Application of TREM2 gene in the preparation of markers for diagnosing podocyte injury

Abstract

The present invention relates to the field of biotechnology, and specifically discloses the use of the TREM2 gene in the preparation of a marker for diagnosing podocyte injury. The present invention has found through research that TREM2 is upregulated when podocytes are injured, and the change in the expression level of the TREM2 gene can be used as a marker for diagnosing podocyte injury, providing a new method for podocyte diagnosis. The present invention further found that gallic acid, which can bind to the TREM2 protein, can treat podocyte injury by affecting the expression level of TREM2, thereby providing a new method for the treatment of podocyte injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of the TREM2 gene in preparing markers for diagnosing podocyte injury. Background Art

[0002] The prevalence of chronic kidney disease (CKD) is on the rise. Glomerulosclerosis is an important pathological feature of CKD, involving lesions of endothelial cells, mesangial cells, and podocytes. These pathological changes are related to the infiltration of cytokines, chemokines, and inflammatory cells. Cell pyroptosis caused by abnormal activation of the immune system is also involved in the pathogenesis of CKD. Studies have shown that specific genetic variants are associated with an increased risk of CKD. However, the signaling factors that specifically regulate podocyte pyroptosis and immune activation of renal tissue have not yet been identified.

[0003] Triggering receptor expressed on myeloid cells-2 (TREM2) is considered to be a pathologically induced immune signaling hub. TREM2 is a receptor for a variety of ligands, most of which are markers of tissue damage. Under physiological conditions, TREM2 activity is low, while in pathological conditions, the TREM2 signaling pathway becomes an immune signaling hub that senses tissue damage and inhibits damage. TREM2 has long been a focus of attention in Alzheimer's disease, but there is still no relevant research showing that TREM2 plays an important role in podocyte disease.

[0004] Podocyte injury is the main cause of proteinuria in kidney disease. Podocytes are the outermost layer of the glomerular filtration membrane, with almost no proliferation ability. They rely on highly differentiated foot processes to form and maintain the glomerular filtration barrier. Cell pyroptosis has received increasing attention due to its association with innate immunity and disease. For a long time, pyroptosis has been considered to be caspase-1-induced monocyte death. Later, it was reported that caspase-1 or caspase-11 / 4 / 5 were activated in this process, GSDMD was cleaved, the N-terminal domain oligomerized, pores were formed in the cell membrane, and cell membrane rupture was induced.

[0005] It is necessary to study the mechanism of pyroptosis in podocytopathy to identify new diagnostic biomarkers to help treat and diagnose podocytopathy and to find targeted therapeutic drugs. Summary of the invention

[0006] In podocytosis, pyroptosis and immune dysregulation are abnormal, but the correlation is still unclear. Therefore, the present invention aims to explore the potential mechanism of pyroptosis-related genes in podocytosis and their relationship with immune inflammatory response, identify new diagnostic biomarkers to help treat and diagnose podocytosis, and find targeted therapeutic drugs.

[0007] The present invention utilizes GEO, MSigDB databases and Immport databases to screen immune-related core target genes that induce podocyte pyroptosis in CKD, performs in vivo experiments to verify the differential abundance of target genes through Western blot, immunohistochemistry and immunofluorescence, and utilizes a variety of kidney disease animal models for in vivo experimental verification. Core compounds are further screened through molecular docking methods based on the Coremine medical database, and potential drugs for improving the immune and inflammatory mechanisms of CKD are identified through in vitro verification.

[0008] Specifically, the present invention has found that podocyte pyroptosis is related to various pathogenic factors represented by immunity and inflammation. After precise analysis, 7 hub pyroptosis genes (NLRP1, NLRP3, TP53, IL1A, IL1B, BAX and TREM2) can reliably distinguish CKD from CON. TREM2 is a core target gene related to CKD and pyroptosis immunity. Based on in vivo experimental studies, the present invention also found that the hub pyroptosis gene TREM2 is highly expressed in a variety of kidney disease animal models, and TREM2 is upregulated when podocytes are injured, confirming that TREM2 plays an important role in the pathogenesis of a variety of kidney diseases. Based on hub pyroptosis genes and enriched immune pathways, it was found that gallic acid and quinic acid can affect podocyte pyroptosis, among which gallic acid can improve the podocyte pyroptosis caused by adriamycin (Adriamycin, ADR) stimulation by combining with TREM2, and is a potential drug for improving the immune and inflammatory mechanisms of CKD.

[0009] The specific technical solutions of the present invention are as follows:

[0010] The present invention provides the use of the TREM2 gene (NCBI accession numbers: GSE124622 and GSE185226) in preparing markers for diagnosing podocyte injury and the use of the TREM2 gene in preparing products for diagnosing podocyte injury.

[0011] In the application of the present invention, the product is a reagent, a kit or a chip.

[0012] In the application of the present invention, podocyte injury is diagnosed by detecting the expression level of the TREM2 gene in the kidney tissue to be tested.

[0013] If the expression level of the TREM2 gene increases (significantly increased compared to the negative control - normal kidney tissue), it is judged that podocyte damage has occurred in the tested kidney tissue.

[0014] The present invention also provides a product for diagnosing podocyte injury, the product comprising a reagent for detecting the expression level of the TREM2 gene. Preferably, the product is a kit.

[0015] The present invention also provides the use of a substance that reduces the expression of the TREM2 gene or TREM2 protein in the preparation of a product for treating podocyte injury. Preferably, the substance is gallic acid or quinic acid.

[0016] The present invention also provides a product for treating podocyte injury, which includes a substance that reduces the expression of the TREM2 gene or TREM2 protein; preferably, the substance is gallic acid or quinic acid.

[0017] The beneficial effects of the present invention are at least:

[0018] The present invention provides a new marker for diagnosing podocyte injury, provides a new way to identify podocyte injury, and provides a new idea for finding a treatment method for podocyte injury. The present invention further provides a new method for treating podocyte injury based on the newly discovered marker for podocyte injury, providing a new way for treating podocyte injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Identification results of immune-related core target genes of podocyte pyroptosis, where A: differential pyroptosis genes of podocytes. B: protein interaction visualization results. C: chromosomal location of differential pyroptosis genes. D: clustering heat map of differential pyroptosis genes. E: GO enrichment analysis results of differential genes.

[0020] Figure 2 The results of the observation of the adriamycin-induced podocyte injury model, where A: the urine albumin / creatinine ratio of rats in the control group and adriamycin-treated group; the error bars represent SD, and *** represents P < 0.001. B: the ultramicroelectron microscopic results of the kidneys of rats in the control group and adriamycin-treated group, and the foot processes are indicated by arrows.

[0021] Figure 3 The results of in vivo experimental validation of TREM2, the immune-related core target gene of podocyte pyroptosis, where A: Western blot results of TREM2 in the rat model of doxorubicin nephropathy. B: Immunohistochemical observation results in two renal models. C: qPCR results of TREM2 mRNA in the nephrotic syndrome group caused by doxorubicin treatment. D: Statistical analysis results of TREM2 Western blot in the rat model of doxorubicin nephropathy. E: Semi-quantitative analysis results of immunohistochemistry in two renal models. In the figure, ** represents P < 0.01.

[0022] Figure 4 The localization results of TREM2 and GSDMD on glomerular podocytes. The enlarged views of the dotted areas in the figure are shown in the rightmost column of figures, and the white arrows in the figures indicate the positive areas.

[0023] Figure 5Docking results of TREM2 with gallic acid (GA) and quinic acid (IA).

[0024] Figure 6 The results show the effect of gallic acid alone (left) and after stimulation with doxorubicin (right) on the survival rate of MPC-5 cells. In the figure, ns means no significant difference, ** means P<0.01, and *** means P<0.001.

[0025] Figure 7 The results show the effect of gallic acid on the expression of TREM2 gene and protein in podocytes stimulated by doxorubicin. In the figure, ns means no significant difference, and ** means P < 0.01.

[0026] Figure 8 The results show the effect of gallic acid on doxorubicin-stimulated podocyte TREM2 and GSDMD. In the figure, ** represents P < 0.01.

[0027] Fig. 9 Correlation analysis and ROC analysis results between TREM2 and WT1. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0030] Example 1

[0031] In this example, markers of podocyte pyroptosis were studied, as follows:

[0032] 1. Methods

[0033] 1.1 Data Source

[0034] GSE124622 and GSE185226 data were obtained from the NCBI high-throughput data analysis platform using the search term "Doxorubicin and podocytes". The data submitted by Nihalani D et al. were updated on Sep 22, 2021, and were analyzed using Illumina HiSeq 2500 (Homo sapiens) and chip platform GPL16791. The data submitted by Zhan P et al. were updated on Oct 06, 2021, and were analyzed using Illumina NovaSeq 6000 (Homo sapiens) and chip platform GPL24676. A total of 10 samples were collected, including 5 samples from doxorubicin-treated human podocytes and 5 samples from control human podocytes.

[0035] 1.2 Screening of pyroptosis-related genes

[0036] The “Limma” package of R software extracted the expression levels of pyroptosis-related genes in the combined dataset. Differential genes were screened from CKD and CON samples and defined as differentially expressed pyroptosis-related genes (DE-PGs). The selection criteria were |log2FC|>1, adjust P-value>0.05. Heatmaps were created using the “HeatMap” package of R software, and DE-PGs were plotted on chromosome location maps using the “Circos” package of R software. Protein network interaction analysis was performed using STRING and cytoscape.

[0037] 1.3 Animal model establishment and sample collection

[0038] Twenty-four 8-week-old SPF SD male rats weighing 180±10g were selected and adaptively raised for 1 week. They were raised under conditions of 12-hour light and dark alternation, temperature of 26±2℃, and humidity of 40%-60%, with free access to food and water. Twelve rats were randomly divided into the normal group and injected with normal saline through the tail vein. The remaining 12 rats were injected with doxorubicin (6.0mg / kg) through the tail vein to prepare a rat model of renal injury, and the samples were collected after 6 weeks. The animal experiments were approved by the Ethics Committee of the China-Japan Friendship Hospital. All procedures were performed in accordance with the 8th edition of the Guiding Principles for the Care and Use of Laboratory Animals of the National Academy of Sciences of the United States. All rats were fasted for 12h before sampling. The animals were then weighed and anesthetized. Rats were killed by cervical dislocation, and the bilateral kidneys of rats in each group were separated. One part was immersed in 4% paraformaldehyde for histopathological and immunohistochemical observations, another part was frozen for immunohistochemical fluorescence analysis, and the rest was frozen at -80°C for real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot experiments, and 24-hour urine was collected to detect the urinary albumin to creatinine ratio (UACR). The STZ model was constructed by the research group in the previous experiment. For details, see: Li N, Zhao T, Cao Y, et al. Tangshen Formula Attenuates Diabetic Kidney Injury by Imparting Anti-pyroptotic Effects via the TXNIP-NLRP3-GSDMD Axis. Front Pharmacol. 2020; 11: 623489.

[0039] 1.4 Transmission electron microscopy

[0040] After the renal tissue was fixed in electron microscopy fixative for 4 h, 1% osmium acid (0.5 mL) was added to each sample, followed by dehydration in alcohol-acetone, embedding in Epon812, polymerization in a 60°C oven for 48 h, ultrathin sectioning (70 nm), and lead citrate staining. The podocyte foot processes and basement membrane were observed under a transmission electron microscope, and the imaging system collected images for analysis.

[0041] 1.5Quantitative Real-time polymerase chain reaction(qRT-PCR)

[0042] Renal tissue was ground on ice and used a high-purity RNA isolation kit (83913; Roche Life Sciences, Indianapolis, IN, USA). The RNA concentration and purity were assessed using a NanoDrop 2000c-UV-Vis spectrophotometer (Thermo Fisher). An Applied Biosystems high-capacity cDNA reverse transcription kit (4368814; ThermoFisher) was used. The mRNA expression of the target gene was detected using a real-time fluorescence quantitative PCR system (A28575; QuantStudio 5, Thermo Fisher). The primer sequences are shown in Table 1.

[0043] Table 1(SEQ ID No.1-4)

[0044]

[0045] 1.6 Western Blot

[0046] Kidney tissue was added with Ripa (R0010, Solebao, China) and protease inhibitors and ground on ice. Ultrasonic cell disruptor (SLC-2006, Xinzhi, China) was used for 10 min, and then centrifuged at 12000g at 4°C for 5 min. The protein concentration was measured by BCA protein concentration assay kit (PC0020, Solebao). 4X loading buffer (YaZyme) was added to prepare 10% PAGE gel, and SDS-PAGE electrophoresis was performed. The membrane was transferred to PVDF membrane at a constant current and blocked with 5% skim milk for 1 h. The internal reference β-actin (4967S, CST) and TREM2 (A10482, Abclonal) were incubated at 4°C overnight, and the membrane was washed with TBST for 10 min three times. HRP secondary antibody, goat anti-rabbit IgG (HRP) (ab205718), goat anti-mouse IgG (HRP) (ab205719) were used for gel imaging analysis by Bio-Rad.

[0047] 1.7 Immunohistochemistry and immunofluorescence detection

[0048] The tissue sections were dewaxed into water, immersed in sodium citrate at high temperature for antigen repair, 3% hydrogen peroxide to eliminate endogenous catalase, incubated with primary antibody at 4°C overnight, the primary antibody dilution ratio was TREM2 (1:100), washed 3 times with PBS and then incubated with secondary antibody, washed 3 times with PBS and then color developed, and image J was used for semi-quantitative analysis of the staining results.

[0049] Immunofluorescence detection used frozen sections, which were fixed with paraformaldehyde after rewarming at room temperature, blocked with 5% BSA, and incubated with primary antibodies at 4°C overnight. The primary antibody dilution ratio was TREM2 (1:100), GSDMD (1:100), and Nephrin (1:200). After washing with PBS three times, the sections were incubated with fluorescent secondary antibodies goat anti-rabbit IgG (H+L) Alexa Fluor 488 labeled secondary antibodies (1:400) and Goat anti-Pig IgG (H+L) Secondary Antibody at 37°C. After washing with PBS three times, the sections were sealed with anti-fluorescence quenching sealing agents. Images were collected and analyzed using a laser confocal microscope.

[0050] 1.8 Molecular docking

[0051] BIOVIA Discovery Studio Visualizer 4.5 software (Dassault Systèmes, San Diego, CA, USA) was used. DE-PGs, GO enrichment, and immune-related BPs were imported into the Coremine Medical database (http: / / www.coremine.com / medical / ) to screen for drugs with potential immunomodulatory effects, and the nuclear magnetic resonance (NMR) structure of TREM2 (UniProt ID: Q99NZC2) was downloaded from the RCSB protein database (https: / / www.rcsb.org). Molecular docking was performed using the CDOCKER module in Discovery Studio. Other parameters were set to default values.

[0052] 1.9 ROC analysis

[0053] The pROC package in R language was used to calculate and draw ROC curves, the roc.test function was used for ROC test, and the multcomp package was used for overall test among multiple groups. The formulas for calculating sensitivity and specificity were: sensitivity = true positive / (true positive + false negative); specificity = true negative / (true negative + false positive).

[0054] 1.10 Statistical analysis

[0055] R software (version 4.0.5) was used for analysis. The two groups were compared by Wilcoxon test to determine significance. Graphpad Prism8.3 software was used for analysis of the in vivo experiment. The quantitative data were expressed as mean ± standard deviation, and one-way analysis of variance was used for comparison between groups. P < 0.05 was *, P < 0.01 was **, and P < 0.05 indicated that the difference was statistically significant.

[0056] 2. Results

[0057] 2.1 Identification of immune-related core target genes of podocyte pyroptosis

[0058] After normalizing the GSE124622 and GSE185226 gene chip data, the two sets of data and the core differentially expressed genes related to immunity / pyroptosis are as follows Figure 1 As shown in A, the results of establishing protein network interactions are shown in Figure 1 As shown in B, the chromosome distribution results are as follows Figure 1 As shown in C. The representation data of the 8 DE-PGs were applied to the machine learning algorithm of the RF classifier. All genes were sorted by "decreased average accuracy" and "decreased average Gini coefficient". The larger these two values ​​are, the closer the relationship between DE-PGs and CKD is. The RF screening results showed that 7 DE-PGs were significant, including NLRP1, TP53, IL1A, NLRP3, TREM2, IL1B and BAX, and their clustering heat map showed that 6 differential genes were expressed upward and 1 differential gene was expressed downward. The results are shown in Figure 1 The results of GO enrichment analysis of differentially expressed genes are shown in Figure 1 E in.

[0059] 2.2 Doxorubicin can cause podocyte damage and proteinuria

[0060] To establish a podocyte injury model, rats were injected with doxorubicin via the tail vein. Podocyte injury in the model rats was observed under transmission electron microscopy. The results showed that compared with the control group, the podocytes in the model group lost their normal structural morphology, and the fusion of the foot processes disappeared. Figure 2 B in the figure. UACR increased significantly after 2 weeks of modeling. Figure 2 A in.

[0061] 2.3 In vivo experimental verification of TREM2, the core immune-related target gene of podocyte pyroptosis

[0062] qPCR and Western blot were used to verify the adriamycin nephropathy rat model, and it was found that the TREM2 transcription and protein levels in the model group rats were significantly increased ( Figure 3 In order to further explore the role of TREM2 in other types of kidney disease, the present invention used IHC to verify it in the STZ+unilateral nephrectomy animal model, and the results were shown in Figure 3 In B, it was found that TREM2 was significantly upregulated in both CKD and DKD (see Figure 3 E).

[0063] 2.4 Expression of TREM2 and GSDMD in glomeruli

[0064] Nephrin is a marker of the glomerular filtration barrier, and GSDMD is a marker of cell pyroptosis. Immunofluorescence localization revealed that TREM2 expression increased around the glomerular filtration barrier in the model group, the edges of nephrin were blurred, and the expression abundance decreased. The expression of nephrin around the high-expression area of ​​GSDMD in the model group was consistent with the above, which indirectly indicated that TREM2 was a marker of tissue damage and was related to podocyte pyroptosis. Figure 4 .

[0065] 2.5 TREM2 and small molecule docking results

[0066] The top ten compounds ranked in "anti-inflammatory and anti-pyroptosis" in the Coremine Medical database were selected for molecular docking of core disease targets. Gallic acid (GA) and quinic acid (IA) were found to have good binding efficiency. The corresponding three-dimensional structures were drawn and output in PDB format. Figure 5 .

[0067] 2.6 Effect of Gallic Acid on Cell Survival Rate

[0068] The results of MPC-5 cell viability test showed that gallic acid at a dose below 4 μg / ml had no significant effect on podocyte cell survival rate. Figure 6 Left figure in the figure. After administration of 0.4μg / ml doxorubicin, the cell survival rate decreased significantly (P<0.01). 4μg / ml gallic acid improved the decrease in podocyte survival rate caused by doxorubicin (P<0.01). The results are shown in Figure 6 Right picture in .

[0069] 2.7 Effect of gallic acid (administered at 4 μg / ml) on doxorubicin-stimulated TREM2 gene and protein expression in podocytes

[0070] qRCP and Western blot results are as follows Figure 7 As shown in the data, doxorubicin stimulation increased the expression levels of TREM2 mRNA and protein in mouse podocytes (P<0.01). Gallic acid had no significant effect on TREM2 gene expression (P=0.67), but gallic acid could significantly downregulate TREM2 protein expression (P<0.01).

[0071] 2.8 Effect of Gallic Acid on Doxorubicin-Stimulated TREM2 and GSDMD in Podocytes

[0072] The immunofluorescence colocalization results are shown in Figure 8 , which showed that under doxorubicin stimulation, the expression intensity and abundance of TREM2 and GSDMD in mouse podocytes increased significantly, and gallic acid could downregulate the expression of TREM2 and GSDMD.

[0073] 2.9 TREM2 predictive value analysis

[0074] To observe the correlation between TREM2 and podocytes, the correlation analysis between TREM2 and the number of podocyte marker WT1 positive cells was performed. The results are shown in Fig. 9 , which showed that high expression of TREM2 would lead to podocyte loss. WT1 is a podocyte marker. TREM2 was negatively correlated with the number of WT1-positive cells (podocytes) in the glomerulus, with a correlation coefficient of -0.88 (P<0.05).

[0075] The ROC curve of TREM2 was drawn, and the predictive value of TREM2 was evaluated by calculating the area under the ROC curve (AUC). The results showed that the AUC was 0.808, the sensitivity was 0.818, and the specificity was 0.909. TREM2 had a good predictive value as a biomarker for podocytosis.

[0076] In order to reveal the molecular mechanism by which pyroptosis affects the immune response and activation of immune pathways in patients with kidney disease, the present invention uses bioinformatics to find that pyroptosis and immune response-related genes (NLRP1, NLRP3, TP53, IL1A, IL1B, BAX and TREM2) are closely related to podocyte injury. NLRP1 is mainly expressed in skin and airway epithelial cells, which can sense toxin-induced ribose toxicity stress response (RSR), direct hyperphosphorylation of specific disordered linker regions, and induce cell pyroptosis through MAP3K20 / ZAKα kinase and its downstream effector p38. NLRP3 is mainly expressed in blood cells, and forms inflammasomes with PYD to directly activate cell pyroptosis. TP53 is highly expressed in tumor cells as a tumor suppressor gene and is associated with a variety of programmed death modes. Interleukin1Alpha (IL1A) and IL1B are secreted factors that are mostly expressed by white blood cells such as macrophages or interstitial cells, often accompanied by cell pyroptosis release. BAX is a classic protein that regulates apoptosis. Many studies in recent years have shown that BAX can activate cell pyroptosis through CASP3 / GSDME and induce immune inflammatory responses.

[0077] TREM2 has an immunoglobulin-like extracellular domain, a transmembrane region, and a short cytoplasmic tail. In the brain, it is mainly expressed by microglia and has been shown to control two signaling pathways: regulating phagocytosis and inhibiting inflammatory reactivity. The in vivo experiments of the present invention were verified, and the results showed that in the CKD and DKD animal models, TREM2 in the kidneys of rats in the model group was significantly increased, confirming that TREM2 plays an important role in the pathogenesis of various kidney diseases, especially podocyte injury caused by doxorubicin stimulation. These results indicate that the imbalance between pyroptosis-driving genes, inhibitory genes, and marker genes is potentially related to a variety of potential pathogenic factors of kidney disease, among which immunity and inflammation are the main related factors, which is crucial for finding multiple targets through pyroptosis intervention. Therefore, targeting the immune response associated with podocyte pyroptosis is a potential strategy to improve the treatment efficiency of CKD patients.

[0078] The present invention also predicts small molecule compounds related to the hub DE-PG and enriched immune pathways, and identifies possible binding sites of proteins and ligands through molecular docking, thereby clarifying the relationship between drug-protein interactions at the molecular level. The results show that gallic acid and quinic acid have good binding efficiency with the TREM2 encoded protein. The results of in vitro experiments show that gallic acid improves the decreased survival rate of podocytes caused by doxorubicin, and can significantly downregulate TREM2 protein expression, thereby reducing GSDMD protein expression. It shows that gallic acid can improve podocyte pyroptosis caused by doxorubicin stimulation by binding to TREM2.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. The use of reagents for quantitative detection of the TREM2 gene in the preparation of podocyte injury diagnostic products, and the podocyte injury is diagnosed by detecting the expression level of the TREM2 gene in the kidney tissue to be tested.

2. The use according to claim 1, characterized in that: The product is a reagent.

3. The use according to claim 1, characterized in that: The product is a kit or a chip.

4. The use according to any one of claims 1 to 3, characterized in that: If the expression level of the TREM2 gene is significantly higher than that of normal kidney tissue, it is judged that podocyte damage has occurred in the tested kidney tissue.

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