FGF2 biomarker as target for diagnosing and treating autosomal dominant polycystic kidney disease and use thereof

Abstract

The present invention relates to an FGF2 biomarker as a target for diagnosing and treating autosomal dominant polycystic kidney disease (ADPKD) and use thereof. Specifically, in the present invention, it was confirmed that an FGF2 gene was increased in the cells and tissues of patients with ADPKD, which is one of the kidney diseases in which fibrosis and cysts occur, and it was suggested that the expression of FGF2 secreted from cyst cells of ADPKD patients is involved in fibrosis by interacting with fibroblasts. In addition, it was verified at the cellular level in ADPKD patients that reducing FGF2 expression by using FGF2 siRNA had effects on cell proliferation and FGF2 secretion, and it was verified at the cellular level that conditioned medium under conditions in which FGF2 expression was reduced by using FGF2 siRNA had effects on fibroblast proliferation, wound healing, and fibrosis-related markers. Therefore, it is expected that the changes in FGF2 expression in the kidneys, discovered in the present invention, can be used in the development of novel therapeutic agents for polycystic kidney disease and fibrosis, and are also expected to be usable as biomarkers for diagnosing and treating ADPKD and fibrosis.

Description

FGF2 Biomarker as Diagnostic and Therapeutic Target for Autosomal Dominant Polycystic Kidney Disease and Its Uses

[0001] The present invention relates to an FGF2 biomarker as a diagnostic and therapeutic target for autosomal dominant polycystic kidney disease (ADPKD) and its use.

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease, with an incidence rate of at least 1 in 500 to 1,000 people. Compared to healthy individuals, ADPKD is characterized by the formation of numerous fluid-filled cysts in both kidneys and a 4 to 8-fold increase in kidney size. To date, there is no effective clinical treatment for polycystic kidney disease, so most cases progress to end-stage renal disease. In human polycystic kidneys, cyst formation is accompanied by increased cell proliferation of cystic epithelial cells and apoptosis.

[0003] ADPKD requires management from the early stages of the disease because it not only causes structural and functional defects in the kidneys but can also be accompanied by various extrarenal complications. Since the causative gene was identified, various changes and abnormalities in intracellular signaling pathways have been revealed, leading to attempts at drug therapy to prevent disease progression. However, there is currently no known cure for ADPKD, and recent research directions for treatment are focused on reducing morbidity and mortality caused by complications. To achieve this, the development of technology capable of diagnosing ADPKD early using non-invasive methods is required.

[0004] The object of the present invention is to provide a biomarker composition for diagnosing ADPKD comprising an FGF2 protein or a gene encoding the same.

[0005] In addition, another objective of the present invention is to provide a composition for diagnosing ADPKD comprising, as an active ingredient, a preparation capable of measuring the expression level of the FGF2 protein or the gene encoding it.

[0006] In addition, another objective of the present invention is to provide an ADPKD diagnostic kit comprising the above composition.

[0007] In addition, another objective of the present invention is to provide a method for providing information necessary for diagnosing ADPKD, comprising the step of measuring the expression level of the FGF2 protein or the gene encoding it.

[0008] In addition, another objective of the present invention is to provide a composition for the prevention, improvement, or treatment of ADPKD comprising an inhibitor of the expression or activity of the FGF2 protein or the gene encoding it as an active ingredient.

[0009] In addition, another objective of the present invention is to provide a screening method for ADPKD therapeutic agents comprising the step of selecting a test substance in which the expression or activity level of the FGF2 protein or the gene encoding it is reduced.

[0010] To achieve the above objective, the present invention provides a biomarker composition for diagnosing ADPKD comprising an FGF2 protein or a gene encoding the same.

[0011] In addition, the present invention provides a composition for diagnosing ADPKD comprising, as an active ingredient, a preparation capable of measuring the expression level of the FGF2 protein or the gene encoding it.

[0012] In addition, the present invention provides an ADPKD diagnostic kit comprising the above composition.

[0013] In addition, the present invention provides a method for providing information necessary for diagnosing ADPKD, comprising the steps of: (1) measuring the expression level of an FGF2 protein or a gene encoding it from a sample isolated from a patient; (2) comparing the expression level of the FGF2 protein or the gene encoding it with a control sample; and (3) determining that the FGF2 protein or the gene encoding it is higher than the control sample.

[0014] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of ADPKD comprising an inhibitor of the expression or activity of the FGF2 protein or the gene encoding it as an active ingredient.

[0015] In addition, the present invention provides a health functional food for the prevention or improvement of ADPKD comprising an inhibitor of the expression or activity of the FGF2 protein or the gene encoding it as an active ingredient.

[0016] In addition, the present invention provides a screening method for ADPKD therapeutic agents comprising: (1) a step of contacting a test substance with isolated ADPKD patient cells; (2) a step of measuring the degree of expression or activity of an FGF2 protein or a gene encoding it in the ADPKD patient cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of expression or activity of the FGF2 protein or a gene encoding it is reduced compared with a control sample.

[0017] The present invention relates to an FGF2 biomarker as a diagnostic and therapeutic target for autosomal dominant polycystic kidney disease and its use. Specifically, the present invention confirmed that the FGF2 gene was increased in patient cells and tissues of autosomal dominant polycystic kidney disease, which is one of the kidney diseases in which fibrosis and cysts occur, and suggested that the expression of FGF2 secreted from cyst cells of autosomal dominant polycystic kidney disease is involved in fibrosis by interacting with fibroblasts. Furthermore, the invention verified at the level of autosomal dominant polycystic kidney patient cells that reducing FGF2 expression using FGF2 siRNA had an effect on cell proliferation and FGF2 secretion, and verified at the cellular level that reducing FGF2 expression using FGF2 siRNA had an effect on cell proliferation, wound healing, and fibrosis-related markers in fibroblasts through a conditioned culture medium. Accordingly, the changes in FGF2 expression in the kidney discovered in this invention can be utilized in the development of new therapeutic agents for polycystic kidney disease and fibrosis capable of alleviating fibrosis and regulating FGF2 expression. Furthermore, it is expected that this can be presented as one of the bio-diagnostic markers for diagnosing autosomal dominant polycystic kidney disease and fibrosis, as well as as a biomarker applicable to the development of therapeutic agents.

[0018] Figure 1 shows the results of the analysis of FGF2 expression in tissue from a patient with autosomal dominant polycystic kidney disease compared to tissue from a normal patient.

[0019] Figure 2 shows the results of the analysis of the expression of FGF2, vimentin, α-SMA, and TGF-beta1 in tissues of patients with autosomal dominant polycystic kidney disease compared to normal patient tissues (A and B), and the results of the analysis after reduction of FGF2 in cells of patients with autosomal dominant polycystic kidney disease using siRNA (C to E).

[0020] Figure 3 shows the degree of FGF2 secretion in serum-free conditioned culture medium in normal cells and cells of patients with autosomal dominant polycystic kidney disease (A) and the results of cell proliferation in fibroblasts exposed to said culture medium (B), FGF2 secretion in serum-free culture medium after reducing FGF2 in cells of patients with autosomal dominant polycystic kidney disease through FGF2 siRNA (C), cell proliferation in fibroblasts exposed to said culture medium, the degree of wound healing and the results of expression analysis of FGF2, vimentin, α-SMA, Collagen, TGF-beta, and N-cadherin (DG).

[0021] The present invention provides a biomarker composition for diagnosing ADPKD comprising an FGF2 protein or a gene encoding the same.

[0022] In this specification, the term “diagnosis” includes determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object with a specific disease or condition, or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0023]

[0024] In addition, the present invention provides a composition for diagnosing ADPKD comprising, as an active ingredient, a preparation capable of measuring the expression level of the FGF2 protein or the gene encoding it.

[0025] More specifically, a preparation capable of measuring the expression level of the FGF2 protein or the gene encoding it may be a primer or probe that specifically binds to the FGF2 gene, an antibody, peptide, aptamer, or compound that specifically binds to the FGF2 protein, but is not limited thereto.

[0026]

[0027] In addition, the present invention provides an ADPKD diagnostic kit comprising the above composition.

[0028]

[0029] In this specification, the term “primer” refers to a short nucleic acid sequence having a short free 3’ hydroxyl group, capable of forming base pairs with a complementary template, and acting as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0030] In this specification, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA. It is labeled to confirm the presence or absence and expression level of a specific mRNA. Probes may be constructed in the form of oligonucleotide probes, single-strand DNA probes, double-strand DNA probes, RNA probes, etc. The selection of appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.

[0031] In this specification, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed toward an antigenic site. An antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody may be prepared according to conventional methods in the art. The forms of the antibody include polyclonal antibodies or monoclonal antibodies, and include all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

[0032] In this specification, the term "peptide" has the advantage of high binding affinity to target substances and does not undergo denaturation even during heat or chemical treatment. Additionally, due to its small molecular size, it can be attached to other proteins to be used as a fusion protein. Specifically, since it can be attached to high-molecular-weight protein chains, it can be used as a diagnostic kit and a drug delivery material.

[0033] In this specification, the term "aptamer" refers to a type of polynucleotide composed of a special kind of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and can bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to antigenic substances just like antibodies, but because they are composed of polynucleotides that are more stable than proteins, have a simple structure, and are easy to synthesize, they can be used as a substitute for antibodies.

[0034]

[0035] In addition, the present invention provides a method for providing information necessary for diagnosing ADPKD, comprising the steps of: (1) measuring the expression level of an FGF2 protein or a gene encoding it from a sample isolated from a patient; (2) comparing the expression level of the FGF2 protein or the gene encoding it with a control sample; and (3) determining that the FGF2 protein or the gene encoding it is higher than the control sample.

[0036] Specifically, the method for measuring the gene expression level described above uses RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips, but is not limited thereto.

[0037] Specifically, the method for measuring the protein expression level described above uses Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chips, but is not limited thereto.

[0038]

[0039] In this specification, the term “sample isolated from a patient” includes, but is not limited to, samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ from the control group in the expression level of the FGF2 gene or FGF2 protein, which is the biomarker of the present invention.

[0040]

[0041] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of ADPKD comprising an inhibitor of the expression or activity of the FGF2 protein or the gene encoding it as an active ingredient.

[0042] More specifically, the inhibitor of expression or activity of the FGF2 protein or the gene encoding it may be any one selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA) and short hairpin RNA (shRNA), compounds, peptides, aptamers, and antibodies that specifically bind to the FGF2 protein or the gene encoding it, but is not limited thereto.

[0043] Preferably, the pharmaceutical composition may alleviate fibrosis by reducing cell proliferation, reducing wound healing, and reducing fibrosis markers in fibroblasts, but is not limited thereto.

[0044] The pharmaceutical composition of the present invention may include chemical substances, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or complex formulation of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients, and said adjuvants may include solubilizing agents such as excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, or flavoring agents. The pharmaceutical composition of the present invention may preferably be formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients for administration. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0045] The pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable liquids, as well as sustained-release formulations of the active compound. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intra-arterial, intraperitoneal, intramuscular, intra-arterial, intraperitoneal, intrasternal, transdermal, nasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes. The effective amount of the active ingredient in the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Accordingly, it may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health status, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and concurrently used drugs. Although not limited thereto, for example, in the case of an adult, when administered once or several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg in the case of a compound, 0.1 ng / kg to 10 g / kg in the case of a polypeptide, protein or antibody, and 0.01 ng / kg to 10 g / kg in the case of an antisense nucleotide, siRNA, shRNAi, or miRNA.

[0046]

[0047] In addition, the present invention provides a health functional food composition for the prevention or improvement of ADPKD comprising an inhibitor of the expression or activity of FGF2 protein or a gene encoding it as an active ingredient.

[0048] The health functional food composition of the present invention may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage, and the health functional food composition may be used in combination with other foods or food additives in addition to the active ingredient, and may be used appropriately according to conventional methods. The amount of the mixture of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health, or therapeutic treatment.

[0049] The effective dose of the active ingredient contained in the above health functional food composition may be used in accordance with the effective dose of the above pharmaceutical composition, but in the case of long-term consumption for the purpose of health and hygiene or health control, it may be less than the above range, and it is certain that the active ingredient may be used in an amount greater than the above range because there is no problem in terms of safety.

[0050] There are no special restrictions on the types of health foods mentioned above, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0051]

[0052] In addition, the present invention provides a screening method for ADPKD therapeutic agents comprising: (1) a step of contacting a test substance with isolated ADPKD patient cells; (2) a step of measuring the degree of expression or activity of an FGF2 protein or a gene encoding it in the ADPKD patient cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of expression or activity of the FGF2 protein or a gene encoding it is reduced compared with a control sample.

[0053] Preferably, the ADPKD therapeutic agent may inhibit fibrosis, but is not limited thereto.

[0054]

[0055] The term "test substance" used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. The sample includes, but is not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0056] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0057]

[0058] <Experimental Example>

[0059] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0060]

[0061] 1. Human cell

[0062] Normal cells were obtained from ATCC's RPTEC / hTERT, and cells from patients with autosomal dominant polycystic kidney disease were obtained from Seoul National University Hospital.

[0063]

[0064] 2. Cell Culture

[0065] Normal cells were cultured in Dulbecco's modified Eagle medium (DMEM) / F-12 containing G418 (0.1 mg / ml), 1% FBS, and penicillin-streptomycin. Cells from patients with autosomal dominant polycystic kidney disease were cultured in Dulbecco's modified Eagle medium (DMEM) containing G418 (0.1 mg / ml), 10% FBS, and penicillin-streptomycin. Fibroblasts were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS and penicillin-streptomycin.

[0066]

[0067] 3. Transfection

[0068] FGF2 siRNA was purchased from Bioneer, and FGF2 siRNA was transfected into cells of patients with autosomal dominant polycystic kidney disease for 48 hours using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen).

[0069]

[0070] 4. Real-time Polymerase Chain Reaction (qRT-PCR, qPCR)

[0071] RNA was extracted from normal cells and cells of patients with autosomal dominant polycystic kidney disease using the Nucleospin kit (MACHEREY-NAGEL). The extracted RNA was quantified using Nanodrop. LightCycler ® mRNA expression levels were confirmed by PCR amplification using the fluorescent qPCRBIO SyGreen Blue mix (PCRBIOSYSTEMS) via the 96 System (Roche). The primers used for PCR were purchased from Bionics.

[0072] Statistical analysis was performed in GraphPad Prism 5.0.

[0073]

[0074] 5. FGF2 Enzyme-Linked Immunoassay (ELISA)

[0075] The level of FGF2 secretion was confirmed using a Human FGF2 ELISA kit (R&D system). After culturing normal cells and cells from patients with autosomal dominant polycystic kidney disease for 24 hours, serum-free conditioned culture medium was obtained and evaluated.

[0076] Cells from patients with autosomal dominant polycystic kidney disease were transfected with siRNA of fibroblast growth factor 2 (FGF2) for 48 hours and cultured in a serum-free medium for 24 hours. Afterward, serum-free conditioned cultures were obtained and evaluated.

[0077] Statistical analysis was performed in GraphPad Prism 5.0.

[0078]

[0079] 6. SnRNA sequencing

[0080] FGF2 expression in patients with autosomal dominant polycystic kidney disease was analyzed against normal patients using KIT Statistics (https: / humphreyslab.com / SingleCell / ). Darker colors indicate higher cell expression, while larger circles indicate a greater number of cell clusters.

[0081]

[0082] 7. Immunofluorescence (IF)

[0083] The expression levels and locations of FGF2 and vimentin were determined in tissues from normal patients and patients with autosomal dominant polycystic kidney disease using immunofluorescence. After rehydrating the dehydration slides, heat was applied using a Borg Decloaker RTU (BD1000G1; Biocare Medical) to restore antigens. Blocking was performed for 10 minutes using a mix of normal horse serum (Vector Laboratories) and cold PBS. The blocking mix containing the primary antibody was applied to the tissue and stored at 4°C for 24 hours. The primary antibody was washed off, and the blocking mix was applied to the fluorescence-conjugated secondary antibody at room temperature for 2 hours. The tissue was mounted using mounting solution (Vector Laboratories) for microscopic observation.

[0084]

[0085] 8. Cellular Immunofluorescence (ICC)

[0086] Place a cover glass in a 6-well plate and collect 5×10 cells of autosomal dominant polycystic kidney disease patients. 4 Cells were arranged in wells. Cells from patients with autosomal dominant polycystic kidney disease were transfected with siRNA of fibroblast growth factor 2 (FGF2) and cultured for 48 hours. After removing the culture medium, the cells were fixed with 10% neutral buffered formalin at room temperature for 10 minutes and washed with PBS. They were stored at 4°C for 24 hours in a permeabilizing solution containing the primary antibody. After washing with the primary antibody, a mixture of the fluorescence-conjugated secondary antibody and the permeabilizing solution was inoculated at room temperature for 2 hours. For microscopic observation, the cells were mounted on fluorescence mounting medium (S3023; Dako).

[0087] Place a cover glass in a 6-well plate and add 5×10⁶ fibroblasts. 4 Cells were arranged in wells. Fibroblasts were exposed to 10% FBS-conditioned culture medium obtained by transfecting fibroblast growth factor 2 (FGF2) siRNA into cells from patients with autosomal dominant polycystic kidney disease and culturing them for 48 hours. After removing the culture medium, cells were fixed with 10% neutral buffered formalin at room temperature for 10 minutes and washed with PBS. They were stored at 4°C for 24 hours in a permeabilizing solution containing the primary antibody. After washing the primary antibody, a mixture of the fluorescence-conjugated secondary antibody and the permeabilizing solution was attached at room temperature for 2 hours. Cells were mounted on fluorescence mounting medium (S3023; Dako) for microscopic observation.

[0088] Statistical analysis was performed in GraphPad Prism 5.0.

[0089]

[0090] 9. Cell proliferation analysis (CCK8 proliferation assay)

[0091] Cells from patients with autosomal dominant polycystic kidney disease were seeded in a 96-well plate. Cells from patients with autosomal dominant polycystic kidney disease were transfected with siRNA of fibroblast growth factor 2 (FGF2) and cultured. Cell proliferation was evaluated at different time points.

[0092] NIH3T3 fibroblasts were plated in a 96-well plate. After microscopically photographing the cells at 0 hours prior to exposure to the serum-free conditioned medium, the fibroblasts were exposed to the serum-free conditioned medium, and cell proliferation analysis was evaluated at different time points.

[0093] Statistical analysis was performed in GraphPad Prism 5.0.

[0094]

[0095] 10. Wound healing assay

[0096] Wound healing analysis was evaluated using serum-free conditioned medium.

[0097] NIH3T3 fibroblasts in a 24-well plate at a rate of 5×10 4 500 μL was spread into the cells / wells. Wounds were created by scraping at even intervals using a 1000p tip. Wound healing was analyzed by taking microscopic images at the same location to observe changes in wound healing at 0 hours (without exposure to serum-free conditioned culture) and after 24 hours of exposure to serum-free conditioned culture.

[0098] Statistical analysis was performed in GraphPad Prism 5.0.

[0099]

[0100] <Example>

[0101] When analyzing intracellular expression in tissues of patients with autosomal dominant polycystic kidney disease, it was confirmed through IF staining that FGF2 expression was increased in cyst cells compared to normal patient tissues (Fig. 1A).

[0102] It was confirmed through human snRNA+ scATAC-seq data that the expression of FGF2 was increased in tissues of patients with autosomal dominant polycystic kidney disease compared to tissues of normal patients (Fig. 1B).

[0103]

[0104] IF staining confirmed that the expression of vimentin in cyst cells was increased in the tissues of patients with autosomal dominant polycystic kidney disease compared to normal patient tissues (Fig. 2A).

[0105] It was confirmed by qRT-PCR that mRNA expression of FGF2, vimentin, α-SMA, and TGF-beta1 was significantly increased in cells of patients with autosomal dominant polycystic kidney disease compared to normal cells (Fig. 2B).

[0106] It was confirmed by qRT-PCR that when FGF2 was reduced in cells of patients with autosomal dominant polycystic kidney disease using FGF2 siRNA, the mRNA expression of FGF2, vimentin, α-SMA, and TGF-beta1 was significantly reduced compared to the control (Fig. 2C).

[0107] Cell proliferation analysis confirmed that cell proliferation was significantly reduced compared to the control when FGF2 siRNA was used to reduce cells from patients with autosomal dominant polycystic kidney disease (Fig. 2D).

[0108] It was confirmed through cell immunofluorescence that vimentin was significantly reduced compared to the control when FGF2 siRNA was used to reduce cells of patients with autosomal dominant polycystic kidney disease (Fig. 2E).

[0109]

[0110] We intended to determine the level of FGF2 secretion in serum-free conditioned cultures of normal cells and cells from patients with autosomal dominant polycystic kidney disease. Through FGF2 ELISA, it was confirmed that the secretion of FGF2 was significantly increased in the culture medium of patients with autosomal dominant polycystic kidney disease compared to normal cells (Fig. 3A).

[0111] As a result of analyzing cell proliferation by exposing NIH3T3 fibroblasts to serum-free conditioned cultures of normal cells and cells from patients with autosomal dominant polycystic kidney disease, respectively, it was confirmed that cell proliferation was significantly increased in fibroblasts exposed to serum-free conditioned cultures of patients with autosomal dominant polycystic kidney disease compared to serum-free conditioned cultures of normal cells (Fig. 3B).

[0112] After reducing FGF2 levels in cells from patients with autosomal dominant polycystic kidney disease using FGF2 siRNA, the cells were cultured in a serum-free medium for 24 hours to obtain a serum-free conditioned medium. Changes in FGF2 secretion were confirmed by FGF2 ELISA, and it was found that FGF2 secretion was significantly reduced in the serum-free conditioned medium with reduced FGF2 levels compared to the control (Fig. 3C).

[0113] When NIH3T3 fibroblasts were exposed to a serum-free conditioned culture medium under the same conditions as in Fig. 3C, it was confirmed that cell proliferation was significantly reduced in fibroblasts exposed to the culture medium with reduced FGF2 (Fig. 3D).

[0114] For the analysis of wound healing, NIH3T3 fibroblasts were exposed to a serum-free culture medium under the same conditions as the cell proliferation analysis. It was confirmed that the degree of wound healing was significantly reduced in fibroblasts exposed to a culture medium with reduced FGF2 (Fig. 3E).

[0115] FGF2 was reduced in cells from patients with autosomal dominant polycystic kidney disease using FGF2 siRNA, and a 10% FBS-conditioned culture medium was obtained after culture. NIH3T3 fibroblasts were exposed to the 10% FBS-conditioned culture medium to check for changes in mesenchymal markers. PCR confirmed that the mRNA expression of FGF2, vimentin, α-SMA, Collagen, TGF-beta, and N-cadherin was significantly reduced in fibroblasts exposed to the culture medium with reduced FGF2 (Fig. 3F).

[0116] FGF2 was reduced in cells from patients with autosomal dominant polycystic kidney disease using FGF2 siRNA, and a 10% FBS-conditioned culture medium was obtained after culture. NIH3T3 fibroblasts were exposed to the 10% FBS-conditioned culture medium to confirm changes. Cell immunofluorescence confirmed that vimentin expression in fibroblasts exposed to the culture medium with reduced FGF2 was significantly reduced (Fig. 3G).

[0117]

[0118] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing autosomal dominant polycystic kidney disease (ADPKD) comprising FGF2 protein or a gene encoding the same.

2. A composition for diagnosing ADPKD comprising, as an active ingredient, a preparation capable of measuring the expression level of FGF2 protein or the gene encoding it.

3. ADPKD diagnostic composition according to claim 2, wherein the preparation capable of measuring the expression level of the FGF2 protein or the gene encoding it is a primer or probe that specifically binds to the FGF2 gene, or an antibody, peptide, aptamer, or compound that specifically binds to the FGF2 protein.

4. An ADPKD diagnostic kit comprising the composition of paragraph 2 or 3. 5.(1) A step of measuring the expression level of FGF2 protein or the gene encoding it from a sample isolated from a patient; (2) a step of comparing the expression level of the FGF2 protein or the gene encoding it with a control sample; and (3) A method for providing information necessary for diagnosing ADPKD, comprising the step of determining ADPKD when the expression level of the FGF2 protein or the gene encoding it is higher than that of a control sample.

6. A pharmaceutical composition for the prevention or treatment of ADPKD comprising, as an active ingredient, an inhibitor of the expression or activity of FGF2 protein or a gene encoding it.

7. A pharmaceutical composition for the prevention or treatment of ADPKD according to claim 6, wherein the inhibitor of expression or activity of the FGF2 protein or the gene encoding it is any one selected from the group consisting of an antisense nucleotide, a small interfering RNA (siRNA) and a short hairpin RNA (shRNA) that specifically binds to the FGF2 protein or the gene encoding it, a compound, a peptide, an aptamer, and an antibody.

8. A pharmaceutical composition for the prevention or treatment of ADPKD according to claim 7, characterized in that the pharmaceutical composition alleviates fibrosis by reducing cell proliferation, reducing wound healing, and reducing fibrosis markers in fibroblasts.

9. A health functional food composition for the prevention or improvement of ADPKD comprising an inhibitor of the expression or activity of FGF2 protein or a gene encoding it as an active ingredient. 10.(1) A step of contacting isolated ADPKD patient cells with a test substance; (2) A step of measuring the degree of expression or activity of the FGF2 protein or the gene encoding it in ADPKD patient cells that have been contacted with the above test substance; and (3) A method for screening ADPKD therapeutic agents, comprising the step of selecting a test substance in which the expression or activity of the FGF2 protein or the gene encoding it is reduced compared to a control sample.

11. A screening method for ADPKD therapeutic agents according to claim 10, wherein the above-mentioned ADPKD therapeutic agent inhibits fibrosis.

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