Application of urine S100A8 in diagnosis and treatment of atherosclerotic renal artery stenosis

Abstract

The invention relates to application of urine S100A8 in preparation of reagents for diagnosis, differential diagnosis, disease monitoring, mechanism research and the like of atherosclerotic renal artery stenosis. At present, diagnosis of atherosclerotic renal artery stenosis in China mainly depends on imaging examination, and is high in price and strong in subjectivity. Urine is a collection of whole body metabolites, urine collection is simple, convenient and noninvasive, the complexity and abundance of urine protein components are far lower than those of blood, and the detection rate of protein markers can be remarkably increased. Through screening and verification, it is found that compared with a healthy control group and a mild atherosclerotic renal artery stenosis patient, the expression of S100A8 in urine of a moderate and severe stenosis patient is increased, and the S100A8 can be used for diagnosis, identification, monitoring and prognosis evaluation of atherosclerotic renal artery stenosis.

Description

Technical Field

[0001] This invention relates to the application of S100A8 in urine in the preparation of reagents for the diagnosis, differential diagnosis, disease monitoring and mechanism research of atherosclerotic renal artery stenosis. Background Technology

[0002] With the aging population and the widespread use of vascular imaging technology, renal artery stenosis (RAS) is increasingly being observed in clinical practice. RAS is a major cause of hypertension and / or renal insufficiency. Studies have found that 80%–90% of RAS cases are caused by renal artery atherosclerosis. If left untreated, renal function gradually deteriorates, leading some patients to end-stage renal disease, resulting in a rise in the proportion of patients requiring dialysis from 8% in the 1980s to 16.5%. Timely detection and appropriate treatment of renal artery stenosis can correct renal function in some patients with ischemic kidney damage and improve previously difficult-to-control renal hypertension. However, atherosclerotic renal artery stenosis (ARAS) often does not present with obvious clinical symptoms, and patients are often discovered incidentally during physical examinations or when seeking medical attention for other reasons. The probability of diagnosis based solely on clinical clues is very low.

[0003] Currently, the diagnosis of ARAS mainly relies on imaging examinations, including renal artery ultrasound, CT angiography, magnetic resonance angiography, and renal artery angiography. The first three non-invasive imaging examinations are still the first choice, but these examinations involve radiation and the results are highly subjective. Renal artery angiography is an invasive examination, and patient compliance is poor. Therefore, finding a simple and rapid method for diagnosing the disease is crucial.

[0004] Urine proteins contain a wealth of information, reflecting subtle changes in different systems and organs of the body. The main task of proteomics is to identify proteins present in cells, tissues, or the body, and to analyze their structure, molecular function, and biological pathways using various techniques to interpret the information they carry. Urine proteomics, the study of the proteome in urine, is one of the most studied areas of biomarker research in recent years.

[0005] This invention investigated the changes in S100A8 protein in the urine of ARAS patients. LC-MS / MS (Liquid Chromatography Tandem-mass Spectrometry) was used to screen for differentially expressed proteins in the urine of ARAS patients. Validation showed that, compared with healthy controls and patients with mild ARAS, patients with moderate to severe ARAS had elevated S100A8 expression in their urine, which can be used for the diagnosis, identification, monitoring, and prognostic assessment of ARAS. Summary of the Invention

[0006] The purpose of this invention is to provide an application of urinary protein S100A8 in the preparation of formulations for ARAS diagnosis, differential diagnosis, disease monitoring and mechanism research.

[0007] Preferably, the amino acid sequences of S100A8 in the urine are as shown in SEQ ID NO.1-NO.4 (isoform a: 1 mslvsclsed lkvlffrwgk svgimltele kalnsiidvy hkyslikgnfhavyrddlkk). 61 lletecpqyi rkkgadvwfk eldintdgav nfqeflilvi kmgvaahkks heeshke isoform b: 1 mslvsclsed lvlffrwgks vgimltelek alnsiidvyh kyslikgnfhavyrddlkkl 61 letecpqyir kkgadvwfke ldintdgavn fqeflilvik mgvaahkksh eeshke isoform c:1 mwgksvgiml telekalnsi idvyhkysli kgnfhavyrd dlkklletecpqyirkkgad 61 vwfkeldint dgavnfqefl ilvikmgvaa hkksheeshk e isoform d:1mltelekalnsiidvyhkyslikgnfhavyrddlkklletecpqyirkkgadvwfkeldi 61 ntdgavnfqe flilvikmgv aahkkshees hke).

[0008] Preferably, the preparation is a kit for detecting the expression level of S100A8 in the urine of ARAS patients.

[0009] Preferably, the kit includes an immunization method for antigen-antibody reaction and a kit thereof, such as one or more aptamer antibodies or antibody fragments that can specifically bind to S100A8.

[0010] Preferably, the kit further includes any one or a combination of several of the following components: solid support, diluent, control, standard, quality control, detection antibody, second antibody, second antibody diluent, luminescent reagent, washing solution, colorimetric solution, and stop solution.

[0011] Preferably, the standard includes S100A8 standard and humanized labeled antibody standard; more preferably, the quality control includes S100A8 quality control and humanized labeled antibody quality control; more preferably, the solid-phase carrier includes microparticles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, microplates or affinity membranes, and other carriers with equivalent functions.

[0012] Preferably, the solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, or a similar carrier.

[0013] The inventors first collected urine samples from healthy individuals and ARAS patients, and used LC-MS / MS to collect hypoxia-related proteomic information in the urine, further validating the expression of target proteins in the urine. The results showed that S100A8 was highly expressed in the urine of ARAS patients compared to healthy individuals. Receiver operating characteristic (ROC) curve analysis showed that S100A8 has good sensitivity and specificity in the diagnosis and monitoring of ARAS.

[0014] The following describes a preferred embodiment in detail with reference to the accompanying drawings: Attached Figure Description

[0015] Figure 1 Expression of S100A8 in urine in mild ARAS, moderate to severe ARAS and normal controls.

[0016] Figure 2 This is an evaluation of the diagnostic and monitoring efficacy of S100A8 in urine for ARAS. Detailed Implementation

[0017] Example 1 Selection of research subjects and specimen collection Patients with a confirmed diagnosis of ARAS were selected as the experimental group and further divided into two subgroups based on imaging results: Group A (mild stenosis, renal artery diameter stenosis <50%), n=66; Group B (moderate to severe stenosis, renal artery diameter stenosis ≥50%), n=10 in each subgroup. Thirty-four patients admitted for hypertension during the same period, with no atherosclerotic changes in the renal arteries on imaging examinations, served as the disease control group (N group). Urine samples were collected from these patients, and urinary proteomics information was obtained using LC-MS / MS technology. Differentially expressed proteins were compared between the experimental and control groups. Functional analysis of differentially expressed proteins in urine was performed using GO and KEGG bioinformatics analysis, and the differentially expressed protein S100A8 was identified.

[0018] Example 2 Enzyme-linked immunosorbent assay (ELISA) was used to verify the expression of S100A8 in urine. Patients with a confirmed diagnosis of ARAS were selected as the experimental group and divided into two subgroups based on imaging results: Group A (mild stenosis, renal artery diameter stenosis <50%), n=44; Group B (moderate to severe stenosis, renal artery diameter stenosis >50%), n=14. Thirty patients admitted for hypertension during the same period with no atherosclerotic changes in the renal arteries on imaging examination served as the disease control group (N group). Urine samples were collected from these patients, and S100A8 protein was verified using ELISA (Enzyme-linked immunosorbent assay). The verification results showed that compared with the mild ARAS group and the disease control group, the expression level of urinary protein S100A8 was significantly increased in the moderate to severe ARAS group (P=0.003). Figure 1 As shown.

[0019] Example 3 Evaluation of the diagnostic monitoring efficacy of urine S100A8 ROC curves were constructed based on the S100A8 data from the validation group, such as... Figure 2 As shown, the area under the ROC curve for S100A8 in diagnosing ARAS is 0.822, with a sensitivity of 84.6% and a specificity of 69.7%, indicating that S100A8 has good auxiliary diagnostic value for ARAS.

[0020] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. Application of urinary S100A8 protein marker in the preparation of formulations for diagnosis, differential diagnosis, disease monitoring and mechanism research of atherosclerotic renal artery stenosis.

2. The application according to claim 1, characterized in that, The amino acid sequence of the urine S100A8 is shown in SEQ ID NO:1-4.

3. The application according to claim 1, characterized in that, The formulation is used to detect the expression level of S100A8 in the urine of patients with atherosclerotic renal artery stenosis.

4. The application according to claim 3, characterized in that, in, The preparation includes an antigen-antibody detection immunoassay reagent; preferably, it includes a urine antigen-antibody detection immunoassay reagent.

5. The application according to claim 4, characterized in that, The reagent kit prepared by the reagent includes an immunoassay method for antigen-antibody reaction and a kit thereof, which can specifically bind to one or more of aptamer antibodies or antibody fragments.

6. The application according to claim 5, characterized in that, The kit also includes any one or a combination of several of the following components: solid support, diluent, control, standard, quality control, detection antibody, secondary antibody, secondary antibody diluent, luminescent reagent, washing solution, colorimetric solution, and stop solution.

7. The application according to claim 6, characterized in that, The standards include S100A8 standards and humanized labeled antibody standards; the quality control products include S100A8 quality control products and humanized labeled antibody quality control products; the solid-phase carriers include microparticles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, microplates or affinity membranes, and other carriers with equivalent functions.

8. The application according to claim 6, characterized in that, The solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, or a similar carrier.

Images