Application of TAX1BP1 function enhancer in preparation of medicine for preventing or treating acute kidney injury

Abstract

The invention discloses application of a TAX1BP1 function enhancer in preparation of a medicine for preventing or treating acute kidney injury, and belongs to the technical field of biological medicine. The invention also provides a pharmaceutical composition for treating the cisplatin-induced acute kidney injury. The pharmaceutical composition comprises the TAX1BP1 function enhancer and a pharmaceutically acceptable carrier. According to the application, the autophagy process of renal tubular cells can be remarkably activated through lentivirus overexpression TAX1BP1, cell death caused by cis-platinum is reduced, renal function indexes of mice with acute kidney injury (AKI) are improved, and the application provides a new strategy for clinical treatment of chemotherapy-related acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a TAX1BP1 function enhancer in the preparation of a medicament for preventing or treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) refers to the sudden loss of renal excretory function. AKI is an acute kidney disease characterized by slow deterioration or persistent renal dysfunction associated with irreversible loss of renal cells, accompanied by a rapid rise in serum creatinine, decreased urine output, or both, and potentially leading to chronic kidney disease. AKI occurs in approximately 10%–15% of hospitalized patients and in over 50% of intensive care unit patients. The mortality rate associated with AKI far exceeds that of breast cancer, heart failure, or diabetes and has remained high over the past 50 years, rising to 42% overall in patients with AKI and 46% in those requiring renal replacement therapy, with hypotension and shock being the primary causes. Risk factors for AKI are numerous, including severe illness, acute infection, sepsis, malaria, severe trauma, hypovolemia, elderly age, preexisting chronic kidney disease, acute organ failure, major surgery (including cardiac surgery), exposure to nephrotoxic drugs and opportunistic infections in the ICU, leukemia or cancer chemotherapy, delayed graft function after renal transplantation, autoimmune diseases with rapidly progressive renal damage, cholesterol crystal embolism, and urinary tract obstruction. However, there is still a lack of effective treatment options for AKI induced by these risk factors. Therefore, exploring the pathogenesis of AKI is of great significance for clinical treatment.

[0003] TAX1BP1 (Tax1-binding protein 1) was first identified in a yeast two-hybrid screen as an interactor of the human T-lymphotropic virus 1 (HTLV-1) Tax oncoprotein. TAX1BP1 was also identified in an independent yeast two-hybrid screen as a binding protein for the zinc finger protein TNFAIP3 / A20 (TNF-α-induced protein 3) and the E3 ubiquitin ligase TRAF6 (TNF receptor-associated factor 6). Overexpression of TAX1BP1 has been documented to inhibit TNF (tumor necrosis factor)-induced apoptosis and to serve as a substrate for CASP-induced cleavage. In addition to regulating cell death pathways, TAX1BP1 has also been implicated in NF-κB (nuclear factor of enhancer of kappa light polypeptide genes in B cells) signaling. IL1β (interleukin 1β) stimulation promotes the interaction between TRAF6 and TAX1BP1. However, TAX1BP1 does not activate the NF-κB or MAPK / JNK (mitogen-activated protein kinase) pathways. TAX1BP1-deficient mouse embryonic fibroblasts (MEFs) exhibit enhanced and sustained NF-κB signaling upon stimulation with TNF or IL1β. The specific mechanisms implicating TAX1BP1 in cisplatin-induced acute kidney injury and its potential use as a therapeutic agent for cisplatin-induced acute kidney injury have not been reported. Summary of the Invention

[0004] The object of the present invention is to provide the use of a TAX1BP1 function enhancer in the preparation of a drug for preventing or treating acute kidney injury, so as to solve the problems existing in the above-mentioned prior art. The present invention demonstrates the protective effect of TAX1BP1 in cisplatin-induced AKI. By overexpressing TAX1BP1 via lentivirus, it was found that the autophagy process in cisplatin-induced acute kidney injury can be induced, thereby protecting the survival of renal tubular cells. It was further found that TAX1BP1 can reduce the expression of serum creatinine and serum urea in mice with acute kidney injury, promote the occurrence of autophagy in a cisplatin-induced acute kidney injury model, thereby inhibiting the death of renal tubular epithelial cells and slowing the development of acute kidney injury.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of a TAX1BP1 function enhancer in preparing a medicament for preventing or treating acute kidney injury, wherein the TAX1BP1 function enhancer is used to activate the autophagy pathway of renal tubular cells.

[0007] In the present invention, TAX1BP1 function enhancers refer to substances that can increase the expression level or biological activity of TAX1BP1 protein, including but not limited to: TAX1BP1 protein itself, nucleic acid sequences encoding the protein (such as DNA, RNA, vector), and small molecule compounds that can upregulate its expression.

[0008] Furthermore, the acute kidney injury is induced by cisplatin.

[0009] Furthermore, the TAX1BP1 function enhancer includes: TAX1BP1 protein or a functional fragment thereof, a nucleic acid molecule encoding TAX1BP1 protein, or a compound that upregulates TAX1BP1 expression or activity.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

[0011] Furthermore, the nucleic acid molecule is loaded into a lentiviral vector pLV4LTR.

[0012] The present invention provides a pharmaceutical composition for treating cisplatin-induced acute kidney injury, comprising a TAX1BP1 function enhancer and a pharmaceutically acceptable carrier.

[0013] Furthermore, the TAX1BP1 function enhancer includes a lentiviral vector pLV4LTR-TAX1BP1.

[0014] Furthermore, the TAX1BP1 function enhancer includes TAX1BP1 protein, and the nucleotide sequence of the TAX1BP1 protein is shown in SEQ ID NO.1.

[0015] The present invention also provides a use of a reagent for detecting the expression level of TAX1BP1 in preparing an in vitro sample detection kit, wherein the kit is used to analyze the expression level of TAX1BP1 in a renal tissue sample.

[0016] The present invention discloses the following technical effects:

[0017] The experiments of the present invention have verified that overexpression of TAX1BP1 by lentivirus can induce the autophagy process in cisplatin-induced acute kidney injury, thereby protecting the survival of renal tubular cells; TAX1BP1 can reduce the expression of serum creatinine and serum urea in mice with acute kidney injury, promote the occurrence of autophagy in the cisplatin-induced acute kidney injury model, and then inhibit the death of renal tubular epithelial cells, slow down the development of acute kidney injury, etc. The present invention is expected to provide a new clinical drug for the treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is the plasmid map of pLV4LTR;

[0020] Figure 2 The infection efficiency of TAX1BP1 lentivirus; scale bar: 50 μm;

[0021] Figure 3 To detect the efficiency of TAX1BP1 overexpression;

[0022] Figure 4 The effect of TAX1BP1 overexpression on autophagy in cisplatin AKI cell model was detected by immunoblotting; A is the effect of TAX1BP1 overexpression on autophagy in cisplatin AKI cell model detected by immunoblotting; B is the statistical plot after immunoblotting grayscale analysis;

[0023] Figure 5 For CCK8 experiments, the toxic effect of overexpressed TAX1BP1 on cisplatin-AKI cells was detected;

[0024] Figure 6 Immunofluorescence analysis of the effect of overexpression of TAX1BP1 on LC3 in the cisplatin-induced AKI cell model; scale bar: 100 μm;

[0025] Figure 7 Calcein / PI staining was used to detect the cytotoxic effect of overexpressed TAX1BP1 on cisplatin-induced AKI cells; scale bar: 100 μm;

[0026] Figure 8 The effect of overexpression of TAX1BP1 on the expression levels of serum creatinine and serum urea in AKI mice; A is the expression of TAX1BP1 and autophagy markers in the cisplatin AKI cell model detected by immunoblotting; B is the statistical plot after grayscale analysis of immunoblotting;

[0027] Figure 9 Immunoblotting was used to detect the effect of TAX1BP1 overexpression on autophagy in the cisplatin-induced AKI mouse model (A) and PAS staining was used to observe the effect of TAX1BP1 on renal tissue (B);

[0028] Figure 10AS staining was used to observe the effect of Tax1bp1 on renal tissue (A); immunohistochemistry was used to detect the effect of overexpression of Tax1bp1 on LC3 in the cisplatin AKI mouse model (B); immunofluorescence staining was used to detect the effect of overexpression of Tax1bp1 on LC3 expression in the cisplatin AKI model (C); Scar: 100 μm; representative transmission electron microscopy images of autophagosomes after overexpression of Tax1bp1 in AKI mice (D); low-magnification and high-magnification images are shown simultaneously; red arrows indicate autophagic structures; scale bar: 100 μm. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The experimental materials and reagents used in the present invention are as follows:

[0035] Forty healthy male C57BL / 6 mice were purchased from the Animal Experiment Center of Chongqing Medical University. The mice were housed in an SPF-grade animal room, fed a standard diet, and randomly divided into four groups of 10 mice each.

[0036] BCA kit, RIPA protein lysis buffer (strong), 4% paraformaldehyde and trypsin cell freezing solution, protease inhibitors, separation gel, stacking gel, virus packaging plasmid, transfection reagent, Calcein / PI Cell Viability / Cytotoxicity Assay Kit (C2015M), and pLV4LTR overexpression vector were all purchased from Beyotime Biotechnology Co., Ltd.; sodium pentobarbital was purchased from RT, Germany; TBST buffer solution was purchased from Saiguo Biotechnology Co., Ltd.; glycine and SDS were purchased from Solebo Biotechnology Co., Ltd.; Opti-DMEM, DMEM, and PBS buffer culture medium were purchased from Sigma, Germany; fetal bovine serum was purchased from Gibco, USA; PMSF was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; PDVF membrane was purchased from Millipore; ECL chemical developer was purchased from ThermoFisher Scientific; LC3B antibody, TAX1BP1 antibody, and Beclin1 antibody were purchased from Abmart; anti-rabbit IgG antibody, P53 antibody, and anti-mouse IgG antibody were purchased from Cell Signaling. Technology; GAPDH antibody was purchased from Proteintech; cisplatin was purchased from Solarbio Biotechnology; TAX1BP1 lentivirus was purchased from Qingke Biotechnology; Boost Detection Reagent was purchased from Biosharp Biotechnology Co., Ltd.; substrate colorimetric solution was purchased from Biosharp Biotechnology Co., Ltd.; NRK cells and 293T cells were purchased from Bohui Biotechnology Co., Ltd.; 1X citrate repair solution was purchased from Biosharp Biotechnology Co., Ltd. Preparation of cisplatin solution: Weigh an appropriate amount of cisplatin and dissolve it in physiological saline to make a solution with a concentration of 1 mg / mL. Mix by pipetting. The cisplatin solution should be prepared and used immediately.

[0037] Other reagents not specifically described in the present invention are all commercial products.

[0038] Example 1 Establishment of cisplatin-induced acute kidney injury mouse model

[0039] Forty healthy male C57BL / 6 mice were randomly divided into four groups, with 10 mice in each group.

[0040] To establish an AKI mouse model, male C57BL / 6 mice (8 weeks old) were intraperitoneally injected with 3% sodium pentobarbital (30 mg / kg) followed by 20 mg / kg of cisplatin. A control group received the same volume of saline intraperitoneally. Mice were sacrificed 12, 24, 48, and 72 hours after cisplatin administration. Blood was collected for serum creatinine and urea analysis, and tissue sections were embedded for pathological sectioning and stained with hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Total protein was extracted from selected tissues, and the expression levels of the corresponding proteins were determined.

[0041] For drug and gene overexpression experiments, C57BL / 6 mice were injected with TAX1BP1 lentivirus through the tail vein, and saline was injected as a control group. Then, the mice were killed 12, 24, 48, and 72 hours after administration, and serum creatinine and serum urea were measured. Tissues were embedded for pathological sections and stained with HE and IHC. Total protein was extracted from some tissues, and the expression levels of the corresponding proteins were detected.

[0042] Example 2 Renal function test

[0043] The blood collected from the mice in each treatment group in Example 1 was placed in a 4°C refrigerator for 1 h, then centrifuged at 3000 rpm / min at 4°C for 15 min. The supernatant was gently removed and transferred to 1.5 mL EP (stored at -20°C). The expression levels of serum creatinine and serum urea in the blood of mice in each experimental group were measured using an automatic analyzer.

[0044] Example 3 HE pathological staining of kidney tissue

[0045] (1) HE staining tissue fixation and sectioning: First, the tissue samples were routinely paraffin-embedded. The kidney tissues of the experimental mice in each treatment group were fixed in 4% paraformaldehyde solution for 24 h, then washed, dehydrated and paraffin-embedded, and then sectioned using a paraffin microtome with a thickness of about 5 μm.

[0046] (2) Dewaxing of HE-stained samples: Place the tissue sample sections to be tested in xylene and soak them for 10 minutes, repeating three times. Xylene can also play a transparent role, making the sections easier to observe;

[0047] (3) Hydration of HE-stained samples: The tissue sample to be tested, which had been soaked in xylene, was first placed in anhydrous ethanol for 10 min, the xylene was removed, and then the sample was placed in 95%, 90%, 80%, and 70% ethanol for 5 min each to achieve full hydration, and then washed once with distilled water;

[0048] (4) Hematoxylin staining, differentiation and anti-blueing: Use PBS to soak and wash, 5 minutes each time, wash 3 times. Then add 100 μL of hematoxylin to the slice and stain for 10 minutes. Wash with distilled water. Then, use 1% hydrochloric acid ethanol for differentiation. After differentiation is completed, wash with distilled water again. Then, use 0.6% ammonia water to turn blue, and rinse with tap water;

[0049] (5) Eosin staining and dehydration: Add eosin staining solution to the sections and stain thoroughly for 3 minutes. After staining, perform gradient dehydration with 80%, 95%, and anhydrous ethanol. Dehydrate with 80% ethanol for 5 seconds; dehydrate with 95% ethanol for 5 minutes, twice; dehydrate with anhydrous ethanol for 5 minutes, twice;

[0050] (6) Air-drying and sealing of sections: Soak the dehydrated sections in xylene twice for 5 min each time, then air-dry the sections and seal them with neutral gum;

[0051] (7) Microscopic examination, image acquisition and analysis.

[0052] Example 4 IHC staining of kidney tissue

[0053] First, tissue samples were routinely paraffin-embedded. Kidney tissues of experimental mice in each treatment group were fixed in 4% paraformaldehyde solution for 24 hours, then washed, dehydrated, and paraffin-embedded. The sections were then sliced using a paraffin microtome with a thickness of approximately 5 μm.

[0054] (1) Dewaxing / Rehydration Dewaxing / Hydration Steps:

[0055] Incubate the sections in xylene wash solution three times for 5 minutes each time; incubate the sections in 100% ethanol twice for 10 minutes each time; incubate the sections in 95% ethanol twice for 10 minutes each time; wash the sections with dH2O twice for 5 minutes each time.

[0056] (2) Antigen retrieval:

[0057] For Citrate: Immerse sections in 1X Citrate Unmasking Solution and heat in a microwave until boiling; maintain sub-boiling temperature (95-98°C) for 10 minutes. Cool sections on the bench for 30 minutes.

[0058] (3) Dyeing:

[0059] Wash the sections three times with dH2O for 5 minutes each; incubate the sections in 3% hydrogen peroxide solution for 10 minutes; wash the sections twice with dH2O for 5 minutes each; wash the sections with wash buffer for 5 minutes; add 100-400 μL of the preferred blocking solution to each section and block at room temperature for 1 hour; remove the blocking solution, and then add 100-400 μL of the primary antibody diluted in the recommended antibody diluent to each section and incubate overnight at 4°C; Equilibrate Boost Detection Reagent to room temperature; remove the antibody solution and wash the sections 3 times with wash buffer for 5 minutes each; add 1-3 drops as needed. Cover the sections with Boost Detection Reagent and incubate in a humidified chamber at room temperature for 30 minutes; wash the sections three times with wash buffer for 5 minutes each; apply 100-400 μL of substrate colorimetric solution to the slide and immerse the sections in dH2O; wash the sections twice with dH2O for 5 minutes each; dehydrate the sections by incubating the sections in 95% ethanol twice for 10 seconds each, then repeating the incubation in 100% ethanol twice for 10 seconds each, and finally repeating the incubation in xylene twice for 10 seconds each; mount the sections with a coverslip and neutral gum.

[0060] Example 5 Western blotting experiment

[0061] (1) Protein sample extraction, the operation steps are as follows: Carefully pour out the culture medium in the culture bottle. Then add 2-3mL PBS with a pipette, rinse gently, and discard the PBS. Repeat the washing twice. Add 1mL fresh PBS, scrape the cells with a cell scraper, transfer the cell suspension to a 1.5mL centrifuge tube, centrifuge at 2000rpm for 5min, and discard the supernatant. Add an appropriate amount of lysis buffer containing protease inhibitors, lyse on ice for 30min, and shake once every 5min to fully lyse the cells. At the same time, ultrasound can be used for lysis to increase the lysis efficiency. Then, centrifuge the cell lysate at 13000g for 15min. Transfer the supernatant to a new 1.5mL tube and then measure the concentration.

[0062] (2) SDS-PAGE electrophoresis: first prepare a 10% concentration separating gel, then prepare a 5% concentration stacking gel;

[0063] (3) Protein loading;

[0064] (4) Protein electrophoresis;

[0065] (5) Protein transfer to PVDF membrane (wet transfer);

[0066] (6) Blocking with 5% skim milk in TBST at room temperature for 1 h. This blocking step helps reduce nonspecific primary antibody binding and lower background;

[0067] (7) Immunoreaction: Discard the blocking solution, add 10 mL of 1×TBST for washing, shake on a shaker for 10 min, and repeat 3 times. Dilute the antibody with 5% BSA to an appropriate concentration of 1:1000-1:2000, add the primary antibody dilution dropwise to the membrane to evenly cover it, and place it in a 4°C refrigerator overnight;

[0068] (8) ECL color development;

[0069] (9) Image analysis: Use corresponding software to analyze the grayscale value of the target band and calculate the expression of the target protein.

[0070] Example 6 Cell experiment

[0071] NRK cells and 293T cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin solution in a constant temperature incubator at 5% CO2 and 37°C.

[0072] Cell recovery: Remove NRK cells or 293T cells from the liquid nitrogen tank and rapidly thaw in a clean 37°C water bath. Once completely thawed, transfer the cells to a 1.5mL EP tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 1mL of complete DMEM medium. After pipetting and vortexing, transfer the cells to a T25 culture flask, add an appropriate amount of medium, and culture in a 5% CO2, 37°C incubator. After overnight culture, allow the cells to adhere to the wall, replace with fresh medium. When the cell density reaches 90%, passage and subsequent cell experiments can be performed.

[0073] Cell cryopreservation: Cryopreserve cells when they are growing well. When the cell density reaches approximately 90%, discard the culture medium and wash twice with an appropriate amount of PBS. Then, add 1 mL of trypsin to digest the cells. Once the cells become round and fall off, add complete culture medium to terminate the digestion. Transfer the cells to a 1.5 mL EP tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add 1 mL of cell freezing solution, gently pipette to evenly distribute, and transfer the cells to cryovials. Place the cryovials in a cryovial containing ice-cold methanol and freeze at -80°C for 48 hours. Transfer the tubes to a liquid nitrogen tank for long-term storage.

[0074] Example 7 Cisplatin-induced NRK cell AKI model

[0075] Cisplatin was diluted to 20 μM with culture medium, and NRK cells were seeded in 6 cm dishes and stimulated continuously with 20 μM cisplatin for 12 h, 24 h, 48 h, and 72 h. Total protein was then extracted to detect the expression of autophagy-related proteins.

[0076] Example 8 Lentivirus packaging and construction of stable strains

[0077] (1) When the 293T cells grow to a density of 80% to 90%, the cells need to be passaged to maintain a good growth state. Wash the cells with PBS, then add 1 mL of trypsin to digest the cells, collect the digested cells, centrifuge at 1000 rpm for 5 minutes, and then add complete culture medium to resuspend. Add an appropriate amount of cells to the culture dish and place the culture dish back in an incubator at 37°C, 5% CO2, and 95% relative humidity for culture;

[0078] (2) Before transfection, 293T cells were passaged on 10 cm culture dishes and incubated in a 37°C incubator containing 5% CO2 for 8–24 h. Transfection was initiated when the cell density reached approximately 80% after attachment.

[0079] (3) Transfection, prepare a mixture of packaging plasmid, target plasmid and transfection reagent. The steps are as follows: a. Take a sterile 1.5mL EP tube, add 500μL serum-free Opti-DMEM, add 8μg target TAX1BP1 plasmid and 8μg virus packaging plasmid (psPAX2: pMD2.G = 1:1), and mix thoroughly; b. Take a sterile 1.5mL EP tube, add 500μL serum-free Opti-DMEM, dissolve 50μL lipo3000 transfection reagent in Opti-DMEM, mix thoroughly, and let it stand for 5 minutes; c. Add the transfection reagent dilution dropwise to the plasmid dilution and let it stand at room temperature for 20 minutes. Replace the cells with fresh DMEM culture medium, add the mixture to the culture dish, mix gently, and continue to culture. Transfect overnight, wash with PBS, and add 10mL fresh culture medium for culture.

[0080] (4) Collect the virus. Collect the supernatant 24, 48, and 72 hours after transfection. Concentrate the virus by ultracentrifugation at 30,000 rpm and 4°C for 2 hours. Carefully remove the supernatant. Resuspend the virus in 100 μL PBS. Aliquot and store in a -80°C refrigerator.

[0081] (5) Transduction: Take an appropriate amount of 293T cells according to the virus titer and infect them for 24 hours each time, twice. Add polybrene at a final concentration of 8 μg / mL to improve the infection efficiency of the lentivirus. After the infection is completed, replace the culture medium with fresh one.

[0082] (6) Screening for stable cell lines: 48 hours after infection, for viruses carrying the GFP reporter gene, the GFP expression efficiency can be observed by fluorescence microscopy. For viruses carrying the puromycin resistance gene, fresh complete culture medium containing an appropriate concentration of puromycin is used to screen for stably transduced cell lines. 3 μg / mL puromycin is added to screen for positive cells. After about a week, protein expression is detected.

[0083] Example 9 Cell activity and cytotoxicity detection experiment

[0084] The Calcein / PI Cell Viability and Cytotoxicity Assay Kit is a dual-fluorescence staining method for detecting the viability of animal cells using Calcein-AM (calcein) and Propidium Iodide (PI). Calcein AM stains live cells, producing green fluorescence, while Propidium Iodide stains dead cells, producing red fluorescence.

[0085] a. Inoculation of cells: Cells were plated on a cell slide and treated with cisplatin for 48 hours before immunofluorescence staining.

[0086] b. Washing: For adherent cells, aspirate the culture medium, wash the cells once with PBS, and aspirate the supernatant (phenol red or serum may interfere with the detection of the kit, so try to completely remove the culture medium);

[0087] c. Staining: Add an appropriate volume of Calcein AM / PI working solution. Typically, add 100 μL per well of a 96-well plate, 250 μL per well of a 24-well plate, 500 μL per well of a 12-well plate, and 1 mL per well of a 6-well plate. Incubate at 37°C in the dark for 30 minutes.

[0088] d. Detection: After incubation, observe the staining effect under a confocal microscope, obtain images, and analyze cell viability and cytotoxicity.

[0089] Example 10 CCK8 assay to detect cytotoxicity

[0090] (1) Collect cells in the logarithmic growth phase and add 100 μL to each well of a 96-well plate to a cell density of 10,000 cells / well.

[0091] There were 5 replicate wells in each group;

[0092] (2) Place the culture plate in a 5% CO2 incubator and culture the cells at 37°C for 24 h;

[0093] (3) Add cisplatin to each well of the culture plate and continue incubating in the incubator for 48 hours. If the substance to be tested is oxidizing or reducing, fresh culture medium can be replaced before adding CCK-8 to eliminate the effect of the substance to be tested. If the effect is relatively small or there is no effect, the culture medium can be left unchanged and the blank absorption after adding the drug to the culture medium can be directly subtracted;

[0094] (4) Add 20 μL of CCK-8 solution to each well. Try not to create bubbles during the addition process to avoid affecting the OD value reading.

[0095] (5) Continue incubating in the cell culture incubator for 1-2 hours;

[0096] (6) Measure the absorbance of each well at 450 nm using an enzyme-labeled instrument.

[0097] Example 11 Statistical analysis method

[0098] ImageJ software was used to analyze the grayscale values of protein bands in the protein immunoblotting results. Quantitative data were presented as mean ± standard deviation (SD), and statistical analysis was performed using GraphPad Prism 9. p < 0.05 was considered statistically significant.

[0099] Experimental results

[0100] 1. TAX1BP1 enhances autophagy in cisplatin-induced AKI model of renal tubular epithelial cells, thereby alleviating renal injury

[0101] The TAX1BP1 gene was constructed into the pLV4LTR overexpression vector ( Figure 1 ), the nucleotide sequence of the TAX1BP1 gene is shown in SEQ ID NO. 1, and then lentiviral packaging and concentration were performed to obtain lentiviral particles, and the titer was determined, and the titer was 2×10 8 TU / mL.

[0102] SEQ ID NO.1:

[0103]

[0104] NRK cells were infected with pLV4LTR-TAX1BP1 lentivirus and the fluorescence intensity of the cells was observed under a fluorescence microscope. It was found that the lentivirus infection efficiency was about 90% ( Figure 2 ). Then, the cells were screened with puromycin to obtain a NRK / Lenti-TAX1BP1 stably overexpressing cell line.

[0105] The efficiency of TAX1BP1 overexpression in NRK cells was detected. Immunoblotting results showed that Lenti-TAX1BP1 could significantly upregulate the expression of TAX1BP1 in NRK cells ( Figure 3 Overexpression of TAX1BP1 in NRK cells can upregulate the expression of Beclin1 and LC3II induced by cisplatin, indicating that overexpression of TAX1BP1 can promote the activation of autophagy in AKI cell models ( Figure 4 In the cisplatin-induced NRK cell AKI model, the expression of LC3 was detected by immunofluorescence staining. The results showed that cisplatin increased the expression of LC3, and overexpression of TAX1BP1 enhanced the expression of LC3 induced by cisplatin, and the autophagic flux was significantly increased ( Figure 6 ). It shows that TAX1BP1 can activate the autophagy process in the cisplatin-induced AKI model. CCK8 experiments showed that cisplatin significantly inhibited the activity of NRK cells, and overexpression of TAX1BP1 could reverse the toxic side effects of cisplatin on NRK cells ( Figure 5 ).

[0106] Calcein / PI cell activity and cytotoxicity detection kit staining was used. The experimental results showed that after 48 hours of cisplatin induction, NRK cell death was promoted (PI red staining), while overexpression of TAX1BP1 reduced the toxic effect of cisplatin on NRK cells ( Figure 7 ).

[0107] 2. TAX1BP1 enhances autophagy in mice, thereby reducing renal tubular epithelial cell death and alleviating cisplatin-induced renal tissue damage

[0108] The present invention found that overexpression of TAX1BP1 in an AKI mouse model can reduce the expression of serum creatinine and serum urea ( Figure 8 ), the effect of TAX1BP1 overexpression on autophagy in cisplatin-induced AKI mouse model was detected by immunoblotting. The results showed that overexpression of TAX1BP1 in AKI mouse model could enhance the expression of Beclin1 and LC3II induced by cisplatin, indicating that overexpression of TAX1BP1 can promote the activation of autophagy in AKI model ( Figure 9The PAS staining results of the mouse kidney tissue also showed the same results. Overexpression of TAX1BP1 can improve the damage of kidney tissue caused by cisplatin ( Figure 10 ). Tissue immunohistochemistry and immunofluorescence results also confirmed the above results. Overexpression of TAX1BP1 can enhance the expression of Beclin1 and LC3 induced by cisplatin ( Figure 10 ).

[0109] The results showed that overexpression of TAX1BP1 enhanced autophagy and alleviated renal injury in a cisplatin-induced AKI model of renal tubular epithelial cells. In summary, in cisplatin-induced acute kidney injury, TAX1BP1 can enhance cisplatin-induced autophagy, thereby reducing renal tubular epithelial cell death and alleviating cisplatin-induced renal tissue damage.

[0110] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a TAX1BP1 function enhancer in the preparation of a drug for preventing or treating acute kidney injury, characterized in that: The TAX1BP1 function enhancer is used to activate the autophagy pathway of renal tubular cells.

2. The use according to claim 1, characterized in that The acute kidney injury was induced by cisplatin.

3. The use according to claim 1, characterized in that The TAX1BP1 function enhancer includes: TAX1BP1 protein or its functional fragment, a nucleic acid molecule encoding TAX1BP1 protein, or a compound that upregulates TAX1BP1 expression or activity.

4. The use according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

1.

5. The use according to claim 3, characterized in that The nucleic acid molecule is loaded into the lentiviral vector pLV4LTR.

6. A pharmaceutical composition for treating cisplatin-induced acute kidney injury, characterized in that: The pharmaceutical composition includes a TAX1BP1 function enhancer and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The TAX1BP1 function enhancer includes a lentiviral vector pLV4LTR-TAX1BP1.

8. The pharmaceutical composition according to claim 6, characterized in that The TAX1BP1 function enhancer includes TAX1BP1 protein, and the nucleotide sequence of the TAX1BP1 protein is shown in SEQ ID NO.

1.

9. Use of a reagent for detecting TAX1BP1 expression level in preparing an in vitro sample detection kit, characterized in that: The kit is used to analyze the expression level of TAX1BP1 in kidney tissue samples.

Images