Agent for preventing or treating tubulointerstitial injury

Abstract

To provide a technique for preventing or treating tubulointerstitial injury.SOLUTION: The present invention discloses a method to screen for an agent for preventing or treating tubulointerstitial injury, the method including the step of determining whether a test substance inhibits the functions of Mitotic Spindle Positioning (MISP) protein, wherein the inhibiting the functions of MISP protein by the test substance shows the test substance being a candidate for the agent for preventing or treating tubulointerstitial injury.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a preventive or therapeutic agent for tubulointerstitial injury. More specifically, the present invention relates to a method for screening a preventive or therapeutic agent for tubulointerstitial injury and a preventive or therapeutic agent for tubulointerstitial injury.

Background Art

[0002] In developed countries, as the number of patients with chronic kidney disease increases, the huge medical costs associated with its prognosis have become a problem, and the development of treatment methods has become an urgent need. However, there is still no effective treatment method for chronic kidney disease. Currently, there is only conservative treatment to suppress proteinuria by blood pressure control, blood sugar control, diet therapy, etc. Regarding chronic kidney disease, genome-wide association analysis has been performed in humans, and various disease susceptibility genes have been identified. However, in chronic kidney disease, which is a multifactorial genetic disease, the contribution of these genes is extremely small and no new drugs have been developed. In chronic kidney disease caused by various causative diseases, tubulointerstitial injury is a common pathological condition. In addition, tubulointerstitial injury strongly correlates with renal function decline. Therefore, in the treatment of chronic kidney disease, treatment to stop the progression of tubulointerstitial injury is effective.

[0003] By the way, the Mitotic Spindle Positioning (MISP) protein is known to have an important function in spindle orientation and mitotic progression (see, for example, Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] Against this background, an object of the present invention is to provide a technique for preventing or treating tubulointerstitial damage. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A method for screening for a preventive or therapeutic agent for tubulointerstitial damage, comprising a step of determining whether a test substance inhibits the function of MISP protein, wherein the test substance's inhibition of the function of MISP protein indicates that the test substance is a candidate for a preventive or therapeutic agent for tubulointerstitial damage. [2] The screening method described in [1], wherein the step of determining whether a test substance inhibits the function of MISP protein comprises the steps of: applying a hypoxic stimulus to proximal tubule epithelial cells in the presence of the test substance; and quantifying the expression level of the MISP gene or MISP protein in the cells; and a decrease in the expression level of the MISP gene or MISP protein in the presence of the test substance compared to the absence of the test substance indicates that the test substance inhibits the function of MISP protein. [3] The screening method according to [2], wherein the hypoxic stimulation is carried out by adding antimycin A to the culture medium. [4] The screening method described in [1], wherein the step of determining whether the test substance inhibits the function of the MISP protein includes a step of measuring the amount of binding between the MISP protein and the PTK2 protein in the presence of the test substance, and a decrease in the amount of binding between the MISP protein and the PTK2 protein in the presence of the test substance indicates that the test substance inhibits the function of the MISP protein. [5] The screening method described in [1], wherein the step of determining whether a test substance inhibits the function of MISP protein includes a step of measuring the amount of binding between MISP protein and CDC42 protein in the presence of the test substance, and a decrease in the amount of binding between MISP protein and CDC42 protein in the presence of the test substance indicates that the test substance inhibits the function of MISP protein. [6] The screening method described in [1], wherein the step of determining whether a test substance inhibits the function of a MISP protein comprises the steps of culturing cells expressing a MISP gene or a MISP protein in the presence of the test substance and quantifying the expression level of the MISP gene or the MISP protein in the cells, and a decrease in the expression level of the MISP gene or the MISP protein in the presence of the test substance compared to the absence of the test substance indicates that the test substance inhibits the function of the MISP protein. [7] A preventive or therapeutic agent for tubulointerstitial damage, the active ingredient of which is a functional inhibitor of MISP protein. [8] The preventive or therapeutic agent for tubulointerstitial damage described in [7], wherein the functional inhibitor is an siRNA, shRNA or morpholino oligo against the MISP gene, a binding inhibitor between MISP protein and PTK2 protein, or a binding inhibitor between MISP protein and CDC42 protein. [Effects of the Invention]

[0007] The present invention can provide a technique for preventing or treating tubulointerstitial damage. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing the signal transduction pathway in which MISP is involved. [Figure 2] (a) and (b) are photographs showing representative results of immunochemical staining in Experimental Example 2. (c) is a graph showing the results of quantification of the MISP-positive area of proximal tubule cells based on the results of immunochemical staining in Experimental Example 2. [Figure 3] (a) to (c) are graphs showing the results of immunochemically staining renal tissue sections of nephrosis model mice with anti-MISP antibody and detecting and quantifying the MISP-positive regions of proximal tubular cells using ImageJ in Experimental Example 2. [Figure 4] (a) and (c) are graphs showing the results of quantitative real-time RT-PCR of the mRNA of the MISP gene in Experimental Example 3. (b) and (d) are photographs showing the results of Western blotting of NGAL protein in Experimental Example 3. [Figure 5] (a) is a photograph showing the results of SDS-PAGE in Experimental Example 5. (b) is a graph obtained by quantifying the results of (a). [Figure 6] Survival curves of Tns2 knockout mice (Tns2nph) and double knockout mice of the MISP gene and the Tns2 gene (MISPKOTns2nph) measured in Experimental Example 5. [Figure 7] [[ID=1"3]](a) and (b) are photographs showing typical results of immunochemical staining in Experimental Example 5. (c) is a graph showing the results of quantifying the MISP-positive regions of proximal tubular cells based on the results of immunochemical staining in Experimental Example 5. [Figure 8] (a) and (b) are typical microscopic photographs of renal tissue sections in Experimental Example 6. (c) is a graph obtained by quantifying the thickness of tubular epithelial cells measured in Experimental Example 6. (d) is a photograph showing the results of Western blotting of NGAL protein in Experimental Example 6. [Figure 9] (a) and (b) are typical microscopic photographs showing the results of immunochemical staining of renal tissue sections in Experimental Example 7. (c) is a photograph showing the results of Western blotting of NGAL protein in Experimental Example 7. (d) is a graph obtained by quantifying the expression level of NGAL protein in Experimental Example 7. [Figure 10] (a) to (d) are typical microscopic photographs showing the results of immunochemical staining of human renal tissue sections in Experimental Example 8. [Figure 11](a) to (d) are graphs showing the results of measuring the ratio of MISP-positive renal tubules in Experimental Example 8.

Mode for Carrying Out the Invention

[0009] [Notation of Gene Names and Protein Names] In this specification, human genes and human proteins shall be represented by capital letters of the alphabet. Also, mouse genes shall be represented by a capital letter at the beginning and small letters of the alphabet thereafter. Also, mouse proteins shall be represented by capital letters of the alphabet. However, in some cases, human genes, mouse genes, human proteins, and mouse proteins may be represented without strictly distinguishing them.

[0010] [Method for Screening a Preventive or Therapeutic Agent for Tubulointerstitial Injury] In one embodiment, the present invention provides a method for screening a preventive or therapeutic agent for tubulointerstitial injury, which includes a step of determining whether a test substance inhibits the function of MISP protein, and the fact that the test substance inhibits the function of MISP protein indicates that the test substance is a candidate for a preventive or therapeutic agent for tubulointerstitial injury.

[0011] Tubulointerstitial injury is a disease in which damage occurs in the tissue (interstitium) between renal tubules. Chronic kidney disease is caused by various causative diseases such as diabetic nephropathy, chronic glomerulonephritis, and hypertensive nephrosclerosis. In the early stage of the course, it shows pathological changes specific to the primary disease, but in many cases, tubulointerstitial injury is a common pathological condition of chronic kidney disease.

[0012] Therefore, a preventive or therapeutic agent for tubulointerstitial injury can be equivalently referred to as a preventive or therapeutic agent for chronic kidney disease, etc.

[0013] The NCBI accession numbers for the human MIPS gene mRNA are NM_173481.4, etc., and the NCBI accession numbers for the human MIPS proteins are NP_775752.1, XP_011525988.1, XP_011525987.1, etc. The NCBI accession number for the mouse MIPS gene mRNA is NM_030218.2, and the NCBI accession number for the mouse MIPS protein is NP_084494.1.

[0014] In the screening method of this embodiment, the test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, and the like.

[0015] (First embodiment) The screening method of the first embodiment comprises the steps of applying a hypoxic stimulus to proximal tubular epithelial cells in the presence of a test substance and quantifying the expression level of the MISP gene or MISP protein in the proximal tubular epithelial cells, and a decrease in the expression level of the MISP gene or MISP protein in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a candidate agent for preventing or treating tubulointerstitial damage.

[0016] The proximal tubule epithelial cells may be cells derived from a human or a non-human animal, and may be cells derived from an established cell line, primary cells, or cells induced to differentiate from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells).

[0017] Hypoxic stimulation can be achieved, for example, by adding antimycin A to the medium, culturing in a sealed container, culturing using an oxygen scavenger, or culturing under hypoxic conditions using a multi-gas incubator.

[0018] As will be described later in the examples, the inventors have clarified that when proximal tubular epithelial cells are subjected to hypoxia stimulation, the expression level of the MISP gene or MISP protein increases, and the expression of the NGAL protein, which is a tubular injury marker, also increases.

[0019] Therefore, in the presence of a test substance, a preventive or therapeutic agent for tubulointerstitial injury can be screened using as an index a decrease in the expression level of the MISP gene or MISP protein as compared with the control.

[0020] (Second Embodiment) The screening method of the second embodiment includes a step of measuring the binding amount between the MISP protein and the PTK2 protein in the presence of a test substance, and a decrease in the binding amount between the MISP protein and the PTK2 protein in the presence of the test substance indicates that the test substance is a candidate for a preventive or therapeutic agent for tubulointerstitial injury.

[0021] Figure 1 is a schematic diagram showing a signal transduction pathway in which MISP is involved. As shown in Figure 1, the MISP protein is known to bind to the FAK protein (PTK2 protein). Therefore, a substance that inhibits this binding can be said to be a candidate for a preventive or therapeutic agent for tubulointerstitial injury.

[0022] The test substance is the same as in the first embodiment. The binding between the MISP protein and the PTK2 protein may be measured at the cell level or at the protein level. The binding amount between the MISP protein and the PTK2 protein can be measured, for example, by immunoprecipitation, binding measurement using BIAcore (GE Healthcare), or the like.

[0023] The NCBI accession numbers of the mRNA of the human PTK2 gene are NM_001199649.2, NM_001316342.2, NM_001352694.2, etc., and the NCBI accession numbers of the human PTK2 protein are XP_024302971.1, XP_016869177.1, NP_001339681.1, etc.

[0024] In addition, the NCBI accession number of the mRNA of the mouse PTK2 gene is NM_007982.2, etc., and the NCBI accession number of the mouse PTK2 protein is NP_032008.2, etc.

[0025] (Third Embodiment) The screening method of the third embodiment includes a step of measuring the binding amount between the MISP protein and the CDC42 protein in the presence of a test substance, and a decrease in the binding amount between the MISP protein and the CDC42 protein in the presence of the test substance indicates that the test substance is a candidate for a prophylactic or therapeutic agent for tubulointerstitial injury.

[0026] Figure 1 is a schematic diagram showing a signal transduction pathway involving MISP. As shown in Figure 1, the MISP protein is known to bind to the CDC42 protein. Therefore, a substance that inhibits this binding can be said to be a candidate for a prophylactic or therapeutic agent for tubulointerstitial injury.

[0027] The test substance is the same as in the first embodiment. The binding between the MISP protein and the CDC42 protein may be measured at the cell level or at the protein level. The binding amount between the MISP protein and the CDC42 protein can be measured, for example, by immunoprecipitation, binding measurement using Biacore (GE Healthcare), etc.

[0028] The NCBI accession numbers of the mRNA of the human CDC42 gene are NM_001039802.2, NM_001791.4, NM_044472.3, etc., and the NCBI accession numbers of the human CDC42 protein are NP_001034891.1, NP_001782.1, NP_426359.1, etc.

[0029] In addition, the NCBI accession numbers of the mRNA of the mouse CDC42 gene are XM_030253139.1, etc., and the NCBI accession numbers of the mouse CDC42 protein are XP_030108999.1, etc.

[0030] (Fourth Embodiment) The screening method of the fourth embodiment includes the steps of culturing cells expressing the MISP gene or MISP protein in the presence of a test substance, and quantifying the expression level of the MISP gene or MISP protein in the cells, and in the presence of the test substance, a decrease in the expression level of the MISP gene or MISP protein as compared to the absence of the test substance indicates that the test substance is a candidate for a prophylactic or therapeutic agent for tubulointerstitial injury.

[0031] As described above, a substance that reduces the expression of the MISP gene can be said to be a candidate for a prophylactic or therapeutic agent for tubulointerstitial injury. Therefore, a prophylactic or therapeutic agent for tubulointerstitial injury can be screened by the screening method of the present embodiment.

[0032] The test substance is the same as in the first embodiment. Examples of cells expressing the MISP gene or MISP protein include A549, SK-BR-3, HaCaT, hTCEpi, MCF7, SiHa, CAPAN-2, CACO-2, HeLa, T-47d, HBEC3-KT, RT4, A-431, PC-3, RPTEC TERT1, U-2 OS, EFO-21, Hep G2, Daudi, hTERT-HME1, SCLC-21H, HMC-1, U-2197, etc.

[0033] The expression level of the MISP gene can be quantified by, for example, RNA-Seq, quantitative real-time RT-PCR, etc. The expression level of the MISP protein can be quantified by, for example, Western blotting, ELISA, immunostaining, etc.

[0034] [Preventive or therapeutic agent for chronic kidney disease] In one embodiment, the present invention provides an agent for preventing or treating tubulointerstitial damage, which contains a functional inhibitor of MISP protein as an active ingredient.

[0035] As described later in the Examples, the inventors have demonstrated that knocking out the MISP protein inhibits the progression of tubulointerstitial damage in nephropathy model mice and reduces the expression of NGAL protein, a tubular injury marker. Therefore, MISP function inhibitors can be used as agents for preventing or treating tubulointerstitial damage.

[0036] Furthermore, as described later in the Examples section, the inventors have demonstrated that mice with the Misp gene knocked out do not exhibit significant phenotypes. This result indicates that inhibiting the function of the MISP protein has few side effects. It also indicates that inhibiting the function of the MISP protein is a useful therapeutic target for chronic kidney disease.

[0037] Examples of the function inhibitor include (i) siRNA, shRNA, or morpholino oligo against the MISP gene, (ii) a substance that inhibits the binding between the MISP protein and the PTK2 protein, and (iii) a substance that inhibits the binding between the MISP protein and the CDC42 protein.

[0038] (siRNA, shRNA or morpholino oligo against MISP gene) siRNA, shRNA, and morpholino oligos are inhibitory nucleic acids that can suppress the translation of MISP gene mRNA. These inhibitory nucleic acids can be prepared by well-known methods.

[0039] Morpholino oligos are nuclease-resistant and stable, allowing them to be sterilized in an autoclave. Furthermore, the Tm value of morpholino oligos and RNA is slightly higher than that of natural DNA and RNA, indicating the formation of stable bonds. Furthermore, morpholino oligos have strong affinity for RNA, allowing them to bind to the desired sequence regardless of the secondary structure of the target mRNA, facilitating effective sequence design. Furthermore, morpholino oligos are highly water-soluble, making them easy to prepare. Furthermore, morpholino oligos have the advantage of not binding nonspecifically to proteins.

[0040] (Inhibitor of the binding between MISP protein and PTK2 protein) As shown in Figure 1, the MISP protein is known to bind to the FAK protein (PTK2 protein). Therefore, substances that inhibit this binding can be said to be functional inhibitors of the MISP protein.

[0041] (A substance that inhibits the binding between MISP protein and CDC42 protein) As shown in Figure 1, the MISP protein is known to bind to the CDC42 protein. Therefore, substances that inhibit this binding can be said to be functional inhibitors of the MISP protein. [Example]

[0042] The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to these examples.

[0043] [Experimental Example 1] (Search for genes related to tubulointerstitial damage) The inventors analyzed the gene expression database Nephroseq (https: / / www.nephroseq.org / resource / login.html) and discovered the Mitotic Spindle Positioning (MISP) gene as a gene whose expression level is elevated in renal diseases such as human membranous glomerulonephritis and mouse diabetic nephropathy. However, the relationship between MISP and nephropathy has not been known until now.

[0044] [Experimental Example 2] (Study on the relationship between MISP gene expression and the pathology of nephropathy) We investigated the relationship between MISP gene expression and the pathology of nephropathy. First, to identify the expression site of the MISP gene, we performed immunochemical staining using nephropathy model mice. The following four types of nephropathy model mice (all males) were used for the experiment.

[0045] Adriamycin-induced nephropathy model mouse This nephropathy model is a mouse model in which adriamycin (ADR) is administered to BALB / c mice to induce glomerular damage and subsequent tubulointerstitial damage. ADR (10 mg / kg) was administered via the tail vein of 8-week-old BALB / c mice (male). Kidneys were harvested 14 days after administration and subjected to immunochemical staining.

[0046] <Tensin2-deficient mouse model> This nephropathy model mouse lacks the Tensin2 gene and progresses through glomerular damage, tubulointerstitial damage, and chronic kidney disease, eventually progressing to end-stage renal failure. Kidneys were harvested at 10 weeks of age and subjected to immunochemical staining.

[0047] <Anti-nephrin antibody-induced nephropathy mouse model> This nephropathy model is a model mouse that is administered with an anti-nephrin antibody (see Takeuchi K., et al., New anti-nephrin antibody mediated podocyte injury model using a C57BL / 6 mouse strain. Nephron, 138(1), 71-87, 2018), inducing glomerular injury and subsequent tubulointerstitial injury. An anti-nephrin antibody (1.5 mg / mouse) was administered via the tail vein to 12-week-old FVB / NJ mice. Kidneys were collected 12 weeks after antibody administration and subjected to immunohistochemical staining.

[0048] 《Model Mice of Renal Ischemia-Reperfusion Induced Renal Insufficiency (IR)》 This nephropathy model is a model that induces tubular injury due to renal ischemic injury. 10-week-old FVB / NJ mice were used. The left kidney was removed 10 days before ischemia induction. Ten days after left kidney removal, the renal artery and vein of the right kidney were clamped with a clamp to maintain an ischemic state for 30 minutes. After 30 minutes of ischemia, the clamp was removed, the skin was sutured, and the kidneys were collected 24 hours later and subjected to immunohistochemical staining.

[0049] 《Immunohistochemical Staining》 Renal tissue sections of each model mouse were immunohistochemically stained with an anti-MISP antibody and developed with 3,3’-diaminobenzidine (DAB). Subsequently, the MISP-positive area of proximal tubular cells was detected and quantified using ImageJ.

[0050] Figure 2(a) is a photograph showing representative results of immunohistochemical staining of renal tissue sections of control mice. The scale bar is 100 μm. Figure 2(b) is a photograph showing representative results of immunohistochemical staining of renal tissue sections of adriamycin nephropathy model mice. The scale bar is 100 μm. In Figure 2(b), "*" indicates proximal tubules with severe injury.

[0051] Figure 2(c) is a graph showing the result of quantifying the MISP-positive region in proximal tubular cells based on the results of immunochemical staining. The vertical axis indicates the relative value of the area of the MISP-positive region. In Figures 2(a) to (c), "Control" indicates the result of control mice, and "ADR" indicates the result of adriamycin nephropathy model mice.

[0052] As a result, it was revealed that the expression of MISP protein was significantly increased in the proximal tubular epithelium with severe injury in the kidney tissue sections of adriamycin nephropathy model mice (P<0.001).

[0053] Figures 3(a) to (c) are graphs showing the results of immunochemically staining kidney tissue sections of Tensin2-deficient model mice, anti-nephrin antibody-induced nephropathy model mice, and renal ischemia-reperfusion-induced renal insufficiency (IR) model mice with anti-MISP antibody and detecting and quantifying the MISP-positive region in proximal tubular cells using ImageJ.

[0054] In Figure 3(a), "WT" indicates the result of control wild-type mice, and Tns2 nph indicates the result of Tensin2-deficient model mice. In Figure 3(b), "Control" indicates the result of control mice, and "Anti-Nephrin" indicates the result of anti-nephrin antibody-induced nephropathy model mice. In Figure 3(c), "Control" indicates the result of control mice, and "IR" indicates the result of renal ischemia-reperfusion-induced renal insufficiency (IR) model mice. In Figures 3(a) to (c), the vertical axis indicates the relative value of the area of the MISP-positive region. Also, "P<0.001" indicates that there is a significant difference at P<0.001.

[0055] As a result, all of Tensin2-deficient model mice, anti-nephrin antibody-induced nephropathy model mice, and renal ischemia-reperfusion-induced renal insufficiency (IR) model mice showed the same results as adriamycin nephropathy model mice.

[0056] These immunochemical staining results confirmed that MISP protein expression was significantly increased in injured proximal tubular epithelium. Combined with the results of the database analysis in Experimental Example 1, this suggests that increased expression of the MISP gene or protein is a common phenomenon in various nephropathies.

[0057] [Experimental Example 3] (Examination of the dynamics of MISP expression in proximal tubular epithelial cell lines) Since MISP was found to be expressed in the proximal tubular epithelium, we investigated the dynamics of MISP expression using a proximal tubular epithelial cell line.

[0058] We investigated the in vitro expression of MISP in mouse renal tubular epithelial cell line MuRTE61 and human renal tubular epithelial cell line HK-2 using an ischemic injury model in which antimycin A (AMA) was added to induce chemical hypoxia. No significant cell loss or cell death was observed at 0, 3, or 6 hours after AMA addition. Cells were harvested 6 hours after AMA addition, and protein and RNA were extracted.

[0059] Next, the mRNA level of MISP was quantified by quantitative real-time RT-PCR (ΔΔCt method), and the expression of NGAL protein, a marker of renal tubular injury, was detected by Western blot.

[0060] Figure 4(a) is a graph showing the results of quantitative real-time RT-PCR of MuRTE61 cells. In Figure 4(a), "Control" indicates the result for control cells to which antimycin A (AMA) was not added, and "AMA" indicates the result for cells to which antimycin A (AMA) was added. The vertical axis of the graph indicates the relative expression level of the MISP gene, with the expression level of glyceraldehyde-3-phosphate dehydrogenase set to 1. Furthermore, "P<0.001" indicates a significant difference at P<0.001.

[0061] Figure 4(b) is a photograph showing the results of Western blotting of MuRTE61 cells. In Figure 4(b), "NGAL" indicates the results of detecting the NGAL protein, and "GAPDH" indicates the results of detecting the glyceraldehyde-3-phosphate dehydrogenase protein.

[0062] Figure 4(c) is a graph showing the results of quantitative real-time RT-PCR of HK-2 cells. In Figure 4(c), "Control" indicates the result for control cells to which antimycin A (AMA) was not added, and "AMA" indicates the result for cells to which antimycin A (AMA) was added. The vertical axis of the graph indicates the relative expression level of the MISP gene, with the expression level of glyceraldehyde-3-phosphate dehydrogenase set to 1. Furthermore, "P<0.001" indicates a significant difference at P<0.001.

[0063] Figure 4(d) is a photograph showing the results of Western blotting of HK-2 cells. In Figure 4(d), "NGAL" indicates the results of detecting NGAL protein, and "GAPDH" indicates the results of detecting glyceraldehyde-3-phosphate dehydrogenase protein.

[0064] As a result, it was revealed that the expression of MISP mRNA increased along with the expression of NGAL protein, a marker of renal tubule injury. These results demonstrate that the expression of MISP gene or protein increases in response to injury, not only in vivo but also in vitro.

[0065] [Experimental Example 4] (Phenotype analysis of MISP knockout mice) To investigate whether MISP is involved in kidney damage, we performed phenotypic analysis using MISP knockout mice. Knockout mice were generated by genome editing using CRISPR / Cas9 in fertilized eggs of FVB mice. The results showed that MISP knockout mice did not exhibit significant phenotypes. This result indicates that knockdown of the MISP gene has few side effects.

[0066] [Experimental Example 5] (Phenotype analysis of MISP gene and Tensin2 gene knockout mice) MISP knockout mice (hereinafter referred to as "MISP KO The Tensin2 (Tns2) gene deletion mutation (hereafter referred to as "Tns2 nph A nephropathy model mouse carrying the Tns2 mutation (hereinafter referred to as "Tns2 nph We created mice (sometimes called "false-skinned mice") and performed phenotypic analysis.

[0067] Specifically, MISP knockout mice (MISP KO ), Tns2 knockout mice (Tns2 nph ), MISP gene and Tns2 gene double knockout mice (hereinafter referred to as "MISP KO Tns2 nph Urine was collected from each mouse at 8 weeks of age, and kidneys were collected at 10 weeks of age.

[0068] To compare the severity of nephropathy, albumin in urine at 8 weeks of age was detected by SDS-PAGE and the amount of albumin in urine was quantified using ImageJ.

[0069] Figure 5(a) is a photograph showing the results of detecting urinary albumin by SDS-PAGE, and Figure 5(b) is a graph showing the numerical results of Figure 5(a).

[0070] As a result, double knockout mice (MISP) KO Tns2 nph) are Tns2 knockout mice (Tns2 nph ) compared with the control group, the amount of albumin in urine was significantly reduced (P<0.001).

[0071] Subsequently, a 20% weight loss was defined as the humane endpoint, and a survival curve was created. nph ), and MISP gene and Tns2 gene double knockout mice (MISP KO Tns2 nph ) survival curve. nph " indicates the results of Tns2 knockout mice, and "Double" indicates the results of double knockout mice of the Tns2 gene (MISP). KO Tns2 nph ) The horizontal axis indicates time (weeks), and the vertical axis indicates survival rate (%).

[0072] As a result, MISP KO Tns2 nph Mice, Tns2 nph Compared to mice, the average lifespan was increased by approximately three times, and the progression of tubulointerstitial damage was clearly suppressed.

[0073] Next, Tns2 nph Mouse and MISP KO Tns2 nph To compare the renal tubule injury in mice, kidney tissue sections were immunochemically stained with anti-NGAL antibody (catalog number ab63929, Abcam) to compare the expression levels of NGAL, a tubule injury marker. The MISP-positive area of proximal tubule cells was quantified using ImageJ.

[0074] Figure 7(a) shows Tns2 nph Figure 7(b) shows a representative result of immunochemical staining of mouse kidney tissue sections. The scale bar is 50 μm. Figure 7(b) shows the MISP KO Tns2 nphPhotographs showing representative results of immunochemical staining of mouse kidney tissue sections. The scale bar is 50 μm. In Figures 7(a) and (b), "*" indicates a proximal tubule with severe damage.

[0075] Figure 7(c) is a graph showing the results of quantifying the MISP-positive area of proximal tubule cells based on the results of immunochemical staining. The vertical axis shows the relative value of the area of the MISP-positive area. In Figures 7(a) to (c), "Tns2 nph " is Tns2 nph "Double" indicates a mouse result, and "MISP" indicates a mouse result. KO Tns2 nph The results are from mice.

[0076] As a result, MISP KO Tns2 nph In mouse kidney tissue sections, Tns2 nph The expression of NGAL protein was significantly decreased in the MISP-treated kidney tissue sections compared with that in the control mice (P<0.001). KO Tns2 nph In mice, Tns2 nph This shows that tubulointerstitial damage was significantly reduced compared to mice.

[0077] [Experimental Example 6] (Effects of MISP knockout on anti-nephrin antibody-induced nephropathy in mouse models) We generated a mouse model of anti-nephrin antibody-induced nephropathy using MISP knockout mice and performed phenotypic analysis. Glomerular and tubulointerstitial injury was induced in the mice by administering an anti-nephrin antibody (see Takeuchi K., et al., New anti-nephrin antibody mediated podocyte injury model using a C57BL / 6 mouse strain. Nephron, 138(1), 71-87, 2018).

[0078] 12-week-old wild-type FVB mice and MISP knockout mice (FVB-MISP)KO ) Anti-nephrin antibody (1.5 mg / mouse) was administered via the tail vein. Kidneys were collected 12 weeks after antibody administration.

[0079] Chronically injured tubular epithelial cells are known to have a reduced epithelial thickness, and this phenomenon is called tubular epithelial thinning.

[0080] Therefore, the thickness of tubular epithelial cells was measured to quantify tubular injury. Cells with clear cell membranes and nuclei in one tubule were selected and the thickness (length) was measured. In addition, proteins were extracted from kidney tissue (renal cortex), and Western blotting was performed using an anti-NGAL antibody (catalog number "ab63929", Abcam). Also, the bands obtained by Western blotting were quantified using ImageJ.

[0081] Figures 8(a) and (b) are representative micrographs of kidney tissue sections. Figure 8(a) is a photograph of a kidney tissue section of wild-type FVB mice, and Figure 8(b) is a photograph of a kidney tissue section of MISP KO mice. In Figures 8(a) and (b), the scale bar is 50 μm. Also, "*" indicates the injured and thinned proximal tubules.

[0082] Figure 8(c) is a graph quantifying the thickness of tubular epithelial cells. In Figure 8(c), the vertical axis indicates the thickness (relative value) of tubular epithelial cells. Also, "Control" indicates the result without anti-nephrin antibody administration, and "Anti-Nephrin" indicates the result with anti-nephrin antibody administration. Also, "WT" indicates the result of wild-type FVB mice, and "MISP KO " indicates the result of MISP KO mice. Also, "****" indicates a significant difference at P < 0.0001, and "n.s." indicates no significant difference.

[0083] Figure 8(d) is a photograph showing the results of Western blotting. In Figure 8(d), lane 1 shows the results of WT mice not administered with anti-nephrin antibody, lane 2 shows the results of WT mice administered with anti-nephrin antibody, lane 3 shows the results of MISP KO mice not administered with anti-nephrin antibody, and lane 4 shows the results of MISP KO mice administered with anti-nephrin antibody. Also, "NGAL" indicates the result of detecting the NGAL protein, and "GAPDH" indicates the result of detecting the glyceraldehyde-3-phosphate dehydrogenase protein.

[0084] As a result, in wild-type FVB mice, the thickness of the renal tubular epithelial cells decreased upon administration of anti-nephrin antibody, whereas in MISP KO mice, no significant decrease was observed. Also, it was revealed that knockout of the MISP gene decreased the expression level of NGAL when anti-nephrin antibody was administered.

[0085] The above results indicate that in MISP KO mice, the tubulointerstitial injury caused by anti-nephrin antibody was significantly reduced compared to wild-type mice.

[0086] [Experimental Example 7] (Examination of the effect of MISP knockout in a mouse model of renal ischemia-reperfusion-induced renal insufficiency (IR)) A mouse model of renal ischemia-reperfusion-induced renal insufficiency (IR) was prepared using MISP knockout mice, and phenotypic analysis was performed. Tubular injury was induced by renal ischemia injury.

[0087] Ten-week-old wild-type FVB mice and MISP knockout mice (FVB-MISP KO ) were used. The left kidney was removed 10 days before ischemia induction. Ten days after left kidney removal, the renal artery and vein of the right kidney were clamped with a microvascular clamp to maintain an ischemic state for 30 minutes. After 30 minutes of ischemia, the clamp was released, the skin was sutured, and the kidneys were collected 24 hours later.

[0088] Immunochemical staining and Western blotting were performed using an anti-NGAL antibody (Catalog number "ab63929", Abcam). Also, the bands obtained by Western blotting were quantified using ImageJ.

[0089] Figures 9(a) and (b) are representative micrographs showing the results of immunochemical staining of kidney tissue sections. Figure 9(a) is a photograph showing the results of wild-type FVB mice, and Figure 9(b) is a photograph showing the results of MISP KO mice. In Figures 9(a) and (b), the scale bar is 50 μm. Also, "WT" indicates the results of wild-type FVB mice, and "MISP KO " indicates the results of MISP KO mice.

[0090] Figure 9(c) is a photograph showing the results of Western blotting. In Figure 9(c), "WT" indicates the results of wild-type FVB mice, and "MISP KO " indicates the results of MISP KO mice. Also, "Control" indicates the results without renal ischemia-reperfusion injury, and "I / R" indicates the results with renal ischemia-reperfusion injury. Also, "NGAL" indicates the results of detecting the NGAL protein, and "GAPDH" indicates the results of detecting the glyceraldehyde-3-phosphate dehydrogenase protein.

[0091] Figure 9(d) is a graph showing the quantified expression level of the NGAL protein. In Figure 9(c), the vertical axis indicates the expression level (relative value) of the NGAL protein. Also, "WT" indicates the results of wild-type FVB mice, and "MISP KO " indicates the results of MISP KO mice. Also, "Sham" indicates the results without renal ischemia-reperfusion injury, and "IR" indicates the results with renal ischemia-reperfusion injury. Also, "P<0.05" indicates that there is a significant difference at P<0.05, and "n.s." indicates that there is no significant difference.

[0092] As a result, in wild-type FVB mice, renal ischemic injury significantly increased the expression of NGAL protein, whereas MISP KO In mice, no significant increase in NGAL protein expression was observed even after renal ischemic injury.

[0093] These results suggest that the MISP KO This shows that tubulointerstitial damage caused by renal ischemic injury was significantly reduced in mice.

[0094] [Experimental Example 8] (Study using human samples) To clarify whether the increased expression of MISP observed in nephropathy model mice is also observed in humans, renal tissue sections from patients with different underlying nephropathy, namely, IgA nephropathy, focal segmental glomerulosclerosis, and tubulointerstitial nephritis, were immunochemically stained with anti-MISP antibody and stained with 3,3'-diaminobenzidine (DAB).

[0095] Figure 10(a) is a representative photograph showing the results of immunochemical staining of a renal tissue section from a normal part of the kidney (Control). Figure 10(b) is a representative photograph showing the results of immunochemical staining of a renal tissue section from a patient with IgA nephropathy (IgA). Figure 10(c) is a representative photograph showing the results of immunochemical staining of a renal tissue section from a patient with focal segmental glomerulosclerosis (FSGS). Figure 10(d) is a representative photograph showing the results of immunochemical staining of a renal tissue section from a patient with tubulointerstitial nephritis (TIN). In Figures 10(a) to (d), the scale bar indicates 100 μm.

[0096] In addition, Fig. 11(a) is a graph showing the ratio of MISP-positive tubules in renal tissue sections of IgA nephropathy (IgA). "Control" indicates the results in renal tissue sections of normal parts of the kidney. Fig. 11(b) is a graph showing the ratio of MISP-positive tubules in renal tissue sections of focal segmental glomerulosclerosis (FSGS). "Control" indicates the results in renal tissue sections of normal parts of the kidney. Fig. 11(c) is a graph showing the ratio of MISP-positive tubules in renal tissue sections of tubulointerstitial nephritis (TIN). "Control" indicates the results in renal tissue sections of normal parts of the kidney.

[0097] As a result, it was revealed that the expression of MISP protein was enhanced in the proximal tubular epithelium in all nephrotic tissues. This result indicates that the enhanced expression of MISP observed in nephrotic model mice is also recognized in humans. Also, combined with the analysis results of nephrotic model mice, it was considered that the enhanced expression of MISP is a common event in various nephropathies.

Industrial Applicability

[0098] According to the present invention, a technique for preventing or treating tubulointerstitial damage can be provided.

Claims

1. A step of determining whether a test substance inhibits the function of Mitotic Spindle Positioning (MISP) protein is included, wherein the test substance inhibiting the function of MISP protein indicates that the test substance is a candidate for a prophylactic or therapeutic agent for tubulointerstitial injury, A screening method for a prophylactic or therapeutic agent for tubulointerstitial injury.

2. The step of determining whether a test substance inhibits the function of MISP protein is a step of applying hypoxia stimulation to proximal tubular epithelial cells in the presence of the test substance, and a step of quantifying the expression level of MISP gene or MISP protein in the cells, and in the presence of the test substance, a decrease in the expression level of the MISP gene or MISP protein as compared to the absence of the test substance indicates that the test substance inhibits the function of MISP protein, The screening method according to Claim 1.

3. The screening method according to Claim 2, wherein the hypoxia stimulation is performed by adding antimycin A to the medium.

4. The step of determining whether a test substance inhibits the function of MISP protein is a step of culturing cells expressing MISP gene or MISP protein in the presence of the test substance, and a step of quantifying the expression level of MISP gene or MISP protein in the cells, and in the presence of the test substance, a decrease in the expression level of the MISP gene or MISP protein as compared to the absence of the test substance indicates that the test substance inhibits the function of MISP protein, The screening method according to Claim 1.

5. A prophylactic or therapeutic agent for tubulointerstitial injury, comprising a function inhibitor of MISP protein as an active ingredient, wherein the function inhibitor is siRNA, shRNA or morpholino oligo against MISP gene.

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