The Role And Mechanism Of DNA Methylation In Podocyte Injury Of Type 2 Diabetic Nephropathy

BACKGROUNDDiabetes (Diabetes mellitus, DM) is a systemic metabolic disease with serious hazard to human health and is currently facing.a major health problem. Diabetic nephropathy (DN) is one of the major and common microvascular complications of diabetes, which is an important cause of death in patients with type 2 diabetes, and also contributed to a major cause of end-stage renal disease(ESRD). The pathogenesis of DN is very complex, involving the role of hemodynamic abnormalities, release of inflammatory mediators, oxidative stress and apoptosis and other factors, but the exact mechanism is still not fully understood.Glomerulus filtration barrier is composed of glomerular endothelial cells (GeCs), the glomerular basement membrane (GBM), and podocytes. Glumerular podocyte is a terminally differentiated cells, which is attached to the outside of the GBM, is an important component of the GBM, and play an important role in maintaining the glomerular filtration barrier structure and function.Previouly studies have shown that podocyte play a key role in the progression of proteinuria associated kidney diseases, including DN. Podocyte has been a target for the study of proteinuria associated with DN,therefore, it is significant to clarify the molecular mechanism of podocyte injury.Recent clinical and experimental studies demonstrate the occurrence of a metabolic memory of prior exposure to hyperglycemia, resulting in persistently increased risk for diabetic complications, including nephropathy, long after glucose normalization. This finding suggests a potential role for epigenetic mechanisms apart from genetic predisposition in the etiology of diabetes and its complicationsEpigenetics is a new branch of genetics.It is defined as mitotically and meiotically stable and heritable changes in gene expression that do not involve a change in the DNA sequence. Epigenetics mainly involve DNA methylation and histone modifications. DNA methylation is associated with gene regulation, which is an important transcriptional modification. DNA methylation is the transfer of methyl (group), provided by methyl S-adenosyl methionine (S-adenosyl-methionine), to the DNA molecule deoxycytidine (cytosine, C) No.5 of the pyrimidine ring carbon atom, making it into a methylated deoxycytidine (dmC) under the action of DNA methylation enzyme (DNA-methyltransferase, Dnmt). The majority of the mammalian genome dmC followed by a guanine (guanine, G) formed dmCpG. Previously studies have shown that DNA methylation is inversely with gene expression, gene expression reduced when hypermethylation, and gene expression increased when demethylation.DNA methylation is also involved in many other biological processes besides gene transcriptional regulation, such as genomic imprinting, X chromosome inactivation, and cell differentiation. In addition, DNA methylation is reversible inhibited by DNA methyltransferase (Dnmt), which can lead to reduced levels of DNA methylation, thus promoting gene transcription.To explore the pathogenesis of chronic kidney disease from the DNA methylation aspects has been become a hotspot in the field of kidney disease. Ingrosso D et al found that DNA hypomethylation is obviously present in patients with end-stage renal failure, and DNA methylation status are associated with the levels of homocysteine.Hyperhomocysteinaemia affects gene expression by epigenetic changes^171. This indicated the toxic action of homocysteine can be mediated by macromolecule hypomethylation. Recent Bechtel W et al [18] also conducted a study of DNA methylation on renal fibrosis. The study demonstrated hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmtl in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmtl+/-heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney.Recently, In proximal tubular cells, mesangial cells and peripheral blood cells of DN were also found abnormal DNA methylation.study the pathogenesis of DN from the perspective of DNA methylation provides a new idea for the cause of such diseases, has also brought new opportunities for the prevention and treatment of DN.METHODSPart one Effect of DNA methylation inhibitor 5-azacytidine (5-Aza) on type 2 diabetic db/db mice.Animal groups:The body weight, diet, drinking water, glucose and urine albumin/creatinine ratio were measured in eighteen C57BLKS/J db/db mice with 12-week-old and twelve wild-type BKS mice with the same genetic background. After spontaneous type 2 diabetic nephropathy mouse were successfully established, these mice were randomly divided into five groups:BKS group of mice; BKS+5-Aza (2mg/kg wt,3 times/week) group; db/db mice group; db/db+5-Aza. (lmg/kg wt) group; db/db+5-Aza (2mg/kg wt) group.5-Aza was injected intraperitoneally to the db/db (n=6) and BKS (n=6) mice with mentioned above doses at three times a week for 8 weeks. The other half of the db/db and BKS mice were given the same volume of PBS without 5-Aza, respectively. Fasting blood glucose was measured in tail-vein blood using one touch ultra glucometer and and Test Strip (Lifescan, Milpitas, CA) after 6 h of fasting weekly. Body weight was obtained at one week intervals. For urine collection, individual mice were caged once 2 weeks in a metabolic cage for 24 h. After 8 weeks of treatment, mice were anaesthetized (10% chloral hydrate; 0.25ml/50g i.p.) and blood samples were obtained from the retro-orbital venous plexus for determination of the plasma concentration of urea nitrogen, creatinine, serum lipids. Heart, liver, and kidney tissues were weighed. The samples were frozen and kept in liquid nitrogen until use.Kidney tissue specimens were stained with periodic acid Schiff (PAS) for light microscopic examination. The number of podocytes per glomerulus was determined based on the WT1 immunohistochemistry.The glomerular podocyte injury was checked by electron microscopy. Podocyte slit diaphragm proteins, including nephrin, and podocin expression were analyzed in the renal cortex of diabetic db/db mice and wild type BKS mice using western blot.Part two DNA methyltransferases (Dnmts) and nucler factor Sp1 and NF-κB p65 expression in renal tissues of spontaneous type 2 diabetes db/db miceDnmtl, Dnmt3a and Dnmt3b mRNA expression were quantified by quantitative real-time PCR (qRT-PCR).Frozen section was stored at -80℃ until use.The expression of Dnmtl, Sp1 and NF-κB p65 protein were analyzed in glomerular podocyte by means of confocal immunofluorescence microscopy. Dnmtl protein expression in renal tissue was analyzed using Western blot.Part three Culturing, identification and dividing subgroup of podocyte treated with or without High glucose1. Mouse podocytes culture:The conditionally immortalized mouse podocyte cell line (MPC) was kindly provided by DrJochen Reiser (Rush University Medical Center, Chicago, USA). Cells were grown to 80% confluence in type-I collagen (Shengyou Biotechnology, China)-coated flasks at 33℃ in RPMI-1640(Gibco BRL, Gaithersburg, USA) supplemented with 10%fetal bovine serum(FBS, Gibco BRL, USA) and 50 U ml-1 γ-interferon (IFN-γ, ProSpec, Israel).To induce differentiation, podocytes were reseeded and cultured at 37℃ in 50 cm 2 culture dish coated with type-Ⅰ collagen in the absence of IFN-γ for 10 to 14 days. To quiescent the cells, differentiated podocytes were serum-starved for 24 h, and then treated with normal glucose (NG,5.3 mM) or high glucose (HG,30 mM) or NG (5.3 mM) plus mannitol (24.7 mM; osmolality control) for 24,48 or 72 hours before experimentation.2. Identification and morphology observation of differentiated podocyte The cultured podocyte in RPMI 1640 with INF-gamma at 33℃and the differentiated podocyte in DMEM without INF-gamma at 37℃for 10-14 days were checked by contrast phase microscope. The variance of podocytes morphology in both conditions was observed. The skeleton protein synaptopodin is expressed in differentiated podocytes. However, it is not expressed in undifferentiated cells.3. The differentiated podocytes were treated as follows: 1) NG group:The podocytes were cultured in DMEM medium with 5.3mM Glucose for 24,48 and 72 hours respectively. 2) HG group:The podocytes were cultured in DMEM medium with 30mM Glucose for 24,48 and 72 hours respectively. 3) HG+5-Aza group:The podocytes were cultured in DMEM medium with 30mM Glucose and 5-Aza (10μM) for 48 hours; 4) HG+Dnmtl-siRNA group:The podocytes were cultured in DMEM medium with 30mM Glucose and 50 nM Dnmtl-siRNA for 48 hours; 5) HG+Sp1-siRNA group:The podocytes were cultured in DMEM medium with 30mM Glucose and 50nM Spl-siRNA for 48 hours; 6) HG+p65-siRNA group:The podocytes were cultured in DMEM medium with 30mM Glucose and 50nM p65-siRNA for 48 hours; 7) HG+Con-siRNA group:The podocytes were cultured in DMEM medium with 30mM Glucose and 50 nM non targeting-siRNA for 48 hours; 8) HG+M1 Group:The podocytes were cultured in DMEM medium with 30mM Glucose for 48 hours and 0.1 μM MithramycinA for 24 hours; 9) HG+M2 group:The podocytes were cultured in DMEM medium with 30mM Glucose for 48 hours and 0.5μM MithramycinA for 24 hours; 10) HG+M3 group:The podocytes were cultured in DMEM medium with 30mM Glucose for 48 hours and 1μM MithramycinA for 24 hours; 11) HG+B1 group:The podocytes were cultured in DMEM medium with 30mM Glucose for 48 hours and 0.5μM BAY 11-7082 for 24 hours; 12) HG+B2 group:The podocytes were cultured in DMEM medium with 30mM Glucose for 48 hours and 1μAY 11-7082 for 24 hours; 13) HG+B3 group:The podocytes were cultured in DMEM medium with 30mM Glucose for 48 hours and 2μM BAY 11-7082 for 24 hours;Part four DNA methyltransferases (Dnmts) and nucleoprotein Sp1 and NF-κB p65 expression in cultured podocytes with different interventionsDnmt1, Dnmt3a and Dnmt3b mRNA expression in cultured podocytes with differentinterventions were quantified by quantitative real-time PCR (qRT-PCR). The protein expression Dnmtl, Sp1 and NF-κB p65 were analyzed in cultured podocyte using confocal immunofluorescence microscopy and western blot.Part five Identified podocyte activity.Transwell migration assay:Transwell cell culture inserts (pore size 8μm; Costar Corporation, Corning, NY) were coated with type-I collagen, rinsed once with PBS and placed in DMEM medium containing the reagent that needed to be tested in the lower compartment. For each experiment,1 ×104 differentiated podocytes treated with different interventions were seeded in the inserts and allowed to migrate for 48 h while being incubated at 37℃. Non-migratory cells were removed from the upper surface of the membrane, and migrated cells were fixed with cold methanol and stained with Crystal Violet Solution (Sigma-Aldrich). The number of migrated cells was counted using phasecontrast microscopy with a×10 objective on an ECLIPSE TS 100 microscope (Nikon, Tokyo, Japan) in the centre of a membrane (one field). The data presented represent the mean ±SEM of four independent experiments.Part six Podocyte monolayer filtration barrier function assessment.Albumin Influx Assay:A simple albumin influx assay was adapted to evaluate the filtration barrier function of podocyte monolayer, as described previously.41’43 Briefly, podocytes (5×103) were seeded onto the collagen-coated transwell filters (3-μm pore; Corning, New York, NY) in the top chamber and cultured under differentiating conditions. After 10 days, podocytes were serum-starved overnight. Pretreated with or without 5-Aza-2’-deoxycytidine (10μM) for 30min or Dnmtl-siRNA (50nM) for 5 hours, podocytes then treated with or without 30mM glucose for 48 hours. Cells were washed twice with PBS supplemented with 1mM MgCl2 and 1mM CaCl2 to preserve the cadherin-based junctions. The top chamber was then refilled with 0.15 ml of RPMI 1640 and the bottom chamber with 1 ml of RPMI 1640 supplemented with 40 mg/ml of BSA and incubated at 37℃ for 6 hours, and a small aliquot of medium from top chamber was collected for albumin concentration measurement by using a bicinchoninic acid protein assay kit (Biocolor Bioscience & Technology Company, China).Part seven Co-ImmunoprecipitationNuclear proteins from cells were extracted using the Nuclear Protein Extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Protein concentration was quantified by the BCA Assay kit (Keygen Biotech, Nanjing, China). Co-immunoprecipitation experiments were performed using the Pierce Co-Immunoprecipitation Kit (26149, Pierce; Rockford, IL) as per the manufacturer’s instructions. Briefly,300μg of protein lysates were pre-cleared using a control agarose resin to minimize non specific binding. These lysates were then applied to columns containing 5μg immobilized Spl antibody covalently linked to an amine-active resin and incubated overnight at 4℃. Equal volumes of the lysates were also applied to columns containing control resin and processed the same as the antibody coupling resin for negative controls. The co-immunoprecipitate was then eluted and analyzed by SDS-PAGE along with the input controls.Part eight Chromatin immunoprecipitation-quantitative real-time PCR (ChlP-qPCR)Approximately 2×107cells were treated with 1% formaldehyde (final concentration) to crosslink proteins to DNA and lysed as per manufacturer’s recommendations using Millipore EZ ChIP Assay (Upstate Biotechnology Inc., Billerica, MA). Chromatin was sheared by sonication to fragments of 500-800 bp in length.After centrifugation with 12000g at 4℃for 5 minutes, the Protein A+G Agarose/Salmon Sperm DNA was added to the supernatants and incubated overnight to remove non-specific binding. Spin at 12000 g for 1 minute at 4℃. Remove the supernatants from each sample to new tubes and the anti-Spl (Santa Cruz, CA) antibody or Non-immune IgG antibody was added to the supernatants and incubated overnight, followed by addition of Protein A+G Agarose/Salmon Sperm DNA at 4 ℃, and slowly rotated for 60 minutes to precipitate protein recognition of Spl antibody or the corresponding protein complexes. The protein complexes were then successively eluted, crosslinked reversed and purified. Purified DNA samples were then amplified in Stratagene MX3000P System (Agilent technologies, Palo Alto, USA), and quantitated in triplicate by SYBR Green Q-PCR (Applied Biosystems, Foster City, CA) using forward and reverse primer sequences for the mice Dnmtl promoter(-119～+102bp).Part nine Statistical analysisAll values are expressed as mean ±SEM. Statistical analysis was performed using the statistical package SPSS for Windows Ver.17.0 (SPSS, Inc., Chicago, IL, USA). Differences between two groups were analyzed using Student’s t-test with a two-tailed test of significance. Multiple comparisons among the groups were conducted by one-way analysis of variance with Bonferonni’s/Turkey test for homogeneity of variance, or Dunnett’s T3-test for heterogeneity of variance. Significance level test for a=0.05,P-values less than 0.05 were considered significant.RESULTS1. Polyuria, polydipsia, polyphagia and obese are significant observed in db/db mice with 12 weeks of age compared with normal BKS mice. Compared to normal BKS mice, fasting blood glucose and urine protein to creatinine ratio were also significantly higher in db/db mice (P<0.001). These indicate that the model of diabetic nephropathy was success.2. DNA methylation inhibitor of 5-azacytidine significantly reduced proteinuria, lowered serum creatinine, triglycerides (TRIG), total cholesterol (CHOL), low-density lipoprotein (LDL), and inhibition of mesangial matrix and basement membrane thickening, reduced foot process fusion, increased the number of podocytes and the expression of podocyte slit diaphragm proteins, including nephrin and podocin. However, metabolic parameters such as weight, diet, water and blood glucose are obviously not changed.3. Treatment with DNA methylation inhibitor 5-.Aza-2’-deoxycytidine significantly increased the expression of podocyte slit diaphragm proteins including nephrin and podocin in podocytes cultured with High glucose, and inhibited enhanced motility as a result of high glucose treatment, attenuated high glucose induced albumin influx, and improved podocyte filtration barrier.4. Increased expression of Dnmtl, Sp1 and NF-κB p65 were observed in spontaneous type 2 diabetes db/db mice and in vitro cultured podocytes incubated with high glucose. Dnmtl protein expression was also significantly increased in glumerular podocyte of patients with type 2 diabetic nephropathy. DNA methylation inhibitor 5-aza-2’-deoxycytidine, Sp1 inhibitor MithramycinA and NF-κB p65 inhibitor BAY 11-7082 inhibited the increased Dnmtl expression in pococytes treated with high glucose.5. Co-immunoprecipitation analysis demonstrated that Spl and NF-κB p65 interact in the nucleus of podocytes. Chromatin immunoprecipitation analysis showed Spl binding to Dnmtl promoter region (-119-+102 bp). Sp1 inhibitor MithramycinA and Spl-siRNA can effectively inhibited the increased Dnmtl expression in podocytes treated with high glucoseCONCLUSIONS1. In vivo studies have shown that DNA methylation inhibitor significantly reduced proteinuria, improved renal function, reduced podocyte injury, delaying the progression of type 2 diabetic nephropathy.2. In vitro studies have shown that DNA methylation inhibitor upregulated the expression of podocyte slit diaphragm proteins including nephrin and podocin in podocytes cultured with high glucose, and inhibited enhanced motility as a result of high glucose treatment, attenuated high glucose induced albumin influx, and improved podocyte filtration barrier.3. Inhibition of DNA methylation can ameliorate glomerular pathologies and podocyte injury in diabetic state, and thus suggest that aberrant DNA methylation is involved in DN and podocyte injury. DNA inhibitor might be a new therapeutic avenue for the treatment of DN. Sp1/NF-κB p65-Dnmtl pathway may be exploited as a therapeutic target for protecting against podocyte injury of DN.