Method for activating mitochondrial autophagy and relieving TECs damage based on mouse TECs and human HK-2 cell UMI-77 in AKI state

Abstract

The invention discloses a method for activating mitochondrial autophagy and relieving TECs injury based on mouse TECs and human-derived HK-2 cells UMI-77 in an AKI state, and belongs to the technical field of methods for relieving TECs injury, in-vivo animal experiments are divided into four groups, namely a Sham group, a UMI-77 group, a UIR group and a UMI-77 + UIR group. Firstly, pathological HE staining is used for observing mouse kidney morphological changes, the result shows that compared with UIR group mice, the conditions of renal tubule expansion and deformation of UMI-77 + UIR group mice are improved, inflammatory cell infiltration and exudation are reduced, then histochemical staining, western blot and real-time quantitative PCR are used for detecting the expression condition of a renal tubule injury index Kim-1, and the result shows that compared with the UIR group mice, the expression condition of the renal tubule injury index Kim-1 is reduced. The Kim-1 expression of the mice in the UMI-77 + UIR group is reduced (* * Plt; the results show that the UMI-77 can relieve the kidney injury of the mouse in the AKI state.

Description

Technical Field

[0001] The present invention relates to a method for reducing TECs damage, in particular to a method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state, belonging to the technical field of methods for reducing TECs damage. Background Art

[0002] The mitochondrial density and oxygen consumption of the kidney are second only to the heart. Mitochondria constantly undergo fission and fusion, biogenesis and mitophagy to control the morphology, quantity and quality of mitochondria. However, mitochondria are easily damaged during AKI. The core pathophysiological feature of renal ischemia-reperfusion injury (IRI) is a complex cascade of oxidative damage, which leads to tubular damage and is accompanied by changes in mitochondrial ultrastructure. In the IRI-induced AKI model, mitochondrial fragmentation and rupture can be observed in TECs, followed by the massive release of apoptotic factors, reactive oxygen species (ROS), cytochrome C, and mtDNA, which further induce inflammation and accelerate cell death. Increasing evidence shows that mitochondrial dysfunction plays a vital role in the occurrence and development of AKI, and regulating mitochondrial function is also considered an important entry point for the prevention and treatment of AKI.

[0003] The role of mitophagy activation in IRI-induced TECs damage remains controversial;

[0004] Maintaining mitochondrial homeostasis is important for cells to maintain activity and perform their physiological functions. Mitochondrial autophagy can selectively remove defective mitochondria to maintain cell homeostasis and function. Many studies have reported the beneficial role of mitophagy in IRI-induced AKI. BNIP3 has the dual function of regulating cell death and mitophagy [4]. Using Bnip3 and Pink1 knockout mouse models, Tang et al. demonstrated that in oxygen-glucose deprivation reperfusion (OGD-R) and IRI models, Bnip3 deficiency reduced mitophagy, leading to the accumulation of damaged mitochondria and increased ROS production, leading to cell death and inflammation [5]. Tang et al. also demonstrated that PINK1-PRKN-mediated mitophagy plays an important role in mitochondrial quality control, TECs survival and renal function in in vitro and in vivo models of ischemic AKI [6]. Knockdown of PINK1 inhibited mitophagy and reduced the cytoprotective effect of ischemic preconditioning (IPC) in rat proximal TECs treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) and in an IRI mouse model, suggesting that mitophagy also plays an important role in the protective effect of IPC.

[0005] However, Liu et al. reported that c-MYC can upregulate RTKN through lncRNA-MEG3 to activate mitophagy and trigger the Wnt / β-catenin pathway, thereby aggravating IRI-AKI.

[0006] Therefore, although the mainstream view is that mitochondrial autophagy activation has a protective effect on TECs damage during the development of AKI, there has been a lack of direct evidence that positive activation of mitochondrial autophagy can reduce TECs damage. In addition, the timing, degree and duration of mitochondrial autophagy activation may also have different effects. How to regulate the activity of mitochondrial autophagy to maximize its protective effect still needs further study.

[0007] UMI-77 may be an effective activator of mitophagy in IRI-AKI, but its activation mode, related targets and mechanisms remain unclear.

[0008] UMI-77 is a BH3 mimetic of myeloid cell leukemia-1 (MCL-1). UMI-77 has a strong binding affinity with MCL-1 protein, which can disrupt the interaction between MCL-1 / Bax protein and induce cancer cell apoptosis at high concentrations. A high-throughput screening of 2024 FDA-approved drugs or candidate drugs using HEK293T cell lines stably expressing mt-Keima found that UMI-77 can effectively induce mitochondrial autophagy without triggering apoptosis at sublethal doses.

[0009] Our preliminary experiments found that in the HR model, UMI-77 can effectively reduce the decrease in TECs activity and apoptosis caused by hypoxia; in addition, in C57 mice treated with UMI-77, the damage to TECs in the mice kidneys was alleviated 5 days after UIR.

[0010] The above results indicate that UMI-77 has a protective effect on renal TECs during IRI-AKI, and we speculate that this effect is achieved by activating mitochondrial autophagy.

[0011] Subsequently, we used in vivo and in vitro AKI models to further explore the activation effect of UMI-77 on mitochondrial autophagy, clarify its effective mode of action and specific mechanism, and help develop new AKI prevention and treatment measures. Summary of the invention

[0012] The main purpose of the present invention is to provide a method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in the AKI state.

[0013] The purpose of the present invention can be achieved by adopting the following technical solutions:

[0014] A method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in the AKI state, comprising the following steps:

[0015] Step 1: Select C57 mice aged 8-10 weeks and divide them into 4 groups: Sham group, UMI-77 group, UIR group and UMI-77+UIR group, with 6-8 mice in each group;

[0016] Step 2: Intraperitoneal injection of UMI-77 and UIR-AKI model;

[0017] Step 3: Collect kidney specimens;

[0018] Step 4: Preparation of paraffin specimens and common pathological staining;

[0019] Step 5: Histochemical staining and immunofluorescence staining of paraffin sections of mouse kidney tissue;

[0020] Step 6: Western blotting of mouse kidney tissue;

[0021] Step 7: Real-time quantitative PCR;

[0022] Step 8: Cell grouping, plating and drug treatment;

[0023] Step 9: CCK8 method was used to detect the activity of HK-2 cells in each group, western blotting method was used to detect the apoptosis of HK-2 cells in each group was used, flow cytometry was used to detect the apoptosis of HK-2 cells in each group, and in situ electron microscopy was performed;

[0024] Step 10: Prepare 3-MA solution;

[0025] Step 11: Flow cytometry and laser confocal microscopy were used to detect the changes of MMP in HK-2 cells;

[0026] Step 12: Knock down the MCL-1 molecule in HK-2 cells with small interfering siRNA;

[0027] Step 13: Detect cell mtROS using laser scanning confocal microscopy;

[0028] Step 14: Detection of cellular ROS by flow cytometry;

[0029] Step 15: Use statistical methods.

[0030] 2. A method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 1, characterized in that: in step 2, UMI-77 is intraperitoneally injected one day before modeling;

[0031] After high-speed centrifugation of 10 mg of UMI-77 powder, 200 μl of DMSO was added according to the instructions to prepare a 2% DMSO stock solution with a concentration of 50 mg / ml;

[0032] According to the ratio of 2% DMSO stock solution: 30% PEG300: ddH2O = 1:8:41, prepare a working solution with a concentration of 1 mg / ml, and use it immediately after preparation;

[0033] The weight of mice was weighed and recorded, and mice in the UMI-77 group and the UMI-77+UIR group were intraperitoneally injected with UMI-77 10 mg / kg;

[0034] In the sham group and UIR group, DMSO was used to replace the 2% DMSO stock solution, and the control solvent was prepared according to the ratio of DMSO: 30% PEG300: ddH2O = 1:8:41, and it was used immediately after preparation and intraperitoneal injection.

[0035] UIR-AKI modeling;

[0036] The mice were weighed and recorded, and the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 μl / g);

[0037] Mice in the UIR and UMI-77+UIR groups were fixed on a surgical board in a prone position;

[0038] The skin of the left kidney area of ​​the mouse was prepared and disinfected, and an incision of about 1 cm was made parallel to the spine;

[0039] The kidney was exposed, and after the renal artery was dissected, the left renal artery was clamped with a micro artery clip, and the kidney turned purple-black;

[0040] During this period, the mice were placed on a 37°C constant temperature blanket, and the incision was covered with moist gauze;

[0041] After 35 minutes, the artery clamp was released, and the entire kidney turned bright red;

[0042] Finally, the left kidney was returned to its original position, and the subcutaneous and skin incisions were sutured in sequence;

[0043] The sham-operated group mice underwent the same surgical procedures as the modeling group, except that the renal pedicle was not clamped;

[0044] After the mice regained consciousness, they were intraperitoneally injected according to the injection formula of the previous day, and the intraperitoneal injection was continued every day until the fifth day after surgery.

[0045] 3. A method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 under AKI state according to claim 1, characterized in that: after the mouse is anesthetized successfully in step 3, the mouse is killed after cardiac blood sampling, and the heart is perfused with ice PBS, the kidney capsule is quickly separated and the kidney is taken out, washed with physiological saline, and the kidney is transversely cut to retain a piece of kidney tissue for making paraffin specimens with a thickness of 2-3 mm, which is fixed in 4% paraformaldehyde solution;

[0046] A 1cm×1cm×3cm piece of kidney tissue was cut from the cortex, placed in the previously prepared electron microscopy fixative, and then sent for examination;

[0047] The remaining kidney tissue was placed in a 1.5 ml enzyme-inactivated EP tube, quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator for storage;

[0048] Step 4: paraffin block preparation;

[0049] Fixed mouse kidney tissue blocks were immersed in 4% paraformaldehyde solution for 24 h;

[0050] Dehydrated tissue blocks were sequentially immersed in 75% ethanol for 40 min, 85% ethanol for 40 min, 95% ethanol for 40 min × 2 times, and anhydrous ethanol for 30 min × 2 times;

[0051] The transparent kidney tissue was immersed in xylene solution twice, the first time for 15 min and the second time for 10 min;

[0052] Wax-immersed tissue blocks were immersed in pre-melted paraffin I for 30 min and paraffin II for 60 min, with the temperature of the paraffin being 60°C to 65°C;

[0053] Embedding: Place the tissue block into the embedding mold with melted paraffin and wait for it to solidify;

[0054] Slice making;

[0055] Use a paraffin slicer to cut out 5 μm paraffin slices, spread, slice and fish out the slices in warm water at 42°C, bake the paraffin slices in a 60°C oven to dry, and store them in a 4°C refrigerator for later use;

[0056] Specific operation steps of HE staining;

[0057] Dewaxing and rehydration: Paraffin sections were sequentially immersed in xylene for 15 min × 2, anhydrous ethanol for 10 s, 95% alcohol for 10 s, and double distilled water for 5 min;

[0058] Hematoxylin staining for 2 minutes, adjusting the time according to the color depth, and washing with running water for 5 minutes;

[0059] 1% hydrochloric acid alcohol for 2 seconds, rinse with running water;

[0060] Ammonia water turns blue for 5-6 seconds, then rinse with running water;

[0061] Stain with eosin for 2 minutes, adjust the time according to the color depth, and rinse with running water;

[0062] After natural air drying or oven drying, add appropriate amount of neutral gum to seal the slide.

[0063] 4. The method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 under AKI state according to claim 1, characterized in that: in step 5, dewaxing and rehydrating: after the paraffin sections are dried in a 60°C oven, they are sequentially immersed in xylene for 15 min×2, anhydrous alcohol for 5 min×2, 95% alcohol for 5 min×2, 75% alcohol for 5 min, and double distilled water for 5 min;

[0064] Microwave antigen repair: Heat the modified sodium citrate antigen repair liquid to boiling at high heat in a microwave oven, put the slices in and repair at medium-high heat for 10 minutes, cool naturally to room temperature, and soak in PBS solution 3 times, 2 minutes each time;

[0065] Protein blocking: draw a circle around the tissue with a histochemical pen, add 20 μl 5% BSA to each tissue, and incubate at room temperature for 30 min;

[0066] Primary antibody incubation: add 20 μl of primary antibody to each tissue and incubate overnight in a humidified box at 4°C.

[0067] Secondary antibody incubation: rewarm for 30 minutes, soak in PBS solution for 5 minutes × 3 times, add secondary antibody PBS buffer to dilute at a dilution ratio of 1:200, and incubate at room temperature in the dark for 1 hour;

[0068] If the histochemical staining continues with DAB color development, the fluorescent staining continues with DAPI nucleus staining;

[0069] DAB color development: soak in PBS solution for 5 min × 3 times, use DAB1:20 for color development, observe appropriate color development under the microscope, and terminate with tap water;

[0070] Stain with hematoxylin for 5 minutes, air-dry the slides, seal the slides with neutral resin, bake dry and wait for filming;

[0071] DAPI nuclear staining: soak in PBS solution for 5 min × 3 times, add DAPI, incubate at room temperature in the dark for 5 min, soak in PBS solution for 5 min × 3 times, add anti-fluorescence quencher to seal the slides, and store in a 4°C refrigerator waiting for laser confocal imaging.

[0072] 5. The method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 4, characterized in that: in step 6, tissue protein extraction and concentration determination;

[0073] The mouse kidney tissue was taken out from the -80℃ refrigerator, a small piece of tissue was quickly cut off on ice, and about 30 mg of the remaining mouse kidney tissue was returned to the -80℃ refrigerator for storage, and placed in 2.0 ml cryotubes. 200 μl of protein lysis buffer was added to each tube in the ratio of RIPA: PMSF: phosphatase inhibitor = 100: 1: 1, and mixed thoroughly. Sterilized steel beads were added, and the tissue was ground in a low-temperature grinder. The machine setting parameters were: -50℃, frequency 60 Hz, time 1 min, standing on ice for 30 min, 13200 rpm, 4℃, centrifugation for 15 min, and the supernatant was aspirated into a 1.5 ml centrifuge tube;

[0074] The protein concentration of each sample was detected by BCA method, and the concentration of each sample was balanced with lysis buffer;

[0075] Protein denaturation;

[0076] Prepare the protein supernatant and 5× protein loading buffer in a ratio of 4:1. Mix thoroughly and heat in a 100°C dry bath for 15 minutes to fully denature the protein. After cooling to room temperature, store in a -20°C refrigerator for later use.

[0077] Sample loading and electrophoresis;

[0078] Prepare the separation gel, wait for the gel to solidify for 20 minutes, then add the concentrated gel and insert the tooth comb;

[0079] 20 μg of protein sample was loaded into each well;

[0080] Add 1.5μl Mark to the left side of the sample well and 2.5μl Mark to the right side. Turn on the power supply and set the constant voltage to 80V. When the blue band on the gel runs to the bottom of the separation gel, stop the electrophoresis.

[0081] Transfer membrane;

[0082] Activate the PVDF membrane in methanol solution, assemble the transfer clip, and place it in a transfer tank filled with pre-cooled transfer solution;

[0083] Cover the transfer tank, turn on the power, set the constant voltage to 100V, and transfer the membrane in an ice bath for 110 minutes. The transfer time is determined by the protein molecular weight.

[0084] Protein blocking;

[0085] The transferred PVDF membrane was washed in PBS-T solution and blocked with 5% skim milk powder at room temperature for 1 h.

[0086] Primary antibody incubation;

[0087] After protein blocking, prepare the primary antibody according to the dilution ratio in the instruction manual or literature, add the corresponding protein band to the primary antibody and incubate at 4°C overnight;

[0088] Secondary antibody incubation;

[0089] The protein bands incubated at 4°C were taken out and reheated at room temperature for 30 min. The primary antibody was recovered and washed with PBS-T solution for 5 min × 3. The secondary antibody was added and incubated at room temperature for 1 h, and then washed with PBS-T solution for 5 min × 3.

[0090] exposure;

[0091] After the developer was thoroughly mixed in a 1:1 ratio, the PVDF membrane was immersed in the developer and then placed in the Azure C500 multifunctional molecular imaging system for development.

[0092] 6. The method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 under AKI state according to claim 1, characterized in that: in step eight, HK-2 cells are divided into a Control group, a UMI-77 group, a HR group, and a UMI-77+HR group;

[0093] According to the cell culture steps, the cells were made into a cell suspension, inoculated at an appropriate cell density and cultured in a 37°C, 5% CO2 cell culture incubator;

[0094] The next day, the UMI-77 group and the UMI-77+HR group were prepared with 2% FBS medium containing UMI-77 at a final concentration of 5 μM to stimulate HK-2 cells;

[0095] After 3 h, the HK-2 cells in the HR group and UMI-77+HR group were placed in a 5% CO2, 1% O2 incubator for 24 h of hypoxia;

[0096] The cells were removed from the hypoxic incubator, replaced with complete culture medium, and placed in a 37°C, 5% CO2 incubator for reoxygenation for 3 h before subsequent testing.

[0097] 7. A method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 1, characterized in that: 10 μl of CCK-8 solution is added to each well of a 96-well plate, and care is taken not to generate bubbles;

[0098] Incubate in a 37°C, 5% CO2 incubator for 1-4 hours;

[0099] Detection: Detect the absorbance value of each well with an enzyme-labeled instrument at a wavelength of 450 nm;

[0100] In the 6-well plate, pour out the culture medium and use a pipette to suck out the remaining culture medium;

[0101] Add appropriate amount of ice PBS to each well for washing, and repeat twice;

[0102] Prepare the lysis buffer at a ratio of RIPA:PMSF:phosphatase inhibitor = 100:1:1 and use it immediately after preparation;

[0103] Add 100-200 μl of RIPA lysis buffer to each well, scrape with a clean scraper, and lyse on ice for 7 min;

[0104] Scrape again and lyse on ice for 30 min to fully lyse the cells;

[0105] After lysis, use a pipette to transfer the lysate to a 1.5 ml EP tube;

[0106] Centrifuge at 4°C, 12,000 rpm for 15 min, take the supernatant, and determine the protein concentration by BCA method;

[0107] Protein denaturation: Add RIPA lysis buffer, balance each group of protein samples, add 5× protein loading buffer at a volume ratio of 4:1, mix well and heat in a 100℃ dry bath for 15 minutes to fully denature the protein. After cooling to room temperature, store in a -20℃ refrigerator for later use;

[0108] Pour off the culture medium on the 12-well plate, wash with PBS for 2 min×2, aspirate the wash solution, digest the cells with 100 μl / well of trypsin for 2 min, add 1 ml of culture medium containing 10% FBS to terminate the digestion, and collect the cell suspension into the corresponding flow tube;

[0109] Annexin V staining: centrifuge the collected cell suspension at 1200 rpm for 5 min, discard the supernatant, add 1 ml of PBS, resuspend, centrifuge at 1200 rpm for 5 min, and discard the supernatant again, being careful not to discard the cell pellet;

[0110] Add 400 μl of 1× Annexin V Binding Solution, 5 μl of Annexin V, FITC conjugate and 5 μl of PI Solution to each flow tube, shake and mix, and incubate at room temperature in the dark for 15 min;

[0111] Determination of apoptosis rate and statistical analysis: 20,000 cells were collected from each tube of stained cell suspension using a flow cytometer to calculate the total apoptosis rate;

[0112] Take a sapphire substrate with words written on the back as a mark;

[0113] Can be sterilized with alcohol or ultraviolet light;

[0114] Treat with poly-lysine before use to increase cell adhesion;

[0115] Place the sapphire substrate in a 96-well plate with the front side (non-writing side) facing upwards, and culture the cells on the front side of the sapphire substrate;

[0116] After treatment of each group, the culture medium was aspirated, electron microscopy fixative was added, and the samples were sent for examination.

[0117] 8. The method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 7, characterized in that: in step 10, according to the recommended table in the instruction manual, 37 mg 3-MA is weighed and dissolved in 5 ml PBS, fully shaken and mixed, heated to facilitate dissolution, and prepared into a 50 mM storage solution;

[0118] -MA was used at a working concentration of 5 mM;

[0119] Before the cells were placed in the hypoxic incubator, 3-MA and UMI-77 were added together to the serum-free medium of the corresponding treatment groups, followed by hypoxia.

[0120] 9. The method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 8, characterized in that: in step 11, the cells are digested and resuspended in 500 μl cell culture medium;

[0121] According to the sample volume, mix JC-1 (200X): ultrapure water: JC-1 staining buffer (5X) at a ratio of 1:160:4, vortex vigorously to fully dissolve and mix, and the mixed solution is the JC-1 staining working solution;

[0122] Add 500 μl of JC-1 staining working solution to each tube, invert several times to mix, and incubate at 37°C for 20 min;

[0123] During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) with JC-1 staining buffer (5X) and distilled water at a ratio of 1:4;

[0124] After incubation at 37°C, centrifuge at 1200 rpm and 4°C for 3-4 min to pellet the cells and discard the supernatant;

[0125] Wash twice with JC-1 staining buffer (1X): add 1 ml of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 1200 rpm, 4°C for 3-4 min to pellet the cells, and discard the supernatant;

[0126] After resuspending with an appropriate amount of JC-1 staining buffer (1X), laser confocal microscopy and flow cytometry were used to analyze the changes in MMP in each group of cells.

[0127] Beneficial technical effects of the present invention:

[0128] The present invention provides a method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 under AKI state. The in vivo animal experiment is divided into 4 groups, namely Sham group, UMI-77 group, UIR group and UMI-77+UIR group. First, the changes in the morphology of mouse kidneys were observed by pathological HE staining. The results showed that compared with the mice in the UIR group, the dilation and deformation of the renal tubules in the mice in the UMI-77+UIR group were improved, and the infiltration and exudation of inflammatory cells were reduced. Subsequently, the expression of Kim-1, an indicator of renal tubular damage, was detected by histochemical staining, protein immunoblotting, and real-time quantitative PCR. The results showed that compared with the mice in the UIR group, the expression of Kim-1 in the mice in the UMI-77+UIR group decreased (**P<0.01). The above results show that UMI-77 can reduce the renal damage of mice under AKI state. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 This is an exploded view of the overall three-dimensional structure of a device according to a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0130] Figure 2 This is an enlarged view of the structure at A of a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0131] Figure 3 A side cross-sectional view of a first telescopic rod according to a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0132] Figure 4 This is an enlarged view of the structure at B of a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0133] Figure 5 A top view of a heat-absorbing tube clamping assembly according to a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0134] Figure 6 This is an enlarged view of the structure at position C of a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0135] Figure 7 A schematic diagram of the structure of a lens-free snap-fit ​​assembly according to a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 in an AKI state according to the present invention;

[0136] Figure 8 This is an enlarged view of the structure at position D of a preferred embodiment of a method for activating mitochondrial autophagy and alleviating TECs damage based on mouse TECs and human HK-2 cells UMI-77 under AKI state according to the present invention. DETAILED DESCRIPTION

[0137] In order to make the technical solution of the present invention more clear and specific to those skilled in the art, the present invention is further described in detail below in conjunction with embodiments and drawings, but the implementation manner of the present invention is not limited thereto.

[0138] Animal grouping;

[0139] C57 mice aged 8-10 weeks were selected and divided into 4 groups: Sham group, UMI-77 group, UIR group and UMI-77+UIR group, with 6-8 mice in each group.

[0140] Intraperitoneal injection of UMI-77 and UIR-AKI model;

[0141] (1) Intraperitoneal injection of UMI-77 was performed one day before modeling;

[0142] After high-speed centrifugation of 10 mg UMI-77 powder, 200 μl DMSO was added according to the instructions to prepare a 2% DMSO stock solution with a concentration of 50 mg / ml; the working solution was prepared at a concentration of 1 mg / ml according to the ratio of 2% DMSO stock solution: 30% PEG300: ddH2O = 1:8:41, and was used immediately. The mice were weighed and recorded, and UMI-77 group and UMI-77+UIR group mice were intraperitoneally injected with UMI-77 (10 mg / kg). For the mice in the sham group and UIR group, DMSO was used instead of 2% DMSO stock solution, and the control solvent was prepared according to the ratio of DMSO: 30% PEG300: ddH2O = 1:8:41, and was used immediately for intraperitoneal injection.

[0143] UIR-AKI modeling

[0144] The mice were weighed and recorded, and 1% sodium pentobarbital (10 μl / g) was injected intraperitoneally to anesthetize the mice. The mice in the UIR group and UMI-77+UIR group were fixed on the surgical board in a prone position. The skin of the left kidney area of ​​the mice was prepared and disinfected, and an incision of about 1 cm was cut parallel to the spine. The kidney was exposed, and after the renal artery was stripped, the left renal artery was clamped with a micro artery clamp, and the kidney turned purple-black. During this period, the mice were placed on a 37°C constant temperature blanket and the incision was covered with moist gauze. After 35 minutes, the artery clamp was released, and the entire kidney turned bright red. Finally, the left kidney was returned to its original position, and the subcutaneous and skin incisions were sutured in sequence. Except for not clamping the renal pedicle, the rest of the surgical operations of the sham group mice were consistent with those of the modeling group. After the mice woke up, they were injected intraperitoneally according to the injection formula of the previous day, and the intraperitoneal injection was continued every day until the fifth day after surgery.

[0145] Kidney specimen collection

[0146] After the mice were anesthetized successfully, they were killed after blood was collected from the heart, and the heart was perfused with ice PBS. The kidney capsule was quickly separated and the kidney was taken out. After washing with saline, the kidney was cut horizontally and a piece of kidney tissue was retained for making paraffin specimens with a thickness of 2-3mm. It was fixed in 4% paraformaldehyde solution. The kidney tissue with a cortex of 1cm×1cm×3cm was cut and placed in the pre-prepared electron microscope fixative, and then sent for examination. The remaining kidney tissue was placed in a 1.5ml enzyme-inactivated EP tube, quickly frozen in liquid nitrogen, and then transferred to a -80℃ refrigerator for storage.

[0147] Paraffin specimen preparation and general pathological staining

[0148] (1) Preparation of paraffin blocks

[0149] ① Fixation: Mouse kidney tissue blocks were immersed in 4% paraformaldehyde solution for 24 hours.

[0150] ② Dehydration: Soak the tissue blocks in 75% ethanol for 40 minutes, 85% ethanol for 40 minutes, 95% ethanol for 40 minutes × 2 times, and anhydrous ethanol for 30 minutes × 2 times.

[0151] ③ Transparency: Immerse the kidney tissue in xylene solution twice, the first time for 15 minutes and the second time for 10 minutes.

[0152] ④ Wax immersion: Immerse the tissue block in pre-melted paraffin I for 30 minutes and paraffin II for 60 minutes. The temperature of the paraffin should be 60℃~65℃.

[0153] ⑤Embedding: Use melted paraffin to put the tissue block into the embedding mold and wait for it to solidify.

[0154] (2) Slice production

[0155] Paraffin slices (5 μm) were cut using a paraffin slicer, spread, sliced ​​and fished out in warm water (42°C), and the paraffin slices were dried in a 60°C oven and stored in a 4°C refrigerator for later use.

[0156] (3) Specific steps of HE staining

[0157] ① Dewaxing and rehydration: paraffin sections were sequentially soaked in xylene (15 min × 2), anhydrous ethanol (10 s), 95% alcohol (10 s) and double distilled water (5 min).

[0158] ② Stain with hematoxylin for 2 minutes (adjust the time according to the depth of color), and rinse with running water for 5 minutes.

[0159] ③1% hydrochloric acid alcohol for 2s, rinse with running water.

[0160] ④The ammonia water turns blue for 5-6 seconds and then rinse with running water.

[0161] ⑤ Stain with eosin for 2 minutes (adjust the time according to the depth of color), and rinse with running water.

[0162] ⑥ After natural air drying or oven drying, add appropriate amount of neutral gum to seal the slide.

[0163] Histochemical and Immunofluorescence Staining of Mouse Kidney Paraffin Sections

[0164] (1) Dewaxing and rehydration: After the paraffin sections were dried in a 60°C oven, they were immersed in xylene (15 min × 2), anhydrous alcohol (5 min × 2), 95% alcohol (5 min × 2), 75% alcohol (5 min), and double distilled water (5 min) in sequence.

[0165] (2) Microwave antigen repair: Heat the modified sodium citrate antigen repair liquid to boiling using high heat in a microwave oven, put the slices in and repair them using medium-high heat for 10 min. After cooling naturally to room temperature, soak in PBS solution three times, each time for 2 min.

[0166] (3) Protein blocking: Draw a circle around the tissue with a histochemical pen, add 20 μl of 5% BSA to each tissue, and incubate at room temperature for 30 min.

[0167] (4) Primary antibody incubation: Add 20 μl of primary antibody (diluted with PBS solution; the primary antibody concentration should be referred to the instructions or literature) to each tissue and incubate in a humidified box at 4°C overnight.

[0168] (5) Secondary antibody incubation: Rewarm for 30 min, soak in PBS solution (5 min x 3 times), add secondary antibody (diluted with PBS buffer, dilution ratio 1:200), and incubate at room temperature in the dark for 1 h. If histochemical staining is performed, proceed to step (6); if fluorescent staining is performed, proceed to step (7).

[0169] (6) DAB color development: Soak in PBS solution (5 min × 3 times), use DAB (1:20) for color development, observe appropriate color development under the microscope, and stop with tap water. Stain with hematoxylin for 5 min, air-dry the slides, seal the slides with neutral resin, bake dry and wait for filming.

[0170] (7) DAPI nuclear staining: soak in PBS solution (5 min × 3 times), add DAPI, incubate at room temperature in the dark for 5 min, soak in PBS solution (5 min × 3 times), add anti-fluorescence quencher to seal the slide, and store in a 4°C refrigerator waiting for laser confocal imaging.

[0171] Western blotting of mouse kidney tissue

[0172] (1) Tissue protein extraction and concentration determination

[0173] Take out the mouse kidney tissue from the -80℃ refrigerator, quickly cut a small piece of tissue on ice, about 30mg (the remaining mouse kidney tissue is returned to the -80℃ refrigerator for storage), put it into a 2.0ml cryotube, add 200μl protein lysis buffer to each tube (configured according to the ratio of RIPA:PMSF:phosphatase inhibitor = 100:1:1, and mix thoroughly), add sterilized steel beads, grind the tissue with a low-temperature grinder (machine setting parameters are: -50℃, frequency 60Hz, time 1min), stand on ice for 30min, 13200rpm, 4℃, centrifuge for 15min, and draw the supernatant into a 1.5ml centrifuge tube. The protein concentration of each sample was detected by the BCA method, and the concentration of each sample was balanced with the lysis buffer.

[0174] (2) Protein denaturation

[0175] Prepare the mixture in a ratio of 4:1 for protein supernatant and 5× protein loading buffer. Mix thoroughly and heat in a 100°C dry bath for 15 min to fully denature the protein. After cooling to room temperature, store in a -20°C refrigerator for later use.

[0176] (3) Sample loading and electrophoresis

[0177] Prepare the separation gel, wait for the gel to solidify (about 20 minutes), then add the concentrated gel and insert the tooth comb. Load 20μg of protein sample into each well. Add 1.5μl Mark to the left side of the loading well and 2.5μl Mark to the right side, turn on the power supply, constant voltage 80V, and stop electrophoresis when the blue band on the gel runs to the bottom of the separation gel.

[0178] (4) Transfer

[0179] Place the PVDF membrane in methanol solution for activation, assemble the transfer clip, and place it in the transfer tank filled with pre-cooled transfer solution. Cover the transfer tank lid, turn on the power supply, constant voltage 100V, and transfer the membrane in an ice bath for 110 minutes (the transfer time is determined by the protein molecular weight).

[0180] (5) Protein blocking

[0181] The transferred PVDF membrane was washed in PBS-T solution and blocked with 5% skimmed milk powder at room temperature for 1 h.

[0182] (6) Primary antibody incubation

[0183] After protein blocking, prepare the primary antibody according to the dilution ratio in the instruction manual or literature, add the primary antibody to the corresponding protein band and incubate at 4°C overnight.

[0184] (7) Secondary antibody incubation

[0185] The protein bands incubated at 4°C were taken out, rewarmed at room temperature for 30 min, the primary antibody was recovered, and the strips were washed with PBS-T solution for 5 min×3. The secondary antibody was added and incubated at room temperature for 1 h, and then washed with PBS-T solution for 5 min×3.

[0186] (8) Exposure

[0187] After the developer was thoroughly mixed in a 1:1 ratio, the PVDF membrane was immersed in the developer and then placed in the Azure C500 multifunctional molecular imaging system for development.

[0188] Real-time quantitative PCR

[0189] Mouse kidney specimens frozen at -80°C were taken, and the mRNA expression level of the gene was detected by real-time quantitative method, and analyzed by PCR instrument and LightCycler 480 amplification instrument. The specific operation steps are as follows:

[0190] (1) Primer design and synthesis

[0191] The primer sequences were synthesized by Sangon Biotech Co., Ltd. The primer sequences are as follows (Table 2.1)

[0192] Table 2.1 Primer sequences

[0193]

[0194]

[0195] Note: F: Forward Sequence; R: Reverse Sequence

[0196] Total RNA extraction

[0197] ① Sample grinding: Take out the mouse kidney tissue from the -80℃ refrigerator, quickly cut off a small piece of tissue on ice (the remaining tissue is stored in the -80℃ refrigerator), put it into a 2.0ml cryotube, add 1ml Trizol, add sterilized steel beads, grind the tissue with a low-temperature grinder (the machine setting parameters are: -50℃, frequency 60Hz, time 1min), and transfer the homogenate to an enzyme-inactivated 1.5ml EP tube.

[0198] ② Extraction of total RNA: Add 200 μl of chloroform to the Trizol sample collected above, shake vigorously for 15 seconds, let stand at room temperature for 15 minutes, and then centrifuge (12300 rpm, 4°C, 15 minutes), and draw the upper aqueous phase into a new enzyme-free 1.5 ml EP tube; add isopropanol (isopropanol: upper aqueous phase = 1:1), mix thoroughly, let stand at room temperature for 25 minutes, and then centrifuge (12300 rpm, 4°C, 10 minutes).

[0199] ③ Wash RNA precipitate: aspirate the supernatant, add 1 ml of 75% alcohol, turn upside down; centrifuge (12300 rpm, 4°C, 5 min), discard the alcohol, and wash twice.

[0200] ④Dissolve RNA: Blot dry with filter paper, absorb the remaining alcohol with a pipette, dry at room temperature (about 5-10 minutes), add DEPC water (depending on the amount of extracted RNA), store in a -80℃ refrigerator or directly perform reverse transcription.

[0201] ⑤ Detect RNA purity and concentration: Measure the absorbance (OD) at 260nm and 280nm wavelengths on a spectrophotometer. A ratio of OD260 / OD280 of 1.8 to 2.0 indicates that the RNA purity is high. Record the RNA concentration.

[0202] (3) Reverse transcription reaction

[0203] Use Takara reverse transcription kit to reverse transcribe total RNA into cDNA. Prepare each reaction solution on ice. First, prepare the genomic DNA removal reaction solution (Table 2.2) and incubate at room temperature for 5 minutes; then prepare the reverse transcription reaction solution (Table 2.3). After loading the sample, put it into the PCR instrument for reverse transcription. The reverse transcription reaction conditions are 25℃, 10min, 42℃, 15min, 85℃, 5min. After obtaining total cDNA, perform real-time quantitative PCR directly or store it in a -20℃ refrigerator.

[0204] Table 2.2 Preparation of genomic DNA removal reaction solution

[0205]

[0206] Note: x means that the maximum amount of Total RNA used in a 20μl reaction system is 1μg

[0207] Table 2.3 Preparation of reverse transcription reaction solution

[0208]

[0210] (4) Real-time quantitative PCR

[0211] Prepare a 10 μl PCR reaction solution as per Table 2.4. Mix all reagents except cDNA thoroughly in advance. Add 9 μl of premixed reagents to each well of a 96-well plate. Finally, add 1 μl of cDNA. Repeat the procedure for each sample in 3 replicate wells. Keep the whole process on ice.

[0212] Table 2.4 Preparation of PCR reaction solution

[0213]

[0214] The amplification conditions were pre-denaturation (95°C, 30s) → annealing (95°C, 5s), extension (60°C, 34s) for 40 cycles → obtaining melting curves and amplification curves.

[0215] (5) Real-time quantitative PCR data processing

[0216] After the reaction is completed, the LightCycler 480 amplification instrument can analyze and calculate the results. The obtained Ct value refers to the number of cycles required for the fluorescence signal to reach the set threshold. The relative expression of the mRNA of the analyzed gene is calculated using the relative quantitative method 2-ΔΔCt.

[0217] Cell grouping, plating and drug treatment

[0218] (1) HK-2 cells were divided into Control group, UMI-77 group, HR group and UMI-77+HR group.

[0219] (2) According to the cell culture steps, the cells were prepared into a cell suspension, inoculated at an appropriate cell density and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0220] (3) The next day, the UMI-77 group and the UMI-77+HR group were prepared with 2% FBS culture medium containing UMI-77 at a final concentration of 5 μM and used to stimulate HK-2 cells.

[0221] (4) After 3 hours, the HK-2 cells in the HR group and the UMI-77+HR group were placed in a 5% CO2, 1% O2 incubator for 24 hours of hypoxia. The cells were removed from the hypoxic incubator, replaced with complete culture medium, and placed in a 37°C, 5% CO2 incubator for 3 hours of reoxygenation before the subsequent tests.

[0222] CCK8 assay to detect the activity of HK-2 cells in each group

[0223] (1) In a 96-well plate, add 10 μl of CCK-8 solution to each well (be careful not to create bubbles).

[0224] (2) Place in a 37°C, 5% CO2 incubator and incubate for 1-4 hours.

[0225] (3) Detection: Use an ELISA reader to detect the absorbance (OD) value of each well at a wavelength of 450 nm.

[0226] 2.2.10 Western blotting of HK-2 cells

[0227] (1) In the 6-well plate, pour out the culture medium and use a pipette to aspirate the remaining culture medium.

[0228] (2) Add appropriate amount of ice-cold PBS to each well for washing and repeat twice.

[0229] (3) Prepare lysis buffer at a ratio of RIPA:PMSF:phosphatase inhibitor = 100:1:1 and use it immediately.

[0230] (4) Add RIPA lysis buffer (100-200 μl / well) to each well, scrape with a clean scraper, and lyse on ice for 7 min. Scrape again and lyse on ice for 30 min to fully lyse the cells.

[0231] (5) After lysis, transfer the lysate to a 1.5 ml EP tube using a pipette. Centrifuge at 4°C, 12,000 rpm for 15 min, take the supernatant, and determine the protein concentration using the BCA method.

[0232] (6) Protein denaturation: Add RIPA lysis buffer to balance the protein samples of each group, add 5× protein loading buffer at a volume ratio of 4:1, mix thoroughly, and heat in a 100°C dry bath for 15 min to fully denature the protein. After cooling to room temperature, store in a -20°C refrigerator for later use.

[0233] Flow cytometry was used to detect the apoptosis of HK-2 cells in each group

[0234] (1) Pour off the culture medium on the 12-well plate, rinse with PBS (2 min × 2), aspirate the rinsing solution, digest the cells with 100 μl / well of trypsin for 2 min, add 1 ml of culture medium containing 10% FBS to terminate the digestion, and collect the cell suspension into the corresponding flow tube.

[0235] (2) Annexin V staining: Centrifuge the collected cell suspension at 1200 rpm for 5 min, discard the supernatant, add 1 ml of PBS, resuspend, centrifuge at 1200 rpm for 5 min, and discard the supernatant again. Be careful not to discard the cell pellet. Add 400 μl of 1× Annexin V Binding Solution, 5 μl of Annexin V, FITC conjugate and 5 μl of PI Solution to each flow tube, shake and mix, and incubate at room temperature in the dark for 15 min.

[0236] (3) Determination of apoptosis rate and statistical analysis: 20,000 cells were collected from each tube of stained cell suspension using a flow cytometer to calculate the total apoptosis rate.

[0237] In situ electron microscopy

[0238] (1) Take a sapphire substrate with the word "up" written on the back as a mark.

[0239] (2) It can be sterilized by alcohol or ultraviolet light.

[0240] (3) Treat with poly-lysine before use to increase cell adhesion.

[0241] (4) Place the sapphire substrate in a 96-well plate with the front side (non-writing side) facing upward, and culture the cells on the front side of the sapphire substrate.

[0242] (5) After treatment of each group, aspirate the culture medium, add electron microscopy fixative, and send for examination.

[0243] 3-MA solution preparation

[0244] (1) According to the instructions, weigh 37 mg of 3-MA and dissolve it in 5 ml of PBS. Vortex thoroughly to mix, heat to aid dissolution, and prepare a 50 mM stock solution.

[0245] (2) The working concentration of 3-MA was 5 mM. Before the cells were placed in the hypoxic incubator, 3-MA and UMI-77 were added to the serum-free medium of the corresponding treatment group, followed by hypoxia.

[0246] Flow cytometry and laser confocal microscopy were used to detect the changes of MMP in HK-2 cells

[0247] (1) Digest the cells and resuspend them in 500 μl of cell culture medium.

[0248] (2) According to the sample volume, mix JC-1 (200X): ultrapure water: JC-1 staining buffer (5X) in a ratio of 1:160:4, vortex vigorously to fully dissolve and mix, and the mixed solution is the JC-1 staining working solution.

[0249] (3) Add 500 μl of JC-1 staining working solution to each tube, invert several times to mix, and incubate at 37°C for 20 min.

[0250] (4) During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) by mixing JC-1 staining buffer (5X) with distilled water at a ratio of 1:4.

[0251] (5) After incubation at 37°C, centrifuge at 1200 rpm and 4°C for 3-4 min to pellet the cells and discard the supernatant.

[0252] (6) Wash twice with JC-1 staining buffer (1X): Add 1 ml of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 1200 rpm and 4°C for 3-4 min to pellet the cells, and discard the supernatant.

[0253] (7) After resuspending with an appropriate amount of JC-1 staining buffer (1X), the changes in MMP of each group of cells were analyzed by laser confocal microscopy and flow cytometry.

[0254] Knockdown of MCL-1 in HK-2 cells by small interfering siRNA

[0255] (1) The cells were divided into two groups, siControl group and siMCL-1 group.

[0256] (2) Transfection system

[0257] Table 2.5 Small interfering transfection system

[0258]

[0259] Seeding cells.

[0260] Dilute the corresponding volume of siRNA to 200 μl Mix by pipetting up and down.

[0261] After vortexing the transfection reagent for 5 seconds, add the corresponding volume of transfection reagent to 200 μl of siRNA and shake to mix.

[0262] Incubate at room temperature for 10-30 minutes to allow siRNA and transfection reagent to form transfection complexes. Do not exceed 30 minutes.

[0263] During complex formation, growth medium was removed and the corresponding volume of fresh pre-warmed complete medium was added to each well.

[0264] Add 200 μl of transfection mixture to the cells and shake gently to mix.

[0265] After incubation at room temperature for 24 h, the knockdown efficiency was verified by western blotting and real-time quantitative PCR as described above. After the knockdown efficiency was determined to be above 85%, the cells were treated according to the UMI-77+HR group to detect mtROS.

[0266] Laser scanning confocal microscopy to detect cellular mtROS

[0267] (1) According to the instructions, add 13 μl DMSO to a tube of MitoSOX probe (50 μg) to prepare a 5 mM MitoSOX stock solution.

[0268] (2) Dilute MitoSOX (5 mM) with PBS to prepare a 2.5 μM MitoSOX working solution.

[0269] (3) Add 1 ml of MitoSOX working solution to each confocal culture dish, and then add 1 μl of Hoechst33342 live cell nuclear staining solution to each dish, and incubate in a 37°C cell culture incubator for 30 min.

[0270] (4) Aspirate the MitoSOX working solution, wash twice with PBS, add 1 ml of DMEM culture medium to each well, and observe the cells under a laser scanning confocal microscope within 2 hours.

[0271] Flow cytometry detection of cellular ROS

[0272] (1) Inoculate cells and perform HR after drug stimulation

[0273] (2) Dilute DCFH-DA (10 mM) with DMEM medium to prepare a 10 μM DCFH-DA working solution;

[0274] (3) Aspirate the cell culture medium from each well, add the prepared DCFH-DA working solution (500 μl / well), and incubate in a 37°C cell culture incubator for 20 min;

[0275] (4) Wash the cells twice with DMEM medium, collect the cells and detect DCFH using flow cytometry.

[0276] Statistical methods

[0277] All data were analyzed using Graphpad Prism 8.0.2 statistical software. The independent sample t test was used to compare the two groups of normally distributed data; the MannWhitney test was used to compare the two variables of non-normally distributed quantitative data; all statistical methods considered the difference statistically significant when P < 0.05.

[0278] Figure 1(B) The effect of UMI-77 on TECs injury in the kidneys of C57 mice after UIR treatment. HE staining was used to detect the renal morphology and structure of C57 mice in each group under AKI state. (C) Protein immunoblotting was used to detect the protein expression level of Kim-1 in the renal cortex of C57 mice in each group. (D) Real-time quantitative PCR was used to detect the mRNA expression level of Kim-1 in the renal cortex of C57 mice in each group. Note: **P<0.01 compared with the UMI-77+UIR group in the UIR group. Scale bar: 100μm.

[0279] UMI-77 alleviates mitochondrial damage in TECs in mouse kidneys under AKI condition;

[0280] We used electron microscopy to detect the morphological and structural changes of mitochondria in the kidney TECs of mice in each group. The results showed that the mitochondria in the kidney TECs of mice in the UIR group were swollen and deformed, the mitochondrial cristae were broken, and vacuoles appeared in the mitochondria; while the mitochondrial morphology of most of the kidney TECs of mice in the UMI-77+UIR group was basically normal, and the mitochondrial cristae were basically intact ( Figure 1 ). This suggests that UMI-77 can alleviate mitochondrial damage in TECs in mouse kidneys under AKI conditions.

[0281] Figure 2 To show the effect of UMI-77 on mitochondrial damage in kidney TECs of C57 mice after UIR treatment, the mitochondrial morphology and structure of kidney TECs of C57 mice in each group were observed by electron microscopy. Scale bar: 1 μm.

[0282] UMI-77 upregulates mitophagy in TECs of mouse kidneys under AKI condition

[0283] We found that UMI-77 can reduce the damage to TECs in the kidney under AKI. In addition, some studies have shown that UMI-77 can activate mitochondrial autophagy. In order to explore whether UMI-77 can reduce the damage to TECs in AKI by activating mitochondrial autophagy, we first used the fluorescence co-staining of TOM20 and LAMP1 to detect the mitochondrial autophagy of TECs. The immunofluorescence results showed that compared with mice in other groups, the renal TOM20 and LAMP1 fluorescence co-staining of mice in the UMI-77+UIR group increased ( Figure 3 A). We then used electron microscopy to observe the mitochondrial autophagy in the TECs of the mouse kidney cortex. The electron microscopy results showed that compared with the mice in other groups, the mice in the UMI-77+UIR group showed mitochondrial autophagy ( Figure 3 B). These results suggest that UMI-77 can upregulate mitophagy in TECs in mouse kidneys under AKI conditions.

[0284] Figure 3 UMI-77 upregulates mitochondrial autophagy in TECs of C57 mouse kidney after UIR treatment

[0285] (A) Immunofluorescence was used to detect the co-localization of TOM20 and LAMP1 in TECs of mice in each group. Scale bar: 10 μm. (B) Electron microscopy was used to observe the mitochondrial autophagy in TECs of kidneys of mice in each group. The red rectangles indicate the mitochondrial autophagy. Scale bars: 1 μm and 500 nm, respectively.

[0286] UMI-77 reduces apoptosis of HK-2 cells under HR condition

[0287] The in vitro HK-2 cell experiment was divided into 4 groups, namely the Control group, UMI-77 group, HR group and UMI-77+HR group. We first used Annexin V and PI double staining flow cytometry to analyze the effect of UMI-77 on HK-2 cell apoptosis under HR state. In the flow cytometer, the fourth quadrant is the normal cell population, the first quadrant is the necrotic cell population, the second quadrant is the late apoptotic cell population, and the third quadrant is the early apoptotic cell population. The sum of the early apoptosis rate and the late apoptosis rate can be obtained to obtain the total apoptosis rate. The results showed that compared with the HR group, the cell apoptosis in the UMI-77+HR group was reduced ( Figure 4 A). We then used Western blotting to detect the effect of UMI-77 on the expression of apoptotic protein Bax in HK-2 cells under HR conditions. The results also showed that compared with the HR group, the expression of apoptotic protein Bax in the UMI-77+HR group decreased ( Figure 4 B). At the same time, we used CCK8 method to detect the effect of UMI-77 on the activity of HK-2 cells in HR state. The results showed that compared with the HR group, the cell viability of the UMI-77+HR group was increased ( Figure 4 C). The above results indicate that UMI-77 can reduce the apoptosis of HK-2 cells under HR state.

[0288] Figure 4 The effect of UMI-77 on apoptosis of HK-2 cells after HR treatment;

[0289] (A) Annexin V and PI double staining flow cytometry analysis of the effect of UMI-77 on apoptosis of HK-2 cells under HR state.

[0290] (B) Western blotting was used to detect the expression level of apoptotic protein Bax in HK-2 cells. (C) CCK-8 assay was used to detect the effect of UMI-77 on the viability of HK-2 cells under HR state. Note: *P<0.05, ***P<0.001.

[0291] UMI-77 alleviates mitochondrial damage in HK-2 cells under HR state;

[0292] Subsequently, we used electron microscopy to observe the changes in mitochondrial morphology and structure in HK-2 cells and found that compared with the HR group, the mitochondrial deformation of HK-2 cells in the UMI-77+HR group was significantly improved, and the mitochondrial cristae remained basically intact ( Figure 4 ).

[0293] Figure 5 The effect of UMI-77 on mitochondrial damage in HK-2 cells after HR treatment;

[0294] The mitochondrial morphology and structure of HK-2 cells in each group were observed by electron microscopy. Scale bar: 500 nm.

[0295] UMI-77 upregulates the level of mitochondrial autophagy in HK-2 cells under HR state;

[0296] We found that UMI-77 can reduce the damage of HR state to HK-2 cells and their mitochondrial morphology. In addition, in the previous mouse kidneys, we observed that UMI-77 can activate mitochondrial autophagy in the mouse kidneys under AKI state. In order to further verify whether UMI-77 can also activate mitochondrial autophagy in HK-2 cells under HR state, we also used TOM20 and LAMP1 fluorescence co-staining to detect mitochondrial autophagy in HK-2 cells. The immunofluorescence results showed that compared with other groups, the co-staining of TOM20 and LAMP1 in the UMI-77+HR group mice increased ( Figure 6 A), then we used electron microscopy to observe the mitochondrial autophagy in HK-2 cells. The electron microscopy results showed that compared with other groups, mitochondrial autophagy occurred in HK-2 cells in the UMI-77+HR group. ( Figure 6 B), these results suggest that UMI-77 can upregulate the level of mitophagy in HK-2 cells under HR state.

[0297] Figure 6 UMI-77 upregulates mitochondrial autophagy in HK-2 cells after HR treatment

[0298] Immunofluorescence was used to detect the co-localization of TOM20 and LAMP1 in HK-2 cells of each group. Scale bar: 10 μm. (B) Electron microscopy was used to observe the mitochondrial autophagy of HK-2 cells of each group. The red rectangles are the mitochondrial autophagy. Scale bar: 1 μm in the upper image and 500 nm in the enlarged image.

[0299] UMI-77 activates mitophagy to alleviate TECs damage in AKI

[0300] In order to further clarify that UMI-77 improves the damage of AKI state to TECs by activating mitochondrial autophagy, we used the autophagy inhibitor 3-MA to detect whether it can weaken or offset the protective effect of UMI-77 on TECs in AKI state. We divided HK-2 cells into Control group, HR group, UMI-77+HR group and UMI-77+3-MA+HR group, and used Annexin V and PI double staining flow cytometry to detect the apoptosis of HK-2 cells in each group. The results of flow apoptosis cytometry showed that compared with the Control group, the apoptosis rate of cells in the HR group increased significantly; compared with the HR group, the apoptosis rate of cells in the UMI-77+HR group decreased; and compared with the UMI-77+HR group, the apoptosis rate of cells in the UMI-77+3-MA+HR group increased ( Figure 6 A and B).

[0301] Figure 7 UMI-77 activates mitophagy and reduces TECs apoptosis;

[0302] (A) Annexin V and PI double staining flow cytometry analysis of the effect of UMI-77 on TECs apoptosis after 3-MA treatment under HR state. (B) Flow cytometry apoptosis analysis was repeated multiple times, and the apoptotic cells in each group were statistically analyzed. Note: **P<0.01, ***P<0.001.

[0303] Next, we used the JC-1 kit to detect the MMP of each group of HK-2 cells. In normal mitochondria, JC-1 forms polymers (JC-1aggregates) in the mitochondrial matrix, producing strong red fluorescence; when MMP decreases, it is a manifestation of early apoptosis, and JC-1 exists in the cytoplasm in monomeric form (JC-1monomers), producing green fluorescence. Therefore, according to the changes in JC-1 fluorescence, the changes in cellular MMP can be judged. The results of JC-1 fluorescence staining showed that compared with the Control group, the red fluorescence of HK-2 cells in the HR group weakened and the green fluorescence increased; compared with the HR group, the green fluorescence of cells in the UMI-77+HR group weakened and the red fluorescence increased; compared with the UMI-77+HR group, the red fluorescence of cells in the UMI-77+3-MA+HR group weakened and the green fluorescence increased ( Figure 7 A). Similarly, the results of JC-1 flow cytometry also showed that compared with the Control group, the JC-1 green fluorescence / red fluorescence of HK-2 cells in the HR group increased; compared with the HR group, the JC-1 green fluorescence / red fluorescence of cells in the UMI-77+HR group decreased; and compared with the UMI-77+HR group, the green fluorescence / red fluorescence of cells in the UMI-77+3-MA+HR group increased. ( Figure 8B and C). The above results indicate that the use of the autophagy inhibitor 3-MA in the AKI state will weaken the protective effect of UMI-77 on TECs in the AKI state, further clarifying that UMI-77 improves the damage of AKI state to TECs by activating mitochondrial autophagy.

[0304] Figure 8 UMI-77 activates mitophagy to maintain MMP stability in TECs

[0305] (A) JC-1 fluorescent probe was used to label the living cells in each group, and the fluorescence expression of JC-1 polymer (red) and JC-1 monomer (green) was observed under a confocal microscope to detect the MMP of HK-2 cells. (B) JC-1 fluorescent probe was used to label the living cells in each group, and the fluorescence ratio of JC-1 monomer (green) and JC-1 polymer (red) was analyzed by flow cytometry to reflect the changes in MMP of HK-2 cells. (C) JC-1 flow cytometry was repeated many times, and the fluorescence ratio of JC-1 monomer (green) and JC-1 polymer (red) in each group of cells was statistically analyzed. Note: **P<0.01, ***P<0.001. Scale bar: 5μm.

[0306] The above description is only a further embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which belong to the protection scope of the present invention.

Claims

1. A method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state, characterized by: The steps include: Step 1: Select C57 mice aged 8-10 weeks and divide them into 4 groups: Sham group, UMI-77 group, UIR group and UMI-77+UIR group, with 6-8 mice in each group; Step 2: Intraperitoneal injection of UMI-77 and UIR-AKI model; Step 3: Collect kidney specimens; Step 4: Preparation of paraffin specimens and common pathological staining; Step 5: Histochemical staining and immunofluorescence staining of paraffin sections of mouse kidney tissue; Step 6: Western blotting of mouse kidney tissue; Step 7: Real-time quantitative PCR; Step 8: Cell grouping, plating and drug treatment; Step 9: CCK8 method was used to detect the activity of HK-2 cells in each group, western blotting method was used to detect the apoptosis of HK-2 cells in each group was used, flow cytometry was used to detect the apoptosis of HK-2 cells in each group, and in situ electron microscopy was performed; Step 10: Prepare 3-MA solution; Step 11: Flow cytometry and laser confocal microscopy were used to detect the changes of MMP in HK-2 cells; Step 12: Knock down the MCL-1 molecule in HK-2 cells with small interfering siRNA; Step 13: Detect cell mtROS using laser scanning confocal microscopy; Step 14: Detection of cellular ROS by flow cytometry; Step 15: Use statistical methods.

2. According to claim 1, a method for activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state, characterized in that: In step 2, UMI-77 was injected intraperitoneally one day before modeling; After high-speed centrifugation of 10 mg of UMI-77 powder, 200 μl of DMSO was added according to the instructions to prepare a 2% DMSO stock solution with a concentration of 50 mg / ml; According to the ratio of 2% DMSO stock solution: 30% PEG300: ddH2O = 1:8:41, prepare a working solution with a concentration of 1 mg / ml, and use it immediately after preparation; The weight of mice was weighed and recorded, and mice in the UMI-77 group and the UMI-77+UIR group were intraperitoneally injected with UMI-77 10 mg / kg; In the sham group and UIR group, DMSO was used to replace the 2% DMSO stock solution, and the control solvent was prepared according to the ratio of DMSO: 30% PEG300: ddH2O = 1:8:41, and it was used immediately after preparation and intraperitoneal injection. UIR-AKI modeling; The mice were weighed and recorded, and the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 μl / g); Mice in the UIR and UMI-77+UIR groups were fixed on a surgical board in a prone position; The skin of the left kidney area of ​​the mouse was prepared and disinfected, and an incision of about 1 cm was made parallel to the spine; The kidney was exposed, and after the renal artery was dissected, the left renal artery was clamped with a micro artery clip, and the kidney turned purple-black; During this period, the mice were placed on a 37°C constant temperature blanket, and the incision was covered with moist gauze; After 35 minutes, the artery clamp was released, and the entire kidney turned bright red; Finally, the left kidney was returned to its original position, and the subcutaneous and skin incisions were sutured in sequence; The sham-operated group mice underwent the same surgical procedures as the modeling group, except that the renal pedicle was not clamped; After the mice regained consciousness, they were intraperitoneally injected according to the injection formula of the previous day, and the intraperitoneal injection was continued every day until the fifth day after surgery.

3. The method of activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 1, characterized in that: After the mice were anesthetized successfully in step 3, blood was collected from the heart of the mice and then they were killed. The hearts were perfused with iced PBS, the kidney capsule was quickly separated and the kidney was taken out. After washing with saline, the kidney was cut transversely and a piece of kidney tissue was retained for making paraffin specimens with a thickness of 2-3 mm, which were fixed in 4% paraformaldehyde solution. A 1cm×1cm×3cm piece of kidney tissue was cut from the cortex, placed in a pre-prepared electron microscopy fixative, and then sent for examination; The remaining kidney tissue was placed in a 1.5 ml enzyme-inactivated EP tube, quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator for storage; Step 4: paraffin block preparation; Fixed mouse kidney tissue blocks were immersed in 4% paraformaldehyde solution for 24 h; Dehydrated tissue blocks were sequentially immersed in 75% ethanol for 40 min, 85% ethanol for 40 min, 95% ethanol for 40 min × 2 times, and anhydrous ethanol for 30 min × 2 times; The transparent kidney tissue was immersed in xylene solution twice, the first time for 15 min and the second time for 10 min; Wax-immersed tissue blocks were immersed in pre-melted paraffin I for 30 min and paraffin II for 60 min, with the temperature of the paraffin being 60°C to 65°C; Embedding: Place the tissue block into the embedding mold with melted paraffin and wait for it to solidify; Slice making; Use a paraffin slicer to cut out 5 μm paraffin slices, spread, slice and fish out the slices in warm water at 42°C, bake the paraffin slices in a 60°C oven to dry, and store them in a 4°C refrigerator for later use; Specific operation steps of HE staining; Dewaxing and rehydration: Paraffin sections were sequentially immersed in xylene for 15 min × 2, anhydrous ethanol for 10 s, 95% alcohol for 10 s, and double distilled water for 5 min; Hematoxylin staining for 2 minutes, adjusting the time according to the color depth, and washing with running water for 5 minutes; 1% hydrochloric acid alcohol for 2 seconds, rinse with running water; Ammonia water turns blue for 5-6 seconds, then rinse with running water; Stain with eosin for 2 minutes, adjust the time according to the color depth, and rinse with running water; After natural air drying or oven drying, add appropriate amount of neutral gum to seal the slide.

4. The method of activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 1, characterized in that: Dewaxing and rehydration in step 5: After the paraffin sections are dried in a 60°C oven, they are sequentially soaked in xylene for 15 min×2, anhydrous alcohol for 5 min×2, 95% alcohol for 5 min×2, 75% alcohol for 5 min, and double distilled water for 5 min; Microwave antigen repair: Heat the modified sodium citrate antigen repair liquid to boiling at high heat in a microwave oven, put the slices in and repair at medium-high heat for 10 minutes, cool naturally to room temperature, and soak in PBS solution 3 times, 2 minutes each time; Protein blocking: draw a circle around the tissue with a histochemical pen, add 20 μl 5% BSA to each tissue, and incubate at room temperature for 30 min; Primary antibody incubation: add 20 μl of primary antibody to each tissue and incubate overnight in a humidified box at 4°C. Secondary antibody incubation: rewarm for 30 minutes, soak in PBS solution for 5 minutes × 3 times, add secondary antibody PBS buffer to dilute at a dilution ratio of 1:200, and incubate at room temperature in the dark for 1 hour; If the histochemical staining continues with DAB color development, the fluorescent staining continues with DAPI nucleus staining; DAB color development: soak in PBS solution for 5 min × 3 times, use DAB1:20 for color development, observe appropriate color development under the microscope, and terminate with tap water; Stain with hematoxylin for 5 minutes, air-dry the slides, seal the slides with neutral resin, bake dry and wait for filming; DAPI nuclear staining: soak in PBS solution for 5 min × 3 times, add DAPI, incubate at room temperature in the dark for 5 min, soak in PBS solution for 5 min × 3 times, add anti-fluorescence quencher to seal the slides, and store in a 4°C refrigerator waiting for laser confocal imaging.

5. The method of activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 4, characterized in that: Extraction and concentration determination of tissue protein in step 6; The mouse kidney tissue was taken out from the -80℃ refrigerator, a small piece of tissue was quickly cut off on ice, and about 30 mg of the remaining mouse kidney tissue was returned to the -80℃ refrigerator for storage, and placed in 2.0 ml cryotubes. 200 μl of protein lysis buffer was added to each tube in the ratio of RIPA: PMSF: phosphatase inhibitor = 100: 1: 1, and mixed thoroughly. Sterilized steel beads were added, and the tissue was ground in a low-temperature grinder. The machine setting parameters were: -50℃, frequency 60 Hz, time 1 min, standing on ice for 30 min, 13200 rpm, 4℃, centrifugation for 15 min, and the supernatant was aspirated into a 1.5 ml centrifuge tube; The protein concentration of each sample was detected by BCA method, and the concentration of each sample was balanced with lysis buffer; Protein denaturation; Prepare the protein supernatant and 5× protein loading buffer in a ratio of 4:

1. Mix thoroughly and heat in a 100°C dry bath for 15 minutes to fully denature the protein. After cooling to room temperature, store in a -20°C refrigerator for later use. Sample loading and electrophoresis; Prepare the separation gel, wait for the gel to solidify for 20 minutes, then add the concentrated gel and insert the tooth comb; 20 μg of protein sample was loaded into each well; Add 1.5μl Mark to the left side of the sample well and 2.5μl Mark to the right side. Turn on the power supply and set the constant voltage to 80V. When the blue band on the gel runs to the bottom of the separation gel, stop the electrophoresis. Transfer membrane; Activate the PVDF membrane in methanol solution, assemble the transfer clip, and place it in a transfer tank filled with pre-cooled transfer solution; Cover the transfer tank, turn on the power, set the constant voltage to 100V, and transfer the membrane in an ice bath for 110 minutes. The transfer time is determined by the protein molecular weight. Protein blocking; The transferred PVDF membrane was washed in PBS-T solution and blocked with 5% skim milk powder at room temperature for 1 h. Primary antibody incubation; After protein blocking, prepare the primary antibody according to the dilution ratio in the instruction manual or literature, add the corresponding protein band to the primary antibody and incubate at 4°C overnight; Secondary antibody incubation; The protein bands incubated at 4°C were taken out and reheated at room temperature for 30 min. The primary antibody was recovered and washed with PBS-T solution for 5 min × 3. The secondary antibody was added and incubated at room temperature for 1 h, and then washed with PBS-T solution for 5 min × 3. exposure; After the developer was thoroughly mixed in a 1:1 ratio, the PVDF membrane was immersed in the developer and then placed in the Azure C500 multifunctional molecular imaging system for development.

6. The method of activating mitophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 1, characterized in that: In step eight, HK-2 cells were divided into Control group, UMI-77 group, HR group, and UMI-77+HR group; According to the cell culture steps, the cells were made into a cell suspension, inoculated at an appropriate cell density and cultured in a 37°C, 5% CO2 cell culture incubator; The next day, the UMI-77 group and the UMI-77+HR group were prepared with 2% FBS medium containing UMI-77 at a final concentration of 5 μM to stimulate HK-2 cells; After 3 h, the HK-2 cells in the HR group and UMI-77+HR group were placed in a 5% CO2, 1% O2 incubator for 24 h of hypoxia; The cells were removed from the hypoxic incubator, replaced with complete culture medium, and placed in a 37°C, 5% CO2 incubator for reoxygenation for 3 h before subsequent testing.

7. The method of activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 1, characterized in that: In a 96-well plate, add 10 μl of CCK-8 solution to each well, being careful not to create bubbles; Incubate in a 37°C, 5% CO2 incubator for 1-4 hours; Detection: Detect the absorbance value of each well with an enzyme-labeled instrument at a wavelength of 450 nm; In the 6-well plate, pour out the culture medium and use a pipette to suck out the remaining culture medium; Add appropriate amount of ice PBS to each well for washing, and repeat twice; Prepare the lysis buffer at a ratio of RIPA:PMSF:phosphatase inhibitor = 100:1:1 and use it immediately after preparation; Add 100-200 μl of RIPA lysis buffer to each well, scrape with a clean scraper, and lyse on ice for 7 min; Scrape again and lyse on ice for 30 min to fully lyse the cells; After lysis, use a pipette to transfer the lysate to a 1.5 ml EP tube; Centrifuge at 4°C, 12,000 rpm for 15 min, take the supernatant, and determine the protein concentration by BCA method; Protein denaturation: Add RIPA lysis buffer, balance each group of protein samples, add 5× protein loading buffer at a volume ratio of 4:1, mix well and heat in a 100℃ dry bath for 15 minutes to fully denature the protein. After cooling to room temperature, store in a -20℃ refrigerator for later use; Pour off the culture medium on the 12-well plate, wash with PBS for 2 min×2, aspirate the wash solution, digest the cells with 100 μl / well of trypsin for 2 min, add 1 ml of culture medium containing 10% FBS to terminate the digestion, and collect the cell suspension into the corresponding flow tube; Annexin V staining: centrifuge the collected cell suspension at 1200 rpm for 5 min, discard the supernatant, add 1 ml of PBS, resuspend, centrifuge at 1200 rpm for 5 min, and discard the supernatant again, being careful not to discard the cell pellet; Add 400 μl of 1× Annexin V Binding Solution, 5 μl of Annexin V, FITC conjugate and 5 μl of PI Solution to each flow tube, shake and mix, and incubate at room temperature in the dark for 15 min; Determination of apoptosis rate and statistical analysis: 20,000 cells were collected from each tube of stained cell suspension using a flow cytometer to calculate the total apoptosis rate; Take a sapphire substrate with words written on the back as a mark; Can be sterilized with alcohol or ultraviolet light; Treat with poly-lysine before use to increase cell adhesion; Place the sapphire substrate in a 96-well plate with the front side (non-writing side) facing upwards, and culture the cells on the front side of the sapphire substrate; After treatment of each group, the culture medium was aspirated, electron microscopy fixative was added, and the samples were sent for examination.

8. The method of activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 7, characterized in that: In step 10, according to the instructions, weigh 37 mg of 3-MA and dissolve it in 5 ml of PBS, shake it thoroughly, heat it to aid dissolution, and prepare a 50 mM storage solution; -MA was used at a working concentration of 5 mM; Before the cells were placed in the hypoxic incubator, 3-MA and UMI-77 were added together to the serum-free medium of the corresponding treatment groups, followed by hypoxia.

9. The method of activating mitochondrial autophagy and reducing TECs damage based on mouse TECs and human HK-2 cells UMI-77 in AKI state according to claim 8, characterized in that: In step 11, cells were digested and resuspended in 500 μl of cell culture medium; According to the sample volume, mix JC-1 (200X): ultrapure water: JC-1 staining buffer (5X) at a ratio of 1:160:4, vortex vigorously to fully dissolve and mix, and the mixed solution is the JC-1 staining working solution; Add 500 μl of JC-1 staining working solution to each tube, invert several times to mix, and incubate at 37°C for 20 min; During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) with JC-1 staining buffer (5X) and distilled water at a ratio of 1:4; After incubation at 37°C, centrifuge at 1200 rpm and 4°C for 3-4 min to pellet the cells and discard the supernatant; Wash twice with JC-1 staining buffer (1X): add 1 ml of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 1200 rpm, 4°C for 3-4 min to pellet the cells, and discard the supernatant; After resuspending with an appropriate amount of JC-1 staining buffer (1X), laser confocal microscopy and flow cytometry were used to analyze the changes in MMP in each group of cells.

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