Application of glycocalyx injury marker SDC-1 as biomarker of ANCA-related vasculitis kidney affected active period

Abstract

The invention relates to the technical field of biology, in particular to application of a glycocalyx injury marker SDC-1 serving as a biomarker of ANCA-related vasculitis in the renal inflection active period. The invention discloses a biomarker capable of being used for early diagnosis and treatment scheme selection of ANCA-related vasculitis in the active period of renal inflection. The invention discloses a specific detection method for early diagnosis of ANCA-related vasculitis in the active period of renal inflection. According to the invention, high-precision and non-invasive early diagnosis of ANCA-related vasculitis in the active period of renal inflection can be realized, and the method has important significance for judging and predicting the progress of ANCA-related vasculitis renal inflection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the glycocalyx damage marker SDC-1 as a biomarker for active renal involvement in ANCA-associated vasculitis. Background Technology

[0002] Renal involvement is common in patients with ANCA-associated vasculitis (AAV), occurring in approximately 70% of patients with granulomatous polyangiitis (GPA) and nearly 90% of patients with microscopic polyangiitis (MPA). Renal involvement typically manifests as ANCA-associated glomerulonephritis (AAGN), generally characterized by rapidly progressive glomerulonephritis. In MPA-ANCA positive patients, renal function may continue to deteriorate, leading to acute renal failure in the early stages of the disease. Pathologically, it presents as necrotizing vasculitis and crescentic glomerulonephritis, with cellular crescents being a histological feature of active renal disease and poor prognosis, requiring intensive immunosuppressive therapy. Pathological confirmation requires renal biopsy; however, AAV predominates in elderly patients, often with multiple underlying diseases or severe conditions that preclude renal biopsy. Therefore, exploring non-invasive biomarkers to predict the active phase of AAGN is of significant clinical importance for guiding treatment and predicting prognosis.

[0003] Although the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA) in AAV has been confirmed in animal studies, limitations remain in assessing disease activity. Only 64% of AAV patients show a timely correlation between continuous changes in ANCA titers and changes in disease status. In clinical practice, some patients with active AAV have low ANCA levels, while some patients in remission exhibit persistently elevated ANCA levels. Therefore, new biomarkers are needed to provide more precise information about renal involvement and disease activity. Summary of the Invention

[0004] To overcome the problems existing in the prior art, the present invention aims to provide the application of the glycocalyx damage biomarker SDC-1 as a biomarker for the active phase of ANCA-associated vasculitis (ANCA)-related renal involvement. Using the GEO database in R language, bioinformatics was employed to screen for RNA-seq-related biomarkers and differentially expressed genes in renal tissues associated with AAV renal involvement. Immunocytocyte infiltration analysis revealed a significant association between macrophage 2 (M2) and AAV. Intersection of differentially expressed genes with M2-related genes yielded M2-AAV-related genes. PPI interaction analysis categorized M2-AAV-related genes into those associated with systemic inflammatory response and M2 macrophages, and those associated with endothelial cell damage and glycocalyx interaction. Pathway enrichment revealed that M2-AAV-related genes focused on glycocalyx interaction. A prospective AAV cohort validation using the glycocalyx damage biomarker SDC-1 showed a significant increase in plasma SDC-1 levels during the active phase of MPA. Therefore, plasma SDC-1 can serve as a novel biomarker for assessing the active phase of ANCA-associated vasculitis-related renal involvement.

[0005] To achieve the above and other related objectives, a first aspect of the present invention is to provide the use of SDC-1 for preparing or screening products for detecting active ANCA-associated vasculitis with renal involvement.

[0006] In one implementation, SDC-1 is used as a biomarker.

[0007] Specifically, the ANCA-associated vasculitis renal involvement detection product is used for the assessment of the active phase of ANCA-associated vasculitis renal involvement and the selection of treatment plans.

[0008] In this invention, the detection product for active phase of ANCA-associated vasculitis with renal involvement typically includes a reagent that specifically recognizes SDC-1. The reagent that specifically recognizes SDC-1 can be selected from reagents that specifically recognize the SDC-1 protein.

[0009] In one embodiment, a reagent that specifically recognizes the SDC-1 protein is prepared or screened based on the protein sequence of the SDC-1, thereby serving as a reagent for detecting active renal involvement in ANCA-associated vasculitis.

[0010] In one embodiment, the reagent that specifically recognizes the SDC-1 protein is an antibody or ligand of the SDC-1 protein.

[0011] In one embodiment, the antibody includes a monoclonal antibody or a polyclonal antibody.

[0012] A second aspect of the present invention is the use of a reagent that specifically recognizes SDC-1 for the preparation of a kit for detecting active renal involvement in ANCA-associated vasculitis.

[0013] Specifically, the ANCA-associated vasculitis renal involvement active phase detection kit is used for the assessment of the active phase of ANCA-associated vasculitis renal involvement and the selection of treatment regimens.

[0014] In one embodiment, the reagent that specifically recognizes SDC-1 is selected from reagents that specifically recognize the SDC-1 protein.

[0015] In one embodiment, the reagent that specifically recognizes the SDC-1 protein is an antibody or ligand of the SDC-1 protein.

[0016] In one embodiment, the antibody includes a monoclonal antibody or a polyclonal antibody.

[0017] In one embodiment, the kit is applied to a human.

[0018] In one embodiment, the sample for the kit is taken from the bodily fluids of the subject of the application. As some embodiments of the invention, the sample for testing is taken from the blood or plasma of the subject of the application.

[0019] Since SDC-1 can also serve as a marker of glycocalyx damage and is associated with other diseases, combining plasma SDC-1 levels with traditional risk factors for active MPA can make the screening results for active ANCA-associated vasculitis with renal involvement more reliable.

[0020] A third aspect of the present invention is to provide a kit for detecting active ANCA-associated vasculitis with renal involvement, the kit comprising at least a reagent that specifically recognizes SDC-1.

[0021] In one embodiment, the ANCA-associated vasculitis renal involvement active phase detection kit is used for the determination of the active phase of ANCA-associated vasculitis renal involvement and the selection of treatment regimens.

[0022] In one embodiment, the reagent that specifically recognizes SDC-1 is selected from reagents that specifically recognize the SDC-1 protein.

[0023] In one embodiment, the reagent that specifically recognizes the SDC-1 protein is an antibody or ligand of the SDC-1 protein.

[0024] In one embodiment, the antibody includes a monoclonal antibody or a polyclonal antibody.

[0025] In one implementation, the kit is used on humans.

[0026] In one embodiment, the sample for the kit is taken from the bodily fluids of the subject of the application. As some embodiments of the invention, the sample for testing is taken from the blood or plasma of the subject of the application.

[0027] A fourth aspect of the present invention is to provide a method for detecting active renal involvement in ANCA-associated vasculitis, including detecting the expression level of SDC-1 in the body of the subject.

[0028] In one embodiment, SDC-1 is derived from bodily fluids. As some embodiments of the invention, SDC-1 is derived from blood samples or plasma samples.

[0029] In one implementation, the subject of the detection is a person.

[0030] Preferably, the target population for this detection method can include different groups, such as healthy individuals, patients with microscopic polyangiitis (MPA), patients with nephritis, and individuals suspected of having ANCA-associated vasculitis. Body fluid samples are collected from the subjects to be tested, and the concentrations of biomarkers in the body fluid samples are quantitatively detected.

[0031] Specifically, the sample for the detection method is taken from the bodily fluids of the subject being tested. In some embodiments of the present invention, the sample is taken from the blood or plasma of the subject being tested.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. This invention uses bioinformatics technology to screen differentially expressed genes related to kidney involvement in ANCA-associated vasculitis, namely COL1A1, COL1A2, CD163, FN1, and MS4A6A. Pathway enrichment analysis of these differentially expressed genes revealed that they focus on glycocalyx interactions. The key target of glycocalyx interactions, SDC-1, was selected as a potential target for validation.

[0034] 2. This invention, through prospective AAV cohort validation, found that SDC-1 levels were significantly elevated during MPA activity, and that SDC-1 has high diagnostic efficacy and clinical significance.

[0035] 3. Compared to the gold standard renal biopsy, it is more non-invasive, especially for patients with absolute contraindications to renal biopsy or those in relatively urgent situations, and can help determine the active phase of renal involvement in ANCA-related vasculitis. Attached Figure Description

[0036] Figure 1 The data is displayed as a volcano plot and Venn diagram showing differentially expressed genes related to AAV and MCD. (AC) Volcano plot of differentially expressed genes related to AAV and MCD in GSE 192994, GSC104948, and GSE108109 datasets, with red representing upregulated genes and blue representing downregulated genes; (D) Venn diagram of the dataset intersection.

[0037] Figure 2 The images show the immune cell infiltration analysis. (A) Immune cell infiltration analysis of GSE 192994 dataset; (B) Immune cell infiltration analysis of GSE108109 dataset; (C) Immune cell infiltration analysis of GSC104948 dataset; In the figures, ** indicates that there is a statistically significant difference between the two groups, P < 0.01; * indicates that there is a statistically significant difference between the two groups, P < 0.05.

[0038] Figure 3 The diagram shows the key gene identification and AUC analysis. (A) Venn diagram of differentially expressed genes and macrophage-related genes; (B) Interaction diagram of PPI genes in the intersection of differentially expressed genes and macrophage-related genes; (C) AUC analysis diagram of intersection genes COL1A1, COL1A2, CD163, FN1, and MS4A6A; (D) AUC analysis diagram of intersection gene binding; (E) Pathway enrichment analysis diagram of intersection genes.

[0039] Figure 4 Receiver Operating Characteristic (ROC) curve of plasma SDC-1 predicting active renal involvement in MPA. Detailed Implementation

[0040] Through research, this invention has for the first time discovered that glycocalyx-related damage proteins exhibit significant differences in renal tissue during the active phase of ANCA-associated vasculitis in humans, and that plasma SDC-1 expression levels are significantly elevated under a microscope in patients with active polyangiitis.

[0041] Based on the inventors' new findings, plasma SDC-1 levels can be conveniently detected via liquid biopsy. These results can be used to evaluate the degree of kidney involvement in ANCA-associated vasculitis and help determine the active phase of ANCA-associated vasculitis-related kidney involvement.

[0042] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0043] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0044] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECμLAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECμLAR RBIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; the series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATIN STRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECμLAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0045] This invention utilizes the GEO database in R language to screen RNA-seq-related biomarkers and differentially expressed genes in renal tissues affected by avascular endothelial atrophy (AAV) using bioinformatics. Immune cell infiltration analysis revealed a significant association between macrophage 2 (M2) and AAV. Intersection of differentially expressed genes with M2-related genes yielded M2-AAV-related genes. PPI interaction analysis categorized these genes into those associated with systemic inflammatory response and M2 macrophages, and those associated with endothelial cell damage and glycocalyx interaction. Pathway enrichment revealed that M2-AAV-related genes focused on glycocalyx interaction. Validation was performed using the glycocalyx damage marker SDC-1, showing a significant increase in plasma SDC-1 levels during periods of MPA activity.

[0046] To better understand the present invention, the following specific embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following examples.

[0047] Example 1: Differential Gene Screening and Functional Verification

[0048] Screening of differentially expressed genes

[0049] Through the public GEO database ( https: / / www.ncbi.nlm.nih.gov / geo / Analysis was performed to screen for RNA-seq-related biomarkers in renal tissue associated with AAV-related kidney involvement. The renal pathology was necrotizing vasculitis, while the control group had minimal change glomerulonephritis (MCD), meaning no obvious abnormalities were observed in the renal tissue under light microscopy. Differentially expressed genes were screened. AAV and MCD data were extracted from three microarray databases: GSE108109, GSE104948, and GSE192994, and normalized. GSE108109 identified 66 differentially expressed genes, including 51 upregulated genes and 15 downregulated genes. Figure 1 C). GSE104948 identified 331 differentially expressed genes, of which 230 were upregulated and 101 were downregulated. Figure 1 B). GSE192994 identified a total of 356 differentially expressed genes, of which 212 were upregulated and 144 were downregulated. Figure 1 A). For example Figure 1 As shown in D, the Venn diagram indicates that the three databases intersect, revealing a total of 9 differentially expressed genes, including complement factor 3 (C3), complement C3a receptor 1 (C3AR1), hemoglobin scavenger receptor (CD163), type I collagen α1 (COL1A1), type I collagen α2 (COL1A2), four-degree transmembrane protein 3 (MS4A6A), high-affinity immunoglobulin Epsilon receptor subunit γ (FCER1G), and formyl peptide receptor 3 (FPR3).

[0050] Immune cell infiltration analysis

[0051] This invention utilizes the bioinformatics algorithm of CIBERSORT (https: / / cibersortx.stanford.edu / ) to quantify the relative proportion of infiltrating immune cells in the AAV gene expression profile. Immune cell infiltration analysis was performed using 22 immune cell subtypes (LM22) and microarray datasets GSE192994, GSE 108109, and GSE 104948. The results are as follows: Figure 2 As shown in AC, macrophage 2 (M2) was significantly associated with AAV (Wilcoxon rank-sum test, P<0.05). The M2-related genes from the intersection of GSE108109 and GSE104948 were intersected with differentially expressed genes related to AAV renal involvement; the results are as follows. Figure 3 As shown in Figure A, a total of 5 differentially expressed genes were found, including COL1A1, COL1A2, CD163, FN1, and MS4A6A.

[0052] PPI Interaction Analysis

[0053] PPI protein interaction network analysis was performed on the above 5 differentially expressed genes, such as... Figure 3 As shown in B, they were divided into two subgroups: MS4A6A and CD163 were associated with acute inflammatory response and M2 macrophages, while FN1, COL1A1, and COL1A2 were associated with endothelial cell damage and glycocalyx interaction.

[0054] Diagnostic value of differentially expressed genes in ANCA-associated vasculitis

[0055] To verify the predictive value of the differentially expressed genes, bioinformatics analysis was used to predict the AUC values ​​of AAV using COL1A1, COL1A2, CD163, FN1, and MS4A6A. The results are as follows: Figure 3 As shown in CD, the AUC values ​​of the five differentially expressed genes were 0.955, 0.952, 0.948, 0.969, and 0.750, respectively. The combined AUC value of these indicators predicted AAV to be 1. A higher AUC value indicates greater diagnostic efficacy and accuracy for that indicator. WebGestalt (https: / / www.webgestalt.org / ) pathway enrichment analysis was performed on the five differentially expressed genes, and the results are as follows: Figure 3 As shown in E, the differentially expressed genes mainly focus on glycocalyx interactions. Considering that SDC-1 (Syndecan-1) is a component of the glycocalyx, it is speculated that SDC-1 may be suitable as a biomarker for the active phase of renal involvement in ANCA-associated vasculitis.

[0056] Example 2: Prospective AAV Cohort Validation

[0057] All participants were informed of the research process and the purpose of the specimens and signed informed consent forms. Demographic and clinical data for all participants were obtained through questionnaires, physical examinations, and blood tests. There were no statistically significant differences in general patient information.

[0058] Prospective AAV queue

[0059] A total of 40 patients with microscopic polyangiitis (MPA) were included, comprising 20 patients in the active phase and 20 patients in the remission phase. Additionally, 20 healthy controls and 15 IgA controls were included. Baseline analysis showed no significant gender difference among the groups, but the MPA group was older (P<0.001, Table 1). During the active phase of MPA, serum creatinine (Scr) was elevated, eGFR was low, and C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were elevated (P<0.001).

[0060] Plasma SDC-1 levels were detected using an ELISA kit (abcam, ab47352).

[0061] A) Add 100 μL of SDC-1 capture antibody to 10 mL of carbonate-coated buffer solution, and add 100 μL to a microplate and incubate overnight at 4°C.

[0062] B) Discard the liquid, pat dry with absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry with absorbent paper, and repeat the washing process twice.

[0063] C) Add 250 μL of Saturation Buffer and seal the well at room temperature for 2 hours. Discard the liquid and let it air dry on the workbench for 24 hours.

[0064] D) Set up standard wells and sample wells. Add 100 μL of standard at different concentrations to each standard well and add 100 μL of diluted sample to each sample well.

[0065] E) Add 50 μL of biotin-labeled detection antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min.

[0066] F) Discard the liquid, pat dry with absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry with absorbent paper, and repeat this washing process 5 times.

[0067] G) Add 100 μL of HRP-streptavidin dilution buffer to each well, incubate at 37°C for 20 min, and wash 3 times.

[0068] H) Add 100 μL of TMB colorimetric reagent to each well and incubate at 37°C in the dark for 15 min.

[0069] I) Add 100 μL of stop solution H2SO4 to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0070] Plotting the standard curve: In an Excel worksheet, plot the linear regression curve of the standard with the concentration of the standard on the x-axis and the corresponding OD value on the y-axis. Calculate the concentration value of SDC-1 for each sample according to the curve equation.

[0071] Experimental results

[0072] SDC-1 levels were significantly elevated during the active phase of MPA, and significantly higher than those during MPA remission, in nephritis controls, and in healthy controls (84.9±10.1 vs. 39.4±6.9 vs. 47.4±9.2 vs. 27.0±2.3 ng / ml, P<0.001). The area under the curve (AUC) for SDC-1 in predicting the active phase of MPA was 0.866 (95% CI = 0.813–0.959, P<0.001). Using plasma SDC-1 levels >40.2 ng / ml as the diagnostic criterion for the active phase of MPA, the sensitivity and specificity were 90.0% and 78.9%, respectively. These data indicate that SDC-1 is suitable as a biomarker for the active phase of ANCA-associated vasculitis with renal involvement.

[0073] Table 1. Baseline Characteristics of the AAV Validation Queue

[0074]

[0075]

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of SDC-1 for the preparation or screening of products for the detection of active ANCA-associated vasculitis with renal involvement.

2. The use according to claim 1, characterized in that, SDC-1 is used as a biomarker, and / or the ANCA-associated vasculitis renal involvement active phase detection product is used for the determination of ANCA-associated vasculitis renal involvement active phase and the selection of treatment regimens.

3. The use according to claim 2, characterized in that, The ANCA-associated vasculitis renal involvement detection product includes reagents that specifically identify SDC-1.

4. The use according to claim 3, characterized in that, The reagent that specifically recognizes SDC-1 is selected from reagents that specifically recognize proteins. Preferably, the reagent that specifically recognizes SDC-1 is selected from antibodies or ligands that specifically recognize the SDC-1 protein.

5. The use of reagents that specifically recognize SDC-1 in the preparation of a kit for detecting active renal involvement in ANCA-associated vasculitis.

6. The use according to claim 5, characterized in that, The reagent that specifically recognizes SDC-1 is selected from reagents that specifically recognize the SDC-1 protein.

7. The use according to claim 6, characterized in that, The reagent that specifically recognizes SDC-1 is selected from antibodies or ligands that specifically recognize the SDC-1 protein.

8. A kit for detecting active renal involvement in ANCA-associated vasculitis, comprising at least a reagent that specifically recognizes SDC-1.

9. The reagent kit according to claim 8, characterized in that, The reagent that specifically recognizes SDC-1 is selected from reagents that specifically recognize the SDC-1 protein.

10. The kit according to claim 9, characterized in that, The reagent that specifically recognizes the SDC-1 gene is selected from antibodies or ligands that specifically recognize the SDC-1 protein.

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