A biomarker for membranous nephropathy and its application

Abstract

This invention provides the use of a reagent for detecting Effector CD4+ T cells in peripheral blood of subjects in the preparation of products for diagnosing membranous nephropathy, lupus nephritis, or ANCA-associated vasculitis. This invention also provides the use of peripheral blood Effector CD4+ T cells in the preparation of diagnostic models for membranous nephropathy, lupus nephritis, or ANCA-associated vasculitis. This invention detects this biomarker by flow cytometry, and the results show that Effector CD4+ T cells are significantly elevated in patients with membranous nephropathy, effectively distinguishing membranous nephropathy from healthy controls (validation set AUC 0.8098), and can help differentiate membranous nephropathy from lupus nephritis (validation set AUC 0.8686) and ANCA-associated vasculitis (validation set AUC 0.8968), and its level is correlated with the refractory nature of membranous nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a detection marker, specifically a peripheral blood cell biomarker for membranous nephropathy (MN) and its application in diagnosis, differential diagnosis and disease assessment. Background Technology

[0002] Membranous nephropathy is a common cause of nephrotic syndrome in adults, characterized by the deposition of immune complexes on the epithelial side of the glomerular basement membrane. Currently, the gold standard for diagnosing membranous nephropathy still relies on renal biopsy pathology. However, renal biopsy is an invasive procedure with certain risks, such as bleeding and infection, and it is not suitable for or acceptable to all patients. Furthermore, it is difficult to use for frequent disease monitoring.

[0003] Clinically, the diagnosis of kidney disease also requires differentiation from other kidney diseases with similar clinical manifestations, such as lupus nephritis (LN) and ANCA (anal nephritis). Antineutrophil cytoplasmic antibodies Acute vasculitis (AAV) and other related conditions. Accurate differential diagnosis is crucial for guiding treatment plans.

[0004] Furthermore, some patients with membranous nephropathy do not respond well to conventional treatment and progress to refractory membranous nephropathy. Identifying these patients and providing early intervention is crucial for improving prognosis. Therefore, there is an urgent clinical need to develop minimally invasive, easy-to-use, highly specific and sensitive biomarkers for the early auxiliary diagnosis of membranous nephropathy, differential diagnosis from other kidney diseases, and assessment of disease severity or prognosis. This would reduce reliance on renal biopsies and provide patients with more timely and accurate individualized treatment strategies. Summary of the Invention

[0005] In view of the above-mentioned technical problems in the prior art, the present invention provides a biomarker for membranous nephropathy and its application. This biomarker and its application aim to solve the technical problem that the gold standard for the diagnosis of membranous nephropathy, lupus nephritis or ANCA-associated vasculitis in the prior art relies on invasive renal biopsy, and lacks effective non-invasive differential diagnosis and disease assessment methods.

[0006] This invention provides the use of a reagent for detecting Effector CD4+ T cells in the peripheral blood of a subject in the preparation of products for the diagnosis of membranous nephropathy, lupus nephritis, or ANCA-associated vasculitis.

[0007] Furthermore, the diagnosis includes distinguishing patients with membranous nephropathy from healthy individuals by detecting the expression level of Effector CD4+ T cells in peripheral blood.

[0008] Furthermore, the diagnosis includes differentiating membranous nephropathy from lupus nephritis or ANCA-associated vasculitis by detecting the expression level of Effector CD4+ T cells in peripheral blood.

[0009] Furthermore, the diagnosis includes assessing the risk of refractory membranous nephropathy by detecting the expression level of Effector CD4+ T cells in peripheral blood.

[0010] Furthermore, the product is a flow cytometry detection antibody combination, kit, or chip.

[0011] Furthermore, the detection process is as follows:

[0012] (1) Obtain peripheral blood samples from the subjects and dilute them with PBS;

[0013] (2) Isolate peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation (e.g., Ficoll);

[0014] (3) Perform erythrocyte lysis on isolated peripheral blood mononuclear cells;

[0015] (4) Resuspend cells in flow cytometry staining buffer, add Fc receptor blocker, and then incubate with antibodies labeled with one or more fluorescent dyes to identify marker combinations for Effector CD4+ T cells;

[0016] After incubation, the cells were washed to remove unbound antibodies, and then flow cytometry was used for detection and analysis.

[0017] Furthermore, the antibody combination may include, for example, antibodies against markers such as CD3, CD4, CD25, and CD127.

[0018] Furthermore, the excitation and emission wavelength range of the fluorescent dye of the fluorescently labeled antibody is 300 nm to 810 nm.

[0019] Furthermore, the fluorescent dye is any one or a combination of two or more of APC, percp-cy5.5, PE, FITC, PE-CY7, APC-CY7, BV421, and BV510.

[0020] This invention also provides the use of peripheral blood Effector CD4+ T cells as diagnostic markers in the preparation of products for the diagnosis of membranous nephropathy, lupus nephritis, or ANCA-associated vasculitis.

[0021] This invention also provides the use of peripheral blood Effector CD4+ T cells in the preparation of diagnostic models for diagnosing membranous nephropathy, lupus nephritis, or ANCA-associated vasculitis, wherein the proportion of peripheral blood Effector CD4+ T cells in patients with membranous nephropathy is significantly higher than that in patients with lupus nephritis, ANCA-associated vasculitis, and healthy individuals.

[0022] This invention aims to assist in the diagnosis and differential diagnosis of membranous nephropathy, lupus nephritis, or ANCA-associated vasculitis in a minimally invasive manner by detecting the expression levels of specific immune cell subsets in the peripheral blood of patients, and to assess their relationship with the refractory nature of the disease, thus providing a new auxiliary means for clinical practice.

[0023] Compared with existing technologies, the present invention has the following beneficial technical effects:

[0024] 1) Non-invasive and convenient: This invention avoids the trauma and risks of traditional kidney biopsy by detecting cell markers in peripheral blood. It is simple to operate, easy for patients to accept and clinically promoted.

[0025] 2) Early diagnosis and auxiliary diagnosis: This invention found that Effector CD4+ T cells were significantly elevated in patients with membranous nephropathy, which could effectively distinguish patients with membranous nephropathy from healthy controls (training set AUC: 0.8511, validation set AUC: 0.8098), providing new evidence for the early diagnosis and auxiliary diagnosis of membranous nephropathy.

[0026] 3) Differential Diagnostic Capability: The Effector CD4+ T cell marker in this invention can not only diagnose membranous nephropathy, but also effectively assist in differentiating membranous nephropathy from lupus nephritis (training set AUC: 0.8540, validation set AUC: 0.8686) and membranous nephropathy from ANCA-associated vasculitis (training set AUC: 0.8363, validation set AUC: 0.8968), which helps in the accurate differentiation of different kidney diseases in clinical practice.

[0027] 4) Potential for disease assessment: This invention found that the Effector CD4+ T cell level in patients with refractory membranous nephropathy was significantly higher than that in non-refractory patients, suggesting that this biomarker may be used to assess the risk of refractory membranous nephropathy or guide treatment.

[0028] 5) The biomarkers and their detection methods provided by this invention offer new technical means for non-invasive auxiliary diagnosis, differential diagnosis and disease assessment of membranous nephropathy, and have important clinical application value. Attached Figure Description

[0029] Figure 1 The gating and analytical methods for flow cytometry detection are shown.

[0030] Figure 2 This study compares the expression levels of Effector CD4+ T cells in peripheral blood of healthy controls (HC), patients with membranous nephropathy (MN), lupus nephritis (LN), and ANCA-associated vasculitis (AAV), and includes information on the composition of the study cohorts (HC 57, MN 100, LN 176, AAV 73; training set 70%, validation set 30%).

[0031] Figure 3 This study compared the expression levels of Effector CD4+ T cells in peripheral blood between patients with refractory membranous nephropathy and those with non-refractory membranous nephropathy.

[0032] Figure 4 ROC curve analysis results (training set AUC and validation set AUC) for Effector CD4+ T cell diagnosis of membranous nephropathy (relative to healthy controls).

[0033] Figure 5 ROC curve analysis results (training set AUC and validation set AUC) of Effector CD4+ T cells in the differential diagnosis of membranous nephropathy and lupus nephritis.

[0034] Figure 6 ROC curve analysis results (training set AUC and validation set AUC) of Effector CD4+ T cells in the differential diagnosis of membranous nephropathy and ANCA-associated vasculitis. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0041] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0042] Example 1

[0043] I. Research Subjects

[0044] The individuals included in the study were: 57 healthy controls (HC), 100 patients with membranous nephropathy (MN), 176 patients with lupus nephritis (LN), and 73 patients with ANCA-associated vasculitis (AAV). All enrolled patients met the corresponding diagnostic criteria. All individuals were divided into a training set (70%) and a validation set (30%) for model construction and evaluation.

[0045] All data from the subjects were obtained with the approval of the ethics review committee and complied with the regulations of the national ethics review system.

[0046] II. Sample Collection and Processing

[0047] 1. Sample collection: Collect 2-3 mL of peripheral venous blood from the subject and use EDTA anticoagulation tubes for anticoagulation.

[0048] 2. PBMC separation: Dilute EDTA-anticoagulated whole blood with an equal volume of PBS and slowly add it onto an equal volume of Ficoll-Paque PLUS separation solution. Centrifuge at a horizontal speed (e.g., 1800-2000 rpm, 20-25 minutes, room temperature, then turn off the centrifuge and brake).

[0049] 3. PBMC collection and washing: Carefully aspirate the mononuclear cell (PBMC) layer into a new centrifuge tube, add PBS to resuspend and centrifuge to wash the cells (e.g., 1500 rpm, 5-10 minutes).

[0050] 4. Red blood cell lysis: Discard the supernatant, add an appropriate amount of red blood cell lysis buffer (e.g., 1X RBC Lysis Buffer) to lyse the red blood cells, incubate for several minutes, then add PBS to stop the lysis, and centrifuge to wash the cells.

[0051] 5. Cell resuspending: Discard the supernatant, resuspend the cells in flow cytometry staining buffer (e.g., PBS containing BSA and NaN3), and adjust the cell concentration to approximately 1-5 × 10^6 cells / mL.

[0052] III. Flow Cytometry Detection

[0053] 1. Fc receptor blockade: Take the cell suspension with the adjusted concentration, add an appropriate amount of Fc receptor blocker (such as HumanTruStain FcX™) to each tube, and incubate at room temperature for 5-10 minutes.

[0054] 2. Antibody staining: According to the experimental design, add a combination of fluorescently labeled antibodies targeting the surface markers of the target cells.

[0055] For the detection of Effector CD4+ T cells: Staining with antibodies containing a combination of anti-CD3, anti-CD4, anti-CD25, and anti-CD127 can be used to differentiate the total CD4+ T cell population and further identify the Effector subset. All antibodies are added at the recommended concentration, gently mixed, and incubated at 4°C in the dark for 25-30 minutes.

[0056] 3. Washing and resuspending: After incubation, wash the cells with flow cytometry staining buffer, centrifuge and discard the supernatant. Resuspend the cells with an appropriate amount of flow cytometry staining buffer.

[0057] 4. Analytical Processing: Transfer the cell suspension to flow cytometry tubes and perform analysis using a flow cytometer (such as BD FACSCanto II, Beckman CytoFLEX, etc.). Set appropriate voltage and compensation, and collect a sufficient number of cell events for analysis.

[0058] IV. Data Analysis and Results

[0059] 1. Gating and analytical methods for flow cytometry detection ( Figure 1 ).

[0060] The steps are as follows: Step 1: Identify Lymphocytes (top left): FSC-A vs SSC-A. This is the first step in flow cytometry analysis, aiming to identify the lymphocyte population from all cells. The X-axis (FSC-A) represents the forward scattering angle, approximately equal to the cell size. The Y-axis (SSC-A) represents the lateral scattering angle, approximately equal to the cell granularity or internal complexity. Based on the typical characteristics of cell size and granularity, a cell population is identified and labeled as Lymphocytes, accounting for 25.9% of the total cells. This forms the basis for all subsequent analyses. Step 2: Remove Single Cells (top center): FSC-A vs FSC-H. This step ensures that we are analyzing single cells, not two or more cells stuck together (called "adhesive bodies" or "hybrids"). Single cells and adhesive cells can be separated by analyzing the area (FSC-A) and height (FSC-H) of the signal. The cell population circled on the diagonal in the figure is labeled "Single Cells," accounting for 98.3% of the lymphocytes. Subsequent analysis will focus solely on these single cells. Step 3: Identifying CD4+ T cells (CD4T) (top right): CD3 vs CD4. This step identifies the CD4+ T cells (also called helper T cells) of interest within the single lymphocytes. The X-axis (CD3) is a universal surface marker for all T cells. The Y-axis (CD4) is a specific surface marker for helper T cells. The cell population selected in the figure is CD3+ and CD4+, labeled CD4T. This cell population accounts for 48.3% of the previous level of cells (single cells). Step 4: Differentiating between effector T cells and regulatory T cells (TEFF vs TREG) (bottom right): CD25 vs CD127. This is a crucial step, further dividing the CD4+ T cells obtained in the previous step into two main functional subsets: effector T cells (TEFF) and regulatory T cells (TREG). Both the X-axis (CD25) and Y-axis (CD127) are important immune cell surface receptors. A typical characteristic of effector T cells (TEFF) is high CD127 expression. The circled TEFF in the figure represents 90.9% of CD4+ T cells. A typical characteristic of regulatory T cells (TREG) is high CD25 expression and low or no CD127 expression. The circled TREG in the figure represents 5.54% of CD4+ T cells.Here, TEFF specifically refers to effector T cells, which are derived from CD4+ T cells.

[0061] 1. Level of expression ( Figure 2 Flow cytometry results showed that the proportion of Effector CD4+ T cells in the peripheral blood of patients with membranous nephropathy (MN) was significantly higher than that of healthy controls (HC), and also significantly higher than that of patients with lupus nephritis (LN) and ANCA-associated vasculitis (AAV).

[0062] This study quantitatively analyzed the proportion of peripheral blood Effector CD4T cells in four groups, revealing a key feature of membranous nephropathy (MN) at the cellular immune level (see Table 1 for details).

[0063] (1) The MN group showed a consistent and significantly high level: the median proportion of effector CD4+ T cells in the membranous nephropathy patient group (n=100) was as high as 44.0%, with a mean of 43.7%. The data distribution was relatively concentrated, with the core population (i.e., the middle 50% of patients, IQR) ranging from 38.4% to 48.1%. This level significantly exceeded all other control groups, showing a unique and concentrated high expression pattern. Healthy control (HC) group (n=57): As a baseline reference, the average cell level in this group was 26.3%, with a median of 27.3%. Its core range (21.5% - 32.1%) clearly defined a normal physiological range. Lupus nephritis (LN) group (n=176): Although both are severe autoimmune nephropathy, the numerical characteristics of this group (mean 29.0%, median 29.6%) were very close to those of the healthy control group, without the significant increase seen in the MN group. ANCA-associated vasculitis (AAV) group (n=73): As another important disease control, the numerical distribution of this group (mean 29.0%, median 29.2%) was also relatively consistent with the healthy control and lupus nephritis groups.

[0064] (2) Clear intergroup boundaries indicate that the indicator has strong discriminative potential: We found a clinically significant phenomenon: the lowest 25% of patients in the membranous nephropathy group (whose effector CD4+ T cell ratio was still >38.4%) still had significantly higher cell levels than the median levels of all three control groups (approximately 27-29%). This indicates that there is a clear numerical "gap" between the membranous nephropathy patient group and the non-membranous nephropathy group (including healthy individuals and other kidney disease patients). The effector CD4+ T cell ratio is not only a statistically significant indicator, but also has the potential to become a powerful biomarker that can be directly used for clinical decision-making to distinguish membranous nephropathy from kidney damage and health status caused by other etiologies. In summary, the research data strongly demonstrate that the significant upregulation of the peripheral blood effector CD4+ T cell ratio is a key and specific immunological feature that distinguishes membranous nephropathy from lupus nephritis, ANCA-associated vasculitis, and healthy individuals, providing solid data support for the innovation and practicality of this invention.

[0065] Table 1: Summary of descriptive statistics on Effector CD4+ T cell levels in each cohort

[0066]

[0067] 2. Diagnostic efficacy ( Figure 4 ):

[0068] For the aforementioned research subjects, data analysis was performed using R software. For different diagnostic objectives, 70% of the data was used to construct the training set, and 30% was used to construct the validation set. ROC curves were constructed... Figure 4 The ability of Effector CD4+ T cells to distinguish between patients with membranous nephropathy (MN) and healthy controls was evaluated. Results showed that the proportion of Effector CD4+ T cells exhibited excellent diagnostic value in differentiating MN patients from healthy controls. Based on the training set data, the Youden index method was used to determine the optimal diagnostic cutoff. The model and the cutoff were subsequently evaluated on an independent internal validation set. The diagnostic efficacy evaluation results of the model showed high accuracy and reliability; specific performance metrics are shown in the table below.

[0069] Table 2: Summary of Effector CD4+ T Performance Evaluation in Diagnosing Membranous Nephropathy

[0070]

[0071] (Note: The optimal diagnostic threshold determined based on the training set is 0.5834)

[0072] Results Interpretation: Extremely High Discriminative Power: The model's AUC values ​​reached 0.9393 and 0.9510 on the training and validation sets, respectively. AUC is the gold standard for evaluating the superiority of diagnostic tests, and this result strongly demonstrates that the Effector CD4+ T cell level detected by this invention can very accurately distinguish membranous nephropathy patients from healthy individuals. Ideal Balance of Sensitivity and Specificity: At the optimal diagnostic threshold, the model exhibited 83.33% sensitivity (ability to correctly identify patients) and 82.35% specificity (ability to correctly identify healthy individuals) on the independent validation set. This high level of numerical values ​​indicates that the method of this invention can not only effectively avoid missed diagnoses in clinical applications but also significantly reduce the misdiagnosis rate, achieving the ideal standard for clinical diagnostic tools. Robust Generalization Ability: Notably, the model's performance metrics on the validation set (especially AUC and sensitivity) even surpassed those on the training set. This clearly shows that the diagnostic model of this invention does not suffer from overfitting and possesses extremely strong generalization ability and stability, indicating that it can maintain a high level of diagnostic performance when facing a wider and more diverse unknown patient population. In summary, this specific embodiment, through rigorous data analysis and model validation, fully demonstrates the important role of detecting peripheral blood Effector CD4+ T cell levels in the diagnosis of membranous nephropathy. The method provided by this invention possesses extremely high accuracy, excellent sensitivity and specificity, and robust reliability, offering clinicians a novel, non-invasive, and highly efficient auxiliary diagnostic tool for membranous nephropathy, with significant technical value and broad application prospects.

[0073] Furthermore, in current clinical practice, anti-phospholipase A2 receptor (Anti-PLA2R) antibody is the most widely used and recognized serological marker for diagnosing primary membranous nephropathy (MN). Clinically, an "Anti-PLA2R antibody titer >2 RU / ml" is typically used as the positive diagnostic criterion for membranous nephropathy. Although Anti-PLA2R has high specificity, some clinically diagnosed membranous nephropathy patients still test negative for Anti-PLA2R antibodies, leading to missed diagnoses. Therefore, developing novel biomarkers that can compensate for the shortcomings of existing technologies and provide higher comprehensive diagnostic efficacy has significant clinical need and value. To verify the superiority of the Effector CD4+ T cell detection method provided in this invention compared to existing gold standard technologies, we conducted a comparative study. The diagnostic performance of the two methods was evaluated separately. Anti-PLA2R used the clinically recognized >2 RU / ml as a fixed diagnostic criterion; while the method of this invention used an optimal diagnostic threshold generated by data-driven analysis. The performance comparison results of the two methods on independent validation sets are shown in the table below.

[0074] Table 3: Comparison of Diagnostic Efficacy between the Invention and Existing Technologies

[0075]

[0076] The advantages of this invention, as clearly demonstrated by the direct comparison in the table above, are primarily reflected in the following key aspects: Higher overall diagnostic efficacy: The AUC value of this invention (0.9510) is higher than that of Anti-PLA2R (0.9167). This higher AUC value directly proves that this invention possesses a stronger and more accurate ability to distinguish between patients and healthy individuals, representing the superiority of the technology. Better sensitivity, effectively reducing missed diagnoses: The sensitivity of this invention reaches 86.67%, superior to Anti-PLA2R's 83.33%. This means that in clinical applications, this invention can identify more true membranous nephropathy patients, especially those who may test negative for Anti-PLA2R. Furthermore, our study has preliminarily explored the possibility of combining this invention with Anti-PLA2R. Data shows that when the two are combined to construct a combined diagnostic model, its diagnostic efficacy can be further improved (combined model AUC = 0.9941, sensitivity = 96.67%, specificity = 100.00%). This discovery not only reaffirms the independent value of the biomarker of this invention, but also reveals its potential as a powerful supplement to the existing diagnostic system. It is expected to be combined with Anti-PLA2R to jointly build the next generation of ultra-high-precision diagnostic standards for membranous nephropathy.

[0077] 3. Differential diagnostic efficacy ( Figure 5 , 6 ):

[0078] (1) Differentiate between membranous nephropathy (MN) and lupus nephritis (LN) Figure 5 ).

[0079] Results: The proportion of CD4+ T cells in the Effector demonstrated excellent and balanced differential diagnostic performance in distinguishing between MN and LN. The AUC values ​​in the training and validation sets were as high as 0.8540 and 0.8686, respectively, showing extremely strong discriminative ability. In the validation set, all performance indicators were excellent: sensitivity was 76.7%, specificity was 86.5%, and accuracy reached 82.9%. The extremely high specificity (86.5%) is a major highlight of this application, indicating that this method can accurately exclude LN patients, thereby greatly avoiding clinical misdiagnosis.

[0080] (2) Differentiate between membranous nephropathy (MN) and ANCA-associated vasculitis (AAV) Figure 6 ).

[0081] Results: The proportion of CD4+ T cells in the effector remained effective in distinguishing between MN and AAV. The AUC values ​​on the training and validation sets reached 0.8363 and 0.8968, respectively. On the validation set, the model achieved ultra-high sensitivity of 86.7% and excellent specificity of 80.9%, with an overall accuracy of 84.3%.

[0082] 4. Relationship with intractable diseases ( Figure 3 )

[0083] Refractory membranous nephropathy refers to a specific clinical subtype in which proteinuria persists or recurs after more than 6 months of standard first-line immunosuppressive therapy (such as adequate doses of corticosteroids combined with cyclophosphamide or rituximab). These patients typically have poor treatment response and a poor prognosis, posing a significant challenge in clinical practice. Therefore, identifying biomarkers that can identify these high-risk patients early and enabling early intervention is crucial for improving their long-term renal survival.

[0084] This invention further reveals the application value of Effector CD4+ T cells as a key biomarker for identifying refractory membranous nephropathy. Current clinical practice lacks effective indicators for early and accurate prediction of whether patients with membranous nephropathy will develop resistance to standard treatment regimens, and the detection reagents and methods provided in this invention precisely fill this technological gap.

[0085] To verify this application of the invention, we constructed a study cohort of patients with membranous nephropathy who had follow-up data for more than 6 months. The cohort included 30 patients with clinically diagnosed refractory membranous nephropathy (“refractory group”) and 52 patients with non-refractory membranous nephropathy (“non-refractory group”). Using the reagents and methods provided in this invention, the percentage of Effector CD4+ T cells among lymphocytes in the peripheral blood of all subjects was quantified. Statistical analysis clearly showed a significant difference between the two groups. Specifically, the average Effector CD4+ T cell percentage in the refractory group was 45.29% (standard deviation 9.98%), significantly higher than the average of 40.10% (standard deviation 5.53%) in the non-refractory group. Independent samples t-test confirmed that this difference was highly statistically significant (t(39.46) = 2.62, p = 0.0124). These results strongly demonstrate that a significant increase in Effector CD4+ T cell levels in peripheral blood is an important indicator of the tendency of membranous nephropathy to become refractory. Therefore, the detection reagents and their supporting detection methods provided by this invention can effectively distinguish high-risk patients with refractory tendencies through quantitative assessment of the cellular level, thereby providing key decision-making basis for clinicians to formulate early and individualized intervention strategies and treatment plans, and have significant clinical application value.

[0086] In summary, the peripheral blood Effector CD4+ T cells provided by this invention show great potential in the auxiliary diagnosis, differential diagnosis, and disease assessment of membranous nephropathy, and are expected to provide valuable non-invasive detection methods for clinical use. The above description is merely a preferred embodiment of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. Detection of Effector CD4 in peripheral blood of subjects + The use of T-cell reagents in the preparation of products for diagnosing membranous nephropathy, specifically the detection of Effector CD4 in the peripheral blood of subjects. + The T-cell reagent contains antibodies against CD3, CD4, CD25, and CD127 markers, and the diagnosis includes detection of Effector CD4 in peripheral blood. + The expression level of T cells can distinguish patients with membranous nephropathy from healthy individuals, or differentiate membranous nephropathy from lupus nephritis or ANCA-associated vasculitis.

2. The use according to claim 1, characterized in that, The product is a flow cytometry detection antibody combination, kit, or chip.

3. The use according to claim 1, characterized in that, The testing process is as follows: (1) Obtain peripheral blood samples from the subjects and dilute them with PBS; (2) Peripheral blood mononuclear cells were separated by density gradient centrifugation; (3) Perform erythrocyte lysis on isolated peripheral blood mononuclear cells; (4) Resuspend cells in flow cytometry staining buffer, add Fc receptor blocker, and then incubate with antibodies labeled with one or more fluorescent dyes to identify Effector CD4. + A combination of T cell biomarkers; (5) After incubation, the cells were washed to remove unbound antibodies, and then flow cytometry was used for detection and analysis.

4. The use according to claim 3, characterized in that, The excitation and emission wavelengths of the fluorescent dye are in the range of 300 nm to 810 nm.

5. The use according to claim 3, characterized in that, The antibody combination comprises antibodies targeting CD3, CD4, CD25, and CD127 markers; the fluorescent dye is any one or a combination of two or more of APC, percp-cy5.5, PE, FITC, PE-CY7, APC-CY7, BV421, and BV510.

6. Effector CD4 in peripheral blood + The use of T cells as diagnostic markers in the preparation of products for diagnosing membranous nephropathy, peripheral blood Effector CD4 in patients with membranous nephropathy. + The proportion of T cells was significantly higher in patients with lupus nephritis, ANCA-associated vasculitis, and healthy individuals.

7. Effector CD4 in peripheral blood + The use of T cells in preparing diagnostic models for membranous nephropathy, peripheral blood Effector CD4 in patients with membranous nephropathy + The proportion of T cells was significantly higher in patients with lupus nephritis, ANCA-associated vasculitis, and healthy individuals.

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