Use of rnf130 activators in the preparation of medicaments for the prevention and / or treatment of acute kidney injury

Abstract

The application discloses an 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 medicines.The application provides an application of the RNF130 activator in preparation of a medicine for preventing or treating kidney ischemia-reperfusion injury, and the RNF130 activator can improve kidney function of a mouse with kidney ischemia-reperfusion injury, improve levels of blood creatinine, urea nitrogen and cystatin C, reduce levels of kidney injury markers KIM-1 and LCN2, and reduce tubular injury, thereby playing a preventive and therapeutic role on kidney ischemia-reperfusion injury, and providing a new target for preparation of a medicine for preventing and treating acute kidney injury.

Description

TECHNICAL FIELD

[0001] The application relates to 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. BACKGROUND

[0002] Acute kidney injury (AKI) is a common clinical symptom, refers to rapid appearance of kidney injury and hypofunction in a short time caused by various reasons, the occurrence rate of AKI is very high, and the occurrence of AKI is closely related to chronic kidney disease and adverse prognosis of severe patients, and the severe stage is acute renal failure (ARF), and renal replacement therapy (RRT) needs to be accepted. Timely intervention in the initial stage of AKI can maximize the alleviation of kidney injury and promote the recovery of kidney function. Therefore, it is of great significance to develop effective AKI disease-related treatment and prevention drugs

[0003] RNF130 (Ring Finger Protein 130) is a mammalian homolog of Drosophila protease-related domain E3 ligase, and is also a kind of protein with E3 ubiquitin ligase activity; it plays a role in various biological processes, including regulation of apoptosis, cell cycle and signal transduction; RNF130 can control the selective degradation of proteasome to protein, and can also catalyze the transfer of ubiquitin to target protein, quickly modify and regulate target protein, so as to endow target protein with specificity, modify protein and regulate its cell localization.

[0004] At present, RNF130 can be used for the treatment of pulmonary fibrosis, the regulation of blood lipid level and the development of drugs for autoimmune diseases, but the role of RNF130 in renal ischemia-reperfusion injury and its mechanism have not been reported. SUMMARY

[0005] In order to solve the above technical problems, the application provides application of an RNF130 activator in preparation of a medicine for preventing or treating renal ischemia-reperfusion injury; it is found that RNF130 expression in the kidney tissue of a mouse with renal ischemia-reperfusion injury is 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 application is to provide application of RNF130 as a therapeutic target in preparation of a medicine for preventing or treating acute kidney injury.

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

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

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

[0010] In one embodiment, the medicament improves the renal injury and / or renal histopathological changes induced by renal ischemia-reperfusion injury; and reduces the expression level of KIM-1, LCN2, which are markers of renal injury.

[0011] A second object of the present application is to provide the use of an activator of RNF130 in the preparation of a medicament for preventing or treating acute kidney injury, wherein the activator of RNF130 includes an agent that enhances the expression of RNF130 gene and / or an agent that enhances the expression of RNF130 protein.

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

[0013] A third object of the present application is to provide the use of RNF130 gene or protein in the screening of a medicament for preventing or treating acute kidney injury.

[0014] In one embodiment, the use includes verifying whether a medicament can be used for preventing or treating renal ischemia-reperfusion injury by using the expression of RNF130 gene or protein.

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

[0016] A fourth object of the present application is to provide a medicament for preventing or treating acute kidney injury, wherein the medicament contains an activator of RNF130 gene and / or its expression product.

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

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

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

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

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

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

[0023] In an embodiment, the adjuvant includes any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, humectants, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents, or buffers.

[0024] In an embodiment, the carrier is one or more of viruses, liposomes, nanoparticles.

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

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

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

[0028] Advantages of the present application

[0029] The RNF130 activator can improve the renal function of mice with renal ischemia-reperfusion injury, improve the levels of creatinine, urea nitrogen, and cystatin C, reduce the levels of kidney injury markers KIM-1 and LCN2, and reduce renal tubular injury, thereby preventing and treating renal ischemia-reperfusion injury, and providing a new target for preparing drugs for preventing and treating acute kidney injury. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Serum indicators of mice;

[0031] Figure 2 Expression of RNF130 in the kidneys of mice;

[0032] Figure 3 H&E and PAS staining of the kidneys of mice and renal tubular injury score;

[0033] Figure 4 Expression levels of kidney injury markers Kim-1 and Lcn2 mRNA in mice. Detailed Implementation

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

[0035] Raw materials used in the examples:

[0036] The AAV-GP-12N vector was purchased from Gemma Gene.

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

[0038] Test method:

[0039] 1. Different treatment methods for renal ischemia-reperfusion:

[0040] Sham-operated group (AAV-NULL group): Six C57BL / 6J mice were randomly selected and acclimatized for one week (1-7 days). At six weeks of age, the mice were injected via the tail vein with a titer of 2×10⁻⁶. 9 GC empty plasmid virus; after feeding for another six weeks, the mice were anesthetized with 2% isoflurane gas. Once the mice were in a stable state of breathing and under anesthesia, a small incision was made along the costovertebral angle on their backs, and the kidneys were squeezed out of the body along the direction of the incision. After 45 minutes, the kidneys were returned without ischemia-reperfusion treatment. After the above sham surgery was completed, the mice were fed for another 24 hours before being euthanized.

[0041] Renal ischemia-reperfusion group (I / R+AAV-NULL group): Six C57BL / 6J mice were randomly selected and acclimatized for one week (1-7 days). At six weeks of age, the mice were injected via the tail vein with a titer of 2×10⁻⁶. 9 GC empty plasmid virus; after six weeks of feeding, mice were anesthetized with 2% isoflurane gas after injection. Once the mice's breathing was stable and they were under anesthesia, a small incision was made along the costovertebral angle on their backs. The kidneys were squeezed out of the body along the incision direction, and the renal arteries and veins were carefully separated and identified. The arterial clamps were used to close the kidneys. Within 1 minute of clamping, the color of the mouse's kidneys darkened to black, indicating successful ischemia. Each mouse underwent renal ischemia for 45 minutes. Afterward, the arterial clamps were released, and blood reperfusion was allowed. The color of the kidneys changed from dark red to reddish, indicating successful reperfusion. Both kidneys of each mouse were clamped simultaneously. After the above reperfusion model was completed, the mice were fed for another 24 hours before being sacrificed.

[0042] Control group (AAV-RNF130 group): Six C57BL / 6J mice were randomly selected and acclimatized for one week (1-7 days). At six weeks of age, the mice were injected via the tail vein with a titer of 2×10⁻⁶. 9GC adeno-associated virus RNF130-AAV9; After feeding for six weeks, 2% isoflurane gas anesthesia was used, and the mouse was put into a state of anesthesia after breathing smoothly. A small opening was made along the rib spine angle of the back, and the kidney was squeezed out in the body along the opening direction. After 45 min, the kidney was returned without ischemia reperfusion treatment. After the above sham operation was completed, the mouse was killed after feeding for 24 h.

[0043] RNF130 treatment group (I / R+AAV-RNF130 group): 6 C57BL / 6J mice were randomly selected, and after adaptive feeding for 1 week (1-7 days), the mice were injected with 2×10 9 GC adeno-associated virus RNF130-AAV9; After feeding for six weeks, 2% isoflurane gas anesthesia was used, and the mouse was put into a state of anesthesia after breathing smoothly. A small opening was made along the rib spine angle of the back, and the kidney was squeezed out in the body along the opening direction. After 45 min, the kidney was returned without ischemia reperfusion treatment. After the above sham operation was completed, the mouse was killed after feeding for 24 h.

[0044] 2, Collection of mouse serum samples:

[0045] (1) Anesthetize the mouse: deeply anesthetize the mouse with sodium pentobarbital;

[0046] (2) Blood collection: use the orbital venous blood collection method, and collect the mouse blood into a centrifuge tube;

[0047] (3) Separate the serum: let the blood sample stand at room temperature for 2 hours, then place it in a 4°C refrigerator overnight, and finally centrifuge the sample to separate the serum. Transfer the serum 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 for observing and analyzing the morphological structure of mouse kidney tissue. The specific operation method is as follows:

[0050] (1) Sample fixation: remove the mouse kidney and fix it with 10% buffered formalin solution to maintain the integrity of the tissue structure;

[0051] (2) Tissue processing: The fixed kidney tissue is dehydrated and embedded. First, the kidney tissue is dehydrated by soaking it in increasing concentrations of alcohol to remove water. Then, the dehydrated kidney tissue is transparentized using xylene to make the tissue compatible with paraffin;

[0052] (3) Section preparation: The transparentized kidney tissue is embedded in paraffin to prepare paraffin blocks. Subsequently, the paraffin blocks are cut into 10 μm thick tissue sections using a microtome, and the sections are transferred to glass slides;

[0053] (4) Staining treatment: The sections are subjected to HE staining treatment. First, the sections are placed in a dye basic acid hydrolysis staining solution, which contains hematin dye and eosin dye. Under basic conditions, the eosin dye binds to the cell nucleus DNA to stain it red; under acidic conditions, the hematin dye stains the cytoplasm and intercellular matrix light blue. After a certain period of staining, the sections are transferred to a dehydration solution to remove excess dye;

[0054] (5) Mounting and observation: The sections are covered with transparent adhesive bubbles and observed using a microscope. After HE staining, the mouse kidney tissue sections show red nuclei, light blue cytoplasm and intercellular matrix, and the tissue structure, cell morphology and tissue type can be observed and analyzed under a microscope.

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

[0056] 1. Construction of RNF130 agonist

[0057] According to the mRNA (NM_021540.4) of the RNF130 gene, cDNA is reverse transcribed (the amino acid sequence is shown in SEQ ID NO. 1), and the target gene fragment is obtained by PCR amplification; the target gene fragment is 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 insertion of the RNF130 gene is used as a control.

[0058] 2. Animal model detection

[0059] (1) Virus construction

[0060] The overexpression plasmid AAV-GP-12N-RNF130 prepared in step 1 is used to construct the adeno-associated virus RNF130-AAV9 by entrusting GenScript to obtain the adeno-associated virus RNF130-AAV9 for animal experiments; the empty plasmid is used as a control to obtain the plasmid AAV-NULL.

[0061] (2) Animal experiment

[0062] Male C57BL / 6 mice were purchased from Beijing Sipuofu Biotechnology Co., Ltd. All mice were housed in specific pathogen-free micro-isolation cages, free to eat and drink water, and the animals were raised in a room with controlled temperature and humidity, with a 12-hour light / dark cycle.

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

[0064] Table 1: Mouse experiment grouping

[0065]

[0066] 3. Result detection

[0067] (1) Serum indicators

[0068] The mouse eyeball blood was taken, centrifuged to prepare serum, and the changes of blood creatinine, urea nitrogen and cystatin C were detected. The results are shown in Figure 1 , which shows that under the action of ischemia-reperfusion, the levels of blood creatinine, urea nitrogen and cystatin C in the I / R+AAV-NULL group mice are significantly higher than those in the AAV-NULL group, but the renal injury indicators of the I / R+RNF130 group are significantly decreased. It can be seen that the use of RNF130 can significantly improve the three renal function indicators.

[0069] (2) Kidney injury results

[0070] The mouse kidney was made into paraffin section for immunohistochemical staining, and the results are shown in Figure 2 , which shows that the expression level of RNF130 in the kidney tissue of the I / R+AAV-NULL group mice is significantly lower than that of the AAV-NULL group mice.

[0071] (3) Kidney staining results

[0072] The results of H&E staining and PAS staining of kidney paraffin sections are shown in Figure 3 , which shows that the I / R+AAV-NULL group mice have more vacuoles in the renal tubules than the AAV-NULL group mice, the lumen of the renal tubules is dilated, and is accompanied by shedding of the brush border. It can be seen that the degree of renal pathological injury is increased; while the renal tubular epithelial cell shedding of the I / R+AAV-RNF130 group mice is reduced, and the shedding of the renal tubular brush border is also reduced. It can be seen that RNF130 alleviates the ischemia-reperfusion-induced renal pathological injury.

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

[0074] The results of the expression levels of KIM-1 and LCN2 in the kidney tissue are shown in Figure 4As shown in the results, the expression levels of the kidney 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 of mice; and the expression levels of KIM-1 and LCN2 genes in the kidneys of mice in the I / R+AAV-RNF130 group were significantly down-regulated compared with those in the I / R+AAV-NULL group.

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

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

[0077]

[0078] While the application has been described by way of example and in terms of the preferred embodiment, it is to be understood that certain modifications can be made to the disclosed apparatus without departing from the scope of the application, and the scope of the application should be determined not by the embodiment but by the appended claims.

Claims

1. Use of an adeno-associated virus (AAV9) overexpressing RNF130 in the preparation of a drug for preventing or treating acute kidney injury induced by renal ischemia-reperfusion injury.

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

3. Use according to claim 1, characterized in that, The drug reduces the expression level of kidney injury markers KIM-1 and LCN2.

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