Use of a reagent for detecting trem2 in the preparation of a diagnostic kit for chronic kidney disease osteoporosis

Abstract

The present application relates to the field of in vitro diagnostic reagents, and particularly relates to the use of a reagent for detecting TREM2 in the preparation of a chronic kidney disease osteoporosis diagnostic kit. The present application finds that the TREM2 signaling pathway is closely related to the common pathological background of chronic kidney disease and osteoporosis. Therefore, TREM2 can be used as an expression feature and a molecular diagnostic marker in chronic kidney disease-related osteoporosis, thereby enabling early diagnosis of chronic kidney disease osteoporosis. Based on this, the present application provides the use of a reagent for detecting TREM2 in the preparation of a chronic kidney disease osteoporosis diagnostic kit, which has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, and more specifically to the use of reagents for detecting TREM2 in the preparation of diagnostic kits for osteoporosis in chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is a progressive kidney dysfunction caused by various etiologies. Its main pathological features include glomerulosclerosis, renal interstitial fibrosis, renal tubular atrophy, and vascularization, ultimately leading to nephron loss and irreversible kidney failure. CKD patients often experience multiple complications, among which osteoporosis is a core manifestation of CKD-mineral and bone disorder (CKD-MBD). Its pathological mechanisms involve secondary hyperparathyroidism, vitamin D metabolism disorders, calcium and phosphorus imbalances, osteoblast-osteoclast coupling dysregulation, and inflammatory microenvironment remodeling, resulting in decreased bone mineral density, bone microstructure destruction, and a significantly increased risk of fractures. CKD-related osteoporosis signifies severe dysfunction of the bone-kidney axis; therefore, early diagnosis and targeted intervention of CKD osteoporosis are crucial for slowing CKD progression and improving patient prognosis.

[0003] Currently, dual-energy X-ray absorptiometry (DXA), quantitative CT (QCT), and serum bone turnover markers (such as osteocalcin and type I collagen C-terminal peptide) are commonly used to assess osteoporosis in CKD patients. However, these techniques mainly reflect macroscopic changes in bone mineral density or bone metabolism and cannot sensitively capture microscopic lesion information during the early inflammatory-immune microenvironment remodeling stage of CKD osteoporosis. Biomarkers, on the other hand, can quantitatively assess the biological process of the disease, reflect bone-kidney axis interactions, and serve as potential therapeutic targets. Their expression levels dynamically change with the activity or progression of CKD osteoporosis. Therefore, screening for highly sensitive and specific CKD osteoporosis biomarkers can help achieve early and accurate diagnosis and targeted therapy.

[0004] Biomarkers, as molecular indicators reflecting the development and progression of diseases, play a crucial role in early disease screening, progression monitoring, and the discovery of therapeutic targets. In recent years, researchers have reported various biomarkers related to bone metabolism, such as osteocalcin, type I collagen C-terminal peptide (CTX), and bone-specific alkaline phosphatase (BALP). However, their low sensitivity and poor specificity limit their diagnostic efficacy in the context of chronic kidney disease (CKD), necessitating the screening of novel molecular biomarkers with higher specificity.

[0005] Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor mainly expressed on myeloid cells (such as macrophages and dendritic cells), and it is widely involved in regulating inflammatory responses, phagocytosis, and tissue repair processes. Current technology has not reported a direct association between TREM2 and CKD-related osteoporosis, nor has it systematically validated its specificity, sensitivity, and clinical application value as a diagnostic marker for CKD-related osteoporosis. Summary of the Invention

[0006] The purpose of this invention is to provide a novel biomarker for screening osteoporosis in chronic kidney disease, and the use of the detection reagent of this biomarker in the preparation of a chronic kidney disease osteoporosis screening kit.

[0007] Use of reagents for detecting TREM2 in the preparation of diagnostic kits for osteoporosis in chronic kidney disease.

[0008] Preferably, the TREM2 is myeloid cell trigger receptor 2, UniProt numbered Q9NZC2.

[0009] Preferably, the reagent is a reagent for detecting the expression level of TREM2 in human body fluid samples.

[0010] Preferably, the body fluid sample is serum.

[0011] Preferably, the reagent is a reagent for detecting TREM2 protein levels, selected from reagents used in immunohistochemical detection, immunofluorescence detection, western blot detection, ELISA detection, chemiluminescent immunoassay, immunochromatography, enzyme-linked immunospot assay, or protein chip detection methods.

[0012] Preferably, the reagent is a reagent for detecting TREM2 nucleic acid levels, selected from qPCR detection reagents, digital PCR detection reagents, RT-PCR detection reagents, or nucleic acid hybridization detection reagents.

[0013] Preferably, the diagnostic kit is used to distinguish between patients with chronic kidney disease and osteoporosis and healthy individuals.

[0014] Preferably, the threshold for TREM2 expression level that distinguishes between patients with osteoporosis due to chronic kidney disease and healthy individuals is 2735.32 pg / ml.

[0015] Preferably, the diagnostic kit is used to distinguish between patients with chronic kidney disease and osteoporosis and patients with chronic kidney disease who have not developed osteoporosis.

[0016] Preferably, the threshold for TREM2 expression in patients with chronic kidney disease and osteoporosis is 2735.32 pg / ml, which distinguishes them from chronic kidney disease patients without osteoporosis.

[0017] The key to this invention lies in determining that the expression level of TREM2 in human serum is significantly correlated with osteoporosis in chronic kidney disease. Therefore, screening for osteoporosis in chronic kidney disease can be performed by detecting the expression level of TREM2 in human serum. As for the specific methods for detecting the expression level of TREM2 in human serum, various methods disclosed in the prior art can be used. This invention specifically employs methods such as Western blotting (WB), ELISA, immunofluorescence, and qPCR, but is not limited to these methods. Any method capable of detecting the expression level of TREM2 protein or TREM2 nucleic acid can be used for screening for osteoporosis in chronic kidney disease.

[0018] This invention provides a novel biomarker for effective screening of osteoporosis in patients with chronic kidney disease. The advantages of this invention lie in its ability to achieve early screening, high specificity, and the use of serum as the test sample, resulting in minimal harm to patients. Therefore, this invention has promising application prospects.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed description of specific embodiments further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 The trend of serum TREM2, Ca ion and P ion concentrations in the normal group and CKD patients at each stage (CKD stage 3-5) measured by the test kit in Experiment Example 1 is shown. Among them, (A) serum TREM2 level; (B) Ca ion level; (C) P ion level; (D) parathyroid hormone level; (E) bone mineral density T value.

[0022] Figure 2 The ROC curves for serum TREM2 and osteoporosis status in the normal group and CKD patients at each stage (CKD stages 3-5) measured by the test kit in Experiment Example 1 are shown, with AUC=0.9984. p<0.01, p<0.0001.

[0023] Figure 3The results are from the experimental model (CKD-MBD model) constructed in Experiment Example 2 to simulate mineral metabolism disorder and renal function impairment. Among them, (A) Ca ion level; (B) P ion level; (C) uric acid level; (D) urea nitrogen level; (E) albumin level.

[0024] Figure 4 The image shows the 3D reconstruction result of the femur of the CKD-MBD model in Experiment Example 2.

[0025] Figure 5 The images show histological staining of the CKD-MBD model in Experiment Example 2, including (A) HE staining of mouse kidneys; (B) Masson staining of mouse kidneys; and (C) HE, Masson, TRAP, Safranin O-Fix Green staining and Acid Fuchs Red-Methylene Blue staining of mouse femurs.

[0026] Figure 6 The results of TREM2 multimodal detection in the CKD-MBD model in Experiment Example 2 are shown below. (A) Serum TREM2 concentration in mice; (B) TREM2-PCR results; (C) TREM2-WB results; (D) Immunofluorescence of TREM2 femoral sections; (E) Scatter plot showing the correlation between serum TREM2 levels and bone mineral density (Tb.BMD). p<0.0001.

[0027] Figure 7 The results of clinical validation of TREM2 in Experiment Example 2 demonstrate its diagnostic efficacy in distinguishing between CKD osteoporosis patients and healthy individuals. In this example, A represents the linear regression results of bone mineral density T-score and serum TREM2 level, and B represents the ROC curves of serum TREM2 and osteoporosis status for the normal group and patients in each stage of CKD (CKD stages 3-5), with AUC=0.96. Detailed Implementation

[0028] Example 1: Detection kit and its usage method I. Components of the Reagent Kit This embodiment provides detection kits for several detection methods, including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), and quantitative real-time PCR (QPCR). The components of each kit are as follows: 1. Western Blot (WB) Detection Kit Table 1 2. Enzyme-linked immunosorbent assay (ELISA) kit Table 2 3. Immunofluorescence (IF) detection kit Table 3 4. Real-time quantitative PCR (qPCR) detection kit Table 4 II. Instructions for using the reagent kit The usage steps for the above reagent kits are as follows: (1) WB: Take tissue or cells, add sample lysis buffer (containing protease inhibitor), lyse on ice for 30 min, centrifuge and collect the supernatant, quantify by BCA method, and then perform SDS-PAGE electrophoresis (10% PAGE gel, Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: PG222). After electrophoresis, transfer wet or semi-dry to PVDF membrane (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: FFP39), block with blocking buffer (5% skim milk powder, reconstituted) at room temperature for 1 h, add TREM2 specific primary antibody (polyclonal, 1:1000 diluted with standard & sample dilution buffer) and incubate overnight at 4℃. The next day, wash the membrane with concentrated washing buffer (25×) diluted to 1× for 3×5 min, and add enzyme-labeled secondary antibody (HRP-Goat) (Anti-Rabbit, 1:5000 diluted with standard and sample diluent) Incubate at room temperature for 1 hour, then wash the membrane with 1× washing solution for 3×5 minutes. Add an equal volume of chemiluminescent substrate solution A + solution B to the membrane, expose and analyze the gray values ​​of the TREM2 bands using a chemiluminescence imager. (2) ELISA: Take out the pre-coated ELISA plate with TREM2 specific capture antibody and equilibrate to room temperature. Reconstitute the lyophilized TREM2 recombinant protein standard with standard & sample dilution buffer and serially dilute to establish a 0–100 ng / mL standard curve. Add 50 μL of standard or test sample to each well, incubate at 37℃ for 60 min, spin dry, and wash the plate 4 times with concentrated washing buffer (25×) to 1×. Add 100 μL of biotinylated detection antibody (100× diluted 1:100 with biotinylated antibody dilution buffer) to each well, incubate at 37℃ for 45 min, wash the plate 4 times, add 100 μL of concentrated HRP enzyme conjugate (100× diluted 1:100 with enzyme conjugate dilution buffer) to each well, incubate at 37℃ for 30 min, wash the plate 5 times, add 100 μL of substrate solution (TMB) to each well, and develop color in the dark for 10–15 min. Finally, add 50 μL of reaction stop solution (2M) The reaction was terminated with H2SO4. The absorbance value was read within 5 minutes using a microplate reader at 450nm / 630nm dual wavelengths and quantified using a four-parameter method.

[0029] (3) Immunofluorescence: Tissue or cell slides were routinely fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with blocking solution (5% skim milk powder) at room temperature for 1 h, and then fluorescently labeled TREM2 antibody (FITC labeled, 1:200–1:300 diluted with standard and sample diluent) was added directly and incubated at 4°C overnight. The next day, the slides were washed with concentrated washing buffer (25×) to 1× for 3×5 min, counterstained with DAPI anti-fluorescence quenching mounting solution (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0131) and then mounted. The TREM2 green fluorescence signal was observed under a fluorescence microscope.

[0030] (4) QPCR: Take the sample and add RNA extraction solution (containing RNase inhibitor) to extract total RNA according to the conventional column method or TRIzol method. Take 1–2 μg of RNA and use a reverse transcription kit to synthesize cDNA. The 20 μL qPCR reaction system contains 2 μL cDNA template, 1 μL LTREM2 qPCR primer-probe mixture, 10 μL fluorescent PCR Mix, and 7 μL nuclease-free water. The reaction conditions are 95℃ pre-denaturation for 5 min, followed by 95℃ for 10 s and 60℃ for 30 s (fluorescence collection) for a total of 40–45 cycles. At the same time, GAPDH or ACTB internal control is set. After the experiment, the relative expression level of TREM2 is calculated by the 2-ΔΔCt method. The methods provided in this embodiment have all been verified to detect TREM2. However, the core of the technical solution of this invention lies in the technical discovery that "TREM2 is a specific biomarker for osteoporosis in chronic kidney disease". All diagnostic techniques based on "specific recognition of TREM2 (protein / nucleic acid), signal amplification, and quantitative analysis" are within the scope of protection of this patent.

[0031] The technical solution of the present invention will be further explained through experiments below.

[0032] Experimental Case 1 clinically confirmed that elevated TREM2 levels sensitively reflect mineral imbalances and the early onset of bone abnormalities. In a clinical setting, a subset of CKD patients (stages 3-5) were recruited. Serum TREM2, Ca and P ion concentrations, and bone mineral density T-scores were measured in both the control group and CKD patients at each stage using a diagnostic kit. Results showed: As CKD stages progress, serum TREM2 levels show a gradual upward trend. Figure 1 A). Compared with the normal group, CKD patients had higher levels of Ca2+ ions (…). Figure 1 B), P ions ( Figure 1 C) and parathyroid hormone (PTH) Figure 1 D) levels increase, bone mineral density T-score decreases ( Figure 1E. TREM2 levels have high specificity and sensitivity in reflecting whether osteoporosis is a symptom of chronic kidney disease. Figure 2 ).

[0033] Table 5. Serum TREM2, bone mineral density T-score, calcium ion concentration, phosphorus ion concentration, and parathyroid hormone level in the normal group and patients at different stages of CKD. Note: The contents of this table are as follows: group (001-012 are healthy people, 013-050 are CKD patients, of which 013-025 are CKD-3 patients, 026-038 are CKD-4 patients, and 039-050 are CKD-5 patients), serum calcium (ng / ml), serum phosphorus (ng / ml), serum parathyroid hormone (PTH) (pg / ml), bone mineral density T-score, whether osteoporosis is present, and serum TREM2 (ng / ml).

[0034] The above results indicate that elevated TREM2 levels sensitively reflect early mineral imbalance and bone abnormalities, and can serve as a biomarker for diagnosing osteoporosis in chronic kidney disease. Serum TREM2 ≥ 2735.32 pg / ml can be used as an excellent threshold for distinguishing patients with osteoporosis in chronic kidney disease from healthy individuals, with near-perfect diagnostic efficacy (AUC=0.9984).

[0035] Experiment 2 successfully established a mouse model of mineral metabolism disorder and renal impairment (CKD-MBD) and confirmed that the significant upregulation of TREM2 expression is associated with mineral abnormalities and bone pathology. Mice were fed an adenine-rich diet with high phosphorus (after 2 weeks of normal diet adaptation, mice were fed a diet containing 0.2% adenine for 6 weeks to induce CKD, and then calcification was induced for 6 weeks by a diet containing 0.2% adenine and 1.8% phosphorus).

[0036] The results showed that the levels of Ca and P ions were significantly increased in the model group mice, uric acid and urea nitrogen were increased, and albumin was decreased. Figure 3 ).

[0037] Micro-CT scans revealed sparse trabeculae and abnormal calcification in the model group mice, while the femur structure in the normal group was dense and the trabeculae were intact. Figure 4 ).

[0038] Femoral sections stained with hematoxylin and eosin (HE) Figure 5 C) showed increased bone resorption in the model mice; femoral sections were stained with Masson's staining ( Figure 5 C), showing an increased blue staining area in the model mice; femoral sections were stained with TRAP ( Figure 5C), showing an increase in positive cells in the model mice; femoral sections were stained with Safranin O-Fixgreen (C). Figure 5 C), showing reduced cartilage thickness in the model mice; femoral sections stained with acid fuchsin-methylene blue (C) Figure 5 C), showing that the collagen in the model mice was immature. Kidneys were stained with hematoxylin and eosin (HE). Figure 5 A), showing tubular atrophy in the model mice; kidneys were stained with Masson's staining ( Figure 5 B) shows interstitial fibrosis in the model mice. The trabeculae of the femur in the normal group mice were orderly arranged, without fibrosis or abnormal mineralization. These results confirm that the constructed model is a CKD-MBD model.

[0039] Serum TREM2 concentration in the model group ( Figure 6 A) PCR detection ( Figure 6 B), WB gray value of bone tissue ( Figure 6 C) and IF fluorescence intensity ( Figure 6 D) The test results showed that TREM2 was upregulated at the transcriptional, translational, and protein levels, confirming that TREM2 is closely related to the progression of CKD-MBD. Pearson correlation analysis was performed on serum TREM2 levels and bone mineral density (Tb.BMD) in CKD-MBD model mice. Figure 6 E), and found that TREM2 and Tb.BMD were strongly negatively correlated (r = -0.872, R). 2 = 0.7596, P<0.001), the regression equation was Tb.BMD = -0.008 × TREM2 + 1.2774. This confirms that serum TREM2 levels can accurately reflect the severity of osteoporosis in CKD.

[0040] Further recruitment of patients with chronic kidney disease (CKD stage 3–5) and healthy volunteers (normal group) was conducted to measure their serum TREM2 levels.

[0041] Table 6. Serum TREM2, bone mineral density T-score, calcium ion concentration, phosphorus ion concentration, and parathyroid hormone level in the supplemented normal group and patients at each stage of CKD. Note: The table contains the following information: group (001-010 for healthy individuals, 011-040 for CKD patients, of which 011-020 are CKD-3 patients, 021-030 are CKD-4 patients, and 031-040 are CKD-5 patients), serum calcium (ng / ml), serum phosphorus (ng / ml), serum parathyroid hormone (PTH) (pg / ml), bone mineral density T-score, osteoporosis status, and serum TREM2 (ng / ml).

[0042] The results showed that serum TREM2 levels were generally elevated in CKD patients compared to the healthy group, and gradually increased with the progression of CKD stages. Among them, serum TREM2 levels were further elevated in patients with osteoporosis, suggesting that TREM2 is closely related to the occurrence and development of chronic kidney disease-related osteoporosis. Pearson correlation analysis showed a significant negative correlation between bone mineral density (T-score) and serum TREM2 levels, indicating that as bone mineral density (T-score) increases, the degree of osteoporosis decreases, and serum TREM2 levels gradually decline. Linear regression analysis showed that ( Figure 7 A) The regression equation between bone mineral density T-score and serum TREM2 level is: TREM2 = -942.53×Bone density T-score + 563.7.

[0043] Further plotting of the ROC curve revealed ( Figure 7 (B) Serum TREM2 showed good diagnostic efficacy in distinguishing CKD osteoporosis patients from healthy individuals (AUC=0.96), suggesting that serum TREM2 could serve as a potential diagnostic biomarker for chronic kidney disease-related osteoporosis. The results from the human sample validation were consistent with those from the mouse model experiments, further confirming that serum TREM2 can be used to assess the severity of chronic kidney disease-related osteoporosis.

[0044] In summary, as demonstrated by the above embodiments and experimental examples, the kit of the present invention can screen individuals for chronic kidney disease (CKD) osteoporosis by detecting TREM2 expression levels. High TREM2 levels (compared to healthy individuals or CKD patients without osteoporosis) indicate a higher probability of the patient having CKD osteoporosis. This invention can be used for the early diagnosis of CKD osteoporosis, exhibiting excellent specificity, stability, and variability, providing a valid basis for patients to take relevant treatment measures or make decisions, and showing promising clinical application prospects.

Claims

1. The use of reagents for detecting TREM2 in the preparation of diagnostic kits for osteoporosis in chronic kidney disease.

2. The use according to claim 1, characterized in that: TREM2 is myeloid cell trigger receptor 2, UniProt numbered Q9NZC2.

3. The use according to claim 1, characterized in that: The reagent is used to detect the expression level of TREM2 in human body fluid samples.

4. The use according to claim 3, characterized in that: The body fluid sample was serum.

5. The use according to claim 1, characterized in that: The reagents are for detecting TREM2 protein levels and are selected from reagents used in immunohistochemistry, immunofluorescence, western blot, ELISA, chemiluminescence immunoassay, immunochromatography, enzyme-linked immunospot assay, or protein chip detection methods.

6. The use according to claim 1, characterized in that: The reagent is for detecting TREM2 nucleic acid levels and is selected from qPCR, digital PCR, RT-PCR or nucleic acid hybridization reagents.

7. The use according to claim 1, characterized in that: The diagnostic kit is used to differentiate between patients with chronic kidney disease and osteoporosis and healthy individuals.

8. The use according to claim 7, characterized in that: The threshold for TREM2 expression in patients with osteoporosis due to chronic kidney disease and healthy individuals was 2735.32 pg / ml.

9. The use according to claim 1, characterized in that: The diagnostic kit is used to differentiate between patients with chronic kidney disease and osteoporosis and those with chronic kidney disease who have not developed osteoporosis.

10. The use according to claim 9, characterized in that: The threshold for TREM2 expression in patients with chronic kidney disease and osteoporosis, and those without osteoporosis, was 2735.32 pg / ml.

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