Application of GNPAT as target spot in medicine for treating diabetic nephropathy podocyte damage

Abstract

The invention provides application of GNPAT as a target in a medicine for treating diabetic nephropathy (DKD) podocyte injury, GNPAT molecules are taken as target protein, the medicine reduces podocyte lipid deposition by knocking down the expression level of GNPAT so as to further improve the podocyte injury, and the treatment effect of reducing the DKD podocyte injury by the medicine is achieved. According to the characteristics that expression of GNPAT in renal cortex of diabetic mice and serum and urine of DKD patients is remarkably increased and the characteristics that GNPAT is in positive correlation with related indexes of renal functions, GNPAT serves as a target spot, and the purpose of treatment is achieved after GNPAT is knocked down through a drug for DKD treatment; meanwhile, the GNPAT molecule can be used as a target for drug screening and development, and a compound which can be combined with GNPAT and reduce GNPAT expression is screened and developed, so that a new direction is provided for targeted therapy of DKD podocyte injury.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of basic and clinical medicine, in particular to application of GNPAT as a target point in a drug for treating diabetic kidney disease podocyte injury. BACKGROUND

[0002] Diabetes mellitus (DM) is the primary medical science and public health problem faced by almost all countries in the world. Renal injury caused by DM is called diabetic kidney disease (DKD), which is one of the most common chronic complications of DM. The main clinical features of DKD include renal failure, proteinuria and elevated serum and urine creatinine levels, which can eventually develop into end-stage renal disease, posing a serious threat to human physical health. In the pathological process of DKD, lipid deposition plays an important role. Studies have shown that under the condition of hyperglycemia, abnormal lipid metabolism can lead to lipid deposition and induce podocyte apoptosis, thereby causing renal function damage. Therefore, elucidating the mechanism of DKD podocyte lipid deposition and exploring drug targets that can prevent or delay podocyte lipid deposition are of great significance for improving the clinical treatment strategy of DKD.

[0003] Further, podocytes are components of the glomerular filtration barrier and are the most terminally differentiated type of resident cells in the kidney. Podocyte injury is one of the key factors leading to glomerular disease. Podocyte loss and foot process fusion are involved in the development of DKD renal injury. Normal lipid metabolism is essential for maintaining podocyte function. In the DM state, decreased fatty acid beta-oxidation and increased fatty acid synthesis lead to a significant increase in lipid content in the kidney. Lipid deposition, formation of a large number of lipid droplets and presentation of a foamy change in the podocyte interior have been observed in patients with DKD, obesity-related glomerulopathy and focal segmental glomerulosclerosis, thereby leading to increased podocyte injury. It can be seen that a large amount of lipid deposition exists in the podocyte interior in the pathological process of DKD and plays a very important role.

[0004] Based on the above analysis, the application provides application of GNPAT as a target point in a drug for treating diabetic podocyte injury, GNPAT is screened as a key factor for regulating DKD podocyte lipid metabolism through data-independent acquisition (DIA) proteomics combined with bioinformatics analysis; and the role of GNPAT in DKD podocyte lipid deposition is studied in vivo and in vitro, confirming that GNPAT plays an important role in DKD podocyte lipid deposition. This finding provides a new idea for exploring DKD drug treatment targets and drug screening, and also provides guidance for targeted treatment of DKD podocyte injury. SUMMARY

[0005] In order to solve the above technical problems, the application discloses an application of GNPAT as a target point in a drug for treating diabetic nephropathy podocyte injury.

[0006] In order to achieve the above-mentioned purpose, the application provides an application of GNPAT as a target point in a drug for improving and / or treating DKD podocyte injury.

[0007] Preferably, as the target point of GNPAT, the drug inhibits high-glucose-induced podocyte lipid deposition by knocking down GNPAT; or, the drug can reduce glomerular lipid droplet deposition by knocking down podocyte GNPAT, the expression level of GNPAT is positively correlated with the expression level of lipid droplet marker protein Adipophilin; or, the drug can alleviate glomerular injury by knocking down podocyte GNPAT, the expression level of GNPAT is negatively correlated with the expression level of mouse kidney podocyte marker protein Nephrin; or, the drug can reduce high-glucose-induced podocyte injury by knocking down podocyte GNPAT.

[0008] As another object of the application, the application also provides an application of a GNPAT molecule as a drug target in development or screening of a drug for treating DKD or DKD podocyte injury.

[0009] Preferably, the method for developing or screening the drug comprises using the GNPAT molecule as a target protein, the drug comprises a compound capable of binding to the GNPAT molecule, and the compound improves high-glucose-induced podocyte injury by knocking down GNPAT.

[0010] Preferably, the method for developing or screening the drug comprises using the GNPAT molecule as a target point to design and synthesize a compound, and screening the compound by computer simulation molecular docking experiment, screening a compound capable of binding to GNPAT, and performing activity evaluation.

[0011] Based on the above technical solutions, the application can also provide a drug for treating DKD or DKD podocyte injury, comprising a compound capable of binding to the GNPAT molecule.

[0012] Technical effects of the technical solutions of the application:

[0013] 1. The application finds, through DIA proteomics combined with bioinformatics analysis, that GNPAT is a key factor for regulating DKD podocyte lipid metabolism; it is confirmed that GNPAT plays an important role in DKD podocyte lipid deposition, thereby providing a new idea for DKD podocyte injury in DKD drug treatment by taking GNPAT as a target of podocyte injury drugs.

[0014] 2.The present application realizes the purpose of treatment by knocking down GNPAT as a target and applying it to drugs for the treatment of DKD, through the significant increase in the expression of GNPAT in the kidney cortex of DKD mice and the serum and urine of DKD patients, and the positive correlation between GNPAT and indicators related to kidney function, such as urine microalbumin, blood creatinine, urea nitrogen, etc. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a graph of the changes in body weight, blood glucose, blood lipids and kidney function indicators of db / m and db / db mice of the same age in a typical embodiment of the present application.

[0016] Figures 2-1-2-2 is the degree of kidney fibrosis of db / m and db / db mice of different ages in a typical embodiment of the present application.

[0017] Figure 3 is a graph of basic data analysis of DIA proteomics in a typical embodiment of the present application.

[0018] Figure 4 4-1- Figure 4-4 is a graph of bioinformatics analysis of the important role of GNPAT in DKD kidney lipid deposition in a typical embodiment of the present application.

[0019] Figure 5 is a graph of the analysis of the expression pattern of GNPAT under high glucose conditions in a typical embodiment of the present application.

[0020] Figures 6-1-6-3 is a graph of the analysis of the correlation between GNPAT and DKD in a clinical study in a typical embodiment of the present application.

[0021] Figure 7 is a graph of the establishment of GNPAT knockdown and overexpression cell lines in a typical embodiment of the present application.

[0022] Figures 8-1-8-6 is a graph of the effect of knocking down or overexpressing GNPAT on podocyte injury in a typical embodiment of the present application.

[0023] Figures 9-1-9-4 is a graph of the establishment of podocyte-specific overexpression of GNPAT db / m mice or podocyte-specific knockdown of GNPAT db / db mice in a typical embodiment of the present application.

[0024] Figures 10-1-10-5 is a graph of the effect of podocyte-specific knockdown or overexpression of GNPAT on kidney function in mice in a typical embodiment of the present application.

[0025] Figures 11-1-11-4Figure is a graph showing the effect of podocyte-specific knockdown or overexpression of GNPAT on glomerular injury in a typical embodiment of the present application.

[0026] Figures 12-1-12-3 Figure is a graph showing the lipid deposition in the kidney under high glucose condition in a typical embodiment of the present application.

[0027] Figures 13-1-13-2 Figure is a graph showing the effect of knockdown or overexpression of GNPAT on podocyte lipid deposition in a typical embodiment of the present application.

[0028] Figures 14-1-14-3 Figure is a graph showing the effect of podocyte-specific knockdown or overexpression of GNPAT on lipid deposition in the kidney of mice in a typical embodiment of the present application. DETAILED DESCRIPTION

[0029] The purposes, technical solutions and advantages of the embodiments of the present application are made clearer, and the technical solutions in the embodiments of the present application are described clearly and completely. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0030] The present application provides a use of GNPAT as a target for a drug for treating diabetic podocyte injury. The drug uses GNPAT as a target, and inhibits high glucose-induced podocyte lipid deposition by knocking down GNPAT.

[0031] In some specific embodiments, DIA technology is used to identify the differential proteins in the renal cortex of 24-week-old db / db and db / m mice, bioinformatics analysis is used to mine key factors promoting the occurrence and development of DKD, the expression patterns thereof are verified on four levels of clinic, animal, cell and molecule, and the correlation of GNPAT with the pathological process of DKD is analyzed. The level of the factor in the serum of DKD patients is determined by measuring the serum samples and urine residual proteins of different populations, and the correlation of the factor with the renal function indicators of clinical patients is evaluated by Spearman correlation analysis. The expression of the factor and the expression of the lipid droplet marker protein Adipophilin in the kidney of db / db mice are detected by IHC and Wes, and the correlation between the two is evaluated; the lipid deposition in the kidney of mice is observed by modified oil red O staining, and the results show that the expression level of GNPAT is positively correlated with the expression level of Adipophilin.

[0032] In some embodiments, real-time PCR, Western blot and immunofluorescence are used to detect the expression of the factor in four types of kidney intrinsic cells (podocytes, mesangial cells, renal tubular epithelial cells and glomerular endothelial cells) under high glucose conditions; modified oil red O staining is used to observe the lipid deposition of renal parenchymal cells under high glucose conditions. Wes is used to detect the expression of the podocyte marker protein Nephrin, and it is found that there is a negative correlation between GNPAT and Nephrin, so that the high glucose-induced lipid deposition can be reduced by knocking down GNPAT, and the podocyte injury in DKD can be improved.

[0033] In some embodiments, GNPAT is overexpressed in podocytes cultured under normal sugar concentration, and GNPAT is knocked down in podocytes cultured under high sugar to detect the following indicators: (1) GNPAT expression: Real-time PCR, Western Blot and immunofluorescence methods are used to detect the expression level of GNPAT in podocytes; (2) lipid metabolism related indicators: protein expression levels of lipid synthesis related proteins SREBP1 and FASN; oil red O staining and nile red staining are used to observe the lipid deposition of podocytes; immunofluorescence and Western blot are used to detect the level of Adipophilin, a specific marker of lipid droplets. (3) Podocyte injury indicators: flow cytometry is used to detect the apoptosis of podocytes; Western blot is used to detect the expression of Cleaved-Caspase3, Bcl-2, Bax, Synaptopodin and Nephrin, which are podocyte marker proteins.

[0034] In some embodiments, a kidney parenchyma is injected with an adeno-associated virus containing a podocyte-specific promoter to knock down or overexpress GNPAT, and a podocyte-specific overexpression GNPAT db / m mouse and a podocyte-specific knockdown GNPAT db / db mouse are constructed, and the following indicators are detected: (1) GNPAT expression and distribution: Wes is used to detect the expression of GNPAT in glomeruli; immunohistochemistry and immunofluorescence are used to detect the distribution of GNPAT; (2) renal function indicators: urea nitrogen, creatinine, urine trace albumin, etc.; (3) PAS and PASM staining are used to observe the glycogen deposition and basement membrane thickening in the mesangial region of the glomerulus, and transmission electron microscopy is used to observe the changes in the podocyte foot process and basement membrane thickness of the glomerulus; (4) lipid metabolism related indicators: modified oil red O staining is used to observe the lipid deposition in the glomerulus; immunohistochemistry and Wes are used to observe the expression of Adipophilin, a specific marker of lipid droplets in the glomerulus; finally, Spearman correlation analysis is used to analyze the positive correlation between the expression of GNPAT in the glomerulus and the renal function indicators and the level of Adipophilin.

[0035] The results of the above cell level, animal experiments and clinical analysis show that GNPAT plays an important role in the pathological process of DKD, and knocking down GNPAT can improve the damage of podocytes caused by high glucose, and knocking down GNPAT in podocytes can slow down the thickening of the basement membrane caused by DM and other glomerular damage. Based on the above analysis, GNPAT can be used as a drug target for treating DKD. After the drug binds to GNPAT, the compounds are screened by computer simulation molecular docking experiment, compounds that can bind to PEX16 are screened, and activity evaluation is performed.

[0036] The technical solutions of the present application will be further described in detail below through specific examples. The experimental methods in the following examples without specific conditions are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturers.

[0037] Example 1: Proteomics combined with bioinformatics analysis to screen key factor GNPAT promoting the occurrence and development of DKD

[0038] This example studies the expression pattern of the key factor GNPAT in the pathological process of DKD disease by DIA proteomics technology combined with bioinformatics analysis, at the clinical, animal and cell and molecular levels.

[0039] 1. Reagents: SDS-AGE gel rapid preparation kit, trypsin cell digestion solution, anti-fluorescence quenching sealing agent (containing DAPI), RIPA lysis buffer, PMSF were purchased from Biyun Tian Biotechnology Co., Ltd.; DAB color developing kit, rabbit two-step method detection kit, mouse two-step method detection kit were purchased from Beijing Zhongsanjin Qiao Biotechnology Co., Ltd.; RPMI 1640 Medium, Fetel Bovine Serum were purchased from Nanjing Senbeiga Biotechnology Co., Ltd.; Apoptosis, phosphatase inhibitor, detection kit were purchased from KAIJIBIO; Glycogen PAS staining kit (G1285), Sirius red staining kit (G1470), Masson's trichrome staining kit (G1340), modified oil red O dye were purchased from Beijing Solabio Technology Co., Ltd.; Urea nitrogen (BUN) test kit (urease method), creatinine (Cr) kit (sarcosine oxidase method), malondialdehyde (MDA) test kit, urine protein quantitative kit, mouse urine trace albumin ELISA kit (LP-M03270), Human GNPAT ELISA Kit were purchased from Shanghai Lanpai Biotechnology Co., Ltd.; TRNzol Universal Reagent was purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.; GNPAT Polyclonal antibody was purchased from Proteintech Company; Adipophilin / Perilipin2 Polyclonal antibody was purchased from Proteintech Company; Peroxin 16 Antibody (H-4), Bcl-2 Antibody (C-2), Synaptopodin Antibody (D-9), Anti-beta Actin Antibody were purchased from Santa Cruz Company; cDKDA reverse transcription kit (FSQ-101) was purchased from Japan TOYOBO Company; SYBR Green I Master was purchased from the United States Roche Company; Wes kit (SM-W004-1) was purchased from Punsun Biotechnology Co., Ltd.

[0040] 2 Clinical analysis

[0041] 2.1 Sample collection and grouping: Collect serum samples of healthy people, type 2 diabetes, and DKD patients and group them, respectively, including: healthy control group (NC group), type 2 diabetes group (T2DM group), and DKD group (DKD group).

[0042] Grouping criteria: NC group: fasting blood glucose (FBG) <7.0 mmol / L, HemoglobinA1c (HbA1c) <6.2%, negative urinary protein, serum creatinine (Cr), urea nitrogen (BUN) were normal; T2DM group: FBG >7.0 mmol / L, HbA1c >6.2%, negative urinary protein, CREA was normal; DKD group: FBG >7.0 mmol / L, HbA1c >6.2% or random blood glucose >11.1 mmol / L.

[0043] 2.2 Number of clinical sample collection: 30 healthy people, 30 DM patients and 63 DKD patients were screened according to the above criteria. 3 mL of fasting morning blood (anticoagulated with EDTA) was collected on the same day, centrifuged at 3000 rpm for 15 min, and the supernatant was stored in a -20°C refrigerator for standby.

[0044] 2.3 Detection of clinical serum samples and correlation analysis by ELISA kit

[0045] According to the operation manual of human serum GNPAT ELISA kit of Shanghai Lanpai Biological Technology Co., Ltd., the level of GNPAT in serum was detected.

[0046] Spearman correlation analysis method was used to analyze the correlation of GNPAT in clinical serum samples with renal function indicators: serum creatinine (Scr), BUN, blood uric acid (BUA), alpha 1-microglobulin (alpha 1-MG), cystatin C (Cys C), glomerular filtration rate (eGFR), urinary microalbumin (mAlb), urinary albumin creatinine ratio (UACR), and the correlation coefficient was calculated.

[0047] 2.4 Preparation of clinical serum sample immunoblotting experiment

[0048] NanoDrop One ultramicro spectrophotometer was used to determine the protein concentration of clinical serum samples. After adding 5x bromophenol blue loading buffer, denaturation at 100°C for 10 min, and storage in a -20°C refrigerator for standby.

[0049] According to the molecular weight of the protein to be tested, cut the gel according to the size of the gel, and cut the NC membrane according to the size and length of the gel; according to the sandwich structure of thick filter paper, NC membrane, gel, thick filter paper, clamp in the wet transfer membrane system, add fast transfer solution, constant current condition 400mA, 20min (less than 150 molecular weight) transfer, if the transfer is larger molecular weight protein, the transfer time can be appropriately prolonged; the transferred NC membrane is blocked in 3% BSA for 30min; incubate the NC membrane in the corresponding primary antibody in the refrigerator at 4°C overnight, the next day, wash the NC in PBST for 5min on the shaker for 3 times, add HRP secondary antibody, incubate at room temperature for 1h, wash the membrane in PBST for 5min on the shaker for 2 times, wash in PBS for 5min; immerse the NC membrane in ECL (enhanced chemiluminescence) luminescent solution, and chemiluminescence imaging; analyze the gray value of the band by Image J software, and express the expression amount of the target protein by the ratio of the optical density of the target protein to the internal reference protein.

[0050] 2.5 SDS-PAGE electrophoresis: prepare electrophoresis gel, divided into upper and lower gels, see Table 1 for formula.

[0051] Table 1 Gel thickness and component ratio of electrophoresis gel

[0052]

[0053] Each component is inverted and mixed for 6-8 times before use; lower gel preparation: take equal volume of Resolver A and Resolver B, 2.0 / 2.7 / 4.0mL each, mix well; upper gel preparation: take equal volume of Stacker A and Stacker B, 0.5 / 0.75 / 1.0mL each, mix well, add 40 / 60 / 80ul of APS to the upper gel mixture solution, mix thoroughly immediately, pour into the gel glass plate, and the liquid level is 1.5cm from the upper edge of the glass plate; add 10 / 15 / 20ul of APS to the lower gel mixture solution, mix thoroughly immediately, without waiting for the lower gel to solidify, directly slowly pour into the gel glass plate, insert the comb; after the gel is solidified (usually 15min at room temperature), remove the comb and it can be used for electrophoresis. Gel is loaded into the electrophoresis tank, and electrophoresis solution is added. Electrophoresis conditions: 150V under constant voltage, electrophoresis for 1h10min.

[0054] 3 Animal experiment

[0055] 3.1 Animals: SPF level db / m and db / db mice, purchased from Changzhou Cavens Experimental Animal Co., Ltd.

[0056] 3.2 Mouse feeding and minutes: This example uses male db / m, db / db mice as experimental objects in accordance with the standard of SPF level. The db / db mice (DKD model mice) are fed as the test group, and the db / m mice are used as the control group. Urine, blood and kidney samples of db / m and db / db mice are collected at 8, 16 and 24 weeks of age.

[0057] 3.3 Mouse weight and blood glucose determination: The mice in each group were weighed and FBG was determined the next day after fasting for 8 hours the night before. The tip of the mouse tail was disinfected with an alcohol cotton ball; the blood glucose test paper chip end was inserted into the blood glucose meter; the mouse tail tip was cut off with ophthalmic scissors, and a drop of mouse blood was squeezed into the tail end of the blood glucose test paper, and a clean cotton swab was used to press the mouse tail tip to stop bleeding; the blood glucose meter reading was recorded. 3.4 Mouse blood, urine sample and kidney tissue sample preparation: The mouse 24h urine was collected in a metabolic cage, the urine volume was recorded, and the supernatant was stored at -20°C after centrifugation at 3000 rpm for 15 min at 4°C; the mouse was sacrificed by cervical dislocation, and the two kidneys were removed and decapsulated, and the kidney weight was recorded. Part of the kidney was fixed in 4% paraformaldehyde, and the rest of the kidney was extracted and separated by sieving method, and stored at -20°C for later use.

[0058] Glomerulus isolation steps: The kidney cortex was cut with ophthalmic scissors, and then placed in 3 layers of stainless steel screens with 80, 150 and 300 meshes, respectively. The kidney cortex tissue was gently crushed with a syringe on the uppermost layer, and washed with 4°C D-HANK's solution to filter into the lower screen. The process was repeated. When reaching the last layer, only washing was performed without crushing. Finally, the last layer was washed in reverse, and the solution was collected. The supernatant was discarded after centrifugation at 1200 rpm for 5 min at 4°C, and the glomeruli were obtained.

[0059] 3.5 Mouse blood biochemical index determination

[0060] Determination of blood creatinine (Cr): according to the operation instruction of creatinine Cr kit (No. C011-2) of Nanjing Jiancheng Biological Engineering Institute; determination of blood urea nitrogen (BUN): according to the operation instruction of urea nitrogen BUN kit (No. C013-2) of Nanjing Jiancheng Biological Engineering Institute; determination of blood lipid level: take an appropriate amount of mouse serum to Xuzhou Dongfangrenmin Hospital for determination of mouse blood lipid level; determination of mouse urine microalbumin (MAU / ALB): according to the operation instruction of mouse urine microalbumin ELISA kit (LP-M03270) of Shanghai Lanpai Biological Technology Co., Ltd.; morphological detection of kidney: the previously 4% paraformaldehyde-fixed kidney tissue was entrusted to Wuhan Savel Biological Technology Co., Ltd. for tissue dehydration, embedding and paraffin section, and the tissue section was deparaffinized: xylene I 10 min→xylene II 10 min→100% ethanol 5 min→95% ethanol 5 min→80% ethanol 5 min→70% ethanol 5 min→PBS washing and then used.

[0061] Periodic acid-Schiff (PAS) staining: 1% periodic acid oxidation for 8 min; Schiff reagent for 15 min; hematoxylin restaining for 2 min; 1% hydrochloric acid alcohol differentiation for 5 s; dehydration and mounting. Six non-overlapping fields were observed, and the ratio of purple gray value of positive area of glomerulus to gray value of observation area was analyzed by Image J Pro plus software.

[0062] Masson staining: hematoxylin for 5 min; 1% hydrochloric acid alcohol differentiation for 3 s; staining with acid magenta for 10 min; 5% phosphomolybdic acid differentiation; into acetic acid aqueous solution for 30 s; dehydration and mounting. Six non-overlapping fields were observed, and the ratio of blue gray value of positive area of glomerulus to gray value of observation area was analyzed by Image J Pro plus software.

[0063] Sirius red staining: Weigert iron hematoxylin staining for 10-20 min, Sirius red staining liquid for 1 h, and dehydration and mounting. Six non-overlapping fields were observed, and the ratio of red gray value of positive area of glomerulus to gray value of observation area was analyzed by Image J Pro plus software.

[0064] Modified Oil Red O staining and Nile Red staining: After removing frozen sections from the -20℃ freezer, allow them to equilibrate to room temperature for 30 minutes. Remove the cell slides. Clean with PBS for 5 minutes, then briefly soak in 60% isopropanol to facilitate staining. Stain in modified Oil Red O staining solution (mixed according to the modified Oil Red kit reagent A1: reagent A2 = 3:2 ratio, allowed to stand for 10 minutes, filtered, and used for later use; do not prepare in advance) for 20 minutes in the dark. Remove the Oil Red dye with 60% isopropanol (observe under a microscope to ensure complete removal). Wash with double-distilled water for 5 minutes, stain the nuclei with hematoxylin for 1 minute, rinse with tap water for 5-10 minutes to regain blue color, and rinse thoroughly with double-distilled water. Finally, mount the slides with mounting medium. Alternatively, remove the cell slides, wash with PBS for 5 minutes, stain directly with Nile Red staining solution (0.05 mg / mL) for 15 minutes, wash thoroughly with PBS, and mount with mounting medium.

[0065] 4. Cell Experiment

[0066] 4.1 Cells: Mouse podocytes (MPC5), purchased from BNCC337685, a subsidiary of Beina Biotechnology Co., Ltd.; mouse mesangial cells (MCs), SV40 MES13, purchased from the Cell Bank of the Chinese Academy of Sciences (Catalog No.: GNM21); mouse renal tubular epithelial cells (RTEC), purchased from Shanghai Lianmai Biotechnology Co., Ltd. (Catalog No.: LM858); mouse glomerular endothelial cells (MRGEC), purchased from Beina Biotechnology Co., Ltd. (Catalog No.: 553370, BD).

[0067] 4.2 Cell Culture: Mouse podocytes (MPC5) and mesangial cells (SV40) were cultured in RPMI 1640 Medium (11.1 mmol / L glucose, containing 10% FBS), while glomerular endothelial cells (MRGEC) and renal tubular epithelial cells (MRTEC) were cultured in DMEM low glucose (5.56 mmol / L glucose, containing 10% FBS). Culture conditions: 5% CO2, constant temperature 37℃.

[0068] Subculturing: Discard the old culture medium, wash the cell flask twice with 2 mL of PBS, add 1 mL of 0.25% trypsin digestion solution, and when the cells shrink to a near-round shape, the bottom of the cell flask appears frosty, and the cells have not started to detach, the cell digestion endpoint has been reached. Add 2 mL of complete culture medium to stop digestion, centrifuge at 800 rpm for 3 min, add an appropriate amount of culture medium and mix by pipetting, and subculture according to the experimental purpose. Gently shake the cells in a figure-eight motion to distribute them evenly, and culture in a 5% CO2, 37℃ constant temperature incubator.

[0069] Freezing: The previous steps are the same as cell passage. After the complete medium stops digestion, centrifuge the cells, add serum-free cell freezing solution, mix the suspended cells by blowing, and distribute them into freezing tubes. Immediately after sealing, place them in a -80°C ultra-low temperature refrigerator. If long-term storage is required, move them to a liquid nitrogen tank for storage.

[0070] Recovery: Remove the frozen cells from the -80°C ultra-low temperature refrigerator or liquid nitrogen tank, immediately heat them in a 37°C water bath, and constantly shake them during heating to ensure uniform heating. After the freezing solution is thawed, move it to a clean bench, and transfer the cell freezing solution to a centrifuge tube containing complete medium. Centrifuge at 800 rpm for 3 min, add 5 mL of complete medium to disperse and resuspend the cells, and transfer them to a cell bottle. Gently shake the cells in a V-shaped manner to distribute them evenly, and culture them in a 5% CO2, 37°C incubator.

[0071] 4.3 Cell treatment: When the cells grow to about 40% density, give treatment. MPC5 and SVS40 normal sugar concentration is 11.1 mmol / L, high sugar concentration is 40 mmol / L, MRTEC and MRGEC normal sugar concentration is 5.56 mmol / L, high sugar concentration is 40 mmol / L, simulating DM conditions. The cells are divided into two groups: normal group (NG), high sugar group (HG). Before the cells are treated with high sugar, they are synchronized for 12 h with serum-free medium, and after 48 h of sugar treatment, the cells are scraped with a cell scraper. Centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the cells.

[0072] 4.4 Tissue and cell sample preparation: Add 9 volumes of prepared RIPA lysis buffer (containing 1 mmol / L PMSF and phosphatase inhibitor) to the kidney tissue, and add 9 volumes of prepared NP40 lysis buffer to the cells. Homogenize the tissue or break the cells in an ice water bath, and lyse on ice for 30 min, with vortexing every 10 min. Centrifuge at 12000g for 15 min at 4°C.

[0073] Draw the supernatant to obtain the extracted protein solution, measure the protein concentration of the sample with a NanoDrop One ultramicro spectrophotometer, add 5x bromophenol blue loading buffer, denature at 100°C for 10 min, and store in a -20°C refrigerator for standby.

[0074] According to the instructions provided by Wes full-automatic protein expression analysis system of PrimoSonic Biotech Co., Ltd., process and load the sample. After the instrument performs a Wes hardware self-check, place the capillary tube and the loaded plate into the instrument to start detection. After detection is complete, use Compass for SW software to calculate.

[0075] 5 Extraction of total RNA

[0076] 5.1 Cell and tissue treatment method:

[0077] Cells: rinse the cells with pre-cooled 4℃ PBS for 5 min. Add 1 mL Trizol to each sample, and let it stand on ice for 5 min. Then transfer the Trizol to a nuclease-free 1.5 mL centrifuge tube with a nuclease-free gun tip.

[0078] Tissues: take fresh tissue samples, cut them into 1 mm3 pieces with ophthalmic scissors, and place them in a nuclease-free 1.5 mL centrifuge tube. Add 1 mL Trizol, homogenize on ice, and let it stand for 5 min.

[0079] 5.2 Trizol extraction of RNA: add 200 μL chloroform to 1 mL Trizol lysis solution in each sample, mix gently, and let it stand at room temperature for 5 min. Centrifuge at 4℃, 12000 g for 15 min. Transfer the supernatant to a new nuclease-free 1.5 mL centrifuge tube, taking care not to suck up the lower layer, and record the volume of the supernatant. Add an equal volume of isopropanol, mix, and let it stand at room temperature for 10 min. Centrifuge at 4℃, 12000 g for 15 min. Discard the supernatant, and leave the crescent-shaped RNA at the bottom of the centrifuge tube. Add 75% ethanol, and wash the RNA three times. After discarding the ethanol, air dry, add 20 μL nuclease-free water, and dissolve the RNA in water by vortexing.

[0080] 5.2.1 RNA concentration determination: use a NanoDrop one spectrophotometer to determine the RNA concentration.

[0081] 5.2.2 Real-time quantitative PCR experiment: reverse transcription: prepare the reverse transcription reaction system, and the formula is shown in Table 2.

[0082] Table 2 Components of the reverse transcription reaction system

[0083]

[0084] Reaction conditions: 50℃ for 15 min; 85℃ for 5 s.

[0085] qRT-PCR: configure the qRT-PCR system, and the formula is shown in Table 3.

[0086] Table 3 Components and proportions of the qRT-PCR amplification system

[0087]

[0088] Note: the cDNA should not exceed 1 / 10 of the reaction system.

[0089] Perform the qPCR reaction according to the conditions listed in Table 4.

[0090] Table 4 qPCR reaction program

[0091]

[0092] The primer sequences of the qPCR reaction are as follows, see Table 5.

[0093] Table 5 Primer sequence list

[0094]

[0095] 6 Immunohistochemistry: Paraffin sections were baked at 60°C for more than 60 min, xylene I and II for 10 min each, gradient ethanol dewaxing, anhydrous ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, PBS washing for 5 min; pepsin antigen retrieval at 37°C for 30 min, PBS washing for 3 times, 3 min each time; 3% H2O2 was used to block endogenous peroxidase for 10 min, PBS washing for 3 times, 3 min each time; 5% BSA was used for room temperature blocking for 20 min. GNPAT and Adipophilin primary antibodies were added and incubated at 4°C overnight. The next day, the temperature was restored to room temperature for 30 min, and PBS was washed for 3 times, 3 min each time. The secondary antibody was incubated at room temperature for 1 h, and PBS was washed for 3 times, 3 min each time. DAB staining for an appropriate time, and the staining condition can be observed under a microscope. Water was used to wash clean. Ethanol gradient dehydration, 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, anhydrous ethanol for 5 min, xylene I and II for 10 min each, placed in the fume hood until the smell disappeared, and neutral resin was used for mounting. The positive rate in the glomerulus was analyzed by Image J software.

[0096] 7 Immunofluorescence: The section was dewaxed and dehydrated as in immunohistochemistry, and pepsin antigen retrieval was performed at room temperature for 30 min. PBS was washed for 3 times, 3 min each time. 5% BSA was used for room temperature blocking for 30 min. The primary antibody was added and incubated at 4°C overnight. The next day, the temperature was restored to room temperature for 30 min, and PBS was washed for 3 times, 3 min each time. Fluorescent secondary antibody was added and incubated at room temperature for 1 h, and PBS was washed for 3 times, 3 min each time. The section was mounted with anti-fluorescent quenching mounting medium containing DAPI, and Leica STELLARIS 5 was used for imaging.

[0097] 8 Proteomics analysis

[0098] The kidney samples of normal group (db / m mice, NC) and diabetic nephropathy group (db / db mice, DKD) were collected, and the proteins in the samples were identified and quantified using DIA proteomics technology, and the differentially expressed proteins between DKD group and NC group were screened. Six different mouse kidney tissues were selected from each group, and two kidney tissues were mixed together for detection, and n=3 experiments were performed.

[0099] DIA technology is a combination of data-dependent acquisition (DDA) mass spectrometry technology and multiple reaction monitoring technology (MRM), which collects all ion information. In order to realize the DIA proteomics identification and quantification of the kidney samples of 24-week-old db / m mice and db / db mice, the DDA library construction and DIA analysis of the above experiment were carried out in this embodiment.

[0100] First, the same amount of pooled samples were used as pool samples for HPRP fractionation and LC-MS / MS (QE-HFX_DDA mode) analysis to obtain the DDA library database. Then, each sample was analyzed by LC-MS / MS (DIA mode), and qualitative and quantitative analysis was performed using the DDA database. The mass spectrometry analysis process mainly includes steps such as protein extraction, peptide enzymolysis, chromatographic fractionation, liquid chromatography-tandem mass spectrometry (LC-MS / MS) DDA data acquisition, database retrieval, etc. The formal experiment stage mainly includes steps such as DIA analysis, quality control analysis, qualitative and quantitative result analysis, and bioinformatics analysis. Through the above hierarchical analysis, the protein expression differences between the kidney samples of db / m mice and db / db mice can be more comprehensively and accurately obtained, and the mechanism and treatment direction of DKD can be further explored. After searching the database with the off-line data, the differentially expressed proteins were screened, and the proteome data were subjected to KEGG enrichment analysis, GO function analysis and bioinformatics analysis to screen the module with the largest correlation with the occurrence and development of DKD.

[0101] 9 Statistical analysis: The data were processed using Prism 8.0 statistical software, and the mean ± standard error (Mean ± SEM) was used. The two groups were compared using t-test (two-tailed Student's t-test), and multiple groups were compared using one-way ANOVA. The correlation analysis was performed using Spearman correlation analysis. P<0.05 indicates that the difference is statistically significant.

[0102] 10 Analysis of experimental results

[0103] Figure 1Figure 1 is a graph showing the changes in body weight, blood glucose, blood lipid and kidney function indicators of different age db / m, db / db mice. A: body weight; B: fasting blood glucose (FBG); C: total cholesterol (TC) level; D: triglyceride (TG) level; E: low-density lipoprotein cholesterol (LDL) level; F: creatinine (CRE) level; G: urea nitrogen (BUN) level; H: urinary microalbumin level; I: urinary albumin level. The analysis showed that the kidney function of db / db mice decreased, and the above indicators increased with age, suggesting that the DKD kidney disease process gradually worsened over time.

[0104] Figure 2-1 and Figure 2-2 Figure 2 is a comparative analysis graph of the degree of kidney fibrosis of different age db / m, db / db mice. Among them Figure 2-1 Figure 2A is the mRNA level of type I collagen, and Figure 2B is the mRNA level of IV collagen; Figure 2-2 Figure 2 is a comparative analysis graph of the degree of kidney fibrosis of different age db / m, db / db mice. Among them

[0105] Figure 3 Figure 3 is a basic data analysis graph of DIA proteomics, wherein Figure 3 Figure 3A is a column chart of protein quantitative difference results; Figure 3B is a cluster analysis graph of differentially expressed proteins; and Figure 3C is a volcano plot of differentially expressed proteins. The results showed that DIA proteomics significantly differentially expressed proteins can effectively separate the comparison groups, indicating that the differential expression protein screening can represent the influence of biological processing on the sample.

[0106] Figures 4-1-4-4 Figure 4 is an analysis graph of the possible role of GNPAT in DKD kidney lipid deposition analyzed by bioinformatics in this embodiment. Figure 4-1 Figure 5 is a KEGG enrichment analysis graph of differential proteins; Figure 4-2 Figure 6 is a GO enrichment analysis graph of differential proteins; Figure 4-3 Figure 7 is a differential ratio table (Top10) of differentially expressed proteins in the peroxisome signaling pathway; Figure 4-4The PPI network of the differentially expressed proteins in the peroxisome signaling pathway was constructed. The results showed that the peroxisome-related signaling pathway was the most significant in the KEGG enrichment analysis and GO component analysis, further indicating that the peroxisome-related signaling pathway and lipid metabolism may play an important role in the disease progression of DKD. The PPI network interaction results showed that the connectivity of GNPAT was the second, but the protein differential ratio was the highest (5.25 times), and was much higher than that of PEX14 (0.59 times), suggesting that GNPAT is a key regulator of kidney lipid metabolism in DKD.

[0107] Figure 5 is an analysis diagram of the expression pattern of GNPAT under high glucose conditions; wherein, Figure 5 A of FIG. in which is a diagram of the GNPAT protein level in the kidneys of db / m and db / db mice of different ages; B is a diagram of the GNPAT immunohistochemical results in the kidneys of db / m and db / db mice of different ages; C is a diagram of the GNPAT protein level in different renal parenchymal cells (podocytes, mesangial cells, renal tubular epithelial cells and endothelial cells); D is a diagram of the GNPAT mRNA level in different renal parenchymal cells (podocytes, mesangial cells and renal tubular epithelial cells); E is a diagram of the podocyte GNPAT immunofluorescence results; F is a diagram of the Nephrin expression in the kidneys of db / m and db / db mice of different ages; G is a diagram of the correlation analysis of the GNPAT and Nephrin protein expression levels in the kidneys of DKD mice of different ages. The results showed that compared with db / m mice, the GNPAT protein level in the kidney cortex of db / db mice was significantly increased, and gradually increased with age. Among the four types of renal parenchymal cells, only the GNPAT mRNA and protein expression in the high glucose cultured podocytes was significantly increased. The expression of the podocyte marker protein Nephrin in the kidneys of db / db mice gradually decreased with age, and was negatively correlated with the expression level of GNPAT, indicating that the expression of GNPAT is related to the injury of glomerular podocytes.

[0108] Figures 6-1-6-3 is a clinical study of the correlation of GNPAT with DKD. In which, Figure 6-1 The representative Western Blot band diagrams of human serum GNPAT and Adipophilin proteins, the expression comparison diagrams of serum GNPAT in DKD patients at each stage, the expression comparison diagrams of serum Adipophilin in DKD patients at each stage, the correlation analysis diagram of serum GNPAT level and lipid marker protein Adipophilin in DKD patients, and the expression comparison diagrams of serum GNPAT in DKD patients at each stage detected by ELISA are shown in FIG. Figure 6-2 is a correlation analysis diagram of the expression level of GNPAT in DKD patients and various indicators of clinical renal function; Figure 6-3Figure 2 shows the GNPAT content in urine of clinical patients and the expression of GNPAT in urine residue. The results show that the protein level of GNPAT in serum is significantly increased compared with healthy subjects; and the more severe the DKD stage, the more significant the increase in the expression level of GNPAT. The level of lipid droplet marker protein Adipophilin is positively correlated with the level of GNPAT; the level of GNPAT in serum is correlated with the renal function of DKD. The expression of GNPAT in urine and urine residue proteins of DKD patients is significantly increased, which is related to the pathological process of DKD.

[0109] In summary, the key factor GNPAT in the pathological process of DKD is screened out through the protein interaction network and the differential protein change rate, and it is confirmed that GNPAT is closely related to the damage of podocytes in DKD.

[0110] Example 2 Role of GNPAT in lipid deposition of podocytes in diabetic nephropathy

[0111] This example studies the relationship between the expression level of GNPAT and the lipid deposition of podocytes in DKD at the whole animal, cell and molecular levels, and confirms that GNPAT plays an important role in the pathological process of DKD.

[0112] 1. Reagents: mouse interference empty vector lentivirus, mouse GNPAT interference lentivirus, mouse overexpression empty vector lentivirus, mouse GNPAT overexpression lentivirus, mouse adeno-associated virus empty vector, mouse rAAV-CMV-nephrin-GNPAT+ / + adeno-associated virus, mouse rAAV-CMV-nephrin-GNPAT- / - adeno-associated virus, purchased from Haigai Gene Chemical Technology Co., Ltd.; DyLight 594 donkey anti-rabbit IgG (E032421-01), Dylight 488 donkey anti-mouse IgG (E032211-01) purchased from EARTHOX company in the United States; others are the same as in Example 1.

[0113] 2 Cell experiment

[0114] 2.1 Cell culture: the steps are the same as the cell culture experiment in Example 1.

[0115] 2.2 Transfection and grouping of mouse podocytes: when the MPC5 cells cultured in normal glucose concentration reach 20% to 30% confluence, lentivirus transfection is performed. Within 24 to 48 hours, the transfection efficiency is determined by observing the green fluorescence intensity of the cells under a fluorescence microscope (strong green fluorescence in more than 70% of the cells is considered successful transfection), q-PCR, Western blot and immunofluorescence are used to detect the expression of GNPAT.

[0116] Grouping: normal group (11.1 mmol / L, NG), normal sugar empty vector control group (NG+Vector), normal sugar GNPAT overexpression group (NG+GNPAT OE), high sugar group (40 mmol / L, HG), high sugar empty vector control group (HG+Vector), high sugar GNPAT knockdown group (HG+GNPAT KD).

[0117] 2.3 Cell climbing sheet immunofluorescence experiment: take out the cell climbing sheet, wash with PBS for 3 min, 3 times; fix the cells with 4% paraformaldehyde for 10 min; wash with PBS for 3 min, 3 times; if staining the nucleus, 0.1% Triton can be selected for 20 min to permeabilize the membrane; wash with PBS for 3 min, 3 times; block with 5% BSA for 20 min; after incubation with the first antibody, place it in a wet box and incubate in a refrigerator at 4°C overnight; the next day, warm it at room temperature for 30 min; wash with PBS for 3 min, 3 times; incubate the second antibody in a wet box in the dark at 37°C on a shaking table for 1 h; wash with PBS for 3 min, 3 times; dry the climbing sheet; add 20 uL of DAPI-containing anti-fluorescence quencher dropwise, cover the climbing sheet on the glass slide; take a photo under a fluorescence microscope.

[0118] 3 Animal experiment

[0119] 3.1 Animal feeding: SPF level db / m and db / db mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd.

[0120] Grouping: db / m mice were randomly divided into normal control group (db / m), normal empty virus group (db / m+Veh), and podocyte-specific GNPAT overexpression group (db / m+GNPAT-OE); db / db mice were randomly divided into model control group (db / db), model empty virus group (db / db+Veh), and podocyte-specific GNPAT knockdown group (db / db+GNPAT-KD).

[0121] At the age of 20 weeks, the mice were injected with physiological saline (control group), empty virus (empty virus group), and GNPAT overexpression and knockdown adeno-associated virus into the kidney parenchyma, respectively. The amount of virus injected into the kidney parenchyma of each mouse was 1×10 11 Copies / mL, intraperitoneal injection of penicillin (2 million U / day) for three consecutive days after kidney parenchyma injection, and routine feeding for four weeks for standby.

[0122] 3.2 Renal parenchyma injection: After the mouse was anesthetized with 4% isoflurane using a small animal anesthesia machine, the mouse was continuously supplied with 2% isoflurane for inhalation anesthesia, and then the mouse was fixed on the operation table with the back upward. The approximate position of the kidney was found by pressing the dorsal spinous process, and the mouse was prepared for skin on the back. An incision of 1.5 cm was made on the left kidney with ophthalmic scissors, and the mouse muscle tissue was separated with forceps. The left kidney was gently propped out with both hands. The renal portal was clamped to prevent the virus stock solution from flowing into the bladder. The left kidney was then fixed with forceps, and a 30G injection needle was inserted into the upper, middle and lower pole of the left kidney. 50 μL of liquid (containing 1 x 10 11 Copies / mL of viral particle genome copy number or physiological saline) was injected into the kidney parenchyma, and the injection needle was slowly withdrawn after staying in the kidney for at least 30 s. A small amount of anastomotic glue was immediately dropped on the injection site to prevent liquid from flowing out. The kidney was reset, and the mouse muscle and skin were sutured with absorbable thread. Postoperative care was performed.

[0123] 3.3 Transmission electron microscopy: 1 mm 3 After the renal cortex was fixed in 2.5% glutaraldehyde in the dark, the following steps were performed: first, PBS was used for washing for 15 min, 3 times. Then the sample was immersed in 1% osmium acid for about 1 h. Gradient ethanol dehydration (50%, 60%, 70%, 80%, 90%, anhydrous ethanol) was performed. Next, Epon812 embedding agent was immersed, and anhydrous acetone was dehydrated 3 times. The sample was immersed in a 1:1 mixture of anhydrous acetone + Epon812 embedding agent for 1 h. Then the embedding agent was overn ight, and placed in a 37°C oven for 12 h, a 45°C oven for 12 h, and a 60°C oven for 24 h. Diamond knife sectioning was performed, and 200 mesh copper mesh was used for sectioning. Acetic acid uranyl acetate staining was performed for 30 min, and lead nitrate staining was performed for 30 min. Finally, the sample was observed and photographed under a transmission electron microscope.

[0124] Result analysis:

[0125] Figure 7 is an analysis diagram of the establishment of GNPAT knockdown and overexpression cell lines, wherein, A is the expression of GNPAT mRNA in the knockdown cell line; B is the expression of GNPAT mRNA in the overexpression cell line. The results show that the GNPAT knockdown and overexpression cell model is successfully constructed.

[0126] Figures 8-1-8-6 is the effect of knocking down or overexpressing GNPAT on podocyte damage. Figure 8-1 includes GNPAT mRNA and protein levels; Figure 8-2 is the result of GNPAT and Synaptopodin, a podocyte marker protein, immunofluorescence double staining; Figure 8-3Figures for the analysis of the effect of abnormal GNPAT expression on the level of Nephrin mRNA and the effect of abnormal GNPAT expression on the level of Nephrin protein; Figure 8-4 Figures for the analysis of the effect of abnormal GNPAT expression on the level of Synaptopodin mRNA and the effect of abnormal GNPAT expression on the level of Synaptopodin protein; Figure 8-5 Figures for the analysis of the effect of knocking down or overexpressing GNPAT on the level of apoptosis of podocytes and the effect of knocking down or overexpressing GNPAT on the level of Cleaved-Caspase3 of podocytes; Figure 8-6 Figures for the analysis of the effect of knocking down or overexpressing GNPAT on the level of Bax protein of podocytes and the effect of knocking down or overexpressing GNPAT on the level of Bcl2 protein of podocytes. The above results show that overexpression of GNPAT in podocytes cultured in normal glucose can lead to a decrease in podocyte marker proteins, an increase in the apoptosis rate of podocytes, and an increase in podocyte damage; and knocking down GNPAT can improve the condition of podocyte damage caused by high glucose.

[0127] Figures 9-1-9-4 Figures for the analysis of the establishment of a podocyte-specific overexpression GNPAT db / m mouse model or a podocyte-specific knocking down GNPAT db / db mouse model. Figure 9-1 Figures for the analysis of the effect of injecting 1 x 10 11 Figures for the analysis of the infection efficiency of kidney frozen sections and the infection efficiency after 4 weeks of injection of 1 x 10 Figure 9-2 Figures for the analysis of the infection efficiency of kidney frozen sections and the infection efficiency after 4 weeks of injection of 1 x 10 Figure 9-3 Figures for the analysis of the infection efficiency of kidney frozen sections and the infection efficiency after 4 weeks of injection of 1 x 10 Figure 9-4 Figures for the analysis of the infection efficiency of kidney frozen sections and the infection efficiency after 4 weeks of injection of 1 x 10

[0128] Figures 10-1-10-5 Figures for the analysis of the effect of podocyte-specific knocking down or overexpressing GNPAT on the kidney function of mice.

[0129] wherein, Figure 10-1 Figures for the analysis of the effect of podocyte-specific knocking down or overexpressing GNPAT on the kidney function of mice. Figure 10-2 Figures for the analysis of the effect of podocyte-specific knocking down or overexpressing GNPAT on the kidney function of mice. Figure 10-3 Figures for the analysis of the effect of podocyte-specific knocking down or overexpressing GNPAT on the kidney function of mice. Figure 10-4The graph shows the effect of different concentrations of uric acid on the MAU / ALB ratio; 10⁻⁵ represents the correlation analysis between glomerular GNPAT expression levels and renal function indicators; the experimental results show that podocyte-specific overexpression of GNPAT impairs renal function in db / db mice, while podocyte-specific knockdown of GNPAT improves renal function and reduces proteinuria in db / db mice. Glomerular GNPAT protein expression is correlated with renal function in DKD mice.

[0130] Figures 11-1-11-4 Figure showing the effect of podocyte-specific knockdown or overexpression of GNPAT on glomerular injury; among which... Figure 14-1 Transmission electron microscope image; Figure 14-2 PAS staining and statistical graphs; Figure 14-3 The figures show PASM staining and statistical plots. As can be seen from the figures, the effect of GNPAT intervention on the podocyte marker protein synaptopodin was observed. Overexpression of GNPAT in normal mouse podocytes led to morphological changes in the mouse kidneys and glomerular damage; db / db mice showed severe morphological damage to the kidneys. Knockdown of GNPAT in podocytes, however, alleviated glomerular damage such as basement membrane thickening induced by diabetic nephropathy (DM).

[0131] Figures 12-1-12-3 This is an analysis diagram of kidney lipid deposition under high glucose conditions. Figure 12-1 Oil Red O staining results and Nile Red staining results in different renal parenchymal cells; Figure 12-2 Oil Red O staining results of renal cortex of dbdb mice at different ages and Adipophilin immunohistochemistry of renal cortex of db / db mice at different ages; Figure 12-3 We statistically analyzed the expression levels of Adipophilin protein in the renal cortex of db / db mice at different ages and performed correlation analysis between Wes bands and GNPAT and Adipophilin protein expression levels. The results showed that lipid deposition was particularly significant in podocytes cultured in high glucose, suggesting that podocyte lipid deposition may play an important role in the development of DKD. We also observed a gradual increase in expression with increasing age, indicating that lipid metabolism abnormalities gradually increase with the progression of DKD, suggesting that lipid metabolism abnormalities play a crucial role in the occurrence and development of DKD.

[0132] Figures 13-1-13-2 This is a graph analyzing the effects of knockdown or overexpression of GNPAT on lipid deposition in podocytes. Figure 13-1 The levels of Oil Red O staining and lipid droplet-labeled protein Adipophilin were respectively determined. Figure 13-2The fluorescence chart is Nile red staining. The results show that overexpression of GNPAT can lead to lipid deposition in normal sugar cultured podocytes, and damage the podocytes; knockdown of GNPAT can improve the lipid deposition in podocytes caused by high glucose.

[0133] Figures 14-1-14-3 Fig. 6 is an analysis chart of the influence of podocyte-specific knockdown or overexpression of GNPAT on lipid deposition in mouse kidney. Figure 14-1 Fig. 7 shows oil red O staining and Adipophilin immunohistochemistry; Figure 14-2 Fig. 8 shows a column chart of Adipophilin protein expression level analysis; Figure 14-3 Fig. 9 shows a statistical analysis chart of the correlation between the column chart of GNPAT level in podocyte-specific knockdown or overexpression of GNPAT mice glomerulus and the level of Adipophilin. The results show that overexpression of podocyte GNPAT in db / m mice glomerulus can cause obvious lipid deposition, lipid droplet deposition in db / db mice glomerulus, and knockdown of podocyte GNPAT can reduce lipid droplet deposition in db / db mice glomerulus. The expression of GNPAT is positively correlated with the expression of Adipophilin.

[0134] Conclusion: Upregulation of GNPAT expression leads to lipid deposition in podocytes, damages the podocytes, and thus causes proteinuria in mice; and downregulation of GNPAT expression in podocytes can reduce high glucose-induced lipid deposition in podocytes, alleviate the damage to the podocytes, and reduce the production of proteinuria in diabetic mice. This shows that GNPAT plays an important role in DKD. Based on the above conclusion, it can be known that screening of compounds combined with GNPAT and reducing the expression level of GNPAT can become a candidate drug for treating DKD, and is expected to improve the problem of lipid deposition in podocytes of patients with this kind of kidney disease, reduce the damage, and achieve the treatment effect.

[0135] The above are only preferred embodiments of the present application and are not intended to limit the protection scope of the present application. For those skilled in the art, the present application can have various changes and modifications. Any changes, modifications, replacements, integrations and parameter changes made to these embodiments within the spirit and principles of the present application, which can achieve the same functions without departing from the principles and spirit of the present application, fall within the protection scope of the present application.

Claims

1. The use of GNPAT as a target in drugs for improving and / or treating podocyte damage in diabetic nephropathy.

2. The application according to claim 1, characterized in that, This includes drugs that target GNPAT, which inhibit high glucose-induced lipid deposition in podocytes by reducing GNPAT expression.

3. The application according to claim 2, characterized in that, The drug reduces glomerular lipid deposition by decreasing podocyte GNPAT expression, and the expression level of GNPAT is positively correlated with the expression level of the lipid droplet marker protein Adipophilin.

4. The application according to claim 2, characterized in that, The drug alleviates glomerular damage by reducing GNPAT expression in podocytes.

5. The application according to claim 1, characterized in that, The expression level of GNPAT was negatively correlated with the expression level of Nephrin, a marker protein of mouse kidney podocytes.

6. The application according to claim 1, characterized in that, The drug can alleviate podocyte damage caused by high glucose by reducing podocyte GNPAT.

7. Application of GNPAT molecules as drug targets in the development or screening of drugs for the treatment of diabetic nephropathy or diabetic podocyte damage.

8. The application according to claim 7, characterized in that, The method for developing or screening the drug includes using GNPAT molecules as target proteins, the drug comprising compounds that can bind to GNPAT molecules, the compounds improving high glucose-induced podocyte damage by reducing GNPAT expression.

9. The application according to claim 7, characterized in that, The method for developing or screening the drug includes designing and synthesizing compounds using GNPAT molecules as targets, screening the compounds through computer-simulated molecular docking experiments, screening for compounds that can bind to GNPAT, and evaluating their activity.

10. A drug for treating diabetic nephropathy or podocyte damage in diabetic nephropathy, characterized in that, This includes compounds that can bind to GNPAT molecules.

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