Construction method and application of exacerbated acute kidney injury model

Abstract

The invention discloses a construction method and application of an exacerbated acute kidney injury model, and belongs to the technical field of biomedicine. In particular discloses application of an Atg7 phosphorylation deficient mutant Atg7-T82A / T562A in construction of an exacerbated acute kidney injury (AKI) model. The mutant is delivered to the kidney of a mouse through an adeno-associated virus (AAV), phosphorylation modification of AMPK on Atg7 is specifically blocked, and in combination with an acute renal ischemia (ARI) model, serum creatinine and urea nitrogen levels can be remarkably improved, renal tubular necrosis and apoptosis can be aggravated, autophagosome formation can be reduced, and mitochondrial injury can be induced. The method has high specificity and high efficiency, and provides a key tool for mechanism research, drug toxicity evaluation and treatment strategy development of autophagy-related renal injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a method for constructing an aggravated acute kidney injury model and its application. Specifically, it relates to the application of the phosphorylation-deficient mutant Atg7-T82A / T562A of the Atg7 protein in constructing an acute kidney injury disease model. Background Art

[0002] Acute Kidney Injury (AKI) is a rapid decline in renal function caused by various factors such as ischemia, toxic substances, or inflammation, with high morbidity and mortality. Autophagy, as a key regulatory mechanism of cellular metabolic homeostasis, plays an important role in renal injury repair. Atg7 is an indispensable E1-like enzyme in the autophagy process, responsible for mediating the ubiquitin-like modification of LC3 and Atg12, and promoting the formation of autophagosome membranes. Existing studies have shown that the function of Atg7 is regulated by various post-translational modifications (such as ubiquitination, acetylation), but the specific mechanism of its phosphorylation modification in renal injury remains unclear. Currently, there is a lack of animal models that can simulate or exacerbate renal injury by specifically regulating the phosphorylation state of Atg7, which limits the drug development and mechanism research targeting the autophagy pathway. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for constructing an aggravated acute kidney injury model and its application. By injecting an adenovirus-associated virus carrying a phosphorylation mutant of Atg7, the severity of the acute kidney injury model is significantly aggravated, providing an efficient tool for disease mechanism research and drug screening.

[0004] In order to achieve the above invention purpose, the present invention provides the following technical solutions.

[0005] The present invention discloses a method for constructing an aggravated acute kidney injury model, which is characterized by including the following steps: (a) delivering the phosphorylation-deficient mutant Atg7-T82A / T562A to the kidneys of target animals through an adenovirus-associated virus (AAV); (b) establishing an acute renal ischemia model; (c) detecting the levels of serum creatinine and blood urea nitrogen and the pathological changes of renal tissues to verify the aggravating effect of the model.

[0006] Further, the injection dose of the AAV is 5×10 11 μg / animal.

[0007] The present invention also discloses the application of the aggravated acute kidney injury model constructed by the above-mentioned construction method in the research on the mechanism of autophagy-related renal injury.

[0008] The present invention also discloses the application of the aggravated acute kidney injury model constructed by the above-mentioned construction method in the research of screening drugs related to autophagy-related kidney injury.

[0009] The present invention also discloses a screening platform for evaluating drugs for repairing autophagy-deficient kidney injury, which is characterized in that the platform is based on the aggravated acute kidney injury animal model described in any one of the above, and evaluates the drug effect by detecting the expression of autophagy marker p62, apoptotic protein cleaved-PARP1 or the degree of renal tissue pathological injury.

[0010] Furthermore, the degree of renal tissue pathological injury is evaluated by TTC staining, TUNEL apoptosis detection or transmission electron microscopy to observe the number of autophagosomes and mitochondrial ultrastructure.

[0011] The present invention also discloses the use of the Atg7-T82A / T562A phosphorylation-deficient mutant in the preparation of an aggravated acute kidney injury model.

[0012] The present invention also discloses an adeno-associated virus (AAV) composition comprising a nucleic acid sequence encoding the Atg7-T82A / T562A mutant for delivery to kidney tissue.

[0013] Furthermore, the use of the above-mentioned composition in the preparation of an aggravated acute kidney injury model.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0015] High specificity: By precisely mutating the phosphorylation sites of Atg7, the activation of the autophagy pathway is targeted and inhibited.

[0016] Efficient model establishment: The mutant significantly exacerbates the renal injury phenotype, shortens the model establishment period and improves the experimental repeatability.

[0017] Wide application: Suitable for the research of mechanisms of autophagy-related diseases, drug toxicity assessment and development of treatment strategies. Description of the Drawings

[0018] Figure 1 It is the Western Blot verification result of the Atg7 phosphorylation-deficient mutant T82A / T562A, showing that the expression of the mutant is inhibited.

[0019] Figure 2 It is the comparison of renal function indexes (serum creatinine, blood urea nitrogen) of mice in the acute renal ischemia (ARI) model. The mutant group is significantly higher than the wild-type group (p<0.001). Among them, A is serum creatinine; B is blood urea nitrogen.

[0020] Figure 3For the TUNEL apoptosis detection of renal tissue, the mutant group showed more extensive renal tubular necrosis and apoptosis.

[0021] Figure 4 The transmission electron microscopy images showed that the number of autophagosomes in the mutant group was significantly reduced (red arrow), accompanied by ultrastructural damage such as mitochondrial swelling. Specific implementation manners

[0022] The present invention will be further described in detail below with specific embodiments. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following embodiments, and all technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0023] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0024] I. Construction of AA9 and mouse model.

[0025] Adeno-associated virus (AAV) 9 was purchased from GENECHEM Company. The specifically constructed AAV9 included: wild-type AAV9-Flag-Atg7-WT of Atg7, and mutant AAV9-Flag-Atg7-T82A / T562A. C57BL / 6 mice, male, 12 weeks old, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. AAV was injected by tail vein injection at a dose of 5×10 11 μg AAV. Three weeks after virus injection, a renal infarction model was constructed in the mice. The mice were divided into the following 2 groups: AAV9-Flag-Atg7-WT group and AAV9-Flag-Atg7-T82A / T562A group. The mice were randomly divided into acute renal ischemia (7 mice) and sham operation (7 mice) groups. The specific surgical method was as follows: The hair on the back of the mice was shaved, and the skin and muscle on the left side were disinfected with 70% alcohol cotton swabs. The skin and muscle on the back were incised along the back at the 1 / 3 position, with a size of 1 - 1.5 cm. The kidney was pushed out with a sterile cotton swab to expose the renal pedicle. The adipose tissue around the renal pedicle was removed with forceps, and the left renal pedicle was clamped with a microaneurysm clip for 6 h, and then the tissue was taken. Fresh tissue was stained with TTC. Blood was taken from the eyes of the mice, and serum creatinine and blood urea nitrogen were detected after centrifugation.

[0026] II. Transmission electron microscopy detection.

[0027] The kidneys were removed, briefly rinsed with phosphate-buffered saline (PBS), and immediately fixed in 10% neutral buffered formalin solution at room temperature for 24 h. Subsequently, the samples were placed in glutaraldehyde solution for preparation of transmission electron microscopy sections. Ultra-thin (60 nm) sections were collected on copper grids, stained with 2% uranyl acetate in 50% methanol for 10 min, and then stained with 1% lead citrate for 7 min. Finally, images were taken using a JEEOL JEM1210 transmission electron microscope (JEOL).

[0028] III. Extraction of kidney tissue proteins and Western blotting.

[0029] Add 60 μL of freshly prepared RIPA lysis buffer to each well of a six-well plate, which contains 47.4 μL of RIPA (strong) lysis buffer, 6 μL of protease inhibitor, 6 μL of protein phosphatase inhibitor, and 0.6 μL of PMSF. Lyse on ice for 10 min, then use a pre-chilled cell scraper to scrape the sample into an EP tube. Then centrifuge at 4°C and 15,000 g for 15 min, carefully aspirate the supernatant, and transfer it into a new pre-chilled 1.5 mL EP tube to obtain the protein sample. The protein sample is quantified using the BCA standard curve method, and after adjusting the concentration, it is aliquoted and frozen at -80°C. Use the Bio-Rad SDS-PAGE gel preparation kit to prepare the gel for western blot detection. Specifically (for each gel): Mix the separating gel solution A and B, and the stacking gel solution A and B at a ratio of 1:1 respectively (6 mL of separating gel in total, 2 mL of stacking gel in total). Add 6 μL and 4 μL of TEMED to the separating gel and stacking gel respectively, mix well, and place it in a cool and dark place to let it stand and exhaust air for 10 min. During this period, assemble the relevant equipment for making vertical gels and set it aside. After the air is exhausted, add 30 μL and 10 μL of 30% ammonium persulfate to the separating gel and stacking gel respectively, gently pipette and mix well, and then immediately pour the gels into the gel glass tank in sequence (separating gel at the bottom, stacking gel on top). Then slowly insert a 1.0 mm sample comb, let it stand at room temperature for 30 min. After the gel is completely solidified, remove the glass plate, rinse the residual gel on the surface with tap water, and soak it in deionized water, and store it at 4°C for later use. Install the vertical electrophoresis equipment and the SDS-PAGE gel, pour 1 L of freshly prepared electrophoresis buffer (14.4 g glycine, 3.03 g Tris, 1 g SDS, made up to 1000 mL with deionized water) into the electrophoresis tank, carefully remove the sample comb, and gently pipette the sample wells with a pipette to remove air bubbles and residual gel. Mix the protein solution extracted from the above experiment with 5×SDS loading buffer (4:1), heat at 95°C for 10 min to denature the protein. Then load 20 μg of protein per sample well, and at the same time add 10 μL of prestained protein molecular weight marker protein, and electrophorese at a constant voltage of 200 V for 60 min. After the protein samples are fully separated, remove the vertical electrophoresis tank, cut the SDS-PAGE gel, and transfer it into a protein transfer tank. The installation order of the transfer tank (from bottom to top) is: sponge pad - thickened filter paper - gel - PVDF membrane (0.45 μm, pre-activated with methanol) - thickened filter paper - sponge pad. Pour 1 L of freshly prepared electrotransfer buffer (14.4 g glycine, 3.03 g Tris, 200 mL methanol, made up to 1000 mL with deionized water) into the transfer tank, and place the transfer tank in ice, and transfer at a constant current of 300 mA for 1 h.

[0030] After the transfer is completed, remove the transfer device, cut the PVDF membrane according to the molecular weight of the protein to be tested, rinse it with TBST, and block it with 5% skim milk at room temperature for 2 h. After blocking, rinse the PVDF membrane with TBST, add the primary antibody prepared with 5% skim milk to the corresponding PVDF membrane, and incubate it slowly on a horizontal shaker at 4 °C overnight. The next day, recover the primary antibody, rinse the PVDF membrane 3 times with TBST, 10 min each time. Subsequently, add the secondary antibody prepared with 5% skim milk and incubate it with shaking at room temperature for 2 h, then rinse the PVDF membrane 3 times with TBST, 10 min each time. After washing, soak the PVDF membrane in a new TBST solution for standby.

[0031] Mix equal proportions of ECL luminescent solution A and B to prepare the ECL developing solution and keep it in the dark for standby. Take out the PVDF membrane, blot it dry with absorbent paper, and place it in the gel imager. Drop a little ECL developing solution on the surface of the PVDF membrane so that the membrane is just wetted by the developing solution. Change the imager to the chemiluminescence (CL) mode, expose and observe the results to judge the expression level of the corresponding protein.

[0032] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a model of aggravated acute kidney injury, characterized in that, Comprising the following steps: (a) Delivering the Atg7-T82A / T562A phosphorylation-deficient mutant to the kidneys of the target animal via adeno-associated virus (AAV); (b) Establishing an acute renal ischemia model; (c) Detecting the levels of serum creatinine and blood urea nitrogen and the pathological changes in the renal tissue to verify the exacerbation effect of the model.

2. The method according to claim 1, wherein The injection dose of the AAV is 5×10 11 μg per animal.

3. Use of the acute kidney injury model constructed by the construction method described in claim 1 in the study of the mechanism of autophagy-related kidney injury.

4. Use of the acute kidney injury model constructed by the construction method described in claim 1 in the study of drug screening for autophagy-related kidney injury.

5. A screening platform for evaluating drugs for repairing autophagy-deficient kidney injury, characterized in that, The platform is based on the acute kidney injury animal model described in any one of claims 1 to 2, and evaluates the drug effect by detecting the expression of the autophagy marker p62, the apoptotic protein cleaved-PARP1, or the degree of pathological damage to the renal tissue.

6. The screening platform according to claim 5, wherein The degree of pathological damage to the renal tissue is evaluated by TTC staining, TUNEL apoptosis detection, or transmission electron microscopy to observe the number of autophagosomes and the ultrastructure of mitochondria.

7. Use of the Atg7-T82A / T562A phosphorylation-deficient mutant in the preparation of an acute kidney injury model with exacerbation.

8. An adeno-associated virus (AAV) composition comprising a nucleic acid sequence encoding the Atg7-T82A / T562A mutant for delivery to kidney tissue.

9. Use of the composition according to claim 6 in the preparation of an acute kidney injury model with exacerbation.

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