KR20140124930A - Composition for detecting or treating kidney fibrosis - Google Patents

Abstract

The present invention relates to a composition for diagnosing kidney fibrosis, which contains an agent capable of detecting Runx2, a kit for for diagnosing kidney fibrosis, which contains the composition, a method for providing information to diagnose kidney fibrosis by using the composition or the kit, and a pharmaceutical composition for prevention or treatment of kidney fibrosis, which comprises a Runx2 gene or protein. The kit for diagnosing kidney fibrosis of the present invention can determine whether kidney fibrosis may occur, and prevent or treat kidney fibrosis, thereby being utilized to build a comprehensive kidney fibrosis management system covering prevention and treatment.

Description

Technical Field [0001] The present invention relates to a composition for diagnosing or treating kidney fibrosis,

The present invention relates to a composition for diagnosing or treating renal fibrosis, and more specifically, the present invention relates to a composition for diagnosing renal fibrosis including an agent capable of detecting Runx2, a kit for the diagnosis of renal fibrosis comprising the composition, A method for providing information for diagnosis of renal fibrosis using a composition or a kit, and a pharmaceutical composition for preventing or treating renal fibrosis including the Runx2 gene or protein.

Runt-related transcription factor 2 (Runx2) is known to be important for bone formation and tooth formation, and causes defects in the musculoskeletal system and tooth formation. The role of Runx2 has recently been reported, and has been reported to play an important role in inducing various cancers and vascular smooth muscle cell morphology, suggesting that Runx2 may play a role in non-bone organs.

Recent reports have shown that Runx2 is correlated with transforming growth factor-β (TGF-β), an important factor in the progression of fibrosis. TGF-beta activates Runx2, which differentiates C2C12 cells into osteoblasts. TGF-β also activates Runx2 in osteoblastic cells, suggesting that TGF-β and Runx2 interact in the activation of collagenase 3 promoter activated by TGF-β. It has also been reported that Runx2 and TGF- [beta] cooperate in the generation of type 1 collagen and fibronectin in C2C12 cell line, an allogeneic mesoderm cell line. Conversely, Runx2 has been shown to inhibit TGF-β function. In another report, TGF-β has been shown to induce ALK5 / Smad3 signaling in human dendritic cells through the inflow of class II HDACs by Smad3 in osteoblast differentiation And that the function of Runx2 is suppressed. The Runx2 / Smad3 complex regulates TGF-β negatively and inhibits the expression of CTGF in vascular smooth muscle cells. Increased TGF-β-induced CTGF in the progression of renal fibrosis is an important pathology, and Runx2 has also been shown to regulate the expression of other fibrosis-related genes in addition to CTGF. In addition, Runx2 induces the expression of collagenase enzymes MMP9 and MMP13, while expression of type 1 collagen in osteoblasts and non-osteoblasts, and expression of smooth muscle cell genes such as a-smooth muscle actin in vascular smooth muscle cells . These results from other researchers suggest that Runx2 may have a potential role in the fibrosis progression of tissue. However, the role of Runx2 in the pathogenesis of fibrosis of the tissues including kidney has not been known at all.

Renal fibrosis is an important marker of kidney disease and one of the most common symptoms of end stage renal failure. An unilateral ureteral obstructed animal model induces fibrosis of the kidneys, characterized by damage to tubular cells, epithelial mesoderm cell metastasis, increased inflammatory response, macrophage infiltration, proliferation of interstitial fibrosing cells and decreased renal function .

Under these circumstances, the present inventors have made extensive efforts to develop a preparation capable of preventing or treating fibrosis of the kidney, and as a result, confirmed that Runx2 can be used to prevent or treat renal fibrosis, thus completing the present invention.

It is an object of the present invention to provide a composition for diagnosing renal fibrosis comprising an agent capable of detecting Runx2.

Another object of the present invention is to provide a kit for the diagnosis of kidney fibrosis comprising the above composition.

It is still another object of the present invention to provide a method for providing information for diagnosis of renal fibrosis using the diagnostic composition or kit.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating renal fibrosis comprising the Runx2 gene or protein.

In order to achieve the above object, the present invention provides a method for diagnosing renal fibrosis, comprising the step of measuring an mRNA level of a gene encoding Run-2 (Runt-related transcription factor 2) or a level of a protein expressed therefrom Lt; / RTI >

The term " Runx-related transcription factor 2 (Runx2) " of the present invention is also referred to as CBF-alpha-1 (core-binding factor subunit alpha-1) and is a protein that acts as a transcription factor essential for bone formation during embryonic development The specific amino acid sequence and the nucleotide sequence information of the gene encoding the amino acid sequence can be obtained from a known database such as NCBI's GenBank. Or a protein represented by ABY71243.1.

The term "diagnosing" of the present invention means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis can be interpreted as confirming the onset of renal fibrosis.

The term " kidney fibrosis "of the present invention is defined as a result of various causes such as excessive inflammatory reaction, oxidative stress, and epithelial fibrosis of the kidney tissue, resulting in fibrosis of the kidney tissue, It means symptoms that are lost. The renal fibrosis itself is not only a lesion showing various symptoms such as damage of tubular cells, epithelial mesodermal cell metastasis, increase of inflammatory reaction, inflow of macrophages, proliferation of interstitial fibrosis cells, decrease of kidney function, ≪ / RTI > is known to be an important marker of various renal diseases.

The term "agent for measuring mRNA level of a gene " of the present invention means a preparation used for measuring the level of mRNA transcribed from the target gene in order to confirm the expression of the target gene contained in the sample But are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) And may be a probe or primer capable of specifically binding to a target gene used in methods such as Northern blotting and DNA chip analysis.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. For the purpose of the present invention, the primer is used as a means for detecting mRNA of Runx2, preferably synthesizing cDNA from mRNA obtained from a patient sample, amplifying and detecting the Runx2 gene contained in the cDNA, Primers can be used. The polynucleotide sequence of the primer is not limited as long as it is capable of amplifying the Runx2 gene contained in the cDNA. Preferably, the polynucleotide sequence may be a primer pair comprising the polynucleotide sequences of SEQ ID NOS: 5 and 6, respectively.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or an mRNA. The oligonucleotide probe, For example, in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

The term "agent for measuring the level of protein " of the present invention means a preparation used for measuring the level of a target protein contained in a sample, and is not particularly limited to, but preferably western blotting, , Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay (Immunoprecipitation Assay), Complement Fixation Assay (FACS), FACS and protein chip assay.

The term "antibody" of the present invention means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene can be obtained and can be produced from the obtained protein by a conventional method. For purposes of the present invention, the antibody is used as a means for detecting the Runx2 protein contained in a sample of a patient, and is preferably a polyclonal antibody, a monoclonal antibody or an antigen-binding antibody capable of specifically binding to Runx2 protein Some of them may be included in the antibody of the present invention and not only all the immunoglobulin antibodies may be contained but may also include special antibodies such as humanized antibodies. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') 2, Fv and the like.

The present inventors examined the relationship between Runx2 and renal damage caused by unilateral ureteral obstruction in Runx2 +/- mice, which can easily detect Runx2 expression changes due to ureteral obstruction. As a result, , And that the Runx2 protein can be used as a marker for diagnosing renal fibrosis in a patient.

Specifically, according to one embodiment of the present invention, the relationship between Runx2 and renal injury caused by unilateral ureteral obstruction showed that Runx2 +/- mice, which can detect changes in Runx2 expression due to ureteral obstruction, When the closure was induced, the expression of Runx2 protein decreased (Fig. 1), and collagen deposition, interstitial expansion and renal tubular atrophy were further increased in the kidney (Figs. 2e and 2f).

Accordingly, the composition for diagnosing renal fibrosis of the present invention comprises an agent for measuring the mRNA level of the gene encoding Runx2 or the level of the protein expressed therefrom. In this case, the agent may be a primer or a probe capable of measuring the level of mRNA of a gene encoding Runx2, or may be an antibody capable of measuring Runx2 protein level. The primer may preferably comprise the polynucleotide sequence of SEQ ID NOS: 5 and 6, and the antibody may be a polyclonal antibody, a monoclonal antibody or a fragment of the antibody capable of specifically binding to the Runx2 protein (Fab, F (ab '), F (ab') 2, Fv, etc.).

In another aspect of the present invention, there is provided a kit for diagnosing renal fibrosis comprising the composition.

The kit of the present invention can be used to diagnose the onset of renal fibrosis by measuring the mRNA level of a gene encoding Runx2 or the level of a protein expressed therefrom from a sample of a patient suspected of having renal fibrosis, Alternatively, one or more other component compositions, solutions or devices suitable for the assay method may be included, as well as primers, probes or antibodies for measuring the mRNA level of the gene or the level of the protein expressed therefrom.

As a specific example, a kit for measuring the mRNA expression level of the Runx2 gene of the present invention may be a kit containing necessary elements necessary for performing RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may contain the necessary elements necessary for performing DNA chip analysis. The kit for DNA chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another example, the kit of the present invention may be a kit for analyzing a protein chip for measuring the level of a protein expressed from the Runx2 gene. The kit may include, but is not limited to, A suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like. The substrate is not particularly limited, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass can be used, and the coloring enzyme is not particularly limited The fluorescent substance may be FITC, RITC or the like, and the coloring substrate liquid is not particularly limited, but ABTS (2, 3, 4, 5, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

In another aspect of the present invention, the present invention provides a method for providing information for diagnosis of renal fibrosis using the diagnostic composition or kit. Specifically, the present invention provides a method for diagnosing renal fibrosis, comprising: (a) Measuring the mRNA level of the gene encoding Runx2 or the level of the protein expressed therefrom from the biological sample of the suspected patient; And (b) comparing the measured mRNA level or the level of the protein expressed therefrom with a level measured from a sample of a normal control, and determining that renal fibrosis is likely to occur if the level is significantly decreased.

At this time, the method of measuring the mRNA level of the gene or the level of the protein expressed therefrom is the same as described above.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating renal fibrosis comprising the Runx2 gene or protein.

When the expression of the Runx2 gene is promoted or enhanced, the onset of the renal fibrosis can be prevented or treated. Therefore, the composition comprising the Runx2 gene or protein of the present invention can be used to prevent or treat the onset of renal fibrosis by the possibility of developing renal fibrosis or by administration to an individual who has developed renal fibrosis.

The present inventors have confirmed that the expression of Runx2 protein is reduced upon the onset of the renal fibrosis. Thus, when Runx2 protein is overexpressed, Runx2 protein is overexpressed, Inhibition of TGF-ß signaling and, consequently, inhibition of renal fibrosis induced by TGF-ß.

Specifically, according to one embodiment of the present invention, the reduction of TGF-ß induced epithelial-mesoblast cell metastasis (EMT) in renal tubular epithelial cells overexpressing the Runx2 gene was confirmed by overexpression of Runx2 gene, (Fig. 5b and 5d), and the expression of alpha-smooth muscle actin, an indicator of myofiber fibroblasts, decreases (Figs. 5b and 5e), and expression of collagen-I induced by TGF- (Figs. 6A to 6D). These TGF-ß-induced Smad3 phosphorylation, α-smooth muscle actin expression and collagen-I expression are known to cause fibrosis of renal cells, and consequently overexpression of the Runx2 gene can prevent or treat renal fibrosis .

Accordingly, the pharmaceutical composition for preventing or treating renal fibrosis of the present invention may comprise Runx2 gene or protein. At this time, the Runx2 gene may be contained in itself or inserted into an expression vector so that it can be expressed in a patient's cell. The expression vector is not particularly limited as long as it can express the Runx2 gene. Preferably, the plasmid from E. coli (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmid (pUB110 and pTP5) Yeast plasmids (YEp13, YEp24 and YCp50), lambda-phage (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP), retrovirus, adenovirus, vaccinia virus , Baculovirus and the like can be used. In addition, the Runx2 protein may be a wild-type Runx2 protein, or may be a protein in which a part of the amino acids contained in the wild-type Runx2 protein is mutated. In the case of the mutated protein, All proteins whose amino acids are mutated by deletion, substitution, addition, or the like can be used.

The term "individual" of the present invention refers to all animals, including humans, that have developed or may have developed renal fibrosis.

In the present invention, the route of administration of the pharmaceutical composition may be administered through any conventional route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intraperitoneally, intrapulmonarily, rectally, but not limited to, as desired . In addition, the pharmaceutical composition may be administered by any device capable of moving the active substance into the target cell.

The content of the Runx2 gene or protein contained in the pharmaceutical composition of the present invention is not particularly limited but may be in the range of 0.0001 to 50% by weight, preferably 0.001 to 10% by weight, based on the total weight of the final composition. can do.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablet pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment And the effective dose level refers to the type and severity of the disease, age, sex, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, Can be determined according to well-known factors. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dose of the pharmaceutical composition for preventing or treating renal fibrosis of the present invention may be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, body weight, sex, history, . For example, it is preferred that the composition of the present invention comprising the Runx2 gene is administered at about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg, per adult, The composition of the present invention containing the expression vector is preferably administered at about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, more preferably 0.01 to 1 mg / kg, per adult, The dosage of the composition of the present invention is not particularly limited, but may be administered once a day or divided into several doses.

The use of the kit for the diagnosis of kidney fibrosis of the present invention can confirm whether or not the kidney fibrosis can be caused and can prevent or treat the onset of renal fibrosis. Therefore, the diagnosis, prevention and treatment of kidney fibrosis are encompassed Which can be used to construct an overall kidney fibrosis management system.

Figure 1 shows that unilateral ureteral obstruction reduces Runx2 gene expression in ureteral obstructed kidneys. (a) shows Western blot results of Runx2 expression in unilateral ureteral obstruction and 5 days after laparotomy, (b) shows the amount of Runx2 protein corrected by GAPDH, and each result is expressed as mean ± standard error 4), and UO means ureteral obstruction.
Figure 2 shows that Runx2 gene reduction promotes renal fibrosis induced by unilateral ureter obstruction. (a) shows the results of Western blotting using type 1 and type 3 collagen and α-SMA specific antibodies in unilateral ureteral obstruction and kidney 5 days after laparotomy. (b) (C) shows the amount of expression of type 3 collagen, and (d) shows the amount of expression of the? -SMA protein. The results are expressed as mean ± standard error (population = 4). (e) shows Masson's trichrome staining of ureteral closure kidneys, and collagen was stained blue. (f) shows the result of summing collagen deposition, and (g) shows the result of immunofluorescence staining of the type-III collagen in red. The results are expressed as mean ± standard error (population = 4). UO means ureteral obstruction.
Figure 3 shows that Runx2 gene reduction promotes a renal inflammatory response induced by ureteral obstruction. (a) shows Western blot results using Ly6G and CD68 specific antibodies in unilateral ureteral obstruction and kidney 5 days after laparotomy, (b) shows the amount of Ly6G protein corrected with GAPDH, and (c) Protein expression level. The above results are expressed as mean ± standard error (population = 4), and UO means ureteral obstruction.
Figure 4 shows that Runx2 gene reduction promotes TGF-beta activity induced by ureteral obstruction. (a) shows the results of Western blot using TGF-ß and phosphorylated Smad-3 specific antibody in unilateral ureteral obstruction and kidney 5 days after laparotomy, (b) shows the amount of TGF-ß protein corrected by GAPDH And (c) represents the amount of phosphorylated Smad-3 protein expressed. The above results are expressed as mean ± standard error (population = 4), and UO means ureteral obstruction.
FIG. 5 shows that the overexpression of the Runx2 gene promotes the switch from the renal tubule to the myoblast cells induced by the TGF-β signal. (a) shows Western blot results using Runx2-specific antibodies in MDCK (Madin-Darby canine kidney) cells infected with LacZ or Runx2 gene transfer adenovirus, (b) LacZ or Runx2 gene transfer adenovirus (C) shows the expression level of Runx2 protein corrected by GAPDH. The results of Western blot analysis of Runx2, p-SMAD3 and α-SMA-specific antibodies of cells that did not or did not treat TGF- (D) represents the expression level of the p-SMAD3 protein, (e) represents the expression level of the? -SMA protein, and (f) represents the expression level of the type 1 collagen protein, Mean ± standard error (population = 4).
Figure 6 shows that overexpression of the Runx2 gene inhibits the conversion of kidney tubular cells into TGF- beta signaling fibroblasts. (a) shows the results of PCR with runx2, α-SMA, type 1 collagen and GADPH primers, (b) shows the amount of Runx2 mRNA corrected with GAPDH, (D) represents the amount of type 1 collagen mRNA, and the results are expressed as mean ± standard error (population = 4).
FIG. 7 shows that Runx2 gene overexpression alleviates the activity of TGF-β-induced CAGR reporter gene assay. In MDCK cells infected with LacZ or Runx2 gene transfer adenovirus, the luciferase activity of cells that did not or did not treat TGF-ß was corrected by β-galactosidase activity and then added up. The relative luciferase activity of the control group treated with vehicle was converted to 1. The results are expressed as mean ± standard error (population = 4).
Figure 8 shows that Runx2 gene deletion enhances the TGF-ß signal in mouse embryonic fibroblast (MEF). (a) shows the results of electrophoresis using genomic PCR using Runx2 + / + and MEF primers of Runx2 - / - mice, (b) shows results of phosphorylated Smad-3, Western blot analysis using a specific antibody was performed. Western blot was performed using p-SMAD3 and a-SMA specific antibodies of MEF cells treated or not treated with GAPDH-corrected TGF-ß. (c) shows Western blot results of p-SMAD3 on average of MEF cells treated or not with TAP-β corrected with GAPDH, and (d) Western blot results of α-SMA. The results are expressed as mean ± standard error (population = 4).

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Materials and Methods

Example  1-1: Animal experiments

All animal experiments were conducted according to the guidelines of the IACUC of Kyungpook National University. 8-week-old male normal mice (Runx2 + / +) or lacking mice (nocturnal / C-terminal length reduced; Runx2 +/-) rats were used. Runx2 +/- mice were confirmed by known methods (F. Otto, AP et al., Cell 89 (1997) 765-771) and confirmed genotypes of Runx2 + / + and Runx2 +/- Jeong, et al., J Cell Physiol 217 (2008) 511-517). Rats were fed standard feed and water ad libitum. Each group consisted of at least 4 rats and the laparotomy (control) or ureteral obstruction (UO) proceeded in a known manner (KM Park, et al, J Biol Chem 277 (2002) 2049). Pentobarbital sodium was used to anesthetize the rats (60 mg / kg body wt, intraperitoneal injection, Sigma, St. Louis, MO). Left ureter was removed through the left lateral incision to close the ureter, and the left ureter near the renal pelvis was closed completely using nylon 6-0. Control rats underwent the same procedure except for ureteral obstruction. The kidneys were fixed by injection of PLP solution (4% paraformaldehyde, 75 mM L-lysine, 10 mM sodium periodate, Sigma, St. Louis, MO) It was rapidly frozen in liquid nitrogen.

Example  1-2: Western blot  analysis

Protein samples were obtained from Runx2 (Santa Cruz Biotechnology, Santa Cruz, Calif.), Collagen III (MyBioSource, Inc., San Diego, Calif.), Α-smooth muscle actin (Gibco) and factor VIII (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Smad3 (cell signaling Technology, Inc., Danvers, Mass.), Ly6G (eBioscience, San Diego, Calif.), CD68 (Novus Biologicals, LLC, Littleton, CO) was used for protein electrophoresis. The secondary antibody (DakoCytomation Inc., Carpinteria, Calif.) Conjugated with Horseradish peroxidase was allowed to bind to the primary antibody previously used and the immunoblot was confirmed using chemiluminescent material (Thermo scientific, Rockford, Ill.). The concentration of immune blot was measured using image analysis software ImageJ (NIH).

Example  1-3: Kidney fibrosis  evaluation

PLP solution (2% paraformaldehyde, 75 mM L-lysine, 10 mM sodium periodate; Sigma, St. Louis, MO) was injected into the kidney through the left ventricle for 15 minutes. The extracted kidney was stored at 4 ° C overnight Respectively. The kidneys fixed to PLP were washed with PBS (phosphate buffered saline) solution for 3 minutes for 5 minutes, solidified with paraffin and cut into 2 μm thickness using a microtome (RM2165, Leica, Bensheim, Germany). Paraffin fragments were immunoblotted with rabbit secondary antibody (Vector Laboratories Inc., Burlingame, Calif.) Combined with Texas Red-conjugated primary antibody (abcam, Cambridge, UK) for the identification of Masson's trichrome and type 3 collagen Lt; / RTI > The sections of the kidneys stained in each rat were observed by optical and fluorescence microscopy using a Nikon Microphot-Fx (Nikon Inc., Melville, NY). Collagen deposition was analyzed using Lab Works 4.5 software (Ultra-Violet Products, Cambridge, UK) at 10 randomly chosen sites (0.23 mm2 / field).

Example  1-4: MDCK  Cell culture and Runx2 Overexpression of

Madin-Darby Canine Kidney cells (MDCK, American Type Culture Collection, Manassas, Va.) Of 8-10 generations were treated with 5% FBS (fetal bovine serum; Mediatech Inc., Manassas, Va.) And streptomycin / penicillin / ml (WelGene Inc., Daegu, Korea). For the overexpression of Runx2, LacZ and Runx2 adenoviruses (MOI, inoculated virus / cell number) were inoculated into MDCK cells at a concentration of 10-20%, and in DMEM without FBS and S / P And then replaced with 5% FBS DMEM medium containing S / P. When the cells were 50-60% full, DMEM supplemented with 1% FBS and S / P was replaced. The cells were treated with 5 ng / ml of TGF-β for 48 hours, rinsed with cold PBS, and the cells were pulverized and analyzed by western blot as described above.

Example  1-5: Mouse embryonic fibroblasts ( MEF ) Cell culture

Male Runx2 +/- mice were mated with female Runx2 +/- mice to obtain offspring on Runx2 + / + and Runx2 - / - at 13 days of gestation and primary MEF cells were isolated and cultured using standard protocols [23]. The placenta, head, arm, vertebra, and other organs were removed from the abdomen and then chopped into a 0.25% trypsin / EDTA solution. The embryos were removed from the pregnant rats and the embryos were removed and washed with PBS containing antibiotics. The cells were mixed at 37 ° C for 15 min. Cells were cultured in DMEM supplemented with 15% FBS (Mediatech Inc., Manassas, VA) and streptomycin / penicillin (S / P, antibiotic) 100 u / ml (WelGENE Inc., Daegu, Korea) And the cells from one embryo confirmed their respective genotypes. PCR was performed using Taq DNA polymerase (Fermentas, Glen Burnie, Maryland) and the following primers.

Runx2 + / + f: 5'-CACGGAGCACAGGAAGTTGGGA-3 '(SEQ ID NO: 1)

Runx2 + / + r: 5'-TGAGCGACGTGAGCCCGGTGG-3 '(SEQ ID NO: 2)

Runx2 - / - f: 5'-GCATCGAGCTGGGTAATAAGCGTTGGCAAT-3 '(SEQ ID NO: 3)

Runx2 - / - r: 5'-GACACCAGACCAGGTAATGGTAGCGAC-3 '(SEQ ID NO: 4)

When the cells became 50-60% cold, they were changed to DMEM containing 1% FBS containing S / P, treated with 5 ng / ml of TGF-β for 48 hours, and rinsed with cold PBS, 1-2 western blot analysis was performed.

Example  1-6: Histological evaluation periodic acid Schiff stain  , PAS stain )

PAS staining was performed on tissue fragments solidified with paraffin by the method of Examples 1-3, and kidney sections stained in each of four rats were examined using a Nikon Microphot-Fx (Nikon Inc., Melville, NY) I got an image.

Example  1-7: Semi-quantitative Reverse transcription  Polymerase chain reaction Semi - quantitative reverse  transcription, RT - PCR )

RNAs extracted with RNeasy kit (Qiagen, Valencia, CA) were applied to RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, Maryland) to synthesize cDNA. PCR was performed using the synthesized cDNA as a template and the following primers and Taq DNA polymerase (Fermentas, Glen Burnie, Maryland)

Runx2 f: 5'-CACTATCCAGCCACCTTTACTT-3 '(SEQ ID NO: 5)

Runx2 r: 5'-GATCCTGACGAAGTGCCATAG-3 '(SEQ ID NO: 6)

actin f: 5'-GTGACATCGACATCAGGAAAGA-3 '(SEQ ID NO: 7)

actin r: 5'-GATCCACATCTGCTGGAAGG-3 '(SEQ ID NO: 8)

collagen I f: 5'-ACGGATTCCAGTTCGAGTATG-3 '(SEQ ID NO: 9)

collagen I r: 5'-GTAGGTGACACTGTAGGTGAAG-3 '(SEQ ID NO: 10)

GAPDH f: 5'-AGTCAAGGCTGAGAACGGGAAACT-3 '(SEQ ID NO: 11)

GAPDH r: 5'-TCCACAACATACTCAGCACCAGCA-3 '(SEQ ID NO: 12)

Example  1-8: Transient transfection and luminescence enzyme ( Luciferase ) analysis

Transfection was performed according to the manufacturer's instructions using DharmaFECT (Thermo scientific, Rockford, Ill.). Cells were each cultured at a density of 5 x 10 ⁵ cells / well, and then CAGA-luc, psv beta-gal, and Runx2 were transiently transfected. Twenty-four hours after the transfection, the medium was changed to DMEM supplemented with 1% FBS and S / P, and treated with 5 ng / ml of TGF-β for 16 hours. Cells were harvested using Luciferase Cell Culture Lysis reagent (Promega, Madison, Wis.) And the activity of luciferase was measured by Luciferase assay system (Promega, Madison, WI). All values were analyzed by using β - galactosidase activity.

Example  1-9: Statistical Analysis

Results are expressed as means ± standard error (SE). Statistical differences between groups were calculated using ANOVA, and statistical differences between groups were assessed by analysis of variance using Student t-test. Differences between groups were considered statistically significant with a significance (P value) of less than 0.05.

Example  2: Results

Example  2-1: Unilateral On the ureteral obstruction  In closed kidneys Runx2  Decreased expression

To confirm the relationship between Runx2 and renal injury caused by unilateral ureteral obstruction, Runx2 protein expression was confirmed by western blot technique in ureteral obstructed kidneys. As a result, Runx2 protein was generally expressed in the kidney, and Runx2 +/- mice showed less expression of Runx2 protein than Runx2 + / + (Fig. 1). Runx2 protein expression was reduced by ureteral obstruction in both Runx2 + / + and Runx2 +/- mice, suggesting that Runx2 is regulated by signals induced by ureteral obstruction and may be associated with renal damage . Runx2 expression after urothelial occlusion was significantly lower in Runx2 +/- mice compared to Runx2 + / +.

Example  2-2: Runx2  Promotion of renal fibrosis induced by ureteral obstruction by reduction of gene expression

In order to confirm the role of Runx2 in renal fibrosis induced by ureteral obstruction, we confirmed the fibrosis pattern in Runx2 + / + and Runx2 +/-. Ureteral obstruction increases the fibrosis markers type 1 and type 3 collagen and α-smooth muscle actin. Expression of type 1 and type 3 collagen and α-smooth muscle actin, induced by ureteral obstruction, / + ≪ / RTI > (Figures 2a, 2b and 2d). Similar to these results, in the Runx2 +/- mice compared to Runx2 + / +, collagen deposition, epileptic swelling and renal tubular atrophy increased in the kidneys after ureteral occlusion (Figs. 2e and 2f). These results suggest that Runx2 plays a protective role in kidney fibrosis induced by ureteral obstruction.

Example  2-3: Runx2  Due to decreased gene expression By ureteral obstruction  Promotes inflammatory response in the induced kidney

To investigate the relationship between renal fibrosis and inflammatory response in Runx2 +/-, we examined the expression of Ly6G, an indicator of neutrophils, and CD68, an indicator of monocyte / macrophage. Ureteral obstruction increased both Ly6G and CD68 expression in Runx2 + / + and Runx2 +/-, but increased more in Runx2 +/- than Runx2 + / + (Figs. 3a-3c). In addition, the infiltration of stromal cells was more frequent in Runx2 +/- mice. This suggests that Runx2 inhibits the inflammatory response of kidneys induced by ureteral obstruction.

Example  2-4: Runx2  +/- In mice  Increased renal fibrosis

To determine whether increased renal fibrosis in Runx2 +/- mice was associated with TGF-ß signaling, we measured the amount of active TGF-ß and phosphorylated Smad3. Ureteral obstruction activated TGF-ß and phosphorylated Smad-3 in both Runx2 + / + and Runx2 +/- mice, but compared with Runx2 + / +, TGF-ß activation and Smad-3 phosphorylation (Figs. 4A to 4C). This suggests that RUNX2 inhibits the TGF-ß signal, a factor that induces fibrosis.

Example  2-5: Runx2  Overexpression of the Gene in the Renal Tubules As a fibroblast  Inhibition of conversion differentiation

From the results of Examples 2-1 to 2-4 above (Figs. 1 to 4), it was found that the decrease of Runx2 promotes renal fibrosis induced by uremic closure through TGF-β signal activation. Overexpression of Runx2 may inhibit renal fibrosis ultimately by inhibiting TGF-β signaling. In order to confirm this, we confirmed the reduction of TGF-ß induced epithelial-mesodermal cell metastasis (EMT) in renal tubular epithelial cells overexpressing Runx2 gene. MDCK (Madin-Darby canine kidney) was infected with Runx2 gene transfer adenovirus (Ad-Runx2) or LacZ gene transfer adenovirus (Ad-LacZ) and treated with TGF-ß, well known as a fibrotic factor of renal epithelial cells. Ad-Runx2 at 200 and 400 MOIs efficiently increased Runx2 protein expression in MDCK cells, while Ad-LacZ did not increase Runx2 protein expression (Fig. 5a). Smad3 phosphorylation induced by TGF-ß was greater in Ad-LacZ infected cells than in Ad-Runx2 infected cells (Figures 5b and 5d). This suggests that Runx2 may inhibit the TGF-ß / Smad3 signaling pathway.

In addition, the expression of a-smooth muscle actin, an indicator of myofiber blasts, was also significantly higher in Ad-LacZ infected cells than in Ad-Runx2 infected cells (Figures 5b and 5e). The mRNA levels of α-smooth muscle actin and collagen-I induced by TGF-ß were also significantly higher in Ad-LacZ-infected cells than in Ad-Runx2-infected cells as well as in protein expression results (Figs. 6a to 6d). These results suggest that Runx2 inhibits the conversion of TGF-ß induced kidney cells to fibrotic cells.

Example  2-6: Runx2  gene And active  by TGF -ß-induced CAGA  reporter( reporter gene ) Active relaxation

To confirm the inhibitory role of Runx2 on TGF-β signaling, we examined the reporter gene assay of CAGA, a TGF-β response element. TGF-ß treatment increased CAGA reporter activity in both Ad-LacZ and Ad-Runx2 infected cells. However, the increase in Ad-Runx2 infected cells was minimal and significantly less than in Ad-LacZ infected cells (Figure 7). These results suggest that Runx2 inhibits TGF-β signaling.

Example  2-7: Runx2  Due to gene deletion MEF in As a fibroblast  Facilitating conversion

To investigate the protective role of Runx2 in TGF-ß-induced fibrosis progression, we investigated the response to TGF-ß using mouse embryonic fibroblast (MEF). TGF-ß increased expression of phosphorylated Smad3 and α-smooth muscle actin in both Runx2 + / + and Runx2 - / - mouse embryonic stem cells, but the expression of Runx2 - / - mouse embryonic stem cells was significantly higher than that of Runx2 + / + (Fig. 8). This confirms that Runx2 acts as an inhibitor of TGF-ß induced fibrosis progression.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Composition for detecting or treating kidney fibrosis <130> PA130330 / KR <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Runx2 + / + f primer <400> 1 cacggagcac aggaagttgg ga 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Runx2 + / + r primer <400> 2 tgagcgacgt gagcccggtg g 21 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Runx2 - / - f primer <400> 3 gcatcgagct gggtaataag cgttggcaat 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Runx2 - / - r primer <400> 4 gacaccagac caggtaatgg tagcgac 27 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Runx2 f primer <400> 5 cactatccag ccacctttac tt 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Runx2 r primer <400> 6 gatcctgacg aagtgccata g 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> actin f primer <400> 7 gtgacatcga catcaggaaa ga 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> actin primer <400> 8 gatccacatc tgctggaagg 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> collagen I f primer <400> 9 acggattcca gttcgagtat g 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> collagen I r primer <400> 10 gtaggtgaca ctgtaggtga ag 22 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH f primer <400> 11 agtcaaggct gagaacggga aact 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH r primer <400> 12 tccacaacat actcagcacc agca 24

Claims (12)

A composition for diagnosing renal fibrosis, comprising an agent that measures the mRNA level of a gene encoding Runx-2 (Runx-related transcription factor 2) or the level of a protein expressed therefrom.
The method according to claim 1,
Wherein the agent that measures the level of the gene mRNA comprises a primer or a probe that specifically binds to the gene.
3. The method of claim 2,
Wherein the primer comprises a polynucleotide sequence of SEQ ID NOS: 5 and 6.
The method according to claim 1,
Wherein the agent that measures the level of said protein comprises an antibody specific for said protein.
In Item 4,
Wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, Fab, F (ab ') 2, F (ab') 2, Fv and combinations thereof.
A kit for the diagnosis of kidney fibrosis, comprising the composition of any one of claims 1 to 5.
The method according to claim 6,
Wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
(a) measuring the mRNA level of a gene encoding Runx2 or the level of a protein expressed therefrom from a biological sample of a patient suspected of having an onset of renal fibrosis; And
(b) comparing the measured mRNA level or the level of the protein expressed therefrom with a level measured from a sample of a normal control, and determining that renal fibrosis is likely to occur if the level is significantly decreased, A method of providing information for diagnosis of an irritation.
9. The method of claim 8,
The mRNA level of the gene may be determined by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay (RNase protection method, Northern blotting or DNA chip technology assay.
9. The method of claim 8,
The level of the protein may be determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography, FACS (Immunohistochemical staining), immunofluorescence (immunohistochemical staining), immunoprecipitation assays a fluorescence activated cell sorter analysis and a protein chip technology assay.
A pharmaceutical composition for preventing or treating renal fibrosis comprising Runx2 gene or protein.
10. The method of claim 9,
Wherein the composition further comprises a pharmaceutically acceptable diluent, excipient or carrier.

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