In-vitro analysis method for identifying correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease and application of corresponding specific inhibitor of GLS1 activity and left ventricular hypertrophy

Abstract

The invention relates to an in-vitro analysis method for identifying correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney diseases, which comprises the following steps: firstly, constructing a chronic kidney disease mouse model, carrying out heart space metabonomics detection, carrying out heart tissue enzyme activity detection and heart immunohistochemical analysis on the frozen heart of the mouse, and determining the correlation between GLS1 activity and left ventricular hypertrophy. Normal neonatal mouse myocardial primary cells are extracted and subjected to real-time quantitative PCR detection, protein determination and protease activity detection respectively, then intervened cells are collected with the kit, mitochondrial function determination, metabolic flux determination, ammonia production determination and acetylation determination are performed, and finally clinical observation experiments are performed. The correlation analysis of the left ventricular hypertrophy index of a chronic kidney disease patient and the enzyme activity of kidney-type glutaminase is carried out, the new application of the CB839 targeting GLS1 in resisting the left ventricular hypertrophy of the chronic kidney disease and delaying the progress of the IV-type kidney-heart syndrome is formulated according to the analysis result, and the application has the advantages of developing the new application of drugs and being good in effect of relieving the left ventricular hypertrophy.

Description

Technical Field

[0001] This invention relates to the field of biotherapy technology for chronic kidney disease-myocardial remodeling, and more particularly to an in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) poses a significant threat to human health and the economy due to its high morbidity, high mortality, and expensive treatment costs. According to statistics from the U.S. Kidney Disease Data System in 2022, more than 60% of CKD patients also suffer from cardiovascular disease (CVD). Cardiovascular events remain the leading cause of death among CKD patients (around 30%), and this trend is increasing. In patients with end-stage renal disease (ESRD), the mortality rate caused by CVD can be as high as 58%. However, neither renal replacement therapy (hemodialysis, peritoneal dialysis, etc.) nor treatment plans aimed at controlling blood pressure, correcting electrolyte imbalances, reducing workload, and combating heart failure can completely prevent or reverse the progression of CVD myocardial hypertrophy and fibrosis in CKD patients, nor can they effectively reduce the occurrence of cardiovascular events such as sudden cardiac death.

[0003] Left ventricular hypertrophy (LVH) is one of the core manifestations of cardiovascular complications in CKD patients. It is the result of the combined effects of multiple factors (hypertension, volume overload, anemia, mineral metabolism disorders, toxins, and neurohormonal activation) and can significantly increase the risk of heart attack, heart failure, and even sudden death. However, the mechanism by which CKD patients affect the occurrence of LVH symptoms is not yet clear. Therefore, exploring the pathogenesis of myocardial remodeling in CKD patients is crucial for the treatment of CKD-LVH and can greatly improve the survival probability of CKD patients.

[0004] In the current treatment of chronic kidney disease (LVD), glutaminase (GLS) belongs to the class of hydrolases, specifically a type of amide hydrolase. It drives glutamine breakdown in cancer cells, supporting tumor proliferation and antioxidant defense. Glutaminase includes two isoenzymes: kidney-type glutaminase (GLS1) located on chromosome 2 and liver-type glutaminase (GLS2) located on chromosome 12. GLS1 has high activity in the kidneys and is the main expressed subtype in the heart. Its overexpression can promote glutamate to enter oxidative phosphorylation metabolism via glutamate dehydrogenase 1 (GLUD1), promoting the generation of mitochondrial oxygen free radicals (ROS). Its expression and activity are upregulated in vascular endothelial cell damage caused by pulmonary hypertension, hypertension, etc. Inhibiting GLS1 can alleviate the development of LVH to some extent. However, there is currently no treatment method that targets GLS1 to inhibit the progression of LVH, nor can there be a corresponding treatment to alleviate the symptoms of left ventricular hypertrophy in patients with chronic kidney disease.

[0005] There is an urgent need for an in vitro analytical method to identify the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease, in order to address the aforementioned problems of the lack of effective treatments targeting renal glutaminase, the inability to alleviate multiple events of cardiomyocyte hypertrophy caused by chronic kidney disease, and the inability to significantly reduce the symptoms of left ventricular hypertrophy complications. Summary of the Invention

[0006] In view of this, the present invention proposes an in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease and its application. By inhibiting GLS1 through the specific GLS1 inhibitor CB839, the symptoms of left ventricular hypertrophy caused by chronic kidney disease can be alleviated. This solves the technical problems of the lack of effective GLS1 targeting methods, the inability to alleviate multiple events of cardiomyocyte hypertrophy caused by chronic kidney disease, and the inability to significantly reduce the symptoms of left ventricular hypertrophy complications.

[0007] To achieve the above-mentioned technical objectives, the specific technical solution adopted by the present invention is as follows: An in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease includes the following steps: S1 was used in in vivo mouse experiments with chronic kidney disease. First, a mouse model of chronic kidney disease was constructed, and the mice in the model were fixed. Then, echocardiography was performed on the fixed mice. The cardiac spatial metabolomics of the heart tissue sections of the mice was performed using the detection platform. Finally, the frozen hearts of the mice were subjected to cardiac tissue enzyme activity detection and cardiac immunohistochemical analysis. S2 was used for in vitro cell experiments; Normal neonatal rat myocardial cells were extracted, cultured, transfected, and then processed. After processing, the cells were collected and subjected to real-time quantitative PCR, protein assay, and protease activity assay. Then, the cells after intervention were collected using a kit and subjected to mitochondrial function assay, metabolic flux assay, ammonia production assay, and acetylation assay. S3 will undergo clinical observation trials; Serum was collected from different patients with chronic kidney disease, and the renal type glutaminase activity was detected in the collected serum. Correlation analysis was also performed between left ventricular hypertrophy index and renal type glutaminase activity in patients with chronic kidney disease. S4 Statistical Analysis; Statistical analysis was performed on all quantitative values ​​in the analysis results of step S3 to remove the influence of errors on the analysis results.

[0008] Furthermore, in step S1, the mice are anesthetized with gas before fixation to facilitate fixation. The detection platform uses an in-situ detection platform for metabolites from tissue sections, which facilitates the acquisition of high-throughput, in-situ metabolomics data.

[0009] Furthermore, the cardiac spatial metabolomics detection in step S1 includes the following steps: S1021 First, the heart tissue was pre-processed. The tissue sample was taken out of the -80℃ ultra-low temperature freezer and then placed in the -20℃ freezer overnight to thaw. The tissue was then embedded and sectioned. Frozen tissue sections were continuously cut at a thickness of 10μm and adhered and fixed onto positively charged anti-detachment glass slides. S1022 performs mass spectrometry. Electrospray solvent forms 100μm×100μm charged droplets on the sample slice under the action of high-speed gas flow. The ejected high-speed charged droplets bombard the surface of the sample to be tested. Under the action of solvent extraction, the sample is simultaneously desorbed and ionized. Then, the ionized metabolites enter the mass spectrometer for mass spectrometry scanning and detection through a 50cm long ion transmission tube under the action of vacuum pump negative pressure. The S1023 performs tissue imaging scans, converts the obtained raw data into a universal mass spectrometry file after format conversion, imports it into mass spectrometry imaging software for analysis and reconstruction to form a mass spectrometry image. The high-resolution orbital mass spectrometer, combined with custom-developed high-discrimination imaging software, ensures that the m / z can accurately identify and match metabolites. Then, H&E staining is performed on serial sections, and tissue-related regions are identified by comparing them with H&E-stained tissue sections. Subsequently, metabolite comparison analysis is performed between the two groups of different regions. S1024 performs quality control on the raw data obtained from mass spectrometry. All detected ions are matched with the human metabolome database HMDB, the Metlin metabolite database, and the lipid database. Using precise molecular weight and mass accuracy (<5ppm mass error), combined with isotopic abundance from high-resolution mass spectrometry, a possible list of elemental composition and endogenous metabolites is provided. S1025 used Metaboanalyst 5.0 to analyze the processed metabolomics data.

[0010] Furthermore, step S1, involving the detection of cardiac tissue enzyme activity and the analysis of cardiac immunohistochemistry, includes the following specific steps: S1031 Frozen heart tissue from each group of mice was thawed on ice, and the GLS1 enzyme activity of each sample was detected according to the kit instructions. S1032 Fresh heart tissue from each mouse was frozen and prepared into 8-10 μm thick frozen sections on a microtome, which were then mounted on glass slides; S1033 slices were air-dried at room temperature for 30 minutes. S1034 Gently wash the dried sections in 1×PBS to remove excess freezing medium; S1035 tissue sections were permeated with 0.1% Triton-X100 at room temperature for 10 minutes; S1036 was used to block sections in 5% normal goat serum for 1 hour to reduce nonspecific binding; S1037: Incubate the slide with the anti-GLS1 antibody overnight at 4°C; The next day, after washing the S1038 sections with PBS, they were incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 hour. S1039 cells were finally stained with DAPI to label the nuclei, and the GLS1 signal was observed under a fluorescence microscope.

[0011] Furthermore, the culture and transfection of primary neonatal rat cardiomyocytes in step 2 includes the following steps: Normal neonatal rat cardiomyocytes were extracted from S2011, grown in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and cultured in an incubator at 37°C and 5% CO2. S2012 cells were passaged using 0.25% trypsin-0.02% EDTA at a confluence of 60%–80%. S2013 uses a lentivirus containing GLS1 to transfect cells, following the supplier's instructions, and then further cultured them after transfection. S2014 After transfection, cells were added to 5 μM CB839 and treated for 24 h. S2015 The cells were collected after 24 hours of treatment.

[0012] Furthermore, in step 2, after culturing and transfecting primary myocardial cells from neonatal rats, real-time quantitative PCR detection, protein assay, and protease activity assay are performed, including the following specific steps: S2016 uses the RNA isoPlus reagent to extract total RNA from tissues or cells; S2017 used PrimeScript™ RT Master Mix to reverse transcribe cDNA; S2018 underwent real-time PCR amplification using SYBR Green PCR Master Mix and an ABI 7500 real-time PCR detection system. S2019 uses RIPA buffer containing 1x protease inhibitor and 1x phosphatase inhibitor to homogenize tissue or cell lysate. S2020 The sample was then centrifuged at 5000 rpm for 30 minutes; S2021 used the BCA protein assay kit to determine protein concentration; S2022 equivalent samples were used for GLS1 (1:2000), SLC1A5 (1:500), β-actin (1:500), and then HRP-labeled secondary antibody (1:5000) was added. The S2023 immune response band was visualized using the Amersham Biosciences ECL detection system. S2024 performs optical density analysis by measuring the intensity of the bands and normalizes them to the corresponding β-actin bands using Quantity One software.

[0013] Furthermore, in step S2018, the temperature cycling conditions are 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C. The relative expression level of mRNA is normalized relative to β-actin and calculated using the 2-ΔΔCT method. The required primers are designed and synthesized by Sangon Biotech Co., Ltd.

[0014] Furthermore, the specific steps for mitochondrial function assays, metabolic flux assays, ammonia production assays, and acetylation assays in step 2 include the following: S2031 Cells after intervention were collected using the Seahorse kit, and mitochondrial function was assessed by detecting cellular oxygen consumption using the Seahorse kit. S2032 cells were added to glutamine-free DMEM / F12 medium and 1.25 μM of 13C-labeled glutamine was added to each group. Mitochondria of the cells after intervention were collected according to the kit, and the labeled cells were analyzed by mass spectrometry and isotope analysis. S2033 Extract mitochondria from each group of cells and detect NH3 in each sample according to the kit; S2034 Cell proteins were extracted from each group, and Western blot analysis was performed to determine the degree of acetylation at histone H3K9 and H3K122 sites.

[0015] Furthermore, the specific steps of the clinical observation experiment in step S3 include the following: S301 collected serum from 30 patients with CKD stages 1 to 5. GLS1 enzyme activity was detected according to the kit operation procedure. The correlation between GLS1 enzyme activity and left ventricular hypertrophy indicators in echocardiography was evaluated based on the GLS1 enzyme activity detection results. The left ventricular hypertrophy indicators included LAs, IVSd, LVEDD, LVPWd, LVM, and LVMI. S302 mouse primary cardiomyocytes were incubated with serum, and the cells were collected after 24 hours for cell proliferation analysis, qPCR analysis, WB analysis and GLS1 enzyme activity detection using a real-time cell analysis system.

[0016] Furthermore, the specific steps of the statistical analysis in step S4 include the following: S401 All quantitative values ​​are expressed as mean ± standard error of mean (SEM); S402 used GraphPad Prism to analyze the statistical differences between the two groups using a two-tailed Student t-test; S403 uses one-way ANOVA for comparisons between multiple groups, where P < 0.05 is considered statistically significant.

[0017] The application of a specific inhibitor CB839 in the preparation of drugs that alleviate symptoms of left ventricular hypertrophy in chronic kidney disease. The specific inhibitor CB839 exerts its effect of alleviating symptoms of left ventricular hypertrophy in chronic kidney disease by inhibiting GLS1 activity.

[0018] By adopting the above technical solution, the present invention can also bring the following beneficial effects: 1. This invention discloses an in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease (CKD). This method provides a basis for alleviating CKD-LVH symptoms by inhibiting GLS1 activity with the specific inhibitor CB839. It demonstrates that specific inhibition of GLS1 activity significantly reduces the clinical and pathological manifestations of CKD-LVH, where LVMI is considered a key indicator of LVH severity. Its level is significantly downregulated after treatment, showing a significant effect on the treatment of LVH symptoms. Based on the in vitro analytical method, this invention further expands the new application of the specific inhibitor CB839, providing a theoretical basis for the correlation between GLS1 activity and left ventricular hypertrophy.

[0019] 2. This invention mentions an in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease (CKD). GLS1 is the rate-limiting enzyme of the glutamine hydrolysis pathway and is the main expressed subtype in the heart. Its overexpression can promote glutamic acid (Glu) to enter oxidative phosphorylation metabolism and promote the generation of mitochondrial oxygen free radicals (ROS). Metabolomics confirmed that the activity of the glutamine hydrolysis pathway was significantly upregulated in cardiomyocytes of CKD mice. GLS1 enzyme activity detection, tissue immunohistochemistry, Western blotting, and qPCR analysis proved that GLS1 activity and expression were upregulated in CKD mice and in vitro cardiomyocyte hypertrophy models. It was found that serum GLS1 enzyme activity in CKD patients was positively correlated with multiple indicators of echocardiographic cardiomyocyte hypertrophy. Therefore, by reverse proof, the use of the GLS1-specific inhibitor CB839 can effectively delay LVH in CKD mice and inhibit changes in cardiomyocyte hypertrophy in vitro, thus indicating that CB839 can reduce the occurrence of LVH and delay its development by inhibiting GLS1.

[0020] 3. This invention provides an in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease (CKD). It demonstrates that CB839 can significantly reduce the acetylation levels at H3K9 and H3K122 sites in CKD mouse cardiomyocytes. By downregulating the transcriptional levels of Anp and Bnp, it can effectively improve LVH, effectively inhibit the production of NH3 in cardiomyocytes, and alleviate mitochondrial oxidative respiratory chain damage. This further illustrates that blocking the pharmacological effects of GLS1 through CB839 can inhibit several events related to cardiomyocyte hypertrophy and significantly reduce LVH, which is groundbreaking for the treatment of LVH. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A flowchart of a method for mitigating LVH by using GLS1 to block GLS1 in the invention; Figure 2 This is a diagram showing the cardiac pathological changes in CKD mice (STNx) in Example 1; Figure 3 This is an echocardiographic image showing the changes in CKD mice (STNx) in Example 1; Figure 4 This is a graph showing the enrichment analysis of left ventricular RNA sequencing in CKD mice in Example 1; Figure 5 This is a graph showing the expression of cardiac metabolomics in CKD mice in Example 1; Figure 6 This is a spatial metabolomics distribution map of CKD mice in Example 1; Figure 7 This is a graph showing the GLS1 expression level in the hearts of CKD mice in Example 1. Figure 8 This is a graph showing the GLS enzyme activity in the hearts of CKD mice in Example 1; Figure 9 This is a graph showing the expression level of GLS1 in the heart of a CKD patient in Example 1. Figure 10 This is a graph showing the correlation between GLS1 enzyme activity and patient clinical indicators in Example 1; Figure 11 This is an experimental diagram showing the improvement of left ventricular hypertrophy in CKD mice after intervention targeting GLS1 enzyme activity in Example 1. Figure 12 This is an experimental diagram showing the improvement of left ventricular hypertrophy in CKD mice after intervention targeting GLS1 expression in Example 1. Figure 13 This is a diagram showing the changes in ATP in cardiomyocytes under CKD conditions in Example 1. Figure 14 This is a diagram showing the changes in mitochondrial respiratory capacity in the CKD state in Example 1; Figure 15 This is a diagram showing the ammonia production level of cardiomyocytes under CKD condition in Example 1; Figure 16 This is a diagram showing the changes in mitochondrial oxidative respiratory chain proteins in cardiomyocytes under CKD condition in Example 1. Figure 17 This is a diagram showing the changes in mitochondrial membrane potential levels in cardiomyocytes under CKD conditions in Example 1. Figure 18 This is a diagram showing the changes in the degree of acetylation of cardiomyocytes under CKD condition in Example 1. Detailed Implementation

[0023] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] Furthermore, specific details are provided in the following description to facilitate a thorough understanding of the examples. However, those skilled in the art will understand that the described aspects can be practiced without these specific details.

[0027] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0028] To achieve the above-mentioned technical objectives, the specific technical solution adopted by the present invention is as follows: One embodiment of this application describes an in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease, including the following steps: S1 was used in in vivo experiments on mice with chronic kidney disease. First, a mouse model of chronic kidney disease was constructed, and the mice in the model were fixed. Then, echocardiography was performed on the fixed mice. The cardiac spatial metabolomics of the heart tissue sections of the mice was performed using a detection platform. Finally, cardiac tissue enzyme activity detection and cardiac immunohistochemical analysis were performed on the frozen hearts of the mice.

[0029] In step S1, the mice are anesthetized with gas before fixation to facilitate fixation. The detection platform uses an in-situ detection platform for metabolites from tissue sections, which facilitates the acquisition of high-throughput, in-situ metabolomics data.

[0030] Step S1 involves cardiac spatial metabolomics detection using the commercially available AFADESI-MSI tissue section metabolite in situ detection platform, including the following steps: S1021 First, the heart tissue was pre-processed. The tissue sample was taken out from the -80℃ ultra-low temperature freezer and then placed in the -20℃ freezer overnight to thaw. The tissue was then embedded and sectioned. The frozen tissue sections were continuously cut at a thickness of 10μm and adhered and fixed on positively charged anti-detachment glass slides.

[0031] S1022 performs mass spectrometry measurements. Electrospray ionization (ESI) forms 100μm×100μm charged droplets on the sample slice under the action of a high-speed gas flow. The ejected high-speed charged droplets bombard the surface of the sample to be tested. Under the action of solvent extraction, the sample is simultaneously desorbed and ionized. Then, the ionized metabolites enter the mass spectrometer for mass spectrometry scanning and detection under the negative pressure of the vacuum pump through a 50 cm long ion transmission tube.

[0032] The S1023 performs tissue imaging scans, converts the obtained raw data into a universal mass spectrometry file after format conversion, imports it into mass spectrometry imaging software for analysis and reconstruction to form a mass spectrometry image. The high-resolution orbital mass spectrometer, combined with the custom-developed high-discrimination imaging software MassImager (Δm / z = 0.001), ensures that the m / z can accurately identify and match metabolites. Then, H&E staining is performed on serial sections, and tissue-related regions are identified by comparing them with H&E-stained tissue sections. Subsequently, metabolite comparison analysis is performed between the two groups of different regions.

[0033] S1024 performs quality control on the raw data obtained from mass spectrometry. All detected ions are matched against the Human Metabolome Database (HMDB), the Metlin Metabolite Database, and the Lipid Database. The URLs for the HMDB, Metlin, and Lipid Databases are "Human metabolomedatabase, https: / / www.hmdb.ca", "https: / / metlin.scripps.edu", and "https: / / www.lipidmaps.org", respectively. Using precise molecular weight and mass accuracy (<5 ppm mass error), combined with isotopic abundance from high-resolution mass spectrometry, a list of possible elemental compositions and endogenous metabolites is provided.

[0034] S1025 used Metaboanalyst 5.0 to analyze the processed metabolomics data. The URL for Metaboanalyst 5.0 is "https: / / www.metaboanalyst.ca".

[0035] Step S1, which involves enzyme activity detection and immunohistochemical analysis of cardiac tissue, includes the following specific steps: S1031 Frozen heart tissue from each group of mice was thawed on ice, and the GLS1 enzyme activity of each sample was tested according to the kit instructions.

[0036] S1032 Fresh heart tissue from each mouse was frozen and prepared into 8-10 μm thick frozen sections on a microtome, which were then mounted on glass slides.

[0037] S1033 slices were air-dried at room temperature for 30 minutes.

[0038] S1034 Gently wash the dried sections in 1×PBS to remove excess freezing medium.

[0039] S1035 used 0.1% Triton-X100 to permeate tissue sections at room temperature for 10 minutes.

[0040] S1036 was used to block sections in 5% normal goat serum for 1 hour to reduce nonspecific binding.

[0041] S1037: Incubate the slide with the anti-GLS1 antibody overnight at 4°C.

[0042] The next day, after washing the S1038 sections with PBS, they were incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 hour.

[0043] S1039 cells were finally stained with DAPI to label the nuclei, and the GLS1 signal was observed under a fluorescence microscope.

[0044] S2 was used for in vitro cell experiments. Normal neonatal rat myocardial cells were extracted, cultured, transfected, and then processed. After processing, the cells were collected and subjected to real-time quantitative PCR, protein assay, and protease activity assay. Then, the treated cells were collected using a kit and subjected to mitochondrial function assay, metabolic flux assay, ammonia production assay, and acetylation assay.

[0045] The culture and transfection of primary neonatal rat cardiomyocytes in step 2 includes the following steps: Normal neonatal rat cardiomyocytes were extracted from S2011 and grown in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and cultured in an incubator at 37°C and 5% CO2.

[0046] S2012 cells were passaged using 0.25% trypsin and 0.02% EDTA at a confluence of 60%–80%.

[0047] S2013 cells were transfected using a lentivirus containing GLS1, following the supplier's instructions, and then cultured further after transfection.

[0048] S2014 After transfection, cells were treated with 5 μM CB839 for 24 h.

[0049] S2015 The cells were collected after 24 hours of treatment.

[0050] Step 2 involves culturing and transfecting primary myocardial cells from neonatal rats, followed by real-time quantitative PCR, protein assays, and protease activity detection. The specific steps are as follows: S2016 uses the RNA isoPlus reagent to extract total RNA from tissues or cells.

[0051] S2017 used PrimeScript™ RT Master Mix to reverse transcribe cDNA.

[0052] S2018 involved real-time PCR amplification using a SYBR Green PCR Master Mix and an ABI 7500 real-time PCR detection system. The cycling conditions in step S2018 were: 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute. The relative expression level of mRNA was normalized relative to β-actin and then analyzed using 2... -ΔΔC The T-method calculations were performed using primers designed and synthesized by Sangon Biotech Co., Ltd.

[0053] S2019 uses RIPA buffer containing 1x protease inhibitor and 1x phosphatase inhibitor to homogenize tissue or cell lysate.

[0054] S2020 The sample was then centrifuged at 5000 rpm for 30 minutes.

[0055] S2021 used the BCA Protein Assay Kit to determine protein concentration.

[0056] The S2022 equivalent sample was used for GLS1 (1:2000), SLC1A5 (1:500), β-actin (1:500), and then HRP-labeled secondary antibody (1:5000) was added.

[0057] The S2023 immune response band was visualized using the Amersham Biosciences ECL detection system.

[0058] S2024 performs optical density analysis by measuring the intensity of the bands and normalizes them to the corresponding β-actin bands using Quantity One software.

[0059] The specific steps for mitochondrial function assays, metabolic flux assays, ammonia production assays, and acetylation assays in step 2 include the following: S2031 Cells after intervention were collected using the Seahorse kit, and mitochondrial function was assessed by detecting cellular oxygen consumption using the Seahorse kit.

[0060] S2032 cells were added to glutamine-free DMEM / F12 medium, and 1.25 μM of [unspecified substance] was added to each group. 13 C-labeled glutamine was used to collect mitochondria from cells after intervention according to the kit, and the labeled cells were analyzed by mass spectrometry and isotope analysis.

[0061] S2033 Extract mitochondria from each group of cells and detect NH3 in each sample according to the kit.

[0062] S2034 Cell proteins were extracted from each group, and Western blot analysis was performed to determine the degree of acetylation at histone H3K9 and H3K122 sites.

[0063] S3 is undergoing clinical observation trials: Serum samples were collected from different patients with chronic kidney disease. Renal type glutaminase activity was then measured in the collected serum, and a correlation analysis was performed between left ventricular hypertrophy indices and renal type glutaminase activity in patients with chronic kidney disease.

[0064] The specific steps of the clinical observation experiment in step S3 include the following: S301 collected serum from 30 patients with CKD stages 1 to 5. GLS1 enzyme activity was measured according to the kit operation procedure. The correlation between GLS1 enzyme activity and left ventricular hypertrophy indicators in echocardiography was evaluated based on the GLS1 enzyme activity test results. The left ventricular hypertrophy indicators included LAs, IVSd, LVEDD, LVPWd, LVM, and LVMI.

[0065] S302 mouse primary cardiomyocytes were incubated with serum, and the cells were collected after 24 hours for cell proliferation analysis, qPCR analysis, WB analysis and GLS1 enzyme activity detection using a real-time cell analysis system.

[0066] S4 Statistical Analysis: Statistical analysis is performed on all quantitative values ​​in the analysis results of step S3 to remove the influence of errors on the analysis results. The specific steps of the statistical analysis in step S4 are as follows: S401 All quantitative values ​​are expressed as mean ± standard error of mean (SEM).

[0067] S402 used GraphPad Prism to analyze the statistical differences between the two groups using a two-tailed Student t-test.

[0068] S403 uses one-way ANOVA for comparisons between multiple groups, where P < 0.05 is considered statistically significant.

[0069] The data analysis results obtained using the above methods are as follows: Figures 1 to 6 As shown, where Figure 2 This is a diagram showing the cardiac pathological changes in CKD mice (STNx), which can be seen... Figure 2 The degree of left ventricular hypertrophy gradually worsened with the extension of CKD modeling time (STNx). Figure 3 This is an echocardiographic image of CKD mice (STNx), showing changes that can be observed. Figure 3 The left ventricular hypertrophy index (β-MHC) gradually increased with the extension of CKD modeling time (STNx). Figure 4 The image shows RNA sequencing enrichment analysis in the left ventricle of CKD mice, revealing that multiple metabolic pathways in the mouse heart were disrupted at 18 weeks of STNx modeling. Figure 5 This is a metabolomics expression map of the heart in CKD mice, representing the results of metabolomics data from mouse heart tissue. The map shows the enrichment of multiple metabolites in the heart. Notably, glutamine levels gradually decreased with prolonged CKD modeling (STNx) time, while glutamate levels gradually increased, indicating that the glutamine hydrolysis pathway is gradually activated in the heart of CKD mice. Figure 6 This is a spatial metabolomics distribution map of CKD mice, reflecting the spatial metabolomics data of the mouse heart. Compared with the control group (Sham), the expression of glutamine in the left ventricle of 18-week-old CKD mice (STNx) was significantly downregulated, while the expression of glutamate was significantly upregulated. This further indicates that glutaminolysis is significantly activated in the heart of CKD mice, thus revealing multiple factors affecting the symptoms of left ventricular hypertrophy in CKD mice.

[0070] like Figures 7 to 11 As shown, Figure 7 The image shows the expression level of GLS1 in the heart of CKD mice. It can be seen that the expression level of GLS1 gradually increases with the extension of CKD modeling time (STNx). Figure 8 The image shows the GLS enzyme activity in the heart of CKD mice. It can be seen that the expression level of GLS1 gradually increases with the extension of CKD modeling time (STNx). Figure 9This image shows the expression level of GLS1 in the heart of a CKD patient. Compared with the left ventricle of healthy individuals, CKD patients exhibit significantly enlarged left ventricular cardiomyocytes and a markedly upregulated expression level of GLS1. Figure 10 The correlation plot between GLS1 enzyme activity and patient clinical indicators shows that GLS1 expression is significantly positively correlated with several echocardiographic indicators of left ventricular hypertrophy (LAs, IVSd, LVEDD, LVPWd, LVM, and LVMI). Figure 11 The figure shows the improvement of left ventricular hypertrophy in CKD mice after intervention targeting GLS1 enzyme activity. It reflects the changes in left ventricular hypertrophy in CKD mice treated with CB839. It can be seen that compared with WT mice, the degree of left ventricular hypertrophy in GLS1 knockdown mice was significantly downregulated (manifested as downregulation of myocardial hypertrophy indicators and significant improvement in echocardiographic left ventricular hypertrophy indicators), and Glutaminolysis was significantly inhibited (manifested as downregulation of downstream rate-limiting enzyme GLUD1 expression and downregulation of Gln transporter, etc.), which can prove that the expression level of GLS1 is indeed related to the degree of left ventricular hypertrophy in patients.

[0071] like Figure 12 and 13 As shown, Figure 12 This image shows the improvement in left ventricular hypertrophy in CKD mice after intervention targeting GLS1 expression. It reflects the changes in left ventricular hypertrophy in CKD mice with GLS1 conditional knockdown of cardiomyocytes. Compared with WT mice, the degree of left ventricular hypertrophy in GLS1 conditional knockdown mice was significantly downregulated (manifested as downregulation of myocardial hypertrophy indices and significant improvement in echocardiographic left ventricular hypertrophy indices), and glutaminolysis was significantly inhibited (manifested as downregulation of downstream rate-limiting enzyme GLUD1 expression and downregulation of Gln transporter, etc.). Figure 13 The figure shows the changes in ATP in cardiomyocytes under CKD conditions. It can be seen that ATP production in mouse cardiomyocytes is impaired under high phosphorus (Pi) stimulation. After knocking down GLS1 (GLS1-si), the ATP downregulated by Pi was significantly relieved, showing that controlling the expression level of GLS1 can effectively affect the changes in left ventricular hypertrophy in mice.

[0072] This embodiment also mentions the application of a specific inhibitor CB839 in the preparation of a drug to alleviate the symptoms of left ventricular hypertrophy in chronic kidney disease. The specific inhibitor CB839 exerts its effect of alleviating the symptoms of left ventricular hypertrophy in chronic kidney disease in the drug by inhibiting GLS1 activity.

[0073] like Figures 14 to 18 As shown, Figure 14 The figure shows the changes in mitochondrial respiratory capacity in CKD. It can be seen that the oxygen consumption of mouse cardiomyocytes was significantly reduced after Pi stimulation, and the oxygen consumption reduced by Pi was significantly relieved after CB839 was applied. Figure 15The image shows the ammonia production levels in cardiomyocytes under CKD conditions. High phosphorus (Pi) stimulation significantly increases NH3 production in mouse cardiomyocytes. The Pi-induced increase in NH3 can be effectively reversed by applying CB839 or knocking down GLS1 (GLS1-si). Figure 16 This diagram illustrates the changes in mitochondrial oxidative respiratory chain proteins in cardiomyocytes under CKD conditions. It shows that high phosphorus (Pi) stimulation significantly downregulates mitochondrial complexes in mouse cardiomyocytes. The effects of Pi can be effectively alleviated by applying CB839 or knocking down GLS1 (GLS1-si). Figure 17 The figure shows the changes in mitochondrial membrane potential in cardiomyocytes under CKD condition 1. It can be seen that high phosphorus (Pi) depolarizes mitochondria in mouse cardiomyocytes and increases ROS production. The application of CB839 can effectively reverse the mitochondrial damage caused by Pi. Figure 18 The diagram shows the changes in histone acetylation in cardiomyocytes under CKD conditions. It reveals that high phosphorus (Pi) stimulation significantly upregulated histone acetylation in mouse cardiomyocytes. Application of CB839 or GLS1 knockdown (GLS1-si) effectively reversed Pi-induced histone acetylation. Figure 11 The combination of these factors further demonstrates that intervention with CB839 can effectively inhibit GLS1 activity, thereby alleviating symptoms of left ventricular hypertrophy in chronic kidney disease.

[0074] This invention utilizes a mouse model of chronic kidney disease in vivo to conduct cardiac spatial metabolomics, cardiac histopathological analysis, cardiac function analysis, and immunohistochemical analysis. In vitro, it employs cell culture studies, real-time quantitative PCR detection, protein quantification, and activity analysis. Clinically, it analyzes the correlation and statistical analysis between cardiac function and GLS1 activity in patients. The cell culture studies include metabolic flux assays, mitochondrial function assays, ammonia production assays, and acetylation assays. By utilizing the inhibitory effect of CB839 on GLS1, it inhibits multiple events related to cardiomyocyte metabolic reprogramming and hypertrophy, and significantly reduces left ventricular hypertrophy caused by chronic kidney disease. Based on the analysis results, a new application of CB839 targeting GLS1 is proposed in combating left ventricular hypertrophy in chronic kidney disease and delaying the progression of type IV renal-cardiac syndrome. In summary, this invention has the advantages of pioneering new drug applications and achieving good results in reducing left ventricular hypertrophy.

[0075] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease, characterized in that, The steps include the following: S1 was used in in vivo mouse experiments with chronic kidney disease. First, a mouse model of chronic kidney disease was constructed, and the mice in the model were fixed. Then, echocardiography was performed on the fixed mice. The cardiac spatial metabolomics of the heart tissue sections of the mice was performed using the detection platform. Finally, the frozen hearts of the mice were subjected to cardiac tissue enzyme activity detection and cardiac immunohistochemical analysis. S2 was used for in vitro cell experiments; Normal neonatal rat myocardial cells were extracted, cultured, transfected, and then processed. After processing, the cells were collected and subjected to real-time quantitative PCR, protein assay, and protease activity assay. Then, the cells after intervention were collected using a kit and subjected to mitochondrial function assay, metabolic flux assay, ammonia production assay, and acetylation assay. S3 will undergo clinical observation trials; Serum was collected from different patients with chronic kidney disease, and the renal type glutaminase activity was detected in the collected serum. Correlation analysis was also performed between left ventricular hypertrophy index and renal type glutaminase activity in patients with chronic kidney disease. S4 Statistical Analysis; Statistical analysis was performed on all quantitative values ​​in the analysis results of step S3 to remove the influence of errors on the analysis results.

2. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 1, characterized in that: in step S1, the mice are anesthetized with air before fixation to facilitate mouse fixation; the detection platform adopts a tissue section metabolite in situ detection platform to facilitate the acquisition of high-throughput, in situ metabolomics data.

3. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 2, characterized in that the cardiac spatial metabolomics detection in step S1 includes the following steps: S1021 First, the heart tissue was pre-processed. The tissue sample was taken out of the -80℃ ultra-low temperature freezer and then placed in the -20℃ freezer overnight to thaw. The tissue was then embedded and sectioned. Frozen tissue sections were continuously cut at a thickness of 10μm and adhered and fixed onto positively charged anti-detachment glass slides. S1022 performs mass spectrometry. Electrospray solvent forms 100μm×100μm charged droplets on the sample slice under the action of high-speed gas flow. The ejected high-speed charged droplets bombard the surface of the sample to be tested. Under the action of solvent extraction, the sample is simultaneously desorbed and ionized. Then, the ionized metabolites enter the mass spectrometer for mass spectrometry scanning and detection through a 50cm long ion transmission tube under the action of vacuum pump negative pressure. The S1023 performs tissue imaging scans, converts the obtained raw data into a universal mass spectrometry file after format conversion, imports it into mass spectrometry imaging software for analysis and reconstruction to form a mass spectrometry image. The high-resolution orbital mass spectrometer, combined with custom-developed high-discrimination imaging software, ensures that the m / z can accurately identify and match metabolites. Then, H&E staining is performed on serial sections, and tissue-related regions are identified by comparing them with H&E-stained tissue sections. Subsequently, metabolite comparison analysis is performed between the two groups of different regions. S1024 performs quality control on the raw data obtained from mass spectrometry. All detected ions are matched with the human metabolome database HMDB, the Metlin metabolite database, and the lipid database. Using precise molecular weight and mass accuracy (<5ppm mass error), combined with isotopic abundance from high-resolution mass spectrometry, a possible list of elemental composition and endogenous metabolites is provided. S1025 used Metaboanalyst 5.0 to analyze the processed metabolomics data.

4. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 3, characterized in that the cardiac tissue enzyme activity detection and cardiac immunohistochemical analysis in step S1 include the following specific steps: S1031 Frozen heart tissue from each group of mice was thawed on ice, and the GLS1 enzyme activity of each sample was detected according to the kit instructions. S1032 Fresh heart tissue from each mouse was frozen and prepared into 8-10 μm thick frozen sections on a microtome, which were then mounted on glass slides; S1033 slices were air-dried at room temperature for 30 minutes. S1034 Gently wash the dried sections in 1×PBS to remove excess freezing medium; S1035 tissue sections were permeated with 0.1% Triton-X100 at room temperature for 10 minutes; S1036 was used to block sections in 5% normal goat serum for 1 hour to reduce nonspecific binding; S1037: Incubate the slide with the anti-GLS1 antibody overnight at 4°C; The next day, after washing the S1038 sections with PBS, they were incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 hour. S1039 cells were finally stained with DAPI to label the nuclei, and the GLS1 signal was observed under a fluorescence microscope.

5. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 4, characterized in that the culture and transfection of primary neonatal rat cardiomyocytes in step 2 includes the following steps: Normal neonatal rat cardiomyocytes were extracted from S2011, grown in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and cultured in an incubator at 37°C and 5% CO2. S2012 cells were passaged using 0.25% trypsin-0.02% EDTA at a confluence of 60%–80%. S2013 uses a lentivirus containing GLS1 to transfect cells, following the supplier's instructions, and then further cultured them after transfection. S2014 After transfection, cells were added to 5 μM CB839 and treated for 24 h. S2015 The cells were collected after 24 hours of treatment.

6. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 5, characterized in that, in step 2, after culturing and transfecting primary myocardial cells from neonatal rats, real-time quantitative PCR detection, protein assay, and protease activity detection are performed, including the following specific steps: S2016 uses the RNA isoPlus reagent to extract total RNA from tissues or cells; S2017 used PrimeScript™ RT Master Mix to reverse transcribe cDNA; S2018 underwent real-time PCR amplification using SYBR Green PCR Master Mix and an ABI 7500 real-time PCR detection system. S2019 uses RIPA buffer containing 1x protease inhibitor and 1x phosphatase inhibitor to homogenize tissue or cell lysate. S2020 The sample was then centrifuged at 5000 rpm for 30 minutes; S2021 used the BCA protein assay kit to determine protein concentration; S2022 equivalent samples were used for GLS1 (1:2000), SLC1A5 (1:500), β-actin (1:500), and then HRP-labeled secondary antibody (1:5000) was added. The S2023 immune response band was visualized using the Amersham Biosciences ECL detection system. S2024 performs optical density analysis by measuring the intensity of the bands and normalizes them to the corresponding β-actin bands using Quantity One software.

7. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 6, characterized in that, The specific steps for mitochondrial function assay, metabolic flux assay, ammonia production assay, and acetylation assay in step 2 include the following: S2031 Cells after intervention were collected using the Seahorse kit, and mitochondrial function was assessed by detecting cellular oxygen consumption using the Seahorse kit. S2032 cells were added to glutamine-free DMEM / F12 medium and 1.25 μM of 13C-labeled glutamine was added to each group. Mitochondria of the cells after intervention were collected according to the kit, and the labeled cells were analyzed by mass spectrometry and isotope analysis. S2033 Extract mitochondria from each group of cells and detect NH3 in each sample according to the kit; S2034 Cell proteins were extracted from each group, and Western blot analysis was performed to determine the degree of acetylation at histone H3K9 and H3K122 sites.

8. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 7, characterized in that, The specific steps of the clinical observation experiment in step S3 include the following: S301 collected serum from 30 patients with CKD stages 1 to 5. GLS1 enzyme activity was detected according to the kit operation procedure. The correlation between GLS1 enzyme activity and left ventricular hypertrophy indicators in echocardiography was evaluated based on the GLS1 enzyme activity detection results. The left ventricular hypertrophy indicators included LAs, IVSd, LVEDD, LVPWd, LVM, and LVMI. S302 mouse primary cardiomyocytes were incubated with serum, and the cells were collected after 24 hours for cell proliferation analysis, qPCR analysis, WB analysis and GLS1 enzyme activity detection using a real-time cell analysis system.

9. The in vitro analytical method for identifying the correlation between GLS1 activity and left ventricular hypertrophy in chronic kidney disease according to claim 8, characterized in that, The specific steps of the statistical analysis in step S4 include the following: S401 All quantitative values ​​are expressed as mean ± standard error of mean (SEM); S402 used GraphPad Prism to analyze the statistical differences between the two groups using a two-tailed Student t-test; S403 uses one-way ANOVA for comparisons between multiple groups, where P < 0.05 is considered statistically significant.

10. The application of a specific inhibitor CB839 in the preparation of a drug to alleviate symptoms of left ventricular hypertrophy in chronic kidney disease, characterized in that: The specific inhibitor CB839 exerts its effect in alleviating symptoms of left ventricular hypertrophy in chronic kidney disease by inhibiting GLS1 activity.

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