Application of EGR1 as target in preparation of DKD diagnostic reagent and drug for treating DKD

Abstract

The invention discloses application of EGR1 as a target in preparation of a DKD diagnostic reagent and a medicine for treating DKD, and relates to the technical field of diagnosis and treatment of diabetic kidney diseases. According to the present invention, EGR1 or STING1 is adopted as the target to design the primer so as to detect the gene expression quantity of the to-be-detected sample, such that the DKD diagnosis is achieved; furthermore, EGR1 or STING1 is used as a target to design small interfering RNA to prepare a medicine for treating DKD. It is proved that the expression level of EGR1 in kidney tissue of a patient with diabetic nephropathy is remarkably increased, high expression of EGR1 is related to podocyte damage and inflammation, and EGR1 participates in high-glucose-induced podocyte damage and inflammation by regulating STING1. The specific mechanism of the compound in mediating DKD inflammatory reaction is further verified through in-vivo and in-vitro experiments, and an important theoretical basis is provided for development of accurate diagnostic reagents of diabetic nephropathy and research and development of targeted therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnosis and treatment of diabetic kidney disease, and more particularly to the use of EGR1 as a target in the preparation of DKD diagnostic reagents and DKD treatment drugs. Background Art

[0002] Diabetic kidney disease (DKD) refers to chronic kidney disease caused by diabetes, primarily characterized by a urine albumin / creatinine ratio (UACR) ≥ 30 mg / g and / or an estimated glomerular filtration rate (eGFR) < 60 ml / min / 1.73 m², persisting for more than three months. Data show that 20%-40% of diabetic patients have DKD. Currently, DKD has become the leading cause of end-stage kidney disease (ESKD). Treatment is difficult and expensive, placing a significant financial burden on patients and their families.

[0003] Podocytes, a specialized cell type in the glomerulus, play a central role in maintaining the glomerular filtration barrier, regulating glomerular function, supporting basement membrane stability, and participating in immune regulation. They are also important targets for the research and treatment of various kidney diseases. Podocyte injury leads to increased glomerular permeability, allowing proteins and other mediators to enter the renal tubular lumen, leading to proteinuria and renal dysfunction, and plays a key role in the development and progression of diabetic renal disease (DKD). Therefore, uncovering the key target proteins that regulate podocyte injury is not only of scientific value in elucidating the pathogenesis of DKD but also provides important insights into the development of targeted therapeutic strategies.

[0004] Based on the above background, how to systematically screen key genes closely related to podocyte injury in diabetic nephropathy and use them to build accurate diagnostic systems and innovative treatment targets has become a core scientific issue that urgently needs to be broken through in the current field. Summary of the Invention

[0005] In view of this, the present invention provides the use of EGR1 as a target in the preparation of DKD diagnostic reagents and DKD treatment drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of EGR1 as a target in the preparation of a DKD diagnostic reagent is to detect the EGRI gene expression level of the test sample using the primer combination shown in SEQ ID NO.1 to SEQ ID NO.4.

[0008] Another object of the present invention is to provide a DKD diagnostic product for detecting the expression level of EGR1, including the primer combination shown in SEQ ID NO.1 to SEQ ID NO.4.

[0009] Another object of the present invention is to provide the application of EGR1 as a target in the preparation of drugs for treating DKD, and designing small interfering RNA against the EGR1 gene.

[0010] Preferably, the sequence of the small interfering RNA is as shown in SEQ ID NO.5.

[0011] Another object of the present invention is to provide a preparation for treating DKD, including small interfering RNA with a nucleotide sequence as shown in SEQ ID NO.5.

[0012] Another object of the present invention is to provide the application of STING1 as a target in the preparation of DKD diagnostic reagents, and detecting the expression level of the STING1 gene in a test sample with the primer combination shown in SEQ ID NO.19 to SEQ ID NO.20 and SEQ ID NO.3 to SEQ ID NO.4.

[0013] Another object of the present invention is to provide a DKD diagnostic product for detecting the expression level of STING1, including the primer combination shown in SEQ ID NO.19 to SEQ ID NO.20 and SEQ ID NO.3 to SEQ ID NO.4.

[0014] Another object of the present invention is to provide the application of STING1 as a target in the preparation of drugs for treating DKD, and designing small interfering RNA against the STING1 gene.

[0015] Preferably, the sequence of the small interfering RNA is as shown in SEQ ID NO.21.

[0016] Another object of the present invention is to provide a preparation for treating DKD, including small interfering RNA with a nucleotide sequence as shown in SEQ ID NO.21.

[0017] In the above technical solution, compared with the healthy control group, the expression level of EGR1 in the renal tissue of diabetic kidney disease patients is significantly increased, and its expression intensity is significantly negatively correlated with eGFR and significantly positively correlated with the 24-hour urinary protein quantification.

[0018] Compared with the normal glycome, EGR1 is upregulated in podocytes treated with high glucose. High glucose treatment can lead to downregulation of podocyte injury-related genes synaptopodin and ZO-1, upregulation of Desmin, and upregulation of inflammation-related genes IL-1α, IL-1β, and TNF-α, promoting podocyte injury and inflammation. Knockdown of EGR1 expression can alleviate high glucose-induced podocyte injury and inflammation, manifested as reversal of the downregulation of podocyte injury-related genes synaptopodin and ZO-1, upregulation of Desmin, and upregulation of inflammation-related genes IL-1α, IL-1β, and TNF-α; overexpression of EGR1 has the opposite result, showing promotion of the abnormal expression of podocyte injury- and inflammation-related genes.

[0019] EGR1 is involved in high glucose-induced podocyte injury and inflammatory responses by regulating STING1. STING1 is upregulated in podocytes induced by high glucose, and EGR1 negatively regulates the mRNA and protein expression of STING1. Knockdown of STING1 can alleviate high glucose-induced podocyte injury and inflammatory responses, reversing the abnormal expression of its related genes synaptopodin, Desmin, ZO-1, IL-1α, IL-1β, and TNF-α; overexpression of STING1 has the opposite result, showing promotion of the abnormal expression of podocyte injury- and inflammation-related genes. Rescue experiments further showed that knockdown of STING1 can alleviate the abnormal expression of podocyte injury- and inflammation-related genes induced by overexpression of EGR1.

[0020] EGR1 is upregulated in streptozotocin (STZ)-induced diabetic nephropathy mice. Compared with wild-type mice (WT), STZ-induced diabetic nephropathy mice (WT-STZ) have increased urinary albumin-to-creatinine ratio (UACR), glomerular hypertrophy, increased mesangial matrix, downregulation of glomerular synaptopodin and ZO-1 expression, upregulation of Desmin expression, and at the same time, upregulation of inflammatory factors IL-1β and TNF-α expression, accompanied by infiltration of F4 / 80-positive macrophages, suggesting that STZ induces glomerular injury and inflammatory responses. Compared with the WT-STZ group, the above-mentioned glomerular and inflammation-related injuries in podocyte-specific EGR1 knockout diabetic nephropathy mice (Egr1 KO +STZ) were all improved, suggesting that podocyte-specific knockout of EGR1 can alleviate glomerular injury and inflammation in STZ-induced diabetic nephropathy mice.

[0021] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the application of EGR1 as a target in the preparation of DKD diagnostic reagents and drugs for the treatment of DKD. The present invention finds that the expression level of EGR1 in the renal tissue of patients with diabetic kidney disease is significantly increased, and its expression intensity is significantly negatively correlated with eGFR and significantly positively correlated with the 24-hour urinary protein quantification. The present invention also finds that the high expression of EGR1 is related to podocyte damage and inflammation, and to a certain extent, it can reflect the inflammatory level of DKD. The present invention also finds that EGR1 participates in high-glucose-induced podocyte damage and inflammation by regulating STING1. Further verifying its specific mechanism in mediating the inflammatory response of DKD through in vivo and in vitro experiments is helpful to provide an important theoretical basis for the development of precision diagnostic reagents for diabetic kidney disease and the research and development of targeted therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0023] Figure 1 In the drawings, A is the immunohistochemical staining of EGR1 in the renal tissues of healthy controls and patients with diabetic kidney disease, n = 20; B is the correlation analysis of the expression level of EGR1 in the glomeruli of DKD patients with eGFR and 24-hour urinary protein; C is the co-expression and co-localization of EGR1 and the podocyte-specific marker synaptopodin detected by double immunofluorescence labeling.

[0024] Figure 2 In the drawings, A is the mRNA and protein expression of EGR1 in podocytes in the normal glucose, high glucose, and hyperosmotic treatment groups; B is the knockdown efficiency detection of EGR1 after transfection of human podocytes with EGR1 small interfering RNA; C is the overexpression efficiency detection of EGR1 after transfection of podocytes with an EGR1 overexpression plasmid. n = 3, ***P < 0.001.

[0025] Figure 3In the attached figure, A shows the detection of mRNA and protein expressions of podocyte-related proteins by real-time PCR and Western blot after transfection of human podocytes with small interfering RNA targeting EGR1; B shows the detection of mRNA and protein expressions of podocyte-related proteins by real-time PCR and Western blot after transfection of podocytes with an overexpression plasmid of EGR1; C shows the detection of protein expression of inflammatory factors by real-time PCR and Western blot after transfection of human podocytes with small interfering RNA targeting EGR1; D shows the detection of protein expression of inflammatory factors by real-time PCR and Western blot after transfection of podocytes with an overexpression plasmid of EGR1. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0026] Figure 4 In the attached figure, A shows the detection of the knockdown efficiency of STING1 after transfection of human podocytes with small interfering RNA targeting STING1; B shows the detection of the overexpression efficiency of STING1 after transfection of podocytes with an overexpression plasmid of STING1. n = 3, ***P < 0.001.

[0027] Figure 5 In the attached figure, A shows the detection of the effects of knocking down EGR1 on the mRNA and protein expressions of STING1 in podocytes; B shows the detection of the effects of overexpressing EGR1 on the mRNA and protein expressions of STING1 in podocytes. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0028] Figure 6 In the attached figure, A shows the detection of mRNA and protein expressions of podocyte-related proteins and inflammatory factors by real-time PCR and Western blot after transfection of podocytes with small interfering RNA targeting STING1; B shows the detection of mRNA and protein expressions of podocyte-related proteins and inflammatory factors by real-time PCR and Western blot after transfection of podocytes with an overexpression plasmid of STING1. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0029] Figure 7 In the attached figure, A shows the detection of the effects of knocking down STING1 on the podocyte injury and the expression of inflammatory factors induced by overexpressing EGR1 by real-time PCR; Figure B shows the detection of the effects of knocking down STING1 on the podocyte injury and the expression of inflammatory factors induced by overexpressing EGR1 by Western blot. n = 3, ***P < 0.001.

[0030] Figure 8In the attached figure, A shows the verification of the knockout efficiency of podocyte-specific EGR1; B shows the detection of the expression of EGR1 in the renal tissue of diabetic nephropathy mice by immunohistochemical staining; C shows the changes in blood glucose levels of mice in different groups, n = 6, ***P < 0.001; D shows the changes in the kidney-to-body weight ratio of mice in different groups, n = 6, ***P < 0.001; E shows the changes in the UACR levels of mice in different groups, n = 6. ***P < 0.001 vs WT, ## P < 0.01 vs WT+STZ, ### P < 0.001 vs WT+STZ; F shows the PAS staining results of mice in different groups; G shows the immunofluorescence staining results of podocyte-related proteins Desmin, synaptopodin, and ZO-1 in mice in different groups; H shows the infiltration of F4 / 80-positive cells in the renal tissue of mice in different groups; I shows the protein changes of IL1β and TNF-α in the glomeruli of mice in different groups. Detailed implementation manner

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] Example 1

[0033] Analysis of the expression and localization of EGR1 in the renal tissue of patients with diabetic nephropathy and its correlation with clinical indicators

[0034] 1. Collection of renal tissue samples

[0035] A total of 40 renal tissue samples were included in this study. Among them, 20 cases in the DKD group were obtained from pathological specimens obtained by percutaneous renal biopsy in the Department of Nephrology, Shandong Provincial Hospital from September 2021 to September 2023, and 20 cases in the normal control group were selected from the adjacent normal renal tissues (more than 2 cm away from the tumor margin) after radical resection of renal tumors in patients with renal tumors during the same period. All sample collections were approved by the hospital ethics committee and written informed consent was obtained from patients or their legal representatives.

[0036] 2. Expression and localization of EGR1 in DKD renal tissue

[0037] The expression level of EGR1 in DKD kidney tissue was analyzed by immunohistochemistry, and its clinical association with estimated glomerular filtration rate (eGFR) and 24-hour urinary protein quantification was explored by Spearman correlation test. To further clarify the cellular localization characteristics of EGR1, the co-expression and co-localization of EGR1 and the podocyte-specific marker synaptopodin were detected by immunofluorescence double labeling method.

[0038] (1) Immunohistochemical staining

[0039] Tissue sample fixation and section preparation: After sampling, the tissue samples were immediately placed in formalin for 24 - 48 hours. After fixation, the tissues were dehydrated with gradient ethanol, cleared with xylene, infiltrated with paraffin and embedded, and then the tissues were cut into 4-μm thick continuous sections using a paraffin slicer. The sections were baked on a 60°C constant temperature baking machine for 1 hour. Subsequently, the sections were dewaxed in xylene three times, 15 minutes each time, placed in absolute ethanol twice, 8 minutes each time, dehydrated through 95%, 85%, and 75% gradient ethanol in turn, 5 minutes each time, and finally rinsed with distilled water for standby.

[0040] Antigen retrieval and blocking: The antigen retrieval method can use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). The sections were placed in the antigen retrieval solution, heated to boiling under high pressure, maintained for 5 - 10 minutes and then cooled to room temperature naturally, and then rinsed 3 times with PBS. Incubate with 3% hydrogen peroxide at 37°C for 30 minutes to block the activity of endogenous peroxidase, and then wash 3 times with PBS, 5 minutes each time. Then, 5% BSA or goat serum was dropped on the tissue sections and incubated at 37°C for 30 minutes to block non-specific binding sites.

[0041] Primary antibody incubation and rewarming: After blocking, discard the blocking solution, drop the appropriately diluted EGR1 antibody (primary antibody), and incubate overnight in a wet box at 4°C. The next day, take out the sections and place them in an environment at 37°C for 30 minutes to rewarm.

[0042] Reaction enhancement and secondary antibody incubation: Drop the reaction enhancement solution and incubate the sections at 37°C for 20 minutes. Then, discard the reaction enhancement solution, drop the enhanced enzyme-labeled goat anti-rabbit IgG polymer (secondary antibody), and incubate at 37°C for 30 minutes. After the secondary antibody incubation, wash 3 times with PBS again, 5 minutes each time, to remove unbound antibodies.

[0043] DAB color development and counterstaining: Color development was performed using a DAB kit. Drop the DAB working solution onto the tissue sections, incubate at room temperature and observe the color change. When the tissue labeled with the target protein showed brownish yellow, terminate the reaction and immediately rinse with tap water. Subsequently, counterstain with hematoxylin, stain at room temperature for 3 minutes, and then rinse with running water until the background color was appropriate.

[0044] Dehydration and clearing: After counterstaining, the sections were dehydrated through the following gradient dehydration procedure: 95% ethanol: dehydrate for 30 seconds; absolute ethanol: dehydrate twice, the first time for 30 seconds and the second time for 1 minute; xylene: clear twice, 2 minutes each time.

[0045] Mounting and microscopic observation: After dehydration, neutral balsam was dropped onto the sections for mounting, and a coverslip was gently placed on top, ensuring no air bubbles between the sections and the coverslip. After mounting, the sections were placed under a microscope for observation, and the expression level of the target protein in the tissue was evaluated by optical microscopy. The area percentage or gray value of the positive signal was measured using ImageJ, and statistical analysis was used to further compare the differences between the experimental group and the control group.

[0046] (2) Immunofluorescence staining

[0047] Fixation and section preparation of tissue samples: The method was the same as that for immunohistochemical staining.

[0048] Antigen retrieval and blocking: For antigen retrieval, sodium citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) could be used. The sections were placed in the antigen retrieval solution, heated to boiling under high pressure, maintained for 5 - 10 minutes, and then cooled to room temperature naturally. Then, they were rinsed 3 times with PBS. Permeabilize with 0.5% Triton for 5 minutes, followed by washing 3 times with PBS, 5 minutes each time. After that, 5% BSA was dropped onto the tissue sections and incubated at 37°C for 30 minutes to block non-specific binding sites.

[0049] Primary antibody incubation and rewarming: The method was the same as that for immunohistochemical staining.

[0050] Secondary antibody incubation: After incubation with the primary antibody, the primary antibody solution was discarded, and the sections were washed 3 times with PBS, 5 minutes each time. Then, an appropriately diluted Alexa 594-conjugated donkey anti-rabbit IgG fluorescently labeled secondary antibody was added and incubated at room temperature for 1 hour. [[ID=2,4]]

[0051] DAPI nuclear staining and mounting: After incubation with the secondary antibody, the sections were washed 3 times with PBS, 5 minutes each time. DAPI solution was added to the sections and stained at room temperature for 5 minutes. After staining, the sections were washed 3 times with PBS, 5 minutes each time.

[0052] Mounting: After completing the above steps, the sections were washed thoroughly with PBS buffer, gently blotted dry, and an anti-fluorescence quenching mounting medium was added. A coverslip was placed on the sections to complete the mounting.

[0053] Microscopic observation and data analysis:

[0054] Use a fluorescence microscope to observe and photograph the sections after mounting, and analyze the results based on the fluorescence intensity or the area of the positive signal.

[0055] The results showed that in immunohistochemical staining, compared with the healthy control group, the staining intensity of EGR1 in the renal tissue of patients with diabetic nephropathy was increased, which was positively correlated with 24-hour urinary protein (r = 0.529, P = 0.0165) and negatively correlated with eGFR (r = -0.4587, P = 0.0419). In the results of immunofluorescence staining, the expression of EGR1 was up-regulated in the renal tissue of DKD patients, and EGR1 was co-localized with podocytes (see attached Figure 1 ).

[0056] Example 2

[0057] The role of EGR1 in high glucose-induced podocyte injury and inflammation

[0058] 1. Primer sequences of the target gene and the reference gene:

[0059] EGR1:

[0060] F 5’-TCCCATTTACTCAGCGGCAC-3’, SEQ ID NO.1;

[0061] R 5’-TGGAAACAGGTAGTCGGGGA-3’, SEQ ID NO.2;

[0062] Synaptopodin:

[0063] F 5’-CGCTCACCACACCAACTTCT-3’, SEQ ID NO.6;

[0064] R 5’-TGCTAGAAAGTGGCAGGCTC-3’, SEQ ID NO.7;

[0065] Desmin:

[0066] F 5’-AGGACCGATTTGCCAGTGAG-3’, SEQ ID NO.8;

[0067] R 5’-CTTGAGGTGCCGGATTTCCT-3’, SEQ ID NO.9;

[0068] ZO-1:

[0069] F 5’-CCCCCAACTCAAACCGAAGA-3’, SEQ ID NO.10;

[0070] R 5’-AGATGCTACTTCTGGAGGCTTA-3’, SEQ ID NO.11;

[0071] IL1β:

[0072] F 5’-CAACAAGTGGTGTTCTCCATGTC-3 SEQ ID NO.12;

[0073] R 5’-ACACGCAGGACAGGTACAGA-3’, SEQ ID NO.13;

[0074] TNF-α:

[0075] F 5’-GAGGCCAAGCCCTGGTATG-3’, SEQ ID NO.14;

[0076] R 5’-CGGGCCGATTGATCTCAGC-3’, SEQ ID NO.15;

[0077] MCP-1:

[0078] F 5’-CTCAGCCAGATGCAATCAATG-3’, SEQ ID NO.16;

[0079] R 5’-CTTCTTTGGGACACTTGCTGC-3’, SEQ ID NO.17;

[0080] β-Actin:

[0081] F 5’-CATGTACGTTGCTATCCAGGC-3’, SEQ ID NO.3;

[0082] R 5’-CTCCTTAATGTCACGCACGAT-3’, SEQ ID NO.4.

[0083] 2. Primary antibodies and catalog numbers used in Western blot:

[0084] EGR1 (Proteintech, 55117-1-AP), synaptopodin (Proteintech, 21064-1-AP), ZO-1 (Proteintech, 21773-1-AP), Desmin (Proteintech, 16520-1-AP), IL1β (Proteintech, 16806-1-AP), TNF-α (Proteintech, 60291-1-Ig), IL1α (Proteintech, 16765-1-AP), β-actin (Proteintech, 66009-1-Ig).

[0085] 3. To clarify the role of EGR1 in podocyte injury and inflammation, we treated podocytes with high glucose to simulate the diabetic nephropathy environment in vitro. Further, the podocytes were divided into a normal glucose group (NG, 5.5 mmol / L glucose), a high glucose group (HG, 30 mmol / L glucose), and a hyperosmotic group (HO, 5.5 mmol / L glucose + 24.5 mmol / L mannitol). The mRNA and protein expressions of EGR1 in podocytes were detected by real-time PCR and Western blot. Further, we transfected podocytes with synthetic EGR1 small interfering RNA and constructed overexpression plasmids [the CDS region of the Homo-EGR1 gene (NM_001964.3) was ligated to the pCDNA3.1(+) vector]. The Western blot results showed good knockdown and overexpression efficiencies (see Appendix Figure 2 ).

[0086] EGR1 small interfering sequence:

[0087] EGR1 siRNA: 5’-AGUUUGCCAGGAGCGAUGA-3’, SEQ ID NO.5.

[0088] Negative control: NC siRNA: 5’-UUCUCCGAACGUGUCACGU-3’, SEQ ID NO.18;

[0089] After knocking down and overexpressing EGR1 in podocytes, the expressions of podocyte injury-related genes synaptopodin, ZO-1, Desmin and inflammation-related genes IL1β, IL1α, TNF-α were further detected by real-time PCR and Western blot.

[0090] (1) real-time PCR

[0091] RNA Extraction: After washing the cell samples with PBS buffer, they were collected. For RNA extraction, commercially available TRIzol reagent or RNeasy kit was used and operated according to the instructions. First, the treated samples were added to the TRIzol reagent, mixed well and incubated for 5 min to lyse the cells. Subsequently, chloroform was added for phase separation. After centrifugation, the supernatant was collected, transferred to a new tube and an equal volume of isopropanol was added to precipitate the RNA. After centrifugation, the supernatant was removed, the RNA precipitate was washed with 75% ethanol and centrifuged again. Finally, the precipitate was dissolved in RNase-free water. After the RNA was dissolved, the concentration and purity of the RNA were measured using a spectrophotometer. The ratio of 260 / 280 should be between 1.8 and 2.0 to ensure that the RNA quality meets the experimental requirements.

[0092] Reverse Transcription: The Evo M-MLV Reverse Transcription Kit was used to prepare the reaction system according to the manufacturer's instructions, with a total volume of 20 μL. Among them, 1 μg of total RNA, 4 μL of 5×Evo M-MLV RT Reaction Mix, 2 μL of gDNA Clean Reaction Mix were added, and made up to 20 μL with RNase-free water. The reaction system was incubated at 37 °C for 15 min for reverse transcription reaction. Finally, the reaction system was incubated at 85 °C for 5 seconds to terminate the reverse transcription reaction and activate the enzyme. The reverse-transcribed cDNA could be directly used for the subsequent PCR reaction. If not used immediately, the cDNA could be aliquoted and stored at -20 °C.

[0093] Fluorescence Quantification: The SYBR Green Pro Taq HS Premixed qPCR Kit was used to prepare the PCR reaction system on ice according to the manufacturer's instructions. The reaction system contained 5 μL of 2×SYBR Green PCR MasterMix, 0.2 μL of forward primer and 0.2 μL of reverse primer, 2 μL of template cDNA, and made up to 10 μL with RNase-free water. The PCR reaction procedure was: first pre-denature at 95 °C for 30 seconds, and then perform 40 cycles, each cycle including denaturation at 95 °C for 5 seconds and annealing at 60 °C for 30 seconds. After all cycles were completed, melt curve analysis was performed to confirm the specificity of the PCR product. When analyzing the data, the gene expression level was normalized to β-actin, and the 2 -ΔΔCt method was used for analysis.

[0094] (2) Western blot

[0095] Protein extraction and quantification: For cell samples, first wash the cells twice with PBS to remove residual culture medium. Subsequently, suspend the cells in an appropriate amount of lysis buffer (prepared according to protein lysis buffer: protease inhibitor: phosphatase inhibitor = 100:1:1), mix well and place on ice for 30 min for lysis. During the lysis process, gently pipette and mix every 10 min. After lysis is complete, centrifuge at 4°C and 12,000 g for 15 min, take the supernatant and store it, which is the total protein extract. After protein extraction, use a BCA protein quantification kit to measure the protein concentration to ensure consistent loading amounts for each sample.

[0096] Protein electrophoresis and membrane transfer: After the protein samples are prepared, perform SDS-PAGE electrophoresis to separate the proteins. Take 20 μg of protein, add 5× SDS protein loading buffer (final concentration 1×), heat at 100°C for 5 min to denature the protein and make it linear. After the sample cools, load it onto a 10% SDS-PAGE gel and run the gel at 160 V for about 60 min until the protein bands are separated to an appropriate position. After electrophoresis is complete, transfer the gel to a PVDF membrane. Before membrane transfer, soak the PVDF membrane in methanol for 1 min. Set a constant current of 220 mA, and set the membrane transfer time according to the molecular weight of the protein to be measured.

[0097] Antibody incubation and signal detection: After membrane transfer is complete, block the PVDF membrane with 5% skim milk powder and incubate at room temperature for 1 hour to reduce non-specific binding. After blocking, wash the PVDF membrane 3 times with TBST, 5 min each time. Subsequently, add an appropriately diluted primary antibody and incubate overnight at 4°C. The next day, after recovering the primary antibody, wash the membrane 3 times with TBST, 10 min each time, then add an HRP-labeled secondary antibody and incubate at room temperature for 1 hour. After incubation with the secondary antibody, wash the membrane 3 times again with TBST to remove unbound antibodies. Finally, incubate the membrane with an ECL chemiluminescent substrate and detect the expression of protein bands by exposure in a chemiluminescent imaging system. Perform gray value analysis based on the internal reference protein to quantify the expression level of the target protein.

[0098] The results showed that: compared with the normal glucose group, high glucose treatment could significantly up-regulate the mRNA and protein expression of EGR1 in podocytes. High glucose treatment could lead to down-regulation of the expressions of podocyte-related genes synaptopodin and ZO-1, up-regulation of Desmin expression, and up-regulation of the expressions of inflammation-related genes IL-1α, IL-1β, and TNF-α, promoting podocyte injury and inflammation. Knockdown of EGR1 could reduce high glucose-induced podocyte injury and inflammatory responses and reverse the abnormal expressions of synaptopodin, Desmin, ZO-1, IL-1α, IL-1β, and TNF-α; while overexpression of EGR1 promoted the abnormal expressions of podocyte injury and inflammation-related genes. It is suggested that EGR1 is involved in high glucose-induced podocyte injury and inflammation (see attachmentFigure 3 )。

[0099] Example 3

[0100] It was clarified that EGR1 is involved in high glucose-induced injury and inflammatory response by regulating STING1

[0101] 1. Primer sequences of target gene and internal reference gene:

[0102] STING1:

[0103] F 5’-TACAACAACCTGCTACGGGG-3’, SEQ ID NO.19;

[0104] R 5’-TCTGCTGGGGCAGTTTATCC-3’, SEQ ID NO.20;

[0105] β-Actin:

[0106] F 5’-CATGTACGTTGCTATCCAGGC-3’, SEQ ID NO.3;

[0107] R 5’-CTCCTTAATGTCACGCACGAT-3’, SEQ ID NO.4.

[0108] 2. To clarify the specific mechanism by which EGR1 is involved in high glucose-induced podocyte injury and inflammation, podocytes were divided into an overexpression control group (pCDNA3.1 empty vector, OE-NC) and an EGR1 overexpression group (OE-EGR1, prepared in Example 2). Transcriptome sequencing was performed and the key downstream target gene STING1 of EGR1 was screened out. The mRNA and protein expressions of STING1 in podocytes were detected by real-time PCR and Western blot, and the effect of regulating EGR1 on the expression of STING1 was also detected.

[0109] Furthermore, podocytes were transfected with synthetic STING1 small interfering RNA and the constructed overexpression vector [the CDS region of the Homo-STING1 gene (NM_198282.4) was ligated to the pCDNA3.1(+) vector] respectively. The Western blot results showed that they had good knockdown and overexpression efficiencies (see attached Figure 4 )。

[0110] STING1 small interfering sequence:

[0111] STING1 siRNA: 5’-GGUCAUAUUACAUCGGAUA-3’, SEQ ID NO.21;

[0112] NC siRNA: 5'-UUCUCCGAACGUGUCACGU-3', SEQ ID NO.18.

[0113] After knocking down and overexpressing STING1 in podocytes, the expressions of podocyte injury-related genes synaptopodin, ZO-1, Desmin and inflammation-related genes IL1β, IL1α, TNF-α were detected by real-time PCR and Western blot. Further, through a rescue experiment, the effect of knocking down STING1 on podocyte injury and inflammation induced by overexpression of EGR1 was detected.

[0114] The experimental procedures of real-time PCR and Western blot were the same as those in Example 2.

[0115] The results showed that: compared with the normal glucose group, high glucose treatment could significantly up-regulate the mRNA and protein expressions of STING1 in podocytes; EGR1 positively regulated the expression of STING1, overexpression of EGR1 could significantly up-regulate the expression of STING1, while knocking down EGR1 significantly down-regulated the expression of STING1; knocking down STING1 could alleviate the down-regulation of synaptopodin and ZO-1 expressions, the up-regulation of Desmin expression, and the up-regulation of inflammation-related gene expressions of IL-1α, IL-1β, TNF-α induced by high glucose, and alleviate podocyte injury and inflammation. Overexpression of STING1 promoted the abnormal expressions of podocyte injury and inflammation-related genes. The rescue experiment further showed that knocking down STING1 could alleviate podocyte injury and inflammation induced by overexpression of EGR1. It is suggested that EGR1 is involved in high glucose-induced podocyte injury and inflammation by regulating STING1 (see attached Figure 5 -Attached Figure 7 ).

[0116] Example 4

[0117] Role of EGR1 in glomerular injury and inflammatory response in streptozotocin (STZ)-induced diabetic kidney disease mice

[0118] 1. C57BL / 6J mice were provided by the Experimental Animal Center of Shandong First Medical University. All animal studies were reviewed and approved by the Experimental Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University.

[0119] Establish a DKD model: C57BL / 6J mice were selected. After being fed a high-fat diet for 4 weeks in the STZ group, low-dose STZ 50 mg / kg / day was intraperitoneally injected for 5 consecutive days. Control group mice were intraperitoneally injected with the same dose of citrate buffer. Monitor the blood glucose of the mice's tail vein. If it is greater than 16.7 mmol / l and persistent proteinuria appears, it is regarded as successful DKD mouse modeling.

[0120] Construction of podocyte-specific EGR1 knockout mice: The cre-Loxp technique was used to construct podocyte-specific EGR1 knockout mice. Primary podocytes were isolated and cultured, and the successful construction of podocyte-specific knockout mice was confirmed by Western blot. Further, 8-week-old male mice were randomly divided into (WT, n = 6), podocyte-specific EGR1 knockout group (Egr1 KO , n = 6), STZ injection group (WT+STZ, n = 6), and podocyte-specific EGR1 knockout + STZ injection group (Egr1 KO +STZ, n = 6).

[0121] 2. Blood and urine specimens were collected at the 4th, 8th, 12th, 16th, and 20th weeks after successful mouse modeling. The mice were euthanized, and kidney tissues were collected: ① Primary podocytes were isolated and cultured, and the EGR1 knockout efficiency was verified by Western blot; ② Immunohistochemical staining was performed to detect the expression and localization of EGR1 in mouse kidney tissues; ③ Changes in blood glucose, kidney-to-body weight ratio, UACR, etc. of the mice were detected; ④ PAS staining was used to observe the pathological changes of the mice; ⑤ Immunofluorescence staining of synaptopodin, ZO-1, Desmin, and F4 / 80 was performed to observe glomerular damage and inflammatory infiltration; ⑥ Glomeruli were isolated, and the expression of inflammatory factors IL1β and TNF-α was detected by Western blot.

[0122] Specifically:

[0123] Isolation and culture of glomeruli and primary podocytes: First, the renal capsule was removed, and the cortical tissue was divided into small tissue blocks and incubated in a composite digestive solution containing collagenase and pronase E. After digestion, preliminary separation was performed successively through sieves with different pore sizes (50 mesh, 100 μm), and finally, glomeruli were enriched using a 400-mesh sieve. The enriched glomerular samples were placed in a 37°C constant temperature incubator for adherent culture. On the 5th day of culture, the migrated podocytes were digested and subcultured, and at the same time, the residual glomerular core structure was removed through a 40-μm pore size filter. During the subsequent culture process, podocyte-specific markers (such as WT1, nephrin, etc.) were used for immunofluorescence staining or Western blot detection to confirm cell characteristics.

[0124] PAS staining: ① Baking the slides: Bake the tissue sections at 65 °C for 60 - 90 minutes. ② Deparaffinization and hydration: Place the sections on a copper rack and pass them through the following solutions in sequence: soak in Deparaffinizing Agent Ⅰ for 15 minutes, Deparaffinizing Agent Ⅱ for 15 minutes, absolute ethanol Ⅰ for 8 minutes, absolute ethanol Ⅱ for 8 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. ③ Rinsing: Immerse the sections in distilled water 3 times, 5 minutes each time. ④ Dropwise add the oxidizing agent to the section tissue at room temperature for 8 minutes; after completion, rinse with tap water for 5 minutes, and soak in distilled water again 2 times, 5 minutes each time. ⑤ Dropwise add Schiff reagent to the section tissue and stain in the dark for 20 minutes; after completion, rinse with tap water for 10 minutes. ⑥ Dropwise add hematoxylin staining solution to the section tissue at room temperature for 2 minutes; after completion, rinse with tap water for 10 minutes. ⑦ Dropwise add acidic differentiation solution to the section tissue and differentiate for several seconds; after completion, rinse with tap water. ⑧ Dehydrate and clear step by step, then mount the slides with neutral balsam. ⑨ Observe and take pictures under the microscope.

[0125] The experimental procedures for immunohistochemical staining and immunofluorescence staining are the same as those in Example 1.

[0126] The experimental procedure for Western blot is the same as that in Example 2.

[0127] The results showed that the expression of EGR1 was increased in STZ-induced diabetic nephropathy mice. Compared with wild-type mice (WT), the urinary albumin-to-creatinine ratio (UACR) increased in STZ-induced diabetic nephropathy mice (WT-STZ), glomerular hypertrophy, mesangial matrix proliferation occurred, the expressions of synaptopodin and ZO-1 in glomeruli were down-regulated, the expression of Desmin was up-regulated, and at the same time, the expressions of inflammatory factors IL-1β and TNF-α were up-regulated, accompanied by infiltration of F4 / 80-positive macrophages, indicating that STZ induced glomerular injury and inflammatory responses. However, compared with the WT-STZ group, the above-mentioned glomerular and inflammation-related injuries in podocyte-specific EGR1 knockout diabetic nephropathy mice (Egr1 KO +STZ) were all improved, suggesting that podocyte-specific knockout of EGR1 could alleviate glomerular injury and inflammation in STZ-induced diabetic nephropathy mice (see attached Figure 8 ).

[0128] In this specification, each example is described in a progressive manner. The key point of each example is to illustrate the differences from other examples. The same or similar parts among the examples can be referred to each other.

[0129] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of EGR1 as a target in the preparation of a diagnostic reagent for DKD, characterized in that, Detect the expression level of the EGRI gene in the sample to be tested using the primer combinations shown in SEQ ID NO.1 to SEQ ID NO.

4.

2. A DKD diagnostic product, characterized in that, The product is used to detect the expression level of EGR1 and includes the primer combinations shown in SEQ ID NO.1 to SEQ ID NO.

4.

3. The use of EGR1 as a target in the preparation of drugs for treating DKD, characterized in that, Design small interfering RNA against the EGR1 gene.

4. Use of EGR1 as a target in the preparation of a drug for treating DKD according to claim 3, characterized in that, The sequence of the small interfering RNA is as shown in SEQ ID NO.

5.

5. A preparation for treating DKD, characterized in that, Includes small interfering RNA with a nucleotide sequence as shown in SEQ ID NO.

5.

6. Use of STING1 as a target in the preparation of a diagnostic reagent for DKD, characterized in that, Detect the expression level of the STING1 gene in the sample to be tested using the primer combinations shown in SEQ ID NO.19 to SEQ ID NO.20 and SEQ ID NO.3 to SEQ ID NO.

4.

7. A DKD diagnostic product, characterized in that, The product is used to detect the expression level of STING1 and includes the primer combinations shown in SEQ ID NO.19 to SEQ ID NO.20 and SEQ ID NO.3 to SEQ ID NO.

4.

8. The use of STING1 as a target in the preparation of drugs for treating DKD, characterized in that, Design small interfering RNA against the STING1 gene.

9. Use of STING1 as a target in the preparation of a drug for treating DKD according to claim 8, characterized in that, The sequence of the small interfering RNA is as shown in SEQ ID NO.

21.

10. A preparation for treating DKD, characterized in that, Includes small interfering RNA with a nucleotide sequence as shown in SEQ ID NO.21.

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