Use of reagent for detecting ACVR2b in preparation of diagnostic kit for diagnosing vascular calcification in chronic kidney disease

Abstract

The present application relates to the field of in vitro diagnostic reagents, in particular to the use of reagents for detecting ACVR2b in the preparation of a diagnostic kit for chronic kidney disease vascular calcification, and it is found for the first time that the expression level of ACVR2b is significantly correlated with chronic kidney disease vascular calcification. Therefore, the expression level of ACVR2b can be used for screening of chronic kidney disease vascular calcification. Based on this, the present application provides the use of reagents for detecting ACVR2b in the preparation of a diagnostic kit for chronic kidney disease vascular calcification, which has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnostic reagents, and in particular to the use of a reagent for detecting ACVR2b in the preparation of a diagnostic kit for vascular calcification in chronic kidney disease. BACKGROUND

[0002] Vascular calcification (VC) is a core common pathological link in the progression of various chronic kidney diseases (CKD). Its pathological features include vascular smooth muscle cell phenotypic transformation, inflammatory cell recruitment and infiltration, osteoblast-like and chondroblast-like cell activation and proliferation, calcification matrix (hydroxyapatite crystals, osteonectin, and osteocalcin) deposition, vascular elastic fiber degradation, vascular wall thickening and stiffness, and lumen stenosis, ultimately leading to impaired vascular structure and function. Since chronic kidney disease vascular calcification marks irreversible vascular damage, early diagnosis and treatment based on the cause are critical to delay CKD progression and reduce the risk of cardiovascular complications.

[0003] Current diagnostic techniques for chronic kidney disease vascular calcification (CKD-VC) are mainly divided into three categories:

[0004] I. Diagnostic techniques based on traditional molecular markers (osteoprotegerin OPG + fetuin-A Fetuin-A): Detecting the concentrations of OPG (increased) and Fetuin-A (decreased) in the serum of CKD patients by ELISA, combined with calcium and phosphorus indicators to determine VC.

[0005] II. Diagnostic techniques based on imaging (multi-slice spiral computed tomography MSCT): MSCT scans the abdominal aorta or lower extremity vessels to quantify calcification using the Agatston score (≥10 points is positive) (the attached figure includes equipment, tomographic images, and scoring interface). The relevant basis is the "Chinese Expert Consensus on Diagnosis and Treatment of Vascular Calcification in CKD Patients (2020)".

[0006] III. Diagnostic techniques based on single signaling pathway molecules (bone morphogenetic protein 2 BMP-2): Vascular tissue is obtained by puncture, and BMP-2 expression is detected by immunohistochemistry (IRS ≥6 points indicates VC).

[0007] However, the above prior art still has various problems. Among them, the shortcomings of the traditional molecular marker diagnosis technology are: low specificity, easy to be affected by inflammation, liver and kidney function; diagnosis has lag, can only detect in the middle and late stage of the disease. The shortcomings of the imaging diagnosis technology are: unable to detect early microcalcification, with the risk of missed diagnosis; with radiation hazards; high dependence on equipment, difficult to popularize in primary medical institutions. The shortcomings of single signal pathway molecular diagnosis technology are: invasive detection, with the risk of bleeding and infection; poor specificity, easy to be affected by bone metabolism, kidney fibrosis and other factors.

[0008] Therefore, it is still necessary in the art to develop more new methods for diagnosing CKD-VC, especially biomarkers and methods that can specifically diagnose in the early stage.

[0009] Activin Receptor Type 2B (ACVR2b) is a type II transmembrane receptor of the TGF-β superfamily, which is widely expressed in tissues such as muscle and brain. After binding to activin or myostatin, it forms a complex with type I receptors, phosphorylates SMAD2 / 3 protein, and then enters the nucleus with SMAD4 to regulate target gene expression, affecting cell differentiation and proliferation. This pathway is involved in the negative regulation of neuronal differentiation, hair follicle development and muscle growth, and its dysfunction is related to muscle atrophy, cancer cachexia and other diseases, and is a potential target for treating muscle wasting diseases.

[0010] The prior art has not reported the direct correlation between ACVR2b and CKD-VC, nor has it systematically verified the specificity, sensitivity and clinical application value of ACVR2b as a diagnostic marker for CKD-VC. SUMMARY

[0011] The purpose of the present application is to provide a new biomarker for screening chronic kidney disease vascular calcification, and the use of the detection reagent of the biomarker in the preparation of a chronic kidney disease vascular calcification screening kit.

[0012] The use of the reagent for detecting ACVR2b in the preparation of a chronic kidney disease vascular calcification diagnostic kit.

[0013] Preferably, the ACVR2b is Activin Receptor Type 2B, UniProt number Q13705.

[0014] Preferably, the reagent is a reagent for detecting the expression amount of ACVR2b in a human body fluid sample.

[0015] Preferably, the body fluid sample is serum.

[0016] Preferably, the reagent is a reagent for detecting the level of ACVR2b protein, selected from the group consisting of immunohistochemical detection reagent, immunofluorescence detection reagent, western blot detection reagent, ELISA detection reagent, chemiluminescence immunoassay, immunochromatography or protein chip detection method reagent.

[0017] Preferably, the reagent is a reagent for detecting the level of ACVR2b nucleic acid, selected from the group consisting of QPCR detection reagent, digital PCR detection reagent, RT-PCR detection reagent or nucleic acid hybridization detection reagent.

[0018] Preferably, the diagnostic kit is used to distinguish chronic kidney disease patients with vascular calcification from healthy people.

[0019] Preferably, the threshold of ACVR2b expression for distinguishing chronic kidney disease patients with vascular calcification from healthy people is 16.44 ng / ml.

[0020] Preferably, the diagnostic kit is used to distinguish chronic kidney disease patients with vascular calcification from chronic kidney disease patients without vascular calcification.

[0021] Preferably, the threshold of ACVR2b expression for distinguishing chronic kidney disease patients with vascular calcification from chronic kidney disease patients without vascular calcification is 16.44 ng / ml.

[0022] The key of the present application is that the expression level of ACVR2b in human serum is significantly related to vascular calcification of chronic kidney disease. Therefore, the vascular calcification of chronic kidney disease can be screened by detecting the expression level of ACVR2b in human serum. As for the means for detecting the expression level of ACVR2b in human serum, various means disclosed in the prior art can be used, and the embodiments of the present application specifically use WB, ELISA, immunohistochemistry, immunofluorescence, QPCR and the like for detection, but are not limited to these means. Any method capable of detecting the expression level of ACVR2b protein or ACVR2b nucleic acid can be used for screening of vascular calcification of chronic kidney disease.

[0023] The present application provides a new biomarker, which can realize effective screening of vascular calcification of chronic kidney disease. The advantage of the present application is that early screening can be realized, the specificity is high, and serum can be used as a detection sample, which causes little harm to patients. Therefore, the present application has good application prospect.

[0024] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or change can be made without departing from the above basic technical idea of the present application.

[0025] The foregoing will be further described in greater detail by way of specific examples. This should not be construed as limiting the scope of the above-described subject matter to the examples described in the following detailed description. Any technical solution achieved based on the above-described subject matter falls within the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Experimental results for the construction and verification of a typical calcification model in Experimental Example 1, wherein (A) alizarin red S staining of vascular smooth muscle cells; (B) alizarin red staining positive result proportion; (C) cell calcium content determination results; (D, E) cell a-SMA and Runx-2 PCR results; (F) a-SMA, Runx-2 WB results; (G) a-SMA, Runx-2 band gray value analysis. p<0.05, p<0.001.

[0027] Figure 2 ACVR2b expression detection results of a typical calcification model in Experimental Example 1, wherein (A) ACVR2b WB results; (B) WB band gray value statistics; (C) QPCR results. p<0.01, p<0.001.

[0028] Figure 3 Detection results of a CKD vascular calcification mouse model constructed in Experimental Example 2, wherein (A) survival curves of mice in each group (control group: normal feed feeding; model group: CKD vascular calcification model, the animal experiment grouping design in the following result figures is consistent with this); (B) mouse survival curve; (C) mouse kidney picture; (D) mouse kidney Masson staining results; (E) mouse renal function serum creatinine; (F) urea nitrogen. p<0.001.

[0029] Figure 4 Detection results of a CKD vascular calcification mouse model constructed in Experimental Example 2, wherein (A) mouse microCT shows the vascular calcification and ectopic calcium salt deposition of the aorta wall of the model group mice; (B) microCT analysis of the calcium salt deposition volume in the aorta of the mice; (C) serum ALPase content of the mice; (D) serum phosphorus concentration of the mice; (E) serum calcium concentration of the mice. p<0.001.

[0030] Figure 5Detection results of the CKD vascular calcification mouse model constructed in Experimental Example 2, wherein (A) Alizarin red S staining of the whole aorta; (B-D) Von kossa and Alizarin red aorta section results; (E) α-SMA and Runx-2 immunohistochemistry; (F, G) α-SMA, Runx-2 immunohistochemistry positive statistics; (H) α-SMA, Runx-2 WB bands; (I) WB gray value statistics. p<0.05, p<0.001.

[0031] Figure 6 Detection results of the ACVR2b expression of the CKD vascular calcification mouse model constructed in Experimental Example 2, wherein (A) WB band results; (B) WB band gray value analysis; (C) ACVR2b aorta section immunofluorescence; (D) Immunofluorescence result analysis; (E) PCR results; (F) Mouse serum ACVR2b Elisa results. p<0.001, p<0.0001.

[0032] Figure 7 ROC curve of ACVR2b and calcification score when diagnosing different populations in Experimental Example 3, AUC = 1. DETAILED DESCRIPTION

[0033] Example 1: Detection kit and method of use thereof

[0034] I. Kit composition

[0035] The present embodiment provides detection kits for several detection methods such as Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), Immunofluorescence (IF), and Real-Time Fluorescent Quantitative PCR (QPCR). The compositions of various kits are as follows:

[0036] 1. Western Blot (WB) detection kit

[0037] Table 1

[0038]

[0039] 2. Enzyme-Linked Immunosorbent Assay (ELISA) detection kit

[0040] Table 2

[0041]

[0042] 3. Immunohistochemistry (IHC) detection kit

[0043] Table 3

[0044]

[0045] 4. Immunofluorescence (IF) detection kit Table 4

[0046]

[0047] 5. Real-time fluorescent quantitative PCR (QPCR) detection kit

[0048] Table 5

[0049]

[0050] II. Kit usage method

[0051] The usage method steps of the above various kits are as follows:

[0052] (1) WB: reagent extracts total protein; SDS-PAGE electrophoresis (10% separation gel); transfer to PVDF membrane; blocking solution blocking for 1 h; primary antibody (1:1000 dilution) 4°C incubation overnight; washing solution washing 3 times; enzyme-labeled secondary antibody (1:5000 dilution) room temperature incubation for 1 h; after washing, add chemiluminescent substrate; developer detects ACVR2b band gray value.

[0053] (2) ELISA: capture antibody (1 μg / mL) coated enzyme-labeled plate; 4°C incubation for 12 h; blocking solution blocking for 2 h; add sample (50 μL / well); 37°C incubation for 1 h; detection antibody (0.5 μg / mL, enzyme-labeled HRP); 37°C incubation for 45 min; TMB substrate color development for 15 min; reagent stop solution (2 mol / L H2SO4) stop; enzyme-labeled instrument 450 nm reads absorbance value.

[0054] (3) Immunohistochemistry: tissue section deparaffinization; gradient hydration; citrate buffer antigen repair; 3% H2O2 block endogenous enzyme; blocking solution blocking; primary antibody (1:200 dilution) 4°C incubation overnight; enzyme-labeled secondary antibody room temperature incubation for 30 min; DAB color development; hematoxylin re-staining; microscope observation ACVR2b positive staining intensity.

[0055] (4) Immunofluorescence: tissue section fixation (4% paraformaldehyde); 0.1% Triton X-100 permeabilization; blocking solution blocking; antibody fluorescence labeling (1:300 dilution) 4°C incubation overnight; DAPI staining nucleus; fluorescence microscope detects ACVR2b fluorescence signal.

[0056] (5) QPCR: extract RNA; reverse transcription into cDNA; the reaction system contains primer F: 5'-AGGCAACTTCTGCAACGAG-3' (SEQ ID NO. 1, 10 μmol / L); R: 5'-CTTCCGATGACGATACATCCAG-3' (SEQ ID NO. 2, 10 μmol / L), probe (5'-FAM-XXX-BHQ1-3', 5 μmol / L), fluorescent PCR Mix; amplification program: 95°C pre-denaturation 5 min; 95°C denaturation 15 s; 60°C annealing and extension 30 s (40 cycles); real-time fluorescence quantitative analysis.

[0057] The above methods provided by the embodiment have been verified to be able to realize detection of ACVR2b. However, the core of the technical scheme of the application is the technical finding that "ACVR2b is a specific biomarker for vascular calcification of chronic kidney disease", and all diagnostic technologies based on "specific recognition of ACVR2b (protein / nucleic acid), signal amplification and quantitative analysis" belong to the protection scope of the patent.

[0058] The technical scheme of the application is further described below through experiments.

[0059] Example 1 In vitro study confirms that ACVR2b expression is significantly reduced in a sodium phosphate-induced smooth muscle calcification model

[0060] Mouse vascular smooth muscle cells were cultured for 5-6 generations, and a cell calcification model was constructed by inducing the cells with 3 mM inorganic phosphorus NaH2PO4 (abbreviated as Pi) containing 10% fetal bovine serum high-glucose DMEM medium for 7 days. The results showed that:

[0061] Alizarin red S staining observed significant calcium salt deposition in Pi-induced smooth muscle cells Figure 1 A, B), and cell calcium content detection showed that the calcium content of the Pi group increased significantly Figure 1 C); QPCR and WB results showed that the expression of Runx2 in the calcification model cells increased, and the expression of the vascular smooth muscle cell marker molecule α-SMA decreased, verifying that the model was a typical calcification model Figure 1 D-G).

[0062] According to the method described in Example 1, cell protein was extracted and subjected to WB detection. It was found that ACVR2b was significantly reduced Figure 2 A, B); QPCR detection was performed on RNA, and it was found that the expression of ACVR2b was reduced Figure 2 C).

[0063] The above results show that the expression of ACVR2b is significantly increased in calcified vascular smooth muscle cells.

[0064] Example 2 in vivo study confirms significant reduction in expression in a mouse model of vascular calcification

[0065] A CKD vascular calcification mouse model was constructed using adenine + high phosphorus feed (after 1 week of normal feed adaptation, the mice were fed with 0.2% adenine feed for 4 weeks to induce CKD, and then fed with 0.2% adenine + 1.8% phosphorus feed for 10 weeks to induce calcification).

[0066] The results show that the model group has obvious body weight loss and increased mortality ( Figure 3 A, B), the kidneys are significantly atrophied ( Figure 3 C), masson staining shows that the kidneys are significantly fibrotic ( Figure 3 D), the serum urea nitrogen and creatinine levels are significantly increased ( Figure 3 E, F).

[0067] microCT detection found that the thoracic and abdominal aorta wall of the model mice was severely calcified, no calcification was found in the control group, and the model mice had ectopic deposition of calcium salt in multiple parts of the body ( Figure 4 A, B); the serum ALP ( Figure 4 C) and blood phosphorus levels ( Figure 4 D) of the model group mice were significantly increased, and the blood calcium level was significantly decreased ( Figure 4 E).

[0068] The mouse aorta was isolated for alizarin red S staining, which showed that the model mouse aorta had obvious calcium salt deposition ( Figure 5 A); aortic sections were stained with alizarin red S and Von kossa silver, which showed that the aortic calcification of the model group ( Figure 5 B-D); immunohistochemistry and WB results showed that the expression of vascular smooth muscle cell marker molecule a-SMA in the aorta of the model group mice was significantly reduced, and the expression of Runx2, a marker molecule of bone transdifferentiation, was significantly increased ( Figure 5 E-I). The above results confirmed that the constructed model was a typical chronic kidney disease vascular calcification model

[0069] ACVR2b was detected according to the method described in Example 1: vascular tissue protein and RNA were extracted for WB and QPCR detection, and it was found that the expression of ACVR2b was significantly reduced ( Figure 6 A, B, E); immunofluorescence staining was performed on the mouse vascular sections, and the expression of ACVR2b in the vascular sections of the model group was significantly reduced ( Figure 6C, D); ACVR2b ELISA detection was performed on mouse serum, and the serum ACVR2b content of the model group was significantly reduced. The experimental results verify that ACVR2b has high specificity, stability and difference for distinguishing healthy mice from chronic kidney disease vascular calcification model mice Figure 6 F).

[0070] Experimental Example 3 Clinical verification of significantly reduced ACVR2b expression in CKD-VC patients

[0071] Healthy people, CKD patients without vascular calcification (calcification score of 0) and CKD patients with vascular calcification (CKD-VC patients) were recruited in the clinic, and the serum ACVR2b content was detected by the ELISA method described in Example 1. The results showed that the expression level of ACVR2b in the serum of CKD-VC patients was significantly lower than that of the healthy control group and the CKD patients without vascular calcification. The calcification scores and ACVR2b levels of the patients were statistically analyzed (as shown in the following table), and the correlation analysis between the calcification score and the ACVR2b level was performed. It was found that ACVR2b was negatively correlated with the calcification score, and the correlation coefficient was high, indicating that the molecule had good diagnostic efficiency Figure 7 ). The ACVR2b level for the diagnosis of CKD-VC patients can be set according to the specific needs of the clinic or the sensitivity, for example, one preferred way is 16.44 ng / ml.

[0072] Table 6 Detection results of samples in each experimental group

[0073]

[0074] Note: The table content is the clinical sample information (healthy people, CKD patients, CKD patients with vascular calcification), calcification score (AU) and ACVR2b (ng / ml), respectively.

[0075] In summary, through the above examples and experimental examples, it can be seen that the kit of the present application can screen whether the to-be-tested population is a CKD-VC patient by detecting the expression level of ACVR2b: if the ACVR2b level is low (relative to healthy people or CKD patients without vascular calcification), the probability of the patient being a CKD-VC patient is higher. The present application can be used for early diagnosis of clinical CKD-VC, has good specificity, stability and difference, and provides effective basis for patients to take relevant treatment measures or decision-making, and has good clinical application prospect.

Claims

1. The use of reagents for detecting ACVR2b in the preparation of diagnostic kits for vascular calcification in chronic kidney disease, characterized in that: The ACVR2b is activin receptor 2B, UniProt number Q13705.

2. The use according to claim 1, characterized in that: The reagent is used to detect the expression level of ACVR2b in human serum samples.

3. The use according to claim 1, characterized in that: The reagent is for detecting ACVR2b protein levels and is selected from reagents used in immunohistochemical detection, immunofluorescence detection, western blot detection, ELISA detection, chemiluminescent immunoassay, immunochromatography, or protein chip detection methods.

4. The use according to claim 1, characterized in that: The reagent is for detecting ACVR2b nucleic acid levels and is selected from qPCR detection reagents, digital PCR detection reagents, RT-PCR detection reagents, or nucleic acid hybridization detection reagents.

5. The use according to claim 1, characterized in that: The diagnostic kit is used to differentiate between patients with vascular calcification in chronic kidney disease and healthy individuals.

6. The use according to claim 1, characterized in that: The diagnostic kit is used to differentiate between patients with vascular calcification in chronic kidney disease and those without vascular calcification.

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