Application of ACVR2b detection reagent in preparation of chronic kidney disease vascular calcification diagnostic kit

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a reagent for detecting ACVR2b in preparation of a chronic kidney disease vascular calcification diagnostic kit. It is found for the first time that the expression quantity of ACVR2b is remarkably associated with chronic kidney disease vascular calcification. Therefore, the expression quantity of the ACVR2b can be used for screening the vascular calcification of the chronic kidney disease. On the basis, the invention provides application of a reagent for detecting ACVR2b in preparation of a chronic kidney disease vascular calcification diagnostic kit, and the reagent 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 reagents 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 phenotype transformation, inflammatory cell recruitment and infiltration, activation and proliferation of osteoblast-like and chondroblast-like cells, calcification matrix (hydroxyapatite crystals, osteonectin, and osteocalcin) deposition, degradation of vascular elastic fibers, thickening and hardening of the vascular wall, and stenosis of the lumen, ultimately leading to damage to the normal structure and function of blood vessels. Since chronic kidney disease vascular calcification marks irreversible vascular damage, early diagnosis and treatment based on the cause are critical to delay the progression of CKD 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: First, the diagnostic technique based on traditional molecular markers (osteoprotegerin OPG + fetuin-A Fetuin-A): through ELISA method to detect the concentration of OPG (increased) and Fetuin-A (decreased) in the serum of CKD patients, combined with calcium and phosphorus indicators to judge VC.

[0004] Second, the diagnostic technique based on imaging (multi-slice spiral computed tomography MSCT): through MSCT scanning of abdominal aorta or lower limb blood vessels, quantifying calcification by Agatston score (≥10 points as positive) (the attached figure contains equipment, tomographic image, and scoring interface), and the relevant basis is the "Chinese expert consensus on diagnosis and treatment of vascular calcification in CKD patients (2020)".

[0005] Third, the diagnostic technique based on a single signal pathway molecule (bone morphogenetic protein 2 BMP-2): through puncture to obtain vascular tissue, and detect BMP-2 expression by immunohistochemistry (IRS ≥6 points indicates VC).

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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

[0010] 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.

[0011] The use of the reagent for detecting ACVR2b in the preparation of a chronic kidney disease vascular calcification diagnosis kit.

[0012] Preferably, the ACVR2b is Activin Receptor Type 2B, UniProt number Q13705.

[0013] Preferably, the reagent is a reagent for detecting the expression amount of ACVR2b in a human body fluid sample.

[0014] Preferably, the body fluid sample is serum.

[0015] Preferably, the reagent is a reagent for detecting ACVR2b protein levels, selected from immunohistochemical detection reagents, immunofluorescence detection reagents, western blot detection reagents, ELISA detection reagents, chemiluminescent immunoassay, immunochromatography, or reagents used in protein chip detection methods.

[0016] Preferably, the reagent is a reagent for detecting ACVR2b nucleic acid levels, selected from qPCR detection reagents, digital PCR detection reagents, RT-PCR detection reagents, or nucleic acid hybridization detection reagents.

[0017] Preferably, the diagnostic kit is used to distinguish between patients with vascular calcification in chronic kidney disease and healthy individuals.

[0018] Preferably, the ACVR2b expression threshold for distinguishing between patients with vascular calcification in chronic kidney disease and healthy individuals is 16.44 ng / ml.

[0019] Preferably, the diagnostic kit is used to distinguish between patients with vascular calcification in chronic kidney disease and those without vascular calcification in chronic kidney disease.

[0020] Preferably, the ACVR2b expression threshold for distinguishing between patients with vascular calcification in chronic kidney disease and those without vascular calcification is 16.44 ng / ml.

[0021] The key to this invention lies in determining that the expression level of ACVR2b in human serum is significantly correlated with vascular calcification in chronic kidney disease. Therefore, vascular calcification in chronic kidney disease can be screened by detecting the expression level of ACVR2b in human serum. As for the specific methods for detecting the expression level of ACVR2b in human serum, various methods disclosed in the prior art can be used. This invention specifically employs methods such as Western blotting (WB), ELISA, immunohistochemistry, immunofluorescence, and qPCR, but is not limited to these methods. Any method capable of detecting the expression level of ACVR2b protein or ACVR2b nucleic acid can be used for screening vascular calcification in chronic kidney disease.

[0022] This invention provides a novel biomarker capable of effectively screening for vascular calcification in chronic kidney disease. The advantages of this invention lie in its ability to achieve early screening, high specificity, and the use of serum as the test sample, resulting in minimal harm to patients. Therefore, this invention has promising application prospects.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed description of specific embodiments further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 The results show the construction and validation of the typical calcification model in Example 1, including: (A) Alizarin Red S staining of vascular smooth muscle cells; (B) Proportion of positive Alizarin Red staining results; (C) Calcium content determination results in cells; (D, E) PCR results of α-SMA and Runx-2 in cells; (F) WB results of α-SMA and Runx-2; and (G) Gray value analysis of α-SMA and Runx-2 bands. *p<0.05, ***p<0.001.

[0026] Figure 2 The results show the ACVR2b expression detection of the typical calcification model in Experiment 1, including (A) ACVR2b WB results; (B) WB band gray value statistics; and (C) qPCR results. **p<0.01, ***p<0.001.

[0027] Figure 3 The results are for the CKD vascular calcification mouse model constructed in Experiment Example 2. (A) Survival curves of each group of mice (Control group: fed with normal diet; Model group: CKD vascular calcification model; the animal experimental grouping design in the following results figures is consistent with this); (B) Mouse survival curves; (C) Images of mouse kidneys; (D) Masson staining results of mouse kidneys; (E) Serum creatinine for mouse renal function; (F) Blood urea nitrogen. ***p<0.001.

[0028] Figure 4 The results are for the CKD vascular calcification mouse model constructed in Experiment Example 2. (A) MicroCT scan of mice showing vascular calcification and ectopic calcium salt deposition in the aortic wall of the model group mice; (B) MicroCT analysis of the volume of calcium salt deposition in the mouse aorta; (C) Serum ALP enzyme content in mice; (D) Serum phosphorus concentration in mice; (E) Serum calcium concentration in mice. ***p<0.001.

[0029] Figure 5The results of the detection of vascular calcification in the CKD mouse model constructed in Experiment Example 2 are shown below. (A) Alizarin Red S staining of the entire aorta; (B-D) Von Kossa and Alizarinred aortic sections; (E) Immunohistochemistry of α-SMA and Runx-2; (F,G) Statistical analysis of α-SMA and Runx-2 immunohistochemistry positivity; (H) Western blotting (WB) of α-SMA and Runx-2; (I) Statistical analysis of WB gray values. *p<0.05, ***p<0.001.

[0030] Figure 6 The results show the ACVR2b expression detection in the CKD vascular calcification mouse model constructed in Experiment Example 2, including (A) WB band results; (B) WB band gray value analysis; (C) ACVR2b aortic section immunofluorescence; (D) immunofluorescence result analysis; (E) PCR results; and (F) mouse serum ACVR2b ELISA results. ***p<0.001, ****p<0.0001.

[0031] Figure 7 The ROC curves of ACVR2b and calcification scores are shown in Experiment Example 3 when different populations are diagnosed. The ROC curves are AUC=1. Detailed Implementation

[0032] Example 1: Detection kit and its usage method I. Components of the Reagent Kit This embodiment provides detection kits for several detection methods, including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence (IF), and quantitative real-time PCR (qPCR). The components of each kit are as follows: 1. Western Blot (WB) Detection Kit Table 1 2. Enzyme-linked immunosorbent assay (ELISA) kit Table 2 3. Immunohistochemical (IHC) detection kit Table 3 4. Immunofluorescence (IF) Detection Kit (Table 4) 5. Real-time quantitative PCR (qPCR) detection kit Table 5 II. Instructions for using the reagent kit The usage steps for the above reagent kits are as follows: (1) WB: Total protein was extracted with reagents; SDS-PAGE electrophoresis (10% separating gel); transferred to PVDF membrane; blocked with blocking buffer for 1 h; primary antibody (1:1000 dilution) was incubated overnight at 4℃; washed 3 times with washing buffer; enzyme-labeled secondary antibody (1:5000 dilution) was incubated at room temperature for 1 h; after washing, chemiluminescent substrate was added; the gray value of ACVR2b band was detected by a developing instrument. (2) ELISA: Capture antibody (1 μg / mL) is coated onto the microplate; incubated at 4℃ for 12 h; blocking buffer is applied for 2 h; sample (50 μL / well) is added; incubated at 37℃ for 1 h; detection antibody (0.5 μg / mL, enzyme-labeled HRP) is applied; incubated at 37℃ for 45 min; TMB substrate is applied for 15 min; stop the reaction with reagent stop solution (2 mol / L H2SO4); absorbance is read at 450 nm using a microplate reader. (3) Immunohistochemistry: Dewaxing of tissue sections; gradient hydration; antigen retrieval with citrate buffer; blocking of endogenous enzymes with 3% H2O2; blocking with blocking solution; incubation of primary antibody (1:200 dilution) at 4℃ overnight; incubation of enzyme-labeled secondary antibody at room temperature for 30 min; DAB staining; hematoxylin counterstaining; observation of ACVR2b positive staining intensity under a microscope.

[0033] (4) Immunofluorescence: Tissue sections were fixed (4% paraformaldehyde); permeabilized with 0.1% Triton X-100; blocked with blocking solution; antibody fluorescent labeling (1:300 dilution) was incubated overnight at 4℃; nuclei were stained with DAPI; ACVR2b fluorescence signal was detected by fluorescence microscopy. (5) QPCR: RNA was extracted; reverse transcribed into cDNA; the reaction system contained 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), and fluorescent PCR Mix; amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s; 60℃ annealing extension for 30 s (40 cycles); real-time quantitative PCR analysis.

[0034] The methods provided in this embodiment have all been verified to detect ACVR2b. However, the core of the technical solution of this invention lies in the technical discovery that "ACVR2b is a specific biomarker for vascular calcification in chronic kidney disease". All diagnostic techniques based on "specific recognition of ACVR2b (protein / nucleic acid), signal amplification, and quantitative analysis" are within the scope of protection of this patent.

[0035] The technical solution of the present invention will be further explained through experiments below.

[0036] Experimental Example 1: In vitro studies confirmed a significant increase in ACVR2b expression in a sodium dihydrogen phosphate-induced smooth muscle calcification model. Mouse vascular smooth muscle cells, passages 5-6, were cultured and induced for 7 days in 3mM inorganic phosphorus NaH2PO4 (Pi) high-glucose DMEM medium containing 10% fetal bovine serum to construct a cell calcification model. Results showed: Alizarin Red S staining revealed significant calcium salt deposition in Pi-induced smooth muscle cells. Figure 1 Cellular calcium content detection showed that the calcium content in the Pi group was significantly increased (A, B). Figure 1 C); qPCR and WB results showed that Runx2 expression was increased and α-SMA expression, a marker of vascular smooth muscle cells, was decreased in the calcified model cells, verifying that the model was a typical calcification model. Figure 1 D - G).

[0037] Following the method described in Example 1, cellular proteins were extracted and analyzed by Western blotting. A significant increase in ACVR2b was observed (…). Figure 2 A, B); qPCR detection of RNA revealed increased ACVR2b expression ( Figure 2 C).

[0038] The above results indicate that ACVR2b expression is significantly increased in calcified vascular smooth muscle cells.

[0039] In vivo studies in Experiment Example 2 confirmed a significant increase in expression in a mouse model of vascular calcification. A mouse model of CKD vascular calcification was constructed using an adenine-rich and high-phosphorus diet (after one week of normal diet adaptation, mice were fed a diet containing 0.2% adenine for four weeks to induce CKD, and then calcification was induced for 10 weeks using a diet containing 0.2% adenine and 1.8% phosphorus).

[0040] The results showed that the model group experienced significant weight loss and increased mortality. Figure 3 A, B), significant kidney atrophy ( Figure 3 C), Masson staining showed significant renal fibrosis ( Figure 3 D), serum urea nitrogen and creatinine levels were significantly elevated ( Figure 3 E, F).

[0041] MicroCT scans revealed severe calcification of the thoracic and abdominal aortic walls in the model mice, while no calcification foci were observed in the control group. Furthermore, ectopic calcium salt deposits were found in multiple locations throughout the model group mice. Figure 4A, B); Serum ALP in model group mice ( Figure 4 C) and blood phosphorus levels ( Figure 4 D) significantly increased, blood calcium level significantly decreased ( Figure 4 E).

[0042] Alizarin Red S staining was performed on isolated mouse aortas, revealing significant calcium salt deposition in the aorta of the model mice. Figure 5 A); Alizarin Red S staining and Von Kossa silver staining of aortic sections both indicated aortic vascular calcification in the model group ( Figure 5 B-D); Immunohistochemical and Western blot results showed that the expression of α-SMA, a marker of vascular smooth muscle cells, was significantly reduced in the aorta of the model group mice, while the expression of Runx2, a marker of osteogenic differentiation, was significantly increased. Figure 5 E - I). The above results confirm that the constructed model is a typical vascular calcification model of chronic kidney disease. ACVR2b was detected according to the method described in Example 1: Vascular tissue proteins and RNA were extracted and analyzed by Western blotting and qPCR. A significant increase in ACVR2b expression was observed. Figure 6 A, B, E); Immunofluorescence staining of mouse vascular sections showed a significant increase in ACVR2b expression in the model group vascular sections. Figure 6 C, D); ACVR2b ELISA was performed on mouse serum, and the ACVR2b level in the model group was significantly increased. These results validate that ACVR2b has high specificity, stability, and differential efficacy in distinguishing between healthy mice and mice with vascular calcification in chronic kidney disease. Figure 6 F).

[0043] Experimental Case 3 clinically confirmed that ACVR2b expression was significantly increased in CKD-VC patients. Clinical trials recruited healthy individuals, CKD patients without vascular calcification (calcification score of 0), and CKD patients with vascular calcification (CKD-VC patients). Serum ACVR2b levels were measured using the ELISA method described in Example 1. Results showed that the expression level of ACVR2b in the serum of CKD-VC patients was significantly lower than that in the healthy control group and the CKD patient group without vascular calcification. The calcification score and ACVR2b level were statistically analyzed (as shown in the table below). Correlation analysis between calcification score and ACVR2b level revealed a positive correlation between ACVR2b and calcification score, with a high correlation coefficient (AUC=1), indicating that this molecule has good diagnostic efficacy. Figure 7 The ACVR2b level used for the diagnosis of CKD-VC patients can be set according to clinical needs for specificity or sensitivity; for example, a preferred approach is 16.44 ng / ml.

[0044] Table 6. Detection results of samples from each experimental group Note: The contents of this table are clinical sample information (healthy individuals, CKD patients, and CKD patients with vascular calcification), calcification score (AU), and ACVR2b (ng / ml).

[0045] In summary, as demonstrated by the above embodiments and experimental examples, the kit of the present invention can screen individuals for CKD-VC by detecting the expression level of ACVR2b: if the ACVR2b level is low (relative to healthy individuals or CKD patients without vascular calcification), the probability of the patient having CKD-VC is higher. This invention can be used for the early clinical diagnosis of CKD-VC, exhibiting good specificity, stability, and variability, providing an effective basis for patients to take relevant treatment measures or make decisions, and has promising clinical application prospects.

Claims

1. Use of an agent for detecting ACVR2b for the manufacture of a diagnostic kit for the diagnosis of vascular calcification in chronic kidney disease.

2. Use according to claim 1, characterized in that: Said ACVR2b is Activin Receptor 2B, UniProt number Q13705.

3. Use according to claim 1, characterized in that: Said agent is an agent for detecting the expression level of ACVR2b in a human body fluid sample.

4. Use according to claim 3, characterized in that: Said body fluid sample is serum.

5. Use according to claim 1, characterized in that: Said agent is an agent for detecting the protein level of ACVR2b, selected from the group consisting of an immunohistochemistry detection agent, an immunofluorescence detection agent, a western blot detection agent, an ELISA detection agent, a chemiluminescence immunoassay, an immunochromatography or a protein chip detection method.

6. Use according to claim 1, characterized in that: Said agent is an agent for detecting the nucleic acid level of ACVR2b, selected from the group consisting of a QPCR detection agent, a digital PCR detection agent, an RT-PCR detection agent or a nucleic acid hybridization detection agent.

7. Use according to claim 1, characterized in that: Said diagnostic kit is used to distinguish between patients with vascular calcification in chronic kidney disease and healthy people.

8. Use according to claim 7, characterized in that: The threshold value of ACVR2b expression level for distinguishing between patients with vascular calcification in chronic kidney disease and healthy people is 16.44 ng / ml.

9. Use according to claim 1, characterized in that: Said diagnostic kit is used to distinguish between patients with vascular calcification in chronic kidney disease and patients with chronic kidney disease who have not developed vascular calcification.

10. Use according to claim 9, characterized in that: The threshold value of ACVR2b expression level for distinguishing between patients with vascular calcification in chronic kidney disease and patients with chronic kidney disease who have not developed vascular calcification is 16.44 ng / ml.

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