Method for judging chronic kidney disease by detecting urine migration body

Abstract

The invention relates to a method for judging chronic kidney disease by detecting urine migrators. Specifically, the invention provides an application of a detection agent of a migration body, the detection agent is used for preparing a detection reagent or a kit, and the detection reagent or the kit is used for evaluating whether a certain object suffers from the chronic kidney disease or the susceptibility of the certain object suffers from the chronic kidney disease. The invention also provides a corresponding detection kit and diagnostic equipment. Researches show that the migration body disclosed by the invention has high sensitivity and specificity, can be used as a marker of chronic kidney disease occurrence risk, and is used for auxiliary examination and early diagnosis of chronic kidney disease.

Description

Technical Field

[0001] The present invention belongs to the field of clinical medicine, and particularly relates to a method for judging chronic kidney disease by detecting urinary migrasomes. Background Art

[0002] The kidney is an important organ of the human body, which is not only responsible for urine formation, but also has multiple functions such as clearing and discharging toxins in the body, reabsorbing nutrients and secreting hormones. Once there is a problem with the kidney, it will not only cause damage to the original functions of the kidney, but also lead to abnormal functions in multiple systems such as the digestive system, respiratory system, cardiovascular system and nervous system, seriously reducing the quality of life of patients and even posing a life threat.

[0003] At present, the most important biological indicators for diagnosing chronic kidney disease include serum creatinine, urea nitrogen, and the related estimated glomerular filtration rate (eGFR) and urine protein level. However, these indicators are affected by multiple factors, such as gender, age, eating habits and infection status, and cannot effectively reflect the degree of kidney damage and fibrosis in the early stage of kidney disease. At present, the diagnosis of kidney disease still relies on the pathological histological test results obtained by kidney puncture biopsy. Although this classic test method can clarify the pathological type and lesion degree of kidney damage, it has many limitations. For example, for patients with too severe conditions or patients who need follow-up, kidney puncture biopsy is not a repeatable diagnostic method; especially as an invasive operation with multiple complication risks, it is not applicable to large-scale disease screening in clinical practice. In addition, this examination also requires expensive pathological test equipment and specially trained technical personnel. Since kidney-related diseases have a long course and high treatment costs, once kidney diseases enter the irreversible stage, patients not only need to take medicine for a long time to maintain their lives, and their quality of life is greatly affected, but they may also lose their labor ability, which will not only increase the treatment burden on patients, but also greatly increase the social medical costs of the country. In addition, when the eGFR and serum creatinine levels are within the normal range, it is impossible to detect whether the subject has chronic kidney disease or the susceptibility to chronic kidney disease. Therefore, there is an urgent need in the market for methods and means that can diagnose chronic kidney disease early and perform dynamic detection.

[0004] In summary, there is an urgent need in this field to develop a convenient, fast, accurate and efficient method for judging whether a subject has chronic kidney disease by detecting urinary migrasomes. This method can be used not only for the diagnosis of chronic kidney disease, but also for judging the curative effect during the treatment of chronic kidney disease. Summary of the Invention

[0005] The object of the present invention is to provide a convenient, fast, accurate and efficient method for judging whether a subject has chronic kidney disease by detecting urinary migrasomes.

[0006] In a first aspect of the present invention, a use of a migrator-activated protein kinase (MAPK) detection agent is provided for preparing a detection reagent or a kit for assessing whether a subject has chronic kidney disease (CKD) or is susceptible to CKD.

[0007] Wherein, the chronic kidney disease is selected from the group consisting of membranous nephropathy (MN), diabetic nephropathy (DN) and IgA nephropathy (IgA).

[0008] In another preferred embodiment, the chronic kidney disease is membranous nephropathy (MN).

[0009] In another preferred embodiment, the subject satisfies the following conditions:

[0010] (Z1) eGFR is 80–125 mL / min / 1.73 m 2 , preferably 85-110 mL / min / 1.73 m 2 , more preferably 90-105 mL / min / 1.73 m 2 , and / or

[0011] (Z2) The blood creatinine level is 0.50-1.50 mg / dl, preferably 0.75-1.25 mg / dl.

[0012] In another preferred embodiment, the subject is a person who has a family member suffering from chronic kidney disease or has had chronic kidney disease.

[0013] In another preferred embodiment, the subject is a patient who has suffered acute renal injury.

[0014] In another preferred embodiment, the subject is not a patient with acute kidney disease.

[0015] In another preferred embodiment, the subject is a patient in CKD-I stage.

[0016] In another preferred embodiment, the detection reagent or kit is also used for typing chronic kidney disease.

[0017] In another preferred embodiment, the detection includes urine detection, serum detection, platelet detection, and detection of tissue or cell samples.

[0018] In another preferred embodiment, the test is a urine test.

[0019] In another preferred embodiment, the migrator is a urine migrator.

[0020] In another preferred embodiment, the cells are podocytes of the kidney.

[0021] In another preferred embodiment, the detection reagent comprises a specific binding molecule for the migrasome, a specific amplification primer, a probe or a chip.

[0022] In another preferred embodiment, the specific binding molecule comprises a specific antibody, a solid-phase carrier with or bound to lectin on its surface.

[0023] In another preferred embodiment, the specific antibody is selected from the group consisting of: an antibody against TSPAN4, an antibody against Integrinα5β1, a PIGK antibody, an NDST1 antibody, a CPQ antibody, an EOGT antibody, or a combination thereof.

[0024] In another preferred embodiment, the solid-phase carrier comprises: solid particles, a microfluidic chip, a cellulose acetate membrane, a nylon membrane, agarose microbeads.

[0025] In another preferred embodiment, the solid-phase carrier comprises magnetic beads or non-magnetic microspheres.

[0026] In another preferred embodiment, the lectin is coupled to the surface of the solid-phase carrier by a chemical bond.

[0027] In another preferred embodiment, the lectin is selected from the group consisting of: wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), or a combination thereof.

[0028] In another preferred embodiment, the specific antibody or the specific binding molecule is conjugated with or carries a detectable label.

[0029] In another preferred embodiment, the detectable label is selected from the group consisting of: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

[0030] In another preferred embodiment, the detection includes: flow cytometry, fluorescence imaging technology, fluorescence immunoassay reading technology.

[0031] In a second aspect of the present invention, there is provided a detection kit, which comprises:

[0032] (a) A detection reagent, wherein the detection reagent comprises:

[0033] (a1) A first detection reagent for detecting migrasomes;

[0034] (a2) A second detection reagent for detecting eGFR;

[0035] (a3) Optionally, a third detection reagent for detecting serum creatinine; and

[0036] (a4) Optionally, a fourth detection reagent for detecting autoantibodies against podocyte proteins such as PLA2R, THSD7A, etc.;

[0037] (b) a label or instructions indicating that the kit is used to assess a subject's susceptibility to chronic kidney disease.

[0038] In another preferred embodiment, the detection kit is also used for typing chronic kidney disease.

[0039] In another preferred embodiment, the detection includes pre-judgment (prediction), typing detection, and prognosis detection.

[0040] In another preferred embodiment, the classification includes dividing chronic kidney disease into membranous nephropathy (MN), diabetic nephropathy (DN) and IgA nephropathy (IgA).

[0041] In another preferred embodiment, the classification includes dividing chronic kidney disease into CKD-I stage and CKD-II stage.

[0042] In another preferred embodiment, the classification includes dividing chronic kidney disease into specific and secondary types.

[0043] In another preferred embodiment, the antibody or protein detection reagent for detecting migratoria includes a specific antibody against migratoria protein.

[0044] In another preferred embodiment, the detection is a blood sample detection and / or a serum sample detection.

[0045] In another preferred embodiment, the label or instructions indicate the following:

[0046] When the ratio of the number of migratory bodies in the urine of a test subject to the number of migratory bodies in a normal person is ≥1.5, more preferably ≥2, it indicates that the test subject has a high risk of developing chronic kidney disease.

[0047] In another preferred embodiment, the label or instructions state the following:

[0048] (i) when the number of migratory bodies in the urine of the test subject is greater than 1390 MFI (mean fluorescence intensity), it indicates that the test subject has a high risk of developing chronic kidney disease; and

[0049] (ii) When the number of migratory bodies in the urine of the test subject is less than 1390 MFI (mean fluorescence intensity), it indicates that the risk of the test subject developing chronic kidney disease is low.

[0050] In another preferred embodiment, the method for detecting the number of migratory bodies is as described in Example 1.

[0051] In a third aspect of the present invention, a method for assessing whether a subject has chronic kidney disease or is susceptible to chronic kidney disease is provided, the method comprising:

[0052] (a) Provide a test sample from a subject,

[0053] wherein the subject meets the following conditions:

[0054] (Z1) eGFR is 80 - 125 mL / min / 1.73m 2 , preferably 85 - 110 mL / min / 1.73m 2 , more preferably 90 - 105 mL / min / 1.73m 2 ; and / or

[0055] (Z2) Serum creatinine level is 0.50 - 1.50 mg / dl, preferably 0.75 - 1.25 mg / dl;

[0056] (b) Detect the number of migrasomes in the test sample; and

[0057] (c) Compare the content of migrasomes determined in step (b) with a control reference value,

[0058] wherein when the content of migrasomes in the sample is higher than the control reference value, it indicates that the subject has a high risk of chronic kidney disease.

[0059] In another preferred embodiment, the test sample includes a urine sample of the subject.

[0060] In another preferred embodiment, the method is non - diagnostic and non - therapeutic.

[0061] In another preferred embodiment, the control reference value is a cut - off value.

[0062] In a fourth aspect of the present invention, a diagnostic device is provided, and the diagnostic device includes:

[0063] (a) An input module, the input module being configured to input the content of migrasomes in a test sample of a subject; wherein the subject is an object meeting the following conditions:

[0064] (Z1) eGFR is 80 - 125 mL / min / 1.73m 2 , preferably 85 - 110 mL / min / 1.73m 2 , more preferably 90 - 105 mL / min / 1.73m 2 ; and / or

[0065] (Z2) Serum creatinine level is 0.50 - 1.50 mg / dl, preferably 0.75 - 1.25 mg / dl;

[0066] (b) Chronic kidney disease diagnosis - Chronic kidney disease classification module, the chronic kidney disease diagnosis - chronic kidney disease classification module is configured to: based on the content of the migrasome, diagnose and analyze whether the subject has chronic kidney disease, and / or classify and analyze chronic kidney disease, and obtain a diagnosis analysis result and / or a classification analysis result; and

[0067] (c) Output module, the output module is configured to output the diagnosis analysis result and / or the classification analysis result.

[0068] In another preferred embodiment, the diagnostic device further includes (d) detection module, the detection module is configured to detect the number of migrasomes.

[0069] In another preferred embodiment, the output module includes: a printer, a display, a screen, a mobile phone, a PAD, or a combination thereof.

[0070] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Description of the Drawings

[0071] Figure 1 Shows the content of urinary migrasomes in patients with chronic kidney disease. Among them, Figures A - C respectively show the content of migrasomes, serum creatinine, and eGFR in the urine of normal people (NOR) and patients with chronic kidney disease (CKD), and Figure D shows the ROC curve of urinary migrasomes (red line), serum creatinine (blue line), and eGFR (purple line) in differentiating normal people from chronic kidney disease.

[0072] Figure 2 Figures A - 2C respectively show the content of migrasomes, serum creatinine, and eGFR in the urine of normal people, patients with diabetic nephropathy, membranous nephropathy, IgA nephropathy, lupus nephritis, purpuric nephritis, minimal change nephropathy, and patients with chronic kidney disease due to other reasons. Figure 2 Figure D shows the ROC curve of urinary migrasomes (red line) in differentiating normal people from patients with diabetic nephropathy, membranous nephropathy, IgA nephropathy, lupus nephritis, purpuric nephritis, minimal change nephropathy, and patients with chronic kidney disease due to other reasons (other).

[0073] Figure 3 Shows the content of urinary migrasomes in CKD patients at different stages, where Figure 3 Figure A shows the content of urinary migrasomes in normal people and CKD patients at different stages, Figure 3 Figures B - 3D respectively show the content of migrasomes, serum creatinine, and eGFR in the urine of normal people and CKD - stage I patients. Figure 3E shows the ROC curves of urine migratoria (red line), serum creatinine (blue line), and eGFR (purple line) in distinguishing normal subjects from CKD-I patients. Figure 3 F shows that the level of migratoria is significantly increased in CKD patients. Figure 3 G-3H showed that the eGFR and serum creatinine levels in CKD patients were almost unchanged compared with those in normal subjects. Figure 3 I shows the area under the receiver operating characteristic curve (ROC) of migratoria, serum creatinine, and eGFR in differentiating CKD patients with eGFR ≥ 80 from normal controls.

[0074] Figure 4 A-4C show the levels of migratsomes, eGFR, and serum creatinine in normal subjects and chronic kidney disease patients with normal serum creatinine, respectively. Figure 4 D shows the ROC curve of urine migratoria in differentiating normal subjects from patients with different types of CKD stage I.

[0075] Figure 5 Figure A shows the migratoria content in urine of normal subjects, DN stage I-IV, and DN-ESRD patients. Figure 5B shows the migratoria content in urine of normal subjects and MN stage I-IV patients. Figure 5 C shows the migratoria content in urine of normal subjects and patients with IgA stage I-IV. Figure 5 D shows the working curve of urine migrator for distinguishing DN-I stage, MN-I stage, and IgA-I stage.

[0076] Figure 6 Schematic diagram showing the chemiluminescence detection of urinary podocyte-derived migrasomes.

[0077] Figure 7 Figure A shows the content of migratoria in the urine of patients with chronic kidney disease and healthy subjects, expressed as OD values. Figure 7B shows the ROC curve of migratoria in differentiating healthy subjects from patients with chronic kidney disease.

[0078] Figure 8 A shows the fluorescence signal intensity (FITC) of autoantibodies in autoantibody-positive and autoantibody-negative migrators. Figure 8 B shows the ROC curve of the migratoria in distinguishing autoantibody-positive (positive) from autoantibody-negative (negative) patients. Figure 8 C shows the linear relationship between the concentration of membranous nephropathy autoantibodies (Autoantibody concentration) detected by ELISA and the concentration of autoantibodies (Fluorescence intensity) detected by urine migration body.

[0079] Figure 9 The experimental flow chart showing the use of WGA-coupled magnetic beads to capture migratsomes in urine as markers for the detection of autoantibodies in membranous nephropathy. DETAILED DESCRIPTION

[0080] After extensive and in-depth research, the inventors unexpectedly developed a highly sensitive and specific marker for detecting specific chronic kidney diseases. Specifically, the present invention combines a lectin-coupled solid support that specifically recognizes migrasome surface proteins with a test sample. This solid support efficiently captures migrasomes in the sample, which are then labeled with migrasome-specific antibodies to identify the number of migrasomes.

[0081] The present invention is based on the fact that the migrator of the present invention is more accurate in diagnosing chronic kidney disease than the existing blood creatinine and eGFR.

[0082] the term

[0083] As used herein, the term "sample" or "specimen" refers to material specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. A sample may consist entirely or in part of biological material from a subject.

[0084] As used herein, the terms "markers of chronic kidney disease of the present invention" and "markers of chronic kidney disease" all refer to "migrasomes".

[0085] As used herein, the term "reference value" refers to a value that is statistically correlated with a particular result when compared to the results of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of studies comparing the number of migratory bodies with known clinical results. In the Examples herein, some such studies are shown. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values can also be determined by considering the circumstances and results particularly relevant to the patient's medical history, genetics, age, and other factors.

[0086] In the present invention, the reference value refers to a cut-off value, preferably 1390 MFI (mean fluorescence intensity, used to indicate the number of migratory bodies in the serum of patients with chronic kidney disease).

[0087] In a preferred embodiment, the method for detecting the number of migratory bodies of the present invention is as described in Example 1.

[0088] Kidney disease

[0089] According to the histological classification scheme of glomerular diseases of the World Health Organization (WHO) in 1995, kidney diseases are classified as follows: (1) Primary glomerulonephritis (PGN), including IgA nephropathy (IgAN), membranous nephropathy (MN), minimal change disease (MCD), mesangial proliferative glomerulonephritis (MsPGN), membranoproliferative glomerulonephritis type I and III (MPGN I&III), endocapillary proliferative glomerulonephritis (EnPGN), C3 glomerulonephritis, dense deposit disease (DDD), etc.; (2) Secondary glomerulonephritis (SGN), mainly divided into immune-mediated diseases, tumor metabolic diseases and infectious diseases. Among them, immune-mediated diseases include lupus nephritis (LN), Henoch-Schönlein purpura nephritis (HSPN), renal damage caused by vasculitis, renal damage related to Sjögren's syndrome, anti-glomerular basement membrane nephropathy, renal damage related to rheumatoid arthritis, etc., tumor metabolic diseases include diabetic nephropathy (DN), hypertensive renal damage, obesity-related renal damage (ORG), renal amyloidosis, monoclonal immunoglobulin deposition disease (MIDD), etc., and infectious diseases include hepatitis B-related glomerulonephritis, hemorrhagic fever with renal syndrome, hepatitis C-related glomerulonephritis and other diseases; (3) Tubulointerstitial diseases (TIN) include acute interstitial nephritis (AIN), chronic interstitial nephritis (CIN), acute tubular necrosis (ATN), aristolochic acid nephropathy, Bartter's syndrome, reflux nephropathy, Gitelman's syndrome, etc.; (4) Hereditary kidney diseases include thin basement membrane nephropathy (TNMN), Alport syndrome, lipoprotein glomerulopathy (LPG), Fabry's disease, etc.; (5) Other cases with unclear diagnosis or unable to be classified.

[0090] Migrasome

[0091] As described herein, a migrasome is a single-membrane vesicle structure with a diameter of 0.5 - 2 μm generated by the contractile filaments at the cell tail during cell directed migration. Migrasomes contain a large number of bioactive substances such as nucleic acids, proteins, fats, etc., and play an important role in cell-to-cell communication, participating in and regulating various physiological and pathological activities. Migrasomes exist in blood and various body fluids, such as serum, urine, etc. The content of migrasomes in some blood or body fluids is closely related to certain diseases (such as diabetic nephropathy, etc.).

[0092] Estimated glomerular filtration rate (eGFR)

[0093] As described herein, the estimated glomerular filtration rate (eGFR) refers to the ability of the two kidneys to generate filtrate per unit time (usually 1 min), and the normal value for adults is (80 - 125) mL / min / 1.73m 2 . GFR is the main indicator for evaluating renal function and is also the main basis for the diagnosis and staging of chronic kidney disease.

[0094] The estimated glomerular filtration rate (eGFR) is a key parameter used to assess kidney function. Based on the eGFR, CKD patients are divided into five stages: eGFR ≥ 90 is CKD-I; 60 ≤ eGFR ≤ 89 is CKD-II; 30 ≤ eGFR ≤ 59 is CKD-III; 15 ≤ eGFR ≤ 29 is CKD-IV; and eGFR < 15 is CKD-V, which represents end-stage renal disease (ESRD).

[0095] Serum creatinine

[0096] As described in this article, creatinine is a metabolic product of muscle in the body. Every 20g of muscle produces 1mg of creatinine. Creatinine is primarily excreted from the body through glomerular filtration. A creatinine level higher than normal is called high creatinine. Creatinine includes both blood and urine creatinine.

[0097] Serum creatinine is commonly used to measure kidney function. Generally speaking, normal serum creatinine levels are between 0.50 and 1.50 mg / dL. A serum creatinine level exceeding 1.50 mg / dL indicates kidney damage, leading to renal insufficiency and renal failure. A level above 1.50 mg / dL indicates inflammatory damage, 2.10 mg / dL indicates renal impairment, and 5.10 mg / dL indicates renal failure. A serum creatinine level exceeding 8.00 mg / dL indicates advanced uremia.

[0098] Detection Methods

[0099] Based on the content of the chronic kidney disease risk marker migrator in a tissue sample or a urine sample, the present invention also provides a corresponding method for determining a specific chronic kidney disease.

[0100] Representative diseases associated with migratoria include, but are not limited to, renal diseases such as membranous nephropathy (MN), diabetic nephropathy (DN), and IgA nephropathy (IgA).

[0101] In addition, the migratoria capturing carrier of the present invention can efficiently and specifically capture migratoria in a sample, and when used in conjunction with flow cytometry for detection, can quickly, simply and accurately detect the types and quantities of migratoria in a sample.

[0102] In a preferred example, the inventors constructed a lectin-coupled solid phase carrier that can specifically recognize the surface protein of the migratoria, and used this solid phase carrier to efficiently capture migratoria in various body fluids such as cell culture fluid, urine and blood. The migratoria were then labeled with migratoria-specific antibodies to identify the type and content of the migratoria.

[0103] Detection Kits

[0104] Based on the correlation between chronic kidney disease risk markers and the occurrence of specific chronic kidney diseases, chronic kidney disease risk markers can be used as markers for judging the risk of specific chronic kidney diseases.

[0105] The present invention also provides a diagnostic kit for judging the risk of chronic kidney disease occurrence or classifying chronic kidney disease. The kit contains a detection reagent, and the detection reagent includes:

[0106] (a1) A first detection reagent for detecting migrasomes;

[0107] (a2) A second detection reagent for detecting eGFR;

[0108] (a3) Optionally, a third detection reagent for detecting serum creatinine; and

[0109] (a4) Optionally, a fourth detection reagent for detecting autoantibodies against podocyte proteins such as PLA2R and THSD7A.

[0110] In another preferred embodiment, the kit further includes a label or an instruction manual.

[0111] Diagnostic device

[0112] Based on the method provided by the present invention for evaluating the susceptibility of a subject to chronic kidney disease, the present invention also provides a corresponding diagnostic device.

[0113] In the present invention, the content of migrasomes in the test sample of the subject can be input by manual input or by automated collection. Typically, the content input module of the migrasomes is selected from the group consisting of: an immune cell typing collector, a scanner, a keyboard, a tablet computer (PAD), a smart phone, or a combination thereof.

[0114] Preferably, in the present invention, the chronic kidney disease diagnosis - chronic kidney disease classification module is configured to: based on the content of the migrasomes, diagnose and analyze whether the subject has chronic kidney disease, and / or classify chronic kidney disease, and obtain a diagnosis analysis result and / or a classification analysis result;

[0115] In the present invention, representative output modules include (but are not limited to): a display, a printer, a tablet computer (PAD), a smart phone.

[0116] The main advantages of the present invention include:

[0117] (a) Compared with existing indicators for judging chronic kidney disease such as serum creatinine and glomerular filtration rate (eGFR), the present invention uses urine samples. The migrasomes of the present invention can be used as markers for kidney diseases, and have the characteristics of being faster, more convenient, low - cost, and highly sensitive.

[0118] (b) The migrasome can be used as a biomarker for the early diagnosis of different types of chronic kidney disease.

[0119] (c) The migrasome of the present invention can be used as a biomarker for the diagnosis of early chronic kidney disease.

[0120] (d) The chemiluminescence method of the present invention has the advantages of being simple, easy to operate, etc. Detecting migrasomes by the chemiluminescence method can be used as a biomarker for the diagnosis of chronic kidney disease.

[0121] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight percentage and weight parts.

[0122] Example 1 Capture of migrasomes in urine by WGA-conjugated magnetic beads as a biomarker for kidney injury

[0123] 1. Collect urine samples from volunteers in the hospital physical examination center and the nephrology department. Within 24 hours, centrifuge at 4000g for 20 minutes at 4 °C to remove cell debris. Add 5 μL of WGA-conjugated magnetic beads to 1 mL of urine and incubate on a mixer at room temperature for 1 h.

[0124] 2. Place the EP tube on a magnetic separation rack to magnetically separate and remove the supernatant. Add 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA and wash 3 times, then resuspend in 100 μL of PBS solution containing 1% BSA and incubate at room temperature for 30 min.

[0125] 3. Subsequently, place the EP tube in the magnetic separation rack to enrich the magnetic beads and discard the supernatant. Resuspend in 100 μL of PBS solution containing 0.1% BSA.

[0126] 4. Add 0.5 μL of TSPAN4 antibody (abcam, catalog number ab181995, rabbit source) to the EP tube, incubate on a mixer at room temperature for 1 h. Subsequently, place the EP tube in the magnetic separation rack to enrich the magnetic beads and discard the supernatant. Add 1000 μL of PBS solution containing 0.1% BSA and wash 3 times, then resuspend in 250 μL of PBS solution containing 0.1% BSA.

[0127] 5. Add 0.5 μL of Alexa Fluor 647-donkey anti-rabbit fluorescent secondary antibody to the EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, resuspend in 300 μL of PBS solution and detect using a fluorescence microscope and a flow cytometer.

[0128] 6. After the flow cytometry test results are obtained, evaluate the kidney condition of the test samples according to TSPAN4 and screen for subjects who may have kidney damage.

[0129] 7. Obtain the clinical pathological diagnosis information of the subjects from the hospital, compare the consistency between the kidney damage condition judged by TSPAN4 fluorescence and the pathological diagnosis results, and observe whether urinary migrasomes can be used as markers for the diagnosis of kidney diseases.

[0130] Table 1 Pathological information of normal subjects (CTL) and patients with chronic kidney disease (CKD)

[0131]

[0132]

[0133] Result 1.1

[0134] The test results are as Figure 1 shown in A. There are basically no migrasomes in the urine of normal subjects, while the urine of CKD patients contains abundant migrasomes. Serum creatinine, an index commonly used to judge chronic kidney disease, is also elevated in CKD patients ( Figure 1 B), and the estimated glomerular filtration rate (eGFR) decreases ( Figure 1 C).

[0135] Further analysis of the receiver operating characteristic curve (ROC curve) of the subjects found that the area under the ROC curve for differentiating CKD patients from normal subjects by urinary migrasomes was 0.9591, the area under the ROC curve for differentiating CKD patients from normal subjects by serum creatinine was 0.5814, and the area under the ROC curve for differentiating CKD patients from normal subjects by eGFR was 0.5725 ( Figure 1 D).

[0136] It can be seen from this that diagnosing chronic kidney disease by migrasomes is more accurate than the existing serum creatinine and eGFR.

[0137] Result 1.2

[0138] The collected CKD patients included those with diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy (IgA), and chronic kidney disease patients due to other reasons (other).

[0139] Furthermore, comparing different types of CKD, consistent with Result 1.1, the content of migrasomes in the urine of patients with diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy (IgA), and chronic kidney disease patients due to other reasons (other) was significantly increased compared with that of normal people (NOR), while there was not much difference among the various diseases ( Figure 2 A).

[0140] Serum creatinine, an index commonly used to judge chronic kidney disease at present, was also increased in various CKD patients ( Figure 2 B), and the estimated glomerular filtration rate (eGFR) decreased ( Figure 2 C).

[0141] The results of the receiver operating characteristic curve showed that the areas under the ROC curves for differentiating DN patients, MN patients, IgA patients, chronic kidney disease patients due to other reasons (other), and normal people by urinary migrasomes were 0.8831, 0.9438, 0.9851, 0.9642, and 0.8492, respectively ( Figure 2 D).

[0142] Result 1.3

[0143] The results of flow cytometry detection were as Figure 3 shown in A. The content of migrasomes in the urine of CKD patients increased from stage I to stage II of CKD, while it showed a downward trend from stage II to stage V, but was higher than that of normal people. Since the vast majority of migrasomes in urine come from podocytes, and podocytes will be slowly lost during the disease progression of CKD patients, the change in the content of migrasomes is basically consistent with the change of podocytes, the most important functional cells in the kidney, in CKD.

[0144] When CKD stage I patients who are almost impossible to accurately diagnose clinically were compared with normal people alone, it was found that urinary migrasomes were significantly increased in CKD stage I patients ( Figure 3 B), while serum creatinine and eGFR showed almost no change in CKD stage I patients and normal people ( Figure 3 C-D).

[0145] Further analysis of the ROC curve found that the area under the ROC curve for differentiating CKD-I and NOR by urinary migrasomes was 0.9516, the area under the ROC curve for differentiating chronic kidney disease and normal people by serum creatinine was 0.6945, and the area under the ROC curve for differentiating chronic kidney disease and normal people by eGFR was 0.7600Figure 3 E).

[0146] Since the estimated glomerular filtration rate (eGFR) in normal individuals is (80 - 125) mL / min / 1.73 m 2 . We selected patients with eGFR ≥ 80 among CKD patients for comparison and analysis with normal individuals. The analysis results showed that migrasomes were significantly increased in CDK patients ( Figure 3 F), while serum creatinine and eGFR showed almost no change between CKD stage I patients and normal individuals ( Figure 3 G - H). Further analysis of the ROC curve found that the area under the ROC curve for urine migrasomes to distinguish patients with eGFR ≥ 80 among CKD patients from normal individuals (NOR) was 0.9535, the area under the ROC curve for serum creatinine to distinguish patients with eGFR ≥ 80 among CKD patients from normal individuals (NOR) was 0.6530, and the area under the ROC curve for eGFR to distinguish patients with eGFR ≥ 80 among CKD patients from normal individuals (NOR) was 0.6718 ( Figure 3 I).

[0147] It can be seen from this that urine migrasomes can be used as biomarkers for the early diagnosis of chronic kidney disease.

[0148] Result 1.4

[0149] In order to compare the advantages and disadvantages of migrasomes and serum creatinine in differentiating chronic kidney disease (CKD), the present invention selected patients with serum creatinine within the normal range of 0.50 - 1.50 mg / dl among CKD patients for analysis.

[0150] The analysis results showed that migrasomes were significantly increased in CKD patients ( Figure 4 A), while serum creatinine and eGFR showed almost no change between CKD stage I patients and normal individuals ( Figure 4 B - C).

[0151] Further analysis of the ROC curve found that the area under the ROC curve for urine migrasomes to distinguish patients with normal serum creatinine among CKD patients from normal individuals was 0.9195, the area under the ROC curve for serum creatinine to distinguish patients with normal serum creatinine among CKD patients from normal individuals was 0.5058, and the area under the ROC curve for eGFR to distinguish patients with normal serum creatinine among CKD patients from normal individuals was 0.5220 ( Figure 4 D).

[0152] It can be seen from this that urine migrasomes can be used as biomarkers for the early diagnosis of chronic kidney disease.

[0153] Result 1.5

[0154] To further identify the changes in the levels of urinary migrasomes in the urine of patients with different types of CKD, we analyzed the levels of urinary migrasomes in patients with diabetic nephropathy (DN), membranous nephropathy (MN), and IgA nephropathy (IgA) at different stages.

[0155] The results were as Figure 5 shown in A-5C. Consistent with the previous results, during the progression from stage I to stage II in patients with DN, MN, and IgA, the levels of urinary migrasomes increased, while during the stage from II to V (ESRD), they decreased due to the loss of podocytes.

[0156] The diagnosis of different types of chronic kidney disease has always been a clinical challenge. Analyzing the ROC curve, it was found that the working curves for differentiating early-stage patients with DN, MN, and IgA nephropathy (DN-I, MN-I, IgA-I) using urinary migrasomes were 0.9987, 0.9587, and 0.9891 ( Figure 5 D).

[0157] The above results indicate that urinary migrasomes can be used as early diagnostic markers for different types of chronic kidney disease.

[0158] Example 2 Detection of migrasomes in urine by chemiluminescence as a marker of kidney injury

[0159] 1. Purchase NHS magnetic beads with a particle size of 2 μm (Solarbio, catalog number M2450). Take 500 μL of the magnetic bead suspension into a 1.5 mL EP tube. Place the EP tube in a magnetic separation rack to enrich the magnetic beads and remove the supernatant. Add 1 mL of pre-cooled Washing Buffer A at 4 °C to the 1.5 mL EP tube and vortex for 15 s to mix the magnetic beads evenly. Place the EP tube in the magnetic separation rack to enrich the magnetic beads and remove the supernatant.

[0160] 2. After dialysis, dissolve the TSPAN4 antibody (abcam, catalog number ab181995, rabbit source) with Coupling Buffer to prepare a protein solution with a concentration of 3.0 mg / mL. Add 500 μL of the prepared TSPAN4 antibody solution to the EP tube in the first step and vortex for 30 s to mix it evenly. Vortex the EP tube for 15 s and place it on a mixer to mix at room temperature for 2 h. If the mixture is not uniform, then within the first 30 min, take down the EP tube and vortex for 15 s every 5 min. Thereafter, take down the EP tube and vortex for 15 s every 15 min.

[0161] 3. Place the EP tube in the magnetic separation rack to enrich the magnetic beads and remove the supernatant. Add 1 mL of Blocking Buffer to the EP tube and vortex for 30 s. Place the EP tube in the magnetic separation rack to enrich the magnetic beads and discard the supernatant.

[0162] 4. Repeat step 3 four times. Add 1 mL of Blocking Buffer to the EP tube, vortex for 30 seconds, and incubate the tube in a mixer at room temperature for 2 hours. Place the tube in a magnetic separation rack to collect the magnetic beads and discard the supernatant. Add 1 mL of ultrapure water to the tube, mix thoroughly, collect the magnetic beads using a magnetic rack, and discard the supernatant.

[0163] 5. Add 1 mL of PBS (pH 7.2) to the EP tube, mix thoroughly, collect the beads using a magnetic rack, and discard the supernatant. Repeat this procedure twice, then resuspend the beads in 500 μL of PBS, mix thoroughly, and store at 4°C until used. Note: The final concentration of the protein-coupled beads is 10 mg / mL.

[0164] 6. Urine samples were randomly collected from volunteers at the hospital's physical examination center and nephrology department. Within 24 hours, the samples were centrifuged at 4000g for 20 minutes at 4°C to remove cellular debris. 1 mL of urine was added with 5 μL of magnetic beads conjugated to TSPAN4 antibody (abcam, cat. no. ab181995, rabbit source) and incubated at room temperature on a mixer for 1 hour.

[0165] 7. Place the EP tube on a magnetic separation rack to remove the supernatant. Wash three times with 1000 μL of PBS solution containing 0.1% BSA (pH 7.2). Resuspend in 100 μL of PBS solution containing 1% BSA and incubate at room temperature for 30 minutes.

[0166] 8. Then place the EP tube in a magnetic separation rack to collect the magnetic beads, discard the supernatant, and resuspend in 100 μL of PBS solution containing 0.1% BSA.

[0167] 9. Add 5 μL of anti-podocalyxin antibody (recombinant anti-PODXL antibody [PcMab-47] (ab264542), mouse monoclonal antibody, purchased from Abcam) to the beads. Incubate on a mixer at room temperature for 1 hour. Then, place the EP tube in a magnetic separation rack to collect the beads and discard the supernatant. Wash three times with 1000 μL of PBS containing 0.1% BSA and resuspend in 250 μL of PBS containing 0.1% BSA.

[0168] 10. Add 5 μL of the secondary antibody, horseradish peroxidase-conjugated anti-mouse monoclonal antibody (Goat Anti-Mouse IgG H&L (HRP) (ab205719)), to the EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, and discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, add 0.1 ml of TMB substrate solution to the EP tube and react at 37 °C for 30 min (Substrate chromogenic solution A: 13.6 g of sodium acetate, 1.6 g of citric acid, 0.3 ml of 30% hydrogen peroxide, add distilled water to 500 ml. Substrate chromogenic solution B: 0.2 g of disodium ethylenediaminetetraacetate, 0.95 g of citric acid, 50 ml of glycerol, 0.15 g of 3,3',5,5'-tetramethylbenzidine (TMB), add distilled water to 500 ml. Mix solution A and solution B at a ratio of 1:1 before use to obtain the TMB substrate solution).

[0169] 11. Add 0.05 ml of 2 M sulfuric acid to the EP tube to terminate the reaction. Then use a detector to measure the OD value at 450 nm.

[0170] Results

[0171] The results showed that urinary exosomes could be detected by chemiluminescence method, and the content of urinary exosomes in CKD patients was significantly higher than that in normal people ( Figure 7 A).

[0172] Further analysis of the receiver operating characteristic curve found that the area under the ROC curve was 0.9200 when urinary exosomes were used to distinguish chronic kidney disease (CKD) from normal people (NOR) ( Figure 7 B).

[0173] It can be seen from this that the exosomes detected by chemiluminescence method can be used as a biomarker for the diagnosis of chronic kidney disease.

[0174] Example 3 Use of TSPAN4-conjugated magnetic beads to capture exosomes in urine as a biomarker for the detection of autoantibodies in membranous nephropathy

[0175] 1. Repeat steps 1-8 in Example 2.

[0176] 2. Add 5 μL of goat anti-mouse IgG H&L antibody (Proteintech, CoraLite594–conjugated Goat Anti-Mouse IgG(H+L), Cat No.SA00013-3) and 5 μL of goat anti-human IgG H&L antibody (Proteintech, Fluorescein(FITC)–conjugated Affinipure Goat Anti-Human IgG(H+L), Cat No.SA00003-12) into an EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, add 1000 μL of PBS solution containing 0.1% BSA to the EP tube and wash 3 times, then resuspend in 300 μL of PBS solution, and detect using a fluorescence microscope and a flow cytometer.

[0177] 3. After the flow cytometry detection, judge the expression level of autoantibodies in the tested patients with membranous nephropathy according to the fluorescence intensity (FITC fluorescence intensity) of human autoantibodies (anti-phospholipase A2 receptor (PLA2R), thrombospondin 7A (THSD7A)) on the surface of podocyte-derived migrasomes.

[0178] 4. Obtain the clinical pathological diagnosis information of the subjects from the hospital. Compare the content of autoantibodies detected by the hospital with the content of autoantibodies detected by urine migrasomes using a flow cytometer, and observe whether urine migrasomes can be used as a marker for the diagnosis of autoantibodies in membranous nephropathy.

[0179] Results

[0180] Thirty-eight patients with primary membranous nephropathy were analyzed in the present invention, and the specific pathological information is shown in Table 2.

[0181] The analysis results showed that urine podocyte-derived migrasomes could distinguish autoantibodies ( Figure 8 A).

[0182] The results of the receiver operating characteristic curve showed that the area under the ROC curve was 0.9200 when urine migrasomes distinguished between autoantibody-positive and autoantibody-negative patients ( Figure 8 B), and the concentration of autoantibodies measured by urine migrasomes was positively correlated with the concentration of autoantibodies measured by ELISA, and the linear correlation coefficient R 2 = 0.8576 ( Figure 8 C).

[0183] It can be seen from this that the migrasomes in urine captured by TSPAN4-conjugated magnetic beads can be used as a marker for the detection of autoantibodies in membranous nephropathy.

[0184] Table 2 Pathological information of patients with primary membranous nephropathy

[0185]

[0186]

[0187] Discussion

[0188] Liquid biopsy is a new technology for diagnosing and dynamically observing diseases by monitoring substances such as proteins, DNA, and RNA in cells or tissues in a pathological state in body fluids such as blood, saliva, and urine. Currently, the four main sources of biomarkers for liquid biopsy are circulating tumor cells (CTC), circulating tumor DNA (ctDNA), circulating RNA, and extracellular vesicles. Migrasomes are single-membrane vesicle structures with a diameter of 0.5 - 2 μm produced by the contractile filaments at the cell tail during cell directed migration. Migrasomes contain a large number of bioactive substances such as nucleic acids, proteins, and fats, play an important role in transmitting signals between cells, mediating intercellular communication, and are involved in and regulate a variety of physiological and pathological activities. Studies have shown that podocytes and other kidney-specific cells that make up the kidney will release a large number of migrasomes into urine during kidney injury, and the proteins and nucleic acids in urinary migrasomes will change accordingly with the type and severity of kidney injury.

[0189] Membranous nephropathy is one of the most common pathological manifestations of adult nephrotic syndrome and is the second leading cause of primary glomerular diseases in China. Approximately 70% of patients with membranous nephropathy present with persistent and recurrent proteinuria, which can cause kidney damage and endanger life. Approximately 1 / 3 of patients with membranous nephropathy can spontaneously remit and gradually improve, and about 1 / 3 of patients will develop end-stage renal disease after 5 - 10 years, while the remaining patients present with persistent proteinuria.

[0190] Membranous nephropathy can be divided into two categories: idiopathic and secondary. Among them, idiopathic membranous nephropathy (IMN) accounts for 70% - 80%. Idiopathic membranous nephropathy mainly refers to the change in the glomerular basement membrane without a clear cause; secondary membranous nephropathy (SMN) accounts for 20% - 30%, and is generally secondary to autoimmune diseases such as systemic lupus erythematosus (SLE), infections (hepatitis B / C virus infection), malignant tumors, and drug poisoning. The pathogenesis and etiology of IMN are not yet clear, and there is currently a lack of specific laboratory indicators for diagnosis.

[0191] It has been found that autoantibodies against podocyte proteins such as anti-phospholipase A2 receptor (PLA2R) and thrombospondin 7A (THSD7A) are detected in the sera of adult patients with IMN, while autoantibodies against podocytes such as serum PLA2R and THSD7A are rarely found in patients with secondary membranous nephropathy and other glomerular diseases. Therefore, autoantibodies against podocyte proteins such as PLA2R and THSD7A in serum can be used as biomarkers for the diagnosis of membranous nephropathy.

[0192] Currently, there are already kits that use synthetic proteins such as PLA2R and THSD7A as antigens to detect autoantibodies by chemiluminescence. However, due to the large molecular weights of proteins such as PLA2R and THSD7A and the presence of multiple repetitive domains, in vitro synthesis is difficult and costly. Even more troublesome is that each protein needs to be synthesized separately and a detection method needs to be designed for detection. This is not only cumbersome to operate but also costly. The migrasomes derived from urine podocytes contain most of the proteins on the surface of podocytes. Therefore, the migrasomes derived from urine podocytes of patients with membranous nephropathy can be used as antigens for autoantibody detection.

[0193] Combined with the urine migrasome capture detection method developed in the present invention, the inventors have developed a detection method for autoantibodies in membranous nephropathy, such as capturing migrasomes in urine based on TSPAN4-conjugated magnetic beads ( Figure 9 ).

[0194] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A use of a detection agent for a migrator, characterized in that: For preparing a detection reagent or a kit for evaluating whether a subject has chronic kidney disease or is susceptible to chronic kidney disease, Wherein, the chronic kidney disease is selected from the group consisting of membranous nephropathy (MN), diabetic nephropathy (DN) and IgA nephropathy (IgA).

2. The use according to claim 1, characterized in that The object is an object that meets the following conditions: (Z1) estimated glomerular filtration rate (eGFR) of 80 to 125 mL / min / 1.73 m 2 , preferably 85-110 mL / min / 1.73 m 2 , more preferably 90-105 mL / min / 1.73 m 2 , and / or (Z2) Serum creatinine level is 0.50-1.50 mg / dl, preferably 0.75-1.25 mg / dl.

3. The use according to claim 1, characterized in that, The detection includes urine detection, serum detection, platelet detection, and detection of tissue or cell samples.

4. The use according to claim 1, characterized in that, The detection reagents include specific binding molecules of migratory bodies, specific amplification primers, probes or chips.

5. The use according to claim 4, characterized in that, The specific binding molecules include specific antibodies and solid phase carriers with lectins coupled or bound to the surface.

6. The use according to claim 1, wherein The detection includes: flow cytometry, fluorescence imaging technology, and fluorescence immunoassay reading technology.

7. A detection kit, characterized in that, The kit contains: (a) a detection reagent, wherein the detection reagent comprises: (a1) a first detection reagent for detecting migrating bodies; (a2) a second detection reagent for detecting eGFR; and (a3) optionally a third detection reagent for detecting blood creatinine; (a4) optionally a fourth detection reagent for detecting autoantibodies against podocyte proteins such as PLA2R and THSD7A; (b) a label or instructions indicating that the kit is used to assess a subject's susceptibility to chronic kidney disease.

8. The detection kit according to claim 7, characterized in that, The label or instructions shall state the following: (i) when the number of migratory bodies in the urine of the test subject is greater than 1390 MFI (mean fluorescence intensity), it indicates that the test subject has a high risk of developing chronic kidney disease; and (ii) When the number of migratory bodies in the urine of the test subject is less than 1390 MFI (mean fluorescence intensity), it indicates that the risk of the test subject developing chronic kidney disease is low.

9. A method for assessing whether a subject has chronic kidney disease or is susceptible to chronic kidney disease, characterized in that: The method comprises: (a) providing a test sample from a subject, The subject meets the following conditions: (Z1) eGFR is 80–125 mL / min / 1.73 m 2 , preferably 85-110 mL / min / 1.73 m 2 , more preferably 90-105 mL / min / 1.73 m 2 ; and / or (Z2) serum creatinine level of 0.50 to 1.50 mg / dl, preferably 0.75 to 1.25 mg / dl; (b) detecting the amount of migratory bodies in the test sample; and (c) comparing the amount of migrating bodies determined in step (b) with a control reference value, Wherein, when the content of the migrator in the sample is higher than the control reference value, it indicates that the subject has a high risk of suffering from chronic kidney disease.

10. A diagnostic device, characterized in that, The diagnostic equipment includes: (a) an input module configured to input the content of migratory bodies in a test sample of a subject; wherein the subject is a subject that satisfies the following conditions: (Z1) The eGFR is 80 to 125 mL / min / 1.73 m 2 , preferably 85 to 110 mL / min / 1.73 m 2 , more preferably 90 to 105 mL / min / 1.73 m 2 ; and / or (Z2) serum creatinine level of 0.50 to 1.50 mg / dl, preferably 0.75 to 1.25 mg / dl; (b) a chronic kidney disease diagnosis-chronic kidney disease typing module, wherein the chronic kidney disease diagnosis-chronic kidney disease typing module is configured to: diagnose and analyze whether the subject has chronic kidney disease based on the content of the migrator, and / or perform typing analysis on chronic kidney disease, and obtain diagnostic analysis results and / or typing analysis results; and (c) an output module, wherein the output module is configured to output the diagnostic analysis result and / or typing analysis result.

Images