Peripheral blood marker for acute pyelonephritis

Abstract

The invention belongs to the field of biological medicine industry, and discloses an acute pyelonephritis peripheral blood marker. The inventor finds and verifies that the average value of LRG1 in blood is higher than that of a normal person during acute pyelonephritis for the first time, LRG1 serving as a peripheral blood marker of acute pyelonephritis has high sensitivity and specificity, and treatment of acute pyelonephritis can be guided more effectively.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine industry, specifically relating to a peripheral blood marker for acute pyelonephritis. Background Technology

[0002] Pyelonephritis (PN) is a common urinary tract infection, primarily characterized by inflammation of the renal pelvis and renal parenchyma. Based on the duration of the illness, pyelonephritis is classified into acute and chronic pyelonephritis. Its causes are complex, including urinary tract obstruction, urethral instrumentation, weakened immune system function, pregnancy, and unhealthy lifestyle habits. High-risk groups include pregnant women, the elderly, those with weakened immune systems, and those with long-term indwelling catheters. While pyelonephritis itself does not directly cause death, untreated pyelonephritis can lead to serious complications such as sepsis and renal failure, thus endangering the patient's life.

[0003] Clinical manifestations: Clinical symptoms of acute pyelonephritis include back pain, urinary tract irritation symptoms such as urinary frequency, urgency, and dysuria, as well as systemic symptoms such as fever, chills, nausea, and vomiting. In severe cases, complications such as pyuria, hematuria, and renal insufficiency may occur.

[0004] Diagnosis: This usually involves taking a medical history, physical examination, and necessary imaging studies to confirm the diagnosis and determine the severity.

[0005] Medical history taking: The doctor will ask the patient in detail about the symptoms, duration, and triggering factors.

[0006] Physical examination: red throat, fever, and rapid heart rate.

[0007] Imaging examinations: Imaging techniques such as X-rays, ultrasound, CT, or MRI can help identify the shape of the kidneys.

[0008] Laboratory tests showed elevated white blood cell count in urine and bacterial growth in urine culture.

[0009] Prevention and Treatment: Use appropriate anti-infective drugs and treat symptoms. Avoid holding urine. Treatment for pyelonephritis typically includes antibiotics, adequate hydration, and pain and fever control. Acute pyelonephritis usually requires intravenous antibiotics, followed by oral therapy. Chronic pyelonephritis may require long-term antibiotic treatment and treatment for urinary tract obstruction. The prognosis is generally good, especially for patients who receive timely treatment. Preventive measures include maintaining good personal hygiene, timely treatment of urinary tract infections, and avoiding urinary tract obstruction.

[0010] Research and treatment techniques for pyelonephritis have continuously advanced with the development of medicine, particularly in the research and application of antibiotics. Furthermore, treatment methods for urinary tract obstruction, such as minimally invasive surgery and extracorporeal shock wave lithotripsy, have provided more options for the treatment of pyelonephritis. With a deeper understanding of kidney diseases, more personalized treatment plans for pyelonephritis may emerge in the future.

[0011] The relationship between acute pyelonephritis and peripheral blood proteins Pyelonephritis causes related symptoms in patients, and every symptom has a material basis, involving changes in gene expression and proteins. Proteins are the functional executors of genes, and the study of protein structure, location, and protein-protein interactions will provide a direct foundation for elucidating the nature of life phenomena. Almost all physiological and pathological processes, as well as the effects of drugs and environmental factors, depend on proteins and cause changes in the proteome. Before any disease manifests any perceptible symptoms, certain proteins have certainly changed. Therefore, identifying key and marker proteins for various diseases is of great significance for disease diagnosis, pathological research, and drug screening. Leucine-rich repeat (LRR) proteins are a family of proteins consisting of eight leucine-rich repeat sequences. Studies have shown that LRR proteins are transmembrane proteins that mainly participate in normal physiological activities as cell adhesion molecules or ligand-binding proteins.

[0012] Leucine-rich α2-glycoprotein 1 (LRG1) is a member of the leucine-rich repeat family and belongs to the LRR protein family. Previous studies have shown that LRG1 participates in important physiological and pathological processes, such as protein-protein interactions, signal transduction, and cell adhesion.

[0013] Currently, various proteins with leucine-rich repeat structures have been found in the nervous system. In some neurodegenerative diseases, decreased concentrations of LRG1 in cerebrospinal fluid (CSF) have been detected, and human LRG1 expression has been observed in the anatomically examined cerebral cortex. Previous studies have performed proteomic analysis on CSF samples from healthy individuals and patients with idiopathic normal pressure hydrocephalus (INPH), finding a specific increase in LRG1 expression in the CSF of INPH patients. Therefore, LRG1 has potential value in the clinical diagnosis of INPH. Abnormal LRG1 expression is associated with the development of various tumors, such as glioma, lung cancer, colon cancer, epithelial ovarian cancer, and hepatocellular carcinoma.

[0014] In studies of retinopathy caused by vascular remodeling, LRG1 was found to promote endothelial cell mitosis and angiogenesis. This hypothesis was further confirmed in in vitro human vascular endothelial cell (HUVEC) lumen formation assays. Providing recombinant human LRG1 as a medium resulted in a significant increase in lumen formation and branching, while anti-LRG1 antibodies significantly blocked lumen formation. Other studies have shown that LRG1's angiogenic activity is not limited to the eye and may play important roles in other major biological processes, including tumors and immune responses. The regulatory role of LRG1 on the TGF-β1 signaling pathway provides strong evidence for the function of LRG1. LRG1 is a very promising therapeutic target for controlling ocular angiogenesis and may play an important role in other diseases such as cancer and atherosclerosis.

[0015] LRG1's function may be related to its LRR structure. LRG is thought to regulate the function of other proteins, such as cytochrome C, by binding to them, thereby limiting the function of some proteins. Gliomas originate from glial cells and are the most common tumors of the central nervous system, accounting for 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors. Related experiments have shown that LRG1 plays an important role in promoting the proliferation, migration, and invasion of glioma cells, and these functions are achieved by mediating the transforming growth factor β1 (TGF-β1) signaling pathway. Furthermore, LRG1 can promote endothelial cell proliferation and angiogenesis through the TGF-β signaling pathway, suggesting that LRG1 may be involved in the occurrence and development of astrocytic malignancies. Previous studies have shown that inhibiting LRG1 expression inhibits the in vitro growth of glioma cells and delays the occurrence of gliomas in xenograft mouse models, providing evidence for the tropism of LRG1 on glioma pathological changes. LRG1 is associated with atherosclerosis and microcirculatory disturbances, and may serve as a biomarker for vascular injury in different vascular beds. LRG1 has the potential role in the early identification of vascular disease risk, but the pathological basis and mechanisms underlying these associations require further investigation. Altering LRG1 activity could be considered as a therapeutic potential in vascular diseases, particularly in minimally invasive surgery and endovascular revascularization. LRG1 expression is upregulated after myocardial infarction, and inhibiting post-infarction cardiac remodeling is a novel cardiovascular protective factor; therefore, LRG1 may become a therapeutic target for cardiovascular diseases. LRG1 gene deletion exacerbates post-infarction cardiac dysfunction and increases myocardial fibrosis. In LRG1-deficient mice, impaired smad1 / 5 / 8 pathways lead to reduced capillary density at the infarct margin, and LRG1 gene ablation further exacerbates the reduction in capillary density after myocardial infarction in a cardiac remodeling model. Furthermore, our study shows elevated blood LRG1 levels after cardiopulmonary resuscitation in patients with cardiac and respiratory arrest, which may be associated with brain and vascular injury caused by cardiac and respiratory arrest. The mechanism of elevated LRG1 levels in the blood is consistent with that in patients with cerebral infarction and myocardial infarction.

[0016] On the other hand, LRG1, as an angiogenesis factor, may have considerable potential for the treatment of vascular diseases by regulating the smad1 / 5 / 8 pathway, but its specific regulatory mechanism is still unclear.

[0017] The inventor's prior Chinese patent application CN117110626A discloses that the average LRG1 level in the blood is higher in individuals with carotid artery disease than in normal individuals, and that the detachment of plaques from carotid vessels increases more significantly as the disease progresses. As a peripheral blood biomarker for dangerous carotid artery disease, it exhibits high sensitivity and specificity and can be used in conjunction with ultrasound results for the diagnosis of carotid artery disease. CN115856325A discloses that the level of peripheral blood LRG1 is closely related to lower limb arterial injury. By detecting the level of LRG1 in peripheral blood, lower limb arterial injury, especially lower limb arteriosclerosis or plaque, lower limb arterial stenosis, thrombosis, or occlusion, can be well predicted. It is particularly effective in predicting lower limb arteriosclerosis or plaque, lower limb arterial stenosis, thrombosis, or occlusion caused by hypertension, hyperlipidemia, or hyperglycemia. CN110261617A discloses that LRG1 can be used as a peripheral blood biomarker for cerebral hemorrhage. CN120539428A discloses that LRG1 can be used as an auxiliary diagnostic reagent for acute pyelonephritis.

[0018] In the existing technology, no research has shown that LRG1 is associated with acute pyelonephritis. Summary of the Invention

[0019] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a peripheral blood marker for acute pyelonephritis.

[0020] The technical solution adopted in this invention is: The first aspect of the present invention provides: Application of LRG1 quantitative reagent in the preparation of diagnostic or auxiliary diagnostic reagents for acute pyelonephritis.

[0021] In some instances, the LRG1 quantification reagent is an ELISA reagent, an immunofluorescence reagent, or a mass spectrometry analysis reagent.

[0022] In some instances, the samples tested by the LRG1 quantitative reagent are peripheral blood.

[0023] A second aspect of the present invention provides: A diagnostic or auxiliary diagnostic system for acute pyelonephritis, comprising: The LRG1 quantification device is used to quantify the amount of LRG1 in a sample. An analytical device was used to determine the risk of acute pyelonephritis based on LRG1 levels. The result output device is used to output the analysis results of the analysis device.

[0024] In some instances, the LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

[0025] In some cases, high LRG1 levels indicate a high risk of acute pyelonephritis.

[0026] In some cases, high LRG1 levels refer to levels exceeding 5 ng / mL in peripheral blood samples.

[0027] In some instances, the samples tested by the LRG1 quantitative reagent are peripheral blood.

[0028] The beneficial effects of this invention are: The inventors' research first discovered and verified that the average value of LRG1 in the blood during acute pyelonephritis is higher than that in normal people. LRG1, as a peripheral blood marker for acute pyelonephritis, has high sensitivity and specificity, which is beneficial for more effectively guiding the treatment of acute pyelonephritis. Detailed Implementation

[0029] The first aspect of the present invention provides: Application of LRG1 quantitative reagent in the preparation of diagnostic or auxiliary diagnostic reagents for acute pyelonephritis.

[0030] LRG1 quantification reagents can be any conventional protein quantification reagents, with no special requirements. In some examples, the LRG1 quantification reagent is an ELISA reagent, an immunofluorescence reagent, or a mass spectrometry analysis reagent. ELISA reagents are preferred for LRG1 quantification because they are easy to use and the results meet the requirements.

[0031] In some instances, peripheral blood is used as the sample for LRG1 quantification. Peripheral blood is readily available and widely accepted.

[0032] A second aspect of the present invention provides: A diagnostic or auxiliary diagnostic system for acute pyelonephritis, comprising: The LRG1 quantification device is used to quantify the amount of LRG1 in a sample. An analytical device was used to determine the risk of acute pyelonephritis based on LRG1 levels. The result output device is used to output the analysis results of the analysis device.

[0033] There are no special requirements for the type of LRG1 quantification device, as long as it can effectively quantify the amount of LRG1. In some examples, the LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

[0034] In some cases, high LRG1 levels indicate a high risk of acute pyelonephritis.

[0035] In some cases, high LRG1 levels refer to levels exceeding 5 ng / mL in peripheral blood samples.

[0036] In some instances, the sample is peripheral blood.

[0037] The technical solution of the present invention will be further explained below with reference to experiments.

[0038] Clinical sample screening and collection: Fifty cases of acute pyelonephritis within the past 3 years were collected. Gender and age were recorded. All patients underwent blood pressure, blood lipids, blood glucose, liver function, kidney function, coagulation parameters, ECG, and other examinations. All cases were confirmed by ultrasound, CT, and neurophysiological testing. Sixty age- and gender-matched healthy individuals were selected as controls. Specimen collection: 5 mL of venous blood was collected from patients meeting the inclusion criteria. After standing for 15 min, the blood was centrifuged at 3000 rpm for 15 min to extract serum. The serum was then placed in sterile cryovials and stored at -80°C for research.

[0039] Study of blood samples from case patients Serum separation. The examination method is as follows: serum and cells are separated using a high-speed centrifuge.

[0040] Main equipment, materials, and reagents: homogenizer, high-speed centrifuge, 3kDa ultrafiltration centrifuge tubes, ICP-MS. The water used in the experiment was ultrapure water with a concentration of 18.2 MΩ / cm. Sample pretreatment methods: 1) Fully automated biochemical analyzer (one each of Bekmann DXC800 and AU400), 0.01% electronic analytical balance (Shimadzu), microplate reader, Roche electrochemiluminescence automated immunoassay analyzer (Elecsys 2010), specific protein analyzer (imported), purified water system (MEDICA60), electric thermostatic water bath (DK-600), low-temperature high-speed centrifuge (Sigma), ultra-low temperature freezer (Thermo Fisher Scientific), low-temperature freezer (HFC350, Germany), etc. The above equipment conditions fully guarantee the implementation of this project.

[0041] Statistical methods: The t-test was used to compare the means of each group. SPSS statistical software was used, and P < 0.05 was considered statistically significant.

[0042] result: The normal range is determined by adding two standard deviations to the mean. For the Shenzhen-made LRG1 test kit (LRG1 Shenzhen), the upper limit of normal is 5 ng / ml. In a study of 50 cases of acute pyelonephritis, with a cutoff value of 5 ng / ml, 45 results exceeded the cutoff value, resulting in 5 false negatives (sensitivity 90%). Among 60 normal individuals, 12 exceeded the cutoff value (false positive 20%), with a specificity of 80%.

[0043] The changes in peripheral blood LRG1 in acute pyelonephritis are shown in Table 1.

[0044] Table 1: Peripheral blood LRG1 changes in acute pyelonephritis (X ± SD, unit ng / ml) Number of examples LRG1 / ng / ml normal people 60 3.2 ± 1.1 Acute pyelonephritis 50 16.6 ± 3.8 The level of acute pyelonephritis was significantly higher than that in normal individuals (P<0.001).

[0045] Sensitivity and specificity in diagnosing acute pyelonephritis Sensitivity = (Number of true positives / (Number of true positives + Number of false negatives)) * 100%, the percentage of patients correctly diagnosed. Specificity = (Number of true negatives / (Number of true negatives + Number of false positives)) * 100%, the percentage of individuals correctly identified as not being patients. False positive rate = Number of false positives / Number of negative results (gold standard) False negative rate = Number of false negatives / Number of gold standard positives The sensitivity and specificity of LRG1 in diagnosing acute pyelonephritis are shown in Table 2.

[0046] Table 2: Sensitivity and specificity of LRG1 in diagnosing acute pyelonephritis Number of examples Correct number Error count Sensitivity False negative rate Specificity False positive normal people 60 48 12 / / 80% 20% Acute pyelonephritis 50 45 5 90% 10% / / Table 3 shows the changes in LRG1 before and after treatment for acute pyelonephritis.

[0047] Table 3: Changes in LRG1 levels before and after treatment for acute pyelonephritis Number of examples LRG1 / ng / ml normal people 60 3.2 ± 1.1 Before treatment of acute pyelonephritis 50 16.6 ± 3.8 After treatment of acute pyelonephritis 32 3.9 ± 1.6 Note: Of the 50 patients with acute pyelonephritis, 32 underwent follow-up examinations after treatment, while the other 18 did not return to the hospital for follow-up examinations.

[0048] After treatment for acute pyelonephritis, LRG1 levels were significantly reduced compared to before treatment (P<0.001), and were comparable to normal levels (P>0.05).

[0049] The above experimental results indicate that the average LRG1 level in the blood of patients with acute pyelonephritis is higher than that of normal individuals, and LRG1 has high sensitivity and specificity as a peripheral blood marker for acute pyelonephritis. In patients with acute pyelonephritis after treatment, the LRG1 level in their peripheral blood significantly decreased, approaching normal levels. Therefore, based on the LRG1 content in peripheral blood and other known methods, acute pyelonephritis can be diagnosed effectively, or its cure status can be determined, providing quantifiable evidence for accurate medication and adjustment of treatment plans.

[0050] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

[0051] References: [1] Tessa Gordon 1, NasimAmirjani, David C Edwards, K Ming Chan,Briefpost-surgical electrical stimulation accelerates axon regeneration andmusclereinnervation without affecting the functional measures in carpaltunnel syndrome patients. Trial Exp Neurol. 2010 May;223(1):192-202. [2] Proteomic analysis ofhippocampus in mice following long-termexposure to low levels of copper. Qian Sun, Ming Ying, Quan Ma, Zhijun Huang,Liangyu Zou, Jianjun Liu, ZhixiongZhuang, Xifei Yang. Toxicol. Res.,2016,4,28 [3] Serum proteomic analysisreveals potential serum biomarkers foroccupational medicamentosa-like dermatitis caused by trichloroethylene PeiwuHuang a,d,1, Xiaohu Ren a,1,Zhijun Huang c,1, Xifei Yang a, Wenxu Hong a,Yanfang Zhang b, Hang Zhang a,d, Wei Liu a, Haiyan Huang a, Xinfeng Huang a,Desheng Wua, Linqing Yang a, HaiyanTang b, Li Zhou a, Xuan Li a, Jianjun Liu。Toxicology Letters 2014,6,21(sci 3.75) [4] 1. Ditochondrial proteomics caused by long-term low –dose copper exposure in mouse cortex. Xuemei Lin, Gang Wei, Zhijun Huang, etc. Toxicology Letter, 2016 [5] Huang Zhijun, Bai Chengwen, Wang Shuiyun, et al. Distribution of coagulation factor VII R353Q genotype in healthy Han Chinese in South China [J]. Practical Preventive Medicine, 2007, 14(004):1007-1008. [6] Huang Zhijun, Gan Meilian, Huang Lulu, et al. Research progress on the application of gene chips in the diagnosis and treatment of cardiovascular diseases [J]. Chinese Journal of Medical Engineering, 2005, 13(5):3. [7] Huang Zhijun (Review), Gan Meilian (Review), Huang Lulu (Review), et al. Application of DNA molecular genetic markers RFLP, STR, and SNP in coagulation factor genotyping [J]. Chinese Journal of Misdiagnosis, 2006.

Claims

1. Application of LRG1 quantitative reagent in the preparation of diagnostic or auxiliary diagnostic reagents for acute pyelonephritis.

2. The application according to claim 1, characterized in that, The LRG1 quantitative reagents are ELISA reagents, immunofluorescence reagents, and mass spectrometry analysis reagents.

3. The application according to claim 1, characterized in that, The sample tested by the LRG1 quantitative reagent is peripheral blood.

4. A diagnostic or auxiliary diagnostic system for acute pyelonephritis, comprising: The LRG1 quantification device is used to quantify the amount of LRG1 in a sample. An analytical device was used to determine the risk of acute pyelonephritis based on LRG1 levels. The result output device is used to output the analysis results of the analysis device.

5. The diagnostic system for acute pyelonephritis according to claim 4, characterized in that, The LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

6. The diagnostic system for acute pyelonephritis according to claim 4, characterized in that, High levels of LRG1 indicate a high risk of acute pyelonephritis.

7. The acute pyelonephritis diagnostic system according to claim 6, characterized in that, High LRG1 content refers to its content in peripheral blood samples exceeding 5 ng / mL.

8. The diagnostic system for acute pyelonephritis according to claim 4, characterized in that, The sample was peripheral blood.