WO2024041257A1 - Method for diagnosing chronic kidney disease by means of detecting urinary migrasome - Google Patents

Abstract

A method for diagnosing chronic kidney disease by means of detecting urinary migrasome. Specifically, provided is the use of a detection agent of the migrasome for the preparation of a detection reagent or a kit, wherein the detection reagent or the kit is used for evaluating whether an object has chronic kidney disease or a susceptibility to chronic kidney disease. Further provided are a corresponding detection kit and diagnostic equipment. Research shows that the migrasome used has high sensitivity and specificity, can be used as a marker for the risk of developing chronic kidney disease and is used for the auxiliary examination and early diagnosis of chronic kidney disease.

Description

A method for diagnosing chronic kidney disease by detecting urinary migrants Technical field

The invention belongs to the field of clinical medicine, and specifically relates to a method for judging chronic kidney disease by detecting urine migration bodies.

Background technique

The kidney is an important organ of the human body. It is not only responsible for the formation of urine, but also has many functions such as removing and expelling toxins from the body, reabsorbing nutrients, and secreting hormones. Once there is a problem with the kidneys, it will not only destroy the original functions of the kidneys, but also cause dysfunction in multiple systems such as the digestive tract, respiratory system, cardiovascular system, and nervous system, seriously reducing the patient's quality of life and even causing Danger to life.

According to epidemiological statistics, the number of chronic kidney disease cases worldwide exceeds 850 million, resulting in more than two million deaths every year. China is one of the countries with the highest incidence of chronic kidney disease. The incidence rate of chronic kidney disease is as high as 10.8%, and the number of patients with chronic kidney disease exceeds 130 million. In addition, due to environmental pollution, population aging and other factors, the incidence of chronic kidney disease is increasing at a rate of 10% year by year.

Currently, the most important biological indicators for diagnosing chronic kidney disease include serum creatinine, urea nitrogen, and related glomerular filtration rate (eGFR) and urine protein levels. However, these indicators are affected by many factors, such as gender, age, dietary habits and infection status, and cannot effectively reflect the degree of kidney damage and fibrosis in the early stages of kidney disease. At present, the diagnosis of kidney disease still relies on the pathological and histological test results obtained by renal puncture biopsy. Although this classic detection method can clarify the pathological type and degree of kidney damage, it has many limitations, such as for patients with severe disease. For patients, or patients who need follow-up, renal biopsy is not a reproducible diagnostic method; especially as an invasive procedure with multiple risks of complications, it is not clinically suitable for large-scale disease screening. check. In addition, this examination also requires expensive pathological testing equipment and specially trained technicians. Since kidney-related diseases have a long course and are expensive to treat, once kidney disease enters the irreversible stage, patients not only need to take long-term medication to maintain life, which greatly affects their quality of life, but may also lose their ability to work. This will not only increase the patient's burden The treatment burden will cause patients to become "poor due to illness" and will also greatly increase the country's social medical costs. In addition, when eGFR and serum creatinine levels are within the normal range, it is impossible to detect whether the subject has chronic kidney disease or is susceptible to chronic kidney disease. Therefore, the market is in urgent need of methods and means for early diagnosis and dynamic detection of chronic kidney disease.

In summary, there is an urgent need in this field to develop a convenient, fast, accurate, and efficient method for determining whether a subject suffers from chronic kidney disease by detecting urinary migrants. This method can be used not only for the diagnosis of chronic kidney disease, but also for the judgment of efficacy during the treatment of chronic kidney disease.

Contents of the invention

The purpose of the present invention is to provide a convenient, fast, accurate and efficient method for determining whether a subject suffers from chronic kidney disease by detecting urine migrants.

In a first aspect of the present invention, there is provided the use of a migratory detection agent for preparing detection reagents or kits for evaluating whether a subject suffers from chronic kidney disease. (CKD) or susceptibility 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).

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

In another preferred example, the object is an object that meets the following conditions:

(Z1) eGFR is 80～125mL/min/1.73m ² , preferably 85～110mL/min/1.73m ² , more preferably 90～105mL/min/1.73m ² , and/or

(Z2) The blood creatinine level is 0.50～1.50mg/dl, preferably 0.75～1.25mg/dl.

In another preferred embodiment, the subject is a person who has chronic kidney disease in his family or has had chronic kidney disease.

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

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

In another preferred embodiment, the subject is a patient in CKD stage I.

In another preferred embodiment, the detection reagent or kit is also used to classify chronic kidney disease.

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

In another preferred embodiment, the detection is urine detection.

In another preferred embodiment, the migration body is a urine migration body.

In another preferred embodiment, the cells are renal podocytes.

In another preferred embodiment, the detection reagent includes a specific binding molecule of the migratory body, a specific amplification primer, a probe or a chip.

In another preferred embodiment, the specific binding molecule includes a specific antibody and a solid-phase carrier coupled or bound to a lectin on its surface.

In another preferred embodiment, the specific antibody is selected from the following group: anti-TSPAN4 antibody, anti-Integrinα5β1 antibody, PIGK antibody, NDST1 antibody, CPQ antibody, EOGT antibody or a combination thereof.

In another preferred embodiment, the solid phase carrier includes: solid particles, microfluidic chips, glass cellulose membranes, nylon membranes, and agarose beads.

In another preferred embodiment, the solid phase carrier includes magnetic beads or non-magnetic microspheres.

In another preferred embodiment, the lectin is coupled to the surface of the solid carrier through chemical bonds.

In another preferred example, the lectin is selected from the following group: wheat germin (WGA), concana lectin (ConA), Peanut agglutinin (PNA), or combinations thereof.

In another preferred embodiment, the specific antibody or specific binding molecule is coupled to or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the detection includes: flow cytometry, fluorescence imaging technology, and fluorescence immunoreading technology.

In a second aspect of the present invention, a detection kit is provided, said kit containing:

(a) Detection reagents, wherein the detection reagents include:

(a1) A first detection reagent for detecting migrating bodies;

(a2) A second detection reagent for detecting eGFR;

(a3) a third detection reagent optionally used to detect blood creatinine; and

(a4) A fourth detection reagent optionally used for detecting PLA2R, THSD7A and other autoantibodies against podocyte proteins;

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

In another preferred embodiment, the detection kit is also used to classify chronic kidney disease.

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

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

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

In another preferred embodiment, the typing includes dividing chronic kidney disease into specific and secondary.

In another preferred embodiment, the detection reagent for detecting migrant antibodies or proteins includes specific antibodies against migrant proteins.

In another preferred embodiment, the detection is blood sample detection and/or serum sample detection.

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

When the ratio of the number of migrants in the urine of the test subject to the number of migrants in normal people is ≥ 1.5, and preferably ≥ 2, it indicates that the risk of developing chronic kidney disease in the subject is high.

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

(i) When the number of migrants in the urine of the subject is >1390 MFI (mean fluorescence intensity), it indicates that the subject has a high risk of developing chronic kidney disease; and

(ii) When the number of migrants in the urine of the subject is <1390MFI (mean fluorescence intensity), it indicates that the subject has a low risk of developing chronic kidney disease.

In another preferred embodiment, the method for detecting the number of migrants is as described in Example 1.

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

(a) provide a test sample from the subject,

Wherein the subject meets the following conditions:

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

(Z2) Serum creatinine level is 0.50～1.50mg/dl, preferably 0.75～1.25mg/dl;

(b) detect the number of migrants in the test sample; and

(c) Compare the content of the migrant measured in step (b) with the control reference value,

Wherein, when the content of migrating bodies 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.

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

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

In another preferred example, the control reference value is a cut-off value.

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

(a) Input module, the input module is configured to input the content of migrants in the test sample of the subject; wherein the subject is an object that meets the following conditions:

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

(Z2) Serum creatinine level is 0.50～1.50mg/dl, preferably 0.75～1.25mg/dl;

(b) Chronic kidney disease diagnosis-chronic kidney disease typing module, the chronic kidney disease diagnosis-chronic kidney disease typing module is configured to: based on the content of the migrants, determine whether the subject suffers from chronic kidney disease. Diagnostic analysis, and/or typing analysis of chronic kidney disease, and obtaining diagnostic analysis results and/or typing analysis results; and

(c) Output module, the output module is configured to output the diagnostic analysis results and/or typing analysis results.

In another preferred embodiment, the diagnostic device further includes (d) a detection module configured to detect the number of migrating bodies.

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

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

Description of drawings

Figure 1 shows the migratory body content in urine of patients with chronic kidney disease. Among them, Figures A-C show the contents of urinary migrants, serum creatinine and eGFR in normal people (NOR) and chronic kidney disease (CKD) patients respectively. Figure D shows the urine migrant (red line), serum creatinine (blue Line) and eGFR (purple line) ROC curves in distinguishing normal people from chronic kidney disease.

Figures 2A-2C respectively show the contents of migratory bodies, serum creatinine and eGFR in the urine of normal people, diabetic nephropathy, membranous nephropathy, IgA nephropathy, lupus nephritis, purpura nephritis, renal minimal change disease and patients with chronic kidney disease due to other causes. . Figure 2D shows the role of urine migrants (red line) in distinguishing normal individuals from patients with diabetic nephropathy, membranous nephropathy, IgA nephropathy, lupus nephritis, purpura nephritis, renal minimal change disease, and patients with chronic kidney disease (other). ROC curve.

Figure 3 shows the urinary migratory content of CKD patients at different stages. Figure 3A shows the urinary migratory content of normal people and CKD patients at different stages. Figures 2B-2D show the urinary migratory contents of normal people and CKD-stage I patients respectively. Contents of migrating bodies, serum creatinine and eGFR. Figure 2E shows the ROC curves of urine migrants (red line), serum creatinine (blue line), and eGFR (purple line) in distinguishing normal people from CKD-stage I patients.

Figures 4A-4C show the contents of migratory bodies, eGFR and serum creatinine in normal subjects and chronic kidney disease patients with normal serum creatinine, respectively. Figure 4D shows the ROC curve of urinary migrants in distinguishing normal people from patients with different types of CKD stage I.

Figure 5A shows the migratory body content in the urine of normal subjects, DN-I stage-IV and DN-ESRD stage patients. Figure 5B shows the migratory body content in the urine of normal people and MN stage I-IV patients. Figure 5C shows the migratory body content in the urine of normal people and IgA stage I-IV patients. Figure 5D shows the working curve for urinary migrants to distinguish DN-I stage, MN-I stage, and IgA-I stage.

Figure 6 shows a schematic diagram of the chemiluminescence method for detecting urinary podocyte-derived migratory bodies.

Figure 7A shows the content of migrating bodies in the urine of chronic kidney disease patients and normal people, expressed as OD values. Figure 7B shows the ROC curve of migratory bodies in distinguishing normal people from chronic kidney disease.

Figure 8A shows the autoantibody fluorescence signal intensity (FITC) in autoantibody-positive (positive) and autoantibody-negative (negative) migrants. Figure 8B shows the ability of the migrants to differentiate between autoantibody-positive (positive) and autoantibody-negative (negative) migrants. ROC curve for patients, Figure 8C shows the linear relationship between the concentration of membranous nephropathy autoantibodies (Autoantibody concentration) detected by ELISA and the autoantibody concentration (Fluorescence intensity) detected by urine migration.

Figure 9 shows the experimental flow chart of using WGA-coupled magnetic beads to capture migratory bodies in urine as markers for membranous nephropathy autoantibody detection.

Detailed ways

After extensive and in-depth research, the inventor unexpectedly developed a highly sensitive and highly specific marker for detecting specific chronic kidney disease for the first time. Specifically, the present invention will be able to specifically recognize the surface of migrating bodies The lectin-coupled solid-phase carrier of the protein is mixed with the sample to be tested, and the solid-phase carrier is used to efficiently capture the migrants in the sample to be tested. The migrants are then labeled with a migrant-specific antibody to identify the number of migrants. .

The diagnosis of chronic kidney disease by the migratory body of the present invention is more accurate than the existing blood creatinine and eGFR. On this basis, the present invention was completed.

the term

The term "sample" or "sample" as used herein refers to material specifically associated with a subject from which specific information about the subject can be determined, calculated or inferred. The sample may consist in whole or in part of biological material from the subject.

As used herein, the terms "marker of chronic kidney disease of the present invention" and "marker of chronic kidney disease" both refer to "migrant".

As used herein, the term "reference value" refers to a value that is statistically related to a specific result when compared to the analytical result. In a preferred embodiment, the reference value is determined based on statistical analysis of studies comparing the number of migrants with known clinical outcomes. In the Examples of this article, some of these studies are shown in part. 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 may also be determined by considering conditions and outcomes that are particularly relevant to the patient's medical history, genetics, age, and other factors.

In the present invention, the reference value refers to the cut-off value, preferably 1390 MFI (mean fluorescence intensity, used to represent the number of migrants in the serum of patients with chronic kidney disease).

In a preferred embodiment, the method for detecting the number of migrants of the present invention is as described in Example 1.

kidney disease

With reference to the World Health Organization (WHO) 1995 histological classification scheme for glomerular diseases, 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 types I and III (MPGN I & III), endocapillary proliferative glomerulonephritis (EnPGN), C3 glomerulonephritis Glomerulonephritis, dense deposit disease (DDD), etc.; (2) Secondary glomerulonephritis (SGN), which is mainly divided into immune-mediated diseases, tumor metabolic diseases and infectious diseases, among which immune-mediated diseases include lupus Nephritis (LN), Henoch-Schönlein purpura nephritis (HSPN), vasculitis renal damage, Sjogren's syndrome-related renal damage, anti-glomerular basement membrane nephropathy, rheumatoid arthritis-related renal damage, etc., and tumor metabolic diseases include Diabetic nephropathy (DN), hypertensive renal damage, obesity-related renal damage (ORG), renal amyloidosis, monoclonal immunoglobulin deposition disease (MIDD), etc. Infectious diseases include hepatitis B-related nephritis and epidemic hemorrhagic fever Renal damage, hepatitis C-related nephritis and other diseases; (3) Tubulointerstitial diseases (TIN) include acute interstitial nephritis (AIN), chronic interstitial nephritis (CIN), acute tubular necrosis (ATN), Aristolochia Acid nephropathy, Bartter's syndrome, Reflux nephropathy, Gitelman's syndrome, etc.; (4) Hereditary kidney diseases include thin basement membrane nephropathy (TNMN), Alport syndrome, lipoprotein nephropathy (LPG), Fabry's disease, etc.; (5) Other unknown diagnoses or unclassifiable cases.

Migrants

As described in this article, migrasomes are single-layer membrane vesicle structures with a diameter of 0.5 to 2 μm produced by contractile filaments at the cell tail during directional cell migration. Migrants contain a large number of biologically active substances such as nucleic acids, proteins, fats, etc., which play an important role in the intercellular communication process and participate in and regulate a variety of physiological and pathological activities. Migrants are found in blood and various body fluids, such as serum, urine, etc. The content of migrants in some blood or body fluids is closely related to certain diseases (such as diabetic nephropathy, etc.).

glomerular filtration rate (eGFR)

As described in this article, glomerular filtration rate (eGFR) refers to the ability of two kidneys to produce filtrate per unit time (usually 1 min). A normal adult is (80-125) mL/min/1.73m ² . GFR is the main indicator for evaluating renal function and the main basis for the diagnosis and staging of chronic kidney disease.

Glomerular filtration rate (eGFR) is an important parameter used to judge kidney function. According to the glomerular filtration rate, CKD patients are divided into five stages: eGFR ≥ 90 is CKD stage I; 60 ≤ eGFR ≤ 89 is CKD stage II; 30 ≤ eGFR ≤ 59 is CKD stage III; 15 ≤ eGFR ≤29 is CKD-IV stage; eGFR<15 is CKD-V stage, that is, end-stage renal disease (ESRD).

Serum creatinine

As mentioned in this article, creatinine is a product of muscle metabolism in the human body. Every 20g of muscle metabolism can produce 1 mg of creatinine. Creatinine is mainly excreted outside the body through glomerular filtration. A creatinine value that is higher than the normal value is called high creatinine. Creatinine includes blood creatinine and urine creatinine.

Serum creatinine is commonly used to measure kidney function. Generally speaking, the normal value of blood creatinine is: 0.50-1.50 mg/dl. When blood creatinine exceeds 1.50 mg/dl, it means kidney damage, renal insufficiency, and renal failure. Above 1.50mg/dl is the inflammatory injury stage, 2.10mg/dl is the renal damage stage, 5.10mg/dl is the renal failure stage, and serum creatinine value exceeding 8.00mg/dl indicates late stage (uremia).

Detection method

Based on the content of chronic kidney disease risk marker migrants in tissue samples or urine samples, the present invention also provides corresponding methods for determining specific chronic kidney disease.

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

In addition, the migrating body capture carrier of the present invention can efficiently and specifically capture migrating bodies in the sample. Flow cytometry can quickly, easily and accurately detect the type and number of migrants in a sample.

In a preferred example, the inventors constructed a lectin-coupled solid-phase carrier that can specifically recognize migratory surface proteins, and used the solid-phase carrier to efficiently capture peptides in various body fluids such as cell culture fluid, urine, and blood. Migrants are then labeled by migratory-specific antibodies to identify the migratory type and content.

Detection kit

Based on the correlation between chronic kidney disease risk markers and the occurrence of specific chronic kidney disease, chronic kidney disease risk markers can be used as markers to determine the risk of specific chronic kidney disease.

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

(a1) A first detection reagent for detecting migrating bodies;

(a2) A second detection reagent for detecting eGFR;

(a3) a third detection reagent optionally used to detect blood creatinine; and

(a4) A fourth detection reagent optionally used for detecting autoantibodies against podocyte proteins such as PLA2R and THSD7A.

In another preferred embodiment, the kit further includes a label or instructions.

diagnostic equipment

Based on the method provided by the present invention for assessing a subject's susceptibility to chronic kidney disease, the present invention also provides corresponding diagnostic equipment.

In the present invention, the content of the migrating body in the subject's test sample can be input using a manual input method or an automated collection method. Typically, the migratory body content input module is selected from the following group: an immune cell typing collector, a scanner, a keyboard, a tablet computer (PAD), a smart phone, or a combination thereof.

Preferably, in the present invention, the chronic kidney disease diagnosis-chronic kidney disease classification module is configured to: perform a diagnostic analysis on whether the subject suffers from chronic kidney disease based on the content of the migratory body, and/ Or perform typing analysis on chronic kidney disease and obtain diagnostic analysis results and/or typing analysis results;

In the present invention, representative output modules include (but are not limited to): monitors, printers, tablet computers (PAD), and smart phones.

The main advantages of the present invention include:

(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, and the migrated body of the present invention can be used as a marker of kidney disease. It has the characteristics of faster, more convenient, low cost and high sensitivity.

(b) Migrants can be used as early diagnostic markers for different types of chronic kidney disease.

(c) The migratory body of the present invention can be used as a marker for early diagnosis of chronic kidney disease.

(d) The chemiluminescence method of the present invention has the advantages of simplicity, ease of operation, etc. The detection of migrating bodies by the chemiluminescence method can be used as a marker for the diagnosis of chronic kidney disease.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

Example 1 WGA-coupled magnetic beads capture migrating bodies in urine as markers of kidney damage

1. Collect urine samples from volunteers in the hospital physical examination center and nephrology department. Within 24 hours, centrifuge at 4000g and 4°C for 20 minutes to remove cell debris. Add 5 μL of WGA-coupled magnetic beads to 1 mL of urine and mix at room temperature. Incubate on the instrument for a total of 1 hour.

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

3. Then place the EP tube in the magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Resuspend in 100 µL of PBS containing 0.1% BSA.

4. Add 0.5 μL TSPAN4 antibody (abcam, Cat. No. ab181995, rabbit source) to the EP tube, place it on a mixer and incubate at room temperature for 1 hour. Then place the EP tube in a magnetic separation rack to enrich the magnetic beads and discard the supernatant. Add 1000 μL of PBS solution containing 0.1% BSA and wash three times, and then resuspend in 250 μL of PBS solution containing 0.1% BSA.

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

6. After the flow cytometry test results are completed, the kidney condition of the test sample is evaluated according to TSPAN4, and the subject is screened for patients with kidney damage.

7. Obtain the clinical pathological diagnosis information of the subjects from the hospital, and compare the consistency of the kidney damage judged by TSPAN4 fluorescence with the pathological diagnosis results, and observe whether urine migrants can be used as markers for the diagnosis of kidney disease.

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

Result 1.1

The test results are shown in Figure 1A. There are basically no migrating bodies in the urine of normal people, but there are abundant migrating bodies in the urine of CKD patients. Serum creatinine, an indicator commonly used to judge chronic kidney disease, is also increased in CKD patients (Figure 1B), and glomerular filtration rate (eGFR) is decreased (Figure 1C).

Further analysis of the receiver operating characteristic curve (ROC curve) found that the area under the ROC curve when urine migrants distinguished CKD patients from normal people was 0.9591, and the area under the ROC curve when serum creatinine distinguished CKD patients from normal people was The area under the ROC curve for eGFR to distinguish CKD patients from normal people was 0.5725 (Figure 1D).

It is known from this that the diagnosis of chronic kidney disease through migrating bodies is more accurate than the existing blood creatinine and eGFR.

Result 1.2

The collected CKD patients include diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy (IgA), and patients with chronic kidney disease due to other causes (other).

Furthermore, comparing different types of CKD, it was found that, consistent with result 1.1, patients with diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy (IgA) and other causes of chronic kidney disease (other) were compared with normal people (NOR). , the migratory content in urine increased significantly, but there was not much difference between the various diseases (Figure 2A).

Serum creatinine, an indicator commonly used to judge chronic kidney disease, is also increased in various types of CKD patients (Figure 2B), and glomerular filtration rate (eGFR) is decreased (Figure 2C).

The results of the receiver operating characteristic curve show that the areas under the ROC curve of urine migrants to distinguish DN patients, MN patients, IgA patients, patients with chronic kidney disease due to other causes (other), and normal people are 0.8831, 0.9438, 0.9851, and 0.9642 respectively. ,0.8492 (Figure 2D).

Result 1.3

The flow cytometry test results are shown in Figure 3A. The migratory body content in the urine of CKD patients increased from CKD stage I to II, and showed a downward trend from stage II to V, but they were all higher than normal. people. Since the vast majority of migratory bodies in urine come from podocytes, and podocytes are slowly lost during the progression of the disease in CKD patients, the change in migratory body content is related to the role of podocytes, the most important functional cells of the kidney, in CKD. The changes are basically the same.

Comparing CKD-I stage patients, who are almost impossible to accurately diagnose clinically, with normal people alone, it was found that urinary migratory substances were significantly increased in CKD-I stage patients (Figure 3B), while serum creatinine and eGFR were significantly higher in CKD-I stage patients. There were almost no changes between patients and normal subjects (Fig. 3C-D).

Further analysis of the ROC curve revealed that the area under the ROC curve when urinary migrants distinguished CKD-I from NOR was 0.9516, the area under the ROC curve when serum creatinine distinguished chronic kidney disease from normal people was 0.6945, and the ROC curve when eGFR distinguished chronic kidney disease from normal people. The lower area is 0.7600 (Figure 3E).

Because the normal human glomerular filtration rate (eGFR) is (80-125) mL/min/1.73m ² . We extracted patients with eGFR ≥ 80 among CKD patients and compared them with normal people. The analysis results showed that migratory bodies increased significantly in CDK patients (Figure 3F), while serum creatinine and eGFR had almost no changes between CKD stage I patients and normal subjects (Figure 3G-H). Further analysis of the ROC curve revealed that the area under the ROC curve was 0.9535 when urine migrants distinguished CKD patients with eGFR ≥ 80 from normal individuals (NOR), and serum creatinine distinguished CKD patients with eGFR ≥ 80 from normal individuals (NOR). The area under the ROC curve was 0.6530, and the area under the ROC curve when eGFR distinguished patients with eGFR ≥ 80 in CKD patients from normal controls (NOR) was 0.6718 (Figure 3I).

It is known from this that urinary migrants can be used as biomarkers for early diagnosis of chronic kidney disease.

Result 1.4

In order to compare the advantages and disadvantages of migrants and serum creatinine in distinguishing chronic kidney disease (CKD), the present invention extracts patients with chronic kidney disease whose serum creatinine is within the normal range of 0.50-1.50 mg/dl for analysis.

The analysis results showed that migratory bodies increased significantly in CKD patients (Figure 4A), while serum creatinine and eGFR had almost no changes between CKD stage I patients and normal subjects (Figure 4B-C).

Further analysis of the ROC curve revealed that the area under the ROC curve when urine migrants distinguished CKD patients with normal serum creatinine from normal people was 0.9195, and when serum creatinine distinguished CKD patients with normal serum creatinine from normal people, the area under the ROC curve was 0.9195. 0.5058, and the area under the ROC curve when eGFR distinguished patients with normal serum creatinine from normal subjects in CKD patients was 0.5220 (Figure 4D).

It is known from this that urinary migrants can be used as biomarkers for early diagnosis of chronic kidney disease.

Result 1.5

In order to further identify the changes in urinary migratory content in the urine of patients with different types of CKD, we analyzed the urinary migratory content of patients with diabetic nephropathy (DN), membranous nephropathy (MN) and IgA nephropathy (IgA) at different stages.

The results are shown in Figures 5A-5C, consistent with previous results, in DN patients, MN patients, and IgA patients in stage I During the progression to stage II, urinary migratory body content increases, while in the stage from stage II to stage V (ESRD), it decreases due to the loss of podocytes.

The diagnosis of different types of chronic kidney disease has always been a clinical difficulty. Analysis of the ROC curve revealed that the working curves of urine migrants when distinguishing early DN patients, early MN patients, and early IgA nephropathy patients (DN-I, MN-I, and IgA-I) were 0.9987, 0.9587, and 0.9891 respectively (Figure 5D) .

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

Example 2 Chemiluminescence method to detect migrating bodies in urine as markers of kidney damage

1. Purchase NHS magnetic beads with a particle size of 2 μm (Solarbio, Cat. No. M2450), take 500 μL magnetic bead suspension in a 1.5 mL EP tube, place the EP tube in a magnetic separation rack, enrich the magnetic beads, and remove the supernatant . Add 1 mL of 4°C pre-cooled Washing Buffer A to the 1.5 mL EP tube and vortex for 15 seconds to mix the magnetic beads evenly. Place the EP tube in a magnetic separation rack, enrich the magnetic beads, and remove the supernatant.

2. TSPAN4 antibody (abcam, catalog number ab181995, rabbit source) was dialyzed and dissolved in 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 seconds to mix evenly. Vortex the EP tube for 15 seconds, place it on the mixer, and mix at room temperature for 2 hours. If the mixing is uneven, remove the EP tube and vortex for 15 seconds every 5 minutes 30 minutes before the reaction. Thereafter, every 15 min, remove the EP tube and vortex for 15 s.

3. Place the EP tube in the magnetic separation rack, enrich the magnetic beads, and remove the supernatant. Add 1mL Blocking Buffer to the EP tube, vortex for 30 seconds, place the EP tube in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant.

4. Repeat step 3 four times. Add 1mL Blocking Buffer to the EP tube, vortex for 30 seconds, and place the EP tube in a mixer for room temperature reaction for 2 hours. Place the EP tube in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Add 1 mL of ultrapure water to the EP tube, mix thoroughly, use a magnetic stand to enrich the magnetic beads, and discard the supernatant.

5. Add 1mL of PBS solution (pH 7.2) to the EP tube, mix thoroughly, use a magnetic stand to enrich the magnetic beads, and discard the supernatant. After repeating this operation twice, resuspend in 500 μL PBS solution, mix thoroughly, and store at 4°C for later use. Note: The final magnetic bead concentration of coupled protein is 10mg/mL.

6. Randomly collect urine samples from volunteers in the hospital physical examination center and nephrology department. Within 24 hours, centrifuge at 4000g and 4°C for 20 minutes to remove cell debris. Take 1mL of urine and add 5μL of conjugated TSPAN4 antibody (abcam, product number ab181995, rabbit source). ) magnetic beads and incubate them on a mixer for 1 hour at room temperature.

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

8. Then place the EP tube in the magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Resuspend in 100 µL of PBS containing 0.1% BSA.

9. Add 5 μL of human podocyte-specific protein podocalyxin antibody purchased from Abcam (recombinant Anti-PODXL antibody [PcMab-47] (ab264542), mouse monoclonal antibody), place it on the mixer and incubate at room temperature for 1 hour, and then Finally, place the EP tube in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Add 1000 μL of PBS solution containing 0.1% BSA and wash three times, and then resuspend in 250 μL of PBS solution containing 0.1% BSA.

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

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

result

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

Further analysis of the receiver operating characteristic curve revealed that the area under the ROC curve when urinary migrants distinguished chronic kidney disease (CKD) from normal individuals (NOR) was 0.9200 (Figure 7B).

It is known from this that the migrated bodies detected by chemiluminescence method can be used as markers for the diagnosis of chronic kidney disease.

Example 3 TSPAN4-coupled magnetic beads capture migratory bodies in urine as markers for the detection of autoantibodies in membranous nephropathy

1. Repeat steps 1-8 in Example 2.

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

3. After the flow cytometry test results are completed, the test results are determined based on the fluorescence intensity (FITC fluorescence intensity) of human autoantibodies (anti-phospholipase A2 receptor (PLA2R), thrombospondin 7A (THSD7A)) on the surface of the podocyte-derived migrating body. Autoantibody expression levels in patients with membranous nephropathy.

4. Obtain the clinical pathological diagnosis information of the subjects from the hospital. Compare the autoantibody content detected by the hospital with the autoantibody content detected by flow cytometry in urine migrants to observe whether urine migrants can serve as membranes Markers for the diagnosis of nephropathy autoantibodies.

result

The present invention analyzed 38 cases of patients with primary membranous nephropathy. The specific pathological information is shown in Table 2.

The analysis results showed that urinary podocyte-derived migrants were able to distinguish autoantibodies (Fig. 8A).

The results of the receiver operating characteristic curve showed that the area under the ROC curve when urine migrants distinguished autoantibody-positive and negative patients was 0.9200 (Figure 8B), and the autoantibody concentration measured by urine migrants was consistent with the autoantibody concentration measured by ELISA. The antibody concentration showed a positive correlation, with a linear correlation coefficient R ² =0.8576 (Figure 8C).

It is known from this that TSPAN4-coupled magnetic beads capture migrating bodies in urine and can be used as markers for the detection of autoantibodies in membranous nephropathy.

Table 2 Pathological information of patients with primary membranous nephropathy

discuss

Liquid biopsy is a new technology for diagnosing and dynamically observing diseases by monitoring proteins, DNA, RNA and other substances in cells or tissues in a pathological state in body fluids such as blood, saliva, urine, etc. Currently, the four main sources of biomarkers in liquid biopsy are circulating tumor cells (CTC), circulating tumor DNA (ctDNA), circulating RNA and extracellular vesicles. Migrasomes are single-layer membrane vesicle structures with a diameter of 0.5 to 2 μm produced by contractile filaments at the cell tail during directional cell migration. Migrants contain a large number of biologically active substances such as nucleic acids, proteins, fats, etc., which play an important role in transmitting signals between cells, mediating intercellular communication, and participating in and regulating a variety of physiological and pathological activities. Studies have shown that kidney-specific cells such as podocytes that make up the kidney will release a large number of migrants into the urine when the kidney is injured, and the proteins and nucleic acids in the urine migrants will vary depending on the type and severity of the kidney injury. corresponding changes.

Membranous nephropathy is one of the most common pathological manifestations of adult nephrotic syndrome and the second leading cause of primary glomerular disease in my country. About 70% of patients with membranous nephropathy present with persistent and recurring proteinuria, which can cause kidney damage and be life-threatening. About 1/3 of patients with membranous nephropathy can spontaneously resolve and gradually improve. About 1/3 of patients develop end-stage renal disease after 5 to 10 years, and the remaining patients show persistent proteinuria.

Membranous nephropathy can be divided into two categories: idiopathic and secondary. Among them, idiopathic membranous nephropathy (Idiopathic membranous nephropathy) membranous nephropathy (IMN) accounts for 70% to 80%. Idiopathic membranous nephropathy mainly refers to changes in the glomerular basement membrane without a clear cause; secondary membranous nephropathy (SMN) accounts for 20% to 30% and is generally secondary to systemic lupus erythematosus (Systemic lupus erythematosus). lupus er ythematosus, SLE) and other autoimmune diseases, infections (hepatitis B/C virus infection), malignant tumors and drug poisoning, etc. The pathogenesis and etiology of IMN are still unclear, and specific laboratory indicators for diagnosis are currently lacking.

Studies have found that anti-phospholipase A2 receptor (PLA2R), thrombospondin 7A (THSD7A) and other autoantibodies against podocyte proteins were detected in the serum of adult patients with IMN. Serum PLA2R, THSD7A and other autoantibodies against podocytes are rarely found in patients with the disease. Therefore, autoantibodies against podocyte proteins such as PLA2R and THSD7A in serum can be used as biomarkers for the diagnosis of membranous nephropathy.

Currently, there are kits that use artificially synthesized PLA2R, THSD7A and other proteins as antigens to detect autoantibodies through chemiluminescence. However, due to the large molecular weight of proteins such as PLA2R and THSD7A and the presence of multiple repeating domains, in vitro synthesis is difficult and costly. What's more troublesome is that each protein needs to be synthesized separately and a detection method designed to detect it. Not only is the operation troublesome, but the cost is high. Migrants derived from urinary podocytes contain most of the proteins on the surface of podocytes, so migratory bodies derived from urinary podocytes in patients with membranous nephropathy can be used as antigens for autoantibody detection.

In combination with the urine migratory capture detection method developed in the present invention, the inventors developed a detection method for membranous nephropathy autoantibodies, for example, based on TSPAN4-coupled magnetic beads to capture migratory bodies in urine (Figure 9).

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 (22)

1. The use of a migrating body detection agent, characterized in that it is used to prepare detection reagents or kits, and the detection reagents or kits are used to evaluate whether a subject (subject) suffers from chronic kidney disease (CKD) or suffers from chronic kidney disease (CKD). Susceptibility 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 satisfies the following conditions:

   (Z1) Glomerular filtration rate (eGFR) is 80~125mL/min/ ^1.73m2 , preferably 85~110mL/min/ ^1.73m2 , more preferably 90~105mL/min/ ^1.73m2 , and /or

   (Z2) Serum creatinine level is 0.50～1.50mg/dl, preferably 0.75～1.25mg/dl.
3. The use according to claim 1, characterized in that the subject is a person who has chronic kidney disease in his family or has suffered from chronic kidney disease.
4. The use according to claim 1, wherein the subject is a patient who has suffered acute renal injury.
5. The use according to claim 1, wherein the CKD includes hereditary CKD or recurrent CKD.
6. The use according to claim 1, wherein the CKD is stage I CKD.
7. The use according to claim 1, characterized in that the detection reagent or kit is also used to classify chronic kidney disease.
8. The use according to claim 1, wherein the detection includes urine detection, serum detection, platelet detection, and detection of tissue or cell samples.
9. The use according to claim 1, wherein the detection reagent includes a specific binding molecule of the migratory body, a specific amplification primer, a probe or a chip.
10. The use according to claim 9, characterized in that the specific binding molecules include specific antibodies and solid-phase carriers with lectins coupled or bound to their surfaces.
11. The use according to claim 10, wherein the specific antibody is selected from the following group: anti-TSPAN4 antibody, anti-Integrinα5β1 antibody, PIGK antibody, NDST1 antibody, CPQ antibody, EOGT antibody, or a combination thereof.
12. The use according to claim 10, characterized in that the solid phase carrier includes: solid particles, microfluidic chips, glass cellulose membranes, nylon membranes, and agarose beads.
13. The use according to claim 1, wherein the detection includes: flow cytometry, fluorescence imaging technology, and fluorescence immunoreading technology.
14. A detection kit, characterized in that the kit contains:

    (a) Detection reagent, wherein the detection reagent includes:

    (a1) A first detection reagent for detecting migrating bodies;

    (a2) A second detection reagent optionally used to detect eGFR;

    (a3) A third detection reagent optionally used to detect blood creatinine; and/or

    (a4) A fourth detection reagent optionally used to detect autoantibodies against podocyte proteins such as PLA2R, THSD7A, etc.; and

    (b) A label or instructions indicating that the kit is used to assess a subject's susceptibility to chronic kidney disease.
15. The detection kit according to claim 14, characterized in that the label or instructions indicate the following:

    When the ratio of the number of migrants in the urine of the test subject to the number of migrants in normal people is ≥ 1.5, and preferably ≥ 2, it indicates that the risk of developing chronic kidney disease in the subject is high.
16. The detection kit according to claim 14, characterized in that the label or instructions indicate the following:

    (i) When the number of migrants in the urine of the subject is > the reference value (preferably 1390 MFI (mean fluorescence intensity)), it indicates that the subject has a high risk of developing chronic kidney disease; and

    (ii) When the number of migrating bodies in the urine of the subject is less than the reference value (preferably 1390 MFI (mean fluorescence intensity)), it indicates that the risk of developing chronic kidney disease in the subject is low.
17. A method for assessing whether a subject suffers from chronic kidney disease or is susceptible to chronic kidney disease, characterized in that the method includes:

    (a) Provide test samples from subjects;

    (b) detect the number of migrants in the test sample; and

    (c) Compare the content of the migrant measured in step (b) with the control reference value,

    Wherein, when the content of migrating bodies 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.
18. The method of claim 17, wherein the subject meets the following conditions:

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

    (Z2) The blood creatinine level is 0.50～1.50mg/dl, preferably 0.75～1.25mg/dl.
19. The method of claim 17, which is non-diagnostic and non-therapeutic.
20. A diagnostic device, characterized in that the diagnostic device includes:

    (a) an input module configured to input the content of migrants in the test sample of the subject;

    (b) Chronic kidney disease diagnosis-chronic kidney disease typing module, the chronic kidney disease diagnosis-chronic kidney disease typing module is configured to: based on the content of the migrants, determine whether the subject suffers from chronic kidney disease. Diagnostic analysis, and/or typing analysis of chronic kidney disease, and obtaining diagnostic analysis results and/or typing analysis results; and

    (c) Output module, the output module is configured to output the diagnostic analysis results and/or typing analysis result.
21. The diagnostic device according to claim 20, wherein the subject meets the following conditions:

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

    (Z2) The blood creatinine level is 0.50～1.50mg/dl, preferably 0.75～1.25mg/dl.
22. The diagnostic device of claim 20, further comprising (d) a detection module configured to detect the number or content of migrating bodies in the test sample of the subject.