Application of transcription factor Bach1 in promoting renal ischemia-reperfusion injury and mitochondrial glutathione depletion

Abstract

The invention discloses application of a transcription factor Bach1 in promoting renal ischemia-reperfusion injury and mitochondrial glutathione depletion, and belongs to the technical field of biology. An ischemia-reperfusion renal injury mouse model is adopted, and kidney inflammatory response, renal injury and mitochondrial dysfunction of a mouse can be remarkably improved through BACH1 gene knockout. Cell experiments verify that BACH1 gene silencing can significantly improve mouse kidney injury and kidney inflammatory response, and increase the mitochondrial reduced glutathione content by up-regulating the SLC25A39 content so as to improve mitochondrial dysfunction, BACH1 overexpression can aggravate mouse kidney injury and kidney inflammatory response, inhibit SLC25A39 expression so as to reduce the mitochondrial reduced glutathione content, and BACH1 overexpression can inhibit SLC25A39 expression so as to improve mitochondrial dysfunction. The invention proves that BACH1 has a regulation effect on SLC25A39, and can provide a new intervention target for renal ischemia-reperfusion injury.

Description

Technical Field

[0001] The invention relates to application of transcription factor Bach1 in promoting renal ischemia-reperfusion injury and mitochondrial glutathione depletion, and belongs to the field of biotechnology. Background Art

[0002] Acute renal ischemia-reperfusion injury (I / R) is an important cause of acute kidney injury. The incidence of acute renal ischemia-reperfusion injury is high and is clinically seen in prerenal hypoperfusion or renal vascular disease caused by various causes, including shock, heart failure, renal transplantation, heart, abdominal aorta surgery and malignant hypertension. [1-4] Clinical manifestations include azotemia, water and electrolyte acid-base imbalance, progressive decrease in urine volume or even anuria. In severe cases, multiple organ dysfunctions such as heart, lung, and brain may occur. The mortality rate is as high as 43%-70%. [5-9] ; Some patients develop chronic kidney disease stage 3 or higher, which significantly increases the economic burden on family and social medical insurance [5] Currently, the clinical use of reduced glutathione prodrug N-acetylcysteine ​​or peripheral intravenous supplementation of reduced glutathione is expected to improve renal ischemia-reperfusion injury, but the efficacy is poor and cannot shorten the course of the disease or effectively improve renal prognosis. There is still a lack of targeted treatment methods for the pathogenesis [10 ,11] Mitochondrial glutathione depletion in proximal tubular epithelial cells is a key pathogenic mechanism of acute renal ischemia-reperfusion injury; therefore, intervention targets for mitochondrial glutathione depletion are expected to become a new therapeutic direction to improve the prognosis of acute renal ischemia-reperfusion injury.

[0003] The content of reduced glutathione in mitochondria accounts for about 10-15% of the total amount of reduced glutathione in cells. Mitochondrial reduced glutathione plays an important role in mitochondrial function and cell survival. Mitochondrial reduced glutathione depletion refers to a reduction of more than 40% in mitochondrial reduced glutathione, which leads to a decrease in the activity of enzymes dependent on reduced glutathione in mitochondria, a decrease in the ability to scavenge reactive oxygen species, and further aggravates oxidative stress damage. [12,13] Reduced glutathione (L-gamma-glutamyl-L-cysteinyl-glycine, GSH) is the most abundant low molecular weight thiol in mammalian cells and has a very important antioxidant effect. [14-16]Mitochondrial reduced glutathione depletion participates in the pathological process of acute renal ischemia-reperfusion injury through two pathways: on the one hand, reduced glutathione plays an anti-oxidative stress role by directly removing excess reactive oxygen species (ROS) and exogenous and exogenous electrophilic substances; therefore, when renal tubular epithelial cells produce excessive ROS, resulting in GSH depletion and insufficient antioxidant effect, it will aggravate the oxidative stress damage of the renal tubules. [17-19] Acute renal ischemia-reperfusion injury mainly causes proximal tubular damage, leading to dysfunction of reabsorption and secretion.

[20] Proximal tubular epithelial cells are rich in mitochondria, which produce about 90% of the total ROS generated by the cells during cell metabolism.

[21] , especially during the reperfusion phase after renal ischemia, more reactive oxygen species will be produced, promoting the occurrence of renal tubular oxidative stress injury. In addition, in renal ischemia-reperfusion injury, the energy supply mode of proximal tubular epithelial cells changes from fatty acid oxidation under physiological conditions to anaerobic glycolysis as an energy source. [22,23] , energy production is significantly reduced, which further promotes the occurrence of lipid peroxidation stress injury. Cisplatin-induced acute renal tubular injury

[12] , ROS accumulation and GSH depletion in renal tissue can be seen, and mitochondrial damage occurs, further activating the IL-6 / JAK / STAT3 signaling pathway, promoting lipid peroxidation-related ferroptosis and cell apoptosis, and thus leading to renal tubular epithelial cell damage. In Fabry disease, the β-galactosidase α gene mutation leads to a decrease in the GSH content in renal tissue, inducing the generation of oxidative stress in renal tissue.

[24] It can be seen that glutathione depletion will aggravate the oxidative stress and mitochondrial function damage of the renal tubules.

[0004] On the other hand, GSH indirectly exerts its antioxidant effect by affecting the activity of enzymes in mitochondria, including glutathione peroxidase, glutathione reductase, catalase, and glutathione-S-transferase. When GSH is depleted, enzymes that rely on GSH for biological activity become inactivated, exacerbating acute renal ischemia-reperfusion injury. [25,26] Studies have found that when GSH is deficient, selenocysteine ​​in GPX4 is catalyzed into selenoamide and inactivated, leading to lipid peroxidation and exacerbating acute renal ischemia-reperfusion injury. [27,28] Therefore, the effect of mitochondrial glutathione depletion on mitochondrial function is particularly critical. In summary, mitochondrial glutathione depletion is an important pathogenic mechanism of mitochondrial function damage caused by acute renal ischemia-reperfusion injury. However, no research has been reported on intervention measures that effectively increase mitochondrial glutathione content.

[0005] The transcription factor BACH1 (BTB and CNC homology 1) is widely expressed in various tissues and organs of mammals. It is a member of the basic leucine zipper protein family and is involved in the deterioration of various diseases involving oxidative stress, such as ischemic heart disease, acute lung injury, and osteoporosis. [29-31] However, there are no studies on the mechanism of action of BACH1 on mitochondrial oxidative stress and mitochondrial function in acute renal ischemia-reperfusion injury. There are no studies on the effect of BACH1 on mitochondrial glutathione depletion.

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[0034]

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[0036] The present invention discovered for the first time the mechanism of action of BACH1 in promoting mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury, providing a new idea and method for the prevention and treatment of renal ischemia-reperfusion injury.

[0037] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0038] The first aspect of the present invention includes the use of a BACH1 molecule as a biological marker in renal ischemia-reperfusion injury.

[0039] (1) In one embodiment of the present invention, the BACH1 molecule is any one or more of the following: BACH1 gene, protein and / or mRNA;

[0040] (2) The sources of biomarker detection include blood, urine, renal tissue, and renal tubular epithelial cells.

[0041] The second aspect of the present invention includes the use of a BACH1 inhibitor in preparing a drug or a pharmaceutical composition for treating renal ischemia-reperfusion injury, and the drug composition or the pharmaceutical composition also includes an inhibitor capable of inhibiting BACH1 expression.

[0042] (1) A knockout reagent comprising the BACH1 gene (BACH1 siRNA base sequence: sense: GCCCAUAUGCUUGUGUCAUTT; antisense: AUGACACAAGCAUAUGGGCTT);

[0043] (2) regulatory factors that inhibit BACH1 gene or mRNA expression;

[0044] (3) Reagents that inhibit BACH1 protein activity.

[0045] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicine having at least one of the following functions:

[0046] (1) Improve renal tubular damage induced by renal ischemia-reperfusion injury and improve renal tissue pathological changes;

[0047] (2) reduce the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α induced by renal ischemia-reperfusion injury;

[0048] (3) inhibiting the production of ROS induced by renal ischemia-reperfusion;

[0049] (4) Improve the decrease of mitochondrial GSH induced by renal ischemia-reperfusion injury;

[0050] (5) Improves GSH-dependent enzyme activity in mitochondria induced by renal ischemia-reperfusion injury;

[0051] (6) Improve the mRNA expression and content of glutathione peroxidase 4 (GPX4) in renal ischemia-reperfusion injury;

[0052] (7) Alleviate mitochondrial damage induced by renal ischemia-reperfusion.

[0053] The third aspect of the present invention includes the use of a BACH1 molecule regulating agent in BACH1 inhibiting SLC25A39 expression. The BACH1 molecule regulating agent includes a BACH1 molecule enhancer or a BACH1 molecule inhibitor, wherein the BACH1 enhancer enhances the activity and / or expression of the BACH1 molecule, and the BACH1 molecule inhibitor inhibits the activity and / or expression of the BACH1 molecule.

[0054] (1) The BACH1 molecule includes BACH1 gene, protein and / or mRNA;

[0055] (2) The regulatory agent of the BACH1 molecule includes inhibiting or enhancing the activity and / or expression of the BACH1 molecule, thereby inhibiting or enhancing BACH1 from inhibiting the expression of SLC25A39.

[0056] In one embodiment of the present invention, the regulatory agent of the BACH1 molecule includes: 1) a vector, an overexpression or a knockout agent comprising the BACH1 gene; 2) a regulatory factor that enhances or inhibits the expression of the BACH1 gene or mRNA;

[0057] 3) Agents that enhance or inhibit the activity of BACH1 protein. The regulating agent includes, according to the needs of the specific implementation, any agent in the prior art, as long as it can enhance or inhibit the expression level of BACH1 molecules.

[0058] The fourth aspect of the present invention includes the use of a regulatory agent for preparing a SLC25A39 molecule in a pharmaceutical composition for preventing and / or treating renal ischemia-reperfusion injury.

[0059] (1) The regulatory agent enhances or inhibits the activity and / or expression of the SLC25A39 molecule.

[0060] (2) The SLC25A39 molecule includes SLC25A39 protein, gene and / or mRNA.

[0061] (3) The regulating agent includes: 1) a vector, overexpression or knockout agent containing the SLC25A39 gene;

[0062] 2) regulatory factors that enhance or inhibit SLC25A39 gene or mRNA expression;

[0063] 3) Agents that enhance or inhibit the activity of SLC25A39 protein.

[0064] (4) The regulating agent includes, according to the needs of the specific implementation, the regulating agent can be any agent in the prior art, as long as it can regulate the expression level of the SLC25A39 molecule.

[0065] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicine having at least one of the following functions:

[0066] (1) Improve renal tubular damage induced by renal ischemia-reperfusion injury and improve renal tissue pathological changes;

[0067] (2) reduce the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α induced by renal ischemia-reperfusion injury;

[0068] (3) inhibiting the production of ROS induced by renal ischemia-reperfusion;

[0069] (4) Improve the decrease in mitochondrial GSH content induced by renal ischemia-reperfusion injury;

[0070] (5) Improves GSH-dependent enzyme activity in mitochondria induced by renal ischemia-reperfusion injury;

[0071] (6) Alleviate mitochondrial damage induced by renal ischemia-reperfusion.

[0072] The fifth aspect of the present invention provides a use of a Bach1 inhibitor in the preparation of a drug for treating and / or alleviating mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury.

[0073] The sixth aspect of the present invention provides a use of a Bach1 inhibitor or a SLC25A39 overexpression agent in the preparation of a drug for treating and / or alleviating renal ischemia-reperfusion injury.

[0074] In one embodiment of the present invention, the Bach1 inhibitor is an agent that inhibits the expression of the transcription factor Bach1; the SLC25A39 overexpression agent is an agent that increases the expression level of SLC25A39.

[0075] In one embodiment of the present invention, the sequence of Bach1 is as follows: Gene ID: 571;

[0076] In one embodiment of the present invention, the sequence of SLC25A39 is as follows: Gene ID: 51629;

[0077] In one embodiment of the present invention, the use in preparing a drug having at least one of the following functions:

[0078] (1) Improve renal tubular damage induced by renal ischemia-reperfusion injury and improve renal tissue pathological changes;

[0079] (2) reduce the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α induced by renal ischemia-reperfusion injury;

[0080] (3) inhibiting the production of ROS induced by renal ischemia-reperfusion;

[0081] (4) Improve the decrease of mitochondrial GSH induced by renal ischemia-reperfusion injury;

[0082] (5) Improves GSH-dependent enzyme activity in mitochondria induced by renal ischemia-reperfusion injury;

[0083] (6) Improve the mRNA expression and content of glutathione peroxidase 4 (GPX4) in renal ischemia-reperfusion injury;

[0084] (7) Alleviate mitochondrial damage induced by renal ischemia-reperfusion.

[0085] The seventh aspect of the present invention provides a method for screening a drug for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction, characterized in that the drug can promote the expression of SLC25A39 protein, and the method is:

[0086] In the test group, a test compound is added to the culture system, and the expression level and / or activity of Bach1 in the test group is observed; in the control group, no test compound is added to the same culture system, and the expression level and / or activity of Bach1 in the control group is observed;

[0087] If the expression level and / or activity of Bach1 in the test group is lower than that in the control group, it indicates that the test compound is a drug for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction.

[0088] The eighth aspect of the present invention is to provide a cell model for screening drugs for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction, wherein the drugs can promote the expression of SLC25A39 protein, and the cell model is:

[0089] In the test group, the test compound is added to the culture system, the test compound is co-incubated with HK-2 cells, and the expression level and / or activity of Bach1 in the test group is observed; in the control group, the test compound is not added to the same culture system, the test compound is co-incubated with HK-2 cells, and the expression level and / or activity of Bach1 in the control group is observed;

[0090] If the expression level and / or activity of Bach1 in the test group is lower than that in the control group, it indicates that the test compound is a drug for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction.

[0091] The ninth aspect of the present invention provides any of the following uses of a Bach1 marker and / or a substance for detecting the Bach1 marker:

[0092] (1) Application in screening candidate drugs for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction;

[0093] (2) Application in screening drugs for treating and / or alleviating mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury;

[0094] In one embodiment of the present invention, the product includes a test paper, a probe, a kit, an antibody, and an aptamer;

[0095] In one embodiment of the present invention, the substance for detecting the Bach1 marker includes a reagent for detecting the Bach1 gene or protein content, or a substance binding to the Bach1 protein;

[0096] In one embodiment of the present invention, the substance binding to Bach1 protein includes an antibody, a polypeptide, an aptamer, a protein or a nucleic acid molecule.

[0097] Beneficial Effects

[0098] The present invention adopts a mouse model of renal ischemia-reperfusion injury with bilateral renal artery clamping, and human proximal tubular epithelial cell line is subjected to hypoxia / reoxygenation treatment. It is first found that BACH1 expression is upregulated and SLC25A39 expression is downregulated in the mouse model of renal ischemia-reperfusion injury and human proximal tubular epithelial cells subjected to hypoxia / reoxygenation treatment; after BACH1 gene knockout in mice, renal inflammation, renal function, tubular function, oxidative stress and mitochondrial damage induced by renal ischemia-reperfusion injury can be significantly improved, and SLC25A39 expression is significantly upregulated, and mitochondrial reduced glutathione content is partially restored. After BACH1 gene silencing in human proximal tubular epithelial cells, hypoxia / reoxygenation treatment is performed, and similar results are obtained, which illustrate that BACH1 plays an important role in aggravating renal ischemia-reperfusion injury.

[0099] Further studies have found that there is a binding site between BACH1 and the SLC25A39 promoter region. In vitro experiments have shown that after the proximal tubular epithelial cells are treated with hypoxia / reoxygenation, overexpression of BACH1 can further inhibit the expression of SLC25A39, thereby further increasing the levels of inflammatory factors and oxidative stress indicators, exacerbating mitochondrial glutathione depletion, and aggravating mitochondrial damage. This clarifies the important role of BACH1-SLC25A39 transcriptional regulation in renal ischemia-reperfusion injury, and provides a theoretical basis for targeting BACH1, intervening in SLC25A39, and treating renal ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] In the following figures, I / R refers to renal ischemia-reperfusion injury, H / R refers to hypoxia / reoxygenation, and HK-2 refers to human proximal tubular epithelial cells.

[0101] Figure 1 : BACH1 expression is upregulated in mice with renal ischemia-reperfusion injury. A is the immunohistochemical detection of BACH1 expression in normal tissues adjacent to renal cancer and kidneys with acute tubular necrosis; B is the semi-quantitative analysis of BACH1 immunohistochemical results.

[0102] Figure 2 : BACH1 expression is upregulated in mice with renal ischemia-reperfusion injury. A is the relative quantitative analysis of total BACH1 protein expression and gray value in mouse kidney tissue; B is the result of real-time fluorescence quantitative PCR (qRT-PCR) detection of BACH1 mRNA expression level in mouse kidney tissue; C is the result of BACH1 immunofluorescence and relative quantitative analysis in mouse kidney tissue; D is the result of BACH1 immunohistochemistry and relative quantitative analysis in mouse kidney tissue; E is the result of BACH1 protein expression and relative quantitative analysis in the nucleus.

[0103] Figure 3 : BACH1 expression is upregulated in proximal tubular epithelial cells after hypoxia / reoxygenation. A is the mRNA expression of BACH1 in HK-2 cells after hypoxia / reoxygenation; B is the protein expression of BACH1 and GPX4 in HK-2 cells after hypoxia / reoxygenation and their relative quantitative analysis; C is the nuclear protein expression and relative quantitative analysis of BACH1 in HK-2 cells after hypoxia / reoxygenation; D is the immunofluorescence staining of BACH1 in HK-2 cells after hypoxia / reoxygenation.

[0104] Figure 4 : BACH1 gene knockout improves inflammation and renal function in mice with renal ischemia-reperfusion injury. Among them, AC shows that BACH1 gene knockout reduces the mRNA levels of inflammatory factors in mice with renal ischemia-reperfusion injury; D shows that BACH1 gene knockout reduces the renal tubular injury indicators in mice with renal ischemia-reperfusion injury (urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1)); E shows that BACH1 gene knockout reduces the renal function of mice with renal ischemia-reperfusion injury (serum creatinine (Scr) and urea nitrogen (BUN)); F shows the HE staining of renal tissues in mice with renal ischemia-reperfusion injury in BACH1 gene knockout mice, showing that renal tubular injury is reduced.

[0105] Figure 5: BACH1 gene knockout improves mitochondrial dysfunction in mice with renal ischemia-reperfusion injury. AC is the result of mitochondrial GSH and superoxide dismutase (SOD) content in BACH1 gene knockout mice after renal ischemia-reperfusion injury; DF is the result of mitochondrial reactive oxygen species, adenosine triphosphate (ATP), and renal tissue mitochondrial ultrastructure in BACH1 gene knockout mice after renal ischemia-reperfusion injury.

[0106] Figure 6 :BACH1 gene silencing improves oxidative stress and mitochondrial dysfunction in proximal tubular epithelial cells treated with hypoxia / reoxygenation. A is the expression and relative quantitative analysis of BACH1, SLC25A39, GPX4, and cytochrome C (cyto-c) proteins in HK-2 cells treated with hypoxia / reoxygenation; BE is the result of the changes in mitochondrial GSH content, mitochondrial complex V activity, mitochondrial membrane potential, and mitochondrial reactive oxygen species in HK-2 cells treated with hypoxia / reoxygenation.

[0107] Figure 7 : BACH1 overexpression aggravates inflammation, oxidative stress, and mitochondrial damage in proximal tubular epithelial cells treated with hypoxia / reoxygenation. A is the result of mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α in HK-2 cells treated with hypoxia / reoxygenation; BE is the result of protein expression and relative quantitative analysis of BACH1, SLC25A39, GPX4, and cyto-c in HK-2 cells treated with hypoxia / reoxygenation; FH is the result of changes in mitochondrial GSH content, mitochondrial complex V activity, and mitochondrial membrane potential in HK-2 cells treated with hypoxia / reoxygenation.

[0108] Figure 8 : BACH1 binds to the SLC25A39 promoter region and inhibits its transcription. A is a luciferase reporter gene showing that overexpression of BACH1 in 293T cells inhibits the activity of the SLC25A39 promoter, and the fluorescence intensity decreases; B is EMSA verification of the interaction between BACH1 protein and SLC25A39 DNA. BACH1 inhibits the transcription of SLC25A39 by binding to site 677 in the SLC25A39 promoter region to achieve its transcriptional inhibition effect.

[0109] Fig. 9 :SLC25A39 gene silencing aggravates inflammation of proximal tubular epithelial cells induced by hypoxia / reoxygenation. A is the expression of SLC25A39 mRNA in HK-2 cells; BD are the expression results of IL-1β, IL-6, and TNF-α mRNA in HK-2 cells.

[0110] Fig.10:SLC25A39 gene silencing aggravates mitochondrial dysfunction in proximal tubular epithelial cells treated with hypoxia / reoxygenation. A is the MitoGSH content of HK-2 cells; B is the activity of mitochondrial electron transport chain complex V of HK-2 cells; C is the ATP content of HK-2 cells; D is the expression of SOD2 mRNA in HK-2 cells; E is the mitochondrial membrane potential of HK-2 cells.

[0111] Fig.11 :Construction map of BACH1 gene overexpression plasmid. DETAILED DESCRIPTION

[0112] The technical solution provided by the present invention will be described in detail below in conjunction with specific embodiments, but it should be understood that the following specific implementation methods are only used to illustrate the present invention, and do not limit the present invention in any form.

[0113] The culture medium DMEM / F12 involved in the following examples was purchased from Wuhan Boster Biotechnology Co., Ltd.

[0114] The detection methods involved in the following embodiments are as follows:

[0115] BACH1 protein content detection method:

[0116] Western blot analysis:

[0117] (1) Sample preparation: Weigh 15 mg of kidney tissue, add 120 μL of RIPA (add phosphatase inhibitors and protease inhibitors at a ratio of 1:100), place on ice, ultrasonically disrupt the tissue to make a homogenate, centrifuge at 12000 rpm for 20 min; take the supernatant to measure the protein concentration by BCA method. Transfer the supernatant to another 1.5 mL EP tube, add 30 μL of 5×loading and mix well, denature at 100°C for 10 min, and store at -80°C;

[0118] (2) Glue making: Add 4 mL of lower glue A solution (Novozyme Biotechnology Co., Ltd.), 4 mL of lower glue B solution, and 80 μL APS to each piece of glue, mix well and add to the glue plate, then press the glue with 70% alcohol for 20 minutes, pour out the alcohol and dry. Add 1 mL of upper glue A solution, 1 mL of B solution, and 20 μL APS, mix well and add to the glue plate, and insert a 1.5 mm 15-hole comb;

[0119] (3) Sample loading: Add 1× electrophoresis buffer to the electrophoresis tank and then load the sample. Calculate the sample loading amount based on the protein concentration of the sample:

[0120]

[0121] (4) Electrophoresis: After the sample is loaded, set the voltage: gel pressing: voltage 80V 30min; gel running: voltage 100V 90min;

[0122] (5) Transfer: Assemble the protein gel sandwich in the order of "filter paper-protein gel-NC membrane-filter paper", install the cathode on the protein gel side in the electrotransfer tank, add electrotransfer solution to the electrotransfer tank, and place the electrotransfer tank on ice. Constant current transfer: 300mA 2h;

[0123] (6) Blocking: After the transfer, remove the NC membrane, block it with rapid blocking solution for 15 min, and wash it with TBST three times, each time for 10 min;

[0124] (7) Incubation with primary antibodies: anti-BACH1 antibody (1:1000), anti-cyto-c antibody (1:1000), anti-SLC25A39 antibody (1:1000), anti-GAPDH antibody (1:1000), anti-GPX4 antibody (1:1000), shake at 4°C overnight;

[0125] (8) Incubation with secondary antibody: remove the protein bands, wash with TBST three times, 10 min each time; incubate with secondary antibody (1:5000) for 40 min; wash with TBST three times, 10 min each time;

[0126] (9) Exposure: Add developer solution to the strips in a dark place and expose with a developer. Use ImageJ to calculate the gray value of the protein strips and perform relative quantification:

[0127] The methods of immunohistochemistry, immunofluorescence, and HE staining are as follows:

[0128] 1. Immunohistochemical staining method:

[0129] (1) Place the kidney pathological sections in an oven for 1 h at 60°C;

[0130] (2) Place the pathological sections in xylene I for 10 min, xylene II for 10 min, and xylene III for 10 min;

[0131] (3) After dewaxing, the pathological sections were rehydrated in the following order: 100% ethanol for 2 min, 95% ethanol for 2 min, 90% ethanol for 2 min, 80% ethanol for 2 min, and 70% ethanol for 2 min;

[0132] (4) Soak the slices in PBS and wash on a shaker for 5 min;

[0133] (5) Heat the sodium citrate antigen retrieval solution to 95°C and place the sections in the antigen retrieval solution for 18 minutes;

[0134] (6) After the antigen retrieval solution has cooled, soak the sections in PBS and wash on a shaker three times, 5 min each time;

[0135] (7) Dilute 100× Triton to 1× with PBS and place the slices in 1× Triton for 10 min to permeabilize the membrane;

[0136] (8) Place the slices in PBS and wash three times, 5 min each time;

[0137] (9) Place the slices in 3% hydrogen peroxide solution for 10 min;

[0138] (10) The sections were soaked in PBS and washed three times, 5 min each time;

[0139] (11) Circle the tissue with a tissue pen and drop 5% BSA onto the tissue for blocking for 30 minutes;

[0140] (12) After drying the blocking solution, add SLC25A39 antibody at a dilution of 1:100 and BACH1 antibody at a dilution of 1:200, and place the slide in a humidified chamber at 4°C overnight.

[0141] (13) Place the wet box in a 37°C incubator and rewarm for 45 min.

[0142] (14) After removing the primary antibody, the sections were immersed in PBS and washed three times on a shaker for 5 min each time;

[0143] (15) Drop HRP-labeled secondary antibody onto the tissue, incubate at 37°C for 30 min, and repeat step 10 above;

[0144] (16) Dry the slices, add SABC solution, place in a humidified chamber and incubate at 37°C for 30 min, and repeat step 10 above;

[0145] (17) The prepared DAB solution is used for color development and is added dropwise to the tissue. When the tissue is observed to be stained light yellow-brown, it is rinsed with running water;

[0146] (18) Place the slices in hematoxylin solution for 32 seconds, rinse with running water, then place the slices in 1% hydrochloric acid alcohol for 2 seconds, and rinse with running water until the blue color returns.

[0147] (19) Dehydrate the slides in 70% ethanol for 2 min, 80% ethanol for 2 min, 90% ethanol for 2 min, 95% ethanol for 2 min, and 100% ethanol for 2 min.

[0148] (20) Place the slide in xylene for 10 min, twice;

[0149] (21) Seal the slide with neutral resin, wait for the neutral resin to dry, and then scan the slide with a digital slide scanner.

[0150] 2. Immunofluorescence staining:

[0151] (1) Dewaxing after baking: Dewaxing with 100% xylene solution for 3 times, 10 min each time;

[0152] (2) Gradient rehydration: After dewaxing, the slices were placed in a gradient ethanol solution of 100%-70% for 2 minutes each time. After rehydration, the slices were washed with pure water for 5 minutes;

[0153] (3) Antigen retrieval: The sections were repaired in sodium citrate antigen retrieval solution at 95°C for 18 min. After cooling to room temperature, the sections were removed and washed three times with PBS, each time for 5 min.

[0154] (4) Permeabilization: Prepare 1% immunostaining permeabilization buffer with Triton X-100 (Trion X-100). Permeabilize the slices in 1% Trion X-100 for 10 min. After permeabilization, wash with PBS three times, 5 min each time.

[0155] (5) Removal of endogenous enzymes: Prepare a 3% hydrogen peroxide (H2O2) solution. Place the slices in the 3% H2O2 solution for 10 min. Remove the slices and wash them with PBS three times, 5 min each time.

[0156] (6) Blocking: Use a tissue pen to draw a circle of the same size as the tissue on the slice to enclose the tissue. Add 5% BSA to the tissue and incubate for 30 minutes;

[0157] (7) Incubation with primary antibody: Prepare a primary antibody solution with a certain dilution ratio (the dilution ratio in this study was 1:100) and incubate it in a humidified box at 4°C overnight. The next day, remove the primary antibody solution and wash with PBS three times, 10 min each time;

[0158] (8) Incubation with secondary antibody: Prepare a fluorescent secondary antibody solution with a certain dilution ratio (the dilution ratio in this study is 1:200) in a dark place. Add an appropriate amount (20 μL in this study) of secondary antibody solution to the tissue, place in a humidified box in a dark place, and incubate in a 37°C incubator for 1 h.

[0159] (9) Nuclear staining and sealing: add anti-quenching DAPI solution, incubate at room temperature for 5 min in the dark, and seal the slides;

[0160] (10) Fluorescence imaging: Observe, image and collect images under a Zeiss upright fluorescence microscope. Then use the accompanying ZEN software for imaging analysis.

[0161] 3. HE staining:

[0162] (1) Place the slices on a slice rack and place them in an oven at 62°C for 1 h;

[0163] (2) After drying, place the slices in xylene I to III for 10 min each.

[0164] (3) Rehydration: 100% ethanol for 2 min, 95% ethanol for 2 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and rinse with tap water for 5 min;

[0165] (4) Hematoxylin staining for 5 min, followed by washing with running water;

[0166] (5) Differentiate with 1% hydrochloric acid and alcohol for 3 seconds, then rinse with running water to turn blue;

[0167] (6) Eosin staining for 40 seconds, then rinse with running water;

[0168] (7) Dehydration: 70% ethanol for 2 min, 80% ethanol for 2 min, 90% ethanol for 2 min, 95% ethanol for 2 min, 100% ethanol for 2 min, xylene I to III for 10 min each;

[0169] (8) Seal the slide with neutral resin to avoid bubbles in the tissue area and let it dry;

[0170] (9) Scan the slides with a digital slide scanner.

[0171] Mitochondrial GSH, SOD, ATP, and reactive oxygen species detection methods:

[0172] 1. Mitochondrial Isolation:

[0173] (1) Melt the various solutions in the kit at room temperature, place on ice and mix after melting. Add 1.5 mL of PMSF solvent to PMSF (crystals), dissolve and mix to obtain 1.5 mL of 100 mM PMSF, and store at -20°C. Take an appropriate amount of the corresponding mitochondrial isolation reagent for use, and add PMSF within a few minutes before adding the mitochondrial isolation reagent to the tissue sample to make the PMSF concentration 1 mM;

[0174] (2) Weigh 15 mg of fresh mouse kidney tissue into a 1.5 mL centrifuge tube and wash the tissue once with PBS;

[0175] (3) After washing the mouse kidney tissue, cut the kidney into tissue fragments with a blade in an ice bath;

[0176] (4) Add 150 μL of pre-cooled mitochondrial isolation reagent A or mitochondrial isolation reagent A with PMSF added just before use, and homogenize on an ice bath for about 10 times;

[0177] (5) The kidney tissue homogenate was centrifuged at 600 g and 4°C for 5 min;

[0178] (6) Transfer the supernatant to another centrifuge tube and centrifuge at 11,000 g, 4°C for 10 min;

[0179] (7) Collect the supernatant into another pre-cooled 1.5 mL EP tube, which is the cytoplasmic protein;

[0180] (8) The precipitate is the isolated mitochondria.

[0181] 2. Determination of mitochondrial GSH content:

[0182] Sample processing: The cytoplasm and mitochondria of kidney tissue were separated by using a tissue mitochondrial isolation kit, and the protein concentration was determined. The supernatant after treatment was operated according to Table 1:

[0183] Table 1:

[0184]

[0185] After mixing the assay plate, let it stand for 5 minutes and measure the OD at 405 nm.

[0186] Calculation formula:

[0187] GSH content in serum (μmol / L) = measured OD value - blank OD value x standard tube concentration x sample pretreatment;

[0188] Standard OD value - blank OD value (20 μmol / L) dilution factor (5 times).

[0189] 3. Mitochondrial SOD content detection:

[0190] Sample processing: The cytoplasm and mitochondria of kidney tissue were separated by using a tissue mitochondrial isolation kit, and the protein concentration was determined. The cytoplasm and mitochondria were taken and operated according to Table 2:

[0191] Table 2:

[0192]

[0193]

[0194] The inhibition rate of SOD was calculated according to the formula (%):

[0195]

[0196] Calculate the activity of SOD according to the formula:

[0197] SOD activity (SOD inhibition rate ÷ 50% x reaction system dilution factor ÷ protein concentration of the sample to be tested mgpr

[0198] 4. Mitochondrial ATP content detection:

[0199] Tissue samples: Add lysis buffer at a ratio of about 100-200 μL per 20 mg of tissue, and then homogenize using a glass homogenizer or other homogenization equipment. Adequate homogenization can ensure that the tissue is completely lysed. After lysis, centrifuge at 12000g at 4℃ for 5 minutes and take the supernatant for subsequent determination.

[0200] Cell samples: Remove the culture medium and add 200 μL of lysis buffer to each well of a 6-well plate to lyse the cells. Centrifuge at 12000 g for 5 min at 4°C and take the supernatant for subsequent determination.

[0201] (1) Add 100 μL of ATP detection working solution to the detection wells or detection tubes. Leave at room temperature for 5 minutes to consume all the background ATP, thereby reducing the background. You can add 100 μL of ATP detection working solution to 10-20 detection wells or detection tubes at one time, thus saving time.

[0202] (2) Add 20 μL of sample or standard into the detection hole or detection tube, mix well, and measure the RLU value using a chemiluminescence instrument after at least 2 seconds.

[0203] 5. Detection of reactive oxygen content in mitochondria of kidney tissue:

[0204] (1) Sample processing: Isolate mitochondria using a tissue mitochondrial isolation kit, following the same steps as above;

[0205] (2) Sample tube: Dilute DCFH-DA 1:1000 in culture medium and resuspend the isolated mitochondria incubated at 37°C for 30 min, inverting and mixing every 3-5 min.

[0206] (4) Collect the incubation suspension, centrifuge at 1000 g for 10 min, remove the supernatant and collect the precipitate, wash twice with PBS to fully remove DCFH-DA. Collect the precipitate by centrifugation and resuspend it in PBS;

[0207] (5) The fluorescence intensity was detected by an enzyme-labeled instrument, and the detection wavelength was set as follows: the optimal excitation wavelength was 488 nm and the optimal emission wavelength was 525 nm.

[0208] 6. Cell mitochondrial reactive oxygen species detection:

[0209] (1) Preparation of stock solution: Add 13 μL DMSO to 50 μg MitoSOX Red Mitochondrial Superoxide Indicator to make a 5 mM stock solution;

[0210] (2) Wash cells three times with 1× PBS, 5 min each time;

[0211] (3) Dilute the MitoSOX Red Mitochondrial Superoxide Indicator 5 mM stock solution to 5 μM using HBSS buffer;

[0212] (4) Add 300 μL of working solution to cover the slide and incubate at 37°C in the dark for 10 min;

[0213] (5) Wash cells three times with HBSS buffer, 5 min each time;

[0214] (6) Add 300 μL of live cell DAPI stain to fully cover the cells growing on the slide and incubate at 37°C in the dark for 10 min;

[0215] (7) Wash the cells three times with preheated HBSS buffer for 5 min each time, and add 300 μL 1× PBS for microscopic examination.

[0216] The inclusion criteria and exclusion criteria involved in the following embodiments are:

[0217] Inclusion criteria: acute ischemic tubular injury in renal biopsy; Exclusion criteria: ① severe infection ② rhabdomyolysis ③ tumor patients ④ drug-induced acute tubular injury ⑤ patients who did not sign informed consent.

[0218] Example 1: BACH1 is upregulated in human acute tubular necrosis

[0219] (1) Experimental methods:

[0220] According to the inclusion and exclusion criteria, paraffin sections of renal tissues of 7 patients diagnosed with acute ischemic tubular injury by renal pathological biopsy in the Affiliated Hospital of Jiangnan University from October 2021 to December 2023 were collected, and paraffin sections of normal renal tissues of 4 patients with renal tumors were used as controls, with no history of hypertension or diabetes.

[0221] Immunohistochemistry was used to detect the BACH1 protein content in renal tissue.

[0222] (2) Experimental results:

[0223] The above experimental results are from Figure 1 As shown, the results showed that immunohistochemistry showed that BACH1 expression increased in the acute tubular injury group compared with the control group, and BACH1 nuclear entry increased ( Figure 1 A in Figure 1); Semi-quantitative analysis of BACH1 immunohistochemical staining showed that the average OD of BACH1 was significantly increased in the acute tubular injury group ( Figure 1B) shows that BACH1 is upregulated in human acute tubular necrosis.

[0224] Example 2: BACH1 expression is upregulated in mice with renal ischemia-reperfusion injury

[0225] The specific steps are as follows:

[0226] (1) Experimental methods:

[0227] The specific experimental groups are shown in Table 3, mainly: sham operation group (Sham), acute renal ischemia-reperfusion model group (I / R), and each group had 6 mice.

[0228] Table 3 Experimental groups

[0229] Group Number / Group deal with Sham group 6 The bilateral kidneys were exposed, and the bilateral renal arteries were not clamped. The rest was the same as the model group. Model group (I / R) 6 Expose both kidneys, clamp both renal arteries for 30 minutes and then release them, and collect samples 24 hours later.

[0230] The specific experimental method is:

[0231] Animals: Adult C57BL / 6J male mice aged 8–10 weeks were used, provided by the Animal Center of Jiangnan University School of Medicine. The animal experiments in this study were approved by the Experimental Animal Management Committee of Jiangnan University, with the ethics number JN.No20220930c0481230

[388] .

[0232] Mouse acute renal ischemia-reperfusion injury model (I / R) group: male mice were anesthetized with intraperitoneal injection of 0.5% sodium pentobarbital, and then the hair on the back of the mice was shaved with a small animal shaver; the mice were fixed on the small animal constant temperature operating table with tape in prone position, and the temperature was adjusted to 37°C; the back skin of the mice was disinfected three times with alcohol cotton balls, and then a sterile scalpel was used to make a longitudinal incision 0.5 cm below the bilateral costal angle of the mice, and then the bilateral kidneys were exposed, and the bilateral blood vessels were separated with a glass needle, and then the bilateral renal arteries were clamped with a vascular clamp, and the wound was covered with sterile gauze soaked in physiological saline. After 30 minutes, the bilateral vascular clamps were removed and sutured in layers. Finally, the wound was disinfected with alcohol cotton balls.

[0233] Sham group: Except for not performing bilateral renal artery clamping, the other steps were the same as those of the model group.

[0234] After 24 hours of the above experiment, the eyeballs were removed under anesthesia to collect blood, and bilateral kidneys were removed. One side was used for WB, qRT-PCR, kit detection, etc., and the other kidney was cut in the middle. 1 / 2 of the kidney tissue was fixed with 4% paraformaldehyde and embedded and sliced ​​for immunohistochemistry, immunofluorescence, HE staining, etc.

[0235] (2) Experimental results:

[0236] The above experimental results are from Figure 2 shown.

[0237] The results showed that compared with the Sham group, qRT-PCR showed that BACH1 mRNA in the I / R group increased significantly, which was 1.8 times that of the control group. WB showed that the BACH1 protein content in the I / R group increased by 2.1 times that of the control group ( Figure 2 A, 2B); Further immunohistochemistry and immunofluorescence revealed that BACH1 was elevated in the I / R group, and the main site of elevation was observed in the renal tubules ( Figure 2 C in , D in 2 ), suggesting that BACH is involved in renal ischemia-reperfusion injury in mice.

[0238] Example 3: BACH1 expression is upregulated in proximal tubular epithelial cells after hypoxia / reoxygenation treatment

[0239] (1) Experimental methods:

[0240] Control group (CON): HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum was inoculated into a six-well plate, placed in a normoxic incubator, and cultured in 10% DMEM / F12 medium for the same culture time as the model group.

[0241] Model group (H / R): HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum size was inoculated into a six-well plate, placed in a normoxic incubator, and cultured in 10% DMEM / F12. When the cells grew to 70%, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12, and the plates were placed in a 1% O2, 5% CO2 three-gas incubator for 6 h. After the cells were taken out, the plates were replaced with 10% DMEM / F12 again and cultured in a normoxic incubator for 24 h before sampling.

[0242] Used to check the BACH1 mRNA expression level, and WB and immunofluorescence to detect the BACH1 protein content.

[0243] (2) Experimental results:

[0244] The above experimental results are from Figure 3 shown.

[0245] The results showed that compared with the CON group, the expression of BACH1 mRNA in the H / R group was increased, which was 1.5 times that of the control group ( Figure 3 A) in

[0246] WB and immunofluorescence showed that the BACH1 content increased, and the BACH1 protein content detected by WB was 1.8 times that of the control group ( Figure 3 B in 3, D in 3), and the cell nuclei were extracted for detection, and it was found that the nuclear entry of BACH1 increased in the H / R group ( Figure 3 C).

[0247] Example 4: BACH1 gene knockout improves inflammation and renal function in mice with renal ischemia-reperfusion injury

[0248] The specific steps are as follows:

[0249] (1) Experimental methods:

[0250] The specific experimental groups are shown in Table 4, mainly: sham operation group (Sham), acute renal ischemia-reperfusion model group (I / R), BACH1 gene knockout-Sham group BACH1 - / - , BACH1 knockout-I / R group BACH1 - / - I / R, 6 mice in each group.

[0251] Table 4 Experimental groups

[0252] Group Number / Group deal with Sham group 6 The bilateral kidneys were exposed, and the bilateral renal arteries were not clamped. The rest was the same as the model group. <![CDATA[BACH1 - / - ]]> 6 BACH1 knockout mice, operated as in the Sham group Model group (I / R) 6 Expose both kidneys, clamp both renal arteries for 30 minutes and then release them, and collect samples 24 hours later. <![CDATA[BACH1 - / - I / R]]> 6 BACH1 knockout mice, the operation is the same as I / R

[0253] The specific experimental method is:

[0254] This embodiment BACH1 - / - The mouse breed was commissioned by Beijing Weishanglide Biotechnology Co., Ltd. and was raised in the Experimental Animal Center of Jiangnan University School of Medicine, as 8-10-week-old adult C57BL / 6J male mice. Project number: FW-20181225-01TY.

[0255] Model group (I / R group): Male mice (C57BL / 6J) were anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital, and then the hair on the back of the mice was removed with a small animal shaver; the mice were fixed on the small animal constant temperature operating table with tape in prone position, and the temperature was adjusted to 37°C; the back skin of the mice was disinfected three times with alcohol cotton balls, and then a sterile scalpel was used to make a longitudinal incision 0.5 cm below the bilateral costal angle of the mice, and then the bilateral kidneys were exposed, and the bilateral blood vessels were separated with a glass needle, and then the bilateral renal arteries were clamped with vascular clamps, and the wound was covered with sterile gauze soaked in physiological saline. After 30 minutes, the bilateral vascular clamps were removed and sutured in layers. Finally, the wound was disinfected with alcohol cotton balls.

[0256] Sham group: Except for not performing bilateral renal artery clamping, the other steps were the same as those of the model group (I / R).

[0257] BACH1 - / - Group: The specific treatment method was the same as the sham operation group, except that the male mice (C57BL / 6J) were adjusted to BACH1 gene knockout mice;

[0258] BACH1 - / -I / R group: The specific treatment method was the same as the model group (I / R) group, except that the male mice were adjusted to (C57BL / 6J) BACH1 gene knockout mice;

[0259] 24 hours after the above experiment, urine was collected to detect renal tubular injury indicators KIM-1 and NGAL; under anesthesia, the eyeballs were removed and blood was collected, and then serum was obtained to detect renal function indicators Scr and BUN after centrifugation; bilateral kidneys were removed, and one kidney was used for WB, qRT-PCR, kit detection, etc.; the other kidney was cut in the middle, 1 / 2 was fixed with 4% paraformaldehyde, and renal tissue pathological sections were HE stained.

[0260] Among them, the primers involved in qRT-PCR are as follows:

[0261] Table 5 Primer sequences for mouse real-time fluorescence quantitative PCR (corresponding to inflammatory factors and renal tubular injury index mRNA)

[0262]

[0263] (2) Experimental results:

[0264] The above experimental results are from Figure 4 shown.

[0265] The results showed that compared with the Sham group, the mRNA levels of inflammatory factors IL-6, IL-1β, and TNF-α in the renal tissue of the model group (I / R) were significantly increased (2.6 times, 3.7 times, and 2.6 times that of the control group, respectively). - / - The above indicators in the I / R group were significantly decreased compared with those in the I / R group (IL-6, IL-1β, TNF-α mRNA, BACH1 - / - The expression levels in the renal tissues of the I / R group decreased by 46%, 49.6%, and 40%, respectively), indicating that BACH1 gene knockout can improve inflammation in mice with renal ischemia-reperfusion injury ( Figure 4 AC in );

[0266] Compared with the Sham group, the renal tubular injury index KIM-1 mRNA and urine KIM-1 (kidney injury molecule-1) and NGAL (neutrophil gelatinase-associated lipocalin) levels in the model group (I / R) were significantly increased (urine KIM-1 content increased by 5.1 times that of the control group, and urine NGAL content increased by 10.6 times that of the control group), BACH1 - / - The above indicators in the I / R group were significantly lower than those in the I / R group (KIM-1 mRNA and urine KIM-1 and NGAL levels were compared with the model group, BACH1 - / -The contents in the I / R group were significantly reduced, and the contents of urine KIM-1 and NGAL decreased by 67.0% and 21.7%, respectively), indicating that BACH1 gene knockout can improve renal tubular injury in mice with renal ischemia-reperfusion injury ( Figure 4 D in the

[0267] Compared with the Sham group, the levels of renal injury indicators Scr (serum creatinine) and BUN (blood urea nitrogen) in the I / R group were significantly increased (10.3 and 4.3 times that of the control group, respectively) ( Figure 4 E), HE can show pathological changes of renal tubular damage such as casts, tubular dilatation, and disappearance of brush border ( Figure 4 F), BACH1 - / - The above indexes in the I / R group were significantly improved compared with those in the WT group (BACH1 - / - The Scr level in I / R blood decreased by 84.7% and the BUN level decreased by 49.1%), indicating that BACH1 gene knockout can improve the renal function of mice with renal ischemia-reperfusion injury.

[0268] Example 5: Acute renal ischemia-reperfusion injury induces mitochondrial glutathione depletion, BACH1 gene knockout improves mitochondrial dysfunction in mice with renal ischemia-reperfusion injury and restores some mitochondrial glutathione content

[0269] (1) Experimental methods:

[0270] The mouse modeling method and grouping were the same as in Example 4. After the experiment, bilateral kidneys were taken, and one kidney was used for WB, qRT-PCR, kit detection, etc.; the other kidney was cut in the middle, 1 / 2 was fixed with 4% paraformaldehyde and HE staining of renal tissue pathological sections was performed.

[0271] After mitochondria were extracted from kidney tissue, mitochondrial GSH, SOD, ATP, reactive oxygen species, and mitochondrial transmission electron microscopy were detected.

[0272] (2) Experimental results:

[0273] The above experimental results are from Figure 5 shown.

[0274] The results showed that compared with the sham group (Sham), the mitochondrial GSH, SOD, and ATP in the renal tissue of the model group (I / R) were reduced (60.2%, 44.7%, and 24.9%, respectively), and the mitochondrial reactive oxygen species increased, indicating the presence of oxidative stress and mitochondrial dysfunction. - / - The I / R group showed improvement compared with the I / R group (BACH1 - / -The contents of mitochondrial GSH, SOD, and ATP in renal tissue of the I / R group increased by 470%, 56.4%, and 26.9%, respectively)( Figure 5 AE in );

[0275] Compared with the Sham group, the I / R group showed mitochondrial swelling, vacuole formation, and decreased mitochondrial cristae in the renal tissue of mice, while BACH1 - / - The mitochondrial morphology and structure were significantly improved in the I / R group ( Figure 5 F).

[0276] The above results indicate that BACH1 gene knockout improves oxidative stress and mitochondrial dysfunction in mice with renal ischemia-reperfusion injury.

[0277] Example 6: Hypoxia / reoxygenation induces mitochondrial glutathione depletion in HK-2 cells, and BACH1 gene silencing improves oxidative stress and mitochondrial dysfunction in proximal tubular epithelial cells treated with hypoxia / reoxygenation, and restores some mitochondrial glutathione content

[0278] The specific steps are as follows:

[0279] (1) Experimental methods:

[0280] The specific experimental groups are shown in Table 6 , mainly: CON group, BACH1 si RNA group, H / R group, BACH1 si RNA + H / R;

[0281] Table 6 Experimental groups

[0282] Group Group deal with CON 4 Transfection of BACH1 NC BACH1 siRNA 4 Transfection of BACH1 siRNA H / R 4 BACH1 NC was transfected and subjected to 6-hour hypoxia followed by 24-hour reoxygenation. BACH1 si RNA+H / R 4 Transfected with BACH1 siRNA, hypoxia for 6 hours followed by reoxygenation for 24 hours

[0283] BACH1 NC plasmid and BACH1 siRNA plasmid were obtained from Shanghai Jima Biotechnology Co., Ltd. The specific construction information of BACH1 siRNA is shown in Table 7 below.

[0284] Table 7 BACH1 siRNA base sequence

[0285]

[0286]

[0287] Control group (CON group): HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum was inoculated into a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, they were transfected with BACH1 NC plasmid and finally collected at the same time (the total culture time was the same as that of the H / R group).

[0288] BACH1 si RNA group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum was inoculated into a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, they were transfected with BACH1 si RNA plasmid and samples were collected at the same time (the total culture time was the same as that of the BACH1 si RNA+H / R group).

[0289] H / R group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The cells were inoculated in a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, the BACH1 NC plasmid was transfected. After 24 hours, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12 and placed in a three-gas incubator with 1% O2 and 5% CO2 for 6 hours. After the cells were taken out, the medium was replaced with 10% DMEM / F12 again and placed in a normoxic incubator for 24 hours before the samples were collected.

[0290] BACH1 si RNA+H / R group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The cells were inoculated in a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, they were transfected with BACH1 siRNA plasmid. After 24 hours, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12 and placed in a three-gas incubator with 1% O2 and 5% CO2 for 6 hours. After the cells were taken out, the medium was replaced with 10% DMEM / F12 and placed in a normoxic incubator for 24 hours before sampling.

[0291] Table 8 siRNA / NC transfection steps

[0292]

[0293] Subsequently, WB was used to detect the protein contents of BACH1, SLC25A39, GPX4 (glutathione peroxidase 4), and Cyto-c (cytochrome c), mitochondria were extracted to detect GSH and mitochondrial complex V activity, and immunofluorescence was used to detect MitoSOX and mitochondrial membrane potential.

[0294] (2) Experimental results

[0295] The above experimental results are from Figure 6The results showed that compared with the CON group, the BACH1 content in the H / R group increased (4.7 times that of the control group), and the BACH1 gene silencing effect was verified. The SLC25A39 and GPX4 protein contents decreased (33.7%, 21.2%), and the Cyto-c protein content increased (5.33 times). The data of the BACH1 si RNA + H / R group showed that BACH1 gene silencing reversed the decrease in the SLC25A39 and GPX4 protein contents in the H / R group, as well as the increase in the Cyto-c protein content ( Figure 6 A in

[0296] Compared with the CON group, the H / R group had mitochondrial reactive oxygen species accumulation, mitochondrial GSH decreased by 48.3%, mitochondrial complex V content decreased by 90.5%, and mitochondrial membrane potential abnormalities, indicating mitochondrial damage; after BACH1 gene silencing, hypoxia / reoxygenation treatment (BACH1 si RNA+H / R group) was performed, and the above indicators were improved compared with the H / R group (mitochondrial reactive oxygen species accumulation decreased, mitochondrial GSH increased by 1.94 times, mitochondrial complex V content increased by 7.2 times, and mitochondrial membrane potential increased), indicating that mitochondrial damage was alleviated ( Figure 6 BE in ).

[0297] The above results indicate that BACH1 gene silencing improves oxidative stress and mitochondrial dysfunction in proximal tubular epithelial cells treated with hypoxia / reoxygenation.

[0298] Example 7: BACH1 overexpression aggravates inflammation, oxidative stress, and mitochondrial damage in HK-2 cells treated with hypoxia / reoxygenation. The specific steps are as follows:

[0299] (1) Experimental methods

[0300] The specific experimental groups are shown in Table 9 , mainly: CON group, BACH1 OE group, H / R group, BACH1 OE+H / R;

[0301] Table 9 Experimental groups

[0302] Group Group deal with CON 4 Transfection of BACH1 NC BACH1 OE 4 Transfection of BACH1 overexpression plasmid H / R 4 BACH1 NC was transfected and subjected to 6-hour hypoxia followed by 24-hour reoxygenation. BACH1 OE+H / R 4 Transfection with BACH1 overexpression plasmid, hypoxia for 6 hours followed by reoxygenation for 24 hours

[0303] The BACH1 gene overexpression plasmid and control plasmid were obtained from Shanghai Jikai Biotechnology Co., Ltd. The specific construction information is shown in Table 10 below

[0304] Table 10 BACH1 overexpression plasmid construction information

[0305]

[0306] Control group (CON group): HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5The inoculum was inoculated into a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, they were transfected with BACH1 NC plasmid and finally collected at the same time (the total culture time was the same as that of the H / R group).

[0307] BACH1 OE group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum was inoculated into a six-well plate, placed in a normoxic incubator, and cultured in 10% DMEM / F12 medium. When the cells grew to 70%, they were transfected with BACH1 overexpression plasmid and finally collected at the same time (the total culture time was the same as that of BACH1 OE+H / R group).

[0308] H / R group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The cells were inoculated in a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, the BACH1 NC plasmid was transfected. After 24 hours, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12 and placed in a three-gas incubator with 1% O2 and 5% CO2 for 6 hours. After the cells were taken out, the medium was replaced with 10% DMEM / F12 and placed in a normoxic incubator for 24 hours before the samples were collected.

[0309] BACH1 OE+H / R group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The cells were inoculated in a six-well plate and placed in a normoxic incubator in 10% DMEM / F12 medium. When the cells grew to 70%, the BACH1 OE plasmid was transfected. After 24 hours, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12 and placed in a three-gas incubator with 1% O2 and 5% CO2 for 6 hours. After the cells were taken out, the medium was replaced with 10% DMEM / F12 and placed in a normoxic incubator for 24 hours before the samples were collected.

[0310] Table 11 Transfection steps

[0311]

[0312] Table 12 HK-2 cell real-time fluorescence quantitative PCR primer sequences

[0313]

[0314] Subsequently, the mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α were detected, the protein contents of BACH1, SLC25A39, GPX4, and Cyto-c were detected by WB, mitochondria were extracted to detect GSH and mitochondrial complex V activities, and mitochondrial membrane potential was detected by immunofluorescence.

[0315] (2) Experimental results

[0316] The above experimental results are from Figure 7 The results showed that compared with the CON group, the mRNA expression of inflammatory factors IL-1β, IL-6, and TNF-α in the H / R group was significantly increased (6.7 times, 5.91 times, and 20.1 times, respectively). After BACH1 overexpression and hypoxia / reoxygenation treatment (BACH1OE+H / R), the expression levels of the above inflammatory factors were further increased compared with the CON group (the expression levels of IL-1β, IL-6, and TNF-α mRNA were 18.1 times, 13.6 times, and 78.2 times that of the control group, respectively) ( Figure 7 A in

[0317] Compared with the CON group, the levels of SLC25A39 and GPX4 proteins in the H / R group decreased (22.6% and 22.0%, respectively), and the level of Cyto-c protein increased by 1.4 times. After BACH1 overexpression, the levels of SLC25A39 and GPX4 proteins in the H / R group (BACH1 OE+H / R) further decreased compared with the CON group (45.5% and 60.0%, respectively), and the level of Cyto-c protein increased by 2.1 times ( Figure 7 Compared with the CON group, the mitochondrial GSH in the H / R group decreased by 36.3%, the mitochondrial complex V content decreased by 48.4%, and the mitochondrial membrane potential was abnormal, indicating mitochondrial damage. After BACH1 overexpression and hypoxia / reoxygenation (BACH1 OE+H / R group), the above indicators decreased more significantly than those in the CON group (mitochondrial GSH: 62.9%, mitochondrial complex V content: 72.3%, and mitochondrial membrane potential was significantly reduced), indicating that mitochondrial damage was aggravated ( Figure 7 F in - H in 7).

[0318] The above results indicate that BACH1 overexpression aggravates inflammation, oxidative stress and mitochondrial damage in HK-2 cells treated with hypoxia / reoxygenation.

[0319] Example 8: BACH1 binds to the SLC25A39 promoter region and inhibits its transcription

[0320] The BACH1 overexpression plasmid, negative control plasmid, SLC25A39 promoter plasmid, empty plasmid, and Renilla internal reference plasmid involved in this example were obtained from Shanghai Jikai Biotechnology Co., Ltd. The design of the sense and antisense strands of the promoter region binding site and the mutation of the promoter region binding site in the EMSA experiment were constructed by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0321] (1) Experimental methods:

[0322] 293T cells were transfected with BACH1 overexpression plasmid, negative control plasmid, SLC25A39 promoter plasmid, empty plasmid, and Renilla internal reference plasmid, respectively. 24 hours after transfection, the activity of the target gene was detected using a dual luciferase reporter gene kit.

[0323] For EMSA experiments: obtain the purified protein of the transcription factor, design two complementary chains of the sense chain and the antisense chain of the binding site, use DNA annealing buffer to anneal the two chains to form double-stranded DNA; DNA and protein binding: DNA (20ng): protein (ng) = 1:2 to 1:100, incubate at 20℃ for 30min; prepare non-deformed 6% SDS-PAGE, pre-run the prepared gel at room temperature for 1h, add 1ⅹDNA loading Buffer to the mixture of DNA and protein binding, load the gel, use Thermo EB (1:10000 dilution, diluted with water), and stain at room temperature for 30min; development: Tianneng, UV 302nm.

[0324] EMSA binding reaction grouping:

[0325] Negative control reaction: Nuclease-Free Water 7μl + Nuclease-Free Water 2μl + Nuclear protein or purified transcription factor 0μl + Labeled probe 1μl;

[0326] Sample reaction: Nuclease-Free Water 5μl + Nuclease-Free Water 2μl + Nuclear protein or purified transcription factor 2μl + Labeled probe 1μl;

[0327] Probe cold competition reaction: Nuclease-Free Water 4μl + Nuclease-Free Water 2μl + nuclear protein or purified transcription factor 2μl + unlabeled probe 1μl + labeled probe 1μl;

[0328] Cold competition reaction of mutation probe: Nuclease-Free Water 5μl + Nuclease-Free Water 2μl + nuclear protein or purified transcription factor 2μl + unlabeled mutation probe 1μl + labeled probe 1μl.

[0329] (2) Experimental results:

[0330] The above experimental results are from Figure 8 show:

[0331] (1) Luciferase reporter gene showed that overexpression of BACH1 in 293T cells inhibited the activity of the SLC25A39 promoter and the fluorescence intensity decreased ( Figure 8 A in

[0332] (2) EMSA experiments preliminarily screened five candidate binding sites and found that BACH1 binds to the 677-689 region of the sense strand of the SLC25A39 promoter region;

[0333] (3) To verify the specificity of BACH1 binding to the SLC25A39 promoter, the 677-689 region of the positive strand of the promoter region was mutated. The results showed that the cold competition mutant group still bound, indicating that BACH1 bound to the 677-689 region of the positive strand of the SLC25A39 promoter region ( Figure 8 B).

[0334] The results showed that BACH1 inhibited the transcription of SLC25A39 by binding to site 677 in the promoter region of SLC25A39.

[0335] Example 9: SLC25A39 gene silencing aggravates inflammation in HK-2 cells treated with hypoxia / reoxygenation

[0336] The specific steps are as follows:

[0337] (1) Experimental methods

[0338] The specific experimental groups are shown in Table 13 , mainly: CON group, SLC25A39 si RNA group, H / R group, SLC25A39 si RNA + H / R;

[0339] Table 13 Experimental groups

[0340] Group Group deal with CON 4 Transfection of SLC25A39 NC SLC25A39 siRNA 4 Transfection of SLC25A39 siRNA H / R 4 Transfected with SLC25A39 NC, hypoxia for 6 hours followed by reoxygenation for 24 hours SLC25A39 siRNA+H / R 4 Transfected with SLC25A39 siRNA, hypoxia for 6 hours followed by reoxygenation for 24 hours

[0341] SLC25A39 siRNA and SLC25A39 NC were obtained from Shanghai Jima Biotechnology Co., Ltd. The specific construction information is shown below

[0342] Table 14 SLC25A39 siRNA base sequence

[0343]

[0344] Control group (CON group): HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum was inoculated into a six-well plate and placed in a normoxic incubator under 10% DMEM / F12 conditions. When the cells grew to 70%, they were transfected with SLC25A39 NC plasmid and finally collected at the same time (the total culture time was the same as that of the H / R group).

[0345] SLC25A39 siRNA group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The inoculum was inoculated into six-well plates, placed in a normoxic incubator, and cultured in 10% DMEM / F12. When the cells grew to 70%, they were transfected with SLC25A39 siRNA plasmid and finally collected at the same time (the total culture time was the same as that of the SLC25A39 siRNA+H / R group).

[0346] H / R group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The cells were inoculated in a six-well plate and placed in a normoxic incubator under 10% DMEM / F12 conditions. When the cells grew to 70%, they were transfected with SLC25A39 NC plasmid. After 24 hours, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12 and placed in a three-gas incubator with 1% O2 and 5% CO2 for 6 hours. After the cells were taken out, they were replaced with 10% DMEM / F12 again and placed in a normoxic incubator for 24 hours before sampling.

[0347] SLC25A39 si RNA+H / R group: HK-2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured at 5×10 5 The cells were inoculated in a six-well plate and placed in a normoxic incubator under 10% DMEM / F12 conditions. When the cells grew to 70%, they were transfected with SLC25A39 siRNA plasmid. After 24 hours, the 10% DMEM / F12 medium was replaced with serum-free DMEM / F12 and placed in a three-gas incubator with 1% O2 and 5% CO2 for 6 hours. After the cells were taken out, they were replaced with 10% DMEM / F12 again and placed in a normoxic incubator for 24 hours before sampling.

[0348] Subsequently, cellular RNA was extracted to detect the mRNA expression of SLC25A39 and inflammatory factors.

[0349] (2) Experimental results:

[0350] The above experimental results Fig. 9 show:

[0351] Compared with the CON group, the SLC25A39 mRNA expression in the H / R group was significantly decreased to 42.0%, and the gene silencing effect of SLC25A39 was observed;

[0352] Compared with the CON group, the mRNA expressions of inflammatory factors IL-1β, IL-6, and TNF-α in the H / R group were significantly increased (3.4 times, 4.1 times, and 2.6 times that of the control group, respectively). After SLC25A39 gene silencing and hypoxia / reoxygenation treatment (SLC25A39+H / R group), the expressions of the above inflammatory factors were further increased compared with the CON group (the mRNA expressions of IL-1β, IL-6, and TNF-α were 6.3 times, 5.2 times, and 4.1 times that of the control group, respectively).

[0353] The above results indicate that SLC25A39 gene silencing aggravates inflammation of proximal tubular epithelial cells induced by hypoxia / reoxygenation.

[0354] Example 10: SLC25A39 gene silencing aggravates mitochondrial glutathione depletion in HK-2 cells treated with hypoxia / reoxygenation and aggravates mitochondrial dysfunction

[0355] (1) Experimental methods

[0356] The experimental method and experimental grouping of this group were the same as those of Example 9. Subsequently, mitochondria were extracted to detect GSH, mitochondrial complex V activity, and ATP content, and mitochondrial membrane potential was detected by immunofluorescence.

[0357] (2) Experimental results:

[0358] The above experimental results Fig.10 Shows: Compared with the CON group, the mitochondrial GSH content in the H / R group ( Fig.10 A) decreased by 41.0%, mitochondrial complex V activity ( Fig.10 B) decreased by 38.1%, and ATP content decreased significantly by 66.1% ( Fig.10 C), mitochondrial membrane potential indicates mitochondrial damage ( Fig.10 E), SLC25A39 gene silencing followed by hypoxia / reoxygenation treatment (SLC25A39 siRNA+H / R) further aggravated mitochondrial glutathione depletion (60.5% lower than that in the CON group), further reduced mitochondrial complex V activity, and further decreased ATP content, by 85.0% compared with the CON group. Mitochondrial membrane potential indicated that mitochondrial damage was further aggravated.

[0359] The above results indicate that SLC25A39 gene silencing aggravates mitochondrial dysfunction in proximal tubular epithelial cells treated with hypoxia / reoxygenation.

[0360] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of BACH1 inhibitors in the preparation of drugs for treating and / or alleviating mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury.

2. Use of BACH1 inhibitors in the preparation of drugs for treating and / or alleviating renal ischemia-reperfusion injury.

3. The use according to claim 1 or 2, characterized in that: The BACH1 inhibitors are: (1) knockout agents comprising the BACH1 gene, (2) regulatory factors that inhibit the expression of the BACH1 gene or mRNA, and (3) agents that inhibit the activity of the BACH1 protein.

4. The use according to any one of claims 1 to 3, characterized in that: Application in the preparation of medicines having at least one of the following functions: (1) Improve renal tubular damage induced by renal ischemia-reperfusion injury and improve renal tissue pathological changes; (2) reduce the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α induced by renal ischemia-reperfusion injury; (3) inhibiting the production of ROS induced by renal ischemia-reperfusion; (4) Improve the decrease of mitochondrial GSH induced by renal ischemia-reperfusion injury; (5) Improve GSH-dependent enzyme activity in mitochondria induced by renal ischemia-reperfusion injury; (6) Improve the mRNA expression and content of glutathione peroxidase 4 (GPX4) in renal ischemia-reperfusion injury; (7) Alleviate mitochondrial damage induced by renal ischemia-reperfusion.

5. A method for screening drugs for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction, characterized in that: The drug can promote the expression of SLC25A39 protein, and the method is: In the test group, a test compound is added to the culture system, and the expression amount and / or activity of the Bach1 gene in the test group is observed; in the control group, no test compound is added to the same culture system, and the expression amount and / or activity of the Bach1 gene in the control group is observed; If the expression level and / or activity of the Bach1 gene in the test group is lower than that in the control group, it indicates that the test compound is a drug for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction.

6. A cell model for screening drugs for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction, characterized in that: The drug can promote the expression of SLC25A39 protein, and the cell model is: In the test group, the test compound is added to the culture system, the test compound is co-incubated with HK-2 cells, and the expression amount and / or activity of the Bach1 gene in the test group is observed; in the control group, the test compound is not added to the same culture system, the test compound is co-incubated with HK-2 cells, and the expression amount and / or activity of the Bach1 gene in the control group is observed; If the expression level and / or activity of the Bach1 gene in the test group is lower than that in the control group, it indicates that the test compound is a drug for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction.

7. Any of the following uses of a BACH1 marker and / or a substance for detecting the BACH1 marker: (1) Application in screening candidate drugs for treating mitochondrial glutathione depletion and / or mitochondrial dysfunction; (2) Application in screening drugs for treating and / or alleviating mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury; (3) Application in assessing renal ischemia-reperfusion injury.

8. The use according to claim 7, characterized in that: The products include test strips, probes, kits, antibodies, and aptamers; preferably, the BACH1 marker and / or a substance for detecting the BACH1 marker is used as a biological marker in renal ischemia-reperfusion injury, and the sources of the biomarker detection include blood, urine, renal tissue, and renal tubular epithelial cells; Preferably, the substance for detecting the BACH1 marker includes a reagent for detecting the Bach1 gene or protein content, or a substance that binds to the BACH1 protein; Preferably, the substance binding to BACH1 protein includes an antibody, a polypeptide, an aptamer, a protein or a nucleic acid molecule.

9. A method for studying the function of genes related to promoting mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury, or analyzing the use of BACH1 gene in inhibiting SLC25A39 expression, or analyzing the use of drugs for gene therapy for promoting mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury, characterized in that: The regulating agent of the BACH1 molecule includes an enhancer of the BACH1 molecule or an inhibitor of the BACH1 molecule, wherein the BACH1 enhancer enhances the activity and / or expression of the BACH1 molecule, and the inhibitor of the BACH1 molecule inhibits the activity and / or expression of the BACH1 molecule; Preferably, the BACH1 molecule includes BACH1 gene, protein and / or mRNA; Preferably, the regulatory agent of the BACH1 molecule includes inhibiting or enhancing the activity and / or expression of the BACH1 molecule, thereby inhibiting or enhancing BACH1 inhibition of SLC25A39 expression; Preferably, the regulatory agent of the BACH1 molecule is: 1) Vectors, overexpression or knockout reagents containing BACH1 gene; 2) regulatory factors that enhance or inhibit BACH1 gene or mRNA expression; 3) Agents that enhance or inhibit the activity of BACH1 protein.

10. Use of a regulating agent of SLC25A39 molecule in the preparation of a pharmaceutical composition for preventing and / or treating renal ischemia-reperfusion injury, or a drug for treating and / or alleviating mitochondrial glutathione depletion and mitochondrial dysfunction in renal ischemia-reperfusion injury, characterized in that: The modulating agent enhances the activity and / or expression of the SLC25A39 molecule; Preferably, the SLC25A39 molecule comprises SLC25A39 protein, gene and / or mRNA; Preferably, the regulating reagent comprises: 1) a vector comprising the SLC25A39 gene, an overexpression reagent; 2) regulatory factors that enhance SLC25A39 gene or mRNA expression; 3) Reagents that enhance the activity of SLC25A39 protein.

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