Application of RNF130 activator in preparation of medicine for preventing and / or treating acute kidney injury

Abstract

The invention discloses application of an RNF130 activator in preparation of a medicine for preventing and / or treating acute kidney injury, and belongs to the technical field of biological medicine. The invention provides an application of an RNF130 activator in preparation of a medicine for preventing or treating renal ischemia-reperfusion injury, the RNF130 activator can improve the renal function of a mouse suffering from renal ischemia-reperfusion injury, improve the levels of serum creatinine, urea nitrogen and cystatin C, reduce the levels of kidney injury markers KIM-1 and LCN2, reduce renal tubule injury, improve the renal function of the mouse suffering from renal ischemia-reperfusion injury, improve the renal function of the mouse suffering from renal ischemia-reperfusion injury and improve the renal function of the mouse suffering from renal ischemia-reperfusion injury and the renal function of the mouse suffering from renal ischemia-reperfusion injury. Therefore, the compound plays a role in preventing and treating the kidney ischemia reperfusion injury, and a new target is provided for preparing the medicine for preventing and treating the acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the use of an RNF130 activator in preparing a medicine for preventing and / or treating acute kidney injury, and belongs to the technical field of biomedicine. Background Art

[0002] Acute kidney injury (AKI) is a common clinical symptom. It refers to rapid kidney damage and functional impairment caused by various reasons in a short period of time. The incidence of AKI is very high and is closely related to the occurrence of chronic kidney disease and the poor prognosis of critically ill patients. The severe stage is acute renal failure (ARF), which requires renal replacement therapy (RRT). Timely intervention at the onset of AKI can minimize kidney damage and promote the recovery of renal function. Therefore, it is of great significance to develop effective therapeutic and preventive drugs related to AKI diseases.

[0003] RNF130 (Ring Finger Protein 130) is the mammalian homolog of the Drosophila protease-associated domain E3 ligase and a protein with E3 ubiquitin ligase activity. It plays a role in a variety of biological processes, including regulating apoptosis, cell cycle, and signal transduction. RNF130 can control the selective degradation of proteins by the proteasome, and can also catalyze the transfer of ubiquitin to target proteins, rapidly modify and regulate target proteins, thereby conferring specificity to target proteins, modifying proteins and regulating their cellular localization.

[0004] Currently, RNF130 can be used for the treatment of pulmonary fibrosis, regulation of blood lipid levels and drug development for autoimmune diseases, but there has been no relevant research and reports on the role and mechanism of RNF130 in renal ischemia-reperfusion injury. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an application of an RNF130 activator in the preparation of a drug for preventing or treating renal ischemia-reperfusion injury; the present invention found that the expression of RNF130 in the renal tissue of mice with renal ischemia-reperfusion injury was reduced, which may be related to the development of the disease and may become an effective target for treating renal ischemia-reperfusion injury.

[0006] The first object of the present invention is to provide the use of RNF130 as a therapeutic target in the preparation of a drug for preventing or treating acute kidney injury.

[0007] In one embodiment, the drug increases the expression of the RNF130 gene or protein.

[0008] In one embodiment, the medicament comprises an activator of RNF130.

[0009] In one embodiment, the activator includes an agent that enhances RNF130 gene expression and / or an agent that enhances RNF130 protein expression.

[0010] In one embodiment, the drug improves renal ischemia-reperfusion injury-induced renal injury and / or renal tissue pathological changes; and reduces the expression levels of renal injury markers KIM-1 and LCN2.

[0011] The second object of the present invention is to provide the use of an RNF130 activator in the preparation of a medicament for preventing or treating acute kidney injury, wherein the RNF130 activator comprises an agent for enhancing RNF130 gene expression and / or an agent for enhancing RNF130 protein expression.

[0012] In one embodiment, the RNF130 activator includes an agent that enhances RNF130 gene expression and / or an agent that enhances RNF130 protein expression.

[0013] The third object of the present invention is to provide the use of RNF130 gene or protein in screening drugs for preventing or treating acute kidney injury.

[0014] In one embodiment, the application includes using the expression of the RNF130 gene or protein to verify whether a drug can be used to prevent or treat renal ischemia-reperfusion injury.

[0015] In one embodiment, the drug for preventing or treating renal ischemia-reperfusion injury can increase the expression of RNF130 gene or protein.

[0016] The fourth object of the present invention is to provide a drug capable of preventing or treating acute kidney injury, wherein the drug comprises an activator of the RNF130 gene and / or its expression product.

[0017] In one embodiment, the medicament comprises a pharmaceutically acceptable excipient.

[0018] In one embodiment, the drug is in a pharmaceutically acceptable dosage form.

[0019] In one embodiment, the drug comprises an activator of the RNF130 gene and / or its expression product.

[0020] In one embodiment, the drug is a vector for gene therapy, including vectors for gene editing, gene expression regulation or gene delivery; or the drug is a vector for cell therapy, including stem cells, immune cells or repair cells.

[0021] In one embodiment, the medicament prevents or treats acute kidney injury by increasing the expression of RNF130 gene or protein.

[0022] In one embodiment, the drug contains the above-mentioned RNF130 activator and a pharmaceutically acceptable excipient.

[0023] In one embodiment, the auxiliary material includes any one of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer, or a combination of at least two thereof.

[0024] In one embodiment, the carrier is one or more of a virus, a liposome, and a nanoparticle.

[0025] In one embodiment, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0026] In one embodiment, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0027] In one embodiment, the administration route of the drug includes oral administration, sublingual administration, rectal administration, skin mucosal administration, inhalation administration or injection administration.

[0028] Beneficial Effects of the Invention

[0029] RNF130 activators can improve the renal function of mice with renal ischemia-reperfusion injury, improve the levels of blood creatinine, urea nitrogen, and cystatin C, reduce the levels of renal injury markers KIM-1 and LCN2, and reduce renal tubular damage, thereby preventing and treating renal ischemia-reperfusion injury and providing new targets for the preparation of drugs for the prevention and treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a mouse serum index;

[0031] Figure 2 is the expression level of RNF130 in mouse kidney;

[0032] Figure 3 H&E and PAS staining and tubular injury scoring of mouse kidneys;

[0033] Figure 4 is the mRNA expression level of Kim-1 and Lcn2, the markers of renal injury in mice. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0035] The raw materials used in the examples are:

[0036] AAV-GP-12N vector was purchased from Jima Gene;

[0037] Male C57BL / 6 mice were purchased from Sibeifu Biotechnology Co., Ltd. (Beijing).

[0038] Test method:

[0039] 1. Treatment methods for different groups of renal ischemia-reperfusion:

[0040] Sham operation group (AAV-NULL group): Six C57BL / 6J mice were randomly selected and fed adaptively for one week (1 to 7 days). The titer of AAV-NULL was 2×10 9 GC empty plasmid virus; feed for another six weeks, use 2% isoflurane gas anesthesia, wait for the mouse's breathing to stabilize and enter the anesthesia state, make a small incision along the costal angle of its back, squeeze out the kidney along the direction of the opening, and return the kidney after 45 minutes without ischemia-reperfusion treatment; after the above sham operation is completed, continue feeding for 24 hours and then kill.

[0041] Renal ischemia-reperfusion group (I / R+AAV-NULL group): Six C57BL / 6J mice were randomly selected and fed adaptively for 1 week (1-7 days). When the mice were six weeks old, the titer of the drug was 2×10 9 GC empty plasmid virus; feed for another six weeks, and use 2% isoflurane gas anesthesia after the injection. After the mouse breathes steadily and enters the anesthetized state, make a small incision along the costal angle of its back, squeeze out the kidney in the body along the opening direction, carefully separate and identify the renal artery and vein, and clamp it with an artery clamp. Within 1 minute after clamping, the color of the mouse kidney can be seen to darken and turn black, which is a successful ischemia. Each mouse kidney is ischemic for 45 minutes, and then the artery clamp is released. When the blood is reperfused, the color of the kidney changes from black and red to ruddy, which is a successful reperfusion. Take both kidneys of each mouse and clamp them at the same time. After the above reperfusion modeling is completed, continue feeding for 24 hours, and then kill.

[0042] Control group (AAV-RNF130 group): Six C57BL / 6J mice were randomly selected and fed adaptively for one week (1 to 7 days). The titer of the AAV-RNF130 was 2×10 9GC adeno-associated virus RNF130-AAV9; feed for another six weeks, and use 2% isoflurane gas anesthesia after the gavage. After the mouse's breathing is stable and it enters the anesthesia state, a small incision is made along the costal angle of its back, and the kidney is squeezed out of the body along the direction of the opening. After 45 minutes, the kidney is returned without ischemia-reperfusion treatment; after the above sham operation is completed, continue feeding for 24 hours and then kill.

[0043] RNF130 treatment group (I / R+AAV-RNF130 group): Six C57BL / 6J mice were randomly selected and fed adaptively for one week (1-7 days). When the mice were six weeks old, the titer of the tail vein injection was 2×10 9 GC adeno-associated virus RNF130-AAV9; feed for another six weeks, use 2% isoflurane gas anesthesia, wait for the mouse to breathe steadily and enter the anesthesia state, make a small incision along the costal angle of the back, squeeze the kidney out of the body along the opening direction, carefully separate and identify the renal artery and vein, take the artery clamp to clamp it, within 1 minute after clamping, the color of the mouse kidney can be seen to darken and blacken, which is a successful ischemia. Each mouse kidney is ischemic for 45 minutes, and then the artery clamp is released. When the blood is reperfused, the color of the kidney changes from black and red to ruddy, which is a successful reperfusion. Take both kidneys of each mouse and clamp them at the same time. After the above reperfusion modeling is completed, continue feeding for 24 hours, and then kill.

[0044] 2. Mouse serum sample collection:

[0045] (1) Anesthetized mice: Mice were deeply anesthetized with sodium pentobarbital;

[0046] (2) Blood collection: Orbital venous blood sampling was used to collect the blood of mice into centrifuge tubes;

[0047] (3) Separation of serum: The blood sample was allowed to stand at room temperature for 2 hours, and then placed in a 4°C refrigerator overnight. Finally, the sample was centrifuged to separate the serum, and the serum was transferred to a new centrifuge tube for subsequent analysis.

[0048] 3. HE staining method:

[0049] Mouse kidney HE staining: Mouse kidney HE staining is a commonly used tissue staining method used to observe and analyze the morphological structure of mouse kidney tissue. The specific operation method is as follows:

[0050] (1) Sample fixation: Mouse kidneys were removed and fixed with 10% buffered formalin solution to maintain the integrity of tissue structure;

[0051] (2) Tissue processing: Dehydrate and embed the fixed kidney tissue. First, dehydrate the kidney tissue by soaking it in alcohol with increasing concentrations to remove water. Then, use xylene to make the dehydrated kidney tissue transparent to make the tissue compatible with paraffin.

[0052] (3) Slice preparation: The cleared kidney tissue was embedded in paraffin to prepare a paraffin block. Subsequently, the paraffin block was cut into 10 μm thick tissue sections using a microtome and the sections were transferred to glass slides;

[0053] (4) Staining: Perform HE staining on the sections. First, place the sections in an alkaline-acid hydrolysis staining solution containing heme dye and eosin dye. Under alkaline conditions, eosin dye binds to the cell nucleus DNA and stains it red; under acidic conditions, heme dye stains the cytoplasm and intercellular matrix into light blue. After a certain period of staining, the sections are transferred to a dehydrating solution to remove excess dye.

[0054] (5) Sealing and observation: Seal the sections with transparent plastic foam and observe under a microscope. In the mouse kidney tissue sections stained with HE, the nuclei appear red, and the cytoplasm and intercellular matrix appear light blue. The tissue structure, cell morphology, and tissue type can be observed and analyzed under a microscope.

[0055] Example 1: Application of RNF130 agonists in improving acute kidney injury

[0056] 1. Construction of RNF130 agonist

[0057] According to the mRNA of the RNF130 gene (NM_021540.4), cDNA (amino acid sequence as shown in SEQ ID NO.1) was obtained by reverse transcription, and the target gene fragment was obtained by PCR amplification; the target gene fragment was inserted between EcoR I and BamH I of the plasmid AAV-GP-12N to construct the overexpression plasmid AAV-GP-12N-RNF130; the empty plasmid AAV-GP-12N without the RNF130 gene inserted was used as a control.

[0058] 2. Animal model testing

[0059] (1) Virus construction

[0060] Take the overexpression plasmid AAV-GP-12N-RNF130 prepared in step 1, entrust Jima Gene to construct the adeno-associated virus RNF130-AAV9 for animal experiments; use the empty plasmid as a control to obtain the plasmid AAV-NULL.

[0061] (2) Animal experiments

[0062] Male C57BL / 6 mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. All mice were housed in specific pathogen-free microisolator cages with free access to food and water, and the animals were maintained in a room with controlled temperature and humidity and a 12-h light / dark cycle.

[0063] The therapeutic effect of RNF130 activator on renal ischemia-reperfusion mice was detected, and the groups were shown in Table 1.

[0064] Table 1 Mouse experimental groups

[0065]

[0066] 3. Result detection

[0067] (1) Serum indicators

[0068] Blood was collected from the eyeballs of mice and centrifuged to prepare serum. Changes in serum creatinine, urea nitrogen, and cystatin C were detected. Figure 1 As shown, the results showed that under the action of ischemia-reperfusion, the levels of blood creatinine, urea nitrogen, and cystatin C in the I / R+AAV-NULL group of mice were significantly higher than those in the AAV-NULL group, but the kidney injury indicators in the I / R+RNF130 group were significantly decreased. It can be seen that the use of RNF130 can significantly improve the three renal function indicators.

[0069] (2) Kidney damage results

[0070] Mouse kidneys were prepared into paraffin sections for immunohistochemical staining. Figure 2 As shown, the results showed that the expression level of RNF130 in kidney tissue of I / R+AAV-NULL group mice was significantly decreased compared with that of AAV-NULL group mice.

[0071] (3) Kidney staining results

[0072] The results of H&E staining and PAS staining of kidney paraffin sections are as follows Figure 3 As shown, the results showed that compared with the AAV-NULL group mice, the I / R+AAV-NULL group mice had more vacuoles in the renal tubules, dilated tubular lumina, and brush border shedding, indicating that the degree of renal pathological damage was aggravated; while the I / R+AAV-RNF130 group mice had reduced shedding of renal tubular epithelial cells and brush border shedding, indicating that RNF130 alleviated the ischemia-reperfusion-induced renal pathological damage.

[0073] (4) Expression levels of renal injury markers

[0074] The expression levels of KIM-1 and LCN2 in kidney tissue are shown in Figure 2. Figure 4As shown, the results showed that the expression levels of renal injury biomarkers KIM-1 and LCN2 genes in the I / R+AAV-NULL group of mice were significantly higher than those in the AAV-NULL group; while the expression levels of KIM-1 and LCN2 genes in the kidneys of the I / R+AAV-RNF130 group were significantly downregulated compared with the I / R+AAV-NULL group.

[0075] The above results show that RNF130 activator can alleviate renal damage in mice with renal ischemia-reperfusion injury, improve the levels of blood creatinine, urea nitrogen, and cystatin C, reduce the expression levels of renal injury markers KIM-1 and LCN2, and reduce renal tubular damage, thereby playing a preventive and therapeutic role in renal ischemia-reperfusion injury.

[0076] RNF130 cDNA gene sequence (SEQ ID NO.1):

[0077]

[0078] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of RNF130 as a therapeutic target in the preparation of drugs for preventing or treating acute kidney injury.

2. The use according to claim 1, characterized in that: The drug increases the expression of the RNF130 gene or protein.

3. The use according to claim 1, characterized in that: The drug contains an activator of RNF130.

4. The use according to claim 3, characterized in that: The activator includes an agent that enhances the expression of the RNF130 gene and / or an agent that enhances the expression of the RNF130 protein.

5. The use according to claim 1, characterized in that: The drug improves renal injury and / or renal tissue pathological changes induced by renal ischemia-reperfusion injury; and reduces the expression levels of renal injury markers KIM-1 and LCN2.

6. Use of an RNF130 activator in the preparation of a drug for preventing or treating acute kidney injury, characterized in that: The RNF130 activator includes an agent that enhances the expression of the RNF130 gene and / or an agent that enhances the expression of the RNF130 protein.

7. Application of RNF130 gene or protein in screening drugs for preventing or treating acute kidney injury.

8. A drug capable of preventing or treating acute kidney injury, characterized in that: The drug comprises an activator of the RNF130 gene and / or its expression product.

9. The drug according to claim 8, characterized in that The drug includes pharmaceutically acceptable excipients.

10. The drug according to claim 8, characterized in that The drug is in a pharmaceutically acceptable dosage form.

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