Application of acetylation modification of protein in diagnosis and treatment of diabetic nephropathy

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and relates to application of acetylation modification of protein in diagnosis and treatment of diabetic nephropathy. The research shows that the acetylation modification of the TFAM K76 site is differentially expressed in the diabetic nephropathy, and shows that the TFAM K76 site has good diagnosis and prognosis effects on the diabetic nephropathy; research finds that inflammation and fibrosis in renal tubular epithelial cells and kidney tissues induced by high glucose can be relieved by regulating and controlling the TFAM K76 site, especially inhibiting acetylation modification of the TFAM K76 site, the TFAM K76 site can be used for treating diabetic nephropathy, and small molecular compounds for relieving kidney injury of mice with diabetic nephropathy can be further screened.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and molecular biology, and relates to the application of acetylation modification of proteins in the diagnosis and treatment of diabetic nephropathy. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes. About 30-40% of diabetic patients eventually develop diabetic nephropathy, which has become the leading cause of end-stage renal disease (ESRD). Its characteristic pathological changes include thickening of the glomerular basement membrane, expansion of the mesangial matrix, podocyte damage and tubulointerstitial fibrosis. The clinical manifestations are proteinuria, progressive renal function decline, and eventually renal failure. Although current treatments (such as blood sugar / blood pressure control, RAS inhibitors) can delay progression, some patients still cannot avoid disease deterioration, and treatment strategies targeting new pathological mechanisms are urgently needed.

[0004] At present, the core pathophysiological mechanisms of diabetic nephropathy include the following aspects, such as (1) metabolic abnormalities and oxidative stress: persistent hyperglycemia causes mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) through the polyol pathway, hexosamine pathway and accumulation of advanced glycation end products (AGEs), leading to oxidative damage to glomerular endothelial cells and podocytes, and destroying the integrity of the filtration barrier. (2) Activation of inflammatory signals: hyperglycemia activates macrophage infiltration through pathways such as NF-κB and NLRP3 inflammasomes, releases proinflammatory factors such as IL-6 and TNF-α, and promotes glomerular sclerosis and interstitial fibrosis. (3) Fibrosis process drive: overexpression of transforming growth factor-β1 (TGF-β1) induces epithelial-mesenchymal transition (EMT), stimulates excessive deposition of extracellular matrix (ECM) (such as collagen IV and fibronectin), and ultimately leads to structural remodeling and functional loss of nephrons. (4) Imbalance of epigenetic regulation: Recent studies have found that abnormal acetylation modification of histones / non-histones plays a key role in the progression of diabetic nephropathy by regulating the chromatin accessibility of inflammation- and fibrosis-related genes, and has become an emerging therapeutic target.

[0005] Protein acetylation is an important post-translational modification process, which mainly regulates protein stability, subcellular localization, enzyme activity and interaction by covalently binding acetyl groups to proteins (especially lysine residues). This process is dynamically regulated by acetyltransferases (HATs) and deacetylases (HDACs / Sirtuins), and is closely related to gene expression, metabolic regulation and inflammatory response. Abnormal protein acetylation increases DKD inflammation and oxidative stress. For example, hyperglycemia inhibits Sirtuin-1 activity, increases the acetylation level of NF-κB, p65 and STAT3, increases the release of proinflammatory factors and oxidative stress, and leads to glomerular podocyte damage and proteinuria. Mitochondrial dysfunction is an important feature of DKD. Acetylation affects energy metabolism by regulating metabolic pathways such as oxidative phosphorylation and tricarboxylic acid cycle (TCA). Literature reports show that mitochondrial protein acetylation significantly increases in renal tissue of DKD patients, leading to decreased metabolic adaptability; at the same time, acetylation modification also mediates the fibrosis progression of DKD. For example, histone acetylation (such as H3K18Ac, H4K8Ac) is abnormally elevated in DKD, promoting the expression of profibrotic genes (such as TGF-β, CTGF), and accelerating tubular interstitial fibrosis. Moreover, hyperglycemia-induced histone acetylation changes can persist for a long time (metabolic memory), driving the progression of DKD even after blood sugar is controlled. Therefore, targeting protein acetylation modification is an important strategy to delay the progression of DKD.

[0006] Mitochondrial transcription factor A (TFAM) is a key DNA-binding protein encoded by nuclear genes and transported to the mitochondrial matrix. It is a core molecule that maintains the stability of the mitochondrial genome (mtDNA) and regulates mitochondrial biogenesis. Its structure contains two high-mobility group (HMG-box) domains, which form a nucleic acid-protein complex by specifically binding to mtDNA. TFAM is directly involved in the transcription and replication of mtDNA, and regulates the synthesis of subunits of the respiratory chain complex (such as Complex I, III, and IV) encoded by mitochondria. Studies have reported that TFAM has a variety of post-translational modifications, including phosphorylation, ubiquitination, and acetylation. Among them, TFAM acetylation is an important form of its functional regulation. Acetylation significantly reduces the binding affinity of TFAM to mitochondrial DNA (mtDNA). At the same time, acetylation reduces the expression of key genes in mitochondrial biogenesis by inhibiting the interaction between TFAM and mtDNA, resulting in a decrease in the number of mtDNA copies. In the vascular dementia (VaD) model, dysregulation of the SIRT3 / TFAM pathway leads to excessive TFAM acetylation, which increases mitochondrial damage. Restoring its deacetylation can improve mitochondrial function and alleviate the disease. In acute kidney injury (AKI), GCN5L1-mediated upregulation of TFAM acetylation is associated with mitochondrial damage. Reducing TFAM acetylation by knocking down GCN5L1 can alleviate renal pathological changes. There is increasing evidence that TFAM acetylation plays an important role in regulating mtDNA function and mitochondrial biogenesis. However, no studies have reported on TFAM acetylation in DKD, and its abnormal expression and biological function in the progression of DKD remain largely unclear. Summary of the invention

[0007] The present invention found that (1) acetylation modification of TFAM K76 site significantly increased in DKD patients and mice and high glucose-induced HK-2 cells; (2) acetylation of TFAMK76 site promoted inflammation and mitochondrial damage of HK-2 cells induced by high glucose; (3) acetylation of TFAMK76 site promoted kidney damage and mitochondrial damage in DKD mice; (4) small molecule compounds targeting acetylation of TFAMK76 site were screened; (5) small molecule compounds can reduce inflammation and mitochondrial damage of HK-2 cells induced by high glucose; (6) small molecule compounds can reduce kidney damage and mitochondrial damage in DKD mice. Based on the above research results, the present invention provides the application of protein acetylation modification in the diagnosis and treatment of diabetic nephropathy.

[0008] Specifically, the technical solution of the present invention is as follows: The first aspect of the present invention provides the use of any one of the following a1) to a4) in the preparation of a drug for diagnosing diabetic nephropathy: a1) Acetylation modification of TFAM K76 site in the renal tissue of the subjects; a2) a nucleic acid encoding an acetyltransferase that modifies the acetylation of the TFAM K76 site; a3) Reagents for detecting acetylation modification expression of TFAM K76 site; a4) Reagents for detecting the expression of acetyltransferases encoding acetylated TFAM K76 sites.

[0009] The present invention found through research that the acetylation level of TFAM K76 site increases in DKD tissues and cell lines, and clarified that TFAM K76 acetylation is a marker for the diagnosis and prognosis of DKD, and can thus be used as an important key target for its treatment. Targeting TFAM K76 acetylation can slow down the progression of DKD.

[0010] The subject of the present invention is a human (especially a diabetic patient) or a non-human mammal, and the non-human mammal is a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey, an orangutan, etc.

[0011] In the present invention, the reagent for detecting the acetylation modification expression of the TFAM K76 site can be a reagent based on immunoblotting detection, a reagent based on mass spectrometry detection, a reagent based on enzyme-linked immunosorbent assay detection, and the like.

[0012] The second aspect of the present invention provides the use of an active ingredient in at least one of the following b1) to b4): b1) Preparation of products for reducing inflammation and / or fibrosis of renal tubular epithelial cells; b2) preparing products for reducing mitochondrial damage in renal tubular epithelial cells; b3) preparing products for screening to slow the progression of diabetic nephropathy; b4) Preparation of drugs for treating diabetic nephropathy; The active ingredients include at least the following c1) or c2): c1) Substances that reduce or inhibit the expression level of acetylation modification at the TFAM K76 site; c2) A substance that reduces or inhibits the expression level of a nucleic acid encoding an acetyltransferase that modifies the acetylation of TFAM K76.

[0013] Among them, in b3), the diabetic nephropathy is a disease caused by diabetes and characterized by lesions of kidney tissue (including renal tubular epithelial cells).

[0014] The product of the present invention can be a medicine or an experimental reagent. The experimental reagent can be used for scientific research, such as constructing a diabetic nephropathy model.

[0015] The substance for reducing or inhibiting the expression level of acetylation modification of TFAM K76 site of the present invention can be a compound for reducing or inhibiting the expression level of acetylation modification of TFAM K76 site. Specifically, the molecular formula of the compound is C 21 H 25 N 3 O 2 S, CAS number is 353261-40-2, and its chemical structure is as follows: .

[0016] In some embodiments, the drug is a pharmaceutical composition, further comprising pharmaceutical excipients.

[0017] The pharmaceutical excipients may be carriers, excipients, etc. commonly used in pharmacy. Moreover, according to common methods, the pharmaceutical composition may be prepared into oral preparations, external preparations, suppositories, and sterile injection solutions in the form of granules, powders, sprays, tablets, capsules, suspensions, emulsions, syrups, etc.

[0018] The non-pharmaceutical active ingredients such as carriers and excipients that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards. The excipients include binders, fillers, disintegrants, preservatives, lubricants, and the like.

[0019] In some embodiments, the carriers and excipients include but are not limited to alumina, serum protein, lecithin, maltitol, starch, gum arabic, sucrose, lactose, alginate, glucose, sorbitol, mannitol, magnesium stearate, mineral oil, xylitol, erythritol, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, phosphate, water, etc.

[0020] The medicine of the present invention can be applied to the body by known means. For example, it can be delivered to the corresponding tissue by intravenous systemic delivery or local injection. It can be applied via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0021] In some embodiments, the pharmaceutical composition can be administered to humans and non-human mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, gorillas, and the like.

[0022] The third aspect of the present invention provides a method for treating diabetic nephropathy, the method comprising: administering to a subject a substance that reduces acetylation of TFAM K76 site.

[0023] Beneficial technical effects of one or more of the above technical solutions or implementation schemes of the present invention are: The subject of the present invention is a human (especially a diabetic patient) or a non-human mammal, and the non-human mammal is a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey, an orangutan, etc.

[0024] The present invention reports for the first time the differential expression of acetylation of TFAM K76 site in diabetic nephropathy, indicating that it has a good diagnostic and prognostic effect on diabetic nephropathy; and through research it is found that by regulating the TFAM K76 site, especially inhibiting the acetylation modification of the TFAM K76 site, the inflammation and fibrosis in renal tubular epithelial cells and kidney tissue induced by high glucose can be reduced, and it can be used for the treatment of diabetic nephropathy. At the same time, small molecule compounds specifically targeting the TFAM K76 site are screened out. The screened small molecule compounds can significantly reduce the inflammation and fibrosis in renal tubular epithelial cells and kidney tissue induced by high glucose, and can slow down the kidney damage of diabetic nephropathy mice.

[0025] In summary, the present invention provides a new mechanism for the development and progression of diabetic nephropathy and a promising treatment strategy for patients with diabetic nephropathy, and therefore has great potential for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 The immunoprecipitation in Result 1 of Example 1 of the present invention verifies that the acetylation level of renal tubular epithelial cells increases under high glucose induction.

[0028] Figure 2 The immunofluorescence in Result 1 of Example 1 of the present invention verifies that the acetylation level of TFAM K76 in renal tubular epithelial cells is increased under high glucose induction.

[0029] Figure 3 The immunohistochemical results in Result 1 of Example 1 of the present invention verify that the acetylation level of TFAM K76 in the renal tissue of diabetic patients is increased.

[0030] Figure 4Result 1 of Example 1 of the present invention shows that the acetylation level of TFAM K76 in the kidney tissue of diabetic mice is increased by immunohistochemical verification.

[0031] Figure 5 The Western blot technique in Result 1 of Example 1 of the present invention detected an increase in the acetylation level of TFAMK76 in the kidney tissue of diabetic mice.

[0032] Figure 6 The Western blot technique in Result 2 of Example 2 of the present invention detected the level of TFAM K76 acetylation promoting inflammation and fibrosis in renal tubular epithelial cells.

[0033] Figure 7 The PCR technology in Result 2 of Example 2 of the present invention verifies that TFAM K76 acetylation in renal tubular epithelial cells increases TGF-β levels.

[0034] Figure 8 The Western blot technique in Result 2 of Example 2 of the present invention detected that TFAM K76 acetylation in renal tubular epithelial cells reduced mitochondrial biogenesis.

[0035] Fig. 9 The PCR technology in Result 2 of Example 2 of the present invention verifies that TFAM K76 acetylation in renal tubular epithelial cells inhibits mitochondrial DNA replication.

[0036] Fig.10 The Western blot technique in Result 3 of Example 3 of the present invention detected that TFAM K76 acetylation increased kidney damage in DKD mice.

[0037] Fig.11 The PCR technology in Result 3 of Example 3 of the present invention verifies that TFAM K76 acetylation in DKD mice promotes the transcription of inflammatory factors.

[0038] Fig.12 The PCR technology in Result 3 of Example 3 of the present invention verifies that TFAM K76 acetylation in DKD mice increases the level of chemokines.

[0039] Fig.13 This is a flow chart for screening small molecule compounds according to Example 4 of the present invention.

[0040] Fig.14 The Western blot technique in Result 4 of Example 4 of the present invention was used to screen small molecule compounds that inhibit TFAM K76 acetylation.

[0041] Fig.15 The Western blot technology in Result 4 of Example 4 of the present invention verifies the intervention concentration of small molecule compounds that inhibit TFAM K76 acetylation.

[0042] Fig.16 The protein thermal migration technology in Result 4 of Example 4 of the present invention verifies the binding of small molecule compounds to TFAM.

[0043] Fig.17 The BLI technology in Result 4 of Example 4 of the present invention verifies that the small molecule compound specifically targets TFAM.

[0044] Fig.18 The BLI technology in Result 4 of Example 4 of the present invention verifies that the small molecule compound specifically targets the TFAM K76 site.

[0045] Fig.19 The PCR technology in Example 5 of the present invention verifies that the small molecule compound rescues the mitochondrial DNA replication inhibited by high glucose.

[0046] Fig. 20 The Western blot technology in Result 5 of Example 5 of the present invention verifies that small molecule compounds alleviate high glucose-induced mitochondrial damage.

[0047] Fig.21 The PCR technology in Example 5 of the present invention verifies that small molecule compounds reduce high glucose-induced inflammation and fibrosis.

[0048] Fig. 22 The PCR technology in Example 5 of the present invention verifies that small molecule compounds reduce the increase of TGF-β induced by high glucose.

[0049] Fig.23 The Western blot technique in Example 6 of the present invention detected that small molecule compounds alleviated kidney damage in DKD mice.

[0050] Fig.24 The PCR technology in Example 6 of the present invention verifies that the small molecule compound reduces the level of inflammatory factors in DKD mice.

[0051] Fig.25 The PCR technology in Result 6 of Example 1 of the present invention verifies that the small molecule compound reduces the chemokine level in DKD mice. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0053] Example Materials and methods Cell transfection 24 hours before transfection, seed the cells into culture plates / dishes to a density of 70%-90% confluence at the time of transfection. 1 hour before transfection, replace with fresh complete medium (containing serum). Prepare transfection complexes: A. Dilute DNA: In a sterile EP tube, dilute the plasmid DNA with Opti-MEM medium.

[0054] B. Dilute Lipofectamine 3000 transfection reagent: In another EP tube, dilute Lipofectamine 3000 transfection reagent with Opti-MEM medium.

[0055] C. Mix DNA and transfection reagent: Gently mix the diluted DNA with the diluted Lipofectamine 3000 transfection reagent (volume ratio is 1:1) and let it stand at room temperature for 10-15 minutes to form a DNA-liposome complex. Add the mixture dropwise to the cell culture plate and gently shake the culture plate to mix. Place in a 37°C, 5% CO 2 Continue culturing in the incubator.

[0056] Immunohistochemical staining 1) Dewaxing and hydration: Immerse the sections in xylene I and II for 10 minutes each to dewax. Hydrate with gradient ethanol (100%→95%→80%→70%) for 5 minutes each. Finally, soak in PBS (phosphate buffer solution) for 5 minutes. 2) Antigen repair: Immerse the sections in antigen repair solution (such as pH 6.0 citrate buffer), microwave until boiling, maintain for 10-15 minutes, and cool to room temperature. 3) Block endogenous peroxidase: 3% H 2 O 2 Incubate at room temperature for 10 minutes, rinse with PBS 3 times × 5 minutes. 4) Blocking: Add blocking solution (such as 5% normal goat serum), incubate at room temperature for 30 minutes to reduce non-specific binding. 5) Primary antibody incubation: Aspirate the blocking solution, add diluted primary antibody, incubate at 4°C overnight or at room temperature for 2 hours. Rinse with PBS 3 times × 5 minutes to avoid residue. 6) Secondary antibody incubation: Add HRP or fluorescent labeled secondary antibody, incubate at room temperature for 1 hour. Rinse with PBS 3 times × 5 minutes. 7) DAB color development: Prepare DAB working solution according to the reagent instructions, add it to the slice, and control the color development time under a microscope. Rinse with running water to terminate the reaction. 8) Counterstaining: Stain the cell nucleus with hematoxylin for 1 minute, rinse with running water to return to blue. Dehydrate with gradient ethanol (70%→80%→95%→100%), and make it transparent with xylene. 9) Sealing and observation: Add neutral gum to seal the slice and observe under a microscope.

[0057] RNA extraction and real-time polymerase chain reaction (qRT-PCR) According to the instructions, the present invention used TRIzol reagent to isolate total RNA from LUAD cell lines. cDNA synthesis was then performed using PrimeScript RT Reagent Kit (TaKaPa, Dalian, China) according to the protocol in the instructions. qRT-PCR was performed using SYBR Green Premix EXTaq (TaKaPa, Dalian, China). The primers used are shown in Table 1 below. −ΔΔCt Methods All mRNA expression levels were calculated and normalized to GAPDH expression.

[0058] Table 1 Primer sequences used for qRT-PCR analysis.

[0059] Western Blotting Cells were lysed using RIPA buffer supplemented with protease inhibitors, and protein samples were subsequently separated by SDS-PAGE and transferred to PVDF (Bio-Rad Laboratories) membranes. The membranes were blocked in 5% milk and Tris-buffered saline containing 0.05% Tween 20 for 2 h at room temperature. Target antigens were probed with specific antibodies by incubation at 4 °C overnight. The following antibodies were used: TFAM K76 acetylation antibody (3683; QYAOBI), BAX (50599-2-Ig; Tri-Innovation Biotechnology, Wuhan, China); Bcl-2 (12789-1-AP; Tri-Innovation Biotechnology, Wuhan, China); N-cadherin (22018-1-AP; Tri-Innovation Biotechnology, Wuhan, China), Vimentin (10366-1-AP), KIM-1 (30948-1-AP), and β-actin (81115-1-RR). The membrane was washed in TBST and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Signal detection was performed using an enhanced chemiluminescence (ECL) detection system. The density of the β-actin band was used to normalize the density of the band representing a specific protein.

[0060] Immunofluorescence experiments Cells were seeded onto culture dishes or coverslips. When the cell density reached 60%-80%, the experiment was performed and high glucose was given for cell intervention. The culture medium was aspirated and the cells were gently rinsed with pre-cooled PBS twice for 5 minutes. 4% paraformaldehyde (prepared in PBS) was added and fixed at room temperature for 15-20 minutes. After fixation, the cells were rinsed with PBS three times for 5 minutes. 0.3% Triton X-100 (prepared in PBS) was added and permeabilized at room temperature for 10-15 minutes. PBS was rinsed three times for 5 minutes. 3% BSA blocking solution was added and blocked at room temperature for 30 minutes to reduce non-specific binding. The blocking solution was aspirated, the diluted primary antibody was added and incubated at 4°C overnight. The next day, the cells were rinsed with PBS three times for 5 minutes in the dark, and the fluorescently labeled secondary antibody was added and incubated at room temperature for 1 hour in the dark. PBS was rinsed three times for 5 minutes (operation in the dark). DAPI (fluorescent dye, 1 μg / mL, diluted in PBS) was added and incubated at room temperature for 5-10 minutes in the dark. Rinse with PBS 3 times for 5 minutes (keep away from light). Use filter paper to absorb excess liquid, add anti-fluorescence quenching mounting medium, and cover with coverslip. After drying in the dark, observe with fluorescence microscope or confocal microscope.

[0061] Mitochondrial DNA copy number detection Total DNA was extracted from cells, and the relative mitochondrial DNA copy number was estimated by detecting the mtDNA / nDNA ratio using fluorescent quantitative PCR.

[0062] Virtual screening based on TFAM crystal structure 1) Build a virtual screening model based on the crystal structure of the TFAM complex (PDB code: 3TMM). 2) It has been confirmed that the K76 residue is the key site of TFAM function, so the molecular docking site is defined with K76 as the center, and the corresponding grid file is generated using Maestro software. Then, the existing compound database (5000 small molecule compounds with high structural diversity) is molecularly docked with the grid file. According to the docking score, the top 150 small molecules are selected. Cluster analysis is performed on the 150 small molecules, and based on the results of the cluster analysis, 68 small molecules are finally selected for subsequent activity test research.

[0063] Statistical analysis Data were analyzed and plotted using GraphPad Prism 8. All experiments were performed at least 3 times. Student's t-test was used for comparison between two groups, and analysis of variance (95% CI) was used for multiple comparisons, P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

[0064] Experimental results: Results 1 The acetylation level of TFAM K76 site was significantly increased in DKD patients and mouse tissues.

[0065] In this example, high glucose was used to induce renal tubular epithelial cells, and immunoprecipitation was used to detect that high glucose induced a significant increase in the acetylation of TFAM ( Figure 1 ), but there is no report on TFAM acetylation in diabetic nephropathy. The TFAM K76 site is a key site for regulating TFAM function. Previous studies of the present invention found that TFAM K76 acetylation inhibits mitochondrial biosynthesis and reduces oxidative phosphorylation levels. In the present invention, this example found that high glucose induced a significant increase in TFAM K76 acetylation in renal tubular epithelial cells by cell immunofluorescence technology ( Figure 2 ), and immunohistochemistry revealed that TFAM K76 acetylation was significantly increased in the kidney tissues of DKD patients and DKD mice ( Figure 3 , Figure 4 ). Subsequently, this example detected by Western Blotting technology that TFAM K76 acetylation in the kidney tissue of DKD mice was significantly increased ( Figure 5 ).

[0066] Results 2 Acetylation of TFAM K76 site increased inflammation, fibrosis and mitochondrial damage in renal tubular epithelial cells. In this example, the acetylation and deacetylation of TFAM K76 were simulated by constructing a TFAM K76 site mutation plasmid. Plasmid transfection was used to express TFAM wild type, TFAM K76 acetylation, and TFAM K76 deacetylation in HK-2 cells. Western Blotting technology was used to detect that overexpression of TFAM K76 acetylation significantly increased the fibrosis of HK-2 cells, while overexpression of TFAM K76 deacetylation significantly reduced the fibrosis of HK-2 cells ( Figure 6 ). PCR detection showed that overexpression of TFAM K76 acetylation significantly increased the mRNA level of TGF-β1 in HK-2 cells, while overexpression of TFAM K76 deacetylation significantly reduced the mRNA level of TGF-β1 in HK-2 cells ( Figure 7 ). Acetylation of TFAM K76 site affects mitochondrial biogenesis. In the present invention, overexpression of TFAM K76 acetylation significantly reduced the protein level of electron transport complex subunits in HK-2 cells, while overexpression of TFAM K76 deacetylation significantly increased the protein level of electron transport complex subunits in HK-2 cells ( Figure 8 At the same time, this example detected the mitochondrial DNA (mtDNA) copy number and determined that overexpression of TFAM K76 acetylation significantly reduced the mtDNA copy number in HK-2 cells, while overexpression of TFAM K76 deacetylation significantly increased the mtDNA copy number in HK-2 cells ( Fig. 9 ).

[0067] Results 3 Acetylation of TFAM K76 site increased inflammation and fibrosis in the renal tissue of DKD mice. This example determines the effect of TFAM K76 site acetylation on HK-2 cells. To further verify the role of this site in mice, this example constructs a diabetic nephropathy mouse model. The successfully constructed mice were divided into 4 groups (8 mice in each group), and adeno-associated viruses were injected into the tail vein of the 4 groups of diabetic mice to overexpress TFAM wild type, TFAM K76 acetylation, and TFAM K76 deacetylation in the kidneys. Subsequently, the present invention extracted total protein and total RNA from mouse kidney tissues, and detected through Western Blotting technology that overexpression of TFAM K76 acetylation significantly increased the expression of kidney injury factor-1 (Kim-1) and increased kidney damage in diabetic mice, while overexpression of TFAM K76 deacetylation significantly reduced the expression of kidney injury factor-1 (Kim-1) and alleviated kidney damage in diabetic mice ( Fig.10 In addition, this example also detected the inflammatory factors in mouse tissues by qPCR technology ( Fig.11 ) and chemokines ( Fig.12 ) mRNA expression level, and found that overexpression of TFAMK76 deacetylation significantly reduced the level of inflammation in the renal tissue of diabetic mice. Therefore, this example clarifies the important role of TFAM K76 acetylation in diabetic mice and is a key target in the treatment of diabetic nephropathy.

[0068] Result 4 Screening of small molecule compounds targeting the TFAM K76 acetylation site. The present invention confirms that K76 is a key site of TFAM, and then screens small molecule compounds targeting the K76 site. Fig.13As shown, first, this embodiment builds a virtual screening model based on the TFAM crystal structure (PDB code: 3TMM), defines the molecular docking site with K76 as the center, and then performs molecular docking on the existing compound database (200,000 small molecule compounds with high structural diversity) and the grid file. According to the docking scores, the top 150 small molecules are selected. Next, a cluster analysis is performed on the 150 small molecules, and 68 small molecules are finally selected based on the results of the cluster analysis. Among these 68 small molecule compounds, some compounds were eliminated due to poor drugability, and then the remaining compounds were tested for activity and function. In this embodiment, the affinity between the compounds and TFAM is detected by BLI technology, and it is found that compounds 10, 14, 25, 34, and 35 have high affinity with TFAM protein. Next, this embodiment further screens the functions of the compounds through Western Blotting technology detection, and finds that in a high sugar environment, only compound 14 can significantly reduce the acetylation level of TFAMK76 ( Fig.14 ), and the screened compound 14 was named S14. Moreover, S14 had the most significant downregulation of TFAM K76 acetylation at a concentration of 50 μM ( Fig.15 In HK-2 cells, compound 14 was found to significantly increase the thermal stability of TFAM protein after S14 intervention ( Fig.16 In addition, this example further demonstrates that the affinity of S14 for binding to TFAM protein increases in a concentration gradient-dependent manner using BLI technology ( Fig.17 ), but cannot bind to the TFAM K76R mutant protein ( Fig.18 ), which further illustrates the targeting effect of S14 on the TFAM K76 site.

[0069] Among them, the chemical structure of S14 is: .

[0070] Results 5 Small molecule compounds alleviate high glucose-induced inflammation, fibrosis and mitochondrial damage in renal tubular epithelial cells. In order to further verify the effect of the screened S14, this example gave S14 treatment in high glucose-induced HK-2 cells. qPCR and Western Blotting technology detection showed that S14 treatment significantly increased the mitochondrial DNA (mtDNA) copy number under high glucose induction ( Fig.19 ) and electron transport complex subunit protein levels ( Fig. 20), indicating that S14 treatment increased the mitochondrial biosynthesis of HK-2 cells induced by high glucose. In addition, this example detected the changes in inflammation and fibrosis by qPCR technology and Western Blotting technology, and determined that S14 treatment reduced the fibrosis and apoptosis of HK-2 cells induced by high glucose ( Fig.21 ) and reduced the mRNA level of TGF-β1 in HK-2 cells induced by high glucose ( Fig. 22 ). This example screened out a new small molecule compound for treating diabetic nephropathy based on protein acetylation regulation.

[0071] Results 6 Small molecule compounds alleviated inflammation and fibrosis in renal tissue of DKD mice.

[0072] Subsequently, the present invention used S14 to treat diabetic mice. During the treatment, the present embodiment also set up a dapagliflozin treatment group as a control. After the treatment, the total protein and total RNA of the mouse kidney tissue were extracted respectively. The Western Blotting technique was used to detect that S14 treatment significantly reduced the expression of kidney injury factor-1 (Kim-1) and alleviated the kidney injury of diabetic mice ( Fig.23 At the same time, qPCR technology was used to detect the inflammatory factors in mouse tissues ( Fig.24 ) and chemokines ( Fig.25 ) mRNA expression levels, and found that S14 treatment significantly reduced the level of inflammation in the kidney tissue of diabetic mice. Therefore, this example further confirmed the therapeutic effect of S14 on delaying the progression of renal injury in diabetic mice, which is a newly discovered effective strategy for the treatment of diabetic nephropathy.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of any one of the following a1)-a4) in the preparation of a drug for diagnosing diabetic nephropathy: a1) Acetylation modification of TFAM K76 site in the renal tissue of the subjects; a2) a nucleic acid encoding an acetyltransferase that modifies the acetylation of the TFAM K76 site; a3) Reagents for detecting acetylation modification expression of TFAM K76 site; a4) Reagents for detecting the expression of acetyltransferases encoding acetylated TFAM K76 sites.

2. Use of a substance that reduces or inhibits the expression level of acetylation modification at the TFAM K76 site in at least one of the following b1)-b3): b1) Preparation of products for reducing inflammation and / or fibrosis of renal tubular epithelial cells; b2) preparing products for reducing mitochondrial damage in renal tubular epithelial cells; b3) preparing products for screening to slow the progression of diabetic nephropathy; b4) preparing medicines for treating diabetic nephropathy.

3. The use according to claim 2, characterized in that: In b3), the diabetic nephropathy is a disease caused by diabetes and characterized by lesions in kidney tissue or renal tubular epithelial cells.

4. The use according to claim 2, characterized in that: The product described is a drug or an experimental reagent.

5. The use according to claim 2, characterized in that: The substance that reduces or inhibits the expression level of acetylation modification of TFAM K76 site is a compound that reduces or inhibits the expression level of acetylation modification of TFAM K76 site.

6. The use according to claim 5, characterized in that: The chemical structure of the compound is shown below: 。 7. The use according to claim 2, characterized in that: The medicine is a pharmaceutical composition and also includes pharmaceutical excipients.

8. The use according to claim 7, characterized in that: The pharmaceutical excipients are carriers and / or excipients.

9. The use according to claim 8, characterized in that: The carrier and excipient are one or more of alumina, serum protein, lecithin, maltitol, starch, gum arabic, sucrose, lactose, alginate, glucose, sorbitol, mannitol, magnesium stearate, mineral oil, xylitol, erythritol, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, phosphate, and water.

10. The use according to claim 2, characterized in that: The subjects of administration of the drug are humans and non-human mammals.

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