Oxidative Stress And Apoptosis In Renal Tubular Epithelial Cells Of Diabetic Nephropathy Affected By Abnormal Dna Methylation Of Syk Gene And The Underlying Mechanism Exploration

Background and ObjectiveDiabetic nephropathy（DN）is a major microvascular complication of diabetes characterized by persistent albuminuria and progressive decline in renal function and is the leading cause of end-stage renal disease（ESRD）.The pathogenesis of DN involves complex molecular signaling pathways,including lipotoxicity,advanced glycation end products,oxidative stress,mitochondrial dysfunction,energy change,and apoptosis,which can cause endothelial cell dysfunction,podocyte loss,mesangial cell hyperplasia,a wide range of molecular mechanisms are involved in DN,and the pathogenesis has not yet been elucidated.Proximal tubular cells play an important role in the progression of diabetic nephropathy.Targeting the renal tubular epithelial sodium/glucose co transporter 2（SGLT2）to treat diabetic nephropathy has moved the study of diabetic nephropathy into the era of"diabetic tubulopathy".Renal tubular epithelial cells contain abundant mitochondria,which are the energy source for tubular epithelial cells to maintain transport function.Renal tubular epithelial cells under high glucose stimulation,mitochondria dependent respiration is diminished and reactive oxygen species（ROS）is generated in large amounts,leading to renal tubular epithelial cell injury.Dysfunctional mitochondria,which contribute to many diseases and pathological conditions through the production of excessive reactive oxygen species,are associated with apoptosis and the development of diabetic nephropathy.Spleen tyrosine kinase（Syk）is an intracellular protein tyrosine kinase that binds immune receptors to transduce intracellular signals to initiate inflammatory responses.Syk mediates disease initiation in SLE,vasculitis,arthritis,and other diseases,and inhibition of Syk ameliorates disease progression and provides a therapeutic target for immune related diseases.Epigenetics complements genetic studies by genetic modifications that do not alter the DNA sequence.Metabolic factors exert long-term effects through the mechanism of epigenetic,and high glucose as a non genetic factor causes abnormal epigenetic modification of genes,which eventually leads to the occurrence of DN.It is of great significance to explore the alterations of epigenetics in DN.Based on the above background,this study first analyzed the expression of Syk in patients with diabetic nephropathy,next explored the changes in Syk promoter methylation and the effects on gene expression in diabetic nephropathy model mice,and finally explored whether Syk could be used as a potential therapeutic target to ameliorate diabetic renal damage.Materials and Methods1.Clinical study:clinical and pathological parameters of patients with renal cancer undergoing nephrectomy and patients with diabetic nephropathy diagnosed by renal needle biopsy were collected.The adjacent renal tissues of patients with renal cancer,renal tissue samples of patients with diabetic nephropathy were stained by immunofluorescence and immunohistochemistry,and the relationships between Syk,PKCβand P66Shc and clinical and pathological parameters were analyzed.2.Changes in Syk promoter methylation in mice with diabetic nephropathy:a diabetic model was established by feeding Apo E^-/-mice high-fat high glucose diet combined with low-dose streptozocin（STZ）intraperitoneally.Six healthy SPF grade6-week-old C57BL/6J male mice were assigned as normal control（CON）（n=6）,six healthy SPF grade 6-week-old male Apo E^-/-mice were assigned as non intervention control(Apo E^-/-)（n=6）,and six were subjected to STZ induced diabetes mellitus(Apo E^-/-DM)（n=6）.Syk expression was detected by immunofluorescence and Westernblot,methylation level was detected by BSP.In vitro ox LDL combined with high glucose stimulated renal tubular epithelial cells（HK2 cells）,cell viability was detected by CCK8,Syk protein expression was detected by Westernblot and methylation level was detected by BSP.3.In diabetic nephropathy,Syk expression was regulated by DNA methylation through Sp1:the promo database,Jaspar database predicted that the possible transcription factors for Syk was Sp1 and its binding sites,chip-q PCR was used to verify the level of Sp1 binding to the Syk promoter,HK2 cells were lentivirally transfected to silence Sp1,a kit to detect oxidative stress kinase activity,and Westernblot to detect oxidative stress-related and apoptosis related proteins expression.4.In diabetic nephropathy,Syk inhibitors suppress oxidative stress and apoptosis by downregulating the PKCβ/P66Shc pathway:an Apo E^-/-mouse model of diabetic mellitus was constructed,in which we administered Syk inhibitor intervention,detected biochemical indicators of blood and urine,HE,PAS,Masson staining were performed to assess pathological changes,and the expression of proteins related to oxidative stress and apoptosis were detected.In vitro ox LDL combined with high glucose stimulated HK2 cells,which were given Syk inhibitor,PKCβinhibitor,and PKCβagonist interventions,the expression of oxidative stress-related and apoptosis related proteins was detected by Westernblot,ROS were detected by fluorescence,and apoptosis was analyzed by flow cytometry.Results1.Clinical studies:Compared with adjacent noncancerous kidney tissue,kidney tissue from patients with diabetic nephropathy showed elevated expression of Syk,PKCβ,and P66Shc in tubules,and Syk levels were positively correlated with blood glucose（r=0.356,p=0.047）and urine albumin to creatinine ratio（r=0.613,p=0.001）.PKCβwas positively correlated with urinary albumin to creatinine ratio（r=0.423,p=0.028）,P66Shc was positively correlated with blood glucose（r=0.368,p=0.046）and positively correlated with urinary albumin to creatinine ratio（r=0.49,p=0.009）.2.Changes in Syk promoter methylation in mice with diabetic nephropathy:compared with normal control mice and Apo E^-/-mice,the expression of Syk protein levels and gene levels were increased in Apo E^-/-DM mice,and the promoter region of Syk in Apo E^-/-DM mice exhibited hypomethylation.In vitro ox LDL combined with high glucose stimulated HK2 cells,and compared with NG group（normal glucose group）,ox LDL combined with high glucose stimulated HK2 cells exhibited hypomethylation in the Syk promoter region.3.In diabetic nephropathy,Syk expression is regulated by DNA methylation through Sp1:compared with normal control mice,Sp1 binding to the Syk promoter was increased in Apo E^-/-DM mice,which was accompanied by increased Sp1 expression in Apo E^-/-DM mice.In vitro studies,ox LDL combined with high glucose stimulation in HK2 cells upregulated Sp1 expression in the nucleus,accompanied by increased Sp1binding to the Syk promoter,and silencing Sp1 in HK2 cells inhibited ROS production as well as reduced cell apoptosis.4.In diabetic nephropathy,Syk inhibitors suppressed oxidative stress and apoptosis by downregulating the PKCβ/p66Shc pathway:in vivo studies,a Syk inhibitor reduced the urinary albumin/creatinine ratio,improved mitochondrial functional status,and reduced NGAL expression in Apo E^-/-diabetic mice,while it suppressed NOX,Bax,and cleaved caspase-3expression in tubular epithelial cells of Apo E^-/-diabetic mice,increased NDUFS1,GPX,Bcl-2expression,ameliorated oxidative stress and apoptosis in diabetic tubular cells.In vitro studies,ox LDL combined with high glucose stimulation of HK2 cells were administrated with Syk inhibitor,PKCβinhibitor,PKCβagonist intervention,PKCβinhibitor reduced oxidative stress and apoptosis caused by ox LDL combined with high glucose,PKCβagonist reversed the inhibitory effects of Syk inhibitor,confirming that Syk inhibitor ameliorated diabetic nephropathy by inhibiting PKCβ/P66Shc.Conclusion1.Clinical studies have shown that tubular expression of Syk,PKCβ,and P66Shc was elevated in patients with diabetic nephropathy and that Syk,PKCβ,and p66Shc contributed to the progression of diabetic nephropathy;2.Syk promoter hypomethylation and increased gene expression levels in mice with diabetic nephropathy indicated that DNA methylation affected Syk expression;3.The enhanced binding of Sp1 to the Syk promoter region in mice with diabetic nephropathy suggested that DNA methylation regulated Syk expression through Sp1;4.Syk inhibitors in diabetic nephropathy inhibited renal tubular oxidative stress and apoptosis by downregulating the PKCβ/P66Shc pathway.Conclusively,Syk promoter hypomethylation and increased binding with Sp1promoted its gene expression in diabetic nephropathy,and Syk inhibitors suppressed renal tubular epithelial cell oxidative stress and apoptosis by downregulating the PKCβ/P66Shc pathway,Syk is a potential therapeutic target for diabetic nephropathy.