Prophylactic or therapeutic agent for kidney disease

Abstract

The present invention relates to a prophylactic or therapeutic agent for kidney disease. Provided are: a prophylactic or therapeutic agent for kidney disease, which contains AIM or a partial peptide thereof, or a nucleic acid that contains a nucleotide sequence encoding the AIM or the partial peptide thereof; or a method for screening for a prophylactic or therapeutic agent for kidney disease comprising using an animal obtained by subjecting a non-human mammal deficient in AIM expression to unilateral ureteral obstruction or transient renal ischemia / reperfusion.

Description

This application is a divisional application of Chinese Patent Application No. 201580015029.X, "Prophylactic or Therapeutic Agent for Kidney Disease", filed on February 6, 2015. [TECHNICAL FIELD] The present application relates to a prophylactic or therapeutic agent for kidney disease, etc. [BACKGROUND] Due to changes in living environments in recent years, the number of patients with kidney disease is increasing along with an increasing number of lifestyle-related diseases such as hypertension, diabetes, lipid abnormalities, etc. and metabolic syndrome, and about 1 / 8 of adults in Japan are considered to be patients with chronic kidney disease. As a result of progression of kidney disease, when kidney function decreases to develop kidney failure, kidney dialysis and kidney transplantation become necessary, which forms a serious problem for the QOL and medical economy of patients. In addition, acute kidney failure (or acute kidney disease) is a disease caused by different causes such as renal ischemia, nephrotoxic toxins, sepsis, etc., has a high long-term hospitalization rate and a high mortality rate, and its incidence has rather increased in recent years. It is known that in very rare cases, acute kidney failure does not heal sufficiently, becomes chronic, and turns into chronic kidney failure.

[0001] As a treatment method for chronic kidney disease, drug therapy, diet therapy, and rest therapy are performed. For example, drug therapy is performed using inhibitors, diuretics, active vitamin D preparations, oral carbon adsorbent preparations, potassium adsorbents, phosphorus adsorbents, etc., with the aim of delaying progression of the disease and improving symptoms related to reduced kidney function. However, any treatment method cannot sufficiently stop progression of the disease, and a new treatment method has been desired. In addition, there is no reliable treatment method for acute kidney failure, and rapid development of a treatment method is required.

[0002] Also, kidney disease causes a serious problem in cats. Chronic kidney failure most commonly occurs in old age cats, and it is said that kidney failure is a major cause of death of old age cats. Almost all cats develop urinary tract stones or urinary tract infections at 6-7 years of age, which triggers a decrease in kidney function, suffers from acute kidney failure (or acute kidney disease), and many of them develop chronic kidney failure and die before about 15 years of age. There is also no satisfactory treatment method for kidney disease in cats, and a new treatment method has been required.

[0003] AIM (Apoptosis Inhibitor of Macrophage) is a factor produced particularly by macrophages identified by the present inventors (non-patent literature 1), and has been suggested to be associated with some diseases. For example, the blood concentration of AIM increases due to obesity, AIM is incorporated into adipocytes by endocytosis via CD36, and induces degradation (lipolysis) of accumulated neutral fat, and thus, an association with anti-obesity has been suggested (non-patent literature 2). AIM releases free fatty acids from adipocytes by decomposition of neutral fat, and the released fatty acids induce and maintain chronic inflammation in adipose tissue via stimulation of toll-like receptors. Although metabolic syndrome is based on the acquisition of insulin resistance in obesity, since chronic inflammation in adipose tissue is important, AIM is considered to be associated with metabolic syndrome (non-patent literature 3). Furthermore, the present inventors clarified that obese AIM KO mice fed with a high-calorie diet showed pathologies similar to human NASH pathologies of obesity, fatty liver, liver parenchymal fibrosis, and carcinogenic effects, and reported the possibility of application of AIM to liver diseases (patent literature 1). However, the association between AIM and kidney diseases has not been known so far.

[0004] [Document List] [Patent Literature] Patent Literature 1: WO 2013 / 162021 [Non-Patent Literature] Non-Patent Literature 1: Miyazaki, J Exp Med 189:413-422, 1999 Non-Patent Literature 2: Kurokawa, Cell Metab 11:479-492, 2010 Non-Patent Literature 3: Kurokawa, PNAS 108:12072-12077, 2011. [Summary of Invention] [Problems to be Solved by the Invention] The present application aims to provide a preventive or therapeutic drug for kidney diseases. In addition, the present application aims to provide a new method for evaluating or screening a preventive or therapeutic drug for kidney diseases, and the like, which uses a model mouse of kidney diseases. Furthermore, another object of the present application is to provide a prognosis prediction method for kidney diseases.

[0005] [Means of Solving the Problems] The present inventors monitored the progression of the kidneys of AIM knockout mice that underwent unilateral ureteral obstruction (UUO) or one- nephrectomy followed by transient renal ischemia / reperfusion (IR; ischemia / reperfusion) and made a very interesting finding that acute renal failure occurred first and, as days passed, symptoms observed in chronic kidney disease, such as accumulation of necrotic tubular cells and renal parenchymal fibrosis, and collapse and fibrosis of glomerular structure, appeared. Also, in AIM knockout mice that underwent bilateral transient renal ischemia / reperfusion (IR; ischemia / reperfusion), acute renal failure occurred and it was confirmed that accumulation of necrotic tubular cells and rapid progression of nephropathy associated therewith, and deterioration of the overall condition and high mortality rate. In addition, when AIM was administered to the AIM knockout mice, BUN values improved, renal function rapidly improved, and along therewith, systemic symptoms associated therewith and mortality rate improved. It was thus considered that supplementation of AIM would provide treatment of acute renal failure and prevention or treatment of chronic kidney disease.

[0006] The present inventors have made further studies based on these findings and completed the present application.

[0007] Therefore, the present application provides [1] A prophylactic or therapeutic agent for kidney disease, comprising AIM or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding the same; [2] A prophylactic or therapeutic agent for kidney disease, comprising a drug that induces expression of AIM or a drug that stabilizes AIM; [3] The agent of the preceding [1] or [2], wherein the kidney 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; [4] The agent of any one of the preceding [1] to [3], wherein the kidney disease is acute renal failure or chronic renal failure; [5] The agent of any one of the preceding [1] to [4], wherein the subject of the prophylaxis or treatment is a human; [6] The agent of any one of the preceding [1] to [4], wherein the subject of the prophylaxis or treatment is a cat; [7] The agent of the preceding [6], wherein the AIM or partial peptide thereof binds to cat IgM; [8] The agent of the preceding [7], wherein the AIM or partial peptide thereof that binds to cat IgM contains the SRCR3 domain of mouse-derived AIM; [9] A screening method for a prophylactic or therapeutic agent for kidney disease, the method comprising using an animal obtained by subjecting an AIM expression-deficient non-human mammal to unilateral ureteral obstruction, one-nephrectomy followed by transient renal ischemia / reperfusion, or bilateral transient renal ischemia / reperfusion;

[10] The method according to the preceding [9], comprising the steps of: (1) a step of administering a test substance to an AIM-deficient non-human mammal under conditions of unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion, (2) a step of observing any one or more of the following properties of the AIM-deficient non-human mammal to which the test substance is administered: (i) accumulation of necrotic tubular cells and renal parenchymal fibrosis, (ii) disintegration and fibrosis of glomerular structure, (iii) expression level of inflammatory cytokines in the kidney, (iv) ratio of macrophages in the kidney, (v) BUN value, (vi) survival rate, (3) a step of selecting a test substance that improves any one or more of the above properties compared to those in the case where the test substance is not administered;

[11] The screening method according to the preceding [9] or

[10] , wherein 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;

[12] A method for evaluating the preventive or therapeutic effect of a preventive or therapeutic agent for a renal disease, the method comprising using an animal obtained by subjecting an AIM-deficient non-human mammal to unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion;

[13] The method for evaluation according to the preceding

[12] , comprising the steps of: (1) a step of administering a preventive or therapeutic agent for a renal disease to an AIM-deficient non-human mammal under conditions of unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion, (2) a step of observing any one or more of the following properties of the AIM-deficient non-human mammal to which the preventive or therapeutic agent for a renal disease is administered: (i) accumulation of necrotic tubular cells and renal parenchymal fibrosis, (ii) disintegration and fibrosis of glomerular structure, (iii) expression level of inflammatory cytokines in the kidney, (iv) ratio of macrophages in the kidney, (v) BUN value, (vi) survival rate, (3) a step of evaluating the effect of the preventive or therapeutic agent for kidney disease by comparing any one or more of the above characteristics with those in the case where no preventive or therapeutic agent for kidney disease is administered;

[14] The evaluation method according to the preceding

[12] or

[13] , wherein the kidney disease is acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, a kidney disease associated with a collagen disease, or IgM nephropathy;

[15] A method of predicting the prognosis of a patient with a kidney disease, the method comprising measuring the concentration of AIM in a sample of the subject;

[16] The method according to the preceding

[15] , wherein the sample is blood or serum;

[17] The method according to the preceding

[15] or

[16] , wherein the method of measuring the AIM concentration is an immunological method using an anti-AIM antibody;

[18] The method according to any one of the preceding

[15] to

[17] , wherein the kidney disease is acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, a kidney disease associated with a collagen disease, or IgM nephropathy;

[19] A test method for acute renal failure, the method comprising measuring the concentration of AIM in a sample of the subject;

[20] The test method according to

[19] , wherein the sample is urine;

[21] The test method according to

[19] or

[20] , wherein the method of measuring the AIM concentration is an immunological method using an anti-AIM antibody;

[22] A kit for diagnosis or prognosis prediction of a kidney disease, comprising the following (a) or (b): (a) a nucleic acid probe or a nucleic acid primer that can hybridize to a transcription product of an AIM gene, and / or (b) an antibody against AIM;

[23] The kit according to

[22] , wherein the kidney disease is acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, a kidney disease associated with a collagen disease, or IgM nephropathy;

[24] A method for preventing or treating a kidney disease, the method comprising administering to a subject an effective amount of AIM or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding the same;

[25] A method for preventing or treating a kidney disease, the method comprising administering to a subject an effective amount of a drug that induces the expression of AIM or a drug that stabilizes AIM;

[26] The method of any one of the preceding

[24] or

[25] , wherein the kidney 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;

[27] The method of any one of the preceding

[24] to

[26] , wherein the kidney disease is acute renal failure or chronic renal failure;

[28] The method of any one of the preceding

[24] to

[27] , wherein the subject to be prevented or treated is a human;

[29] The method of any one of the preceding

[24] to

[27] , wherein the subject to be prevented or treated is a cat;

[30] The method of the preceding

[29] , wherein the AIM or partial peptide thereof binds to feline IgM;

[31] An AIM or partial peptide thereof, or a nucleic acid comprising a base sequence encoding the same, for use in the prevention or treatment of a kidney disease;

[32] The AIM or partial peptide thereof, or the nucleic acid comprising a base sequence encoding the same, of the preceding

[31] , wherein the kidney 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;

[33] The AIM or partial peptide thereof, or the nucleic acid comprising a base sequence encoding the same, of the preceding

[31] or

[32] , wherein the kidney disease is acute renal failure or chronic renal failure;

[34] The AIM or partial peptide thereof, or the nucleic acid comprising a base sequence encoding the same, of any one of the preceding

[31] to

[33] , wherein the subject to be prevented or treated is a human;

[35] The AIM or partial peptide thereof, or the nucleic acid comprising a base sequence encoding the same, of any one of the preceding

[31] to

[33] , wherein the subject to be prevented or treated is a cat;

[36] The AIM or partial peptide thereof, or the nucleic acid comprising a base sequence encoding the same, of the preceding

[35] , wherein the AIM or partial peptide thereof binds to feline IgM;

[37] An agent that induces expression of an AIM or an agent that stabilizes an AIM for use in the prevention or treatment of a kidney disease;

[38] The agent that induces expression of an AIM or the agent that stabilizes an AIM of the preceding

[37] , wherein the kidney 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;

[39] The agent that induces expression of an AIM or the agent that stabilizes an AIM of the preceding

[37] or

[38] , wherein the kidney disease is acute renal failure or chronic renal failure;

[40] The drug for inducing expression of AIM or the drug for stabilizing AIM according to any one of the preceding

[37] to

[39] , wherein the subject to be prevented or treated is a human;

[41] The drug for inducing expression of AIM or the drug for stabilizing AIM according to any one of the preceding

[37] to

[39] , wherein the subject to be prevented or treated is a cat;

[42] The drug for inducing expression of AIM or the drug for stabilizing AIM according to the preceding

[41] , wherein the AIM or the partial peptide thereof binds to feline IgM;

[43] Use of AIM or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding the same, or a drug for inducing expression of AIM or a drug for stabilizing AIM for the production of a preventive or therapeutic agent for a kidney disease;

[44] The use according to the preceding

[43] , wherein the kidney disease is acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, a kidney disease associated with collagen disease, or IgM nephropathy;

[45] The use according to the preceding

[43] or

[44] , wherein the kidney disease is acute renal failure or chronic renal failure;

[46] The use according to any one of the preceding

[43] to

[45] , wherein the subject to be prevented or treated is a human;

[47] The use according to any one of the preceding

[43] to

[45] , wherein the subject to be prevented or treated is a cat;

[48] The use according to the preceding

[47] , wherein the AIM or the partial peptide thereof binds to feline IgM.

[0008] [Effects of the Invention] The present application can provide a preventive or therapeutic agent for a kidney disease, which comprises AIM or the like as an active ingredient. In addition, according to the screening method using a kidney disease model mouse according to the present application, a substance effective for the prevention or treatment of a kidney disease can be explored. In addition, using the kidney disease model mouse according to the present application, the effect of a known preventive or therapeutic agent for a kidney disease can be evaluated. Furthermore, the present application can provide a prognosis prediction method and a test method for a kidney disease, which include measuring the concentration of AIM in a sample of a test subject. [BRIEF DESCRIPTION OF DRAWINGS] Figure 1 Azan and hematoxylin simultaneous staining images of normal kidney tissue sections and kidney tissue sections after UUO of AIM KO mice and WT mice subjected to unilateral ureteral obstruction are shown in A, and simultaneous Azan, PAS, and hematoxylin staining images of normal kidney tissue sections and kidney tissue sections after UUO of AIM KO mice and WT mice subjected to unilateral ureteral obstruction are shown in B.

[0009] Figure 2Hematoxylin-eosin staining images of kidney tissue sections of AIM KO mice and WT mice that underwent transient renal ischemia / reperfusion after unilateral nephrectomy.

[0010] Figure 3 F4 / 80 immunostaining images of kidney tissue sections of AIM KO mice and WT mice that underwent transient renal ischemia / reperfusion after unilateral nephrectomy, and a graph showing MCP-1 expression levels of kidney tissue sections of AIM KO mice and WT mice that underwent transient renal ischemia / reperfusion after unilateral nephrectomy. *: P<0.05, **: P<0.01.

[0011] Figure 4 Hematoxylin-eosin staining images of kidney tissue sections of AIM KO mice that underwent transient renal ischemia / reperfusion after unilateral nephrectomy and administration of rAIM or PBS, and BUN values of AIM KO mice and WT mice that underwent transient renal ischemia / reperfusion after unilateral nephrectomy and administration of rAIM or PBS.

[0012] Figure 5 Phase contrast images and immunostaining images of AIM of kidney tissue sections of AIM KO mice that underwent transient renal ischemia / reperfusion after unilateral nephrectomy and administration of rAIM. N: necrotic lesion. Arrow: AIM.

[0013] Figure 6 The graph of FIG. 1 shows urine AIM levels of WT mice that underwent bilateral transient renal ischemia / reperfusion. *: p<0.05.

[0014] Figure 7 The graph of FIG. 2 shows survival rates of AIM KO mice and WT mice that underwent bilateral transient renal ischemia / reperfusion.

[0015] Figure 8 The graph of FIG. 3 shows clinical scores of AIM KO mice and WT mice that underwent bilateral transient renal ischemia / reperfusion. **: p<0.01.

[0016] Figure 9 The graph of FIG. 4 shows BUN values of AIM KO mice and WT mice that underwent bilateral transient renal ischemia / reperfusion. The number of mice that died each day is indicated with +. *: P<0.05, **: P<0.01.

[0017] Figure 10 PAS staining images of kidney tissue sections of AIM KO mice and WT mice that underwent bilateral transient renal ischemia / reperfusion.

[0018] Figure 11Figure 6 shows the ratio of the area of dead cell mass in proximal tubules in kidney tissue sections of AIM KO mice and WT mice subjected to bilateral transient renal ischemia / reperfusion relative to the total area of each section. *: p<0.05, **: p<0.01.

[0019] Figure 12 Figure 7 shows the ratio of mRNA expression of IL-1b and IL-6 in AIM KO mice and WT mice subjected to bilateral transient renal ischemia / reperfusion determined by quantitative RT-PCR. *: p<0.05.

[0020] Figure 13 Figure 8 shows the results of flow cytometric analysis of kidney-derived cells from AIM KO mice and WT mice subjected to bilateral transient renal ischemia / reperfusion at day 7 after IR.

[0021] Figure 14 Figure 9 shows PAS staining images and immunostaining images of kidney tissue sections of WT mice subjected to bilateral transient renal ischemia / reperfusion with anti-AIM antibody.

[0022] Figure 15 Figure 10 shows PAS staining images and immunostaining images of kidney tissue sections of a human who died of acute renal failure caused by renal infarction with anti-AIM antibody.

[0023] Figure 16 Figure 11 shows the concentration of AIM in urine of 3 patients who were transported to a hospital due to human acute kidney injury (AKI), 3 healthy individuals, and 5 WT mice subjected to bilateral IR before IR, 1 day and 7 days after IR measured by ELISA. N.D.: not detected.

[0024] Figure 17 Figure 12 shows PAS staining images and immunostaining images of kidney tissue sections of AIM KO mice subjected to bilateral transient renal ischemia / reperfusion with anti-AIM antibody over time.

[0025] Figure 18 Figure 13 shows phagocytic activity of F4 / 80-positive macrophages isolated from kidneys of AIM-KO mice at day 3 after bilateral IR treated with collagenase by FACS sorter.

[0026] Figure 19 Figure 14 shows phagocytic activity of macrophages obtained by differentiating AIM-KO derived bone marrow cells with M-CSF and isolating by FACS sorter.

[0027] Figure 20Figure 2 shows survival rate of AIM KO mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3.

[0028] Figure 21 Figure 3 shows clinical score of AIM KO mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3. *: P<0.05, **: P<0.01.

[0029] Figure 22 Figure 4 shows BUN values of AIM KO mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3. The number of mice that died every day is indicated with +. *: P<0.05, **: P<0.01.

[0030] Figure 23 Figure 5 shows PAS staining images of kidney tissue sections of AIM KO mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3.

[0031] Figure 24 Figure 6 shows the ratio of the area of dead cell mass in proximal tubules relative to the total area of each section in kidney (7 days after IR) tissue sections of AIM KO mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3. *: p<0.05.

[0032] Figure 25 Figure 7 shows the ratio of mRNA expression of IL-1b and IL-6 in AIM KO mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3, determined by quantitative RT-PCR. *: p<0.05.

[0033] Figure 26 Figure 8 shows BUN values of AIM KO mice subjected to non-fatal bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3. *: p<0.05.

[0034] Figure 27 Figure 9 shows BUN values of WT mice subjected to bilateral transient renal ischemia / reperfusion and administered rAIM or PBS from day 1 to day 3.

[0035] Figure 28 Figure 10 shows PAS staining images and Azan staining images of kidney tissue sections of AIM KO mice and WT mice subjected to non-fatal bilateral transient renal ischemia / reperfusion.

[0036] Figure 29Figure 2 shows the mRNA expression ratios of 4 fibrosis markers in AIM KO mice and WT mice subjected to non-lethal two-sided transient renal ischemia / reperfusion determined by quantitative RT-PCR.

[0037] Figure 30 Figure 3 shows gender-differentiated blood AIM concentrations in healthy humans, diabetic patients without kidney disease, and patients with diabetic chronic kidney failure. *: p<0.05, **: p<0.01.

[0038] Figure 31 Figure 4 shows A: the graph shows the correlation between eGFR (glomerular filtration rate; ml / min / 1.73m 2 ) or blood creatinine level (mg / dl) and blood AIM concentration in chronic kidney failure patients (n=55), B: the graph shows the correlation between blood AIM concentration and change in kidney function 2-3 years from the time of measuring blood AIM concentration in chronic kidney failure patients.

[0039] Figure 32 Figure 5 shows A: Western blot images of AIM present in the serum of dogs (n=3), cats (n=3), and mice, B: Western blot images of rAIM in cats and mice.

[0040] Figure 33 Figure 6 shows A: Western blot images of AIM and IgM on each size fraction of serum derived from a cat injected intravenously with recombinant cat AIM (1 mg), B: Western blot images of AIM and IgM on each size fraction of serum derived from a mouse.

[0041] Figure 34 Figure 7 shows Western blot images of AIM and IgM on each size fraction of serum derived from a cat injected intravenously with recombinant mouse AIM (1 mg).

[0042] Figure 35 Figure 8 shows the cat AIM cDNA sequence (SEQ ID NO: 5), wherein the translation initiation site (atg) and the translation termination site (tga) are shown in bold, the non-coding sequence is shown in italic, the base sequence encoding the leader peptide is underlined with a solid line, and further, the base sequence encoding the peptide sequence between the hinge region between SRCR1 and SRCR2, the hinge region between SRCR2 and SRCR3, and SRCR3 and the translation termination site is underlined with a dotted line.

[0043] Figure 36 Figure 9 shows the hydrophobicity of the leader peptide sequence of the cat AIM protein.

[0044] Figure 37Hydrophobicity of the leader peptide sequence of the human AIM protein is shown.

[0045] Figure 38 Hydrophobicity of the leader peptide sequence of the mouse AIM protein is shown.

[0046] Figure 39 Hydrophobicity of the leader peptide sequence of the dog AIM protein is shown.

[0047] Figure 40 Comparison of the leader peptide (LS) and amino acid sequences of each SRCR and hinge region of human (SEQ ID NO: 2), cat (SEQ ID NO: 4), and mouse (SEQ ID NO: 6) is shown.

[0048] Figure 41 A reduced protein blot image of AIM in serum of a cat using an anti-cat AIM monoclonal antibody is shown, in which individuals 1-4 are individuals detected with AIM, and the AIM value is calculated by comparing the intensity of the signal with that of rcAIM of the control, and individuals 5-7 are individuals not detected with AIM signal (N.D.: not detectable).

[0049] Figure 42 The graph of FIG. 1 shows BUN values and Cre values of cats subjected to two-sided transient renal ischemia / reperfusion.

[0050] Figure 43 Reduced protein blot images of serum or urinary AIM of a cat before and after IR using an anti-cat AIM monoclonal antibody are shown.

[0051] Figure 44 Immunostaining images of kidney tissue sections of a cat subjected to two-sided transient renal ischemia / reperfusion using an anti-AIM antibody are shown.

[0052] Figure 45 The graph of FIG. 1 shows eGFR (glomerular filtration rate; ml / min / m 2 ) of cats subjected to two-sided transient renal ischemia / reperfusion and administered with rAIM or PBS. n = 1 each.

[0053] Figure 46 PAS staining images of kidney tissue sections of a cat subjected to two-sided transient renal ischemia / reperfusion and administered with rAIM or PBS are shown. [DETAILED DESCRIPTION] The present application provides a prophylactic or therapeutic agent for renal disease, which comprises AIM or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding the same.

[0054] The AIM in the present application is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 (amino acid sequence of a human-derived AIM protein) or SEQ ID NO: 4 (amino acid sequence of a cat-derived AIM protein).

[0055] The AIM can be, for example, a protein isolated and purified from macrophages, which are immune cells of a warm-blooded animal (e.g., human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, chicken, etc.). It can also be a protein chemically synthesized or biochemically synthesized in a cell-free translation system. Alternatively, the protein can be a recombinant protein produced from a transformant incorporating a nucleic acid containing a base sequence encoding the above-described amino acid sequence.

[0056] An amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 refers to an amino acid sequence having about 60% or more, preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more homology to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Here, "homology" refers to the ratio (%) of identical amino acid residues and similar amino acid residues in the optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm takes into account the introduction of gaps on one or both sides of the sequence for optimal alignment). "Similar amino acid" means an amino acid having similar physicochemical properties; examples thereof include amino acids classified under the same group, 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 having a hydroxyl group (Ser, Thr), and amino acids having a small side chain (Gly, Ala, Ser, Thr, Met). It is contemplated that such substitution of similar amino acids does not change the phenotype of the protein (i.e., conservative amino acid substitution). Specific examples of conservative amino acid substitution are known in the art and described in different documents (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

[0057] Amino acid sequence homology in the present description can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (Expect value = 10; Allowed gaps; Matrix = BLOSUM62; Filter = OFF). As examples of other algorithms for determining amino acid sequence homology, mention can be made of the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [said algorithm being incorporated in the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [said algorithm being incorporated in the GAP program which is part of the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [said algorithm being incorporated in the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [said algorithm being incorporated in the FASTA program which is part of the GCG software package], etc., which can likewise be preferred to use.

[0058] More preferably, an amino acid sequence which is substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 is an amino acid sequence which is identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 to the extent of about 60% or more, preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.

[0059] As the protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, for example, a protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having an activity substantially identical in quality to the activity of the protein comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 or the like is preferred. Here, the "activity" refers to, for example, an activity of inhibiting apoptosis of macrophages in an atherosclerotic plaque, an activity of maintaining or promoting arteriosclerosis, a fat cell differentiation inhibitory activity, an activity of melting lipid droplets of fat cells, a fat cell reducing activity, a CD36 binding activity, an activity of endocytosis into fat cells, a FAS binding activity, a FAS function inhibitory activity, an anti-obesity activity, and the like. The "substantially identical in quality" means that the activities are qualitatively (for example, physiologically or pharmacologically) the same. Therefore, it is preferred that the above-mentioned activities are mutually equivalent, but the quantitative coefficients of these activities, such as the degree of the activity (for example, about 0.1 to about 10 times, preferably about 0.5 to about 2 times) and the molecular weight of the protein, can be different.

[0060] The above-mentioned activity can be measured by a method known per se.

[0061] Examples of the AIM in the present application also include a protein comprising an amino acid sequence in which (1) 1 or 2 or more (preferably about 1 to 100, preferably about 1 to 50, further preferably about 1 to 10, particularly preferably 1 to several (2, 3, 4, or 5)) amino acids are deleted from the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (2) 1 or 2 or more (preferably about 1 to 100, preferably about 1 to 50, further preferably about 1 to 10, particularly preferably 1 to several (2, 3, 4, or 5)) amino acids are added to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (3) 1 or 2 or more (preferably about 1 to 50, preferably about 1 to 10, further preferably 1 to several (2, 3, 4, or 5)) amino acids are inserted in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (4) 1 or 2 or more (preferably about 1 to 50, preferably about 1 to 10, further preferably 1 to several (2, 3, 4, or 5)) amino acids are substituted with other amino acids in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or (5) an amino acid sequence comprising a combination thereof.

[0062] When the amino acid sequence has been inserted, deleted, or substituted as described above, the position of the insertion, deletion, or substitution is not particularly limited as long as the activity of the protein is maintained.

[0063] The AIM of the present application is preferably a human AIM protein having the amino acid sequence shown in SEQ ID NO: 2 (GenBank Accession No: AAD01446), or a cat AIM protein having the amino acid sequence shown in SEQ ID NO: 4 or a homologue thereof in other mammals [e.g., a mouse homologue registered in GenBank as Accession No: AAD01445, etc.], more preferably a human AIM protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a cat AIM protein consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0064] In the present specification, proteins and peptides are described in accordance with the convention of peptide nomenclature, in which the left end indicates the N-terminal end and the right end indicates the C-terminal end. In the AIM of the present application including a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the C-terminal end can be any one of a carboxyl group (-COOH), a carboxylate (-COO - ), an amide (-CONH2), and an ester (-COOR).

[0065] Here, as R in the ester, a C 1-6 alkyl group such as a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, and an n-butyl group, a C 3-8 cycloalkyl group such as a cyclopentyl group and a cyclohexyl group, a C 6-12 aryl group such as a phenyl group and an α-naphthyl group, a phenyl-C 1-2 alkyl group such as a benzyl group and a phenethyl group, a C 7-14 aralkyl group such as an α-naphthyl-C 1-2 alkyl group such as an α-naphthylmethyl group, a pivaloyloxymethyl group; and the like.

[0066] When the AIM of the present application has a carboxyl group (or a carboxylate) at a position other than the C-terminal end, a protein in which the carboxyl group is amidated or esterified is also included in the protein of the present application. In this case, as the ester, for example, the above-described ester at the C-terminal end or the like is used.

[0067] Further, the AIM of the present application also includes: a protein in which the amino group of the N-terminal amino acid residue is protected with a protecting group (e.g., a C 1-6 acyl group such as a C 1-6 alkanoyl group such as a formyl group and an acetyl group, etc.); a protein in which a glutamine residue that can be produced in vivo after cleavage at the N-terminal end has been converted into pyroglutamic acid; a protein in which a substituent on the side chain of an amino acid in the molecule (e.g., -OH, -SH, an amino group, an imidazolyl group, an indolyl group, a guanidino group, etc.) is protected with a suitable protecting group (e.g., a C 1-6 acyl group such as a C 1-6proteins, conjugated peptides, such as glycopeptides having a sugar chain bonded thereto, and the like.

[0068] The partial peptide of the AIM (hereinafter sometimes simply referred to as "the partial peptide of the present application") can be arbitrary as long as it is a peptide having a partial amino acid sequence of the above-described AIM and having an activity of substantially the same quality as that of the AIM. Here, the "activity of substantially the same quality" is as defined above. Further, the "activity of substantially the same quality" can be measured in the same manner as in the case of the AIM.

[0069] Since the AIM contains 3 SRCR (scavenger receptor cysteine-rich) domains containing a large number of cysteines, each of the SRCR domains can be used as the partial peptide of the present application. Specifically, for example, in the amino acid sequence shown in SEQ ID NO: 2, a partial amino acid sequence containing the SRCR1 domain (amino acid numbers 24-125 of the amino acid sequence shown in SEQ ID NO: 2), the SRCR2 domain (amino acid numbers 138-239 of the amino acid sequence shown in SEQ ID NO: 2), and the SRCR3 domain (amino acid numbers 244-346 of the amino acid sequence shown in SEQ ID NO: 2) respectively, a partial amino acid sequence containing any combination of the SRCR domains, and the like can be used. In addition, in the amino acid sequence shown in SEQ ID NO: 4, a partial amino acid sequence containing the SRCR1 domain (amino acid numbers 24-125 of the amino acid sequence shown in SEQ ID NO: 4), the SRCR2 domain (amino acid numbers 139-239 of the amino acid sequence shown in SEQ ID NO: 4), and the SRCR3 domain (amino acid numbers 244-346 of the amino acid sequence shown in SEQ ID NO: 4) respectively, a partial amino acid sequence containing any combination of the SRCR domains, and the like can be used. The size of the partial peptide of the present application is not particularly limited as long as it contains the above-described functional domain. The partial peptide preferably contains a partial amino acid sequence of not less than 50, more preferably a partial amino acid sequence of not less than 100, and further preferably a partial amino acid sequence of not less than 200. The partial amino acid sequence can be a single continuous partial amino acid sequence, or a plurality of discontinuous partial amino acid sequences connected to each other.

[0070] Further, the C-terminus of the partial peptide of the present application can be a carboxyl group (-COOH), a carboxylate (-COO -), amide ( - CONH2), and ester ( - COOR). Here, examples of R in the ester include those similar to the above examples of AIM. When the partial peptide of the present application has a carboxyl group (or carboxylate) at a position other than the C-terminus, the carboxyl group is amidated or esterified, which is also included in the partial peptide of the present application. As the ester in this case, for example, those similar to the ester at the C-terminus are used, etc.

[0071] Further, the partial peptide of the present application includes, in the same manner as the above AIM, the amino group of the N-terminal amino acid residue can be protected with a protecting group, the glutamine residue at the N-terminus can be converted to pyroglutamic acid, the substituents on the side chains of the amino acids in the molecule can be protected with a suitable protecting group, or the partial peptide can be a synthetic peptide in which a sugar chain is bonded (so-called glycopeptide, etc.), etc.

[0072] The AIM or the partial peptide thereof to be used in the present application can be in the form of a salt. For example, a salt with a physiologically acceptable acid (e.g., inorganic acid, organic acid), base (e.g., alkali metal salt), etc. is used, and a physiologically acceptable acid addition salt is preferred. Useful salts include, for example, a salt with an inorganic acid (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or a salt with an organic acid (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid), etc.

[0073] The AIM can be produced from the above-described mammalian macrophage by a protein purification method known per se. Specifically, the AIM or salt thereof can be prepared by homogenizing the mammalian macrophage, removing the cell debris by low-speed centrifugation, precipitating the fraction containing the cell membrane by high-speed centrifugation of the supernatant, and subjecting the supernatant to chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc.

[0074] The AIM or the partial peptide thereof can also be prepared according to a publicly known peptide synthesis method (hereinafter, in the description of the chemical synthesis, the full-length AIM and the partial peptide thereof are collectively referred to as AIM unless otherwise indicated).

[0075] The method of peptide synthesis can be, for example, either of a solid phase synthesis method and a liquid phase synthesis method. The desired protein can be produced by condensing the partial peptide or amino acid capable of constituting the AIM with the remaining portion, and removing any protecting group that the resulting product can have.

[0076] Here, the condensation and the protecting group removal are performed according to a method known per se, for example, the methods shown in the following (1) and (2) : (1) M. Bodanszky and M. A. Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966) (2) Schroeder and Luebke: The Peptide, Academic Press, New York (1965).

[0077] The AIM thus obtained can be purified or isolated by known purification methods. Here, as examples of the purification methods, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, combinations thereof, and the like can be mentioned.

[0078] When the AIM thus obtained is in a free form, the free form can be converted into a suitable salt form by known methods or similar methods thereof, and, on the other hand, when the AIM in a salt form is obtained, it can be converted into a free form or a form of a different salt by known methods or methods based thereon.

[0079] Further, the AIM can also be produced by culturing a transformant containing a nucleic acid encoding the same, and isolating and purifying the AIM from the obtained culture. The nucleic acid encoding the AIM or a partial peptide thereof can be DNA or RNA, or a DNA / RNA chimera, preferably DNA. Further, the nucleic acid can be double-stranded or single-stranded. In the case of a double-stranded nucleic acid, it can be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of single-stranded, it can be a sense strand (i.e., coding strand) or an anti-sense strand (i.e., non-coding strand).

[0080] Examples of the DNA encoding the AIM or the partial peptide thereof include genomic DNA, cDNA derived from macrophages of a warm-blooded animal (e.g., human, bovine, monkey, equine, porcine, ovine, caprine, canine, feline, guinea pig, rat, mouse, rabbit, hamster, chicken, etc.), synthetic DNA, and the like. The genomic DNA encoding the AIM or the partial peptide thereof can be directly amplified by a polymerase chain reaction (hereinafter referred to as "PCR method") using a genomic DNA fraction prepared from any of the aforementioned animals as a template. The genomic DNA fraction can be prepared from any of the aforementioned cells [e.g., hepatocyte, splenocyte, neural cell, glial cell, pancreatic beta cell, myeloblast, mesangial cell, Langerhans cell, epidermal cell, epithelial cell, goblet cell, endothelial cell, smooth muscle cell, fibroblast, fibrocyte, myocyte, adipocyte, immune cell (e.g., macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synoviocyte, chondrocyte, osteocyte, osteoblast, osteoclast, mammary cell, hepatocyte, or interstitial cell, or a corresponding progenitor cell, stem cell, or cancer cell thereof, etc.] or any tissue in which such cells are present [e.g., brain or any portion thereof (e.g., olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, infralaminar nucleus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (e.g., brown adipose tissue, white adipose tissue), skeletal muscle, etc.]. The cDNA encoding the AIM or the partial peptide thereof can also be directly amplified by the PCR method and reverse transcriptase-PCR (hereinafter referred to as "RT-PCR method") using a total RNA or mRNA fraction prepared from the macrophages as a template, respectively. Alternatively, the genomic DNA and cDNA encoding the AIM or the partial peptide thereof can also be cloned from a genomic DNA library and a cDNA library prepared by inserting a fraction of the aforementioned genomic DNA and total RNA or mRNA into a suitable vector by a colony or plaque hybridization method or a PCR method, etc. The vector for the library can be any of a bacteriophage, a plasmid, a cosmid, a phagemid, etc.

[0081] Examples of the DNA encoding the AIM include DNA comprising a base sequence identical or substantially identical to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, etc.

[0082] As the DNA containing a base sequence identical or substantially identical to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, for example, a DNA containing a base sequence having homology of not less than about 60%, preferably not less than about 70%, more preferably not less than about 80%, particularly preferably not less than about 90% to the base sequence shown in SEQ ID NO: 1 and encoding a protein having substantially the same quality of activity as the above-described AIM or the like is used.

[0083] The base sequence homology in the present specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (Expect value = 10; Allow gaps; Filter = ON; Match score = 1; Mismatch score = -3). As a preferred example of other algorithms for determining base sequence homology, the above-described amino acid sequence homology calculation algorithm can also be mentioned.

[0084] The DNA encoding AIM is preferably a DNA containing a base sequence encoding a human AIM protein shown in the base sequence shown in SEQ ID NO: 1 (GenBank Accession No. AF011429), or a DNA containing a base sequence encoding a cat AIM protein shown in the base sequence shown in SEQ ID NO: 3, or a homolog thereof in other mammals [for example, a mouse homolog registered in GenBank as Accession No. AF011428 or the like].

[0085] The DNA encoding the partial peptide of the present application can be arbitrary as long as it contains a base sequence encoding a peptide containing an amino acid sequence identical or substantially identical to a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Specifically, as the DNA encoding the partial peptide of the present application, (1) a DNA containing a partial base sequence of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or (2) a DNA containing a base sequence having homology of not less than about 60%, preferably not less than about 70%, more preferably not less than about 80%, particularly preferably not less than about 90% to the DNA containing a partial base sequence of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein having substantially the same quality of activity as the above-described AIM or the like is used.

[0086] The DNA encoding the AIM or partial peptide thereof can be cloned by amplifying them by a PCR method using synthetic DNA primers having a portion of the base sequence encoding the AIM or partial peptide thereof, or hybridizing DNA incorporated into a suitable expression vector with a labeled DNA fragment or synthesis encoding a partial or entire region of the AIM. The hybridization can be performed according to a method known per se or a method based thereon, for example, a method described in Molecular Cloning, 2nd Ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989), and the like. When a commercially available library is used, the hybridization can be performed according to the method described in the instruction manual attached thereto. The hybridization can be preferably performed under high stringent conditions.

[0087] As an example of the high stringent conditions, there can be mentioned conditions of a hybridization reaction in 6 x SSC (sodium chloride / sodium citrate) at 45°C, followed by washing once or more times in 0.2 x SSC / 0.1% SDS at 65°C, and the like. A person skilled in the art can easily obtain the desired stringency by appropriately changing the salt concentration of the hybridization solution, the hybridization reaction temperature, the probe concentration, the probe length, the number of mismatches, the hybridization reaction time, the salt concentration of the washing solution, the washing temperature, and the like. When a commercially available library is used, the hybridization can be performed according to the method described in the instruction manual attached to the library.

[0088] The expression vector containing the DNA encoding the AIM or partial peptide thereof can be produced, for example, by excising a desired DNA fragment from the DNA encoding the AIM, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.

[0089] As the expression vector, a plasmid derived from E. coli (for example, pBR322, pBR325, pUC12, pUC13); an animal cell expression plasmid (for example, pAl-ll, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo); an animal viral vector such as a retrovirus, a vaccinia virus, an adenovirus, and the like is used.

[0090] The promoter can be any promoter as long as it is suitable for the host used for expressing the gene.

[0091] For example, when the host is an animal cell, an SRa promoter, an SV40 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, a MoMuLV (Moloney murine leukemia virus) LTR, an HSV-TK (herpes simplex virus thymidine kinase) promoter, and the like are used. Among these, a CMV promoter, an SRa promoter, and the like are preferred.

[0092] When the host is a bacterium belonging to the genus Escherichia, a trp promoter, a lac promoter, a recA promoter, a λ P L A lpp promoter, a T7 promoter, and the like are preferable.

[0093] In addition to the above, useful expression vectors include those optionally carrying an enhancer, a splicing signal, a polyadenylation signal, a selection marker, an SV40 replication origin (hereinafter also referred to as SV40 ori), and the like. As examples of the selection marker, a dihydrofolate reductase (hereinafter also referred to as dhfr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter also referred to as Amp r ), a neomycin resistance gene (hereinafter also referred to as Neo r , G418 resistance), and the like can be mentioned. Specifically, when Chinese hamster cells lacking the dhfr gene and the dhfr gene as the selection marker are used, a thymidine-free medium can also be used to select the target gene.

[0094] If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host can be added (or replaced with a natural signal codon) to the 5'-terminal side of the DNA encoding the AIM or the partial peptide thereof. For example, when the host is the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, and the like are used; when the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like are used.

[0095] The AIM or the partial peptide thereof can be produced by transforming a host with an expression vector containing the above-described DNA encoding the AIM or the partial peptide thereof and culturing the obtained transformant.

[0096] As the host, for example, the genus Escherichia, animal cells, and the like are used.

[0097] As the genus Escherichia, for example, Escherichia coli K12 · DH1 [Proc. Natl. Acad. Sci. USA), Vol. 60, 160 (1968)], Escherichia coli JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], Escherichia coli JA221 [Journal of Molecular Biology, Vol. 120, 517 (1978)], Escherichia coli HB101 [Journal of Molecular Biology, Vol. 41, 459 (1969)], Escherichia coli C600 [Genetics, Vol. 39, 440 (1954)], and the like are used.

[0098] As animal cells, for example, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter referred to as CHO cells), dhfr gene-deficient CHO cells (hereinafter referred to as CHO (dhfr - ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, etc.

[0099] Transformation can be performed based on publicly known methods depending on the host species.

[0100] Escherichia can be transformed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972), Gene, Vol. 17, 107 (1982), and the like.

[0101] Animal cells can be transformed, for example, according to the method described in Saibo Kogaku (Cell Engineering), extraissue 8, Shin Saibo Kogaku Jikken Protocol (New Cell Engineering Experimental Protocol), 263-267 (1995), published by Shujunsha, or Virology, vol. 52, 456 (1973).

[0102] The culture of the transformant can be performed based on publicly known methods depending on the host species.

[0103] As an example of a culture medium for culturing a transformant (whose host is a bacterium belonging to the genus Escherichia), M9 medium supplemented with glucose and casein hydrolysate [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferred. If necessary, a chemical agent such as 3β-indolyl acrylic acid may be added to the culture medium to enhance promoter efficiency.

[0104] The transformant (whose host is a bacterium belonging to the genus Escherichia) is usually cultured at about 15° C. to about 43° C. for about 3 to about 24 hours. If necessary, the culture may be aerated or stirred.

[0105] Useful culture media for culturing transformants whose host is an animal cell include, for example, minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], Dulbecco's modified Eagle's medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI1640 medium [The Journal of the American Medical Association, Vol. 199, 519 (1967)], and medium 199 [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. The pH of the culture medium is preferably about 6 to about 8. Cultivation is usually carried out at about 30° C. to about 40° C. for about 15 to about 60 hours. If necessary, the culture may be aerated or stirred.

[0106] As described above, AIM can be produced inside or outside the transformant cell.

[0107] AIM or a partial peptide thereof can be isolated and purified according to a method known per se from a culture obtained by culturing the above-mentioned transformant.

[0108] For example, when extracting AIM or a partial peptide thereof from the cytoplasm of cultured bacteria or cells, a method is suitably used in which the bacteria or cells are collected from the culture by a known method, suspended in an appropriate buffer solution, and disrupted by means of ultrasonic treatment, lysozyme, and / or freeze-thaw, etc., after which a crude extract of soluble protein is obtained by centrifugation or filtration. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, and a surfactant such as Triton X-100. TM Furthermore, when AIM or a partial peptide thereof is secreted outside the bacteria (cells), a method of separating the culture supernatant from the culture or the like by centrifugation, filtration, or the like is used.

[0109] The separation and purification of AIM or its partial peptides contained in the soluble fraction and culture supernatant thus obtained can be carried out according to methods known per se. Useful methods include solubility-based methods such as salting-out and solvent precipitation; methods primarily based on molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods based on charge differences such as ion exchange chromatography; methods based on specific affinity such as affinity chromatography; methods based on hydrophobicity differences such as reversed-phase high-performance liquid chromatography; and methods based on isoelectric point differences such as isoelectric focusing. These methods can be appropriately combined.

[0110] The presence of the AIM or the partial peptide thereof thus obtained can be confirmed by an enzyme immunoassay, a Western blotting method, etc. using an antibody against the AIM.

[0111] The AIM or the partial peptide thereof or the salt thereof or the nucleic acid comprising a base sequence encoding the AIM or the partial peptide thereof (sometimes denoted as AIM herein) obtained as described above can be provided as a medicament for preventing the onset of a renal disease or for the treatment of a renal disease.

[0112] In the present application, a drug that induces the expression of the AIM and a drug that stabilizes the AIM can also be used to replace the AIM.

[0113] Examples of the drug that induces the expression of the AIM include a compound having AIM transcription activity, etc., and examples of the compound include a transcription factor that can bind to the promoter region of the AIM gene, etc. The present inventors have also found that the AIM is expressed in macrophages. Therefore, as the drug that induces the expression of the AIM, a macrophage differentiation inducer can be mentioned. The macrophage differentiation inducer is not particularly limited as long as it can induce the differentiation of macrophages from progenitor cells such as granulocyte-macrophage colony-forming cells (CFU-GM), macrophage colony-forming cells (CFU-M), etc., and can use, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), etc. The transcription factor, GM-CSF, M-CSF can be proteins isolated and purified from mammalian tissues and cells by the known methods described above, or can be proteins chemically synthesized or biochemically synthesized in a cell-free translation system. Alternatively, they can be recombinant proteins produced from transformants into which nucleic acids comprising base sequences encoding the above-described proteins are introduced.

[0114] Examples of the drug stabilizing AIM include a compound inhibiting degradation of AIM, a compound inhibiting excretion into urine, and the like. Examples of the compound inhibiting degradation include a protease inhibitor, a proteasome inhibitor, and the like. Examples of the protease inhibitor include a serine protease inhibitor (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSGF), aprotinin, a trypsin inhibitor, and the like), a cysteine protease inhibitor (E-64, leupeptin, and the like), and the like. Examples of the proteasome inhibitor include lactacystin, MG-115, MG-132, a proteasome inhibitor I, and the like. Examples of the compound inhibiting excretion into urine include a compound imparting a molecular weight preventing passage through a glomerular basement membrane to AIM. Since IgM binds to AIM (WO 2013 / 162021; Arai et al., Cell Reports 3: 1187-1198, 2013), IgM can be mentioned as the compound inhibiting excretion of AIM into urine. However, since administration of IgM itself is feared to cause side effects in the immune system, it is preferable to use a fusion protein obtained by fusing an Fc fragment of IgM, which is a binding site to AIM, and a protein having a molecular weight at a level preventing filtration by a renal tubule and excretion into urine. Although the protein to be fused is not particularly limited, a protein having less side effect concern is preferable, and, for example, albumin can be used. The binding can be direct binding or via a hinge region. Examples of the hinge region include a tandem FLAG tag. Such a molecule can be produced as a single recombinant protein by linking the respective genes encoding by a conventional method. Although the AIM binding to IgM can be derived from any warm-blooded animal, AIM derived from a cat can be excluded.

[0115] In the examples of the present application mentioned below, the AIM knockout mouse shows symptoms of kidney disease under conditions of unilateral ureteral obstruction, one- kidney nephrectomy followed by transient renal ischemia / reperfusion, or bilateral transient renal ischemia / reperfusion. From the above, it is suggested that AIM, a drug inducing expression of AIM or a drug stabilizing AIM, or a compound capable of replacing the function of AIM, which can be searched by the screening method mentioned below, will prevent the onset and progression of kidney disease and treat kidney disease.

[0116] The administration subject of the pharmaceutical composition of the present application containing AIM, a drug inducing expression of AIM, or a drug stabilizing AIM includes, for example, humans and other warm-blooded animals (for example, mice, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, avians, preferably cats, and the like).

[0117] The kidney disease to be the application target of the pharmaceutical composition of the present application containing AIM, a drug inducing AIM expression, or a drug stabilizing AIM is, for example, acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, a kidney disease associated with collagen disease, or IgM nephropathy, preferably, acute renal failure or chronic renal failure can be mentioned. A representative kidney disease associated with collagen disease is, for example, lupus nephritis.

[0118] The pharmaceutical composition of the present application containing AIM, a drug inducing AIM expression, or a drug stabilizing AIM is low in toxicity, and can be administered to a human or other warm-blooded mammal (for example, a mouse, a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, a bird, preferably a cat, and the like) as a liquid as it is, or as a suitable dosage form of a pharmaceutical composition orally or parenterally (for example, intravascular administration, subcutaneous administration, and the like).

[0119] As examples of the composition for parenteral administration, injections, suppositories, and the like are used; the injections can include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections can be prepared according to publicly known methods. The injections can be prepared, for example, by dissolving, suspending, or emulsifying the above-described AIM of the present application, a drug inducing AIM expression, or a drug stabilizing AIM in a sterile aqueous solution or an oily solution generally used for injections. As examples of the aqueous solution for injection, physiological saline, an isotonic solution containing glucose or another auxiliary drug, and the like, which can be used in combination with an appropriate solubilizing agent, for example, an alcohol (for example, ethanol), a polyhydric alcohol (for example, propylene glycol, polyethylene glycol), a nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like, can be used. As examples of the oily solution, sesame oil, soybean oil, and the like, which can be used in combination with benzyl benzoate, benzyl alcohol, and the like as solubilizing agents, can be used. The prepared injection solution is preferably filled in an appropriate ampoule. The suppositories for rectal administration can be prepared by mixing the above-described AIM of the present application, a drug inducing AIM expression, or a drug stabilizing AIM with a general suppository base.

[0120] As the composition for oral administration, solid or liquid dosage forms, particularly tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, and the like can be mentioned. Such compositions are produced by publicly known methods, and can contain carriers, diluents, or excipients commonly used in the field of pharmaceutical preparation. As examples of the carriers or excipients for tablets, lactose, starch, sucrose, magnesium stearate, and the like can be used.

[0121] The above pharmaceutical composition for parenteral or oral administration is conveniently prepared in a pharmaceutical unit dosage form suitable for the dosage of the active ingredient. As examples of such pharmaceutical unit dosage form, tablets, pills, capsules, injections (ampoules), and suppositories can be mentioned. Preferably, the above AIM of the present application, the drug inducing expression of AIM, or the drug stabilizing AIM can be contained in each pharmaceutical unit dosage form, for example, usually 5 to 500 mg, particularly 5 to 100 mg for injections, or 10 to 250 mg for other dosage forms.

[0122] Although the above prophylactic or therapeutic agent of the present application comprising the AIM, the drug inducing expression of AIM, or the drug stabilizing AIM varies in dosage depending on the administration subject, the target disease, the symptoms, the administration route, etc.; for example, when the agent is used for treating / preventing renal diseases in an adult, the AIM of the present application is conveniently administered at about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, and more preferably about 0.1 to 5 mg / kg body weight, based on a single dose, about 1 to 5 times a day, preferably about 1 to 3 times a day, for about 1 to 21 days, preferably about 1 to 14 days, by intravenous injection. In the case of other modes of parenteral and oral administration, similar dosages can be administered. In the case of particularly severe symptoms, the dosage can be increased depending on the symptoms.

[0123] Each of the above compositions can contain any other active ingredient which does not produce an undesirable interaction when formulated with the above AIM, the drug inducing expression of AIM, or the drug stabilizing AIM.

[0124] In addition, the AIM of the present application, the drug inducing expression of AIM, or the drug stabilizing AIM can be used in combination with other drugs useful for treating renal diseases, such as inhibitors (e.g., angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, calcium antagonists, renin inhibitors, alpha blockers, beta blockers, etc.); diuretics (e.g., carbonic anhydrase inhibitors, loop diuretics, thiazide diuretics, antialdosterone drugs, potassium-sparing diuretics, etc.); active vitamin D3 preparations (e.g., calcitriol, alfacalcidol, maxacalcitol, falecalcitriol, etc.); oral carbon adsorbent preparations (e.g., activated charcoal, etc.); drugs for correcting potassium (e.g., sodium polystyrene sulfonate, etc.); phosphorus adsorbents (e.g., calcium carbonate, calcium acetate, sevelamer hydrochloride, lanthanum carbonate, etc.), red blood cell erythropoiesis stimulating factor preparations (erythropoiesis stimulating agents; ESAs) (e.g., erythropoietin preparations), amino acid infusion preparations, etc. The AIM of the present application, the drug inducing expression of AIM, or the drug stabilizing AIM and the above drugs can be administered to a patient simultaneously or non-simultaneously.

[0125] As demonstrated in the examples mentioned below, when the AIM is not detected in the blood of the cat, it is suggested that the cat AIM does not bind to the cat IgM in the blood and is easily excreted into the urine through the basement layer of the glomerulus. Therefore, the cat AIM does not stably exist in the blood, and is considered to indirectly cause kidney disease. Furthermore, it has been clarified that the mouse AIM can bind to the cat IgM in the blood of the cat, and particularly suggests that the SRCR3 domain of the mouse AIM is important for the binding to the cat IgM. Therefore, the present application also provides a prophylactic or therapeutic agent for kidney disease for administration to a cat, which contains an AIM or a partial peptide thereof that binds to the cat IgM, or a nucleic acid comprising a base sequence encoding the same.

[0126] The AIM that binds to the cat IgM in the present application is a protein that contains an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 4, which can bind to the cat IgM.

[0127] Such a protein can also be, for example, a chemically synthesized or biochemically synthesized protein in a cell-free translation system. Alternatively, the protein can be a recombinant protein produced from a transformant incorporating a nucleic acid comprising a base sequence encoding the above-described amino acid sequence.

[0128] The amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 4 indicates an amino acid sequence having about 60% or more, preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology to the amino acid sequence shown in SEQ ID NO: 4. The "homology" can be as described above. More preferably, the amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 4 is an amino acid sequence having about 60% or more, preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more identity to the amino acid sequence shown in SEQ ID NO: 4. As the protein containing an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 4, for example, a protein containing an amino acid sequence substantially identical to the aforementioned amino acid sequence shown in SEQ ID NO: 4 and having an activity substantially the same quality as that of a protein containing the amino acid sequence shown in SEQ ID NO: 4 or the like is preferred. Here, the "activity substantially the same quality" is as defined above.

[0129] Although the AIM protein that can bind to feline IgM can be any AIM protein, as long as it binds to feline IgM in the blood of a cat, for example, it is preferably a protein containing the SRCR3 domain of a mouse-derived AIM. Specifically, for example, mention can be made of an AIM protein containing the amino acid sequence shown in SEQ ID NO: 6 at amino acid numbers 246-348. The protein containing the SRCR3 domain of a mouse-derived AIM can be a protein in which the SRCR3 domain naturally possessed by the AIM is replaced with the SRCR3 domain of a mouse-derived AIM.

[0130] The partial peptide of the AIM that binds to feline IgM can be any, as long as it is a peptide that has the above-described partial amino acid sequence of the AIM that binds to feline IgM and that has substantially the same quality of activity as the AIM. Here, "substantially the same quality of activity" is as defined above.

[0131] Specifically, for example, in the amino acid sequence shown in SEQ ID NO: 4, a partial amino acid sequence containing the SRCR1 domain (amino acid numbers 24-125 of the amino acid sequence shown in SEQ ID NO: 4), the SRCR2 domain (amino acid numbers 139-239 of the amino acid sequence shown in SEQ ID NO: 4), and the SRCR3 domain (amino acid numbers 244-346 of the amino acid sequence shown in SEQ ID NO: 4) respectively, a partial amino acid sequence containing any combination of the SRCR domains, and the like can also be used. The size of the above-described partial peptide is not particularly limited, as long as it contains the above-described functional domain.

[0132] Examples of the DNA containing a base sequence encoding the AIM that binds to feline IgM or a partial peptide thereof include a DNA containing a base sequence identical or substantially identical to the base sequence shown in SEQ ID NO: 3, and the like.

[0133] As the DNA containing a base sequence identical or substantially identical to the base sequence shown in SEQ ID NO: 3, for example, a DNA containing a base sequence having homology of not less than about 60%, preferably not less than about 70%, more preferably not less than about 80%, particularly preferably not less than about 90% to the base sequence shown in SEQ ID NO: 3 and encoding a protein having substantially the same quality of activity as the aforementioned AIM, and the like is used. Here, "homology" is as described above. The DNA is similarly prepared as in the above-described method.

[0134] As the administration subject of the pharmaceutical composition (which contains the AIM that binds to feline IgM of the present application or a partial peptide thereof, or a nucleic acid containing a base sequence encoding the same), mention can be made of a cat.

[0135] As renal diseases to be used as the pharmaceutical composition (which contains the AIM that binds to feline IgM of the present invention or a partial peptide thereof, or a nucleic acid containing a base sequence encoding them), for example, acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy related to collagen disease or IgM nephropathy can be mentioned, and acute renal failure or chronic renal failure can be mentioned preferably. As a representative renal disease related to collagen disease, lupus nephritis can be mentioned.

[0136] The pharmaceutical composition containing the feline IgM-binding AIM of the present invention or its partial peptide, or nucleic acid encoding the base sequence thereof, is low in toxicity and can be administered orally or parenterally as described above. The dosage form, dosage, etc. are as described above.

[0137] As described above, AIM knockout mice exhibit symptoms of renal disease when subjected to unilateral ureteral obstruction, transient renal ischemia / reperfusion (TIR) ​​after uninephrectomy, or bilateral TIR. This suggests that AIM knockout mice subjected to unilateral ureteral obstruction, transient renal ischemia / reperfusion (TIR) ​​after uninephrectomy, or bilateral TIR can be provided as new mouse models of renal disease. Therefore, the present invention provides a screening method for a preventive or therapeutic agent for renal disease, using an animal obtained by subjecting a non-human mammal deficient in AIM expression to unilateral ureteral obstruction, transient renal ischemia / reperfusion (TIR) ​​after uninephrectomy, or bilateral TIR.

[0138] AIM expression defective non-human mammals means non-human mammals in which the expression of endogenous AIM is inactivated, including AIM KO animals prepared from ES cells of which AIM is knocked out (KO), and AIM expression is inactivated by antisense or RNAi technology. Knockout (KO) means preventing the production of complete mRNA by destroying or removing endogenous genes, while knocking down (KD) means suppressing the expression of proteins from mRNA to inactivate endogenous genes. Hereinafter, the AIM KO / KD animals of the present invention are sometimes referred to as “KO / KD animals of the present invention”. The AIM KO animals of the present invention are disclosed in Miyazaki T. et al. (J. Exp. Med., 189, 413-422, 1999 or WO 2013 / 162021).

[0139] The "non-human mammal" that can be a subject of the present invention is not particularly limited as long as it is a non-human mammal for which a transgenic system has been established; examples include mice, rats, cattle, monkeys, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc. Rabbits, dogs, cats, guinea pigs, hamsters, etc. are preferred; in particular, from the viewpoint of preparing disease model animals, rodents that have a relatively short individual development period and life cycle and are easy to breed are more preferred; in particular, mice (for example, pure strains of C57BL / 6, BALB / c, DBA2, etc., and hybrid strains of B6C3F1, BDF1, B6D2F1, ICR) and rats (for example, Wistar, SD, etc.) are preferred.

[0140] In addition to mammals, birds such as chickens can be used for the same purposes as those of the "non-human mammals" that are the subjects of the present invention.

[0141] A specific method for knocking out AIM is disclosed in the aforementioned Miyazaki T. et al. (J. Exp. Med., 189, 413-422, 1999 or WO 2013 / 162021). As other known general methods, a method comprising the following steps can be preferably used: isolating AIM (genomic DNA) from a non-human mammal as a subject by conventional methods, and, for example, (1) destroying the function of the exon or promoter by inserting another DNA fragment (e.g., a drug resistance gene, a reporter gene, etc.) into the exon portion or promoter region, or (2) using the Cre-loxP system or the Flp-frt system to cut out the entirety or part of AIM to delete the gene, or (3) inserting a stop codon into the protein coding region to prevent translation into a complete protein, or (4) inserting a DNA sequence that terminates gene transcription (e.g., a polyadenylation addition signal, etc.) into the transcription region to prevent synthesis of complete mRNA, integrating a DNA chain having a DNA sequence constructed to subsequently inactivate the gene at the AIM locus of the non-human mammal as a subject by homologous recombination, etc. (hereinafter referred to as a targeting vector).

[0142] Homologous recombination can be achieved by, for example, introducing the above-mentioned targeting vector into embryonic stem cells (ES cells).

[0143] ES cells refer to cells derived from the inner cell mass (ICM) of a blastocyst stage zygote, and can be cultured and maintained while retaining an undifferentiated state in vitro. ICM cells are destined to form the blastula, and are stem cells on which all tissues including germ cells are based. The ES cells used can be established cell lines, or newly established cell lines according to the method of Evans and Kaufman (Nature, Vol. 292, p. 154, 1981). For example, in the case of mouse ES cells, ES cells derived from the 129 mouse strain are currently commonly used, but the immunological background thereof is unclear; for the purpose of replacing it with ES cells of a pure line having an immunologically clear genetic background, or the like, ES cells established from C57BL / 6 mice, or from BDF1 mice (F1 of C57BL / 6 and DBA / 2), in which a small number of oocytes collectable from C57BL / 6 have been improved by crossing with DBA / 2, or the like, can also be suitably used. In addition to the advantages of a high number of collectable oocytes and strong oocytes, BDF1 mice have C57BL / 6 mice as their background; thus, ES cells derived therefrom can be advantageously used, because when a disease model mouse is prepared, the genetic background can be replaced with the genetic background of C57BL / 6 mice by backcrossing with C57BL / 6 mice. ES cells can be differentiated into many types of cells, including parietal muscle, visceral muscle, and cardiac muscle, under appropriate conditions by monolayer culture (up to a high density), or suspension culture (up to formation of cell aggregates) [M. J. Evans and M. H. Kaufman, Nature Vol. 292, p. 154, 1981; G. R. Martin, Proceedings of the National Academy of Sciences, USA (Proc. Natl. Acad. Sci. U.S.A.), Vol. 78, p. 7634, 1981; T. C. Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985]; cells of an AIM expression-deficient non-human mammal obtained by differentiating ES cells into which the targeting vector of the present application has been integrated can be used in in vitro cellular biological studies of AIM.

[0144] For example, if the targeting vector is designed to disrupt the function of an exon or a promoter by inserting another DNA fragment into an exon portion or a promoter region of AIM, the vector can assume, for example, the configuration shown below.

[0145] First, to ensure that another DNA fragment is inserted into the exon or promoter portion of AIM by homologous recombination, the targeting vector needs to contain sequences homologous to each target site (5' arm and 3' arm) 5' upstream and 3' downstream in the other DNA fragment.

[0146] Although the other DNA fragments to be inserted are not particularly limited, ES cells in which a targeting vector having drug resistance or reporter gene activity as an indicator has been integrated into their chromosomes can be selected by using a drug resistance gene or a reporter gene. Examples of drug resistance genes and reporter genes include, but are not limited to, neomycin phosphotransferase II (nptII) gene, hygromycin phosphotransferase (HPT) gene, and β-galactosidase (lacZ) gene, chloramphenicol acetyltransferase (cat) gene, respectively.

[0147] Drug resistance or reporter gene are preferably under the control of the promoter that can play a role in mammalian cells that is arbitrarily selected.For example, viral promoters can be mentioned, such as SV40 early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, murine leukemia virus (MoMuLV) LTR and adenovirus (AdV) derived early promoter and promoter for mammalian constitutive protein gene, such as β-actin gene promoter, PGK gene promoter and transferrin gene promoter etc. However, if drug resistance or reporter gene is inserted into AIM, so that it is placed under the control of the endogenous promoter of AIM, then the promoter that controls the transcription of the gene does not need to be present in the targeting vector.

[0148] The targeting vector preferably has a sequence that terminates transcription of mRNA from a gene downstream of the drug resistance or reporter gene (polyadenylation (polyA) signal, also referred to as a terminator); for example, terminator sequences derived from viral genes or from various mammalian or avian genes can be used. Preferably, the SV40 terminator or the like is used.

[0149] Generally, gene recombination in mammals occurs mainly non-homologously; the introduced DNA is inserted randomly into any selected position on the chromosome. Therefore, it is not possible to effectively select only those clones targeted to the endogenous AIM targeted by homologous recombination by selection (positive selection) based on detection of expression of a drug resistance or reporter gene, etc.; it is necessary to confirm the integration site by DNA hybridization or PCR for all selected clones. Therefore, if, for example, a herpes simplex virus-derived thymidine kinase (HSV-tk) gene conferring ganciclovir sensitivity is joined outside the region homologous to the target sequence of the targeting vector, cells having the vector randomly inserted therein cannot grow on a medium containing ganciclovir because they have the HSV-tk gene, while cells targeted to the endogenous AIM locus by homologous recombination become resistant to ganciclovir and are selected because they do not have the HSV-tk gene (negative selection). Alternatively, if a diphtheria toxin gene, for example, is joined to replace the HSV-tk gene, cells having the vector randomly inserted therein die due to the toxin produced by themselves, so that homologous recombinants can also be selected in the absence of the drug.

[0150] Although any of the calcium phosphate coprecipitation method, electroporation method, liposome transfection method, retrovirus infection method, agglutination method, microinjection method, gene gun (particle gun) method, DEAE-dextran method, etc. can be used for introduction of the targeting vector into the ES cell, the electroporation method is generally selected because of easy handling of a large number of cells, etc. since gene recombination in mammals occurs mainly non-homologously, so that the frequency of obtaining homologous recombinants is low as described above. For electroporation, ordinary conditions used for transfection into animal cells can be used as they are; for example, electroporation can be performed by trypsinizing ES cells in the logarithmic growth phase so as to disperse them into single cells, suspending the cells in a medium to obtain a density of 10 6 to 10 8 cells / ml, transferring the cells to a small cup, adding 10 to 100 μg of the targeting vector, and applying an electric pulse of 200 to 600 V / cm.

[0151] Where ES cells into which the targeting vector is integrated can be determined from chromosomal DNA isolated and extracted from colonies obtained by culturing single cells on feeder cells by DNA hybridization or PCR screening; if a drug resistance gene or a reporter gene is used as the other DNA fragment, it is possible to select transformants at the cell stage using expression thereof as an indicator. For example, if a vector containing the nptll gene as a marker gene for positive selection is used, ES cells after transfection treatment are cultured in a medium containing a neomycin-type antibiotic such as G418, and the resulting resistant colonies are selected as candidates for transformants. If a vector containing the HSV-tk gene as a marker gene for negative selection is used, ES cells are cultured in a medium containing ganciclovir, and the resulting resistant colonies are selected as candidates for homologous recombinants. The obtained colonies are transferred to respective culture plates, and trypsinization and medium exchange are repeated, after which a portion is left for culture, and the remainder is subjected to PCR or DNA hybridization to confirm the presence of the introduced DNA.

[0152] When ES cells in which the introduced DNA is confirmed to be integrated are returned to an embryo derived from a non-human mammal source of the same species, the ES cells are integrated into the ICM of the host embryo to form a chimeric embryo. This is transplanted into a recipient mother (female embryonic recipient), and allowed to continue development, thereby obtaining a chimeric KO animal. If the ES cells contribute to the formation of primordial germ cells that will differentiate into oocytes or sperm in the chimeric animal, a germline chimera will be obtained; by mating thereof, a KO animal in which the expression defect of AIM is genetically maintained can be prepared.

[0153] To prepare a chimeric embryo, there are a method in which early embryos up to the morula stage are adhered and aggregated together (aggregation chimera method), and a method in which cells are microinjected into the blastocoele of a blastocyst (injection chimera method). Although the latter has been traditionally widely practiced in the preparation of chimeric embryos using ES cells, as a method that does not require a micromanipulator and can be easily manipulated, a method in which aggregation chimeras are generated by injecting ES cells into the zona pellucida of 8-cell stage embryos, and a method in which aggregation chimeras are generated by co-culturing ES cell clumps and 8-cell stage embryos from which the zona pellucida has been removed and aggregating them have been practiced recently.

[0154] In all cases, the host embryo can be collected from the female non-human mammal that can be used as the female for oocyte collection for transfection into the zygote, as mentioned in the same manner; for example, in the case of mice, in order to make it possible to determine the percentage contribution of the ES cells to the formation of chimeric mice by coat color, it is preferable that the host embryo be collected from a mouse of a strain that shows a coat color different from that of the strain from which the ES cells are derived. For example, in the case of ES cells derived from the 129 mouse strain (coat color: agouti), C57BL / 6 mice (coat color: black) or ICR mice (coat color: albino) are used as the female for oocyte collection; in the case of ES cells derived from C57BL / 6 or DBF1 mice (coat color: black) or from TT2 cells (F1 derived from C57BL / 6 and CBA (coat color: agouti)), ICR mice or BALB / c mice (coat color: albino) can be used as the female for oocyte collection.

[0155] Since the germ line chimera formation ability depends mainly on the combination of the ES cells and the host embryo, it is more preferable to select a combination that shows a high germ line chimera formation ability. For example, in the case of mice, it is preferable to use a host embryo derived from the C57BL / 6 strain or the like for ES cells derived from the 129 strain, and to use a host embryo derived from the BALB / c strain or the like for ES cells derived from the C57BL / 6 strain.

[0156] It is preferable that the female mouse for oocyte collection be about 4 to about 6 weeks old, and that the male mouse for mating be about 2 to about 8 months old of the same strain. Although mating can be by natural mating, it is preferably performed after administration of a gonadotropin (follicle-stimulating hormone, then luteinizing hormone) to induce superovulation.

[0157] In the case of the blastocyst injection method, a blastocyst embryo (for example, in the case of mice, about 3.5 days after mating) is collected from the uterus of the female for oocyte collection (or an early embryo in the morula stage or before or after collection from the oviduct, which can be cultured in a culture medium (described below) for embryo culture until the blastocyst stage), and ES cells (about 10 to about 15 cells) having the targeting vector introduced therein are injected into the blastocyst cavity of the blastocyst with a micromanipulator, after which the embryo is transplanted into the uterus of a pseudopregnant embryorecipient female non-human mammal. As the embryorecipient female non-human mammal, the non-human mammal that can be used as the embryorecipient female for transfection into the zygote can be used in the same manner.

[0158] In the case of the co-culture method, 8-cell stage embryos and morulae (for example, in the case of mice, about 2.5 days after mating) are collected from the oviduct and uterus of the female used for the collection of eggs (or 8-cell stage or early embryos collected from the oviduct before or after, which can be cultured in the medium for embryo culture (described below) until the 8-cell stage or morula stage), and the zona pellucida is dissolved in acidic Tyrode's solution, after which, the ES cell mass (cell number: about 10 to about 15 cells) integrated with the targeting vector is placed in a droplet of the medium for embryo culture covered with mineral oil, the above-mentioned 8-cell stage embryo or morula (preferably 2 embryos) is further placed, and they are co-cultured overnight. The obtained morula or blastocyst is transplanted into the uterus of the embryo recipient female non-human mammal as described above.

[0159] If the transplanted embryo is successfully implanted and the embryo recipient female is pregnant, the chimeric non-human mammal will be obtained by natural birth or caesarean section. The embryo recipient female who has given birth naturally is allowed to continue nursing; if the pups are delivered by caesarean section, the pups can be nursed by a separately provided female (usually the mated and delivered female non-human mammal) for nursing.

[0160] For selection of the germline chimera, if the sex of the ES cell has been determined, a chimeric mouse of the same sex as the ES cell is first selected (usually, a male chimeric mouse is selected because a male ES cell is used), and then a chimeric mouse showing a high ES cell contribution rate (for example, 50% or more) is selected based on a phenotype such as coat color. For example, in the case of obtaining a chimeric mouse from a chimeric embryo between D3 cells (which are male ES cells derived from the 129 mouse strain) and a host embryo derived from a C57BL / 6 mouse, it is preferable to select a male mouse showing a high percentage of agouti coat color. Whether the selected chimeric non-human mammal is a germline chimera can be determined based on the phenotype of the Fl animals obtained by crossing with an appropriate strain of the same animal species. For example, in the case of the above-mentioned chimeric mouse, agouti is dominant over black; therefore, when a male mouse is crossed with a female C57BL / 6 mouse, if the selected male mouse is a germline chimera, the coat color of the obtained Fl is agouti.

[0161] Usually, the germline chimeric non-human mammal (founder) integrated with the targeting vector thus obtained is obtained as a heterozygote in which the AIM is knocked out in either of the homologous chromosomes. To obtain a homozygote in which the AIM is knocked out in both homologous chromosomes, in the Fl animals obtained as described above, sibling animals of the heterozygote can be crossed. Selection of the heterozygote can be determined by, for example, screening the chromosomal DNA isolated and extracted from the tail of the Fl animal by DNA hybridization or PCR. One quarter of the obtained F2 animals will be homozygotes.

[0162] In another preferred embodiment using viruses as targeting vectors, mention can be made of the following method, which comprises infecting ES cells of a non-human mammal with a virus containing DNA comprising a marker gene for positive selection inserted between 5' and 3' arms and a marker gene for negative selection outside said arms (see, for example, Proceedings of the National Academy of Sciences, USA (Proc. Natl. Acad. Sci. USA), Vol. 99, No. 4, pp. 2140-2145, 2002). For example, when using retroviruses or lentiviruses, the cells are seeded into appropriate culture vessels such as culture dishes, the viral vector is added to the culture medium (polybrene can be co-present, if necessary), the cells are incubated for 1 to 2 days, after which the culture is continued in the presence of the selection drug, as described above, and the cells in which the vector has integrated are selected.

[0163] As to the specific method for knocking down AIM, mention can be made of the following method, which comprises introducing DNA encoding an antisense RNA or siRNA (including shRNA) of AIM using the techniques for preparing transgenic animals known per se, and allowing it to be expressed in the cells of the subject non-human mammal, etc.

[0164] DNA comprising a base sequence complementary to a target region of a desired polynucleotide, i.e., DNA hybridizable to a desired polynucleotide can be referred to as "antisense" to the desired polynucleotide.

[0165] An antisense DNA having a base sequence complementary or substantially complementary to a base sequence of a polynucleotide encoding AIM or a portion thereof can be any antisense DNA as long as it comprises a base sequence complementary or substantially complementary to a base sequence of a polynucleotide encoding AIM or a portion thereof and has an effect of inhibiting the expression of the polynucleotide.

[0166] A base sequence substantially complementary to a polynucleotide encoding AIM is, for example, a base sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more homology to a base sequence of a complementary strand of the polynucleotide of the overlapping region. The base sequence homology herein can be calculated, for example, using the homology calculation algorithm NCBIBLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (Expect value = 10; Allow gaps; Filter = ON; Match score = 1; Mismatch score = -3).

[0167] Specifically, in the entire base sequence of the complementary strand of the polynucleotide encoding the AIM, (a) in the case of antisense DNA intended to suppress translation, antisense DNA having about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more homology with the complementary strand of the base sequence of a portion encoding the N-terminal portion of the AIM (e.g., the base sequence in the vicinity of the initiation codon, etc.) is suitable, and (b) in the case of antisense DNA intended to degrade RNA with RNase H, antisense DNA having about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more homology with the complementary strand of the entire base sequence of the polynucleotide encoding the AIM including introns is suitable.

[0168] Specifically, when the subject non-human mammal is a mouse, mention can be made of antisense DNA comprising a base sequence complementary or substantially complementary to the base sequence registered under GenBank Accession No. AF011428 or a portion thereof, preferably, antisense DNA comprising a base sequence complementary to the base sequence or a portion thereof, etc.

[0169] Antisense DNA having a base sequence complementary or substantially complementary to the base sequence of the polynucleotide encoding the AIM or a portion thereof can be designed and synthesized based on the base sequence information of the DNA encoding the cloned or determined AIM (hereinafter also referred to as "antisense DNA of the present application"). Such antisense DNA is capable of inhibiting the replication and expression of the AIM. Specifically, the antisense DNA of the present application is capable of hybridizing with RNA (mRNA or initial transcription product) transcribed from the AIM, and is capable of inhibiting the synthesis (processing) or function (translation into protein) of the mRNA.

[0170] The target region of the antisense DNA of the present application is not particularly limited in terms of its length, as long as translation into the AIM is inhibited due to hybridization of the antisense DNA; the target region can be the entire sequence or a partial sequence of the mRNA encoding the protein, and is at least about 10 bases in length, and at most the entire sequence of the mRNA or initial transcription product. Specifically, the 5' terminal hairpin loop, 5' terminal 6 base pair repeat, 5' terminal untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3' terminal untranslated region, 3' terminal palindrome region, or 3' terminal hairpin loop, etc. of the AIM can be selected as a preferred target region of the antisense DNA, but any other region of the AIM gene can also be selected as a target. For example, the intron portion of the gene can also be a target region.

[0171] Furthermore, the antisense DNA of the present application can be antisense DNA which not only hybridizes with the mRNA or primary transcription product of AIM to inhibit translation into a protein, but also is capable of binding to AIM as a double-stranded DNA to form a triple strand (triplex) so as to inhibit transcription into RNA. Alternatively, the antisense DNA of the present application can be antisense DNA which forms a DNA:RNA hybrid to induce degradation by RNase H.

[0172] DNA encoding a ribozyme capable of specifically cleaving the mRNA or primary transcription product of AIM (in the case of the primary transcription product, including the intron portion) within the coding region can also be encompassed in the antisense DNA of the present application. One of the most versatile ribozymes is a self-splicing RNA found in infectious RNAs such as viroids and viroids, and hammerhead, hairpin, etc. are known. The hammerhead type exhibits enzymatic activity, has a length of about 40 bases, and can specifically cleave a target mRNA by making several bases (about 10 bases in total) flanking both ends of the hammerhead moiety into a sequence complementary to the desired cleavage site of the mRNA. Because this type of ribozyme uses only RNA as a substrate, it provides an additional advantage of not attacking genomic DNA. If the AIM mRNA itself assumes a double-stranded structure, the target sequence can be made single-stranded by using a hybrid ribozyme prepared by further ligating a sequence derived from a viral nucleic acid which can specifically bind to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, the ribozyme can be a hybrid ribozyme prepared by further ligating a sequence modified from a tRNA which promotes translocation of the transcription product to the cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].

[0173] In this context, a double-stranded RNA consisting of an oligo-RNA (oligo-RNA) homologous to a partial sequence in the coding region of the mRNA or primary transcription product of AIM (in the case of the primary transcription product, including the intron portion) and a complementary strand thereto (i.e., so-called single-stranded interfering RNA (siRNA)) can also be used to prepare the KD animal of the present application. It is known that so-called RNA interference (RNAi), which is a phenomenon in which mRNA homologous to the RNA is degraded when siRNA is introduced into a cell, occurs in nematodes, insects, plants, etc.; since this phenomenon has also been confirmed to occur widely in animal cells [Nature, 411(6836): 494-498 (2001)], siRNA has been used as an alternative technique to ribozymes. siRNA can be appropriately designed using commercially available software (e.g., RNAiDesigner; Invitrogen) based on base sequence information of the mRNA targeted.

[0174] The antisense oligo-DNA and ribozyme of the present application can determine the target sequence of mRNA or primary transcription product by the AIM-based cDNA sequence or genomic DNA sequence, and be prepared by synthesizing the sequence complementary thereto using a commercially available DNA / RNA synthesizer (Applied Biosystems, Beckman, etc.). By inserting the synthesized antisense oligo-DNA or ribozyme downstream of the promoter in the expression vector through a sequence of an appropriate linker (adaptor molecule) used as needed, a DNA expression vector encoding the antisense oligo-RNA or ribozyme can be prepared. Examples of the expression vector that can be preferably used herein include plasmids from E. coli, Bacillus subtilis, or yeast, bacteriophages such as lambda phage, retroviruses such as Moloney leukemia virus, animal or insect viruses such as lentivirus, adeno-associated virus, vaccinia virus, and baculovirus, etc. In particular, plasmids (preferably plasmids from E. coli, Bacillus subtilis, or yeast, particularly plasmids from E. coli) and animal viruses (preferably retroviruses, lentivirus) are preferred. Examples of the promoter include viral promoters such as SV40 early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, mouse leukemia virus (MoMuLV) LTR, and adenovirus (AdV)-derived early promoter, and promoters for mammalian constitutive protein genes such as beta-actin gene promoter, PGK gene promoter, and transferrin gene promoter, etc.

[0175] The DNA expression vector encoding a longer antisense RNA (e.g., a full-length complementary strand of AIM mRNA, etc.) can be prepared by inserting the AIM cDNA cloned by a conventional method downstream of the promoter in the expression vector in the opposite direction through a sequence of an appropriate linker (adaptor molecule) used as needed.

[0176] Meanwhile, the DNA encoding the antisense RNA, siRNA, shRNA, or miRNA can be prepared by separately synthesizing DNA encoding the sense strand and DNA encoding the antisense strand, and inserting them into an appropriate expression vector. As the siRNA expression vector, an siRNA expression vector having a Pol III system promoter such as U6 or Hl can be used. In this case, in an animal cell into which the vector is integrated, the sense strand and the antisense strand are transcribed and annealed to form siRNA. The shRNA can be prepared by inserting a unit containing a sense strand and an antisense strand separated by a length base which allows the formation of an appropriate loop structure (e.g., about 15 to 25 bases) into an appropriate expression vector. As the shRNA expression vector, a shRNA expression vector having a Pol III system promoter such as U6 or Hl can be used. In this case, the shRNA transcribed in an animal cell into which the expression vector is integrated forms a loop by itself, and then is processed by an endogenous enzyme Dicer enzyme or the like to form a mature siRNA. Alternatively, it is also possible to achieve knockdown by RNAi by expressing micro RNA (miRNA) containing the siRNA sequence targeted by using a Pol II promoter. In this case, tissue-specific knockdown is also possible by a promoter showing tissue-specific expression.

[0177] To introduce the expression vector containing the DNA encoding the antisense RNA, siRNA, shRNA, or miRNA of AIM into a cell, a method known per se is appropriately used depending on the target cell. For example, to introduce an early embryo such as a fertilized egg, a microinjection method is used. To introduce an ES cell, a calcium phosphate coprecipitation method, an electroporation method, a lipofection method, a retrovirus infection method, an agglutination method, a microinjection method, a particle gun method, a DEAE-dextran method, or the like can be used. Alternatively, when a retrovirus, a lentivirus, or the like is used as the vector, it is sometimes possible to conveniently achieve transfection by adding the virus to an early embryo or an ES cell, and culturing the embryo or the cell for 1 to 2 days so as to infect the cell with the virus. Regeneration of an individual from an ES cell (establishment of a founder), subculturing (preparation of a homozygote), or the like can be performed as described above with respect to the KO animal of the present application.

[0178] In a preferred embodiment, the expression vector containing the DNA encoding the antisense RNA, siRNA, shRNA, or miRNA of AIM is introduced into an early embryo (fertilized egg) of a non-human mammal as a subject by microinjection.

[0179] DNA microinjection into a zygote can be performed by a conventional method using a generally known device such as a micromanipulator. Briefly, a zygote placed in a droplet of a culture medium for embryo culture is aspirated and immobilized using a holding pipette, and a DNA solution is directly injected into a male or female pronucleus, preferably a male pronucleus, using an injection pipette. The DNA for introduction is preferably used after highly purified using CsCl density gradient ultracentrifugation or an anion exchange resin column, etc. It is also preferable that the DNA for introduction be previously linearized by cleaving a vector portion using a restriction enzyme.

[0180] After the introduction of DNA, the zygote is cultured in a culture medium for embryo culture in 5% gaseous carbon dioxide / 95% atmosphere by a droplet culture method, etc., until the 1-cell stage to the blastocyst stage, after which it is transplanted to the oviduct or uterus of a female non-human mammal that is made pseudopregnant for the purpose of accepting the embryo. The female non-human mammal for accepting the embryo can be any of the same species as the animal from which the early embryo to be transplanted is derived; for example, when mouse early embryos are transplanted, it is preferable to use a female ICR mouse (preferably about 8 to about 10 weeks old), etc. A known method of making the female non-human mammal for accepting the embryo pseudopregnant is, for example, a method comprising mating the female with a male non-human mammal of the same species whose vas deferens is excised (vasectomy) (for example, in the case of a mouse, with a male ICR mouse (preferably about 2 months or older)), and selecting a female that is confirmed to have a vaginal plug.

[0181] The female used for embryo culture can be a female that has spontaneously ovulated, or a female that receives an administered luteinizing hormone-releasing hormone (often abbreviated as LHRH) or an analog thereof to induce fertility before mating with a male whose vas deferens is excised (vasectomy). Examples of the LHRH analog include [3,5-DiI-Tyr 5 ]-LH-RH, [Gln 8 ]-LH-RH, [D-Ala 6 ]-LH-RH, [des-Gly 10 ]-LH-RH, [D-His(Bzl) 6 ]-LH-RH, and ethylamides thereof, etc. The amount of the administered LHRH or analog thereof and the timing of mating with the male non-human mammal after administration vary depending on the species of the non-human mammal. For example, when the non-human mammal is a mouse (preferably an ICR mouse, etc.), it is generally preferable to mate the female mouse with the male mouse about 4 days after administration of the LHRH or analog thereof; the amount of the administered LHRH or analog thereof is generally about 10 to 60 μg per individual, preferably about 40 μg per individual.

[0182] Generally, if the early embryo to be transplanted is at the morula stage or later, the embryo is transplanted into the uterus of the female to receive the embryo; if the early embryo is at a stage prior to the morula stage (e.g., 1 -cell stage to 8-cell stage embryo), the embryo is transplanted into the oviduct. Depending on the developmental stage of the embryo to be transplanted, a female to receive the embryo is appropriately used after a given number of days have passed after becoming pseudopregnant. For example, in the case of mice, a female mouse that has become pseudopregnant for about 0.5 days is preferably used for transplantation of 2-cell stage embryos, and a female mouse that has become pseudopregnant for about 2.5 days is preferably used for transplantation of blastocyst embryos. After the female to receive the embryo is anesthetized (preferably, using Avertin, Nembutal, or the like), laparotomy is performed, the ovary is pulled out, and the early embryo (about 5 to about 10 embryos) suspended in the medium used for embryo culture is injected into the vicinity of the abdominal ostium of the oviduct or the oviduct junction of the uterine horn using a pipette for embryo transplantation.

[0183] When the transplanted embryo is successfully implanted and the embryo recipient female is impregnated, the non-human mammal offspring will be obtained by natural birth or caesarean section. The embryo recipient female that has naturally given birth is allowed to continue nursing; when the offspring is delivered by caesarean section, the offspring can be nursed by a separately provided female for nursing (e.g., in the case of mice, a female mouse (preferably, a female ICR mouse or the like) that has normally mated and delivered).

[0184] It is ensured that the DNA encoding the antisense RNA, siRNA, shRNA, or miRNA of AIM is transferred at the zygote cell stage, so that the introduced DNA will be present in all of the germ line cells and somatic cells of the non-human mammal of the subject. Whether the introduced DNA is integrated in the chromosomal DNA can be determined, for example, by screening the chromosomal DNA isolated and extracted from the tail of the offspring by DNA hybridization or PCR. The presence of the expression vector in the germ line cells of the offspring non-human mammal (F0) obtained as described above means that the expression vector is present in all of the germ line cells and somatic cells of all of the animals in the subsequent generation (F1).

[0185] Generally, the F0 animal is obtained as a heterozygote having the introduced DNA in either homologous chromosome. Different F0 individuals have the introduced DNA randomly inserted on different chromosomes, unless the insertion is by homologous recombination. To obtain a homozygote having the expression vector in both homologous chromosomes, the F0 animal is crossed with a non-transgenic animal to make an F1 animal, and can be crossed with its sibling animal that is a heterozygote having the introduced DNA in either homologous chromosome. If the introduced DNA is integrated only on one locus, 1 / 4 of the F2 animals obtained will be homozygotes.

[0186] In another preferred embodiment using a virus as a vector, as in the case of the KO animal described above, mention can be made of a method comprising infecting an early embryo or ES cell of a non-human mammal with a virus containing DNA encoding an antisense RNA, siRNA, shRNA or miRNA of AIM. When a zygote is used as a cell, it is preferable to remove the zona pellucida before infection. After culturing for 1 to 2 days after infection of the virus vector, in the case of an early embryo, the zygote is transplanted into the oviduct or uterus of a female non-human mammal rendered pseudopregnant as described above for receiving the embryo, or in the case of an ES cell, the zygote is continued to be cultured with the addition of a selection drug as described above, and cells incorporating the vector are selected.

[0187] Furthermore, as described in Proceedings of the National Academy of Sciences, USA (Proc. Natl. Acad. Sci. USA), Vol. 98, pp. 13090-13095, 2001, spermatogonial cells collected from a male non-human mammal are infected with a virus vector during co-culture with STO feeder cells, after which the spermatogonial cells are injected into the vas deferens of a male sterile non-human mammal, and the male sterile non-human mammal is mated with a female non-human mammal, whereby a pup heterozygous-Tg (+ / -) for DNA encoding an antisense RNA, siRNA, shRNA or miRNA of AIM can be effectively obtained.

[0188] The non-human mammal of the present application having a deficiency in expression of AIM gene described in Miyazaki T. et al. (J. Exp. Med., 189, 413-422, 1999 or WO 2013 / 162021) or obtained by the above-mentioned method has the following characteristics under conditions of unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy or bilateral transient renal ischemia / reperfusion: (1) In the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion, necrotic tubular cells accumulate and the renal parenchyma becomes fibrotic compared to the control kidney, (2) In the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion, glomerular structure collapses and the glomerulus becomes fibrotic compared to the control kidney, (3) In the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion, expression of inflammatory cytokines is promoted compared to the control kidney, (4) In the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion, infiltration of macrophages is promoted compared to the control kidney, (5) The blood BUN value of the AIM expression-deficient non-human mammal is higher compared to the control non-human mammal, (6) The survival rate of the AIM expression-deficient non-human mammal is lower than that of a control non-human mammal, (7) The administration of AIM improves the aforementioned (1) to (6). These phenotypes have not been reported at least in the AIM KO mice known from conventional publications. Specifically, they are similar to the pathology of chronic kidney disease associated with acute kidney disease (acute kidney failure) triggered by ureteral compression / obstruction caused by ureteral stones, ascending urinary tract infections, tumors, etc., and chronic kidney disease associated with ischemic kidney disease caused by tumor masses, thrombi, or renal vascular stenosis or obstruction caused by diabetes, hypertension, etc., which is a new finding.

[0189] (1) Necrotic tubular cells accumulate and the renal parenchyma becomes fibrotic in the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion compared to the control kidney (normal kidney, or a kidney without ureteral obstruction or transient renal ischemia / reperfusion, hereinafter the same), which means that by unilateral ureteral obstruction, transient renal ischemia / reperfusion after nephrectomy, or bilateral transient renal ischemia / reperfusion on the AIM expression-deficient non-human mammal of the present application, accumulation of necrotic tubular cells and extensive fibrosis of the renal parenchyma are observed in the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion compared to the control kidney. The accumulation of necrotic tubular cells can be confirmed by hematoxylin-eosin staining of kidney tissue sections, and the fibrosis of the renal parenchyma can be confirmed by simultaneous staining with Azan staining and hematoxylin staining. In the examples mentioned below, a significant difference was observed in the AIM knockout mice compared to the control kidney from day 14 after ureteral obstruction. In addition, a significant difference was observed in the AIM knockout mice compared to the control kidney from day 7 after transient renal ischemia / reperfusion.

[0190] (2) Glomerular structures collapse and glomeruli become fibrotic in the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion compared to the control kidney, which means that by unilateral ureteral obstruction, transient renal ischemia / reperfusion after nephrectomy, or bilateral transient renal ischemia / reperfusion on the AIM expression-deficient non-human mammal of the present application, collapse of glomerular structures and fibrosis of glomeruli are observed in the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion compared to the control kidney. The collapse of glomerular structures can be confirmed by hematoxylin-eosin staining of kidney tissue sections, and the fibrosis of glomeruli can be confirmed by simultaneous staining with Azan staining and hematoxylin staining. In the examples mentioned below, a significant difference was observed in the AIM knockout mice compared to the normal kidney from day 14 after ureteral obstruction. In addition, a significant difference was observed in the AIM knockout mice compared to the normal kidney from day 7 after transient renal ischemia / reperfusion.

[0191] (3) In the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion, expression of inflammatory cytokines is promoted compared with the control kidney, which means that by unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion to the AIM expression deficient non-human mammal of the present application, expression of MCP-I, IL-I β, and IL-6 is promoted in the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion compared with the control kidney. The promotion of expression can be confirmed by, for example, quantitative RT-PCR, Northern blotting method, and the like. In the example mentioned below, a significant difference in MCP-I and IL-6 was observed in the AIM knockout mouse compared with the normal kidney, and a tendency of high expression of IL-I β was also observed.

[0192] (4) In the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion, infiltration of macrophages is promoted compared with the control kidney, which means that by unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion to the AIM expression deficient non-human mammal of the present application, the number of macrophages (Mac-I positive cells) is higher in the kidney subjected to ureteral obstruction or transient renal ischemia / reperfusion compared with the control kidney. The counting of the number of cells can be confirmed by, for example, identifying Mac-I positive cells with a flow cytometer, and the like. In the example mentioned below, it was confirmed that the ratio of macrophages was higher in the AIM knockout mouse compared with the normal kidney.

[0193] (5) The blood BUN value of the AIM expression deficient non-human mammal is higher compared with the control non-human mammal, which means that by unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion to the AIM expression deficient non-human mammal of the present application, the blood BUN value of the AIM expression deficient non-human mammal is higher compared with the control non-human mammal.

[0194] (6) The survival rate of the AIM expression deficient non-human mammal is lower compared with the control non-human mammal, which means that by unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion to the AIM expression deficient non-human mammal of the present application, the survival rate is lower compared with the control non-human mammal.

[0195] (7) AIM administration improves the foregoing (1) to (6), which means that: by unilateral ureteral obstruction, one kidney resection followed by transient renal ischemia / reperfusion or bilateral transient renal ischemia / reperfusion to the AIM expression-deficient non-human mammal of the present application, followed by AIM administration, a significant decrease in BUN value is observed in the accumulation of necrotic renal tubular cells and the disintegration of glomerular structure, and the fibrosis of renal parenchyma and glomerulus associated therewith is significantly improved, the expression of inflammatory cytokines decreases, the survival rate improves, and the infiltration of macrophages is inhibited.

[0196] These findings indicate that the AIM expression-deficient non-human mammal subjected to unilateral ureteral obstruction, one kidney resection followed by transient renal ischemia / reperfusion or bilateral transient renal ischemia / reperfusion is useful as an animal model of renal disease, and can be further used for screening of a prophylactic or therapeutic drug for renal disease. Specifically, the screening method of the present application comprises the following steps: (1) a step of administering a test substance to the AIM expression-deficient non-human mammal under the condition of unilateral ureteral obstruction, one kidney resection followed by transient renal ischemia / reperfusion or bilateral transient renal ischemia / reperfusion, (2) a step of observing any one or more of the following properties of the AIM expression-deficient non-human mammal to which the test substance is administered: (i) accumulation of necrotic renal tubular cells and renal parenchymal fibrosis, (ii) disintegration and fibrosis of glomerular structure, (iii) expression level of inflammatory cytokines in the kidney, (iv) ratio of macrophages in the kidney, (v) BUN value, (vi) survival rate, (3) a step of selecting the test substance that improves any one or more of the above properties by comparison with those in the absence of administration of the test substance.

[0197] In the screening method of the present application, unilateral ureteral obstruction means obstruction of the ureter of one kidney. The ureteral obstruction can induce necrosis of renal tubules and glomeruli in the renal parenchyma of the specific kidney which is obstructed, and subsequently inflammation and fibrosis, and finally functional disorder of the kidney. In addition, one kidney resection followed by transient renal ischemia / reperfusion means that one kidney is previously separated, obstruction in the renal artery of the remaining kidney is induced to cause ischemia after 2 weeks, and relief of the obstruction allows reperfusion of blood flow after 30 min. This transient ischemia causes progression of renal tubular necrosis and mild fibrosis for about 3 days, thereby resulting in a decrease in kidney function. Bilateral transient renal ischemia / reperfusion means obstruction of the renal arteries of both kidneys to induce ischemia without separating one kidney, and relief of the obstruction allows reperfusion of blood flow after 30 min.

[0198] As the test substance to be administered to the AIM expression-deficient non-human mammal, a protein, a peptide, an antibody, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract, plasma, etc. can be used. The timing of administration of the test substance can be before or at the same time as the unilateral ureteral obstruction, the unilateral nephrectomy, the transient renal ischemia / reperfusion or the bilateral transient renal ischemia / reperfusion, or after the observation of the above-mentioned properties after the unilateral ureteral obstruction, the unilateral nephrectomy, the transient renal ischemia / reperfusion or the bilateral transient renal ischemia / reperfusion of the AIM expression-deficient non-human mammal. The method of administration can be oral or parenteral. For oral administration, it can also be administered by mixing with feed or drinking water. As the parenteral administration, intraperitoneal administration, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, administration by drip, etc. can be mentioned. The administration can include single administration or multiple administration.

[0199] After the administration of the test substance, the properties of the AIM expression-deficient non-human mammal to which the test substance is administered are observed generally 3 or 4 days or later, preferably 7 days or later, more preferably 14 days or later. The accumulation of necrotic tubular cells and renal parenchymal fibrosis related thereto can be observed by staining the kidney tissue sections isolated from the kidney of the aforementioned mammal with hematoxylin-eosin or Azan and hematoxylin, and converting the staining level thereof into a numerical value. Similarly to the above, the level of glomerular disintegration and glomerular fibrosis can be observed by staining the aforementioned kidney tissue sections of the isolated kidney with hematoxylin-eosin or Azan and hematoxylin, and converting the staining level thereof into a numerical value. The expression level of inflammatory cytokines in the kidney can be measured by quantitative RT-PCR, etc. Examples of the inflammatory cytokines to be measured here include MCP-1, IL-1β and IL-6. The ratio of macrophages in the kidney can be confirmed by identifying Mac-1 positive cells with a flow cytometer, etc. The BUN value can be observed by measuring the blood urea nitrogen concentration.

[0200] The observation results of the above-mentioned properties obtained as described above are compared with the results in the case where the test substance is not administered. Alternatively, a correlation diagram of the presence or absence of kidney disease and the above-mentioned properties is prepared in advance, and the observation results of the above-mentioned properties obtained can be compared with the correlation diagram. The comparison is preferably made based on the presence or absence of a significant difference.

[0201] When the observation results of the above-described properties obtained are improved as compared with those in the case where the test substance is not administered, the so-called test substance can be selected as a preventive or therapeutic agent for kidney disease. Here, improved means (i) the level of accumulation of necrotic renal tubular cells (the level of hematoxylin-eosin staining, or the level of Azan staining and hematoxylin staining) is significantly lower than the level in the case where the test substance is not administered, (ii) the level of disintegration of glomerular structure (the level of hematoxylin-eosin staining, or the level of Azan staining and hematoxylin staining) is significantly lower than the level in the case where the test substance is not administered, (iii) the expression level of an inflammatory cytokine in the kidney is significantly lower than the level in the case where the test substance is not administered, (iv) the ratio of macrophages in the kidney is significantly lower than the level in the case where the test substance is not administered, (v) the BUN value is significantly lower than the level in the case where the test substance is not administered, and (vi) the survival rate is significantly higher than the level in the case where the test substance is not administered.

[0202] When the test substance selected above is used as a preventive or therapeutic agent for kidney disease, it can be formulated in the same manner as the AIM of the present application, and administered through a similar administration route and in a similar dose. The kidney disease to be targeted as a preventive or therapeutic agent can be similar to those described above.

[0203] In addition, since the AIM expression-deficient non-human mammal can be used as an animal model of kidney disease under conditions of unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion, the mammal can be used in an evaluation method for a preventive or therapeutic drug for kidney disease. Therefore, the present application also provides a method for evaluating the preventive or therapeutic effect of a preventive or therapeutic agent for kidney disease, which comprises using an animal obtained by subjecting an AIM expression-deficient non-human mammal to unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion. Specifically, the evaluation method of the present application comprises the following steps: (1) a step of administering a preventive or therapeutic agent for kidney disease to an AIM expression-deficient non-human mammal under conditions of unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion, (2) a step of observing any one or more of the following properties of the AIM expression-deficient non-human mammal to which the preventive or therapeutic agent for kidney disease is administered: (i) accumulation of necrotic renal tubular cells and renal parenchymal fibrosis, (ii) disintegration and fibrosis of glomerular structure, (iii) expression level of an inflammatory cytokine in the kidney, (iv) ratio of macrophages in the kidney, (v) BUN value, (vi) survival rate, (3) a step of evaluating the effect of the preventive or therapeutic agent for kidney disease by comparing any one or more of the above-mentioned characteristics with those in the case where the preventive or therapeutic agent for kidney disease is not administered.

[0204] The preventive or therapeutic agent for kidney disease to be administered to the AIM expression-deficient non-human mammal in the evaluation method of the present application can be a known preventive or therapeutic agent for kidney disease. Examples thereof include, but are not limited to, inhibitors (e.g., angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, calcium antagonists, renin inhibitors, alpha blockers, beta blockers, etc.); diuretics (e.g., carbonic anhydrase inhibitors, loop diuretics, thiazide diuretics, anti-aldosterone drugs, potassium-sparing diuretics, etc.); active vitamin D3 preparations (e.g., calcitriol, alfacalcidol, maxacalcitol, falecalcitriol, etc.); oral carbon adsorbent preparations (e.g., activated charcoal, etc.); drugs for correcting potassium (e.g., sodium polystyrene sulfonate, etc.); phosphorus adsorbents (e.g., calcium carbonate, calcium acetate, sevelamer hydrochloride, lanthanum carbonate, etc.), red blood cell erythropoiesis stimulating factor preparations (erythropoiesis stimulating agents, ESAs) (e.g., erythropoietin preparations), amino acid infusion preparations, etc. The administration period, administration method, administration frequency, etc. of the preventive or therapeutic agent for kidney disease can be the same as those in the above-mentioned screening method.

[0205] The observation method of the characteristics to be observed by the evaluation method of the present application can be performed according to the description of the above-mentioned screening method. When the observation results of the above-mentioned characteristics obtained by the evaluation method are improved to a greater extent as compared with the case where the preventive or therapeutic agent for kidney disease is not administered, the test substance can be evaluated as having a higher preventive or therapeutic effect as a preventive or therapeutic agent for kidney disease. As used herein, improved means the same as above.

[0206] In the following examples of the present application mentioned below, it was confirmed that the blood AIM concentration of patients with chronic kidney disease is correlated with the kidney function (eGFR: glomerular filtration rate). Specifically, it was confirmed that the kidney function of chronic kidney disease patients having a blood AIM concentration lower than a given level decreases after 2-3 years. The above suggests that by measuring the blood AIM concentration of test subjects, the prognosis of chronic kidney disease patients can be predicted. Thus, the present application provides a method of predicting the prognosis of patients with kidney disease, which comprises measuring the concentration of AIM in a sample of a subject.

[0207] Although the test subject to which the prediction method of the present application is applicable is not particularly limited, there can be mentioned, for example, a test subject having a risk of developing acute renal failure or chronic kidney disease or being suspected of having developed acute renal failure or chronic kidney disease. Although the chronic kidney disease is not limited, it includes, for example, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease, or IgM nephropathy, and the like.

[0208] The sample to be used in the prediction method of the present application is not particularly limited, as long as it is collected from the above-described test subject, and contains an AIM gene product (e.g., RNA, protein, a cleavage product thereof, etc.) to be a measurement target. Examples thereof include body fluids such as blood, plasma, serum, lymph, urine, sweat, saliva, synovial fluid, etc. or fractions thereof, and cells contained therein, particularly macrophages, etc., and preferably, there can be mentioned blood, plasma, and serum.

[0209] The AIM concentration of the sample collected from the test subject can be measured by preparing an RNA (e.g., total RNA, mRNA) fraction from the aforementioned sample, and measuring the transcription product of the AIM gene contained in the fraction. Although the RNA fraction can be prepared by using known methods such as guanidine-CsCl ultracentrifugation, AGPC method, etc., high-purity total RNA can be rapidly and conveniently prepared from trace amounts of macrophages by using a commercially available RNA extraction kit (e.g., RNeasy Mini Kit; manufactured by QIAGEN, etc.). Examples of the method for detecting the transcription product of the AIM gene in the RNA fraction include methods using hybridization (northern blotting, dot blotting, DNA chip analysis, etc.), methods using PCR (RT-PCR, competitive PCR, real-time PCR, etc.), and the like. Quantitative PCR methods such as competitive PCR, real-time PCR, etc. are preferable, because the change in the expression of the AIM gene can be rapidly, conveniently, and highly quantitatively detected from trace amounts of macrophages.

[0210] When northern blotting or dot blotting hybridization is employed, the transcription product of the AIM gene can be measured by using a nucleic acid (probe) capable of hybridizing with the transcription product of the gene. Examples of such a nucleic acid include a nucleic acid capable of hybridizing with a nucleic acid comprising a base sequence (e.g., the base sequence shown in SEQ ID NO: 1) shown by the transcription product of the AIM gene under high stringent conditions. The high stringent conditions are the above-described conditions, etc. More preferably, there can be mentioned a nucleic acid comprising a base sequence complementary to a base sequence (e.g., the base sequence shown in SEQ ID NO: 1) shown by the transcription product of the AIM gene.

[0211] The nucleic acid to be used as a probe may be double-stranded or single-stranded. In the case of double-stranded nucleic acid, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of a single-stranded nucleic acid, an antisense strand may be used. Although the length of the nucleic acid is not particularly limited as long as it can specifically hybridize with the target nucleic acid, for example, it is not less than about 15 bases, preferably not less than about 30 bases. The nucleic acid is preferably labeled with a labeling agent capable of detecting and quantifying the target nucleic acid. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used. As the radioisotope, for example, [ 32 P]、[ 3 H]、[ 14 C], etc. As the above-mentioned enzymes, stable enzymes with high specific activity are preferred; for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc. can be used. Fluorescent substances include, for example, fluorescamine and fluorescein isothiocyanate. Luminescent substances include, for example, luminol, luminol derivatives, luciferin, and lucigenin. Furthermore, biotin-(strept)avidin can be used for probe binding and labeling.

[0212] When using RNA hybridization, the RNA fraction prepared above is separated by gel electrophoresis, transferred to a membrane made of nitrocellulose, nylon, polyvinylidene fluoride, or the like, and hybridized under the aforementioned high stringency conditions in a hybridization buffer containing the labeled probe prepared above. The amount of label bound to the membrane is measured for each band by a suitable method, thereby measuring the expression level of the AIM gene. Alternatively, in the case of dot blot, the expression level of the AIM gene can be measured by subjecting a membrane spotted with the RNA fraction to a hybridization reaction in the same manner and measuring the amount of label in the spot.

[0213] In another preferred embodiment, a quantitative PCR method is used as a method for measuring the AIM concentration. Examples of quantitative PCR include competitive PCR, real-time PCR, and the like.

[0214] The oligonucleotide set used as primers in PCR is not particularly limited, as long as they can each specifically hybridize to the sense strand (coding strand) and the anti-sense strand (non-coding strand) of the transcription product of the AIM gene, and can amplify the DNA fragment sandwiched by them. For example, a set of oligo DNAs each having a length of about 15 to about 100 bases, preferably about 15 to about 50 bases, and designed to amplify a DNA fragment of about 100 bp to 1 kbp can be mentioned. More specifically, as the oligonucleotide set used as primers, a nucleic acid capable of hybridizing to a nucleic acid (anti-sense strand) comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 under high stringent conditions can be mentioned. As used herein, high stringent conditions are as defined above. More preferably, a nucleic acid comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1, and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid can be mentioned.

[0215] In competitive RT-PCR, the amount of the desired DNA is determined by allowing a known amount of another template nucleic acid that can be amplified by the primer set capable of amplifying the desired DNA to coexist in the reaction solution as a competitor to cause a competitive amplification reaction, and comparing the amount of the amplification product. Therefore, when competitive RT-PCR is used, in addition to the above-described primer set, a known amount of a competitor nucleic acid that can be amplified with the primer set and can be distinguished from the amplification product of the target nucleic acid (i.e., the transcription product of the AIM gene) after amplification (e.g., different amplification size, different migration pattern of the fragment after restriction enzyme treatment, etc.) is used. Because amplification occurs competitively when the target nucleic acid and the competitor nucleic acid compete for the primer, the quantitative ratio of the amplification product reflects the quantitative ratio of the initial template. The competitor nucleic acid can be DNA or RNA. In the case of DNA, cDNA is synthesized from the RNA fraction prepared as described above by a reverse transcription reaction, and PCR can be performed in the presence of the above-described primer set and the competitor. In the case of RNA, the competitor is added to the RNA fraction and a reverse transcription reaction is performed, and the above-described primer set is added and PCR is performed. In the latter case, the absolute amount of the initial mRNA can be estimated because the reverse transcription reaction efficiency is also taken into account.

[0216] On the other hand, in real-time PCR, the amount of amplification is monitored in real time using a fluorescent reagent, and a device that integrally contains a thermal cycler and a fluorescence spectrophotometer is necessary. Such a device is commercially available. Depending on the fluorescent reagent to be used, there are several methods, and, for example, a intercalating agent method, a TaqMan TMprobe method, TaqMan method, molecular beacon method, etc. In any case, cDNA is synthesized from the RNA fraction prepared as described above through a reverse transcription reaction, and the above-described primer set and fluorescent reagent (probe) (a reagent (intercalator) that emits fluorescence by binding to double-stranded DNA, such as SYBR Green I, ethidium bromide, etc.), a nucleic acid that can be used as the above-described probe (the nucleic acid is hybridized to the target nucleic acid within the amplification region) (in which both ends are modified with a fluorescent substance (for example, FAM, HEX, TET, FITC, etc.) and a quencher substance (for example, TAMRA, DABCYL, etc.), etc.) (TaqMan TM- probe or molecular beacon probe) are each added to the PCR reaction system. Since the intercalator binds to the synthesized double-stranded DNA and emits fluorescence after irradiation of excitation light, the amount of amplification product can be monitored by measuring the intensity of fluorescence, based on which the amount of initial template cDNA can be inferred. TaqMan TM The probe is an oligonucleotide that can be hybridized to the amplification region of the target nucleic acid, both ends of which are modified with a fluorescent substance and a quencher substance, respectively. It is hybridized to the target nucleic acid during annealing, but is prohibited from emitting fluorescence by the presence of the quencher substance, and emits fluorescence when it is decomposed by the exonuclease activity of the DNA polymerase during elongation, which releases the fluorescent substance. Thus, by measuring the intensity of fluorescence, the amount of amplification product can be monitored, based on which the amount of initial template cDNA can be inferred. The molecular beacon probe is an oligonucleotide that can be hybridized to the amplification region of the target nucleic acid and has a hairpin-type secondary structure, both ends of which are modified with a fluorescent substance and a quencher substance, respectively. When it has a hairpin structure, it does not emit fluorescence due to the presence of the quencher substance, and emits fluorescence when the distance between the fluorescent substance and the quencher substance increases after hybridization to the target nucleic acid during annealing. Thus, the amount of amplification product can be monitored by measuring the intensity of fluorescence, based on which the amount of initial template cDNA can be inferred. Since real-time RT-PCR allows real-time monitoring of the amplification amount of PCR, it does not require electrophoresis and can more rapidly analyze the expression of the AIM gene.

[0217] In another embodiment, the AIM concentration of a sample collected from a test subject can be measured by preparing a protein fraction from the sample and detecting the AIM contained in the fraction. The detection of the AIM can be performed by an immunoassay method (e.g., ELISA, FIA, RIA, Western blotting, etc.) using an antibody against the AIM. Alternatively, the detection of the AIM can also be performed by mass spectrometry such as MALDI-TOF MS, etc.

[0218] Antibodies against AIM can be obtained according to techniques commonly used to produce polyclonal antibodies or monoclonal antibodies and using as immunizing antigen a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a partial amino acid sequence thereof.

[0219] In applying these individual immunoassay methods to the diagnostic method of the present application, it is not necessary to set specific conditions, procedures, etc. The measurement system of AIM can be constructed by those of ordinary skill in the art taking into consideration the conventional conditions and procedures in each method. For details of these general technical means, reference can be made to compilations, books, etc. For example, Hiroshi Irie, ed., "Radioimmunoassay" (published by Kodansha Ltd., 1974), Hiroshi Irie, ed., "Sequel to the Radioimmunoassay" (published by Kodansha Ltd., 1979), Eiji Ishikawa et al., eds., "Enzyme Immunoassay" (published by Igakushoin, 1978), Eiji Ishikawa et al., eds., "Enzyme Immunoassay" (2nd edition) (published by Igakushoin, 1982), Eiji Ishikawa et al., eds., "Enzyme Immunoassay" (3rd edition) (published by Igakushoin, 1987), Methods in ENZYMOLOGY, Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73 (Immunochemical Techniques (Part B)), ibid., Vol. 74 (Immunochemical Techniques (Part C)), ibid., Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press Publishing), etc.

[0220] The prediction method of the present application can specifically be a method comprising the following steps.

[0221] (1) a step of measuring the AIM concentration of a sample of a healthy person and a test subject, (2) a step of comparing the AIM concentration measured in the healthy person and the AIM concentration measured in the test subject.

[0222] As described above, in patients with chronic kidney disease, the renal function of patients with chronic kidney disease and exhibiting a blood concentration of the AIM of the present application lower than a given level decreases after 2 to 3 years. Therefore, as described above, when the measured AIM concentration is lower than the AIM concentration of a healthy person or a given level, it can be judged that the chronic kidney disease of the test subject is very likely to decrease in the future. Alternatively, a correlation diagram of the decrease of chronic kidney disease and the AIM concentration is drawn in advance, and the obtained observation can be compared with the correlation diagram. The comparison is preferably made based on the presence or absence of a significant difference.

[0223] In addition to the above steps (1) and (2), the prediction method of the present application can comprise (3) a step of judging that the chronic kidney disease of the test subject is very likely to decrease in the future when the AIM concentration of the test subject is significantly higher than the AIM concentration of the healthy person.

[0224] Further, in the examples of the present application mentioned below, a significantly higher AIM concentration was found in the urine of patients with acute kidney failure and mice that experienced two-sided transient renal ischemia / reperfusion, as compared with healthy individuals. The above suggests that by measuring the AIM concentration of the urine of a test subject, acute kidney failure can be examined. Therefore, the present application provides a test method for acute kidney failure, which comprises measuring the concentration of AIM in a sample of a test subject.

[0225] Although the test subject to which the test method of the present application is applicable is not particularly limited, for example, a test subject having a risk of developing acute kidney failure or being suspected of having developed acute kidney failure can be mentioned. The sample that can be used for the test method of the present application is as described with respect to the method of predicting the prognosis of a patient with kidney disease, and preferably, urine can be mentioned. In addition, the measurement of the AIM concentration of the sample collected from the test subject is as described with respect to the method of predicting the prognosis of a patient with kidney disease.

[0226] The test method of the present application can specifically be a method comprising the following steps.

[0227] (1) a step of measuring the AIM concentration of a sample of a healthy person and a test subject, (2) a step of comparing the AIM concentration measured in the healthy person and the AIM concentration measured in the test subject.

[0228] As described above, the urine concentration of AIM of the present application is significantly higher in patients with acute renal failure than in healthy persons. Therefore, when the AIM concentration is measured as described above and the result shows a significantly higher concentration than in healthy persons, it can be judged that the test subject has developed acute renal failure. Alternatively, a correlation diagram of the presence or absence of acute renal failure and the AIM concentration is prepared in advance, and the obtained observation can be compared with the correlation diagram. Comparison is preferably made based on the presence or absence of a significant difference.

[0229] In addition to the above steps (1) and (2), the test method of the present application can include (3) a step of judging that the test subject is affected by acute renal failure when the AIM concentration of the test subject is significantly higher than the AIM concentration of healthy persons.

[0230] Furthermore, the present application also encompasses a kit for diagnosis or prognosis prediction of kidney disease. The kit is not particularly limited as long as it is a kit for facilitating the implementation of the aforementioned test method or prediction method of the present application. The kit contains (a) a nucleic acid probe or a nucleic acid primer that can hybridize to the transcription product of the AIM gene, and / or (b) an antibody against AIM.

[0231] When the kit contains two or more of the above-mentioned nucleic acids and / or antibodies, each nucleic acid or antibody can specifically recognize mutually different regions on the base sequence of the AIM gene, or can specifically recognize different epitopes of the translation product of the AIM gene.

[0232] When the kit of the present application contains a reagent that includes the nucleic acid of the aforementioned (a) in its configuration, the nucleic acid of the probe or oligonucleotide of the above-mentioned primer for the test method or prediction method of the present application can be mentioned as a nucleic acid.

[0233] The nucleic acid capable of detecting the expression of the AIM gene can be provided as a solid in a dry state or an alcohol precipitate, or in a dissolved state in water or a suitable buffer (e.g., TE buffer, etc.). When it is used as a labeled probe, the nucleic acid can be provided in a state that is previously labeled with any of the above-mentioned labeling substances, or can also be provided independently of the labeling substance and labeled at the time of use.

[0234] Alternatively, the nucleic acid can also be provided in a state immobilized on a suitable solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene fluoride, etc. Examples of the immobilization method include, but are not limited to, a method comprising the steps of introducing a functional group such as an amino group, an aldehyde group, an SH group, biotin, etc. into the nucleic acid, introducing a functional group capable of reacting with the nucleic acid (e.g., an aldehyde group, an amino group, an SH group, streptavidin, etc.) on the solid phase, and cross-linking the solid phase and the nucleic acid through a covalent bond between the two functional groups, or introducing a polycation-coated solid phase in relation to polyanionic nucleic acids, and immobilizing the nucleic acid by electrostatic binding, etc.

[0235] The nucleic acid included in the kit is particularly preferably configured to be capable of detecting the expression of the AIM gene by the same method (e.g., Northern blotting, dot blotting, DNA array technology, quantitative RT-PCR, etc.).

[0236] When the kit of the present application contains a reagent comprising the antibody of the aforementioned (b) in the configuration, the antibody used in the test method or the prediction method of the present application described above can be mentioned as the antibody.

[0237] In addition to the nucleic acid and the antibody capable of detecting the expression of the AIM gene, the reagent constituting the kit of the present application can also contain other substances necessary for the reaction for detecting the expression of the gene, which do not adversely affect the reaction when coexisting. Alternatively, the reagent can also be provided together with a separate reagent containing other substances necessary for the reaction for detecting the expression of the AIM gene. For example, when the reaction for detecting the expression of the AIM gene is PCR, examples of such other substances include a reaction buffer, dNTP, a thermostable DNA polymerase, etc. When competitive PCR and real-time PCR are used, a competitive nucleic acid, a fluorescent reagent (the aforementioned intercalator, a fluorescent probe, etc.), etc. can also be contained. When the reaction for detecting the expression of the AIM gene is an antigen-antibody reaction, examples of such other substances include a reaction buffer, a competitive antibody, a labeled secondary antibody (e.g., mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc. when the first antibody is a rabbit anti-human AIM antibody), a blocking solution, etc.

[0238] The sequence identification numbers in the sequence table herein show the following sequences.

[0239] [SEQ ID NO: 1] The base sequence of human AIM is shown.

[0240] [SEQ ID NO: 2] The amino acid sequence of human AIM is shown.

[0241] [SEQ ID NO: 3] The base sequence of cat AIM is displayed.

[0242] [SEQ ID NO:4] The amino acid sequence of feline AIM is shown.

[0243] [SEQ ID NO:5] The complementary sequence of the transcript of cat AIM is shown.

[0244] [SEQ ID NO:6] The amino acid sequence of mouse AIM is shown.

[0245] [Example] The present invention will be described in more detail below with reference to the following Examples and Reference Examples, but the present invention is not limited to the following Examples and Reference Examples.

[0246] Example 1: Inhibition of progression of chronic renal failure or renal fibrosis by AIM One of the most commonly used animal models of kidney disease is the unilateral ureteral obstruction (UUO) model. In this case, unilateral ureteral obstruction induces gradual necrosis of the renal tubules and glomeruli in the obstructed renal parenchyma, followed by inflammation and fibrosis, and ultimately functional disorders of the kidney. UUO was performed on AIM knockout mice (AIM-KO) and wild-type mice (WT), and the progression was observed (n=6 each) ( Figure 1 A). The structure of the normal kidney did not differ between WT and AIM-KO. However, when the UUO kidneys were subjected to Azan staining and hematoxylin staining for fiber staining on day 14, WT showed diffuse fibrosis, but a considerable number of glomeruli and renal tubules still maintained normal structure, and many renal tubules were not necrotic. In contrast, AIM-KO showed accumulation of necrotic tubular cells over a wide range, destroyed glomerular structure, and decomposed renal parenchyma structure. Similar results were obtained in all observed mice.

[0247] Similarly, AIM-KO and WT mice were subjected to UUO, and kidneys were observed by HE, PAS, and Azan staining on day 14 (n=6 for each). Figure 1 B). WT shows the fibrosis of diffusion, but a considerable number of glomeruli and renal tubules still maintain normal structure (HE, Azan staining). On the other hand, AIM-KO shows the progress of fibrosis, destroyed glomerular structure and decomposed renal parenchyma structure. PAS staining reveals the necrotic cell mass (PAS positive) that accumulates in a wide range inside and outside the renal tubules of AIM-KO mice. Similar results were obtained in all mice observed.

[0248] Example 2: Inhibition of progression of acute renal failure (until day 7) to chronic kidney disease (becoming chronic renal failure) (day 14) by AIM Another model of kidney disease is the transient renal ischemia / reperfusion (IR) model. Transient renal ischemia / reperfusion involves pre-removal of one kidney, occlusion of the renal artery in the remaining kidney to induce ischemia, and release after 30 min of occlusion to allow reperfusion of blood flow. This transient ischemia causes progression of necrotic tubules and mild fibrosis about 3 days later, resulting in a decrease in kidney function. AIM knockout mice (AIM-KO) and wild-type mice (WT) were each subjected to IR and the progression was observed Figure 2 ). In the WT, after the decrease in kidney function, necrotic cells were removed, non-necrotic tubules rapidly divided, and almost normal tubular structure was recovered after 14 days. The kidney function then became normal. In the AIM-KO, although the initial damage level of the tubules was not different from that of the WT, removal of necrotic cells did not progress, and accumulation of necrotic cells occurred. Since removal of necrotic cells did not progress, division of new tubular cells was inhibited, and progression of secondary inflammation and fibrosis occurred. The experiment was performed with N = 6, and similar results were obtained in all mice. That is, the results from Example 1 and Example 2 indicate that deletion of AIM causes accumulation of necrotic tubular cells and repair of significantly impaired kidney structure and function.

[0249] Example 3: Inhibition of long-term inflammation after acute renal failure (kidney disease becomes chronic) by AIM The transient renal ischemia / reperfusion (IR) performed in Example 2 was applied to wild-type mice (WT) and AIM knockout mice (AIM-KO), and postoperative infiltration of inflammatory macrophages was observed by immunostaining of the macrophage marker F4 / 80 Figure 3 A). On day 3 after the operation, the AIM KO mice showed significantly promoted infiltration of F4 / 80-positive macrophages compared to the WT. On day 14 after the operation, the WT showed reduced infiltration of macrophages, but the AIM KO mice showed further deterioration. Quantitative RT-PCR experiments confirmed that, in line with the progression of macrophage infiltration, expression of MCP-1, one of the inflammatory cytokines, was similarly significantly increased in the kidneys of the AIM KO mice Figure 3 B). The experiment was performed with N = 6, and similar results were obtained in all mice.

[0250] Example 4: Recovery from acute renal failure by AIM The IR performed in Example 2 was applied to AIM-KO mice, and 100 μg of recombinant AIM (rAIM) or PBS (n = 6 each) was intraperitoneally administered at day 3, day 4, and day 7 when the kidney function was temporarily at its lowest, and the progression was observed Figure 4). In the PBS administration group, similar results to those in Example 2 were obtained, and the accumulation of necrotic cells, destruction of glomeruli, etc. progressed thereafter, and renal function (BUN value) was slightly improved from day 3, but did not return to the normal value, and gradually decreased thereafter. However, in the rAIM administration group, after rAIM administration on day 3, the BUN value was significantly improved, and had returned to the normal range on day 7, and further decreased thereafter. Histologically, the necrotic tubular cells were removed by day 7, and the structure of the renal parenchyma also became normal. That is, it was suggested that AIM administration can promote removal of necrotic cells, accelerate regeneration of the tubules, and restore renal function.

[0251] Example 5: Application of AIM attached to necrotic renal tubular epithelial cell mass to AIM-KO mice due to acute renal failure and removal of necrotic lesions Figure 5 , upper panel: In the phase-contrast micrograph, the necrotic lesion is indicated with N. Figure 5 , lower panel: AIM signals (arrow) overlapping with the necrotic lesion were observed. With the passage of time, the necrotic lesion area decreased, and almost only a trace could be confirmed after 12 hours. That is, when renal function was restored with AIM after IR (Example 4), it is considered that attachment of AIM to the necrotic lesion accelerates its removal, with concomitant suppression of inflammation and progression of tissue regeneration.

[0252] Example 6: Appearance of urinary AIM after acute renal failure In the urine of wild-type mice (WT) to which two-sided transient renal ischemia / reperfusion (IR) was applied (in which the renal arteries of both kidneys were occluded to induce ischemia, without isolating one of the kidneys), detection was performed by an ELISA method on day 1 and day 7 after IR Figure 6 ). Almost no AIM was detected in the urine of mice in the normal state (before IR), but a large amount of AIM was detected in the urine on day 1 after IR when the renal disease was most severe. Urinary AIM decreased (on day 7 after IR) along with recovery of the renal disease. Since the urine excreted during the course of the renal disease induced by IR becomes a dilute urine, the urinary AIM value was normalized with the urinary creatinine value.

[0253] Example 7: Worsening of acute renal failure due to deficiency of AIM (survival rate) Two-sided IR was performed on WT and AIM-KO mice, and the survival rate was examined (n = 8 each) Figure 7). On day 7 after IR, survival rate of AIM-KO was 30% or less under the condition that not less than 80% of WT survived, and most of the dead mice died before day 3 after IR.

[0254] Example 8: Clinical score Two-side IR was performed on WT and AIM-KO mice, and clinical score was analyzed over time ( Figure 8 ). Clinical scores on lack of agile movement, eye opacity, decrease in responsiveness to painful stimulation on the tail, poor coat layer (0: no abnormality, 1: mild symptoms, 2: moderate symptoms, 3: severe symptoms) were obtained by accumulation, and their sum was graphically displayed. In WT, the score reached a maximum on day 1 after IR and gradually decreased; however, in AIM-KO, the score remained high.

[0255] Example 9: Renal functional disorder associated with acute renal failure (BUN value) Two-side IR was performed on WT and AIM-KO mice, and BUN, which is a marker of renal function, was measured over time (n = 8) Figure 9 ). Similar to the clinical score in Example 8, BUN reached a peak on day 1 in WT and decreased thereafter; however, in AIM-KO, it increased until day 2 and no significant decrease was observed thereafter.

[0256] Example 10: Renal functional disorder associated with acute renal failure (kidney tissue: PAS staining) Two-side IR was performed on WT and AIM-KO mice, and kidney tissue was analyzed over time by PAS staining Figure 10 ). Similar to the clinical score in Example 8 and the BUN value in Example 9, renal functional disorder reached a peak on day 1 in WT and recovered thereafter, and proximal tubular epithelium with normal renal tubular structure and brush border (brush border; arrow) was regenerated on day 7. However, in AIM-KO, the disorder continued, and PAS-positive dead cell mass accumulated in proximal tubules, and was not removed even on day 7.

[0257] Example 11: Quantification of dead cell mass of renal tubular epithelial cells that became necrotic due to acute renal failure Figure 10 The area of dead cell mass of epithelial cells observed in proximal tubules in Figure 11 ) was quantified by calculating the ratio to the total area of one section (n = 3-5). A graph showing findings similar to those obtained in Examples 8-10 was displayed, and accumulation and residue of dead cell mass were apparent in AIM-KO.

[0258] Example 12: Long-term inflammation (inflammatory cytokines) of acute renal failure treated with AIM WT and AIM-KO mice were subjected to bilateral IR, and RNA was extracted from kidneys before IR and 7 days after IR, and the inflammatory cytokines IL-1β and IL-6 were analyzed by quantitative RT-PCR (n=3 for each) ( Figure 12 ). Both markers showed higher values ​​in AIM-KO compared with WT, suggesting long-term inflammation associated with IR-induced renal tissue destruction.

[0259] Example 13: Treatment of chronic inflammation (infiltrating macrophages) in acute renal failure with AIM WT and AIM-KO mice were subjected to bilateral IR, and kidneys were treated with collagenase on day 7 after IR and analyzed by flow cytometry to examine the ratio of macrophages (Mac-1 positive cells) ( Figure 13 ). Similar to the results of Example 12, the ratio of macrophages in the kidney was higher in AIM-KO than in WT. Three mice were analyzed each, and similar results were obtained. Figure 13 Representative results are shown.

[0260] Example 14: Accumulation of AIM in epithelial cell masses in renal tubules of mice that became necrotic due to acute renal failure Serial kidney sections from WT mice that underwent bilateral IR were subjected to PAS staining (left) and immunostaining using anti-AIM antibody (right) ( Figure 14 ). Accumulation of AIM was observed on many dead cell clusters (white).

[0261] Example 15: Accumulation of AIM in Necrotic Epithelial Cell Masses in Renal Tubules of Mice from Patients with Acute Renal Failure Serial kidney sections from a human patient who died of acute renal failure due to renal infarction were subjected to PAS staining (left) and immunostaining using anti-AIM antibody (right) ( Figure 15 ). Similar to the IR mice in Example 14, accumulation of AIM on many dead cell clusters (white) was observed.

[0262] Example 16: Detection of Urinary AIM in Patients with Acute Renal Failure and Mice with Acute Renal Failure The concentrations of AIM in urine of three patients transported to the hospital due to human acute renal failure (AKI), three healthy individuals, and five WT mice with bilateral IR were analyzed by ELISA before IR, 1 day after IR, and 7 days after IR. Figure 16 AIM is rarely observed in the urine of healthy individuals, but is observed in the urine of AKI patients. In mice, it is not observed before IR, but significant amounts of AIM are observed in the urine 1 day after IR when renal disease becomes apparent, and the amount of AIM decreases on day 7 when renal disease improves.

[0263] Example 17: AIM accumulation causes shrinkage of epithelial cell mass in the renal tubule Two-sided IR was performed on AIM-KO mice, 200 μg of rAIM was administered at day 3 after IR, and kidney sections were obtained over time and subjected to PAS staining and immunostaining with anti-AIM antibody Figure 17 ). Dead cell mass accumulated with AIM rapidly shrank.

[0264] Example 18: In vitro phagocytosis assay (experiment using kidney-derived macrophages) Two-sided IR was performed on AIM-KO mice, collagenase treatment was performed on the kidneys at day 3 after IR, F4 / 80-positive macrophages were isolated using a FACS sorter, and their phagocytic activity was analyzed Figure 18 ). As targets for phagocytosis, human renal tubular cell line HK2 cells were heat-treated to undergo necrosis, labeled with FITC, coated with recombinant AIM (rAIM) or bovine serum albumin (BSA), and the resulting dead HK2 cells were used. As a blank, dead HK2 cells that were not coated with rAIM or BSA were used. Macrophages were incubated with the three types of dead HK2 cells described above, and FITC-positive dead HK2 cells taken up by macrophages were analyzed over time by flow cytometry (FACS). It was confirmed that dead HK2 cells coated with rAIM were phagocytosed much more efficiently by macrophages than dead HK2 cells coated with BSA or dead HK2 cells not coated with them.

[0265] 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 phagocytes Figure 19 ). Since dead HK2 cells coated with BSA and dead HK2 cells not coated with them did not exhibit a difference in the manner of phagocytosis in Example 18, dead HK2 cells not coated were not used in this example. Similar to the results of Example 18, phagocytosis of dead HK2 cells coated with rAIM was increased.

[0266] Example 20: Treatment of acute renal failure in AIM-KO mice by AIM administrationTwo-sided IR was performed on AIM-KO mice, and rAIM (200 μg / mouse) or an equal amount of PBS was intravenously injected from day 1 to day 3 (n = 5-6). The survival rate of mice injected with PBS did not exceed 40% at day 7, but recovered to 100% in mice administered with rAIM Figure 20

[0267] Example 21: Recovery of clinical scores by AIM administration The clinical scores of the mice of Example 20 were studied similarly to those in Example 8 ( Figure 21 ). Significant recovery of clinical scores was observed after rAIM administration.

[0268] Example 22: Restoration of renal function by AIM administration The BUN ( Figure 22 ). A significant decrease in BUN value was observed by rAIM administration.

[0269] Example 23: Removal of Necrotic Epithelial Cell Masses in Renal Tubules by AIM Administration AIM-KO mice were subjected to bilateral IR and intravenously injected with rAIM (200 μg / mouse) or an equal amount of PBS from day 1 to day 3, and the status of the kidneys was analyzed by PAS staining on day 3 and day 7 ( Figure 23 ). In PBS administration, dead cells accumulated in the proximal tubules, but significant removal was observed in mice administered with rAIM, and renal tubular cells with brush borders recovered on day 7.

[0270] Example 24: Quantification of Necrotic Epithelial Cell Masses in Renal Tubules after AIM Administration In the kidneys at day 7 after IR, the expression of β-actin in the glomerulonephritis group was quantified by calculating the ratio relative to the total area of ​​a section. Figure 23 The area of ​​dead cell clusters in the proximal tubules observed in (n = 3 each) Figure 24 A significant decrease in dead cell mass was observed in the rAIM-administered group.

[0271] Example 25: AIM administration reduces inflammatory response (inflammatory cytokines) In the mice of Example 20, RNA was extracted from the kidneys 7 days after IR, and the inflammatory cytokines IL-1β and IL-6 ( Figure 25 ). In the rAIM-administered group, IL-1β and IL-6 decreased compared with the PBS-administered group, and this also suggests that the inflammatory response associated with acute renal failure has been suppressed by rAIM administration.

[0272] Example 26: Effect of AIM on non-fatal (mild) IR-induced acute kidney failure In the two-sided IR performed in Examples 6-25, ischemia was performed for 30 min, and induced acute kidney failure with a high mortality rate in AIM-KO. In this example, ischemia time was shortened (30 min→25 min), acute kidney failure was induced at a level that showed 100% survival rate at day 7 even in AIM-KO, and rAIM or PBS was administered by injection into the jugular vein from day 1 to day 3, similarly to Example 20. Even under such mild conditions in IR, rAIM administration can accelerate the improvement of BUN more (n=5 each) Figure 26

[0273] Example 27: Therapeutic effect of AIM on WT mice Two-sided IR (ischemia for 30 min) was performed on WT mice originally having endogenous AIM, and rAIM or PBS was administered similarly to Example 20 (n=5-6). In WT, the original endogenous AIM restored kidney disease, and rAIM can further accelerate the improvement thereof Figure 27

[0274] Example 28: Chronic kidney failure by deficiency of AIM WT and AIM-KO mice were subjected to mild IR similarly to Example 26, and the state of the kidney at day 28 after IR was analyzed by PAS staining (to observe dead cell mass) and Azan staining (to observe fibrosis) Figure 28

[0275] Example 29: Deficiency of AIM promotes kidney fibrosis RNA was extracted from the kidney of mice in Example 28 at day 28 after IR, and 4 types of fibrosis markers were analyzed by quantitative RT-PCR (n=4 each) Figure 29

[0276] Example 30: Blood AIM value in human patients with diabetic chronic kidney failure ​​​​Blood AIM concentrations were measured for gender discrimination in (1) healthy humans (male: 142, female: 54), (2) diabetic patients without nephropathy (Cre < 1.0 mg / dl) (male: 70, female: 57), and (3) patients with diabetic chronic renal failure (male: 146, female: 54) who were in almost the same age ( Figure 30 ). There were no significant differences in the AIM values between males and females in (1) and (2), but the values were significantly lower in (3).

[0277] Example 31: Correlation between renal function and blood AIM in human patients with chronic kidney disease Blood AIM values were analyzed for patients with chronic kidney disease (CKD), and the correlation between each renal function marker eGFR (glomerular filtration rate) and blood creatinine value was examined (n = 55). As the cause disease of CKD, diabetic nephropathy, glomerulonephritis, hypertensive nephropathy, IgA nephropathy, etc. can be mentioned. The age of the patients (male and female) was not more than 60. As shown in Figure 31 A, a significant correlation between blood AIM values and renal function was found. Furthermore, patients with relatively high AIM values at the time point of this analysis showed improved renal function in a follow-up study 2-3 years later, and patients with low AIM values showed, on the contrary, a decreased renal function ( Figure 31 B). That is, AIM can be a useful marker for predicting not only the present renal function but also the prognosis in CKD patients.

[0278] Example 32: Absence (or significant decrease) of blood AIM in cats Using anti-AIM antibody (Rab2: polyclonal antibody generated by immunizing a rabbit with mouse AIM, which has been purified to detect mouse and human AIM), immunoblotting was performed under reducing conditions on each serum from dog (3), cat (3), and mouse ( Figure 32 A). So, although a signal can be confirmed in dog serum, a signal was hardly detectable in 3 cats. This is not a problem of the antibody, and cat rAIM protein obtained by cloning cat AIM cDNA (integrated into pCAGGS expression vector together with HA tag attached to C-terminus, expressed in HEK293T cells, and purified from culture supernatant using anti-HA antibody column) (see Example 36) can be detected by immunoblotting under reducing conditions using the antibody at the same level as mouse rAIM ( Figure 32 B). From these results, it was found that cats studied in this example express functional AIM mRNA, but hardly contain AIM protein in blood.

[0279] Example 33: Association of cat AIM and IgM in cat blood​ The plasmid (wherein Figure 35 The feline AIM cDNA shown in was inserted into an expression vector) and transfected into HEK293T cells. Recombinant feline AIM (1 mg) purified from its culture supernatant was intravenously injected into a cat (mongrel, male, 2 years and 3 months old). Blood samples were collected after 1 hour and serum was separated. The serum was applied to size fractionation using gel and each fraction was analyzed for AIM and IgM by Western blotting. As in Figure 33 As shown in A, the fractions containing AIM and IgM were clearly different. This result was different from the results of similar fractionation analysis of serum obtained by intravenous injection of mouse AIM into AIM KO mice ( Figure 33 B, Comparative Example) (IgM and AIM fractions are completely matched). That is, AIM initially binds to IgM in the blood and maintains its stability (prior art reference: Arai et al., Cell Reports 3:1187-1198, 2013), resulting in a sustained blood concentration of AIM. However, in cats, since AIM cannot bind to IgM, its stability in the blood cannot be maintained, and therefore, the blood concentration of AIM cannot be maintained.

[0280] Example 34: Binding of Mouse AIM and IgM in Cat Blood Using mouse AIM, a test similar to that of Example 33 was performed. Recombinant mouse AIM (1 mg) was intravenously injected into a cat (mongrel, male 2 years and 3 months old), and blood samples were collected 1 hour later, and serum was separated. The serum was applied to size fractionation using gel, and each fraction was analyzed for AIM and IgM by Western blotting. As in Figure 34 As shown in , the fraction containing mouse AIM completely matches the fraction containing cat IgM. Therefore, unlike cat AIM, it is believed that mouse AIM binds to IgM and is stable in the blood.

[0281] Example 35: Binding Site of Mouse AIM and IgM A variety of C-terminal defective recombinantly modified mouse AIMs were produced. The combination of modified mouse AIM and IgM was confirmed in vitro. Therefore, modified mouse AIMs with partially defective SRCR3 domains showed significantly reduced binding to IgM. Therefore, it was found that the SRCR3 domain of mouse AIM is important for binding to IgM.

[0282] Example 36: Feline AIM cDNA sequence Multiple primers were designed from the predicted sequence of feline CD5L (= AIM) disclosed in NCBI Resources (GenBank Accession No: XM_003999688.1) and full-length feline AIM cDNA was isolated from a cDNA library of feline spleen (SEQ ID NO: 5) Figure 35 ) Among the sequences, specifically, the sequence encoding the leader peptide is quite different from the disclosed sequence.

[0283] Example 37: Hydrophobicity of the leader peptide The hydrophobicity of the leader peptide sequence of the respective AIM proteins of feline, human, mouse, and dog is shown Figures 36-39 ). All of them exhibit sufficient hydrophobicity and satisfy the prerequisite of a secreted protein. The leader peptide of feline was analyzed from the cDNA we isolated and that of dog AIM from the predicted sequence disclosed in NCBI Resources (GenBank Accession No: XM_846487.2).

[0284] Example 38: Comparison of the AIM amino acid sequences of human, feline, and mouse The amino acid sequences were compared among the leader peptide (LS), each SRCR, and the hinge region Figure 40 ). In the figure, the amino acids common to the three are shown with “*”, the amino acids common only to human and feline with “.”, and the amino acids common only to human and mouse with “:”.

[0285] Example 39: Analysis of blood AIM in felines In Example 32, an anti-mouse AIM antibody was used to detect feline AIM. In this example, DNA in which a HA tag was attached to the cDNA of feline AIM was incorporated into an expression vector and transfected into HEK293T cells, thereby producing a recombinant feline AIM protein in which a HA peptide was added to the C terminus (rcAIM-HA), which was used to immunize mice to establish an anti-feline AIM monoclonal antibody. Using the antibody, the blood AIM concentration of different breeds of felines (48 individuals) was analyzed by a reducing Western blot method Figure 41 ). As a control concentration, rcAIM-HA was used. Therefore, regardless of the breed, individuals in which AIM was detected and individuals in which AIM was not detected were found. Among the individuals in which AIM was detected, the average blood AIM concentration of 4 representative individuals was 16.8 μg / ml, which was significantly higher than that of mouse and human, about 5 μg / ml.

[0286] Example 40: Establishment of an IR method for felines A method for inducing acute renal failure by IR was established in cats with high blood AIM concentrations. Under general anesthesia, bilateral renal arteries were occluded with clips laparoscopically for 1 hour and then released. Thereafter, blood and urine were collected over time, and renal function was studied. Figure 42 An overview of blood BUN and Cre levels is shown. Both BUN and Cre levels peaked 12 hours after IR and did not improve until day 7. This suggests that recovery from acute renal failure is highly promiscuous, as in AIM-KO mice.

[0287] Example 41: Analysis of blood and urine AIM in cats after IR In mice, AIM was detected in urine after IR and is thought to accumulate on dead cell masses that clog the renal tubules. Serum and urine of cats after IR were analyzed by reduced Western blot using anti-feline AIM antibodies, and no AIM was detected in cat urine after IR ( Figure 43 ). The amount of AIM in the blood did not show significant changes.

[0288] Example 42: Presence or Absence of AIM Accumulation in Dead Cell Masses of Proximal Tubules in Cats with Induced Acute Renal Failure Cats underwent bilateral IR and were analyzed by immunostaining to determine whether AIM accumulated in necrotic cell masses in the tubules of the kidney on day 3 ( Figure 44 ). Necrotic cell clusters were detected in the proximal renal tubules (white arrow portion); however, no accumulation of AIM was observed in the same portion. In Example 41, AIM was not detected in the urine after IR, suggesting that AIM did not reach the dead cell clusters in the renal tubules.

[0289] Example 43: Effects of AIM Administration on Cats with Induced Acute Renal Failure Similar to the method described in Example 40, a cat (5 years old, female) was subjected to bilateral IR. 24 hours later, an arterial catheter was inserted from the inguinal artery under anesthesia, the catheter tip was advanced to the renal artery, and 25 ml (rAIM: 25 mg) of a solution of rAIM (50 mg) dissolved in PBS (50 ml) was injected into the unilateral renal artery. Another cat (also 5 years old, female) was similarly subjected to IR and catheterization, and 25 ml of PBS alone was injected into the unilateral renal artery. GFR (glomerular filtration rate) normalized by body surface area before IR and 24 hours after rAIM or PBS injection (48 hours after IR) is shown. Figure 45 In cats injected with PBS alone, GFR decreased and renal function declined; however, cats injected with rAIM did not show a decrease in GFR and renal function did not decline.

[0290] Example 44: Effects of AIM Administration on Cats with Induced Acute Renal Failure Similar to the method described in Example 43, cats underwent bilateral IR and renal tissue was analyzed by PAS staining 24 hours after rAIM or PBS injection (48 hours after IR). Figure 46 In cats injected with PBS, necrosis and loss of renal tubular epithelial cells, disruption of tubular architecture, and growth of interstitial tissue were observed. In cats injected with rAIM, renal tubular epithelial cells had recovered, along with the brush border and structure. Furthermore, interstitial tissue was thinner than in cats injected with PBS, and no growth was observed. Histologically, the cure of renal failure by rAIM was evident.

[0291] Example 45: Effects of AIM Administration on Cats AIM or vehicle are applied to 6-8 year old cats continuously every day.Use and measure renal function (BUN value) in 2-4 weeks afterwards.In the AIM application group, the decline of the renal function observed in the vehicle application group is suppressed.Therefore, obviously, AIM can be used for the decline of renal function and the prevention of renal failure in cats.Use the medicine that can control the function of AIM (it contains the partial peptide of AIM with AIM activity) or the medicine that induces AIM to express and replace AIM, obtain similar result.

[0292] Example 46: Effects of Administration of Modified AIM on Cats The SRCR3 domain of cat AIM is changed into the SRCR3 domain of mouse AIM to produce the cat AIM of modification in conjunction with IgM. The AIM of modification or vehicle are applied to 6-8 years old cats continuously every day. Renal function (BUN value) is measured in 2-4 weeks after application. In the AIM application group of modification, the decline of renal function observed in the vehicle application group is suppressed. Therefore, it is obvious that AIM can be used for the decline of renal function and the prevention of renal failure in cats. In addition, because the cat AIM of modification can be combined with the IgM in the blood and is stabilized, effectiveness is obtained by applying the cat AIM of modification of low dosage rather than applying cat AIM. The cat AIM of modification only needs to be attached to cat IgM and is not restricted.

[0293] [Industrial Applicability] The present invention can provide a preventive or therapeutic agent for renal disease, which comprises AIM as an active ingredient. In addition, the renal disease model mouse of the present invention helps to clarify the onset mechanism of renal disease, and according to the screening method using the renal disease model mouse, it is possible to search for a substance effective for the prevention or treatment of renal disease. In addition, using the renal disease model mouse of the present invention, the effect of a preventive or therapeutic agent for known renal disease can be evaluated. In addition, the present invention can provide a method for diagnosing renal disease.

[0294] This application is based on Japanese Patent Application No. 2014-022041 (filed on February 7, 2014), the contents of which are incorporated herein by reference in its entirety.

Claims

1. A preventive or therapeutic agent for renal disease, comprising AIM or a partial peptide thereof, or a nucleic acid comprising a base sequence encoding the same.

2. A preventive or therapeutic agent for renal disease, comprising a drug that induces AIM expression or a drug that stabilizes AIM.

3. The agent according to claim 1 or 2, wherein 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. 4 . The agent according to claim 1 , wherein the renal disease is acute renal failure or chronic renal failure.

5. The agent according to any one of claims 1 to 4, wherein the subject for prevention or treatment is a human.

6. The agent according to any one of claims 1 to 4, wherein the subject to be prevented or treated is a cat. The agent according to claim 6 , wherein the AIM or a partial peptide thereof binds to feline IgM. The agent according to claim 7, wherein the feline IgM-binding AIM or a partial peptide thereof contains the SRCR3 domain of mouse-derived AIM.

9. A method for screening a preventive or therapeutic agent for renal disease, the method comprising using an animal obtained by subjecting a non-human mammal deficient in AIM expression to unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion.

10. The method according to claim 9, comprising the steps of: (1) a step of administering a test substance to a non-human mammal deficient in AIM expression under conditions of unilateral ureteral obstruction, transient renal ischemia / reperfusion after uninephrectomy, or bilateral transient renal ischemia / reperfusion, (2) observing any one or more of the following properties of the AIM expression-deficient non-human mammal to which the test substance is administered: (i) accumulation of necrotic tubular cells and renal parenchymal fibrosis, (ii) disintegration and fibrosis of glomerular architecture, (iii) expression levels of inflammatory cytokines in the kidney, (iv) the ratio of macrophages in the kidney, (v) BUN value, (vi) survival rate, (3) A step of selecting a test substance that improves any one or more of the above-mentioned characteristics compared to those in the case where the test substance is not administered.

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