Application of TREM2 detection reagent in preparation of chronic kidney disease osteoporosis diagnostic kit

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a reagent for detecting TREM2 in preparation of a diagnostic kit for chronic kidney disease osteoporosis. The invention finds that the TREM2 signal channel is closely related to the common pathological background of chronic kidney disease and osteoporosis. Therefore, the TREM2 can be used as an expression characteristic and a molecular diagnosis marker in the chronic kidney disease-related osteoporosis, so that the chronic kidney disease-related osteoporosis can be subjected to early diagnosis. On the basis, the invention provides application of the TREM2 detection reagent in preparation of the chronic kidney disease osteoporosis diagnosis kit, and the application prospect is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnostic reagents, in particular to the use of a reagent for detecting TREM2 in the preparation of a diagnostic kit for chronic kidney disease osteoporosis. BACKGROUND

[0002] Chronic kidney disease (CKD) is a progressive renal dysfunction disease caused by various etiologies, and its main pathological features include glomerulosclerosis, renal interstitial fibrosis, renal tubular atrophy, and vascular rarefaction, ultimately leading to loss of renal units and irreversible failure of renal function. CKD patients often have multiple complications, among which osteoporosis is one of the core manifestations of CKD-mineral and bone disorder (CKD-MBD), and its pathological mechanism involves secondary hyperparathyroidism, vitamin D metabolic disorder, calcium and phosphorus metabolic imbalance, osteoblast-osteoclast coupling disorder, and remodeling of the inflammatory microenvironment, resulting in decreased bone density, destruction of bone microstructure, and significantly increased risk of bone fracture. CKD-related osteoporosis indicates a serious dysfunction of the bone-kidney axis, so early diagnosis of CKD osteoporosis and targeted intervention are key to delaying the progression of CKD and improving patient outcomes.

[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, but these techniques mainly reflect bone density or macroscopic changes in bone metabolism, and cannot sensitively capture micro-lesion information at the early inflammatory-immune microenvironment remodeling stage of CKD osteoporosis. Biomarkers can quantitatively assess the biological process of the disease, reflect the interaction of the bone-kidney axis, and serve as potential therapeutic targets, with their expression levels dynamically changing with the activity or progression of CKD osteoporosis. Therefore, screening of biomarkers with high sensitivity and specificity for CKD osteoporosis can help achieve early and accurate diagnosis and targeted treatment.

[0004] Biomarkers, as molecular indicators reflecting the development of diseases, play an important role in early screening, progression monitoring, and target discovery. In recent years, researchers have reported a variety of bone metabolism-related markers, such as osteocalcin, type I collagen C-terminal peptide (CTX), and bone-specific alkaline phosphatase (BALP), but their low sensitivity and poor specificity limit their diagnostic efficiency in the context of CKD, and there is an urgent need to screen new molecular markers with higher specificity.

[0005] Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor mainly expressed in myeloid cells (such as macrophages, dendritic cells, etc.), which is widely involved in the regulation of inflammatory response, cell phagocytosis and tissue repair process. The prior art has not reported the direct correlation between TREM2 and CKD-related osteoporosis, nor has it systematically verified the specificity, sensitivity and clinical application value of TREM2 as a diagnostic marker for CKD-related osteoporosis. SUMMARY

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

[0007] The use of the reagent for detecting TREM2 in the preparation of a chronic kidney disease osteoporosis diagnostic kit.

[0008] Preferably, the TREM2 is a myeloid cell triggering receptor 2, with UniProt number Q9NZC2.

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

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

[0011] Preferably, the reagent is a reagent for detecting the protein level of TREM2, selected from the group consisting of an immunohistochemical detection reagent, an immunofluorescence detection reagent, a western blot detection reagent, an ELISA detection reagent, a chemiluminescence immunoassay, an immunochromatography, an enzyme-linked immunospot method or a protein chip detection method.

[0012] Preferably, the reagent is a reagent for detecting the nucleic acid level of TREM2, selected from the group consisting of a QPCR detection reagent, a digital PCR detection reagent, an RT-PCR detection reagent or a nucleic acid hybridization detection reagent.

[0013] Preferably, the diagnostic kit is used to distinguish chronic kidney disease osteoporosis patients from healthy people.

[0014] Preferably, the threshold value of the expression amount of TREM2 for distinguishing chronic kidney disease osteoporosis patients from healthy people is 2300 pg / ml.

[0015] Preferably, the diagnostic kit is used to distinguish chronic kidney disease osteoporosis patients from chronic kidney disease patients without osteoporosis.

[0016] Preferably, the threshold value of the expression amount of TREM2 for distinguishing the chronic kidney disease osteoporosis patients from the chronic kidney disease patients without osteoporosis is 2300 pg / ml.

[0017] The key of the present application is that the expression amount of TREM2 in human serum is significantly related to chronic kidney disease osteoporosis. Therefore, the chronic kidney disease osteoporosis can be screened by detecting the expression amount of TREM2 in human serum. As for the means for detecting the expression amount of TREM2 in human serum, various means disclosed by the prior art can be used, and the embodiments of the present application specifically use WB, ELISA, immunofluorescence, QPCR and the like for detection, but are not limited to these means. Any method capable of detecting the expression amount of TREM2 protein or TREM2 nucleic acid can be used for screening chronic kidney disease osteoporosis.

[0018] The present application provides a new biomarker, which can realize effective screening of chronic kidney disease osteoporosis. The advantage of the present application is that early screening can be realized, the specificity is high, and serum can be used as a detection sample, which is low in harm to patients. Therefore, the present application has good application prospect.

[0019] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or change can be made without departing from the above basic technical idea of the present application.

[0020] The above content of the present application will be further described in detail through specific embodiments. However, this should not be understood as the scope of the above subject matter of the present application being limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The serum TREM2, Ca ion and P ion concentration level trends of the normal group and the CKD patients in each period (CKD 3-5 period) measured by the detection kit in experimental example 1 are shown, wherein (A) is the serum TREM2 level; (B) is the Ca ion level; (C) is the P ion level.

[0022] Figure 2 The ROC curve of serum TREM2 and calcium-phosphorus product of the normal group and the CKD patients in each period (CKD 3-5 period) measured by the detection kit in experimental example 1 is shown, AUC=1. **p<0.01, ****p<0.0001.

[0023] Figure 3Experimental results of the model constructed in Experimental Example 1 to simulate mineral metabolism disorder and renal function impairment (CKD-MBD model), wherein (A) Ca ion level; (B) P ion level; (C) uric acid level; (D) urea nitrogen level; (E) albumin level.

[0024] Figure 4 Femur 3D reconstruction image results of the CKD-MBD model in Experimental Example 1.

[0025] Figure 5 Histological staining images of the CKD-MBD model in Experimental Example 1, wherein (A) mouse kidney HE staining; (B) mouse kidney Masson staining; (C) mouse femur HE, Masson, TRAP, Safranin O-fast green staining and acid fuchsin-methylene blue staining.

[0026] Figure 6 TREM2 multi-modal detection results of the CKD-MBD model in Experimental Example 1, wherein (A) mouse TREM2 serum concentration; (B) TREM2-PCR results; (C) TREM2-WB results; (D) TREM2 femur section immunofluorescence; (E) serum TREM2 level and bone mineral density (Tb.BMD) correlation scatter plot results, ****p<0.0001. DETAILED DESCRIPTION

[0027] Example 1: Detection kit and method of use thereof I. Kit composition The present embodiment provides detection kits for several detection methods such as Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunofluorescence (IF), Real-Time Fluorescent Quantitative PCR (QPCR), etc. The compositions of various kits are as follows: 1. Western Blot (WB) detection kit Table 1 2. Enzyme-Linked Immunosorbent Assay (ELISA) detection kit Table 2 3. Immunofluorescence (IF) detection kit Table 3 4. Real-Time Fluorescent Quantitative PCR (QPCR) detection kit Table 4 II. Method of use of the kit The method of use of the above various kits is 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.

[0028] (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.

[0029] (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.

[0030] The technical solution of the present invention will be further explained through experiments below.

[0031] 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 were measured in both the control group and patients at each CKD stage using a diagnostic kit. The 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) and P ions ( Figure 1 C) Levels rise. TREM2 levels have high specificity and sensitivity in reflecting changes in calcium and phosphorus levels. Figure 2 ).

[0032] Table 5. Serum TREM2, Ca, and P ion concentrations, and calcium-phosphorus product in the normal group and patients at different stages of CKD. Note: The table contains the following information: group (001-020 for healthy individuals, 021-040 for CKD patients), serum calcium (units), serum phosphorus (units), calcium-phosphorus product, and serum TREM2 (units).

[0033] The above results indicate that elevated TREM2 levels sensitively reflect early mineral imbalances and bone abnormalities, and can serve as a biomarker for diagnosing osteoporosis in chronic kidney disease. Serum TREM2 ≥ 2300 pg / ml can serve as an excellent threshold for distinguishing patients with chronic kidney disease (with a high risk of osteoporosis) from healthy individuals, with near-perfect diagnostic efficacy (AUC≈1.000).

[0034] 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).

[0035] The results showed that the model group mice had significantly elevated levels of calcium ions and phosphorus, elevated uric acid and blood urea nitrogen, and decreased albumin. Figure 3 ).

[0036] 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 ).

[0037] 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 5 C), 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 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.

[0038] 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 6D) 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) A strong negative correlation was found between TREM2 and Tb.BMD (r = -0.872, R² = 0.7596, P < 0.001), with the regression equation Tb.BMD = -0.008 × TREM2 + 1.2774. This confirms that serum TREM2 levels can accurately reflect the severity of osteoporosis in CKD.

[0039] In summary, as demonstrated by the above embodiments and experimental examples, the kit of the present invention can screen individuals for CKD-MBD by detecting TREM2 expression levels: if TREM2 levels are high (relative to healthy individuals or CKD patients without osteoporosis), the probability of the patient having CKD-MBD is higher. This invention can be used for the early clinical diagnosis of CKD-MBD, exhibiting good specificity, stability, and variability, providing an effective basis for patients to take relevant treatment measures or make decisions, and has promising clinical application prospects.

Claims

1. Use of an agent for detecting TREM2 in the manufacture of a diagnostic kit for chronic kidney disease osteoporosis.

2. Use according to claim 1, characterized in that: Said TREM2 is the Triggering Receptor Expressed on Myeloid Cells 2, UniProt number Q9NZC2.

3. Use according to claim 1, characterized in that: Said agent is an agent for detecting the expression level of TREM2 in a human body fluid sample.

4. Use according to claim 3, characterized in that: Said body fluid sample is serum.

5. Use according to claim 1, characterized in that: Said agent is an agent for detecting the protein level of TREM2, selected from the group consisting of an immunohistochemical detection agent, an immunofluorescence detection agent, a western blot detection agent, an ELISA detection agent, a chemiluminescence immunoassay, an immunochromatography, an enzyme-linked immunospot method or a protein chip detection method.

6. Use according to claim 1, characterized in that: Said agent is an agent for detecting the nucleic acid level of TREM2, selected from the group consisting of a QPCR detection agent, a digital PCR detection agent, an RT-PCR detection agent or a nucleic acid hybridization detection agent.

7. Use according to claim 1, characterized in that: Said diagnostic kit is used to distinguish chronic kidney disease osteoporosis patients from healthy people.

8. Use according to claim 7, characterized in that: The threshold value of the expression level of TREM2 for distinguishing chronic kidney disease osteoporosis patients from healthy people is 2300 pg / ml.

9. Use according to claim 1, characterized in that: Said diagnostic kit is used to distinguish chronic kidney disease osteoporosis patients from chronic kidney disease patients without osteoporosis.

10. Use according to claim 9, characterized in that: The threshold value of the expression level of TREM2 for distinguishing chronic kidney disease osteoporosis patients from chronic kidney disease patients without osteoporosis is 2300 pg / ml.

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