Marker for lupus nephritis and application thereof

Abstract

The invention discloses a lupus nephritis marker and application thereof, and belongs to the technical field of biomarkers. The marker for the lupus nephritis, disclosed by the invention, comprises one or more of CD8, CD38 and PD-1. By detecting specific cell surface markers in patient blood, early diagnosis of LN is assisted in a minimally invasive mode, and based on deep comparative analysis of LN patients and healthy people, the specificity and sensitivity of the biomarkers in LN diagnosis are ensured through screening of the biomarkers. In the aspect of differential diagnosis, a clinician is assisted to distinguish LN from other types of kidney diseases, such as membranous nephropathy (MN) and anti-neutrophil cytoplasm antibody related vasculitis (AAV), by analyzing specific biomarker modes of different nephropathy. The dependence on kidney puncture is reduced, related potential risks and inconvenience are avoided, and meanwhile more accurate and timely diagnosis is provided for an SLE patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarkers, and in particular relates to markers of lupus nephritis and applications thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organ systems, particularly women of childbearing age. Lupus nephritis (LN), one of the most common and serious complications of SLE, affects approximately 50% of SLE patients and is a leading cause of death in SLE patients. Early identification of kidney damage in SLE patients is crucial for improving prognosis.

[0003] Currently, the initial clinical diagnosis of lupus nephritis (LN) primarily relies on indicators such as urine protein, urine protein-to-creatinine ratio, and cellular casts, but pathological biopsy after renal puncture remains the gold standard for diagnosis. However, this invasive procedure is highly invasive for patients, has low patient acceptance, and results are delayed. Before a diagnosis of systemic lupus erythematosus (SLE) is confirmed, urine protein and creatinine testing are essential tools for diagnosing LN. While accurate, 24-hour urine protein quantification is complicated, can lead to poor patient compliance, and sample contamination, which can affect interpretation. Urine creatinine testing is significantly affected by diet and can fluctuate significantly during the day. Therefore, the search for less invasive and simpler diagnostic methods is urgent. Furthermore, numerous immune diseases involving the kidneys are clinically recognized, such as membranous nephropathy (MN) and antineutrophil cytoplasmic antibody-associated vasculitis (AAV), necessitating the need to differentiate LN from other renal diseases when diagnosing early-stage LN.

[0004] Given the limitations of existing diagnostic methods, a new diagnostic approach is needed to reduce reliance on renal puncture, avoid the potential risks and inconveniences associated with it, and provide SLE patients with a more accurate and timely diagnosis. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide markers for lupus nephritis and their applications. The present invention assists in the early diagnosis of LN in a minimally invasive manner by detecting specific cell surface markers in the patient's blood. This not only reduces the dependence on renal puncture, but also avoids the potential risks and inconveniences associated with it, while providing more accurate and timely diagnosis for SLE patients.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides markers for lupus nephritis, including one or more of CD8, CD38, and PD-1.

[0008] In a second aspect, the present invention provides the use of the above-mentioned markers in products for diagnosing lupus nephritis.

[0009] Preferably, the product includes a kit and a chip.

[0010] In a third aspect, the present invention provides the aforementioned markers for evaluating the disease status, disease activity, and treatment responsiveness of lupus nephritis.

[0011] In a fourth aspect, the present invention provides a method for detecting lupus nephritis for non-diagnostic purposes, comprising the following steps:

[0012] (1) Take 2.5 mL of EDTA-anticoagulated whole blood from the patient and dilute it to 5 mL with PBS. Add 5 mL of ficoll and 5 mL of diluted blood sample to a 15 mL centrifuge tube, and then centrifuge.

[0013] (2) After the centrifugation in step (1), aspirate the PBMCs and add PBS to the centrifuge volume of 15 mL;

[0014] (3) After the centrifugation in step (2), remove the supernatant, add 5 mL of red blood cell lysis buffer and gently blow the cells apart. After 2 minutes, add PBS to 15 mL and centrifuge;

[0015] (4) After centrifugation in step (3), remove the supernatant and resuspend the cells in 100 μL flow cytometry staining buffer. Then, filter the cell suspension using a 70-mesh sieve and transfer it to a 1.5 mL eppendorf tube. Add 4 μL HumanTruStainFcX to each tube. TM , incubate at room temperature for 5 min;

[0016] Then, add 3 μL of APC anti-human CD8a Antibody, 3 μL of PE / Cyanine7 anti-human CD38 Antibody, and 3 μL of FITC anti-human CD279 Antibody, mix gently, and incubate at 4°C for 25 min.

[0017] (5) After the incubation in step (4), 1 mL of PBS was added to each tube to wash the unbound antibodies, the supernatant was discarded by centrifugation, 300 μL of PBS was added to each tube, and the tubes were transferred to flow tubes. The fluorescence compensation adjustment was completed on the flow cytometer, and the test results were analyzed. The flow cytometric detection method was used for analysis and detection. If the proportion of PD-1+CD38+CD8+T cells detected was greater than 0.4136% of the total lymphocyte proportion, LN was confirmed, with a sensitivity of 79.10% and a specificity of 84.21%.

[0018] Preferably, the absorption wavelength and emission wavelength of the fluorescent dye are in the range of 300 nm to 810 nm.

[0019] Preferably, the fluorescent dyes include: APC, FITC, and PE-Cy7.

[0020] Contains at least the following beneficial technical effects:

[0021] The present invention assists in the early diagnosis of LN in a non-invasive manner by detecting specific cell surface markers in the patient's blood. Based on in-depth comparative analysis of LN patients and healthy people, through the screening of biomarkers, its specificity and sensitivity in LN diagnosis are ensured. The results show that it has excellent accuracy and sensitivity in distinguishing healthy controls (HC) from other kidney-involving diseases (MN, AAV).

[0022] In differential diagnosis, analysis of biomarker patterns specific to different kidney diseases reveals significant correlations with multiple disease activity assessment indicators. The more severe the kidney damage, the higher the proportion of these cells. Patients with higher levels of these markers are more likely to achieve a good response to treatment and can be used to predict the prognosis of lupus nephritis in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 is a flow cytometry application strategy diagram for three markers.

[0024] Figure 2 The results of flow cytometric analysis of 92 LN patients, 57 HC controls, 100 MN patients, and 73 AAV patients in the examples are shown.

[0025] Figure 3 To analyze the correlation between clinical indicators used to evaluate disease activity in patients with lupus nephritis and CD8+CD38+PD1+T cells.

[0026] Figure 4 The composition is used to predict the prognosis of lupus nephritis to explore the results of CD8+CD38+PD1+T cells in different groups, including patients who responded to treatment (response) and patients who did not respond to treatment (non-response). DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Unless otherwise specified, the "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C.

[0033] Unless otherwise specified, all raw materials and instruments used in the following examples of the present invention are commercially available.

[0034] Example

[0035] Participants: 92 patients with lupus nephritis (LN) and 57 healthy subjects (HC)

[0036] (1) Take about 2.5 mL of EDTA-anticoagulated whole blood from the patient and dilute it to 5 mL with PBS. Add 5 mL of ficoll to a 15 mL centrifuge tube, then slowly layer 5 mL of the diluted blood sample, and then centrifuge at 1440 RPM for 20 min.

[0037] (2) After centrifugation, four layers can be observed in the centrifuge tube: from top to bottom, the plasma and platelet layer, the PBMC layer, the ficol layer, the polymorphonuclear leukocyte layer, and the red blood cell layer. The PBMC (buffy coat layer) is aspirated, of which 70-90% are lymphocytes (70-90%), of which T cells account for the vast majority of lymphocytes (45-70%). PBS is then added to 15 mL, and the tube is centrifuged at 500 x g for 5 min.

[0038] (3) After centrifugation, remove the supernatant and add about 5 mL of red blood cell lysis buffer to gently blow the cells apart. After 2 minutes, add PBS to 15 mL and then centrifuge at 500×g for 5 minutes.

[0039] (4) After centrifugation, remove the supernatant and resuspend the cells in about 100 μL of flow cytometry staining buffer. Then, filter the cell suspension using a 70-mesh sieve and transfer it to a 1.5 mL eppendorf tube and add Human TruStain FcX TM (Fc Receptor Blocking Solution), add 4 μL to each tube and incubate at room temperature for 5 minutes; then add 3 μL of APC anti-human CD8a Antibody, 3 μL of PE / Cyanine7 anti-human CD38 Antibody, and 3 μL of FITC anti-human CD279 (PD-1) Antibody, respectively, mix gently, and incubate at 4°C for 25 minutes.

[0040] (5) After incubation, add 1 mL of PBS to each tube to wash away unbound antibodies and centrifuge at 0.5 x g for 5 min. Discard the supernatant, add 300 μL of PBS to each tube, transfer to a flow cytometer, and perform flow cytometric analysis. Complete fluorescence compensation adjustment and analyze the test results. All test indicators are strictly set up with blank, isotype, FMO, negative, and positive control groups.

[0041] Flow cytometry gating and analysis methods Figure 1 According to the conventional clinical flow cytometer detection method, 2-5×10 6 Resuspend the cells at a concentration of 100 cells / mL, and add the primary antibody (0.1-10 μg / mL) according to the recommended reference value range in the instructions and incubate at room temperature or 4°C in the dark for 30 minutes. This example uses flow cytometry for detection. Commercial companies can provide a variety of primary antibodies with self-fluorescent labels. The principle of antibody selection is to use one fluorophore per channel. The fluorophores between channels are reasonably allocated according to the strength of antigen expression and the fluorophores with small spectral overlap are selected. An optimized fluorophore-antibody pairing scheme can be used.

[0042] 1. The test results of 92 LN patients and 57 HC were analyzed. Figure 2 A confirmed that the ratio of CD8+CD38+PD1+T cells in lupus patients was significantly increased compared with that in healthy controls; 69 LN patients and 22 HC were selected again for flow cytometry testing. The test results of 92 LN patients and 57 HC were used as the training set, and the test results of 69 LN patients and 22 HC were used as the test set. The ROC curve (see Figure 2 B) Demonstrates the effectiveness of CD38+PD-1+CD8+ T cells in distinguishing LNs from HCs.

[0043] 2. 100 MN patients and 73 AAV patients were selected for flow cytometry testing. The test results (see Figure 2 C) This indicates that the CD8+CD38+PD1+T cell index is significantly higher than that of other types of kidney disease, and can effectively distinguish LN, MN, and AAV.

[0044] Figure 2 Figure D shows the receiver operating characteristic (ROC) curve for the differential diagnosis of LN and MN using the above flow cytometry data. 70% of the LN and MN flow cytometry data were used as the training set, and 30% as the test set. The training set consisted of MN (n=70) and LN (n=64), while the validation set consisted of MN (n=30) and LN (n=28). This demonstrates the effectiveness of CD8+CD38+PD1+ T cells in differentiating LN from MN.

[0045] Figure 2 Figure E shows the receiver operating characteristic (ROC) curve for the differential diagnosis of LN and AAV using the above flow cytometry data. 70% of the LN and AAV flow cytometry data were used as the training set, and 30% as the test set. The training set consisted of AAV (n=51) and LN (n=64), while the validation set consisted of AAV (n=22) and LN (n=28). This demonstrates the effectiveness of CD8+CD38+PD1+ T cells in differentiating LN from AAV.

[0046] In summary, the CD8+CD38+PD1+T indicator can be used clinically to evaluate the disease status of lupus nephritis and for early warning and / or differential diagnosis of lupus nephritis.

[0047] 3. We analyzed 92 patients with follow-up data. 48 of them were tested once at baseline and subsequently 86 times, while the remaining 44 patients were not followed up. Therefore, a total of 44+48+86=178 data points were obtained. Correlation analysis of commonly used clinical indicators of lupus nephritis with CD8+CD38+PD1+T cells showed a significant correlation, see Figure 3 .

[0048] Figure 3Middle A represents the correlation between the proportion of CD8+CD38+PD-1+T cells in peripheral blood and SLEDAI score in patients with lupus nephritis (n=175);

[0049] B represents the correlation between the proportion of CD8+CD38+PD-1+T cells in peripheral blood and urine NAGase (U-NAG) in patients with lupus nephritis (n=52);

[0050] C represents the correlation between the ratio of CD8+CD38+PD-1+ T cells in peripheral blood and urine creatinine (Ucr) in patients with lupus nephritis (U-Cr) (n=111);

[0051] D represents the correlation between the ratio of CD8+CD38+PD-1+ T cells in peripheral blood and urinary retinol-binding protein (U-PBP) in patients with lupus nephritis U-RBP (n=80);

[0052] E represents the correlation between the proportion of CD8+CD38+PD-1+T cells in the peripheral blood of patients with lupus nephritis and complement C3 (n=159);

[0053] F represents the correlation between the proportion of CD8+CD38+PD-1+T cells in the peripheral blood of patients with lupus nephritis and complement C4 (n=159);

[0054] G represents the correlation between the proportion of CD8+CD38+PD-1+T cells in the peripheral blood of patients with lupus nephritis and serum albumin (ALB) alb (n=171).

[0055] The above SLEDAI scores, C3, and C4 are indicators for evaluating SLE disease activity; while urine NAG, urine creatinine, urine retinol-binding protein, and serum albumin reflect the degree of kidney damage.

[0056] In summary, through the comparison of clinical indicators, it was found that the more severe the kidney damage, the higher the proportion of CD8+CD38+PD-1+T cells. Therefore, this indicator can be used to clinically evaluate the activity of lupus nephritis patients.

[0057] 4. 48 patients with follow-up data were divided into two groups: patients who responded after treatment (all using conventional clinical treatment methods) (responders) and patients who did not respond after treatment (non-responders). Among them, LN patients responded (n=32) and LN patients were non-responders (n=16). The initial CD38+PD-1+CD8+T cell data of the two groups of patients were analyzed. Figure 4By comparing the baseline data, it can be found that the baseline CD8+CD38+PD1+T ratio of responsive patients was significantly higher than that of non-responsive patients, indicating that if the index of LN patients is higher at the beginning, they are more likely to achieve good treatment responsiveness. This index can be used to predict the prognosis of lupus nephritis in clinical practice.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A marker of lupus nephritis, characterized in that Including one or more of CD8, CD38, and PD-1.

2. Use of the marker according to claim 1 in a product for diagnosing lupus nephritis.

3. The use according to claim 2, characterized in that The products include test kits and chips.

4. The marker of claim 1 is used to evaluate the disease status, disease activity, and treatment responsiveness of lupus nephritis.

5. A method for detecting lupus nephritis for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Take 2.5 mL of EDTA-anticoagulated whole blood from the patient and dilute it to 5 mL with PBS. Add 5 mL of ficoll and 5 mL of diluted blood sample to a 15 mL centrifuge tube, and then centrifuge. (2) After the centrifugation in step (1), aspirate the PBMCs, add PBS to 15 mL, and centrifuge; (3) After the centrifugation in step (2), remove the supernatant, add 5 mL of red blood cell lysis buffer and gently blow the cells apart. After 2 minutes, add PBS to 15 mL and centrifuge; (4) After centrifugation in step (3), remove the supernatant and resuspend the cells in 100 μL flow cytometry staining buffer. Then, filter the cell suspension using a 70-mesh sieve and transfer it to a 1.5 mL eppendorf tube. Add 4 μL HumanTruStainFcX to each tube. TM , incubate at room temperature for 5 min; Then, add 3 μL of APC anti-human CD8a Antibody, 3 μL of PE / Cyanine7 anti-human CD38 Antibody, and 3 μL of FITC anti-human CD279 Antibody, mix gently, and incubate at 4°C for 25 min. (5) After the incubation in step (4), 1 mL of PBS was added to each tube to wash the unbound antibodies, and the supernatant was discarded by centrifugation. 300 μL of PBS was added to each tube, and the tubes were transferred to flow tubes. The fluorescence compensation adjustment was completed on the flow cytometer, and the test results were analyzed. The flow cytometric detection method was used for analysis and detection. If the proportion of PD-1+CD38+CD8+T cells detected was greater than 0.4136% of the total lymphocyte proportion, lupus nephritis was confirmed, with a sensitivity of 79.10% and a specificity of 84.21%.

6. The method according to claim 5, characterized in that The absorption and emission wavelengths of fluorescent dyes range from 300 nm to 810 nm.

7. The method according to claim 6, characterized in that The fluorescent dyes include: APC, FITC, and PE-Cy7.

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