Composition for diagnosing kidney disease using urine samples

Abstract

This invention provides a kidney disease diagnostic composition, a kit, a method for providing information for diagnosis, and a method for providing information for predicting the likelihood of progression to end-stage renal failure, all of which enable the early and simple diagnosis of kidney disease from a urine sample. [Solution] The present invention relates to a composition for diagnosing kidney disease using a urine sample, and more particularly to a urine test composition for diagnosing kidney disease, a kit, and a method for diagnosing kidney disease using the composition, comprising an agent for specifically detecting amphiregulin from a urine sample. The present invention has the advantage of allowing for the simple and non-invasive diagnosis of kidney disease, for which early diagnosis is important, using a urine sample. Furthermore, if the present invention is applied to a patient diagnosed with chronic renal failure, it has the advantage of allowing for the prediction of the possibility of progression to end-stage renal failure.

Description

Technical Field

[0001] The present invention relates to a composition for diagnosing kidney diseases using a urine sample, and more particularly, to a composition for urine examination for diagnosing kidney diseases, a kit, and a method for diagnosing kidney diseases using the composition, which contain an agent for specifically detecting amphiregulin from a urine sample.

Background Art

[0002] In recent years, due to significant changes in lifestyle habits including diet and aging, the incidence of lifestyle-related diseases such as hypertension and diabetes has increased, and along with it, the risk of kidney diseases and even renal failure has increased.

[0003] The kidney is an important organ for maintaining the homeostasis of the internal environment by excreting waste products, regulating body fluids, electrolytes, and acid-base balance. The kidney controls the concentrations of various compounds in the blood such as hydrogen, sodium, potassium, and silicon, and excretes waste products in urine. Any decline in kidney function can interfere with the body's ability to adequately remove metabolites in the blood and disrupt the body's electrolyte balance. A decline or failure of kidney function can be fatal in its symptoms.

[0004] Chronic renal failure is a state in which such kidney function gradually decreases unidirectionally (irreversibly) and the homeostasis of the living body cannot be maintained. Chronic renal failure is known to occur due to diabetic nephropathy, chronic glomerulonephritis, malignant nephrosclerosis, polycystic kidney disease, etc. All kidney diseases are accompanied by fibrosis of the kidney, and ultimately lead to end-stage renal failure. In particular, chronic decline in kidney function is deeply related to the progression of kidney fibrosis, and it is considered that the progression of chronic renal failure can be suppressed by suppressing the progression of fibrosis.

[0005] Generally, renal fibrosis is accompanied by an inflammatory response due to endothelial cell damage, and ultimately, the overproduction of extracellular matrix (ECM) leads to the development of fibrosis. For example, in glomerulosclerosis, it occurs due to protoglomerular endothelial cell damage, which secretes cytokines such as chemokines and growth factors, causing inflammatory responses in monocytes and macrophages. Subsequently, mesangial cells are activated, proliferate, and transform, and an excess of extracellular matrix is ​​produced in mesangial cells and other extracellular matrix-producing cells, leading to fibrosis and ultimately glomerulosclerosis.

[0006] In particular, diabetic nephropathy is the leading cause of chronic renal failure and is one of the serious complications of diabetes. A characteristic feature of diabetic nephropathy is the proliferation of glomerular mesangials, which is mainly associated with increased accumulation of ECM proteins such as type I and type IV collagen, fibronectin, and laminin (Verena Klemis et al., Kidney International (2017) 91, 1374-1385).

[0007] When chronic renal failure worsens to the point where the kidneys can no longer function at all, it progresses to end-stage renal disease (ESRD). Once end-stage renal disease develops, recovery is impossible, and patients with end-stage renal disease need to undergo dialysis (hemodialysis or peritoneal dialysis) or receive a new kidney through transplantation to restore kidney function.

[0008] On the other hand, amphiregulin is known to bind to the epidermal growth factor receptor (EGFR) and activate the epidermal cell receptor pathway (EGFR pathway), thus being involved in cell proliferation. It has been reported that amphiregulin expression can be inhibited by amphiregulin-specific siRNA, and that this has been shown to have therapeutic effects on certain types of breast cancer (Cancer Res 2008;68:225-2265). Furthermore, it has been reported that cell invasion in inflammatory breast cancer can be suppressed using shRNA against amphiregulin (J Cell Physiol 2011 226(10):2691-2701), and that suppressing amphiregulin expression using amphiregulin-specific shRNA inhibits pulmonary artery remodeling in mice exposed to tobacco smoke. It has been reported that amphiregulin is associated with airway smooth muscle (ASM) hyperplasia and angiogenesis, particularly promoting airway remodeling in asthma patients, and that amphiregulin and epidermal growth factor (EGF), which is excessively secreted during tissue remodeling in acute asthma, are involved.

[0009] While diagnostic methods using amphiregulin for chorioamnionitis (WO2008-029664) and inflammatory diseases (US2006-0286586) are publicly known, there are currently no studies that have used amphiregulin to diagnose kidney disease.

[0010] Therefore, recognizing the importance of early diagnosis given that kidney disease becomes difficult to recover from once it progresses to a severe stage, the inventors sought a method for easily diagnosing kidney disease from urine, which can be collected without invasive procedures. They confirmed that kidney disease can be easily diagnosed and the progression of end-stage renal failure can be predicted by checking the amphiregulin concentration in urine, leading to the completion of the present invention. [Overview of the project]

[0011] The object of the present invention is to provide a kidney disease diagnostic composition, a kit, a method for providing information for diagnosis, and a method for providing information for predicting the likelihood of progression of end-stage renal failure, which can be easily and quickly diagnosed from a urine sample.

[0012] To achieve the above objective, the present invention provides a composition for diagnosing kidney disease, comprising an agent for specifically detecting amphiregulin or its mRNA.

[0013] The present invention also provides a kit for diagnosing kidney disease, comprising the aforementioned composition.

[0014] The present invention also provides a method for providing information for the diagnosis of kidney disease, comprising the following steps: (a) The stage of collecting a urine sample from the individual; (b) the step of detecting amphiregulin in the urine sample; and (c) If the amount of amphiregulin detected in the urine sample is 5 pg / mgCr or higher, the individual is judged to have kidney disease.

[0015] The present invention also provides a method for providing information to predict the likelihood of progression of end-stage renal failure, comprising the following steps: (a) The step of collecting a urine sample from a patient with kidney disease; (b) Steps to measure the amount of amphiregulin in the urine sample: and (c) When the measured amount of amphiregulin is 30 pg / mgCr or higher, it is determined that the patient may be at risk of progressing to end-stage renal failure.

[0016] The present invention also provides a composition comprising an agent for specifically detecting amphiregulin for use in the diagnosis of kidney disease.

[0017] The present invention also provides applications for producing reagents for the diagnosis of kidney disease, which include an agent that specifically detects amphiregulin.

[0018] The present invention also provides a method for diagnosing kidney disease, comprising the following steps: (a) Step of collecting a urine sample from an individual; (b) Step of detecting amphiregulin in the urine sample: and (c) Step of determining that the individual has a kidney disease when the amphiregulin detected from the urine sample is 5 pg / mgCr or more.

[0019] The present invention also provides a method for predicting the progression of end-stage renal failure, including the following steps: (a) Step of collecting a urine sample from a patient suffering from a kidney disease; (b) Step of measuring the amount of amphiregulin in the urine sample: and (c) Step of determining that the patient has a possibility of progressing to end-stage renal failure when the measured amount of amphiregulin is 30 pg / mgCr or more.

Brief Description of the Drawings

[0020] [Figure 1] Results showing the mRNA expression rate of amphiregulin in the kidney and lung tissues of normal animals (Normal) and animal models of pulmonary fibrosis (BLM).

[0021] [Figure 2] Results showing the mRNA expression rate of collagen 1 in the kidney and lung tissues of normal animals (Normal) and animal models of pulmonary fibrosis (BLM).

[0022] [Figure 3] Results showing the mRNA expression rate of fibronectin in the kidney and lung tissues of normal animals (Normal) and animal models of pulmonary fibrosis (BLM).

[0023] [Figure 4] Results showing the ratio of amphiregulin / creatinine using ELISA in the urine of normal animals (Normal) and animal models of pulmonary fibrosis (BLM).

[0024] [Figure 5]This shows the results of measuring the albumin / creatinine ratio using ELISA in the urine of normal animals and an animal model of pulmonary fibrosis (BLM).

[0025] [Figure 6A] These are the results of H&E staining or MT staining of lung tissue from normal animals and an animal model of pulmonary fibrosis (BLM).

[0026] [Figure 6B] These are the results of H&E staining or MT staining of kidney tissue from normal animals and an animal model of pulmonary fibrosis (BLM).

[0027] [Figure 7] This graph shows serum urea nitrogen levels in serum samples from normal animals and an animal model of pulmonary fibrosis (BLM).

[0028] [Figure 8] This graph shows creatinine levels in serum samples from normal animals and an animal model of pulmonary fibrosis (BLM).

[0029] [Figure 9] This graph compares serum (A) and urine (B) amphiregulin concentrations in patients diagnosed with IgA nephropathy (immunoglobulin A nephropathy) or diabetic nephropathy who underwent kidney tissue biopsy and exhibited severe minor glomerular change in their renal pathology.

[0030] [Figure 10]This graph compares urinary amphiregulin concentrations in the patient group shown in Figure 9, based on the severity of histological findings associated with renal fibrosis (A: interstitial fibrosis, B: intimal thickening, C: tubular atrophy, C: mesangial expansion).

[0031] [Figure 11] Figure 11A shows the analysis of amphiregulin concentrations in plasma from a normal control group and a group of type 2 diabetes patients. Figure 11B shows the analysis of amphiregulin concentrations in plasma from a group of type 2 diabetes patients further divided into three groups based on the degree of albuminuria. Figure 11C shows the analysis of amphiregulin concentrations in urine from a normal control group and a group of type 2 diabetes patients. Figure 11D shows the analysis of amphiregulin concentrations in urine from a group of type 2 diabetes patients further divided into three groups based on the degree of albuminuria.

[0032] [Figure 12] This is the result of examining the amount of amphiregulin in urine according to the stage of chronic kidney disease in a group of patients who underwent kidney tissue biopsy.

[0033] [Figure 13] This is a Kaplan-Meier survival curve graph comparing the progression of end-stage renal failure based on amphiregulin concentration in a group of patients who underwent kidney tissue biopsy. The urinary amphiregulin concentration was divided into three groups based on tertiles, and named T1, T2, and T3 in descending order of concentration. [Modes for carrying out the invention]

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those commonly understood by skilled experts in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0035] In this invention, we performed kidney tissue examinations on patients suspected of having kidney damage due to various diseases, and confirmed that amphiregulin in the urine of this patient group reflects the degree of fibrosis inside the kidney.

[0036] Therefore, in one aspect, the present invention relates to a composition for diagnosing kidney disease, comprising an agent for specifically detecting amphiregulin or its mRNA.

[0037] In the present invention, the agent may be characterized by its ability to specifically detect amphiregulin from a urine sample.

[0038] In the present invention, the agent for specifically detecting amphiregulin may be an amphiregulin-specific antibody or aptamer, but is not limited thereto. Any agent capable of detecting the presence or absence of amphiregulin mRNA or protein from a urine sample, or measuring its amount, can be used. In the examples of the present invention, an ELISA method using an amphiregulin-specific antibody is used, but a PCR method using primers and / or PNA probes to measure the amount of amphiregulin mRNA could also be used.

[0039] In the present invention, "agent for specifically detecting amphiregulin" means a molecule that can be used to detect a biomarker (i.e., amphiregulin mRNA or protein) whose expression is altered by kidney dysfunction by measuring its expression level. The expression level of the biomarker can be determined by confirming the expression level of the marker mRNA or protein.

[0040] In the present invention, the term "antibody" is a term known in the art and means a specific protein molecule directed to an antigenic site. For the purposes of the present invention, the antibody means an antibody that specifically binds to the biomarker protein of the present invention, and such an antibody may be produced by cloning each gene into an expression vector by conventional methods to obtain the protein encoded by the marker gene, and then producing the obtained protein by conventional methods. This also includes partial peptides that can be produced from the protein, and the partial peptide of the present invention contains at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids.

[0041] The form of the antibodies of the present invention is not particularly limited; polyclonal antibodies, monoclonal antibodies, or any antibodies that have antigen-binding properties are included in the antibodies of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

[0042] The antibodies used for detecting kidney function diagnostic markers in the present invention include not only the complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. Functional fragments of the antibody molecule refer to fragments that possess at least antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv, etc.

[0043] In the present invention, "measurement of protein expression level" refers to the process of confirming the presence and degree of expression of kidney dysfunction marker proteins in a biological sample of an individual in order to diagnose kidney disease. For example, the amount of protein can be confirmed by detecting an antigen-antibody complex using an antibody that specifically binds to the marker protein in question. Examples of specific analytical methods include, but are not limited to, Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip.

[0044] In this invention, “diagnosis” means confirming the presence or characteristics of a pathological state. For the purposes of this invention, the diagnosis may be interpreted as confirming the presence or absence of kidney disease.

[0045] In the present invention, “diagnostic marker, marker for diagnosis, or diagnostic marker” means a substance that can distinguish a sample in which kidney dysfunction has occurred or is likely to occur from a sample from a healthy individual, and the sample from an individual with kidney dysfunction contains polypeptides or nucleic acids (e.g., mRNA, etc.) or proteins in which the diagnostic marker (i.e., amphiregulin) is increased compared to the sample from a healthy individual.

[0046] The protein or genetic information of the biomarker for diagnosing kidney disease provided in this invention can be easily obtained using known gene databases. For example, the amino acid sequence of the biomarker protein amphireglin is registered in the NCBI (National Center for Biotechnology Information), a known gene database, and is as follows.

[0047] Sequence ID 1 MRAPLLPPAPVVLSLLILGSGHYAAGLDLNDTYSGKREPFSGDHSADGFEVTSRSEMSSGSEISPVSEMPSSSEPSSGADYDYSEEYDNEPQIPGYIVDDSVRVEQVVKPPQNKTESENTSDKPKR KKKGGKNGKNRRNRKKKNPCNAEFQNFCIHGECKYIEHLEAVTCKCQQEYFGERCGEKSMKTHSMIDSSSLSKIALAAIAAFMSAVILTAVAVITVQLRRQYVRKYEGEAEERKKLRQENGNVHAIA

[0048] In the present invention, the amphireglin may be characterized by being represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0049] In the present invention, kidney disease may be characterized by being selected from the group consisting of renal fibrosis, renal syndrome, renal cancer, chronic renal failure, diabetic nephropathy, acute pyelonephritis, acute renal failure, end-stage renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjögren's syndrome, Behçet's disease, lupus, candidiasis, hemorrhagic fever with renal syndrome, leptospirosis, legionellosis, autosomal dominant polycystic kidney disease, and hydronephrosis, but is not limited thereto.

[0050] In other words, the present invention relates to a kit for diagnosing kidney disease, comprising the aforementioned composition.

[0051] The kit of the present invention can detect markers by confirming the expression level of marker proteins for the diagnosis of kidney disease. In addition to antibodies that selectively recognize markers in order to measure the expression level of markers for the diagnosis of kidney function, the kit of the present invention may include one or more other component composition solutions or devices suitable for the analytical method.

[0052] As a specific example, the kit for measuring protein expression levels for kidney disease diagnosis in the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. Here, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a slide glass made of glass; the chromogenic enzyme may be peroxidase or alkalin phosphatase; the fluorescent substance may be FITC, RITC, etc.; and the chromogenic substrate solution may be ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine), etc.

[0053] In the present invention, the kit may be, but is not limited to, a protein chip kit or an immunodiagnostic kit.

[0054] In yet another aspect, the present invention relates to a method for providing information for the diagnosis of kidney disease, comprising the following steps: (a) The stage of collecting a urine sample from the individual; (b) Steps to detect amphiregulin in the urine sample: and (c) If the amount of amphiregulin detected in the urine sample is 5 pg / mgCr or higher, the individual is judged to have kidney disease.

[0055] In the present invention, if the amount of amphiregulin detected in a urine sample is 5 pg / mgCr or higher, preferably 6 pg / mgCr or higher, more preferably 6.3 pg / mgCr or higher, and most preferably 6.5 pg / mgCr or higher, the individual may be judged to have kidney disease.

[0056] In the present invention, the detection of amphiregulin may be characterized by an amphiregulin-specific binding reaction using an amphiregulin-specific antibody or aptamer, but is not limited thereto.

[0057] In yet another aspect, the present invention provides a method for detecting amphiregulin from an individual's urine sample to provide information necessary for diagnosing kidney disease.

[0058] Specifically, gene expression can be detected at the protein level, and the separation of proteins from biological samples can be performed using known procedures.

[0059] In this invention, the term "individual" refers to an animal that exists in nature and possesses kidneys as a bodily organ, preferably a mammal, and more preferably a human. Examples of mammals include rats, mice, rabbits, horses, cows, sheep, dogs, cats, monkeys, and humans, but the types of individuals in this invention are not limited by these examples.

[0060] In the present invention, the sample used to detect amphiregulin includes tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine samples in which the mRNA or protein expression levels of amphiregulin differ between normal individuals and individuals with kidney disease, with urine samples being preferred.

[0061] In the present invention, analytical methods for detecting marker proteins include, but are not limited to, Western blotting, ELISA, radioimmunoanalysis, radioimmunodiffusion, Oak-Turrony immunodiffusion, rocket-type electrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chips. These analytical methods allow for a comparison of the amount of antigen-antibody complexes formed in a normal control group with the amount of antigen-antibody complexes formed in individuals suspected of having kidney disease. This enables the determination of whether there is a significant increase in the expression level of kidney disease diagnostic marker genes in protein form, and allows for the prediction or confirmation of whether or not actual kidney disease has developed in individuals suspected of having kidney disease. The antigen-antibody complex refers to a conjugation of a kidney disease diagnostic marker protein and an antibody specific to it, and the amount of antigen-antibody complex formed can be quantitatively measured by the magnitude of the signal of the detection label.

[0062] Protein expression level measurement is preferably performed using the ELISA method. ELISA includes various ELISA methods, such as direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support; indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support; direct sandwich ELISA using yet another labeled antibody that recognizes an antigen in a complex of antibodies and antigens attached to a solid support; and indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody after reacting it with yet another antibody that recognizes an antigen in a complex of antibodies and antigens attached to a solid support. More preferably, detection is performed by a sandwich ELISA method in which an antibody is attached to a solid support and the sample is reacted, and then a labeled antibody that recognizes the antigen of the antigen-antibody complex is attached and enzymatically colored, or a labeled secondary antibody is attached to the antibody that recognizes the antigen of the antigen-antibody complex and enzymatically colored. The degree of complex formation between the kidney disease diagnostic marker protein and the antibody can be confirmed, and the presence or absence of kidney disease can be confirmed.

[0063] Preferably, a Western blot is used with one or more antibodies against the kidney disease diagnostic markers. For example, the whole protein is separated from the sample, separated by size using electrophoresis, and then transferred to a nitrocellulose membrane and reacted with an antibody. The amount of the generated antigen-antibody complex is confirmed using the labeled antibody, thereby confirming the amount of protein produced by gene expression and confirming whether or not kidney dysfunction has occurred. The detection method is performed by examining the expression level of the marker gene in a control group and the expression level of the marker gene in cells where kidney dysfunction has occurred. Protein levels can be expressed as absolute (e.g., μg / ml) or relative (e.g., relative signal intensity) differences of the marker proteins mentioned above.

[0064] Preferably, a protein chip is used in which one or more antibodies against the kidney disease diagnostic markers are arranged at predetermined positions on a substrate and immobilized at high density. For example, a method of analyzing a sample using a protein chip involves separating proteins from the sample, mixing the separated proteins with the protein chip to form an antigen-antibody complex, and interpreting this complex to confirm the presence or degree of protein expression and to confirm whether or not kidney dysfunction has occurred.

[0065] These detection methods allow for the confirmation or prediction of actual kidney dysfunction in patients suspected of having kidney disease by comparing the protein expression levels in a normal control group with the gene expression levels in individuals suspected of having kidney disease. Specifically, by measuring the expression level of the marker of the present invention from a urine sample of an individual suspected of having kidney disease, and then measuring the expression level of the marker of the present invention from a urine sample of a healthy individual and comparing the two, if it is confirmed that the expression level of the marker of the present invention is significantly higher than that of a healthy individual, then it can be predicted that the individual has kidney disease.

[0066] On the other hand, according to the inventors' experimental results, urinary amphiregulin levels accurately reflect basal renal function and can predict the degree of kidney damage at the time of measurement. Furthermore, analysis using follow-up observation data confirmed that it is possible to predict not only the progression of kidney damage at the time of measurement but also the progression of kidney damage in the future. Measuring urinary amphiregulin levels is applicable as a biomarker to predict end-stage renal failure in patients with kidney disease.

[0067] Therefore, in yet another aspect, the present invention provides a method for providing information for predicting the likelihood of progression of end-stage renal failure, comprising the following steps: (a) The step of collecting a urine sample from a patient with kidney disease; (b) Steps to measure the amount of amphiregulin in the urine sample: and (c) When the measured amount of amphiregulin is 30 pg / mgCr or higher, it is determined that the patient may be at risk of progressing to end-stage renal failure.

[0068] In the present invention, if the measured amount of amphiregulin is 30 pg / mgCr or higher, preferably 35 pg / mgCr or higher, more preferably 39 pg / mgCr or higher, and most preferably 39.9 pg / mgCr or higher, the patient may be judged to be at risk of progression to end-stage renal failure.

[0069] In the present invention, the patient suffering from the aforementioned kidney disease may be characterized as a patient with chronic renal failure, but is not limited thereto.

[0070] In the present invention, step (c) above may be characterized by determining that the likelihood of progression to end-stage renal failure is 50%, preferably 60% or more, based on the measurement of urinary amphiregulin, within two years.

[0071] In another aspect, the present invention relates to a composition comprising amphiregulin or an agent for specifically detecting amphiregulin for use in the diagnosis of kidney disease.

[0072] In another aspect, the present invention relates to an application for producing a reagent for the diagnosis of kidney disease that includes an agent for specifically detecting amphiregulin.

[0073] In another respect, the present invention relates to a method for diagnosing kidney disease, comprising the following steps: (a) The stage of collecting a urine sample from the individual; (b) Steps to detect amphiregulin in the urine sample: and

[0074] (c) If the amount of amphiregulin detected in the urine sample is 5 pg / mgCr or higher, the individual is judged to have kidney disease.

[0075] In yet another aspect, the present invention relates to a method for predicting the likelihood of progression of end-stage renal failure, comprising the following steps: (a) The step of collecting a urine sample from a patient with kidney disease; (b) Steps to measure the amount of amphiregulin in the urine sample: and (c) When the measured amount of amphiregulin is 30 pg / mgCr or higher, it is determined that the patient may be at risk of progressing to end-stage renal failure.

[0076] The present invention will be described in more detail below with reference to examples. These examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.

[0077] Example 1. Verification of amphiregulin expression in urine samples from a pulmonary fibrosis animal model with normal kidney function.

[0078] To determine the presence or absence of kidney function abnormalities using urine samples from an animal model of pulmonary fibrosis, we conducted an experiment to detect amphiregulin in the urine samples.

[0079] 1.1. Animal Experimentation Methods

[0080] To create an animal model of pulmonary fibrosis, a 15-unit stock of C57BL / 6 (mouse) bleomycin (BLM) (B5507, Sigma, USA) was dissolved in 5 ml of PBS to a concentration of 3 units / ml. 50 µl of this stock was then administered to C57BL / 6 (mice) via intratracheal instillation, at a dose of 1.5 units / kg relative to the mouse's body weight. After creating the bleomycin-induced pulmonary fibrosis model, the following mediating variables were measured 14 days later: body weight, urinary albumin / creatinine ratio, and serum creatinine. Furthermore, histological analysis was performed on lung and kidney tissue. Fibronectin and collagen 1 were also analyzed to confirm whether fibrosis had occurred in the lung and kidney tissue. Then, gene expression analysis was performed to determine how much amphiregulin was expressed in each tissue.

[0081] 1.2 RNA isolation and expression analysis of fibrotic disease genes and amphiregulin gene

[0082] Lung and kidney tissue from sacrificial mice was obtained and pulverized using a homogenizer. Total RNA was extracted using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea). The extracted RNA was quantified by measuring 1 ug using a spectrophotometer. RNA reverse transcriptase (AccuPower(R) RocketScript) was used. TM RT Premix with o cDNA was prepared using ligo(dT)20 (Bioneer, Korea) according to the manufacturer's instructions. The relative expression rate of total mRNA in each group was analyzed using the prepared cDNA as a template and real-time qPCR with the SYBR green method, as described below. The prepared cDNA was diluted 5-fold with distilled water in each well of a 96-well plate, and 3 μl of the diluted cDNA was used for the mRNA expression analysis of each gene. TM A mixture was prepared by adding 25 μl of (Korea) solution, 19 μl of distilled water, and 3 μl of each gene-specific qPCR primer (10 pmole / μl) (BIONEER, Korea). Meanwhile, to normalize the fibronectin, collagen 1, and amphiregulin genes, the housekeeping gene (HK gene) RPL13A was used as the standard gene. The primer sequences used to confirm gene expression in each tissue are as follows.

[0083] [Table 1]

[0084] As a result, as shown in Figure 1, there was no difference in gene expression between bleomycin-induced mice and healthy mice in kidney tissue, but in lung tissue, the difference in AREG was statistically significantly increased in bleomycin-induced mice. On the other hand, as shown in Figures 2 and 3, to confirm whether fibrosis had progressed in each tissue, we examined collagen 1 and fibronectin, which are markers of fibrosis disease. The results showed that there was no difference in expression of collagen 1 and fibronectin between healthy mice and bleomycin-induced mice in kidney tissue, but in lung tissue, it was confirmed that pulmonary fibrosis induced by bleomycin was statistically significantly increased compared to healthy mice. Therefore, gene expression analysis confirmed that in bleomycin-induced pulmonary fibrosis mice, fibrosis occurred only in lung tissue, and not in kidney tissue.

[0085] 1.3. Detection of amphiregulin and albumin / creatinine ratio in urine samples

[0086] 1-3-1. Mouse amphiregulin ELISA method

[0087] To detect amphiregulin in mouse urine, the Mouse Amphiregulin Duoset ELISA (DY989, R&D Systems, USA) was used. Each well was coated with 100 µl of mouse-captured amphiregulin antibody (DY989, R&D Systems, USA) at a concentration of 800 ng / ml. Then, 100 µl of 1% BSA was added to each well, and the mixture was reacted at room temperature for 1 hour to remove nonspecific reactions other than the sample-antibody reaction. Each well was washed three times with PBST containing 0.05% Tween20 as the washing buffer. Next, 100 µl of mouse urine was added to each well of the coated 96-well plate and reacted at room temperature for 2 hours. After the reaction, the urine samples were removed from each well, and each well was washed three times with PBST containing 0.05% Tween20 as the washing buffer. Then, 100 µl of detection antibody (DY989, R&D Systems, USA) at a concentration of 25.0 ng / ml was added to each well and reacted at room temperature for 2 hours. After the reaction, the detection antibody solution was removed and each well was washed three times with PBST. A secondary antibody, streptavidin-HRP (DY989, R&D Systems, USA), was diluted at a ratio of 1:200 and 100 µl was added to each well and reacted at room temperature for 20 minutes. After the reaction, each well was washed three times with PBST for 5 minutes each. Then, 100 µl of 1-step Ultra TMB (34028, Thermo, USA) was added to each well and reacted at room temperature. After that, 100 µl of sulfuric acid (H2SO4) was added to each well to stop the reaction, and the absorbance of each well was measured at a wavelength of 450 nm using a spectrophotometer. Using the measured absorbance values, an equation was created for the standard curve, and quantitative values ​​for each sample were calculated. As a result, the amphiregulin / creatinine ratio corrected for creatinine levels did not differ significantly between healthy mice and mice with induced pulmonary fibrosis. Furthermore, in the pulmonary fibrosis model with normal renal function, the urinary amphiregulin / creatinine levels were found to be similar to those of healthy mice (Figure 4).

[0088] 1-3-2. Detection of the albumin / creatinine ratio in mouse urine

[0089] The experiment was conducted using an albumin ELISA kit (Excocell, Phila, PA19103, Cat:1011). To measure albumin, urine samples were diluted in a 1:13 ratio with NHE buffer (Excocell, Phila, PA19103, Cat:1011), and 50 μl was added to each well. Then, 50 μl of primary antibody (Excocell, Phila, PA19103, Cat:1011) was added to each well, and the mixture was reacted at room temperature for 1 hour. After that, the mixture was washed 10 times with distilled water, and 100 μl of two-color developer (Excocell, Phila, PA19103, Cat:1011) was added to each well, and the mixture was reacted at room temperature for 20 minutes. The reaction was stopped by adding 100 μl each of two color-developing stoppers (Excocell, Phila, PA19103, Cat:1011), and the absorbance was measured at 450 nm using a spectrophotometer. A calculation formula for the standard curve was created using the measured absorbance values, and the quantitative values ​​for each sample were calculated.

[0090] The reason for measuring creatinine in urine is to correct for variations in urine amphiregulin levels depending on the degree of urine concentration. Therefore, a creatinine ELISA kit (Excocell, Phila, PA19103, Cat:1012) was used to measure creatinine as follows: Urine samples were diluted with distilled water at a ratio of 1:10 and placed in each well. Then, a picrate solution (Excocell, Phila, PA19103, Cat:1012) was prepared, 100 μl was added to each well, and the reaction was allowed to proceed at room temperature. After that, the absorbance was measured primary at 500 nm using a spectrophotometer, and then 100 μl of acid reagent (Excocell, Phila, PA19103, Cat:1012) was added to each well, and the absorbance was measured secondary after 5 minutes. The final result value was obtained by subtracting the secondary absorbance from the primary absorbance. Using the absorbance values ​​measured at creatinine standard concentrations of 10 mg / dL, 3 mg / dL, and 1 mg / dL, a calculation formula was created for the standard curve, and quantitative values ​​for each sample were calculated.

[0091] Furthermore, there was no significant difference in the albumin / creatinine ratio between mice with induced pulmonary fibrosis and healthy mice (Figure 5), indicating that kidney function was maintained normally in mice with induced pulmonary fibrosis.

[0092] 1.4. Histopathological analysis

[0093] Immunohistochemical staining was performed on lung and kidney tissues in the aforementioned animal models for histopathological verification. Animal models were sacrificed in separate groups, and paraffin sections were prepared by tissue fixation, washing, dehydration, clearing, infiltration, embedding, and sectioning. The paraffin sections were thinly sliced ​​using a sectioning machine, and the tissues were attached to slides. The attached tissues were deparaffinized, hydrated, washed, stained, dehydrated, cleared, and mounted, followed by hematoxylin and eosin staining, and also Masson's trichrome staining. Next, tissue staining was performed by the KPNT analytical laboratory.

[0094] As a result, it was found that mice with induced pulmonary fibrosis had more differentiated fibrotic cells in their lung tissue compared to healthy mice, and collagen 1 expression analysis using MT staining confirmed that more collagen was deposited in mice with induced pulmonary fibrosis compared to healthy mice (Figure 6A). However, no significant difference was observed in kidney tissue between healthy mice and mice with induced pulmonary fibrosis; similar tissue conditions were maintained, and no collagen 1 deposition was observed in mice with induced pulmonary fibrosis (Figure 6B).

[0095] 1-5. Measurement of blood urea nitrogen and creatinine levels

[0096] When kidney function deteriorates, substances that are normally excreted through the kidneys accumulate in the body. BUN and creatinine are the most representative of these substances, and their concentrations are known to reflect kidney function well. Therefore, to check kidney function, we measured blood urea nitrogen (BUN) and creatinine levels in serum samples.

[0097] As shown in Figure 7, when comparing blood urea nitrogen levels between bleomycin-induced mice and healthy mice, no statistically significant difference was found. Furthermore, as shown in Figure 8, when comparing creatinine levels, there was no difference between healthy mice and bleomycin-induced mice. Therefore, it was confirmed that renal function is maintained normally in the bleomycin-induced pulmonary fibrosis animal model.

[0098] Example 2. Verification of amphiregulin expression in serum and urine in a group of patients who underwent kidney tissue biopsy.

[0099] Amphiregulin concentrations were measured in the serum and urine of patients who underwent kidney tissue biopsy using the ELISA method. Serum and urine amphiregulin expression was analyzed by comparing patients with minor glomerular change (MI), who have pathological findings similar to those of a normal kidney, with patients with more advanced pathological findings, such as immunoglobulin A nephropathy and diabetic nephropathy. Furthermore, kidney tissue biopsy results were reviewed for these patients, and they were divided into four groups based on the degree of histological findings associated with renal fibrosis. The level of urine amphiregulin expression was then compared among these groups.

[0100] 2-1. Amphiregulin analysis using ELISA in patient serum and urine samples

[0101] To detect amphiregulin from human serum or urine, the Human Amphiregulin Duoset ELISA (DY262, R&D Systems, USA) was used. Specifically, amphiregulin antibody was added to each well of a 96-well plate at a concentration of 2.00 ug / ml, with 100 uls added to each well for coating. Then, 100 uls of 1% BSA was added to each well, and the mixture was reacted at room temperature for 1 hour to suppress nonspecific expression other than the sample-antibody reaction. Each well was washed three times with PBST containing 0.05% Tween20 as the washing buffer. After adding 100 uls of patient serum or urine stock sample to each well and reacting at room temperature for 2 hours, the serum or urine sample was removed from each well, and each well was washed three times with PBST containing 0.05% Tween20 as the washing buffer. Each well was washed three times with PBST, and 100 µl of detection antibody (DY262, R&D Systems, USA) at a concentration of 100 ng / ml was added to each well and reacted at room temperature for 2 hours. After that, the detection antibody solution was removed, each well was washed three times with PBST, and 100 µl of streptavidin-HRP (DY262, R&D Systems, USA) secondary antibody, diluted at a ratio of 1:200, was added to each well and reacted at room temperature for 20 minutes. After the reaction, each well was washed three times for 5 minutes each with PBST, and 100 µl of 1-step Ultra TMB (34028, Thermo, USA) was added to each well and reacted at room temperature. Then, 100 µl of sulfuric acid (H2SO4) was added to each well to stop the reaction. The absorbance of each well at a wavelength of 450 nm was measured using a spectrophotometer, and the measured absorbance values ​​were used to create an equation for the standard curve and calculate the quantitative value for each sample.

[0102] As a result, while there was no significant difference in serum amphiregulin expression among patients with minor glomerular change, immunoglobulin A nephropathy, and diabetic nephropathy, urinary amphiregulin expression was found to be increased in patients with more severe renal pathological findings, particularly in those with immunoglobulin A nephropathy and diabetic nephropathy, compared to patients with minor glomerular change (Figure 9).

[0103] On the other hand, in the aforementioned patient group, histological findings associated with renal fibrosis were compared according to severity, and the patients were divided into groups of no fibrosis (absent), mild, moderate, and severe. The concentration of amphiregulin was then checked from the patients' urine samples. Interstitial fibrosis and intimal thickening significantly increased the expression of urinary amphiregulin in patients in the moderate stage compared to those in the absent stage. Tubular atrophy and mesangial expansion also significantly increased the expression of urinary amphiregulin in patients in the moderate and severe stages compared to those in the absent stage. Therefore, it was found that the more severe the renal fibrosis progresses in kidney tissue, the more the expression of urinary amphiregulin increases (Figure 10).

[0104] Based on the aforementioned tissue examination results, and considering the interstitial fibrosis results, which are a representative measure reflecting renal fibrosis, patients were divided into two groups: those with no renal fibrosis (no interstitial fibrosis observed) and those with confirmed renal fibrosis (interstitial fibrosis observed). Receiver operating characteristic (ROC) curve analysis was performed based on urinary amphiregulin concentration. As a result, the optimal cutoff value for amphiregulin, which maximizes the sum of sensitivity and specificity, was found to be 6.55 pg / mg.

[0105] Therefore, when amphiregulin expressed in individual urine samples was 6.55 pg / mgCr or higher, it was confirmed that renal fibrosis, in particular, was progressing among kidney diseases (sensitivity, 0.84; specificity, 0.78).

[0106] Example 3. Verification of amphiregulin expression in plasma and urine in a group of diabetic patients.

[0107] The amphiregulin expression patterns were examined in plasma and urine samples isolated from a normal control group and patients with type 2 diabetes. Since the severity of diabetic nephropathy in type 2 diabetes patients can be determined by the amount of albuminuria, these patients were divided into three groups based on the degree of albuminuria: normal proteinuria (normoalbuminuria), microalbuminuria (microalbuminuria), and macroalbuminuria (macroalbuminuria). The levels of amphiregulin expression in plasma and urine were then compared for each group. ELISA analysis was performed using the same product (DY262, R&D systems, USA) as described in Example 2-1.

[0108] As a result, as shown in Figure 11, there was no clear difference in plasma amphiregulin concentration between the normal control group and type 2 diabetic patients, nor was there a significant difference in concentration among the albuminuria groups. In contrast, urinary amphiregulin concentration was clearly increased in type 2 diabetic patients compared to the normal control group, and it was observed that in type 2 diabetic patients, as albuminuria worsened, i.e., as the severity of diabetic nephropathy increased, the urinary amphiregulin concentration also increased.

[0109] The amphiregulin-to-creatinine ratio for the experimental group was analyzed as follows:

[0110] [Table 2]

[0111] [Table 3]

[0112] Example 4. Verification of end-stage renal failure progression prediction using urinary amphiregulin concentration in a group of patients who underwent kidney tissue biopsy.

[0113] In a study of 72 patients who underwent kidney tissue biopsy and were diagnosed with minor glomerular change, IgA nephropathy (immunoglobulin A nephropathy), or diabetic nephropathy, urinary amphiregulin was quantified using ELISA in urine samples taken at the time of tissue biopsy. Subsequently, the patients were classified according to the stage of chronic renal failure while their prognosis was observed. The classification of chronic renal failure was based on the KDIGO guidelines, one of the representative guidelines for the diagnosis and treatment of chronic renal failure (Kidney Int Suppl 2012;2013:1-150).

[0114] The urinary amphiregulin concentrations analyzed at the time of tissue examination according to the stage of chronic renal failure were plotted, and this patient group was divided into three groups (T1: 0.598-9.472 pg / mgCr, T2: 9.473-39.904 pg / mgCr, T3: 39.905-597.252 pg / mgCr) based on the tertiles of urinary amphiregulin concentration. Kaplan-Meier survival analysis was performed to confirm whether there was a difference in the probability of future progression to end-stage renal failure among the three groups. The ELISA analysis method was the same as the method described in Example 2-1.

[0115] The results, as shown in Figure 12, showed that chronic new diseases in the patient group who underwent kidney tissue biopsy were distributed across stages 1 to 4. Compared to stage 1-2 patients with relatively good renal function, patients in stages 3-4, who had impaired renal function, measured higher concentrations of urinary amphiregulin. Furthermore, as shown in Figure 13, the Kaplan-Meier survival curves revealed that the T3 group, which showed high urinary amphiregulin concentrations at the time of tissue biopsy, had a significantly higher incidence of end-stage renal failure (ESRD incidence per 1000 person-years: T1, 0.0 [95% CI, 0.0-117.3]; T2, 71.4 [95% CI, 14.2-229.0]; T3, 600.0 [95% CI, 296.3-1095.1]).

[0116] On the other hand, to improve the accuracy of the experiment, we corrected for age, sex, baseline 24hr urine protein (creatine), and baseline GFR. When comparing the combined T1 / T2 tertile group with the T3 group, the progression rate of end-stage renal failure was significantly higher in the T3 group (hazard ratio between T3 group and T1 / T2 group: 11.7 [95% CI 1.6-86.5]). Furthermore, when we analyzed with urinary amphiregulin-to-creatinine ratio as a continuous variable, we found a significant correlation with the development of chronic renal failure even after correcting for these variables (hazard ratio: 5.1 [95% CI 1.814.8] for each increase of 1 in the log urine amphiregulin-to-creatinine ratio). In the group of patients who participated in this experiment, a urinary amphiregulin level of 39.905 pg / mgCr or higher was associated with a 62.2% probability of developing end-stage renal failure after two years (see Figure 13).

[0117] Having described in detail certain aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. [Industrial applicability]

[0118] According to the present invention, there is an advantage in that kidney diseases, for which early diagnosis is important, can be easily diagnosed in a non-invasive manner using a urine sample. Furthermore, if the present invention is applied to patients diagnosed with chronic renal failure, there is an advantage in that the possibility of progression to end-stage renal failure can be predicted in advance.

Claims

1. A diagnostic composition for kidney disease comprising an agent for specifically detecting amphiregulin or its mRNA.

2. The composition for diagnosing kidney disease according to claim 1, characterized in that the agent specifically detects amphiregulin from a urine sample.

3. The composition for diagnosing kidney disease according to claim 1, characterized in that the amphireglin is represented by the amino acid sequence of SEQ ID NO:

1.

4. The kidney disease diagnostic composition according to claim 1, characterized in that the agent for specifically detecting amphiregulin is an amphiregulin-specific antibody or aptamer.

5. The kidney disease diagnostic composition according to claim 1, characterized in that the kidney disease is selected from the group consisting of renal fibrosis, renal syndrome, renal cancer, chronic renal failure, diabetic nephropathy, acute pyelonephritis, acute renal failure, end-stage renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjögren's syndrome, Behçet's disease, lupus, candidiasis, hemorrhagic fever with renal syndrome, leptospirosis, legionellosis, autosomal dominant polycystic kidney disease, and hydronephrosis.

6. A kit for diagnosing kidney disease, comprising the composition according to any one of claims 1 to 4.

7. The kit for diagnosing kidney disease according to claim 6, characterized in that the kit is a protein chip kit or an immunodiagnostic kit.

8. The kidney disease diagnostic kit according to claim 6, characterized in that the kidney disease is selected from the group consisting of renal fibrosis, renal syndrome, renal cancer, chronic renal failure, diabetic nephropathy, acute pyelonephritis, acute renal failure, end-stage renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjögren's syndrome, Behçet's disease, lupus, candidiasis, hemorrhagic fever with renal syndrome, leptospirosis, legionellosis, autosomal dominant polycystic kidney disease, and hydronephrosis.

9. Methods for providing information for the diagnosis of kidney disease, including the following stages: (a) The stage of collecting a urine sample from the individual; (b) Steps to detect amphiregulin in the urine sample: and (c) If the amount of amphiregulin detected in the urine sample is 5 pg / mgCr or higher, the individual is judged to have kidney disease.

10. The method for diagnosing kidney disease according to claim 9, characterized in that the detection of amphiregulin is performed by an amphiregulin-specific reaction using an amphiregulin-specific antibody or aptamer.

11. The method for diagnosing a kidney disease according to claim 9, characterized in that the kidney disease is selected from the group consisting of renal fibrosis, renal syndrome, renal cancer, chronic renal failure, diabetic nephropathy, acute pyelonephritis, acute renal failure, end-stage renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjögren's syndrome, Behçet's disease, lupus, candidiasis, hemorrhagic fever with renal syndrome, leptospirosis, legionellosis, autosomal dominant polycystic kidney disease, and hydronephrosis.

12. Methods for providing information to predict the progression of end-stage renal failure, including the following stages: (a) The step of collecting a urine sample from a patient with kidney disease; (b) Step of measuring the amount of amphiregulin in the urine sample: and (c) When the measured amount of amphiregulin is 30 pg / mgCr or higher, it is determined that the patient may be at risk of progressing to end-stage renal failure.

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