Use of a reagent for detecting tnn in the preparation of a diagnostic kit for diagnosing vascular calcification in chronic kidney disease

Abstract

The present application relates to the field of in vitro diagnostic reagents, in particular to the use of a reagent for detecting TNN in the preparation of a diagnostic kit for chronic kidney disease vascular calcification, and it is found for the first time that the expression level of TNN is significantly correlated with chronic kidney disease vascular calcification. Therefore, the expression level of TNN can be used for screening of chronic kidney disease vascular calcification. Based on this, the present application provides the use of a reagent for detecting TNN in the preparation of a diagnostic kit for chronic kidney disease vascular calcification, 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 TNN in the preparation of diagnostic kits for vascular calcification in chronic kidney disease. Background Technology

[0002] Vascular calcification (VC) is a core common pathological step in the progression of various chronic kidney diseases (CKD). Its pathological features include phenotypic transformation of vascular smooth muscle cells, recruitment and infiltration of inflammatory cells, activation and proliferation of osteoblast-like and chondrocyte-like cells, deposition of calcified matrix (hydroxyapatite crystals, osteopontin, osteocalcin), degradation of vascular elastic fibers, thickening and stiffening of the vessel wall, and narrowing of the lumen, ultimately leading to damage to the normal structure and function of blood vessels. Because vascular calcification in CKD signifies irreversible vascular damage, early diagnosis and treatment targeting the underlying cause are crucial for slowing CKD progression and reducing the risk of cardiovascular complications.

[0003] Current diagnostic techniques for vascular calcification in chronic kidney disease (CKD-VC) are mainly divided into three categories:

[0004] I. Diagnostic techniques based on traditional molecular markers (osteopeptide OPG + fetuin-A): The concentrations of OPG (elevated) and Fetuin-A (decreased) in the serum of CKD patients are detected by ELISA, and VC is determined in combination with calcium and phosphorus indicators.

[0005] II. Diagnostic techniques based on imaging (multi-slice spiral computed tomography, MSCT): Calcification is quantified by scanning the abdominal aorta or lower extremity vessels with MSCT and using the Agatston score (≥10 points is positive) (see attached diagram including equipment, tomographic images, and scoring interface). The relevant basis is the "Chinese Expert Consensus on the Diagnosis and Treatment of Vascular Calcification in CKD Patients (2020)".

[0006] III. Diagnostic techniques based on a single signaling pathway molecule (bone morphogenetic protein 2, BMP-2): Vascular tissue is obtained by puncture, and BMP-2 expression is detected by immunohistochemistry (IRS ≥ 6 points indicates VC).

[0007] However, the aforementioned existing technologies still have various problems. Traditional molecular marker diagnostic techniques suffer from low specificity, are easily affected by inflammation and liver / kidney function, and exhibit diagnostic lag, only detecting in the middle and late stages of disease. Imaging diagnostic techniques are disadvantaged in that they cannot detect early microscopic calcifications, posing a risk of missed diagnoses; they carry radiation hazards; and they are highly dependent on equipment, making them difficult to implement in primary healthcare institutions. Single-signal pathway molecular diagnostic techniques are invasive, carrying risks of bleeding and infection; and have poor specificity, easily affected by factors such as bone metabolism and renal fibrosis.

[0008] Therefore, there is still a need in this field to develop more new methods for diagnosing CKD-VC, especially methods that can make specific diagnoses at an early stage.

[0009] Tenascin-N (TNN), also known as Tenascin-W, is the fourth member of the extracellular matrix glycoprotein family of Tenascin. It consists of an EGF-like repeat sequence, a fibronectin type III domain, and a carboxyl-terminal fibroinogen-like domain (FBG). As a secreted protein, TNN primarily binds directly to Toll-like receptor 4 (TLR4) through its FBG domain, activating the NF-κB signaling pathway and inducing the expression of pro-inflammatory cytokines (such as IL-6 and TNF-α), thus playing a role in the regulation of the immune microenvironment. In the nervous system, TNN promotes hippocampal neurite growth and neuronal migration, participating in neural development and plasticity regulation. Furthermore, TNN is abnormally expressed in extracellular vesicles derived from vascular smooth muscle cells and in liver and ovarian cancer tissues, suggesting its potential role in pathological processes such as tumor progression.

[0010] As the least studied member of the tenascin family, the detailed molecular mechanisms, full picture of physiological functions, and clinical translational value of TNN still need to be elucidated in depth. Current technologies have not reported a direct association between TNN and CKD-VC, nor have they systematically verified its specificity, sensitivity, and clinical application value as a diagnostic biomarker for CKD-VC. Summary of the Invention

[0011] The purpose of this invention is to provide a novel biomarker for screening vascular calcification in chronic kidney disease, and the use of the detection reagent of this biomarker in the preparation of a chronic kidney disease vascular calcification screening kit.

[0012] The use of reagents for detecting TNN in the preparation of diagnostic kits for vascular calcification in chronic kidney disease.

[0013] Preferably, the TNN is tendinin N, UniProt numbered Q9UQP3.

[0014] Preferably, the reagent is a reagent for detecting the expression level of TNN in human body fluid samples.

[0015] Preferably, the body fluid sample is serum.

[0016] Preferably, the reagent is a reagent for detecting TNN protein levels, selected from immunohistochemical detection reagents, immunofluorescence detection reagents, western blot detection reagents, ELISA detection reagents, chemiluminescent immunoassay, immunochromatography, or reagents used in protein chip detection methods.

[0017] Preferably, the reagent is a reagent for detecting TNN nucleic acid levels, selected from qPCR detection reagents, digital PCR detection reagents, RT-PCR detection reagents, or nucleic acid hybridization detection reagents.

[0018] Preferably, the diagnostic kit is used to distinguish between patients with vascular calcification in chronic kidney disease and healthy individuals.

[0019] Preferably, the threshold for TNN expression level that distinguishes patients with vascular calcification in chronic kidney disease from healthy individuals is 525 pg / ml.

[0020] Preferably, the diagnostic kit is used to distinguish between patients with vascular calcification in chronic kidney disease and those without vascular calcification in chronic kidney disease.

[0021] Preferably, the threshold for TNN expression level that distinguishes between patients with vascular calcification in chronic kidney disease and those without vascular calcification is 525 pg / ml.

[0022] The key to this invention lies in determining that the expression level of TNN in human serum is significantly correlated with vascular calcification in chronic kidney disease. Therefore, vascular calcification in chronic kidney disease can be screened by detecting the expression level of TNN in human serum. As for the specific methods for detecting the expression level of TNN in human serum, various methods disclosed in the prior art can be used. This invention specifically employs methods such as Western blotting (WB), ELISA, immunohistochemistry, immunofluorescence, and qPCR, but is not limited to these methods. Any method capable of detecting the expression level of TNN protein or TNN nucleic acid can be used for screening vascular calcification in chronic kidney disease.

[0023] This invention provides a novel biomarker capable of effectively screening for vascular calcification in 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.

[0024] 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.

[0025] 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

[0026] Figure 1 The results show the construction and validation of the typical calcification model in Example 1, including: (A) Alizarin Red S staining of vascular smooth muscle cells; (B) Proportion of positive Alizarin Red staining results; (C) Calcium content determination results in cells; (D, E) PCR results of α-SMA and Runx-2 in cells; (F) WB results of α-SMA and Runx-2; and (G) Gray value analysis of α-SMA and Runx-2 bands. *p<0.05, ***p<0.001.

[0027] Figure 2 The results show the TNN expression detection of the typical calcification model in Experiment 1, including (A) TNN WB results; (B) WB band gray value statistics; and (C) qPCR results. **p<0.01, ***p<0.001.

[0028] Figure 3 The results are for the CKD vascular calcification mouse model constructed in Experiment Example 2. (A) Survival curves of each group of mice (Control group: fed with normal diet; Model group: CKD vascular calcification model; the animal experimental grouping design in the following results figures is consistent with this); (B) Mouse survival curves; (C) Images of mouse kidneys; (D) Masson staining results of mouse kidneys; (E) Serum creatinine for mouse renal function; (F) Blood urea nitrogen. ***p<0.001.

[0029] Figure 4 The results are for the CKD vascular calcification mouse model constructed in Experiment Example 2. (A) MicroCT scan of mice showing vascular calcification and ectopic calcium salt deposition in the aortic wall of the model group mice; (B) MicroCT analysis of the volume of calcium salt deposition in the mouse aorta; (C) Serum ALP enzyme content in mice; (D) Serum phosphorus concentration in mice; (E) Serum calcium concentration in mice. ***p<0.001.

[0030] Figure 5The results are for the CKD vascular calcification mouse model constructed in Experiment Example 2. (A) Alizarin Red S staining of the entire aorta; (C) Von kossa and Alizarinred aortic sections; (B, D) Von kossa and Alizarinred positive areas; (E) α-SMA and Runx-2 immunohistochemistry; (F, G) α-SMA and Runx-2 immunohistochemistry results; (I) α-SMA and Runx-2 WB bands; (H) WB grayscale value statistics. *p<0.05, ***p<0.001.

[0031] Figure 6 The results show the TNN expression detection in the CKD vascular calcification mouse model constructed in Experiment Example 2, including (A) WB band results; (B) WB band gray value analysis; (C) TNN immunofluorescence of aortic sections; (D) Immunohistochemical analysis; (E) PCR results; and (F) Mouse serum TNN ELISA results. ***p<0.001, ****p<0.0001.

[0032] Figure 7 The ROC curves of TNN and calcification integrals are shown in Experiment Example 3 when different populations are diagnosed. The AUC is 0.9909. Detailed Implementation

[0033] Example 1: Detection kit and its usage method

[0034] I. Components of the Reagent Kit

[0035] This embodiment provides detection kits for several detection methods, including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence (IF), and quantitative real-time PCR (qPCR). The components of each kit are as follows:

[0036] 1. Western Blot (WB) Detection Kit

[0037] Table 1

[0038]

[0039] 2. Enzyme-linked immunosorbent assay (ELISA) kit

[0040] Table 2

[0041]

[0042] 3. Immunohistochemical (IHC) detection kit

[0043] Table 3

[0044]

[0045] 4. Immunofluorescence (IF) detection kit

[0046] Table 4

[0047]

[0048] 5. Real-time quantitative PCR (qPCR) detection kit

[0049] Table 5

[0050]

[0051] II. Instructions for using the reagent kit

[0052] The usage steps for the above reagent kits are as follows:

[0053] (1) WB: Total protein was extracted with reagents; SDS-PAGE electrophoresis (10% separating gel); transferred to PVDF membrane; blocked with blocking buffer for 1 h; primary antibody (1:1000 dilution) was incubated overnight at 4℃; washed 3 times with washing buffer; enzyme-labeled secondary antibody (1:5000 dilution) was incubated at room temperature for 1 h; after washing, chemiluminescent substrate was added; the gray value of ACVR2b band was detected by a developing instrument.

[0054] (2) ELISA: Capture antibody (1 μg / mL) is coated onto the microplate; incubated at 4℃ for 12 h; blocking buffer is applied for 2 h; sample (50 μL / well) is added; incubated at 37℃ for 1 h; detection antibody (0.5 μg / mL, enzyme-labeled HRP) is applied; incubated at 37℃ for 45 min; TMB substrate is applied for 15 min; stop the reaction with reagent stop solution (2 mol / L H2SO4); absorbance is read at 450 nm using a microplate reader.

[0055] (3) Immunohistochemistry: Dewaxing of tissue sections; gradient hydration; antigen retrieval with citrate buffer; blocking of endogenous enzymes with 3% H2O2; blocking with blocking solution; incubation of primary antibody (1:200 dilution) at 4℃ overnight; incubation of enzyme-labeled secondary antibody at room temperature for 30 min; DAB staining; hematoxylin counterstaining; observation of TNN positive staining intensity under a microscope.

[0056] (4) Immunofluorescence: Tissue sections were fixed (4% paraformaldehyde); permeabilized with 0.1% Triton X-100; blocked with blocking solution; antibody fluorescent labeling (1:300 dilution) was incubated overnight at 4℃; nuclei were stained with DAPI; ACVR2b fluorescence signal was detected by fluorescence microscopy.

[0057] (5) QPCR: RNA was extracted; reverse transcribed into cDNA; the reaction system contained primers F: 5'-CTGGCGACTGTAGTGGCAAT-3' (SEQ ID NO. 1, 10 μmol / L), R: 3'-ACCTGTTTTGATGCTTGCTCC-3' (SEQ ID NO. 2, 10 μmol / L), probe (5'-FAM-XXX-BHQ1-3', 5 μmol / L), and fluorescent PCR Mix; amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s; 60℃ annealing extension for 30 s (40 cycles); real-time quantitative PCR analysis.

[0058] The technical solution of the present invention will be further explained through experiments below.

[0059] Experimental Example 1: In vitro studies confirmed a significant increase in TNN expression in a sodium dihydrogen phosphate-induced smooth muscle calcification model.

[0060] A cell calcification model was constructed using 5th-6th generation mouse vascular smooth muscle cells, induced for 7 days in DMEM medium containing 3mM inorganic phosphorus NaH2PO4 (Pi) and 10% fetal bovine serum. Results showed:

[0061] Pi-induced calcium salt deposition was observed in Pi-induced smooth muscle cells by S staining. Figure 1 Cellular calcium content detection showed that the calcium content in the Pi group was significantly increased (A, B). Figure 1 C); PCR and WB results showed that Runx2 expression was increased and α-SMA expression, a marker of vascular smooth muscle cells, was decreased in the calcified model cells, verifying that the model was a typical calcification model. Figure 1 D - G).

[0062] Following the method described in Example 1, cellular proteins were extracted and analyzed by Western blotting. A significant increase in TNN was observed (…). Figure 2 A, B); Following the method described in Example 1, qPCR detection was performed on RNA, and TNN expression was found to be increased ( Figure 2 C).

[0063] The above results indicate that TNN expression is significantly increased in calcified vascular smooth muscle cells.

[0064] In vivo studies in Experiment Example 2 confirmed a significant increase in expression in a mouse model of vascular calcification.

[0065] A mouse model of CKD vascular calcification was constructed using an adenine-rich and high-phosphorus diet (after one week of normal diet adaptation, mice were fed a diet containing 0.2% adenine for four weeks to induce CKD, and then calcification was induced for 10 weeks using a diet containing 0.2% adenine and 1.8% phosphorus).

[0066] The results showed that the model group experienced significant weight loss and increased mortality. Figure 3 A, B), significant kidney atrophy ( Figure 3 C), Masson staining showed significant renal fibrosis ( Figure 3 D), serum urea nitrogen and creatinine levels were significantly elevated ( Figure 3 E, F).

[0067] MicroCT scans revealed severe calcification of the thoracic and abdominal aortic walls in the model mice, while no calcification foci were observed in the control group. Furthermore, ectopic calcium salt deposits were found in multiple locations throughout the model group mice. Figure 4 A, B); Serum ALP in model group mice ( Figure 4 C) and blood phosphorus levels ( Figure 4 D) significantly increased, blood calcium level significantly decreased ( Figure 4 E).

[0068] Alizarin Red S staining was performed on isolated mouse aortas, revealing significant calcium salt deposition in the aorta of the model mice. Figure 5 A); Alizarin Red S staining and Von Kossa silver staining of aortic sections both indicated aortic vascular calcification in the model group ( Figure 5 B-D); Immunohistochemical and Western blot results showed that the expression of α-SMA, a marker of vascular smooth muscle cells, was significantly reduced in the aorta of the model group mice, while the expression of Runx2, a marker of osteogenic differentiation, was significantly increased. Figure 5 E - I). The above results confirm that the constructed model is a typical vascular calcification model of chronic kidney disease.

[0069] TNN was detected according to the method described in Example 1: Vascular tissue proteins and RNA were extracted and analyzed by Western blotting and qPCR, revealing a significant increase in TNN expression. Figure 6 A, B, E); Immunofluorescence staining of mouse vascular sections showed a significant increase in TNN expression in the model group vascular sections. Figure 6 C, D); TNN ELISA was performed on mouse serum, and the TNN content in the model group was significantly increased. These results validate that TNN has high specificity, stability, and differential efficacy in distinguishing between healthy mice and mice with vascular calcification in chronic kidney disease. Figure 6 F).

[0070] Experimental Case 3 clinically confirmed that TNN expression was significantly increased in CKD-VC patients.

[0071] Clinical trials recruited healthy individuals, CKD patients without vascular calcification (calcification score of 0), and CKD patients with vascular calcification (CKD-VC patients). Serum TNN levels were detected using the ELISA method described in Example 1. Results showed that TNN expression levels in the serum of CKD-VC patients were significantly higher than those in the healthy control group and the CKD patient group without vascular calcification. The calcification score and TNN level were statistically analyzed (as shown in the table below). Correlation analysis between calcification score and TNN level revealed a positive correlation between TNN and calcification score, with a high correlation coefficient (AUC = 0.9909), indicating that this molecule has good diagnostic efficacy. Figure 7 The TNN level used for the diagnosis of CKD-VC patients can be set according to clinical needs for specificity or sensitivity; for example, a preferred approach is 525 pg / ml.

[0072] Table 6. Detection results of samples from each experimental group

[0073]

[0074] Note: The contents of this table are clinical samples (healthy individuals, CKD patients, and CKD patients with vascular calcification), calcification score (AU), and TNN (pg / ml).

[0075] In summary, as demonstrated by the above embodiments and experimental examples, the kit of the present invention can screen individuals for CKD-VC by detecting the expression level of TNN: if the TNN level is high (relative to healthy individuals or CKD patients without vascular calcification), the probability of the patient having CKD-VC is higher. This invention can be used for the early clinical diagnosis of CKD-VC, 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. The use of reagents for detecting TNN in the preparation of diagnostic kits for vascular calcification in chronic kidney disease, characterized in that: The TNN is tendinin N, UniProt numbered Q9UQP3.

2. The use according to claim 1, characterized in that: The reagent is used to detect the expression level of TNN in human serum samples.

3. The use according to claim 1, characterized in that: The reagents are for detecting TNN protein levels and are selected from immunohistochemical detection reagents, immunofluorescence detection reagents, western blot detection reagents, ELISA detection reagents, chemiluminescent immunoassay, immunochromatography, or protein chip detection methods.

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

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

6. The use according to claim 1, characterized in that: The diagnostic kit is used to differentiate between patients with vascular calcification in chronic kidney disease and those without vascular calcification.

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