Biomarker and biomarker combination for evaluating activity of lupus nephritis disease and application of biomarker and biomarker combination

Abstract

The invention discloses a biomarker and a biomarker combination for evaluating the activity of lupus nephritis diseases and application of the biomarker and the biomarker combination. The biomarker is one of BTN3A2, CCL23 and CKAP4 or a combination of two or more of BTN3A2, CCL23 and CKAP4. The system also comprises an LIFR. The system also comprises a TREM1. According to the present invention, the expression of LIFR, BTN3A2, CCL23, CKAP4 and TREM1 in the circulation of the active lupus nephritis patient detected by using the Olink proteomics is significantly higher than the expression of the LIFR, BTN3A2, CCL23, CKAP4 and TREM1 in the circulation of the inactive lupus nephritis patient, and the activity condition of the lupus nephritis disease can be evaluated by detecting the expression of the marker so as to formulate the more accurate treatment scheme for the lupus nephritis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a biomarker, a combination of biomarkers, and their applications for assessing the activity of lupus nephritis. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a common autoimmune disease. The kidneys are the most frequently affected organ; approximately 7–31% of patients are diagnosed with lupus nephritis at the time of SLE diagnosis, and 5–20% of patients with lupus nephritis progress to end-stage renal disease, seriously impacting public health.

[0003] Active lupus nephritis can lead to severe kidney damage and can also affect other systems. When systemic lupus erythematosus (SLE) progresses to lupus nephritis, the risk of cardiovascular disease increases twofold, and the decline in retinal vascular density and average visual acuity is more significant. When it progresses to uremia, neurological complications may occur due to damage to the nervous system caused by uremic toxins. Active lupus nephritis severely affects all systems of the body and is a major risk factor for morbidity and mortality from SLE.

[0004] Early identification and treatment of lupus nephritis (LN) can significantly impact morbidity and mortality. Therefore, determining disease activity and assessing treatment response are crucial. Renal biopsy is the "gold standard" for diagnosing lupus nephritis, assessing disease activity, evaluating prognosis, and monitoring treatment. However, renal biopsy has limitations; it is an invasive procedure with poor reproducibility. There is an urgent need to find easily measurable biomarkers with high predictive value to reflect disease activity. Studies have identified several biomarkers related to lupus nephritis, but few are truly effective non-invasive biomarkers for assessing renal histology and monitoring activity, such as:

[0005] Leukemia suppressor receptor (LIFR): As a type I cytokine receptor, LIFR participates in immune regulation by activating the JAK / STAT signaling pathway, which plays a key role in LN. Inhibiting JAK / STAT can improve kidney inflammation.

[0006] BTN3A2, a member of the lactolipin subfamily 3, affects adaptive immunity by regulating T cell function (such as inhibiting IFN-γ release). It has been shown to be associated with disease activity and treatment response in rheumatoid arthritis (RA), but its role in LN remains unclear.

[0007] CC chemokine ligand 23 (CCL23): secreted by myeloid cells, it can chemotact T cells and monocytes. Its level is elevated in diseases such as RA and inflammatory bowel disease, and may exacerbate kidney inflammation through the TLR7 / 8 and TNFα pathways.

[0008] Trigger receptor 1 (TREM1) expressed by myeloid cells: As an inflammation amplifier, TREM1 can enhance TLR / NOD-like receptor signaling, promote the release of pro-inflammatory factors, and may be involved in the excessive immune activation of LN.

[0009] Cytoskeleton-associated protein 4 (CKAP4): This protein is associated with the PI3K-AKT pathway and is upregulated in chronic kidney disease. It may accelerate the progression of LN by affecting immune cell function.

[0010] However, given the heterogeneity of lupus nephritis, the differences between detection methods, the problems with existing molecules, and the important role of inflammation and immunity in the disease, it is necessary to use precise techniques to systematically study the molecules in these patients from both inflammatory and immune perspectives, in order to find accurate and sensitive molecular markers that can reflect the activity of lupus nephritis. Summary of the Invention

[0011] To address the aforementioned problems, the present invention aims to provide a biomarker for assessing the disease activity of lupus nephritis.

[0012] A second objective of this invention is to provide a biomarker for assessing the activity of lupus nephritis in the preparation of a kit for assessing the activity of lupus nephritis.

[0013] A third objective of this invention is to provide a product for assessing the disease activity of lupus nephritis.

[0014] To achieve the above objectives, the present invention provides the following technical solution:

[0015] This invention provides a biomarker for assessing the disease activity of lupus nephritis, wherein the biomarker is one, two or more of BTN3A2, CCL23, and CKAP4;

[0016] The aforementioned biomarkers also include LIFR;

[0017] The aforementioned biomarkers also include TREM1.

[0018] Given the high heterogeneity of lupus nephritis, the differences in detection methods, and the crucial role of inflammation and immune responses in disease development, the inventors used Olink proteomics technology to systematically analyze plasma samples from lupus nephritis patients, focusing on detecting inflammation and immune-related molecules. The aim was to screen for novel biomarkers that could assess disease activity. The results showed that the expression levels of five proteins—BTN3A2, CCL23, CKAP4, LIFR, and TREM1—in circulation were significantly higher in patients with active lupus nephritis than in inactive patients and healthy controls (p<0.01). To validate the evaluative value of these candidate biomarkers, the research team expanded the sample size and used a specific human ELISA kit to quantitatively detect these molecules. Statistical analysis revealed:

[0019] The area under the receiver operating characteristic (AUC) curve for BTN3A2 was 0.806; for CCL23, it was 0.780; for CKAP4, it was 0.765; for LIFR, it was 0.815; and for TREM1, it was 0.775.

[0020] The AUC of the BTN3A2 and CCL23 combination was 0.870.

[0021] The AUC of the BTN3A2 and CKAP4 combination was 0.855.

[0022] The AUC of the CCL23 and CKAP4 combination was 0.876.

[0023] The AUC of the BTN3A2 and LIFR combination was 0.900.

[0024] The AUC of the three-molecule combination of BTN3A2, CCL23, and CKAP4 was 0.91.

[0025] The AUC of the four-molecule combination of BTN3A2, CCL23, CKAP4 and LIFR was 0.932.

[0026] The five-molecule combination evaluation model of BTN3A2, CCL23, CKAP4, LIFR and TREM1 showed the best evaluation power, with an AUC of 0.955.

[0027] The above results indicate that the aforementioned molecular markers and combinations of markers have extremely high accuracy in assessing the activity of lupus nephritis and can be used as biomarkers for assessing the activity of lupus nephritis.

[0028] The present invention also provides the use of the biomarkers as described above and combinations thereof in the preparation of a kit for assessing the disease activity of lupus nephritis.

[0029] The expression levels of the biomarkers were obtained by at least one of Olink proteomics, adjacent extension technology, and enzyme-linked immunosorbent assay (ELISA).

[0030] The present invention also provides a product for assessing the disease activity of lupus nephritis, the product comprising a kit for detecting the expression levels of the above-mentioned biomarkers.

[0031] This invention has significant advantages: the biomarkers of this invention can accurately assess the disease activity of patients with lupus nephritis.

[0032] Five molecules were used to assess disease activity in patients with lupus nephritis. The AUC of LIFR was 0.815, the Youden index was 0.518, the sensitivity was 86.67%, and the specificity was 65.12%; the AUC of BTN3A2 was 0.806, the Youden index was 0.560, the sensitivity was 67.35%, and the specificity was 88.64%; the AUC of CCL23 was 0.780, the Youden index was 0.489, the sensitivity was 72.34%, and the specificity was 76.60%; the AUC of CKAP4 was 0.765, the Youden index was 0.407, the sensitivity was 87.50%, and the specificity was 53.19%; and the AUC of TREM1 was 0.775, the Youden index was 0.470, the sensitivity was 72.00%, and the specificity was 75.00%.

[0033] The combination of two molecules, BTN3A2 and CCL23, was evaluated, with an AUC of 0.870, a sensitivity of 82.61%, and a specificity of 80.95%.

[0034] The combination of two molecules, BTN3A2 and CKAP4, was evaluated, with an AUC of 0.855, a sensitivity of 97.87%, and a specificity of 56.82%.

[0035] The combination of CCL23 and CKAP4 was evaluated, and the AUC reached 0.876, with a sensitivity of 80.00% and a specificity of 80.00%.

[0036] The combination of BTN3A2 and LIFR molecules was evaluated, with an AUC of 0.900, a sensitivity of 84.09%, and a specificity of 86.84%.

[0037] The combination of three molecules, BTN3A2, CCL23, and CKAP4, was evaluated, achieving an AUC of 0.911, a sensitivity of 90.91%, and a specificity of 76.19%.

[0038] The combination of four molecules, LIFR, BTN3A2, CCL23, and CKAP4, was evaluated, achieving an AUC of 0.932, a sensitivity of 100.00%, and a specificity of 70.27%.

[0039] The combined assessment of five molecules—LIFR, BTN3A2, CCL23, CKAP4, and TREM1—achieved an AUC of 0.955, a sensitivity of 82.50%, and a specificity of 94.59%. These biomarkers and their combinations can effectively identify the activity level of lupus nephritis patients, enabling more precise treatment plans and overcoming the limitations of existing non-invasive detection methods for active lupus nephritis. Attached Figure Description

[0040] Figure 1 Heatmap of the expression of five molecules BTN3A2, CCL23, CKAP4, LIFR and TREM1 in the active lupus nephritis (ALN) group, inactive lupus nephritis (ILN) group and control (CON) group.

[0041] Figure 2 Differential expression of five molecules, BTN3A2, CCL23, CKAP4, LIFR and TREM1, in ALN1, ILN1 and CON1 among groups;

[0042] Figure 3 The AUC of five molecules BTN3A2, CCL23, CKAP4, LIFR and TREM1 in the ALN1 and ILN1 groups;

[0043] Figure 4 The dynamic changes of five molecules, BTN3A2, CCL23, CKAP4, LIFR and TREM1, during disease remission in lupus nephritis;

[0044] Figure 5 The concentrations of five molecules, BTN3A2, CCL23, CKAP4, LIFR, and TREM1, in ALN2, ILN2, and CON2;

[0045] Figure 6 The concentrations of five molecules, BTN3A2, CCL23, CKAP4, LIFR, and TREM1, at different treatment response points in the follow-up cohort;

[0046] Figure 7 The AUC for distinguishing between the ALN2 group and the ILN2 group for five molecules BTN3A2, CCL23, CKAP4, LIFR and TREM1;

[0047] Figure 8 The correlation between five molecules and clinical indicators of lupus nephritis. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0050] Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment;

[0051] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0052] In this application, the disease activity of LN was assessed based on the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K). Those with SLEDAI-2K ≥12 were in the active group (ALN), and those with SLEDAI-2K ≤8 were in the inactive group (ILN). Age- and sex-matched healthy controls (CON) were also included.

[0053] In this application, the term "active" refers to a SLEDAI-2K score ≥12 for LN patients, and "inactive" refers to a SLEDAI-2K score ≤8 for LN patients.

[0054] In this application, the criteria for evaluating the efficacy of lupus nephritis (LN) are defined according to the "Guidelines for the Diagnosis and Treatment of Lupus Nephritis in China". The baseline represents the time point at which the patient underwent a renal biopsy upon admission. Partial remission (PR) is defined as a decrease in urinary protein exceeding 50% of the baseline value, with urinary protein quantification <3.0g / 24h, serum creatinine (SCr) normal or an increase not exceeding 10% of the baseline value, and serum albumin >30g / L. Complete remission (CR) is defined as urinary protein <0.5g / 24h, serum albumin ≥35g / L, and SCr normal or an increase not exceeding 10% of the baseline value. Non-remission (NR) is defined as treatment failing to achieve CR or PR.

[0055] In this application, the five molecules refer to BTN3A2, CCL23, CKAP4, LIFR, and TREM1.

[0056] The design concept of this invention: Eighteen patients diagnosed with lupus nephritis (LN) by renal biopsy were selected. Among them, nine patients with active LN with a SLEDAI-2K score ≥12 (ALN1 group) and nine patients with inactive LN with a score ≤8 (ILN1 group) were included. Ten healthy controls were also included (CON1 group). The expression changes of LIFR, BTN3A2, CCL23, CKAP4, and TREM1 in the circulation of these patients were detected using Olink proteomics.

[0057] Samples from two prospective LN cohorts (n=20) at different treatment response points were selected. Olink proteomics was used to detect the expression of LIFR, BTN3A2, CCL23, CKAP4, and TREM1 in the prospective cohort circulation. Self-controls of patients before and after treatment were used to verify the role of the five molecules in reflecting LN disease activity.

[0058] A larger sample size of LN patients (n=148) and two follow-up cohorts (n=50) were selected. ELISA was used to detect changes in the expression of LIFR, BTN3A2, CCL23, CKAP4, and TREM1 in circulation. The relationship between the concentrations of these molecules and clinical indicators was analyzed to verify whether these five molecules can serve as novel biomarkers reflecting disease activity in lupus nephritis patients. The specificity and sensitivity of the combined diagnosis and the assessment performance reflecting LN disease activity were calculated.

[0059] Example 1

[0060] Screening of biomarkers for assessing disease activity in lupus nephritis:

[0061] (1) Sample setup

[0062] In this embodiment, 9 patients with active lung disease (LN) with a SLEDAI-2K score ≥12 (ALN1 group) and 9 patients with inactive LN with a score ≤8 (ILN1 group) were selected, along with 10 healthy controls (CON1 group). All 18 patients and 10 healthy controls were female.

[0063] (2) Main reagents

[0064] Olink target 96Inflammation Panel and Olink target 96Immune ResponsePanel

[0065] (3) Sample collection and retrieval

[0066] All selected samples were from the biobank of the National Clinical Research Center for Kidney Diseases.

[0067] The sample collection process is as follows: approximately 3 ml of peripheral venous blood is collected from each patient into an EDTA tube, and plasma is extracted by centrifugation at 3000g for 15 minutes at 4°C. The plasma is then aliquoted and frozen at -80°C for later use.

[0068] (4) Inflammation and immune molecular detection

[0069] S1. Incubation: Patient plasma samples are thawed at 4°C. 1 μL of each sample is then incubated on the panel. Each well of the 96-series panel can simultaneously detect 92 target proteins, with an additional 4 proteins used for quality control.

[0070] S2. Quantitative Detection: Antibodies labeled with oligonucleotides are designed to specifically recognize and bind to 92 target proteins. Two adjacent single-stranded DNA molecules bind complementary to each other and extend to form a double-stranded template. The addition of DNA polymerase triggers DNA polymerization, generating a unique PCR target sequence. The generated DNA sequence is detected and quantified using a microfluidic real-time quantitative PCR instrument.

[0071] S3. Data Transformation and Quality Control (QC): The obtained Ct data undergoes quality control and standardization using internal and external controls. The final detection results are presented as Normalized Protein Expression (NPX) values, with higher values ​​indicating higher protein expression levels. For each sample and detection, NPX is calculated using the following equation:

[0072] 1. Ct(analyte) - Ct(extended control) = dCt(analyte) (to reduce technical variability)

[0073] 2. dCt(analyte) - Ct(median interplate control) = ddCt(analyte) (to improve interplate variability)

[0074] 3. Correction factor (analyte) - ddCt (analyte) = NPX (analyte) (to make the data more intuitive) The correction factor is a fixed variable unique to each test and reagent batch.

[0075] QC steps: First, the standard deviation of the detection / incubation control must be <0.2; second, a warning is issued when the sample NPX deviates from the plate midpoint by >0.3.

[0076] (5) Statistical methods

[0077] Statistical analysis was performed using GraphPad Prism 8 software. Olink Analyze was used to analyze the Olink test results, and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic efficacy of differentially expressed molecules. Based on logistic regression, two or more indicators were combined; the larger the area under the curve (AUC), the higher the diagnostic value of the target. An AUC ≈ 1.0 was considered the ideal indicator; an AUC above 0.8 indicated high accuracy for the target; and an AUC of 0.5 indicated no diagnostic value for the target.

[0078] (6) Results Analysis

[0079] I. Clinical characteristics of active and inactive patients

[0080] In this study, 9 patients with LN were selected from the ALN1 group and 9 patients from the ILN1 group, and 10 patients were selected from the CON1 group. All 18 patients and 10 healthy controls were female. The clinical baseline characteristics of the participants in the three groups are shown in Table 1. There were no differences in gender and age among the participants in the three groups. There were also no statistically significant differences in renal pathology CI score, complement C3 and C4, and platelet count in the ALN1 and ILN1 groups.

[0081] The SLEDAI-2K score of the ALN1 group was higher than that of the ILN1 group; the 24-hour urinary protein quantification was higher than that of the ILN1 group; the renal pathology AI score was significantly higher than that of the ILN1 group; the serum albumin was lower than that of the ILN1 group and the CON1 group; and the serum creatinine was higher than that of the ILN1 group and the CON1 group.

[0082] The renal pathological classification of the ALN1 group was all type IV, while the ILN1 group had 5 cases of type II, 3 cases of type IV, and 1 case of type V. The pathological classifications of the two groups of patients were different.

[0083] Table 1. Clinical characteristics of patients with lupus nephritis and healthy controls.

[0084] ALN1 ILN1 CON1 p-value Age (years) 25.11±7.04 29.67±9.03 26.80±7.71 0.481 Albumin (g / L) 31.57±4.20 37.82±6.75 47.92±1.81 <0.001 Serum creatinine (mg / dl) 1.46±0.60 0.74±0.18 0.71±0.09 <0.001 SLEDAI-2K 16.00(12.00,18.00) 8.00(6.50,8.00) <0.001 24-hour urinary protein (g / d) 3.95(2.56,5.00) 0.88(0.64,2.63) 0.014 Complement C4 (g / L) 0.10(0.07,0.14) 0.05(0.04,0.11) 0.248 Complement C3 (g / L) 0.48±0.15 0.42±0.21 0.496 <![CDATA[Platelet (×10 9 / L)]]> 220.00±46.43 224.30±78.92 0.889 LN pathological classification 0.005 II 0 5 IV 9 3 V 0 1 AI 8.67±3.46 2.78±2.22 0.001 CI 2.67±2.24 1.56±1.13 0.202

[0085] II. Differential Protein Analysis in the Circulation of Active and Inactive LN Patients

[0086] The expression levels of five proteins—BTN3A2, CCL23, CKAP4, LIFR, and TREM1—were detected among the ALN1, ILN1, and CON1 groups. Figure 1As shown in the heatmap, each grid on the horizontal axis represents a patient, and each grid on the vertical axis represents a molecule; the redder the color, the higher the protein expression level. Table 2 shows the relative quantification of five protein molecules in the ALN1, ILN1, and CON1 groups. Pairwise comparisons of the three groups revealed that LIFR, BTN3A2, CCL23, CKAP4, and TREM1 were significantly higher in the ALN1 group than in the ILN1 and CON1 groups, respectively, while there were no significant differences in LIFR, BTN3A2, CCL23, and CKAP4 between the ILN1 and CON1 groups. Figure 2 As shown, TREM1 was significantly elevated in inactive LN patients compared to healthy individuals.

[0087] Table 2. Expression of the five molecules in the ALN1, ILN1, and CON1 groups.

[0088] ALN1 ILN1 CON1 p-value LIFR 3.41±0.35 2.97±0.26 2.77±0.15 <0.001 BTN3A2 4.06±0.60 3.22±0.57 2.79±0.35 <0.001 CCL23 12.24±0.78 11.43±0.58 10.83±0.46 <0.001 CKAP4 6.38±0.63 5.36±0.44 4.88±0.21 <0.001 TREM1 2.73±0.46 2.05±0.58 1.36±0.37 <0.001

[0089] like Figure 3 As shown, ROC curves were plotted for the expression of the five molecules in ALN1 and ILN1. The AUC of LIFR was 0.864, with a 95% confidence interval (CI) of 0.655–1.000 (p = 0.009); the AUC of BTN3A2 was 0.840, with a 95% CI of 0.656–1.000 (p = 0.015); the AUC of CCL23 was 0.840, with a 95% CI of 0.637–1.000 (p = 0.015); the AUC of CKAP4 was 0.914, with a 95% CI of 0.780–1.000 (p = 0.003); and the AUC of TREM1 was 0.827, with a 95% CI of 0.629–1.000 (p = 0.019).

[0090] The AUC of the two molecules BTN3A2 and CCL23 evaluated together was 0.877, 95% CI 0.716-1.000, p = 0.007;

[0091] The AUC of the two molecules BTN3A2 and CKAP4 evaluated together was 0.926, with a 95% CI of 0.805–1.000 and p = 0.002.

[0092] The AUC of the two molecules CCL23 and CKAP4 evaluated together was 0.963, with a 95% CI of 0.880–1.000 and p < 0.001.

[0093] The AUC of the two molecules BTN3A2 and LIFR evaluated together was 0.901, with a 95% CI of 0.761–1.000 and p = 0.004.

[0094] The AUC of the three molecules BTN3A2, CCL23 and CKAP4 evaluated together was 0.975, with a 95% CI of 0.914–1.000 and p < 0.001.

[0095] The AUC of the four molecules LIFR, BTN3A2, CCL23 and CKAP4 evaluated together was 0.975, with a 95% CI of 0.914–1.000 and p < 0.001.

[0096] The combined evaluation AUC of five molecules, BTN3A2, CCL23, CKAP4, LIFR, and TREM1, reached 1.

[0097] In summary, BTN3A2, CCL23, CKAP4, LIFR, and TREM1 were significantly elevated in patients with active lupus nephritis, distinguishing them from inactive patients and healthy individuals. Each molecule had high evaluative value, with AUC > 0.8. The combined evaluation of three and four molecules was > 0.95, and the combined evaluation of five molecules even reached 1.

[0098] Example 2

[0099] Changes in the expression of BTN3A2, CCL23, CKAP4, LIFR, and TREM1 in self-control samples before and after treatment in a prospective cohort of patients with lupus nephritis.

[0100] (1) Sample setup

[0101] This embodiment selects plasma samples, corresponding information at the time of sample collection, and clinical data from patients diagnosed with LN by renal biopsy and included in the prospective cohort between January 1, 2017 and December 31, 2023.

[0102] Twenty patients with lupus nephritis (LN) were selected from a prospective cohort and divided into a complete remission cohort (CR1) and a non-remission cohort (NR1) based on their treatment response. The observation time points for both the CR1 and NR1 cohorts were the same patient's renal biopsy and two follow-up points, with 10 patients in each cohort (n=20).

[0103] (2) Main reagents

[0104] The main reagents are the same as those in Example 1.

[0105] (3) Sample collection and retrieval

[0106] The main reagents are the same as those in Example 1.

[0107] (4) Inflammation and immune molecular detection

[0108] Consistent with the inflammation and immune molecule detection in Example 1.

[0109] (5) Statistical methods

[0110] Repeated measures ANOVA was used to analyze differences in clinical data (24-hour urinary protein, serum creatinine, etc.) at different time points in the prospective cohort. Olink Analyze was used to identify differentially expressed protein (DEPs) between multiple time points in the prospective cohort samples. The Benjamini-Hochberg (FDR) method was used for multiple test corrections, and a p-value <0.05 was considered statistically significant. Short Time-series Expression Miner (STEM) software was used to analyze continuous protein changes across multiple treatments in the cohort.

[0111] (6) Results Analysis

[0112] I. Clinical characteristics of patients in a prospective cohort

[0113] All patients in both the CR1 and NR1 cohorts were female. In the CR1 cohort, the baseline 24-hour urinary protein and serum creatinine were 4.51±2.11 g / d and 1.21±0.68 mg / dL, respectively; at partial remission, they were 0.66±0.45 g / d and 0.75±0.11 mg / dL, respectively; and at complete remission, they were 0.28±0.10 g / d and 0.73±0.07 mg / dL, respectively. Both 24-hour urinary protein and serum creatinine decreased significantly (p<0.001, p=0.002).

[0114] At baseline, urinary protein and serum creatinine in the NR1 cohort were 3.35±2.28 g / d and 1.33±0.88 mg / dL, respectively; at the first ineffective treatment, they were 4.95±2.94 g / d and 1.12±0.73 mg / dL, respectively; and at the second ineffective treatment, they were 3.39±2.76 g / d and 0.93±0.49 mg / dL, respectively. There was no significant change in urinary protein in patients without remission (p=0.293), but serum creatinine decreased significantly (p=0.008).

[0115] Table 3 shows the basic information of patients at each follow-up point in the prospective cohort. The time interval for sample collection in the CR1 and NR1 cohorts was approximately 3 or 6 months.

[0116] Table 3 Clinical characteristics of the prospective cohort

[0117]

[0118] II. Dynamic Changes of Five Molecules with Disease Status

[0119] Table 4 shows the expression levels of BTN3A2, CCL23, CKAP4, LIFR, and TREM1 at different time points in the CR1 and NR1 cohorts. It is evident that these five molecules gradually decreased with disease remission, while in the non-remission cohort, patients showed no significant improvement in their condition, and the expression levels of these five molecules remained largely unchanged. Figure 4 As shown, the trends of five molecules in CR1 cohort patients and the 24-hour urinary protein levels in the CR1 cohort are presented. The left side shows the 24-hour urinary protein quantification at baseline and two follow-up points for CR1 cohort patients, while the right side shows the expression trends of the five molecules at different time points. Time points 1, 2, and 3 represent baseline, partial remission, and complete remission, respectively. The vertical axis represents the NPX value, with the expression value at baseline uniformly set to 0.

[0120] Table 4. Expression of five molecules in the prospective cohort.

[0121]

[0122] Example 3

[0123] Expression and significance of BTN3A2, CCL23, CKAP4, LIFR and TREM1 in plasma of a larger sample size of lupus nephritis patients and follow-up cohort patients.

[0124] (1) Sample setup

[0125] Fifty active patients with SLEDAI-2K ≥ 12 were assigned to the ALN2 group, and 49 inactive patients with SLEDAI-2K ≤ 8 were assigned to the ILN2 group. Forty-nine age- and sex-matched healthy controls (CON2) were also included. Two follow-up cohorts were established: a remission cohort (CR2) and a non-remission cohort (NR2), each with 25 patients. Baseline and CR / NR were used at two time points, consistent with Example 2, with sample collection intervals of approximately 3 / 6 months. All patients had complete and detailed hospitalization records, renal biopsy records, laboratory test results, and regular follow-up records.

[0126] (2) Main reagents

[0127] Human ELISA kit containing five molecules: BTN3A2, CCL23, CKAP4, LIFR, and TREM1.

[0128] (3) Sample collection and retrieval

[0129] The sample collection and retrieval process is consistent with that in Example 1.

[0130] (4) ELISA verification

[0131] S1. Sample preparation: Samples for LIFR detection were diluted 1:2 using the sample diluent provided with the kit. Samples for BTN3A2 detection were diluted 1:5. Samples for CCL23, TREM1, and CKAP4 were tested using the undiluted solution.

[0132] S2. Preparation of standard: Number the EP tubes 1 to 7, add the standard diluent of the required volume as specified in the instructions to each tube, transfer the same volume of standard to tube 1 and mix well, and then transfer the above mixed standard from tube 1 to tubes 2 to 7 for gradient dilution. The LIFR standard gradients were 10000, 5000, 2500, 1250, 625, 312, 156, and 0 pg / ml; the BTN3A2 standard gradients were 960, 480, 240, 120, 60, 30, and 0 pg / ml; the CCL23 standard gradients were 2500, 1250, 625, 312.5, 156, 78, and 39 pg / ml; the TREM1 standard gradients were 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg / ml; and the CKAP4 standard gradients were 1500, 750, 375, 187.5, 93.75, 46.88, and 23.44 pg / ml.

[0133] S3. Sample incubation: Add 100 μL of standard or sample to each well of LIFR plate, and incubate at 37°C for 90 min after sealing; add 50 μL of standard or sample to each well of BTN3A2 plate, and incubate at 37°C for 30 min after sealing. Also set up blank control wells without adding any sample or standard; add 100 μL of standard or sample to each well of CCL23, TREM1 and CKAP4 plates, and incubate at 37°C for 2 h after sealing.

[0134] S4. Antibody Incubation: After sample incubation, discard the liquid. LIFR, CCL23, TREM1, and CKAP 4-well plates do not require washing. Add 100 μL of the corresponding antibody working solution (1:100) to each well, block, and incubate at 37°C for 60 min. For BTN3A 2-well plates, discard the samples and wash with washing buffer (1:30), 300 μL per well, let stand for 30 s, then discard. Repeat 5 times. Add 50 μL of enzyme-labeled reagent to each well except for blank wells, block, and incubate at 37°C for 30 min.

[0135] S5. Avidin-Biotin-Peroxidase Complex Incubation: After antibody incubation, discard the liquid. Wash LIFR, CCL23, TREM1 and CKAP 4-well plates with washing buffer (1:25), 300 μL per well, let stand for 1 min and then discard. Repeat 3 times and then pat dry. Add 100 μL (1:100) of the corresponding peroxidase-labeled avidin working solution to each well, block and incubate at 37°C for 30 min or 60 min.

[0136] S6. Color Development: Discard the liquid in the well plate. Wash the LIFR, CCL23, TREM1, and CKAP4 wells 5 times using the same method. Add 90 μL of TMB colorimetric reagent to each well and incubate at 37°C in the dark for 15-30 min. For the BTN3A2 well plate, discard the enzyme label and wash 5 times using the same method as before. Add 50 μL each of colorimetric solutions A and B to each well, mix well, and incubate at 37°C in the dark for 10 min.

[0137] S7. Terminate color development and read the values: Terminate color development when the four wells with the highest concentration of standard show a clear blue color. Add the corresponding stop solution to each well according to the instructions, and immediately use an ELISA reader to read the OD absorbance value at 450 nm.

[0138] S8. Data Processing: Use the Logistic Curve Fitting 2 (four parameters) function in the ELISA data processing software to fit the curve, with R2>0.999. Input the OD value to calculate the corresponding sample concentration. The final concentration = concentration corresponding to the OD value × dilution factor.

[0139] (5) Statistical methods

[0140] Statistical analysis was performed using GraphPad Prism 8 software.

[0141] (6) Results Analysis

[0142] I. Clinical characteristics of the patient

[0143] The SLEDAI-2K score of the ALN2 group was higher than that of the ILN2 group; 24-hour urinary protein quantification was significantly higher in ALN2 than in ILN2; serum creatinine was higher in ALN2 than in ILN2; albumin was lower in ALN2 than in ILN2; complement C3 and C4 were lower in ALN2 than in ILN2; platelet count was significantly lower in ALN2 than in ILN2; the renal pathological classification of ALN2 was 26 cases of type IV, 10 cases of type V, and 14 cases of type IV+V, while the renal pathological classification of ILN2 was 17 cases of type IV, 27 cases of type V, and 5 cases of type IV+V, with significant differences between the two groups (p<0.001). The AI ​​of ALN2 was higher than that of ILN2; the CI of ALN2 was not different from that of ILN2 (p=0.421).

[0144] The CR2 cohort showed significantly lower 24-hour urinary protein and serum creatinine levels (p<0.001, p=0.003), while the NR2 cohort showed no change (p=0.055, p=0.203).

[0145] Clinical data for ALN2, ILN2, and CON2 are shown in Table 5, and basic information of patients at each follow-up point in the follow-up cohort is shown in Table 6.

[0146] Table 5 Clinical characteristics of ALN2, ILN2 and CON2

[0147] ALN2 ILN2 CON2 p-value Gender (Male / Female) 25 / 25 27 / 22 23 / 26 0.716 Age (years) 30.16±11.98 31.45±11.56 31.67±10.42 0.635 Albumin (g / L) 28.13±6.44 32.42±6.94 0.002 Serum creatinine (mg / dl) 1.11(0.78,1.67) 0.85(0.69,1.21) 0.023 SLEDAI-2K 20.00(18.00,22.00) 6.00(6.00,8.00) <0.001 24-hour urinary protein (g / d) 3.91(2.48,6.92) 2.61(1.33,5.12) 0.026 Complement C4 (g / L) 0.07(0.04,0.13) 0.10(0.10,0.18) <0.001 Complement C3 (g / L) 0.43±0.15 0.73±0.24 <0.001 <![CDATA[Platelets (×10 9 / L)]]> 185.00(115.00,217.80) 206.00(161.00,264.50) 0.013 Kidney pathology classification <0.001 IV 26 17 V 10 27 IV+V 14 5 AI 8.00(6.00,11.25) 3.00(2.00,5.50) <0.001 CI 3.00(1.00,5.00) 3.00(2.00,5.00) 0.421

[0148] Table 6 Clinical characteristics of the follow-up cohort

[0149]

[0150] II. ELISA results for five molecules

[0151] The concentrations of the five molecules in plasma samples from the ALN2, ILN2, and CON2 groups are shown in Table 7. It can be seen that, compared with CON2 and ILN2, LIFR (p<0.001, p<0.001), BTN3A2 (p<0.001, p<0.001), CCL23 (p<0.001, p<0.001), TREM1 (p<0.001, p<0.001), and CKAP4 (p<0.001, p<0.001) were significantly elevated in active patients.

[0152] Table 7. Concentrations of five molecules in plasma samples from the ALN2, ILN2, and CON2 groups.

[0153] ALN2 ILN2 CON2 p-value LIFR (pg / ml) 1284.00(889.00,2186.00) 636.60(477.10,914.00) 562.80(404.40,714.60) <0.001 BTN3A2 (pg / ml) 309.60(262.80,352.40) 239.60(209.90,275.30) 226.40(207.30,278.60) <0.001 CCL23 (pg / ml) 448.20(329.00,594.50) 231.40(136.70,352.80) 114.20(73.40,196.20) <0.001 CKAP4 (pg / ml) 121.60(94.60,151.40) 84.80(67.20,113.20) 57.80(22.00,85.80) <0.001 TREM1 (pg / ml) 517.70(310.00,782.40) 290.30(199.10,399.10) 310.20(209.30,375.20) <0.001

[0154] Table 8 shows the concentrations of the five molecules in plasma samples from the follow-up cohort, illustrating the specific concentrations of the five molecules at renal biopsies and different treatment response points, as well as the p-values ​​for comparison. Figure 6 As shown, the expression levels of the five molecules at baseline and follow-up points in the CR2 and NR2 cohorts all decreased with disease remission. In the NR2 cohort, LIFR, CCL23, TREM1, and CKAP4 remained unchanged, while BTN3A2 increased in the non-remission state.

[0155] Table 8. Concentrations of five molecules in plasma samples from the follow-up cohort.

[0156]

[0157] III. Assessment performance of five molecules in reflecting lupus nephritis activity

[0158] like Figure 7As shown, ROC analysis of plasma concentrations in the ALN2 and ILN2 groups revealed that the AUCs of LIFR (AUC = 0.815, 95% CI: 0.725–0.904, p < 0.001), BTN3A2 (AUC = 0.806, 95% CI: 0.716–0.896, p < 0.001), CCL23 (AUC = 0.780, 95% CI: 0.686–0.875, p < 0.001), TREM1 (AUC = 0.775, 95% CI: 0.683–0.867, p < 0.001), and CKAP4 (AUC = 0.765, 95% CI: 0.671–0.858, p < 0.001) were all > 0.75, indicating high evaluative value.

[0159] The AUC of the two molecular combinations BTN3A2 and CCL23 was 0.870 (95% CI 0.796-0.944, p<0.001).

[0160] The AUC of the two molecular combinations BTN3A2 and CKAP4 was 0.855 (95% CI 0.781-0.930, p<0.001).

[0161] The AUC for the two molecular combinations CCL23 and CKAP4 was 0.876 (95% CI 0.806–0.946, p<0.001).

[0162] The combination of BTN3A2 and LIFR was evaluated, with an AUC of 0.900 (95% CI 0.829–0.970, p < 0.001).

[0163] The combination of three molecules, BTN3A2, CCL23, and CKAP4, was evaluated, with an AUC of 0.911 (95% CI: 0.853–0.968, p<0.001).

[0164] The combination of four molecules, BTN3A2, CCL23, CKAP4, and LIFR, was evaluated, with an AUC of 0.932 (95% CI: 0.881–0.983, p < 0.001).

[0165] The five molecules BTN3A2, CCL23, CKAP4, LIFR and TREM1 were evaluated together, and the AUC was 0.955 (95% CI: 0.916-0.994, p<0.001).

[0166] The maximum Youden index, sensitivity, and specificity for each molecule are shown in Table 9. The Youden index for LIFR is 0.518, with a sensitivity of 86.67% and a specificity of 65.12%; for BTN3A2, it is 0.560, with a sensitivity of 67.35% and a specificity of 88.64%; for CCL23, it is 0.489, with a sensitivity of 72.34% and a specificity of 76.60%; for CKAP4, it is 0.407, with a sensitivity of 87.50% and a specificity of 53.19%; and for TREM1, it is 0.470, with a sensitivity of 72.00% and a specificity of 75.00%.

[0167] The Youden index for the two molecular combinations BTN3A2 and CCL23 was 0.636, with a sensitivity of 82.61% and a specificity of 80.95%.

[0168] The Youden index of the two molecular combinations BTN3A2 and CKAP4 was 0.547, the sensitivity was 97.87%, and the specificity was 56.82%.

[0169] The Youden index for the combination of CCL23 and CKAP4 was 0.600, with a sensitivity of 80.00% and a specificity of 80.00%.

[0170] The Youden index for the combination of BTN3A2 and LIFR was 0.709, with a sensitivity of 84.09% and a specificity of 86.84%.

[0171] The Youden index of the combination of three molecules, BTN3A2, CCL23 and CKAP4, was 0.671, with a sensitivity of 90.91% and a specificity of 76.19%.

[0172] The Youden index for the combination of four molecules, BTN3A2, CCL23, CKAP4, and LIFR, was 0.703, with a sensitivity of 100.00% and a specificity of 70.27%.

[0173] The Youden index of the five molecular combination of BTN3A2, CCL23, CKAP4, LIFR and TREM1 was 0.771, with a sensitivity of 82.50% and a specificity of 94.59%.

[0174] Table 9. Sensitivity and specificity of the five molecules and their combinations evaluated.

[0175] Maximum Oden Index Sensitivity % Specificity% BTN3A2 0.560 67.35 88.64 CCL23 0.489 72.34 76.60 CKAP4 0.407 87.50 53.19 LIFR 0.518 86.67 65.12 TREM1 0.470 72.00 75.00 BTN3A2+CCL23 0.636 82.61 80.95 BTN3A2+CKAP4 0.547 97.87 56.82 CCL23+CKAP4 0.600 80.00 80.00 BTN3A2+LIFR 0.709 84.09 86.84 BTN3A2+CCL23+CKAP4 0.671 90.91 76.19 BTN3A2+CCL23+CKAP4+LIFR 0.703 100.00 70.27 Five molecules 0.771 82.50 94.59

[0176] IV. Correlation between five molecules and clinical indicators of lupus nephritis

[0177] Regarding its correlation with clinical indicators, such as Figure 8As shown in Table 10, all five molecules showed good correlation with SLEDAI-2K. LIFR, BTN3A2, CCL23, and TREM1 were negatively correlated with complement C3. Furthermore, BTN3A2, TREM1, and CKAP4 were also negatively correlated with complement C4. CCL23 was also correlated with serum creatinine and albumin. None of the five molecules were associated with the patient's 24-hour urinary protein or platelet count.

[0178] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A biomarker for assessing disease activity in lupus nephritis, characterized in that: The biomarker is one, two, or a combination of more than one of BTN3A2, CCL23, and CKAP4.

2. The biomarker according to claim 1, characterized in that: The markers also include LIFR.

3. The biomarker according to claim 1 or 2, characterized in that: The markers also include TREM1.

4. The use of the biomarker as described in any one of claims 1-3 in the preparation of a kit for assessing the disease activity of lupus nephritis.

5. The application according to claim 4, characterized in that: The expression levels of the biomarkers were obtained by at least one of Olink proteomics, adjacent extension technology, and enzyme-linked immunosorbent assay (ELISA).

6. A product for assessing disease activity in lupus nephritis, characterized in that: The product includes a kit for detecting the expression level of any of the biomarkers described in claims 1-3.

Images