を阻害し得るものであってもよい。あるいはDNA:RNAハイブリッドを形成してRNaseHによる分解を誘導するものであってもよい。
通常、F0動物は相同染色体の一方にのみ導入DNAを有するヘテロ接合体として得られる。また、個々のF0個体は相同組換えによらない限り異なる染色体上にランダムに挿入される。相同染色体の両方に発現ベクターを有するホモ接合体を得るためには、F0動物と非トランスジェニック動物とを交雑してF1動物を作製し、相同染色体の一方にのみ導入DNAを有するヘテロ接合体の兄妹同士を交雑すればよい。1遺伝子座にのみ導入DNAが組み込まれていれば、得られるF2動物の1/4がホモ接合体となる。
(1)対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓において壊死した尿細管細胞が蓄積し、腎実質が線維化する、
(2)対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓において糸球体構造が崩壊し、糸球体が線維化する、
(3) 対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓において炎症性サイトカインの発現が亢進する、
(4) 対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓においてマクロファージの浸潤が亢進する、
(5)対照非ヒト哺乳動物と比較して、AIM発現不全非ヒト哺乳動物の血中BUN値が高い、
(6)対照非ヒト哺乳動物と比較して、AIM発現不全非ヒト哺乳動物の生存率が低い、
(7)AIM投与によって、前記(1)~(6)が改善する、
を有する。これらの表現型は、従来公知のAIM KOマウスにおいては、少なくとも報告されていない。特に、尿管結石、上行性尿路感染症、あるいは腫瘍等による尿管の圧迫・閉塞を引金とした急性腎障害(急性腎不全)に伴う慢性的な腎障害、および、腫瘤、血栓、あるいは糖尿病、高血圧等による腎血管狭窄・閉塞による虚血性腎障害に伴う慢性的な腎障害の病態と近似したものであることは新たな発見である。
(2)対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓において糸球体構造が崩壊し、糸球体が線維化するとは、本発明のAIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって、対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓において糸球体構造の崩壊と糸球体の線維化が認められることをいう。糸球体構造の崩壊は、例えば、腎組織片をヘマトキシリン・エオジン染色によって確認することができ、糸球体の線維化はAzan染色およびヘマトキシリン染色の同時染色によって確認することができる。後述する実施例においては、AIMノックアウトマウスでは、尿管結紮後14日目から正常腎臓と比べて有意な差が認められた。また、AIMノックアウトマウスでは、一過性腎虚血再灌流後7日目から正常腎臓と比べて有意な差が認められた。
(3) 対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓において炎症性サイトカインの発現が亢進するとは、本発明のAIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって、対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓においてMCP-1、IL-1βおよびIL-6の発現が亢進することをいう。発現の亢進は、例えば、定量的RT-PCRやノーザンブロット法などによって確認することができる。後述する実施例においては、AIMノックアウトマウスでは、MCP-1およびIL-6は正常腎臓と比べて有意な差が認められ、IL-1βについても、発現が高い傾向が認められた。
(4) 対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓においてマクロファージの浸潤が亢進するとは、本発明のAIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって、対照腎臓と比較して、尿管結紮または一過性腎虚血再灌流を行った腎臓においてマクロファージ(Mac-1陽性細胞)の数が多いことをいう。細胞数のカウントは、例えば、フローサイトメーターなどによってMac-1陽性細胞を識別することによって確認することができる。後述する実施例においては、AIMノックアウトマウスでは、正常腎臓と比べてマクロファージの割合が高いことが確認された。
(5)対照非ヒト哺乳動物と比較して、AIM発現不全非ヒト哺乳動の血中BUN値が高値であるとは、本発明のAIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって、対照非ヒト哺乳動物と比較して、AIM発現不全非ヒト哺乳動の血中BUN値が高いことをいう。
(6)対照非ヒト哺乳動物と比較して、AIM発現不全非ヒト哺乳動物の生存率が低いとは、本発明のAIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって、対照非ヒト哺乳動物と比較して、生存率が低くなることをいう。
(7)AIM投与によって、前記(1)~(6)が改善するとは、本発明のAIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行った後、AIM投与によってBUN値に有意な低下が認められることと、壊死した尿細管細胞の蓄積と糸球体構造の崩壊、またそれらに伴う腎実質および糸球体の線維化が、明瞭に改善すること、炎症性サイトカインの発現が低下すること、生存率が改善すること、マクロファージの浸潤を抑制することをいう。
(1)片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行う条件下、AIM発現不全非ヒト哺乳動物に被検物質を投与する工程、
(2)被検物質を投与されたAIM発現不全非ヒト哺乳動物の下記特性のいずれか一項目以上を観察する工程:
(i)壊死した尿細管細胞の蓄積と腎実質の線維化、
(ii)糸球体構造の崩壊と線維化、
(iii)腎臓における炎症性サイトカインの発現量、
(iv)腎臓におけるマクロファージの割合、
(v)BUN値、
(vi)生存率、
(3)被検物質非投与の場合と比較して、前記特性のいずれか一項目以上が改善される被検物質を選択する工程。
(1)片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行う条件下、AIM発現不全非ヒト哺乳動物に腎疾患予防・治療剤を投与する工程、
(2)腎疾患予防・治療剤を投与されたAIM発現不全非ヒト哺乳動物の下記特性のいずれか一項目以上を観察する工程:
(i)壊死した尿細管細胞の蓄積と腎実質の線維化、
(ii)糸球体構造の崩壊と線維化、
(iii)腎臓における炎症性サイトカインの発現量、
(iv)腎臓におけるマクロファージの割合、
(v)BUN値、
(vi)生存率、
(3)前記特性のいずれか一項目以上を腎疾患予防・治療剤非投与の場合と比較して、腎疾患予防・治療剤の効果を評価する工程。
PCRにおいてプライマーとして用いられるオリゴヌクレオチドのセットとしては、AIM遺伝子転写産物のセンス鎖(コード鎖)およびアンチセンス鎖(非コード鎖)とそれぞれ特異的にハイブリダイズすることができ、それらに挟まれるDNA断片を増幅し得るものであれば特に制限はなく、例えば、各々約15~約100塩基、好ましくは各々約15~約50塩基の長さを有し、約100bp~1kbpのDNA断片を増幅するようにデザインされたオリゴDNAのセットが挙げられる。より具体的には、プライマーとして用いられるオリゴヌクレオチドのセットとしては、配列番号:1に示される塩基配列を含む核酸(センス鎖)とハイストリンジェントな条件下でハイブリダイズし得る核酸、及び前記の塩基配列に相補的な塩基配列を含む核酸(アンチセンス鎖)とハイストリンジェントな条件下でハイブリダイズし得る核酸が挙げられる。ここでハイストリンジェントな条件とは前記と同義である。より好ましくは、配列番号:1に示される塩基配列と相補的な塩基配列を含む核酸、及び該核酸の塩基配列に相補的な塩基配列を含む核酸が挙げられる。
尚、AIMに対する抗体は、配列番号:2または配列番号:4に示されるアミノ酸配列と、同一もしくは実質的に同一のアミノ酸配列もしくは部分アミノ酸配列を含む蛋白質を感作抗原として、通常使用されるポリクローナル抗体またはモノクローナル抗体作製技術に従って取得することができる。
(1)健常者および被験者の試料中のAIM濃度を測定する工程、
(2)健常者で測定されたAIM濃度と被験者で測定されたAIM濃度を比較する工程。
(1)健常者および被験者の試料中のAIM濃度を測定する工程、
(2)健常者で測定されたAIM濃度と被験者で測定されたAIM濃度を比較する工程。
(a)AIM遺伝子の転写産物とハイブリダイズし得る核酸プローブまたは核酸プライマー、および/または
(b)AIMに対する抗体
を含有してなる。該キットが2以上の上記核酸および/または抗体を含む場合、各核酸または抗体は互いにAIM遺伝子の塩基配列上の異なる部分を特異的に認識、またはAIM遺伝子の翻訳産物の異なるエピトープを特異的に認識し得るものである。
あるいは、該核酸は、適当な固相に固定化された状態で提供することもできる。固相としては、例えば、ガラス、シリコン、プラスチック、ニトロセルロース、ナイロン、ポリビニリデンジフロリド等が挙げられるが、これらに限定されない。また、固定化手段としては、予め核酸にアミノ基、アルデヒド基、SH基、ビオチンなどの官能基を導入しておき、一方、固相上にも該核酸と反応し得る官能基(例:アルデヒド基、アミノ基、SH基、ストレプトアビジンなど)を導入し、両官能基間の共有結合で固相と核酸を架橋したり、ポリアニオン性の核酸に対して、固相をポリカチオンコーティングして静電結合を利用して核酸を固定化するなどの方法が挙げられるが、これらに限定されない。
〔配列番号:1〕
ヒトAIMの塩基配列を示す。
〔配列番号:2〕
ヒトAIMのアミノ酸配列を示す。
〔配列番号:3〕
ネコAIMの塩基配列を示す。
〔配列番号:4〕
ネコAIMのアミノ酸配列を示す。
〔配列番号:5〕
ネコAIMの転写産物の相補的配列を示す。
〔配列番号:6〕
マウスAIMのアミノ酸配列を示す。
動物を用いた、多用される腎疾患モデルの一つに片側尿管結紮術(UUO)モデルがある。これは、片側の尿管を結紮することにより、結紮を施した腎実質の尿細管および糸球体が、徐々に壊死し、それに引き続き炎症や線維化が生じ、最終的にはその腎臓は機能不全となる。AIM欠損マウス(AIM-KO)と野生型マウス(WT)にそれぞれUUOを施術し、経過を観察した(n=6 for each)(図1A)。正常腎の構造はWT、AIM-KOとも全く差異はなかった。しかし、14日目のUUO腎において、線維を染色するAzan染色と、ヘマトキシリン染色を同時に行うと、WTでは線維化の広がりが見られるが、相当数の糸球体および尿細管は未だ正常構造を保ち、壊死に陥っていない尿細管も多くみられた。それに対してAIM-KOでは、壊死した尿細管細胞が広範囲に蓄積し、糸球体構造も破壊されており、腎実質の構造がもはや崩壊していた。観察した全てのマウスで同様の結果が得られた。
また同様に、AIM-KOとWTマウスにそれぞれUUOを施術し、14日目でHE、PAS、Azan染色によって腎臓を観察した(n=6 for each)(図1B)。WTでは線維化の広がりが見られるが、相当数の糸球体および尿細管は未だ正常構造を保っていた(HE、Azan染色)。一方AIM-KOでは、線維化が進行し、糸球体構造は破壊されており、腎実質の構造がもはや崩壊していた。PAS染色では、AIM-KOマウスで尿細管内外に壊死した細胞塊(PAS陽性)が広範囲に蓄積していた。観察した全てのマウスで同様の結果が得られた。
別の腎疾患モデルに一過性腎虚血再灌流(IR)モデルがある。一過性腎虚血再灌流では、あらかじめ一方の腎臓を摘出しておき、残った腎臓側の腎動脈を結紮し虚血にする。30分後結紮を解除し血流の再灌流を行うが、この一過性の虚血のために尿細管の壊死が軽度の線維化を伴い3日間程度進行し、それに伴い腎機能が悪化する。AIM欠損マウス(AIM-KO)と野生型マウス(WT)にそれぞれIRを施術し、経過を観察した(図2)。WTでは、腎機能の悪化後、壊死した細胞が除去され、壊死しなかった尿細管が急速に分裂し、14日後には、ほぼ正常の尿細管構造を回復した。それと並行して、腎機能も正常化した。ところが、AIM-KOでは、初期の尿細管のダメージの程度はWTと差異は見られないが、壊死した細胞の除去が進まず、壊死細胞が蓄積した。壊死細胞の除去が進行しないため、新しい尿細管細胞の分裂は抑制され、二次的な炎症や線維化が進行した。N=6で実験を行ったが、全てのマウスで同様の結果が得られた。すなわち、実施例1と実施例2の結果から、AIMがないと壊死した尿細管細胞が蓄積し、腎臓の構造・機能修復が著しく損なわれることが明らかとなった。
実施例2で行った一過性腎虚血再灌流(IR)を野生型マウス(WT)およびAIM欠損マウス(AIM-KO)に施し、術後の炎症性マクロファージの浸潤をマクロファージマーカーであるF4/80の免疫染色によって観察した(図3A)。術後3日目ではWTに比べAIM KOマウスでは、F4/80陽性のマクロファージの浸潤が明らかに亢進した。また、術後14日目でWTでは、マクロファージの浸潤は軽減したが、AIM KOマウスでは更に増悪した。このマクロファージ浸潤の経過に伴い、炎症性サイトカインの一つであるMCP-1の発現も同様にAIM KOマウス腎臓で有意に増加していることが、quantitative RT-PCRの実験により明らかになった(図3B)。N=6で実験を行ったが、全てのマウスで同様の結果が得られた。
AIM-KOマウスに実施例2で行ったIRを施し、腎機能が一過性に最も悪化する3日目、および4日目、7日目にそれぞれ組換えAIM(rAIM)またはPBSを100μg腹腔内投与し(n=6 for each)、経過を観察した(図4)。PBSを投与した群では、実施例2の結果と同様に、その後壊死細胞の蓄積や糸球体の破壊など進行し、腎機能(BUN値)も、3日目よりはある程度改善するものの正常値に復旧することはなく、後半徐々に悪化の傾向を見せた。しかし、rAIMを投与した群では、3日目のrAIM投与後にBUN値は有意に改善し、7日目には既に正常範囲に戻り、その後もさらに低下した。組織学的にも、7日目には壊死した尿細管細胞は除去されており、腎実質の構造も正常化した。すなわち、AIM投与は壊死細胞の除去を亢進させ、尿細管の再生を促し、腎機能を回復できることが明らかとなった。
AIM-KOマウスに実施例2で行ったIRを施し、術後rAIM (100μg)を静脈投与し、3、6、12時間後に腎臓の切片を抗AIM抗体で染色した。AIM投与後3時間で、尿細管の壊死部分にAIMが強く付着していることが確認された(図5上段:phase-contrastによる写真中、壊死巣をNで指している。図5下段:AIMシグナル(矢頭)は壊死巣と重なって観察された)。また、時間経過と共に、壊死巣部分は縮小し、12時間後にはほぼ痕跡程度しか確認されなかった。すなわち、AIMによるIR後の腎機能回復(実施例4)は、壊死巣にAIMが付着することによって、その除去を促し、それに伴い炎症の抑制ひいては組織再生が進行していたと考えられる。
一方の腎臓を摘出せず、両方の腎臓の腎動脈を結紮し虚血にする、両側性の一過性腎虚血再灌流(IR)を施した野生型マウス(WT)の尿をIR後1日目、7日目にELISA法によって検出した(図6)。通常状態のマウスの尿にはAIMはほとんど検出されなかった(IR前)が、腎障害が最も激しいIR後一日目では尿中に多量のAIMが検出された。腎障害の回復と共に尿中AIMは減少した(IR後7日目)。なお、IRによる腎障害時は排出される尿は希釈尿となるため、尿中AIM値は尿中クレアチニン値でnormalizationした。
WTおよびAIM-KOマウスに両側性のIRを施し、生存率をみた(n=8 each)(図7)。IR後7日目で、WTは80%以上生存している条件でAIM-KOは30%以下の生存率であり、死亡したマウスのほとんどはIR後3日目までに死亡していた。
WTおよびAIM-KOマウスに両側性のIRを施し、経時的にクリニカルスコアを解析した(図8)。クリニカルスコアは、動きの機敏さの欠如、目の混濁、尾における痛み刺激に対する反応性の低下、毛並の悪さ、についてそれぞれ加点(0:異常なし、1:軽度の症状有、2:中等度の症状有、3:重度の症状有)し、その合計をグラフ化した。WTではIR後一日目にスコアが最大値を示し徐々に軽減したが、AIM-KO ではスコアは高値のままであった。
WTおよびAIM-KOマウスに両側性のIRを施し、腎機能のマーカーであるBUNを継時的に計測した(n=8)(図9)。実施例8のクリニカルスコアと同様に、BUNはWTでは一日目がピークでその後低下するが、AIM-KOでは2日目まで上昇しその後も著明な低下は認められなかった。
WTおよびAIM-KOマウスに両側性のIRを施し、腎組織を継時的にPAS染色で解析した(図10)。実施例8のクリニカルスコアや実施例9のBUN値と同様に、腎機能障害は、WTでは一日目がピークでその後回復し、7日目には正常な尿細管構造とbrush border(冊子縁;矢頭)を持った近位尿細管上皮が再生した。しかしAIM-KOでは、障害は継続し、近位尿細管中にPAS陽性の死細胞塊が蓄積しており7日目に至っても除去されなかった。
図10で認められる近位尿細管中の上皮細胞の死細胞塊の面積を、1切片辺りの全面積に対する割合として算出することによって定量化した(n=3~5)(図11)。実施例8~10から得られる知見と同様の推移を示しており、AIM-KOでは死細胞塊の蓄積・残留が明らかであった。
WTおよびAIM-KOマウスに両側性のIRを施し、IR前およびIR後7日目の腎臓からRNAを抽出し、炎症性サイトカインであるIL-1βおよびIL-6について定量的RT-PCRで解析を行った(n=3 each)(図12)。両方のマーカーについてAIM-KOではWTに比べて高値であり、IRによる腎組織破壊に伴う炎症の遷延が示唆された。
WTおよびAIM-KOマウスに両側性のIRを施し、IR後7日目の腎臓をコラゲナーゼ処理したのちフローサイトメーターで解析することによって、マクロファージ(Mac-1陽性細胞)の割合を調べた(図13)。実施例12の結果と同様に、AIM-KOでは腎臓内のマクロファージの割合がWTに比べ高かった。それぞれ3匹のマウスについて解析を行い、同様の結果を得た。図13は、representative resultを提示している。
両側性のIRを施したWTマウスの腎臓連続切片について、PAS染色(左)と抗AIM抗体による免疫染色(右)を行った(図14)。多くの死細胞塊にAIM(白)が集積していることが認められた。
腎梗塞による急性腎不全で死亡したヒト患者の腎臓連続切片について、PAS染色(左)と抗AIM抗体による免疫染色(右)を行った(図15)。実施例14のIRマウス同様、多くの死細胞塊にAIM(白)が集積していることが認められる。
急性腎不全(AKI)で病院に搬送された患者および健常人を3名ずつ、また両側性のIRを施したWTマウス5匹について、IR前、IR1日後、および7日後の尿について、AIM濃度をELISAによって解析した(図16)。健常人の尿にはAIMはほとんど認められないが、AKI患者の尿には認められた。マウスにおいても、IR前には認められないが、腎障害が著しいIR1日後では有意な量のAIMが尿中に認められ、腎障害が改善した7日目ではAIMの量が減少していた。
AIM-KOマウスに両側性のIRを施し、IR後3日目に200μgのrAIMを投与して経時的に取得した腎臓切片について、PAS染色および抗AIM抗体による免疫染色を行った(図17)。AIMが集積した死細胞塊は速やかに縮小した。
AIM-KOマウスに両側性のIRを施し、IR後3日目の腎臓をコラゲナーゼ処理し、F4/80陽性のマクロファージをFACSソーターを用いて単離した後、その貪食能を解析した(図18)。貪食の標的として、ヒト尿細管細胞株であるHK2細胞を熱処理で壊死させFITCでラベルした後、リコンビナントAIM(rAIM)またはbovine serum albumin (BSA)でコートしたHK2死細胞を用いた。Noneとしては、rAIMでもBSAでもコートしていないHK2死細胞を用いた。マクロファージと上記3種類のHK2死細胞をincubateし、マクロファージ内に取り込まれたFITC陽性のHK2死細胞をフローサイトメーター(FACS)で経時的に解析した。BSAでコートしたHK2死細胞、あるいはコートしていないHK2死細胞に対してrAIMでコートしたHK2死細胞はより高効率にマクロファージに貪食されることが示された。
実施例18と同様の実験を、貪食細胞としてAIM-KO由来の骨髄細胞をM-CSFによって分化させたマクロファージを用いて行った(図19)。実施例18では、BSAでコートしたHK2死細胞とコートしていないHK2死細胞では貪食のされ方に差がなかったので、本実施例ではコートしていないHK2死細胞は使用しなかった。実施例18の結果と同様に、rAIMでコートしたHK2死細胞の貪食が上昇した。
AIM-KOマウスに両側性のIRを施した後、1日目から3日目までrAIM 200μg/マウスまたは等容量のPBSを静脈注射した(n=5-6)。PBSを打ったマウスの生存率は7日目で40%以下であったが、rAIMを投与したマウスでは100%に回復した(図20)。
実施例20のマウスについて、実施例8と同様にクリニカルスコアを検討した(図21)。rAIM投与後からクリニカルスコアの著しい回復を認めた。
実施例20のマウスについて、実施例9と同様に経時的にBUNを測定した(図22)。rAIM投与によりBUN値の有意な低下を認めた。
AIM-KOマウスに両側性のIRを施した後、1日目から3日目までrAIM 200 μg/マウスまたは等容量のPBSを静脈注射し、3日目と7日目の腎臓の状態をPAS染色で解析した(図23)。PBS投与では近位尿細管内死細胞が蓄積しているが、rAIMを投与したマウスでは著しい除去が認められ、7日目にはbrush border (冊子縁)を持つ尿細管細胞が回復した。
IR後7日目の腎臓で、図23で認められるような近位尿細管中の死細胞塊の面積を、1切片辺りの全面積に対する割合として算出することによって定量化した(n=3 each)(図24)。rAIM投与群では死細胞塊の著しい減少が認められた。
実施例20のマウスについて、IR後7日目の腎臓からRNAを抽出し、炎症性サイトカインであるIL-1βおよびIL-6について定量的RT-PCRで解析を行った(図25)。rAIM投与群ではIL-1β、IL-6ともPBS投与群に比較して低下しており、急性腎不全に伴う炎症反応もrAIM投与によって抑制されたことが示唆された。
実施例6~25で実施した両側性のIRでは、虚血を30分間実施し、AIM-KOに対し致死率の高い程度の急性腎不全を誘導した。本実施例では、虚血時間を短くし(30分→25分)、AIM-KOでも7日目での生存率が100%ある程度の腎不全をAIM-KOマウスに誘導し、実施例20と同様に1日目から3日目までrAIMまたはPBSを頸静脈に注射投与した。このようなマイルドな条件のIRにおいても、rAIM投与によってBUNの改善をより加速させることが出来た(n=5 each)(図26)。
もともと内因性のAIMを持っているWTマウスに対し両側性のIR(虚血30分間)を施し、実施例20と同様に、rAIMまたはPBSを投与した(n=5~6)。WTではもともと内因性のAIMによって腎障害は回復するが、rAIMによってその改善をさらに加速できた(図27)。rAIM投与によって、2日目まで上昇していたBUN値が既に低下していることが分かった。
実施例26と同様のマイルドなIRをWTおよびAIM-KO マウスに施し、IR後28日目の腎臓の状態をPAS染色(死細胞塊をみるため)とAzan染色(線維化をみるため)によって解析した(図28)。28日目でもAIM-KOではPAS陽性の死細胞塊がWTに比べて多少残っていた。またAIM-KOでは著明な線維化(Azan陽性)が認められ、尿細管の構造もWTに比べていびつであった。
実施例28のマウスについて、IR後28日後の腎臓からRNAを抽出し、定量的RT-PCRで4種類の線維化マーカーについて解析した(n=4 each)(図29)。AIM-KOではWTに比べ、線維化マーカーが全て上昇した。
年齢がほぼ等しい、(1)健常者(男142、女:54)、(2)腎障害のない(Cre<1.0 mg/dl)糖尿病患者(男:70、女:57)、(3)糖尿病性の慢性腎不全患者(男:146、女:54)、について男女分けて血中AIM濃度を計測した(図30)。男女とも、(1)と(2)ではAIM値に有意差はなかったが、(3)では有意に低かった。
慢性腎臓疾患(CKD)患者の血中AIM値を解析し、個々の腎機能のマーカーであるeGFR(糸球体濾過量)および血中クレアチニン値との相関を調べた(n=55)。CKDの原因疾患としては、糖尿病性腎症、糸球体腎炎、高血圧性腎症、IgA腎症などが挙げられる。また男女混合で患者年齢は60歳以下である。図31Aのように、血中AIM値と腎機能は有意な相関関係を示した。さらに、この解析の時点で比較的AIM値が高い人は、2-3年後の追跡調査で腎機能が改善しており、逆にAIMが低値だった患者は腎機能が悪化していた(図31B)。すなわち、AIMはCKD患者において、現在の腎機能のみならず予後を予測する有用なマーカーとなり得る。
イヌ(3匹)、ネコ(3匹)およびマウスのそれぞれの血清を抗AIM抗体(Rab2:マウスrAIMをウサギに免疫して作製したポリクローナル抗体。これまでマウスおよびヒトAIMを検出することが分かっている)を用い、還元条件で免疫ブロットを行った(図32A)。その結果、イヌ血清にはシグナルが確認できたが、ネコでは3匹ともほとんどシグナルが検出できなかった。これは抗体の問題ではなく、ネコAIM cDNAをクローニングし(実施例36参照)、pCAGGS発現ベクターにC末端にHAタグを付けた形で組み込み、HEK293T細胞に発現させた培養上清から抗HA抗体カラムを用いて精製したネコrAIM蛋白質は、マウスrAIMと同程度に本抗体を用いた還元条件での免疫ブロットで検出することが出来た(図32B)。これらの結果から、本実施例で検討を行ったネコは機能的なAIM mRNAを発現しているが、血中にAIM蛋白質としてほとんど存在していないことが分かった。
図35に示すネコAIM cDNAを発現ベクターに挿入したプラスミドを、HEK293T細胞にトランスフェクションし、その培養上清から精製した組換えネコAIM(1 mg)をネコ(雑種、オス2歳3か月齢)に静注し1時間後に採血し、血清分離した。血清を、ゲルによるサイズ分画を行い、各分画についてAIMとIgMについてウエスタンブロット法により解析した。図33Aに示すように、AIMの存在する分画とIgMの存在する分画は明らかに異なっている。この結果は、マウスAIMをAIM KOマウスに静注した血清を同様に分画解析した結果(図33B、比較例)(IgMとAIMの分画が完全に一致している)とは異なっている。すなわち、本来AIMは、血液中でIgMと結合し、その安定性が保たれる(先行文献:Arai et al., Cell Reports 3: 1187-1198, 2013)結果、AIMの血中濃度が維持される。しかし、ネコではAIMがIgMと結合できないため、血中におけるAIMの安定性が保てず、その結果、AIMの血中濃度が維持できないと考えられる。
マウスAIMを用いて、実施例33と同様な試験を実施した。組換えマウスAIM(1 mg)をネコ(雑種、オス2歳3か月齢)に静注し1時間後に採血し、血清分離した。血清を、ゲルによるサイズ分画を行い、各分画についてAIMとIgMについてウエスタンブロット法により解析した。図34に示すように、マウスAIMの存在する分画とネコIgMの存在する分画は完全に一致した。したがって、ネコAIMと異なり、マウスAIMはIgMと結合し、血中で安定すると考えられる。
C末端を欠損した組換え改変マウスAIMを複数作製した。これらの改変マウスAIMとIgMの結合をin vitroで確認した。その結果、SRCR3ドメインの一部を欠損した改変マウスAIMは、IgMとの結合が著しく低下した。よって、マウスAIMのSRCR3ドメインがIgMとの結合に重要であることがわかった。
NCBI Resourcesに公開されているネコCD5L(=AIM)の予想配列(GenBank Accession No.:XM_003999688.1)をもとにプライマーを複数設計し、ネコ脾臓のcDNAプールからネコAIM cDNAの全長(配列番号:5)を単離した(図35)。配列上、特にリーダーペプチドをコードする配列が、公開されている配列と大きく異なっていた。
ネコ、ヒト、マウス、イヌ、それぞれのAIM蛋白質のリーダーペプチド配列の疎水性を示す(図36~39)。いずれも十分な疎水性を持ち、分泌蛋白質としての条件を満たした。なお、ネコは我々が単離したcDNAから、イヌAIMに関しては、NCBI Resourcesに公開されている予想配列(GenBank Accession No.:XM_846487.2)からリーダーペプチドについて解析した。
リーダーペプチド(LS)、各SRCR、およびヒンジ部分について3者でアミノ酸配列の比較を示す(図40)。図中、3者で共通しているアミノ酸を“*”で、ヒトとネコのみで共通しているものは“.”で、ヒトとマウスでのみ共通しているものは“:”で示した。
実施例32においては、抗マウスAIM抗体を使用してネコAIMの検出を試みたが、本実施例においては、ネコAIMのcDNAにHAタグを連結したDNAを発現ベクターに組み込み、HEK293T細胞に遺伝子導入することによってC末端にHAペプチドを負荷したリコンビナント・ネコAIMタンパク質(rcAIM-HA)を産生した。それをマウスに免疫して、抗ネコAIMモノクローナル抗体を樹立した。その抗体を用いて、様々な系統のネコ(48個体)について血中AIM濃度を還元型のウエスタンブロッティング法により解析した(図41)。濃度のコントロールとしてrcAIM-HAを用いた。その結果、系統に関わらず、AIMが検出される個体と、AIMが検出されない個体がいることが分かった。AIMが検出される個体のうち、代表的な4個体の血中AIM濃度の平均値は16.8μg/mlであり、これはマウスやヒトの約5μg/mlよりも著しく高値であった。
血中AIM濃度が高いネコでIRによる急性腎不全誘導法を確立した。全身麻酔下で腹腔鏡下で両側腎動脈をクランプで1時間結紮したのち開放した。その後血液及び尿を経時的に採取し、腎機能を検討した。図42に血中BUN値とCre値の推移を示す。IR後12時間でBUN値、Cre値は共にピークとなったが、その後7日目まで改善しなかった。すなわちAIM-KOマウスと同じように急性腎不全からの回復が障害されている可能性が高いことが示唆された。
マウスではIR後尿中にAIMが検出され、それが尿細管中に詰まった死細胞塊に集積すると考えられる。IR後のネコの血清および尿を抗ネコAIM抗体を用いて還元型ウエスタンブロッティング法によって解析した結果、ネコではIR後尿中にAIMは検出されなかった(図43)。また血中AIMの量にも明らかな変化は起こらなかった。
ネコに両側性のIRを施し3日目の腎臓について、尿細管内壊死細胞塊にAIMが集積しているかについて免疫染色によって解析した(図44)。近位尿細管内に壊死細胞塊は検出された(白矢印部)が、同箇所にAIMの集積は認められなかった。実施例41において、IR後尿中にAIMは検出されなかったことを示したが、その結果、尿細管中の死細胞塊にAIMが達しなかったことが示唆された。
実施例40の記載の方法と同様に、ネコ(メス5歳)に両側性のIRを施し、24時間後に麻酔下で動脈カテーテルを鼠蹊部動脈より挿入し、腎動脈までカテーテル先端を進め、rAIM 50mgをPBS50mlに溶解した溶液を片側腎動脈にそれぞれ25ml(rAIM:25mg)ずつ注入した。別のネコ(同じくメス5歳)に同様にIRおよびカテーテル挿入を行い、PBSのみを片側腎動脈にそれぞれ25mlずつ注入した。IR前とrAIMまたはPBS注入後24時間(IR後48時間)での体表面積で標準化したGFR(Glomerular Filtration Rate;糸球体濾過量)を示す(図45)。PBSのみ注入したネコでは、GFRが低下し腎機能が低下していたが、rAIMを注入したネコではGFRの低下は見られず、腎機能の低下が起きなかった。
実施例43の記載の方法と同様に、両側性のIRを施し、rAIMまたはPBS注入後24時間(IR後48時間)で腎組織をPAS染色で解析した(図46)。PBSを注入したネコでは尿細管上皮細胞の壊死と脱落、尿細管構造の破壊および間質の増殖が認められたが、rAIM注入したネコでは尿細管上皮細胞は既に回復しており冊子縁も復活し、構造も復元していた。また間質もPBSを注入したネコに比べて薄く、増殖は認められなかった。組織学的にもrAIMによる腎不全の治癒は明らかである。
6~8歳のネコに、AIMまたはvehicleを連日投与する。投与2~4週後に腎機能(BUN値)を測定する。AIM投与群では、vehicle投与群で観察される腎機能の悪化が抑制される。従って、AIMがネコの腎機能悪化、腎不全の予防に有用であることがわかる。また、AIMの代わりに、AIMの機能をアゴニスティックに調節できる薬剤(AIM活性を有するAIMの部分ペプチドを含む)やAIMの発現を誘導する薬剤を使用しても同様の結果が得られる。
ネコAIMのSRCR3ドメインをマウスAIMのSRCR3ドメインに改変し、IgMと結合する改変ネコAIMを作製する。6~8歳のネコに、改変AIMまたはvehicleを連日投与する。投与2~4週後に腎機能(BUN値)を測定する。改変AIM投与群では、vehicle投与群で観察される腎機能の悪化が抑制される。従って、AIMがネコの腎機能悪化、腎不全の予防に有用であることがわかる。また、改変ネコAIMは血中のIgMと結合し、安定化することから、ネコAIMを投与するよりも低用量の改変ネコAIM投与で有効性が得られる。改変ネコAIMは、ネコIgMと結合すれば良く、限定されない。
本出願は、日本で出願された特願2014-022041(出願日:平成26年2月7日)を基礎としており、その内容はすべて本明細書に包含されるものとする。
Claims (48)
- AIMもしくはその部分ペプチド、またはそれらをコードする塩基配列を含む核酸を含有してなる、腎疾患の予防・治療剤。
- AIMの発現を誘導する薬剤またはAIMを安定化させる薬剤を含有してなる、腎疾患の予防・治療剤。
- 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である請求項1または2に記載の予防・治療剤。
- 腎疾患が急性腎不全または慢性腎不全である請求項1~3のいずれか1項に記載の予防・治療剤。
- 予防・治療の対象がヒトである請求項1~4のいずれか1項に記載の予防・治療剤。
- 予防・治療の対象がネコである請求項1~4のいずれか1項に記載の予防・治療剤。
- AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、請求項6に記載の予防・治療剤。
- ネコIgMと結合するAIMもしくはその部分ペプチドが、マウス由来AIMのSRCR3ドメインを含む、請求項7に記載の予防・治療剤。
- AIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって得られる動物を用いる、腎疾患の予防・治療剤のスクリーニング方法。
- 以下の工程を含むことを特徴とする、請求項9に記載のスクリーニング方法:
(1)片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行う条件下、AIM発現不全非ヒト哺乳動物に被検物質を投与する工程、
(2)被検物質を投与されたAIM発現不全非ヒト哺乳動物の下記特性のいずれか一項目以上を観察する工程:
(i)壊死した尿細管細胞の蓄積と腎実質の線維化、
(ii)糸球体構造の崩壊と線維化、
(iii)腎臓における炎症性サイトカインの発現量、
(iv)腎臓におけるマクロファージの割合、
(v)BUN値、
(vi)生存率、
(3)被検物質非投与の場合と比較して、前記特性のいずれか一項目以上が改善される被検物質を選択する工程。 - 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である請求項9または10に記載のスクリーニング方法。
- AIM発現不全非ヒト哺乳動物に片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行うことによって得られる動物を用いる、腎疾患予防・治療剤の予防・治療効果の評価方法。
- 以下の工程を含むことを特徴とする、請求項12に記載の評価方法:
(1)片側尿管結紮、一側性腎摘出後の一過性腎虚血再灌流または両側性の一過性腎虚血再灌流を行う条件下、AIM発現不全非ヒト哺乳動物に腎疾患予防・治療剤を投与する工程、
(2)腎疾患予防・治療剤を投与されたAIM発現不全非ヒト哺乳動物の下記特性のいずれか一項目以上を観察する工程:
(i)壊死した尿細管細胞の蓄積と腎実質の線維化、
(ii)糸球体構造の崩壊と線維化、
(iii)腎臓における炎症性サイトカインの発現量、
(iv)腎臓におけるマクロファージの割合、
(v)BUN値、
(vi)生存率、
(3)前記特性のいずれか一項目以上を腎疾患予防・治療剤非投与の場合と比較して、腎疾患予防・治療剤の効果を評価する工程。 - 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である請求項12または13に記載の評価方法。
- 被検者の試料中のAIM濃度を測定することを含む、腎疾患患者の予後の予測方法。
- 試料が血液または血清である、請求項15に記載の予測方法。
- AIM濃度の測定方法が抗AIM抗体を用いた免疫学的方法である、請求項15または16に記載の予測方法。
- 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である請求項15~17のいずれか1項に記載の予測方法。
- 被検者の試料中のAIM濃度を測定することを含む、急性腎不全の検査方法。
- 試料が尿である、請求項19に記載の検査方法。
- AIM濃度の測定方法が抗AIM抗体を用いた免疫学的方法である、請求項19または20に記載の検査方法。
- 以下の(a)または(b)を含む、腎疾患の診断または予後の予測キット:
(a)AIM遺伝子の転写産物とハイブリダイズし得る核酸プローブまたは核酸プライマー、および/または
(b)AIMに対する抗体。 - 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である請求項22に記載のキット。
- AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸を対象に有効量投与することを含む、腎疾患の予防・治療方法。
- AIMの発現を誘導する薬剤またはAIMを安定化させる薬剤を対象に有効量投与することを含む、腎疾患の予防・治療方法。
- 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、請求項24または25に記載の予防・治療方法。
- 腎疾患が急性腎不全または慢性腎不全である、請求項24~26のいずれか1項に記載の予防・治療方法。
- 予防・治療の対象がヒトである、請求項24~27のいずれか1項に記載の予防・治療方法。
- 予防・治療の対象がネコである、請求項24~27のいずれか1項に記載の予防・治療方法。
- AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、請求項29に記載の予防・治療方法。
- 腎疾患の予防・治療に用いるための、AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸。
- 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、請求項31に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸。
- 腎疾患が急性腎不全または慢性腎不全である、請求項31または32に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸。
- 予防・治療の対象がヒトである、請求項31~33のいずれか1項に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸。
- 予防・治療の対象がネコである、請求項31~33のいずれか1項に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸。
- AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、請求項35に記載のAIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸。
- 腎疾患の予防・治療に用いるための、AIMの発現を誘導する薬剤またはAIMを安定化させる薬剤。
- 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、請求項37に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤。
- 腎疾患が急性腎不全または慢性腎不全である、請求項37または38に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤。
- 予防・治療の対象がヒトである、請求項37~39のいずれか1項に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤。
- 予防・治療の対象がネコである、請求項37~39のいずれか1項に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤。
- AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、請求項41に記載のAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤。
- 腎疾患の予防・治療剤を製造するための、AIMもしくはその部分ペプチドまたはそれらをコードする塩基配列を含む核酸またはAIMの発現を誘導する薬剤またはAIMを安定化させる薬剤の使用。
- 腎疾患が急性腎不全、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、請求項43に記載の使用。
- 腎疾患が急性腎不全または慢性腎不全である、請求項43または44に記載の使用。
- 予防・治療の対象がヒトである、請求項43~45のいずれか1項に記載の使用。
- 予防・治療の対象がネコである、請求項43~45のいずれか1項に記載の使用。
- AIMまたはその部分ペプチドが、ネコIgMと結合することを特徴とする、請求項47に記載の使用。
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SG11201606514SA SG11201606514SA (en) | 2014-02-07 | 2015-02-06 | Preventive or therapeutic agent for kidney disease |
CA2938944A CA2938944C (en) | 2014-02-07 | 2015-02-06 | Apoptosis inhibitor of macrophage (aim) as preventive or therapeutic agent for kidney disease |
KR1020167024671A KR101870246B1 (ko) | 2014-02-07 | 2015-02-06 | 신질환의 예방 또는 치료제 |
CN201580015029.XA CN106102761A (zh) | 2014-02-07 | 2015-02-06 | 用于肾疾病的预防或治疗剂 |
US15/116,907 US10349640B2 (en) | 2014-02-07 | 2015-02-06 | Preventive or therapeutic agent for kidney disease |
IL247111A IL247111B1 (en) | 2014-02-07 | 2015-02-06 | causes the prevention of or treatment of kidney disease |
JP2015561061A JP6460483B2 (ja) | 2014-02-07 | 2015-02-06 | 腎疾患の予防または治療剤 |
EP15746352.2A EP3103466A4 (en) | 2014-02-07 | 2015-02-06 | Preventive or therapeutic agent for kidney disease |
US16/509,161 US11253571B2 (en) | 2014-02-07 | 2019-07-11 | Preventive or therapeutic agent for kidney disease |
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US16/509,161 Continuation US11253571B2 (en) | 2014-02-07 | 2019-07-11 | Preventive or therapeutic agent for kidney disease |
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WO2017022315A1 (ja) * | 2015-08-06 | 2017-02-09 | エーディア株式会社 | 腎疾患の検査方法 |
WO2019097898A1 (ja) | 2017-11-16 | 2019-05-23 | 宮崎 徹 | 変異型aim |
WO2020071318A1 (ja) | 2018-10-01 | 2020-04-09 | 宮崎 徹 | 神経変性疾患治療剤 |
JP2020158437A (ja) * | 2019-03-26 | 2020-10-01 | 宮崎 徹 | 血中フリー体aim増加用組成物 |
WO2023008555A1 (ja) * | 2021-07-30 | 2023-02-02 | 徹 宮崎 | 虚血性疾患の治療剤 |
WO2024014463A1 (ja) * | 2022-07-12 | 2024-01-18 | 徹 宮崎 | 腎臓結石の治療または予防剤 |
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CA2938944C (en) * | 2014-02-07 | 2020-01-28 | Toru Miyazaki | Apoptosis inhibitor of macrophage (aim) as preventive or therapeutic agent for kidney disease |
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KR20160110538A (ko) | 2016-09-21 |
IL247111B1 (en) | 2024-03-01 |
CA2938944C (en) | 2020-01-28 |
EP3103466A4 (en) | 2017-11-22 |
IL247111A0 (en) | 2016-09-29 |
JPWO2015119253A1 (ja) | 2017-03-30 |
CA2938944A1 (en) | 2015-08-03 |
US20170172120A1 (en) | 2017-06-22 |
KR101870246B1 (ko) | 2018-06-22 |
EP3103466A1 (en) | 2016-12-14 |
JP6460483B2 (ja) | 2019-01-30 |
US20190364859A1 (en) | 2019-12-05 |
US10349640B2 (en) | 2019-07-16 |
US11253571B2 (en) | 2022-02-22 |
CN106102761A (zh) | 2016-11-09 |
SG11201606514SA (en) | 2016-09-29 |
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