Application of METTL4 inhibitor in preparation of medicine for treating acute kidney injury or renal fibrosis

Abstract

The invention provides an application of an METTL4 inhibitor in preparation of a medicine for treating acute kidney injury or renal fibrosis, and tests show that the degree of acute kidney injury and renal fibrosis can be reduced by over-inhibition of METTL4, and the degree of acute kidney injury and renal fibrosis is further aggravated by over-expression of METTL4. The result shows that related reagents for inhibiting METTL4 can be used as drugs for treating acute kidney injury or renal fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to application of a METTL4 inhibitor in preparing a medicine for treating acute kidney injury or renal fibrosis. Background Art

[0002] Kidney disease has become a major public health problem worldwide. As a key organ, the kidney maintains the homeostasis of body fluid and electrolyte status by effectively removing metabolic waste. Acute kidney injury (AKI) or chronic kidney disease (CKD) may lead to renal insufficiency and renal fibrosis, which may be accompanied by systemic complications in severe cases, and ultimately require renal replacement therapy such as dialysis and transplantation, thus placing a heavy socioeconomic burden on the healthcare system. Renal fibrosis is a typical feature of all chronic kidney diseases and a sign that the kidneys gradually lose their normal function. Its mechanism has always been a hot topic of research. However, its pathogenesis is complex and the specific pathogenic mechanism is still unclear. Finding molecular therapeutic targets is an urgent problem to be solved in the clinic.

[0003] Therefore, seeking new early biomarkers and therapeutic intervention target genes is crucial to improving acute and chronic kidney disease and slowing down the progression of renal fibrosis. These methods are very useful in helping clinicians find diagnostic and therapeutic indicators. Summary of the invention

[0004] The purpose of the present invention is to provide an application of inhibiting the expression level of METTL4 at the gene or protein level, and inhibiting its function for preparing a drug for treating kidney injury or renal fibrosis. The present application found through experiments that over-inhibition of METTL4 can reduce the degree of acute kidney injury and renal fibrosis, and further overexpression of METTL4 aggravates the degree of acute kidney injury and fibrosis, indicating that related reagents that inhibit METTL4 protein or inhibit its function can be used as drugs for treating kidney injury or renal fibrosis.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, provided is a use of a METTL4 inhibitor in the preparation of a medicament for treating acute kidney injury or renal fibrosis.

[0007] Furthermore, the inhibitor of METTL4 includes:

[0008] Substances that inhibit the expression of METTL4: inhibit the transcription and translation of the METTL4 gene at the DNA level, RNA level or protein level;

[0009] and substances that inhibit the function of METTL4.

[0010] Furthermore, the METTL4 inhibitor comprises at least one of the following components:

[0011] METTL4 inhibitors, antagonists, downregulators, blockers, and blocking agents;

[0012] Knockdown reagent for METTL4.

[0013] Furthermore, the knockout reagent of METTL4 includes: sgRNA, the sequence of which is shown in SEQ ID NO.1-SEQ ID NO.2.

[0014] Furthermore, the acute kidney injury or renal fibrosis includes acute kidney injury, chronic kidney disease and end-stage renal disease.

[0015] In the present invention, the nucleotide sequence of METTL4 may be or include the sequence of NCBI reference sequence: Gene ID: 64863.

[0016] The present invention first discovered that the expression of METTL4 increased in acute kidney injury tissues of humans and mice, and that inhibition of METTL4 could reduce the degree of acute kidney injury and renal fibrosis, while further overexpression of METTL4 aggravated the degree of acute kidney injury and renal fibrosis. This indicates that METTL4 is a damaging factor for acute kidney injury and renal fibrosis. Accordingly, the technology for inhibiting the expression of METTL4 and the technology for inhibiting the function of METTL4 can be applied to the preparation of drugs for treating renal fibrosis and renal injury diseases. The present invention provides a new idea and strategy for treating renal fibrosis and early renal injury diseases.

[0017] In an optional embodiment, the inhibition of METTL4 expression refers to inhibiting the transcription and translation of the METTL4 gene at the DNA level, RNA level or protein level; the inhibition of METTL4 function refers to inhibiting the function of METTL4 by gene therapy technology or small molecule drugs and other technologies.

[0018] Based on the contents disclosed in the present invention, those skilled in the art can easily think of inhibiting the expression of the METTL4 gene or inhibiting its function in a variety of ways, including but not limited to inhibition at the DNA level, RNA level and protein level, or using other similar technical means to inhibit the expression of the gene.

[0019] For example, adenovirus, adeno-associated (AAV) virus, lentivirus, retrovirus, and the regulatory elements constructed on the vector backbone can inhibit the transcription and translation of the METTL4 gene under artificial control, thereby achieving the silencing of the METTL4 gene; using gene editing technology to directly change the coding sequence of the METTL4 gene or regulate epigenetic modification to reduce or increase the gene expression (transcription level or translation level); using vesicles or nanomaterials as delivery technologies for carriers; and using small molecule compounds or drugs to inhibit the biological function of METTL4. Based on this, no matter how to inhibit the expression of the METTL4 gene or inhibit its biological function, it is easy for those skilled in the art to achieve, and it all belongs to the protection scope of the present invention.

[0020] In a second aspect of the present invention, a drug for treating acute kidney injury or renal fibrosis is provided, which comprises a gene delivery drug for inhibiting the expression and function of the METTL4 gene.

[0021] In an optional embodiment, the renal injury or renal fibrosis disease includes but is not limited to acute kidney injury (AKI), chronic kidney disease (CKD) and end-stage renal disease (ESRD), and other related renal diseases caused or resulted from infection, poisons, renal ischemia-reperfusion injury and other factors also fall within the scope of protection of the present invention.

[0022] Furthermore, the drug also includes pharmaceutically acceptable excipients. The excipients include at least one of a filler, a disintegrant, a binder, an excipient, a lubricant, a sweetener or a colorant. The dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections or dispersants.

[0023] In a third aspect of the present invention, there is provided use of METTL4 as a molecular marker in the preparation of a diagnostic and / or prognostic product for renal injury or renal fibrosis.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] The drug for treating kidney injury or kidney fibrosis provided by the present invention improves acute kidney injury and kidney fibrosis by inhibiting the expression of METTL4 or inhibiting the function of METTL4, thereby achieving the effect of treating kidney injury or kidney fibrosis. The present invention provides a new drug for treating renal insufficiency or kidney injury. Currently, there are no related drugs on the market that use this mechanism to treat renal insufficiency or kidney injury. Therefore, the present invention proposes for the first time to use the inhibition of METTL4 to prepare a new target for treating kidney injury and kidney fibrosis, and also provides a new idea for the treatment of kidney injury and chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 Immunohistochemistry was used to detect the expression of METTL4 protein in human renal tissue, and the results showed that the expression of METTL4 was increased in human AKI kidneys.

[0028] Figure 2 Immunohistochemistry was used to detect the expression of METTL4 protein in renal tissue in a mouse acute kidney injury model. The results showed that METTL4 expression was increased in the kidneys of mice with AKI.

[0029] Figure 3 Knockout of Mettl4 in renal proximal tubular cells inhibited renal injury and renal fibrosis in the mouse ischemia-reperfusion model. Figure a shows the results of histopathological analysis, Figure b shows the creatinine level, Figure c shows the BUN level, and Figure d shows protein expression analysis (Western Blot).

[0030] Figure 4 Overexpression of METTL4 in human renal tubular epithelial cells (HK2) increased the expression of the fibrosis marker Fibronectin.

[0031] Figure 5 Schematic diagram of the construction and breeding of Mettl4-CKO mice. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0033] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0035] The application of inhibiting METTL4 in the preparation of a drug for treating renal injury or renal fibrosis diseases will be described in detail below in conjunction with examples and experimental data.

[0036] In the following examples, strains of B6 / JGpt-Mettl4 em1cflox / Gpt mice and B6 / JGpt-Slc34a1-(CreERT2)-EGPF / Gpt mice were purchased from Chengdu Jicui Yaokang Biotechnology Co., Ltd.

[0037] Example 1: Increased expression of METTL4 in human acute kidney injury tissue

[0038] 1. Preparation before the experiment

[0039] 1. Sample collection and processing

[0040] The remaining samples after pathological examination of human kidney tissue were obtained from Zhongnan Hospital of Wuhan University (with ethical approval and informed consent from the patients).

[0041] The wax block tissues were divided into normal kidney tissue (control group) and AKI kidney tissue (experimental group).

[0042] Fix tissues: Immediately fix with 4% paraformaldehyde (dissolved in PBS) at 4°C overnight.

[0043] 2. Tissue Slice Preparation

[0044] Paraffin embedding;

[0045] Dehydrate (gradient ethanol: 70%, 80%, 90%, 95%, 100%).

[0046] Clear (xylene × 2 times, 10 minutes each time).

[0047] Paraffin wax infiltration (melt the paraffin and infiltrate step by step, in an oven at 60°C).

[0048] The blocks were embedded and sectioned (thickness 4-6 μm).

[0049] Cryosectioning (optional): Quickly freeze in liquid nitrogen and then slice, suitable for preserving antigen activity.

[0050] 2. Immunohistochemical staining process

[0051] 1. Dewaxing and hydration: Paraffin sections were dehydrated in xylene (×2), gradient ethanol (100%→70%), and finally washed with PBS three times.

[0052] 2. Blocking: Block with 5% BSA (or normal serum) for 30 minutes to inhibit nonspecific binding.

[0053] 3. Primary antibody incubation: Add METTL4-specific primary antibody and incubate at 4°C overnight or 37°C for 2 hours.

[0054] 4. Secondary antibody incubation: Add HRP-labeled secondary antibody (such as anti-rabbit IgG-HRP, dilution ratio 1:200) for 1 hour at room temperature.

[0055] 5. Color reaction: DAB colorimetric solution (containing 0.03% H 2 O 2 ) Incubate for 5-10 minutes in the dark and observe the signal intensity under a microscope. Optional: TSA fluorescence color development (need to be protected from light).

[0056] 6. Counterstaining and sealing: stain the cell nucleus with hematoxylin (1-2 minutes), differentiate with hydrochloric acid and ethanol, rinse with dilute buffer, and seal with neutral gum.

[0057] 3. Results Analysis

[0058] 1. Image acquisition: Use an optical microscope (×200 or ×400) to capture representative fields of view.

[0059] 2. Semi-quantitative analysis: Calculate the integrated optical density (IOD) or the proportion of positive cells using ImageJ software.

[0060] The control group and the experimental group need to match the tissue area (such as the same cortex / medulla ratio).

[0061] Statistical verification: Student's t-test or ANOVA was used to compare the significance of differences (p < 0.05 was considered significant).

[0062] The results are as follows Figure 1 Shown: METTL4 expression is increased in human AKI kidneys.

[0063] Example 2: Increased expression of METTL4 in acute kidney injury tissues of mice

[0064] 1. Experimental animal modeling and sample processing

[0065] 1. Construction of AKI model

[0066] Ischemia-reperfusion injury (IRI): Bilateral renal arteries were clamped for 30 minutes and the blood flow was restored and then observed for 24-48 hours.

[0067] Cisplatin induction: single intraperitoneal injection of cisplatin (dose adjusted according to body weight, usually ≥15 mg / kg).

[0068] Other models: gentamicin-induced, doxorubicin-induced, etc. (selected according to the research purpose).

[0069] 2. Animal grouping

[0070] There were at least 5-6 animals in each control group (Sham / normal mice) and AKI model group to ensure statistical power.

[0071] 3. Sample collection

[0072] Anesthesia and sampling:

[0073] The mice were anesthetized with isoflurane, and the bilateral kidneys were removed after laparotomy and immediately placed on an ice box for cooling.

[0074] Part of the tissue is used for paraffin sectioning, and the other part can be frozen (-80℃) for subsequent molecular testing.

[0075] 4. Tissue Fixation

[0076] The kidney tissue was fixed with 4% paraformaldehyde (prepared in PBS) for 24 hours (to avoid destruction of antigen epitopes due to prolonged fixation).

[0077] 2. Tissue Section Preparation

[0078] 1. Paraffin embedding

[0079] Dehydration: gradient ethanol (70% → 95% → 100%, 15 min each time).

[0080] Transparency: xylene × 2 times (10 minutes / time).

[0081] Paraffin wax penetration: Soak step by step in a 60℃ oven (soak after the paraffin wax melts).

[0082] Embedding: The tissue block is placed in a mold and sliced ​​(thickness 4-6μm) after the paraffin solidifies.

[0083] 2. Cryosectioning

[0084] Liquid nitrogen quick-frozen tissue, OCT embedded and then sliced ​​(thickness 10-15μm), suitable for detecting membrane proteins or situations where rapid experiments are required.

[0085] 3. Immunohistochemical staining process

[0086] 1. Dewaxing and Hydration

[0087] Paraffin sections were washed three times in sequence with xylene (×2), gradient ethanol (100%→70%), and finally with PBS.

[0088] 2. Antigen Retrieval

[0089] Thermal repair: Microwave heating in citrate buffer (pH 6.0) (medium heat for 2 minutes × 3 times, cooling to room temperature).

[0090] 3. Closure

[0091] Use 5% normal mouse serum (same as the primary antibody, such as rabbit serum for rabbit antibody) to block for 30 minutes to reduce nonspecific binding.

[0092] 4. Primary Antibody Incubation

[0093] METTL4 primary antibody (species specificity needs to be verified, such as rabbit anti-human METTL4 may require cross-reaction testing):

[0094] Refer to the instructions for dilution ratio (usually 1:100-1:500) and incubate at 4°C overnight or at 37°C for 2 hours.

[0095] 5. Secondary Antibody Incubation

[0096] HRP-labeled secondary antibody (e.g. anti-rabbit IgG-HRP, 1:200 dilution) for 1 hour at room temperature.

[0097] 6. Color rendering

[0098] DAB color development: prepared with 0.03% H 2 O 2 DAB solution and incubate for 5-10 minutes in the dark (observe gray-brown signal under a microscope).

[0099] 7. Counterstaining and Sealing

[0100] Hematoxylin staining: counterstain the cell nucleus for 1-2 minutes and differentiate with hydrochloric acid-ethanol.

[0101] Dehydration and sealing: gradient ethanol dehydration, neutral gum sealing.

[0102] IV. Results Analysis

[0103] Image acquisition: Representative fields of view of the cortex and medulla were photographed under a microscope (×200 or ×400). Semi-quantitative analysis was performed using Student's t-test or ANOVA to compare differences between groups (p<0.05 was considered significant).

[0104] The results are as follows Figure 2 As shown, the expression of METTL4 was increased in the kidneys of mice with AKI.

[0105] Example 3: Knockout of Mettl4 inhibits renal injury and renal fibrosis in a mouse ischemia-reperfusion model

[0106] 1. Construction of Mettl4 knockout mice in renal tubular cells

[0107] Mettl4 knockout mice were purchased from Jicui Pharmaceuticals, with the catalog number: Mettl4-flox|StrainNO.T009584Conditional knockout (cKO). Mettl4 knockout mice used CRISPR / Cas9 gene editing technology to knock out the exon 3 to exon 5 region in the Mettl4-203 (ENSMUST00000234990.1) transcript.

[0108] There are three transcripts of the Mettl4 gene in mice. Mettl4-203 (ENSMUST00000234990.1) is one of the specific transcripts. It consists of multiple exons, which are gene segments that encode protein sequences. In this transcript, exon 3 to exon 5 are considered to be the key knockout region. This region contains a coding sequence of 503 base pairs (bp). The coding sequence is the part of DNA that determines the structure and function of the protein. Therefore, knocking out the coding sequence in this region will result in the corresponding protein not being properly synthesized or malfunctioning. Cas9, sgRNA (5'-TACACCGTTGTAATTCGAGC-3', 5'-ACCGTTGTAATTCGAGCAGG-3') and donor vector were co-microinjected into fertilized eggs of C57BL / 6JGpt mice. These fertilized eggs were transplanted into pseudopregnant female mice, and positive F0 generation mice were successfully obtained. The gene editing effect was confirmed by PCR and gene sequencing. Next, the positive F0 mice were mated with C57BL / 6JGpt mice to successfully breed a stable F1 mouse model. F1 mice were bred with each other to obtain Loxp homozygotes (F2). F2 mice were bred with B6 / JGpt-Slc34a1-(CreERT2)-EGPF / Gpt tool mice to obtain Mettl4-CKO and Mettl4-WT (abbreviated as WT) mice. Figure 5 Schematic diagram of the construction and breeding of transgenic mice.

[0109] The following sequence is the target sequence for knockout, where the underlined ones are loxP sites and the red ones are the knocked-out exons.

[0110]

[0111]

[0112]

[0113]

[0114] 2. Ischemia-reperfusion model: 8-week-old male specific knockout mice (Mettl4-CKO) and negative control (WT) mice were randomly divided into 2 groups: sham operation group (Sham) and 35-minute ischemia-reperfusion group (UIRI) for 4 weeks. Mice were intraperitoneally injected with 50–60 mg / kg sodium pentobarbital. The pentobarbital solution was diluted with normal saline to a concentration of 5 mg / ml for injection. The mice were placed on a constant temperature heating blanket with a temperature control range of 36.5–37°C. The mice were placed in a prone position, the skin of the left posterior back was prepared, and the active iodine was disinfected. The muscle layer of the left posterior back was separated layer by layer with scissors, and the left kidney was freed and exposed with forceps and cotton swabs. The surrounding tissue was removed with sharp forceps to expose the renal aorta, and then the renal aorta was clamped with a vascular clamp to induce renal ischemia. The color of the kidney changed from red to dark purple after a few seconds. After 35 minutes, the vascular clamp was released, and the kidney was placed in the mouse body, and the fascia and skin were sutured layer by layer. In the sham operation group, only the ureter was separated and then sutured. After surgery, mice were given 0.2% carprofen 5 mg / kg subcutaneously once a day for 3 consecutive days. After modeling, samples were collected to prepare serum samples, tissue RNA samples, protein samples and pathological sections, and the knockout efficiency, damage and fibrosis degree of METTL4 in Mettl4 ptCKO and WT mice were detected.

[0115] 3. Preparation of Protein Samples

[0116] ① Pre-cool the mortar with liquid nitrogen, take the frozen tissue from the -80℃ refrigerator out to the mortar, add liquid nitrogen while grinding, and keep the temperature low. Put the ground tissue powder into a 1.5ml EP tube. Add 40μl protein lysis buffer (RIPA, Biotech, P0013) and phosphatase inhibitor PMSF (Biyotech, P1048) (RIPA:PMSF=100:1, prepared and used) for every 1mg of tissue. Take out the 6-well plate from the incubator, remove the culture medium, and wash it with PBS three times. Add 200μl RIPA lysis buffer (RIPA:PMSF=100:1, prepared and used) to each well. Then scrape the cells with a cell scraper and collect them in a 1.5ml EP tube.

[0117] ②Use ultrasonic cell disruptor on ice for 10-15 times and place on ice for 30 minutes to lyse the cells.

[0118] ③ Centrifuge at 4℃, 13000rpm for 10min and collect the supernatant.

[0119] ④ Add 5x loading buffer (Biyuntian, P0015) according to the ratio of protein solution: loading buffer = 4:1.

[0120] ⑤ Denature the protein in a metal bath at 95℃ for 5 minutes. Store at 80℃.

[0121] 4. Western blot

[0122] ① Preparation of PAGE gel:

[0123] According to the instructions of the SDS-PAGE 10% kit, separation gel and stacking gel were prepared (SDS-PAGE kit, Yazyme, 03571300). The product content includes separation gel buffer (2×) 250ml, separation gel solution (2×) 250ml, color stacking gel buffer (2×), 80ml stacking gel solution (2×) 80ml, and improved ammonium persulfate solution (APS) 8ml. The prepared 1.5mm gel was used in the experiment. Before making the gel, the glass plate should be cleaned and air-dried naturally.

[0124] When preparing the separation gel (lower gel), first take 4.0 ml of separation gel buffer and separation gel solution, add 80 μl of APS, and mix them thoroughly; inject the solution into the installed 1.5 mm gel glass plate, and then add anhydrous ethanol to cover the separation gel; after the separation gel solidifies, discard the upper layer of anhydrous ethanol and use a single-channel pipette to absorb the excess anhydrous ethanol.

[0125] When preparing the concentrated gel (upper gel), first take 1.0 ml of concentrated gel buffer and concentrated gel solution, then add 20 μl of APS and mix them thoroughly. Inject the solution into the glass plate and insert a 1.5 mm comb with 10 or 15 holes. Avoid bubbles when inserting the comb. After the concentrated gel solidifies, you can use it immediately for sample electrophoresis or store the gel in a 4°C refrigerator.

[0126] ② Electrophoresis: Take out the prepared gel and install it on the electrophoresis stand, paying attention to the positive and negative poles. Place the electrophoresis stand in the electrophoresis tank and pour in the electrophoresis solution, the height of which should exceed the aluminum wire. Pull out the comb vertically upward, pre-run at 100V for 10 minutes, check for leakage, and then add the maker and protein sample. Use 80V voltage for the concentrated gel and 120V for the separated gel. Stop the electrophoresis when the bromophenol blue is 1cm above the bottom of the glass plate.

[0127] ③ Transfer: Soak the transfer clip, filter paper, and sponge in the transfer solution half an hour before transfer. After the electrophoresis is completed, remove the glass plate, gently push the glass plate apart, cut off the concentrated gel and bromophenol blue, and cut the separation gel of the corresponding size according to the number of samples. Gently push the remaining gel onto the filter paper. Cut a PVDF membrane slightly larger than the gel, activate it with methanol, and gently cover it on the gel, expel the bubbles between the filter paper and the gel, and assemble it in the transfer clip in the order of negative electrode, sponge, 3 layers of filter paper, gel, membrane, 3 layers of filter paper, sponge, and positive electrode. Install the clip in the transfer tank, which is filled with pre-cooled 1× electrotransfer solution. Pay attention to the positive and negative electrodes. Transfer the membrane at 100V. The target protein <100KD is transferred for 60min, and the target protein >100kD is determined according to the molecular weight 1KD / 1min to determine the transfer time.

[0128] ④ Blocking: After transfer, take out the PVDF membrane, mark the front and back and the date, and block it in 5% skim milk for experiments at room temperature for 2 hours.

[0129] ⑤ Primary antibody incubation: After blocking, remove the membrane from the skim milk, wash the membrane three times for 5 minutes with 1× TBST, and then prepare antibodies with 1× TBST according to the dilution ratio of β-actin (1:1000, ZENBIO, #340042) and Hoxa5 (1:1000, AFFBIOTECH, #DF4123). Add the primary antibody to the antibody incubation box first and then put the membrane in the box, and incubate it on a shaker in a 4°C refrigerator overnight.

[0130] ⑥ Secondary antibody incubation: On the second day, remove the membrane from the antibody incubation box and wash the membrane three times with 1× TBST, 5 minutes each time. Prepare HRP-labeled secondary antibody (ZENBIO, anti-rabbit 1:5000, #511203; anti-mouse 1:10000, #701051) in 1× TBST according to the dilution ratio and incubate at room temperature for 2 hours.

[0131] ⑦ Color exposure: After the secondary antibody incubation, remove the membrane from the antibody incubation box and wash the membrane three times with 1× TBST, 5 minutes each time. Prepare the electrochemiluminescence (ECL) reagent (Millipore) at a ratio of 1:1, add the exposure solution to the membrane surface, and use the Image Quant LAS 4000mini system for automatic exposure imaging.

[0132] The results are as follows Figure 3 As shown,

[0133] Figure 3 a Histopathological analysis: Shows kidney tissue sections of WT and Mettl4 CKO mice after Sham (sham surgery) and UIRI (renal ischemia-reperfusion injury). WT Sham group: normal kidney structure, no obvious pathological damage. WT UIRI group: typical ischemia-reperfusion injury features (such as tubular epithelial cell necrosis, interstitial edema, inflammatory infiltration). Mettl4CKO UIRI group: the degree of injury is significantly aggravated, which may be manifested as more extensive tubular damage, fibrosis or increased inflammatory markers. This indicates that Mettl4 gene knockout may aggravate UIRI-induced renal tissue damage, suggesting that this gene has a potential protective effect.

[0134] Figure 3Creatinine level in Figure b: Under UIRI conditions, the creatinine level in the Mettl4 CKO group was significantly higher than that in the WT group (P<0.05*, P<0.01), indicating that Mettl4 deficiency leads to worsening of renal function, manifested as more severe tubular damage and decreased filtration function.

[0135] Figure 3 BUN level in Figure c: Under UIRI conditions, the BUN level in the Mettl4 CKO group was significantly higher than that in the WT group (P<0.05*, P<0.01). Consistent with the creatinine results, this indicates that Mettl4 gene knockout exacerbated azotemia (a sign of renal failure) after UIRI.

[0136] Figure 3 d Detected proteins: Fibronectin, Collagen I / IV (fibrosis markers), α-SMA (myofibroblast activation marker), β-actin (internal reference). Under UIRI conditions:

[0137] WT group: The expressions of Fibronectin, Collagen I / IV and α-SMA were slightly increased (possibly related to damage repair).

[0138] Mettl4 CKO group: The expression of the above proteins was significantly upregulated (marked with asterisks), indicating that the fibrosis process was accelerated.

[0139] Under sham conditions: the protein expressions of the two groups were close to the baseline.

[0140] Figure 3 d shows that Mettl4 deficiency leads to abnormal increase in the expression of renal fibrosis markers after UIRI, which may be related to chronic injury or repair disorder.

[0141] In summary, knocking out Mettl4 in renal tubular cells inhibited renal injury and renal fibrosis in the mouse ischemia-reperfusion model.

[0142] 5. Fibrosis marker detection indicators:

[0143] Masson staining was used to detect the morphological changes and collagen precipitation of mouse kidney tissue cells;

[0144] Western blot was used to detect the expression of fibrosis marker proteins, such as Fibronectin, a-SMA, Collagen I and Collagen IV.

[0145] The results are as follows Figure 4 As shown, it was demonstrated that overexpression of METTL4 in human renal tubular epithelial cells (HK2) increased the expression of fibrosis marker Fibronectin.

[0146] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0147] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0148] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of METTL4 inhibitors in the preparation of drugs for treating renal injury or renal fibrosis.

2. The use according to claim 1, characterized in that: The acute kidney injury or renal fibrosis includes acute kidney injury, chronic kidney disease and end-stage renal disease.

3. The use according to claim 1, characterized in that: The METTL4 inhibitors include: Substances that inhibit the expression of METTL4: inhibit the transcription and translation of the METTL4 gene at the DNA level, RNA level or protein level; and substances that inhibit the function of METTL4.

4. The use according to claim 3, characterized in that: The METTL4 inhibitor comprises at least one of the following ingredients: METTL4 inhibitors, antagonists, downregulators, blockers, and blocking agents; Knockdown reagent for METTL4.

5. The use according to claim 1, characterized in that: The drug further comprises pharmaceutically acceptable excipients. The excipients comprise at least one of a filler, a disintegrant, a binder, an excipient, a lubricant, a sweetener or a colorant. The dosage form of the drug comprises at least one of a granule, a tablet, a pill, a capsule, an injection or a dispersant.

6. Use of METTL4 as a target in screening drugs for preventing, alleviating and / or treating renal injury or renal fibrosis, characterized in that: The screening method includes screening substances that can inhibit the expression of METTL4.

7. A drug for treating renal injury or renal fibrosis, characterized in that: The drug includes at least one of a METTL4 inhibitor, an antagonist, a downregulator, a blocker, a blocking agent and a METTL4 knockout agent.

8. Use of METTL4 as a molecular marker in the preparation of diagnostic and / or prognostic products for renal injury or renal fibrosis.

9. Use of METTL4 detection reagents in the preparation of diagnostic and / or prognostic products for renal injury or renal fibrosis.

10. The use according to claim 9, characterized in that: The METTL4 detection reagent includes a METTL4 detection primer or an immunohistochemical detection reagent.

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