Application of histone methyltransferase EZH2 as therapeutic target for podocyte senescence and injury of diabetic nephropathy

Abstract

The invention provides application of histone methyltransferase EZH2 as a therapeutic target of diabetic nephropathy podocyte senescence and injury, and relates to the technical field of biological medicine. It is found that the expression level of EZH2 in podocyte under the high glucose condition is remarkably increased, and conditions are provided for taking EZH2 as a biomarker for diagnosing or evaluating diabetic nephropathy podocyte damage and senescence. Subsequently, it is verified that podocyte damage of db / db mice can be remarkably improved, proteinuria of the db / db mice can be reduced, pathological damage of kidneys can be relieved, glomerular senescence of the db / db mice can be delayed, damage and senescence of podocytes under the high-glucose condition can be remarkably improved, the purpose of protecting the kidneys is achieved, the development of diabetic nephropathy is delayed, and the development of diabetic nephropathy is delayed. Therefore, the EZH2 can be used as a potential target for treating diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of histone methyltransferase EZH2 as a therapeutic target for podocyte aging and damage in diabetic nephropathy. Background Art

[0002] Statistics from the International Diabetes Federation (IDF) show that as of 2021, the number of patients with diabetes mellitus (DM) worldwide has reached 537 million, or 10.5% of adults worldwide suffer from DM. Diabetic nephropathy (DN), as one of the most common microvascular complications of DM, has become the main cause of chronic kidney disease (CKD) in urban tertiary hospitalized patients in my country, eventually leading to end-stage renal disease (ESRD), which may require dialysis or kidney transplantation in the late stage. Typical pathological changes in the kidney structure of DN include thickening of the glomerular basement membrane (GBM), detachment of podocytes and disappearance of foot processes, mesangial expansion, and loss of endothelial pores [4]. Podocytes are terminally differentiated epithelial cells and an indispensable component of the glomerular filtration barrier. The destruction of their morphology and function is closely related to the production of proteinuria in DN patients [5]. The molecular mechanisms involved in podocyte injury in DN include inflammation, cell senescence, and accumulation of advanced glycation end products (AGEs). Cell senescence refers to the loss of cell replication ability. The normal aging process can assist embryonic development, respond to various cellular stresses, and limit tissue damage and tumorigenesis during the life cycle. However, when senescent cells accumulate in damaged tissues, they promote the development of related diseases. In DN, podocytes, renal tubular epithelial cells, and mesangial cells all show senescence. Podocyte senescence may play an important role in various pathogenesis of the kidney by regulating processes such as oxidative stress, mitochondrial dysfunction, pyroptosis, and apoptosis.

[0003] Histone methyltransferase Enhancer of zeste homolog 2 (EZH2) is a functional component of Polycomb repressive complex 2 (PRC2), which can catalyze the trimethylation of lysine 27 of histone H3 (H3K27me3) to inhibit the transcription of target genes. It has been reported that EZH2 is associated with a variety of kidney diseases such as DN, acute kidney injury (AKI) and lupus nephritis. Our team's previous studies have shown that long noncoding RNA plasmacytoma variant translocation 1 (LncRNA PVT1) can promote H3K27me3 recruitment in the promoter region of Forkhead box A1 (FOXA1) and inhibit its transcription by recruiting EZH2, thereby promoting damage and apoptosis of podocytes in DN. So far, a large number of studies have been conducted on the histone modification function of EZH2. However, as the understanding of EZH2 deepens, researchers have found that EZH2 can also regulate biological processes and disease progression by methylating non-histone proteins and affecting protein activity. EZH2 can bind to the signal transducer and activator of transcription 3 (STAT3) protein and increase its methylation level to activate its activity. STAT3 is a key injury mediator in many kidney diseases. STAT3 can be activated by factors such as high glucose levels, AGEs and angiotensin II. Activated STAT3 causes glomerular sclerosis and tubulointerstitial fibrosis by secreting inflammatory and fibrotic factors, ultimately leading to the occurrence of DN. Many studies have shown that both EZH2 and STAT3 can participate in regulating cell senescence. However, in DN, this non-histone modification of EZH2 and its role in podocyte senescence have not been reported. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In order to explore the potential role of EZH2 in DN podocyte senescence, this study used DN in vivo and in vitro models to downregulate EZH2 expression through small molecule inhibitors or genetically regulate EZH2 and STAT3 to investigate the relationship between EZH2 and STAT3 in DN podocytes and the mechanism of action of EZH2 in DN podocyte injury and senescence, providing new therapeutic targets and ideas for the treatment of diabetic nephropathy.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides the use of histone methyltransferase EZH2 as a therapeutic target for podocyte senescence and damage in diabetic nephropathy.

[0009] In a second aspect, the present invention provides the use of a histone methyltransferase EZH2 specific inhibitor in the preparation of a drug for treating podocyte senescence and damage in diabetic nephropathy.

[0010] Specifically, the drug includes a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of the EZH2 gene.

[0011] Specifically, the specific inhibitor is one of a drug inhibitor or a gene inhibitor.

[0012] Specifically, the drug inhibitor is GSK126; the gene inhibitor is Ezh2 ShRNA2.

[0013] In a third aspect, the present invention provides the use of histone methyltransferase EZH2 as a biomarker for diagnosing or evaluating podocyte senescence and damage in diabetic nephropathy.

[0014] Specifically, EZH2 expression levels were upregulated in podocytes under high glucose conditions.

[0015] In a fourth aspect, the present invention provides an in vitro non-therapeutic method for inhibiting podocyte aging and damage in diabetic nephropathy, the method specifically comprising: culturing podocytes in a high-glucose environment in the presence of a specific inhibitor of histone methyltransferase EZH2, thereby inhibiting the degree of damage and aging of podocytes caused by the high-glucose environment.

[0016] (III) Beneficial effects

[0017] We first confirmed that podocytes were damaged and glomerular senescence was present in db / db mice, and that podocytes cultured under high glucose conditions were damaged and aged. On this basis, we found that the expression level of EZH2 in podocytes under high glucose conditions was significantly increased, which provided conditions for EZH2 to be used as a biomarker for diagnosing or evaluating podocyte damage and senescence in diabetic nephropathy. Subsequently, we verified that the use of gene or drug targeted inhibition of EZH2 expression can significantly improve podocyte damage in db / db mice, reduce proteinuria in db / db mice, alleviate renal pathological damage, delay glomerular senescence in db / db mice, significantly improve podocyte damage and senescence under high glucose conditions, achieve the purpose of protecting the kidneys, and delay the progression of diabetic nephropathy. At the same time, we also discovered for the first time that EZH2 increases its activity by binding to STAT3 and catalyzing its lysine methylation, thereby inducing DN podocyte damage and senescence. The present invention provides a theoretical basis for EZH2 to be used as a potential target for the treatment of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :(A) uACR of db / : control mice and db / d diabetic nephropathy model mice was detected, ****P<0.0001vs.db / m group (n=6);(B) Blood glucose levels of the two groups of mice were detected, ****P<0.0001vs.db / m group (n=6);(C) Representative PAS staining images of glomeruli of db / m and db / db mice (40×) to observe the degree of glomerular damage;(D) Typical transmission electron microscopy images of kidney tissues of db / m and db / db mice (8000× and 12000×) to observe the ultrastructure of glomerular GBM (blue arrow) thickness and podocyte foot process (red arrow) etc.

[0019] Figure 2 :(A); Western blotting was used to detect the expression levels of EZH2, podocyte marker proteins (Nephrin, Podocin) and inflammatory factor B7-1 in the isolated glomeruli of the two groups; H3 and β-actin were used as internal references; (B) Grayscale analysis of Western blotting bands in each group, and normalization was performed using H3 or β-actin as internal reference, *P<0.05 vs. db / m group, **P<0.01 vs. db / m group (n=6); (C) Immunofluorescence staining was performed on frozen sections of renal tissue of each group of mice, and Podocin (green) and B7-1 (red) were co-stained to observe their expression and localization.

[0020] Figure 3:(A) Western blotting was used to detect the expression changes of aging-related proteins (p21, Klotho) in the glomeruli of the two groups of mice: H3 and β-actin were used as internal references; (B) Grayscale analysis of Western blotting bands in each group was performed, and H3 or β-actin was used as internal reference for normalization, *P<0.05 vs.db / m group, **P<0.01 vs.db / m group (n=6); (C) Representative images of SA-β-gal staining (40×) were used to observe the aging of the glomeruli of the two groups of mice (yellow arrows: SA-β-gal positive); (D) The paraffin sections of the renal tissues of each group of mice were subjected to immunohistochemical staining of p16INK4A.

[0021] Figure 4 :(A) MPCs were cultured under normal glucose, hypertonic and high glucose conditions, and the cell lysates were collected for Western blotting to observe the expression changes of EZH2, Synaptopodin, Podocin and B7-1:, H3 and β-actin were used as internal references; (B) Grayscale analysis of Western blotting bands in each group was performed, and H3 or β-actin was used as internal reference for normalization, ns: P>0.05 vs. NG group, *P<0.05 vs. NG group, **P<0.01 vs. NG group (n=3); (C) Representative immunofluorescence images showed the expression and localization of EZH2 (green) and DAPI (blue) in MPCs of each group.

[0022] Figure 5 :(A): Western blotting was used to detect the expression changes of cell senescence-related proteins (p21, Klotho) in the podocytes cultured in vitro in each group, with H3 and β-actin as internal references; (B) Grayscale analysis of Western blotting bands in each group, and normalization was performed using H3 or β-actin as internal references, ns: P>0.05 vs. NG group, *P<0.05 vs. NG group (n=3); (C) Representative pictures of SA-β-gal staining, observing the cell senescence of podocytes cultured in each group (yellow arrow: SA-β-gal positive).

[0023] Figure 6:(A) uACR of mice in each group was detected, ns: P>0.05 vs.db / db group, **P<0.01 vs.db / db group (n=6);(B) Blood glucose level of mice in each group was detected (n=6);(C) Representative PAS staining images of glomeruli of mice in each group (40×) were taken to observe the pathological changes of glomeruli;(D) Typical transmission electron microscopy images of renal tissues of mice in each group (8000× and 12000×) were taken to observe the ultrastructural changes such as glomerular GBM thickness (blue arrow) and podocyte foot process (red arrow).

[0024] Figure 7 :(A) Western blotting was used to detect the expression levels of EZH2, podocyte marker proteins (Nephrin, Podocin) and inflammatory injury factor B7-1 in the glomeruli of each group of mice: H3 and β-actin were used as internal references; (B) Grayscale analysis of each group of bands in Western blotting was performed, and H3 or β-actin was used as internal reference for normalization, ns: P>0.05 vs.db / db group, ***P<0.001 vs.db / db group, ****P<0.0001 vs.db / db group (n=6); (C) Immunofluorescence staining was performed on frozen sections of renal tissues of each group of mice, and Podocin (green) and B7-1 (red) were co-stained to observe their expression and localization.

[0025] Figure 8 :(A) Western blotting was used to detect the expression levels of aging-related proteins (p21, Klotho) in the glomeruli of each group of mice: H3 and β-actin were used as internal references; (B) Grayscale analysis of each group of bands in Western blotting was performed, and H3 or β-actin was used as internal reference for normalization, ns: P>0.05 vs.db / db group, **P<0.01 vs.db / db group, ***P<0.001 vs.db / db group (n=6); (C) Representative microscopic images (40×) of SA-β-gal staining of glomeruli of each group of mice, and the aging of glomeruli of each group of mice was observed (yellow arrow: SA-β-gal positive); (D) Typical images (40×) of immunohistochemical staining of p16INK4A in glomeruli of each group of mice.

[0026] Fig. 9:(A) Western blotting was used to detect the expression of STAT3 and pSTAT3 in the kidney of each group of mice. H3 was used as an internal reference;(B) The grayscale analysis of each group of bands in Figure (A) was performed using ImageJ, *P<0.05 vs. db / m group (n=3);(C) Western blotting was used to detect the expression of STAT3 and pSTAT3 in MPC of each group, H3 was used as an internal reference;(D) The grayscale analysis of each group of bands in Figure (C) was performed using ImageJ, ns: P>0.05 vs. NG group, **P<0.01 vs. NG group (n=3).

[0027] Fig.10 :(A) Western blotting was used to detect the inhibitory effects of three ShRNAs on Ezh2 in MPCs, and H3 was used as an internal reference;(B) Under high glucose conditions, podocytes were transfected with Ezh2 ShRNA2 and its control empty vector, and immunofluorescence staining was used to show the expression and localization of EZH2 (green), and DAPI was used to stain the nucleus;(C) Western blotting was used to detect the expression of pSTAT3 and STAT3 in MPCs of each group, and H3 was used as an internal reference;(D) Grayscale analysis of the bands in each group in Figure (C) was performed using ImageJ, ns: P>0.05 vs. HG group, ***P<0.001 vs. HG group (n=3);(E) Western The expression of pSTAT3 and STAT3 in the renal tissues of each group of mice was detected by blotting, and H3 was used as an internal reference; (F) The grayscale analysis of the bands in each group in Figure (E) was performed using ImageJ, ns: P>0.05 vs.db / db group, **P<0.05 vs.db / db group (n=6).

[0028] Fig.11 :(A) Co-IP experiments showed that EZH2 and STAT3 directly bind to each other; (B) Co-IP experiments were performed on MPCs cultured in vitro to detect the levels of STAT3 and its lysine methylation in each group. The Input group was used as a positive control, and the IgG group was used as a negative control; (C) Podocytes were divided into HG group and HG+Ezh2 KD group for Co-IP experiments to detect the levels of STAT3 and its lysine methylation in each group. The Input group was used as a positive control, and the IgG group was used as a negative control.

[0029] Fig.12:(A) uACR of mice in db / db group, db / db+Vehicle group, db / db+Ezh2 KD group, and db / db+Ezh2 KD+Stat3 OE group were detected, *P<0.05, **P<0.01 (n=6); (B) Blood glucose levels of mice in the four groups were detected (n=6); (C) Representative PAS staining images (40×) of glomeruli of db / db mice in the four groups were used to observe the changes of glomerular pathological damage; (D) Typical transmission electron microscopy images of renal tissues of db / db mice in the four groups were used to observe and compare the ultrastructural changes such as the thickness of glomerular GBM (blue arrow) and podocyte foot process (red arrow).

[0030] Fig.13 :(A) The expression levels of pSTAT3, STAT3, Nephrin, Podocin, and Desmin in the kidneys of each group of mice were detected by Western blotting, with β-actin and H3 as internal references; (B) The grayscale analysis of the bands in each group in Figure (A) was performed using ImageJ, and after normalization with β-actin, *P<0.05vs.db / db+Ezh2 KD group, **P<0.01vs.db / db+Ezh2 KD group (n=6); (C) Immunofluorescence staining showed the expression and localization of Podocin (green) and B7-1 (red) in podocytes of each group.

[0031] Fig.14 :(A) Western blotting was used to detect the expression levels of p21 and Klotho, with H3 and β-actin as internal references; (B) Grayscale analysis of the bands in each group in Figure (A) was performed using ImageJ, and after normalization using H3 or β-actin, *P<0.05 vs.db / db+Ezh2 KD group, **P<0.01 vs.db / db+Ezh2 KD group (n=6); (C) Representative images of SA-β-gal staining in each group (40×), observing the aging of the glomeruli of mice in each group (yellow arrow: SA-β-gal positive); (D) p16 in the glomeruli of mice in each group INK4A Representative immunohistochemistry images (40×).

[0032] Fig.15:(A) Western blotting was used to detect the expression levels of pSTAT3, STAT3, Synaptopodin, Podocin, B7-1, p21, and Klotho in the HG group, HG+Vehicle group, HG+Ezh2 KD group, and HG+Ezh2 KD+Stat3 OE group, with H3 and β-actin as internal references;(B) Grayscale analysis of the bands in each group in Figure (A) was performed using ImageJ, and normalized using H3 and β-actin. *P<0.05 vs. HG+Ezh2 KD group, **P<0.01 vs. HG+Ezh2 KD group, ***P<0.001 vs. HG+Ezh2 KD group (n=3);(C) Representative images of SA-β-gal staining in each group, and the senescence of podocytes in each group was observed (yellow arrow: SA-β-gal positive). (D) Statistical analysis of the number of SA-β-gal positive cells in each group. *P<0.05, **P<0.01 (n=3). DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] 1 Experimental methods

[0036] 1.1 Culture and grouping of mouse immortalized podocytes (MPCs)

[0037] (1) Cultivation of MPCs: MPCs were proliferated in a 33°C, 5% CO2 incubator. Fresh 33°C culture medium was replaced every two days. When the cell density reached an appropriate density, the cells were washed with PBS buffer and replaced with 37°C MPC culture medium. The cells were cultured and differentiated in a 37°C, 5% CO2 incubator. After 10-14 days of differentiation and maturation, relevant experimental treatments were given.

[0038] (2) Model group: After the MPCs were differentiated and matured in a 37°C incubator, the MPCs with better growth status were washed with PBS buffer and then replaced with NG, HM, and HG culture media, respectively. After 36 hours of stimulation, the cells were collected for subsequent experiments.

[0039] (3) Adenovirus transfection group: After the MPCs were differentiated and matured in a 37°C incubator, the MPCs with better growth status were randomly divided into the following groups: ①HG group. ②HG+Vehicle group, transfected with an adenovirus control empty vector that knocked down Ezh2 under high glucose conditions. ③HG+Ezh2 KD group, transfected with an adenovirus that knocked down Ezh2 under high glucose conditions. ④HG+Ezh2 KD+Stat3 OE group, simultaneously transfected with adenoviruses that knocked down Ezh2 and overexpressed Stat3 under high glucose conditions. First, a preliminary experiment was conducted to find out the most appropriate MOI value (200) for the transfection virus. In addition, three shRNAs that can knock down Ezh2 were used to transfect MPCs separately, and the sequence with the most obvious knockdown efficiency was selected as the adenovirus used in the Ezh2 KD group. During transfection, thaw the virus on ice in advance, add the required amount of virus to half volume (7.5 ml) of 37°C MPC medium, mix thoroughly, add to 75T culture flask and mark, add the other half of the medium after 4 hours. Replace with fresh 37°C high-glucose MPC medium the next morning, and collect cells 36 hours later for subsequent experiments.

[0040] 1.2 Experimental Animal Grouping

[0041] Eight-week-old male db / db mice and db / m mice were purchased and, after two weeks of adaptive feeding, randomly divided into groups according to the experimental arrangement, with no less than 6 mice in each group. At the end of the observation period, kidney tissues were collected and euthanasia was performed.

[0042] (1) Model group: 6 male db / db mice and 6 male db / m mice aged 10 weeks were not treated. Blood glucose was tested and urine was collected via tail vein. The mice were raised until 22 weeks of age, when the observation endpoint was reached. Kidney tissue specimens were collected as needed and stored under appropriate conditions for subsequent experiments.

[0043] (2) Intraperitoneal injection of drug group: 10-week-old male db / db mice were randomly divided into 3 groups, 6 mice in each group. The groups were as follows: ①db / db group, i.e., control group, without any treatment. ②db / db+β-CD group: According to the body weight of mice, the solvent (SBE-β-CD) was intraperitoneally injected for 8 weeks, 5 days a week. ③db / db+GSK126 group: According to the body weight of mice, the EZH2-specific small molecule inhibitor GSK126 (50 mg / kg) was intraperitoneally injected for 8 weeks, 5 days a week. Blood glucose was tested through the tail vein and urine was collected. At the end of the observation, i.e., at 18 weeks of age, mouse kidney tissue specimens were collected as needed for subsequent experiments.

[0044] (3) Adeno-associated virus (AAV) injection group: 10-week-old male db / db mice were randomly divided into 4 groups, with 6 mice in each group. The groups were as follows: ①db / db group, no treatment. ②db / db+Vehicle group, mice were injected with control empty vector containing podocyte-specific promoter through tail vein at 10 and 16 weeks of age. ③db / db+Ezh2 KD group, mice were injected with Ezh2 knockdown AAV containing podocyte-specific promoter through tail vein at 10 and 16 weeks of age (to maintain gene stability under regulation). ④db / db+Ezh2 KD+Stat3 OE group, mice were injected with AAV containing podocyte-specific promoter to inhibit Ezh2 and overexpress Stat3 through tail vein at 10 and 16 weeks of age. Blood glucose of mice was tested through tail vein and urine was collected. At the end of observation, i.e., at 22 weeks of age, renal tissue specimens of mice were collected for subsequent experiments.

[0045] 1.3 Specimen collection and basic data testing

[0046] (1) Urine sample collection: Collect mouse urine with a volume of no less than 0.2 ml.

[0047] (2) Collection of kidney specimens: The mice were anesthetized with isoflurane, an inhaled anesthetic, and fixed on the operating table. Blood specimens were collected from the right ventricle with a 1 ml syringe. The right auricle of the mice was then cut open and the kidneys were perfused with PBS buffer at the apex of the heart. Part of the renal cortex was taken and cut into 1 mm 3 The tissue blocks were fixed in electron microscope solution; 1 / 2 of the kidney tissue was cut and placed in OCT embedding medium and light microscope solution respectively. After the cortex and medulla of the remaining kidney tissue were separated, the cortex was quickly frozen with liquid nitrogen and stored in a -80℃ refrigerator.

[0048] (3) Blood glucose test: Measure and record the blood glucose of mice through the tail vein. After disinfecting the mouse tail with an alcohol cotton pad, insert the blood collection needle into the tail vein, test with a blood glucose meter, read and record the value, and press with a cotton swab to stop bleeding.

[0049] (4) Urinary albumin / creatinine (uACR) detection: The collected urine samples were used to measure the uACR of mice using a fully automatic analyzer.

[0050] 1.4 Isolation of mouse glomeruli

[0051] The mice were anesthetized and fixed. After being fully exposed, blood samples were collected from the right ventricle with a syringe. The right atrial appendage was cut open and perfused from the apex with PBS containing iron powder. The kidneys on both sides were then quickly separated and placed on ice, and the renal capsule was gently peeled off. The renal cortex was retained and minced and transferred to a 1.5ml sterile ep tube. 1ml of collagenase at a concentration of 3mg / ml and 1μl of DNase were added. Digestion was performed in a 37℃ incubator for 30 minutes, and the digestion was terminated by adding culture medium. The tissue was then placed in a 100μm cell sieve, continuously ground with a sterile tissue grinding rod, and the tissue and sieve were repeatedly rinsed with PBS, and the tissue fluid was collected in a sterile culture dish. After replacing a new cell sieve, the above steps were repeated. The tissue fluid finally obtained was centrifuged at 3000rpm for 10-15 minutes and the supernatant was discarded. After the tissue precipitate was resuspended with 2ml PBS buffer, it was placed in a magnetic stand for adsorption. The supernatant was discarded, and the glomerular precipitate was obtained by gently washing it again with PBS buffer for 3 times, and centrifuged at 3000rpm for 10 minutes.

[0052] 1.5 Detection indicators

[0053] Western blotting (WB) was used to detect marker proteins in cell and tissue samples;

[0054] The cell samples and tissue samples were subjected to immunohistochemical staining, β-galactosidase (SA-β-gal) staining, periodic acid Schiff (PAS) staining, immunoprecipitation and electron microscopy.

[0055] 1.6 Statistical analysis

[0056] Cell experiments were repeated 3 times in each group, and animal experiments were performed in 6 mice per group. ImageJ software was used to analyze the normalized grayscale of WB bands. GraphPad Prism software was used for statistical analysis of differences. If the data conformed to normal distribution, Student's t test was used, and one-way ANOVA analysis of variance was used for comparison between multiple groups. All statistical analysis data were expressed as mean ± standard deviation (mean ± SD), and P < 0.05 was considered statistically significant.

[0057] 2 Results

[0058] EZH2 is elevated in db / db mice, exacerbating podocyte injury and glomerular aging

[0059] 2.1.1 Increased urinary protein excretion and aggravated renal pathological damage in db / db mice

[0060] In this study, spontaneous type 2 diabetic db / db mice were used as an in vivo model of DN, and db / m mice were used as a control group. The blood glucose of the mice was monitored and their urine was collected. The mice were raised to 22 weeks of age and then killed and their kidneys were collected. Figure 1 As shown in AB, when the observation endpoint was reached, the urine albumin / urine creatinine (uACR) of db / db mice increased significantly (P < 0.0001) and blood glucose increased significantly (P < 0.0001) compared with the control db / m mice. Figure 1 C) showed that compared with control mice, the glomeruli of db / db mice were significantly enlarged and the mesangial matrix was significantly deposited. In addition, electron microscopy results ( Figure 1 D, ) showed that the GBM of db / db mice was significantly thickened, and the foot processes were fused and disappeared, indicating that podocytes were damaged.

[0061] 2.1.2 Podocyte damage is aggravated in db / db mice compared with db / m mice

[0062] Western blotting results ( Figure 2 AB) showed that the expression of EZH2 in db / db mice was significantly increased compared with the control group (P < 0.05). In db / db mice, the expression levels of podocyte marker proteins Nephrin and Podocin were significantly reduced compared with the control group (P < 0.01), while the level of inflammatory factor B7-1 was significantly increased (P < 0.05) ( Figure 2 AB). In addition, immunofluorescence results ( Figure 2 C) Also shows that Podocin expression is reduced and B7-1 is increased in db / db mice. These results indicate that podocytes in db / db mice are damaged compared with db / m mice.

[0063] 2.1.3 Glomerular aging is more severe in db / db mice than in db / m mice

[0064] We used Western blotting to detect changes in aging-related proteins (p21, Klotho) in db / m and db / db mice. Figure 3 AB) showed that compared with control mice, the expression of p21 in the kidney tissue of db / db mice was significantly increased (P<0.05), while the level of Klotho was significantly decreased (P<0.01). In addition, SA-β-gal staining showed that compared with db / m mice, the positive area in the glomerulus of db / db mice was significantly increased ( Figure 3 C). Immunohistochemistry ( Figure 3 D) The results showed that p16 INK4AThe expression of β-actin (a typical marker of cellular senescence) was significantly increased in the glomeruli of db / db mice. The above results show that glomerular senescence is aggravated in DN.

[0065] 2.2 EZH2 is elevated in podocytes cultured in high glucose, exacerbating podocyte injury and senescence

[0066] 2.2.1 High glucose leads to increased EZH2 levels and damage in podocytes cultured in vitro

[0067] Western blotting was used to detect the changes in EZH2, podocyte marker proteins, and inflammatory factors in MPCs cultured under high glucose conditions. Consistent with the results of in vivo experiments, Figure 4 AB showed that the expression of EZH2 in MPC of HG group was significantly increased (P<0.01). In addition, compared with the control group (NG group and HM group), the podocyte marker proteins Synaptopodin (P<0.05) and Podocin (P<0.01) in HG group were significantly reduced, and the inflammatory factor B7-1 (P<0.05) was significantly increased ( Figure 4 AB). Immunofluorescence results ( Figure 4 C) also showed that high glucose environment significantly increased the expression level of EZH2 in MPCs.

[0068] 2.2.2 High glucose stimulation causes senescence of podocytes cultured in vitro

[0069] Western blotting Figure 5 AB) showed that compared with the NG and HM groups, the high glucose environment increased the expression of p21 in podocytes (P<0.05) and significantly reduced the anti-aging protein Klotho (P<0.05). In addition, SA-β-gal staining showed that the number of positive cells increased in MPCs cultured in high glucose ( Figure 5 C) These results demonstrate that high glucose can induce senescence of podocytes cultured in vitro.

[0070] Pharmacological inhibition of EZH2 expression reduces podocyte injury and glomerular aging in db / db mice

[0071] 2.3.1 Inhibition of EZH2 expression reduces urinary albumin excretion and renal pathological damage in db / db mice

[0072] The specific small molecule inhibitor of EZH2, GSK126, was intraperitoneally injected into db / db mice for 8 weeks (50 mg / kg). The blood glucose of the mice was monitored and urine was collected. The mice were killed at 18 weeks of age and kidney tissue was collected for subsequent experiments. The results showed that compared with the control db / db group mice and the db / db+β-CD group mice injected with solvent intraperitoneally, the db / db+GSK126 group mice had reduced urinary protein excretion (P < 0.01), but there was no significant difference in blood glucose among the three groups ( Figure 6 AB). PAS results ( Figure 6 C) showed that compared with the db / db group and the db / db+β-CD group, the db / db+GSK126 group had significantly reduced mesangial matrix deposition and glomerular hypertrophy. In addition, electron microscopy results showed that the podocyte foot process disappearance, fusion, and GBM thickening in db / db mice could be alleviated by the inhibition of EZH2 ( Figure 5 D) Thus, inhibition of EZH2 can delay the progression of DN in db / db mice.

[0073] 2.3.2 Inhibition of EZH2 expression reduces podocyte injury in db / db mice

[0074] We then examined the changes in podocyte injury-related indicators in db / db mice after inhibiting EZH2 with GSK126. First, we used Western blotting to detect the expression of EZH2, confirming that GSK126 successfully inhibited the level of EZH2 in db / db mice (P < 0.0001) ( Figure 7 AB). In addition, intraperitoneal injection of GSK126 restored the expression of podocyte marker proteins Nephrin (P < 0.001) and Podocin (P < 0.0001) in db / db mice, and reduced the level of inflammatory injury factor B7-1 (P < 0.001) ( Figure 7 AB). Immunofluorescence results ( Figure 7 C) It was also confirmed that GSK126 could increase the expression of Podocin and reduce the expression of B7-1 in the kidney tissue of db / db mice. These results suggest that inhibition of EZH2 can alleviate podocyte injury in db / db mice.

[0075] 2.3.3 Inhibition of EZH2 expression delays glomerular aging in db / db mice

[0076] Next, we examined the changes in renal aging after inhibiting EZH2 expression in db / db mice using GSK126. Western blotting results ( Figure 8AB) showed that GSK126 could inhibit the expression of p21 in the kidney of db / db mice (P<0.001) and restore the level of Klotho (P<0.01). SA-β-gal staining results showed that the positive area of ​​glomeruli in db / db+GSK126 group mice was reduced compared with the control group (db / db group and db / db+β-CD group) ( Figure 8 C). In addition, p16 INK4A The immunohistochemical results ( Figure 8 D) confirmed that compared with the control group, intraperitoneal injection of GSK126 significantly reduced the expression of p16 in the glomerular nuclei of db / db mice. INK4A The above results prove that GSK126 can delay glomerular aging in db / db mice.

[0077] 2.4 EZH2 can directly methylate STAT3 and increase its activity in podocytes

[0078] 2.4.1 Increased pSTAT3 / STAT3 levels in DN models

[0079] We speculated that EZH2 may promote the progression of DN by regulating STAT3 methylation and affecting its activity. We first detected the changes in STAT3 and pSTAT3 levels in DN models in vivo and in vitro. Results ( Fig. 9 AB) showed that the pSTAT3 / STAT3 ratio in db / db mice was increased compared with that in db / m mice, with a significant statistical difference (P < 0.05). The in vitro experimental results were consistent with the in vivo results. The pSTAT3 / STAT3 ratio in the HG group was higher than that in the control group (NG group and HM group) (P < 0.01) ( Fig. 9 CD).

[0080] 2.4.2 Inhibition of EZH2 reduces pSTAT3 / STAT3 levels in DN

[0081] We inhibited EZH2 expression in MPCs cultured in vitro to observe whether changes in STAT3 and pSTAT3 were related to EZH2 levels. We first transfected MPCs cultured in vitro with three adenoviruses to screen for the virus with the highest knockdown efficiency, and finally selected Ezh2 ShRNA2 as the adenovirus used in the Ezh2 KD group in the cell model ( Fig.10 A). Immunofluorescence results showed the knockdown effect of Ezh2 after Ezh2 ShRNA2 transfection into MPCs ( Fig.10 B). Western blotting results ( Fig.10CD) showed that compared with the HG group and HG+Vehicle group, the pSTAT3 / STAT3 ratio of the HG+Ezh2 KD group decreased (P<0.001), that is, the proportion of activated STAT3 decreased. The results of the in vivo experiment were consistent with the results of the in vitro experiment. Inhibition of EZH2 with GSK126 can reduce STAT3 activity ( Fig.10 EF). This shows that the expression changes of EZH2 can affect the activation of STAT3.

[0082] 2.4.3 EZH2 can bind to STAT3 and regulate its lysine methylation level

[0083] We used Co-IP experiments to verify whether EZH2 and STAT3 bind to each other in MPCs cultured in vitro. Fig.11 A) shows that EZH2 and STAT3 bind to each other at the protein level in podocytes cultured in vitro. Next, we detected the changes in STAT3 methylation levels under high glucose conditions. The results are shown in Figure 2. Fig.11 B shows that the lysine methylation level of STAT3 in MPCs cultured under HG conditions was increased compared with that in the NG group. To verify whether EZH2 can regulate the lysine methylation level of STAT3, we used adenovirus to knock down Ezh2 in podocytes cultured under high glucose conditions. Results ( Fig.11 C) shows that compared with the HG group, the STAT3 lysine methylation level in the HG+Ezh2KD group was significantly decreased, indicating that downregulation of EZH2 can reduce the methylation level of STAT3 lysine. The above results indicate that EZH2 may enhance the activity of STAT3 by increasing the lysine methylation level of STAT3.

[0084] 2.5 Podocyte-specific overexpression of STAT3 can counteract the renal protective effect of EZH2 inhibition in db / db mice

[0085] 2.5.1 Podocyte-specific overexpression of STAT3 can counteract the effect of EZH2 inhibition in reducing urinary albumin and alleviating renal pathological damage in db / db mice

[0086] To verify whether EZH2 mediates podocyte injury and senescence in DN by regulating STAT3, we first performed a rescue experiment in db / db mice. We injected adeno-associated viruses containing podocyte-specific promoters into the tail vein of db / db mice to knock down EZH2 and overexpress STAT3, respectively. When the observation endpoint (22 weeks of age) was reached, the mice were killed and kidney tissues were obtained for subsequent experiments.

[0087] like Fig.12As shown in A, compared with the control group (db / db group and db / db+Vehicle group), the uACR of the db / db+Ezh2 KD group mice was significantly reduced (P<0.05). However, after podocyte-specific overexpression of Stat3, the uACR of the mice increased compared with the db / db+Ezh2 KD group, indicating that podocyte-specific overexpression of Stat3 can reverse the beneficial effect of downregulating Ezh2 to reduce urinary protein excretion (P<0.01), but there was no difference in blood glucose among the groups ( Fig.12 AB). PAS staining results ( Fig.12 C) shows that after Ezh2 in podocytes was inhibited, the degree of glomerular hypertrophy and mesangial matrix accumulation were alleviated. However, after inhibiting Ezh2 and overexpressing Stat3, the glomerular pathological damage was significantly aggravated compared with the db / db+Ezh2 KD group ( Fig.12 B). Transmission electron microscopy results ( Fig.12 D) showed that compared with the control group, the podocyte morphology of the db / db+Ezh2 KD group mice was significantly improved, the foot process fusion was reduced, and the GBM thickness was thinner. However, the podocyte ultrastructure of the db / db+Ezh2 KD+Stat3 OE group mice was significantly damaged compared with the db / db+Ezh2 KD group, and the GBM was significantly thickened ( Fig.12 C). These results suggest that Stat3 overexpression counteracts the renal protective effect of Ezh2 inhibition in db / db mice.

[0088] 2.5.2 Podocyte-specific overexpression of Stat3 can counteract the alleviating effect of Ezh2 inhibition on podocyte injury in db / db mice

[0089] Western blotting results ( Fig.13 AB) showed that, consistent with the above results, podocyte-specific downregulation of Ezh2 increased the expression of podocyte marker proteins Nephrin and Podocin and decreased the level of podocyte injury marker Desmin (P < 0.05). Compared with the db / db + Ezh2 KD group, the podocyte injury in the db / db + Ezh2 KD + Stat3 OE group was aggravated, the expression levels of Nephrin (P < 0.01) and Podocin (P < 0.05) were significantly reduced, and the expression of Desmin was increased (P < 0.01) ( Fig.13 AB). Immunofluorescence staining ( Fig.13 C) showed that downregulation of Ezh2 can increase the expression of Podocin and reduce the expression of inflammatory factor B7-1. The podocyte-specific marker proteins in the db / db+Ezh2 KD+Stat3OE group were significantly lower than those in the db / db+Ezh2 KD group, and the expression of B7-1 was increased ( Fig.13C). The above results indicate that the podocyte-protective effect of podocyte-specific downregulation of Ezh2 is closely related to STAT3.

[0090] 2.5.3 Podocyte-specific overexpression of Stat3 can antagonize the effect of Ezh2 knockdown in delaying renal aging in db / db mice

[0091] Western blotting results ( Fig.14 AB) showed that podocyte-specific targeted inhibition of Ezh2 reduced p21 levels and increased Klotho expression in db / db mice (P < 0.05). Compared with the db / db+Ezh2 KD group, the p21 level in the glomeruli of the db / db+Ezh2KD+Stat3 OE group was significantly increased (P < 0.01), and Klotho was significantly decreased (P < 0.05) ( Fig.14 AB). Representative images of SA-β-gal staining ( Fig.14 C) shows that after inhibiting Ezh2, the positive area in the glomeruli of the db / db+Ezh2 KD group mice was reduced compared with that of the db / db group mice, while the positive area in the glomeruli was significantly increased after overexpression of Stat3. INK4A However, overexpression of Stat3 reversed this effect ( Fig.14 D) These results showed that EZH2 was involved in glomerular senescence in DN by regulating STAT3.

[0092] 2.6 In podocytes cultured in high glucose, overexpression of Stat3 reversed the podocyte-protective effect of inhibiting Ezh2 production

[0093] To further verify whether EZH2 mediates podocyte injury and senescence by regulating STAT3, we knocked down Ezh2 with adenovirus under high glucose conditions and overexpressed Stat3 to observe the changes in podocyte injury and senescence-related indicators. Western blotting results ( Fig.15 AB) showed that, similar to the above results, knocking down Ezh2 increased the expression of podocyte marker proteins Synaptopodin and Podocin and reduced the level of inflammatory factor B7-1, while overexpression of Stat3 offset the effect of inhibiting Ezh2 on alleviating podocyte injury (P < 0.05). Subsequently, we detected the changes in senescence-related proteins in podocytes of each group. Results ( Fig.15AB) showed that compared with the control group, the expression of p21 in MPCs in the HG+Ezh2 KD group decreased, while the expression of Klotho increased (P<0.05). Compared with the HG+Ezh2 KD group, the p21 level in the HG+Ezh2 KD+Stat3 OE group increased significantly (P<0.05), while the Klotho level decreased significantly (P<0.01). SA-β-gal staining ( Fig.15 CD) showed that the number of positive podocytes in the HG+Ezh2 KD group was significantly reduced compared with the control group; the number of positive podocytes in the HG+Ezh2 KD+Stat3OE group was significantly more than that in the HG+Ezh2 KD group, and the difference was statistically significant (P < 0.01). The above results show that the podocyte protection effect produced by knockdown of Ezh2 is reversed by overexpression of Stat3. This shows that STAT3 is a key mediator of podocyte injury and senescence in EZH2-mediated DN.

[0094] 3 Conclusion

[0095] We first confirmed that podocytes were damaged and senescent in db / db mice, and that podocytes cultured under high glucose conditions were damaged and aged. On this basis, we found that the expression level of EZH2 in podocytes under high glucose conditions was significantly increased, which provided conditions for EZH2 to be used as a biomarker for diagnosing or evaluating podocyte damage and senescence in diabetic nephropathy. Subsequently, we verified that the use of gene or drug targeted inhibition of EZH2 expression can significantly improve podocyte damage in db / db mice, reduce proteinuria in db / db mice, alleviate renal pathological damage, delay glomerular aging in db / db mice, significantly improve podocyte damage and senescence under high glucose conditions, achieve the purpose of protecting the kidneys, and delay the progression of diabetic nephropathy. At the same time, we also found for the first time that EZH2 increases its activity by binding to STAT3 and catalyzing its lysine methylation, thereby inducing DN podocyte damage and senescence. The above results indicate that EZH2 can be used as a potential target for the treatment of diabetic nephropathy.

[0096] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of histone methyltransferase EZH2 as a therapeutic target for podocyte senescence and damage in diabetic nephropathy.

2. Application of histone methyltransferase EZH2 specific inhibitors in the preparation of drugs for the treatment of podocyte aging and damage in diabetic nephropathy.

3. The use according to claim 2, characterized in that: The medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of the EZH2 gene.

4. The use according to claim 2, characterized in that: The specific inhibitor is a drug inhibitor or a gene inhibitor.

5. Application of histone methyltransferase EZH2 as a biomarker for diagnosing or evaluating podocyte aging and damage in diabetic nephropathy.

6. An in vitro non-therapeutic method for inhibiting podocyte aging and damage in diabetic nephropathy, the method specifically comprising: culturing podocytes in a high-glucose environment in the presence of a specific inhibitor of histone methyltransferase EZH2, thereby inhibiting the degree of damage and aging of podocytes caused by the high-glucose environment.

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