Mechanism Of CGAS-STING Activation Regulating Podocyte Injury In Diabetic Kidney Disease

BackgroundDiabetic kidney disease is one of the most serious chronic complications of diabetes mellitus and is now the leading cause of chronic kidney disease and end-stage renal disease worldwide.However,there is a lack of effective clinical treatment,so early intervention is extremely important.Podocytes are the most critical part of the glomerular filtration barrier,and podocyte injury is the key to the development,progression and clinical intervention of diabetic kidney disease.Recent studies suggest that renal inflammation is a key factor in the pathogenesis of diabetic kidney disease.cGAS-STING pathway is an important signaling molecule in immunity and inflammation and plays an important role in metabolism-related inflammation by recognizing exogenous DNA and its own abnormal DNA in the cytoplasm,binding to the promoter of target genes and promoting the expression of inflammatory genes.However,whether this pathway is involved in the development of diabetic kidney disease is unclear.Part Ⅰ.Expression and activation of cGAS-STING signaling in the process of kidney injury in DKD miceObjectiveTo investigate the time course of renal function alteration in db/db mice and the activation of cGAS-STING signaling in renal parenchymal cells of db/db mice.Methods1.db/db mice were used as a model of diabetic kidney disease,and db/m mice were used as control.Mice were monitored from 4 weeks old,body weight and fasting glucose were measured weekly,and three mice from each of the two groups were randomly executed every two weeks,and serum and kidney tissues were collected.2.The process of diabetic kidney disease in db/db mice was analyzed comprehensively by HE staining,PAS staining,Masson staining,TUNEL staining,immunohistochemical staining of Nephrin protein,transmission electron microscopy,blood urea nitrogen,blood creatinine and blood lipids,and the results of GTT and ITT.3.Find the cells where the cGAS-STING signaling pathway mainly functions by immunofluorescence co-localization.The activation of cGAS-STING signaling pathway in the kidney injury process in db/db mice was detected by western blotting.Results1.The body weight and fasting glucose of 10-week-old db/db mice were higher than those of the control group,and were accompanied by dyslipidemia,impaired glucose tolerance and impaired insulin sensitivity.2.db/db mice began to show obvious microalbuminuria with normal blood urea nitrogen and creatinine levels at 10 weeks of age.Renal pathology showed obvious glomerular hypertrophy with slight mesangial expansion,without obvious collagen disposition,decreased expression of the podocyte marker protein Nephrin,appearance of widely fused podocytes and irregularly thickened glomerular basement membranes,and increased levels of podocyte apoptosis.3.Activation of the cGAS-STING pathway was detected in the renal cortex of approximately 8-week-old db/db mice,mainly in the podocytes.The levels of cGAS and STING proteins were elevated in the renal cortex,and the levels of TBKI phosphorylation were elevated,while the levels of IRF3 phosphorylation and IFN-βremained unchanged.Conclusion1.Podocyte injury is the main early manifestation of diabetic kidney disease.2.Activation of the renal cGAS-STING pathway in db/db mice is mainly in the podocytes.3.Activation of the renal cGAS-STING pathway in db/db mice precedes podocyte injury.4.The cGAS-STING pathway is activated in a non-classical manner in podocytes without activating IRF3.Part Ⅱ.Exploration of in vivo inhibition of STING to improve podocyte injury in DKD miceObjectiveTo investigate whether inhibition of the cGAS-STING pathway can improve diabetic kidney disease and obesity-related kidney disease.Methods1.7-week-old db/db mice were administered with STING inhibitor C-176 intraperitoneally once a day for 21 days,and changes in body weight,blood glucose and urinary protein were monitored and recorded.Collect serum and kidney tissues to detect renal function and renal morphological changes,and the detection methods were the same as in Part Ⅰ.2.Establish diet-induced obesity mouse(DIO)model,give intraperitoneal injection of STING inhibitor C-176 once every other day for 28 days,monitor and record the changes of body weight,blood glucose and urinary albumin creatinine ratio of mice.Serum and kidney tissues were collected to detect renal function and morphological changes in the kidneys,and the detection methods were the same as in Part Ⅰ.Results1.C-176 inhibited cGAS-STING activation.C-176 intervention did not affect body weight,ITT,GTT,blood lipid results and decreased fasting glucose in db/db mice.During the first week of C-176 treatment,microalbuminuria was significantly reduced and the levels of blood urea nitrogen and blood creatinine were unchanged.Glomerular hypertrophy and mesangial expansion were improved after C-176 treatment,nephrin levels were increased,apoptosis of podocytes was reduced,podocytes were ordered,foot processes were clear,and glomerular basement membrane was homogeneous.2.cGAS-STING pathway is activated in DIO mouse podocytes.C-176 inhibited cGAS-STING pathway activation.DIO mice showed increased body weight,abnormal glucose tolerance and insulin tolerance,elevated fasting glucose,increased urinary albumin creatinine ratio,glomerular hypertrophy and mesangial expansion,decreased nephrin levels,diffuse thickening of glomerular basement membrane,extensive fusion of foot processes,decreased number and increased apoptosis of podocytes in DIO mice.C-176 intervention improved the kidney phenotype of DIO mice.Conclusion1.Inhibition of the cGAS-STING pathway ameliorates podocyte injury in db/db mice.2.Inhibition of the cGAS-STING pathway ameliorates podocyte injury in DIO mice.3.The renoprotective effects of STING inhibition were not simply because they attenuated obesity or systemic metabolism,because:(1)C-176 attenuated renal injury in db/db mice without significantly improving hyperglycemia,dyslipidemia,or obesity;and(2)the renoprotective effects of C-176 in DIO mice preceded its effects on blood glucose levels.Part Ⅲ.Mechanisms of cGAS-STING pathway mediating MPC5 injuryObjectiveTo elucidate whether the protective effect of inhibiting the cGAS-STING pathway against podocyte injury is cell-autonomous and to explore the underlying molecular mechanisms.Methods1.MPC5 injury was induced by PA.cGAS-STING activation was detected by western blotting and immunofluorescence staining,nephrin and podocin levels were detected by western blotting.The level of apoptosis was detected by TUNEL staining and western blotting,and the expression level of inflammatory factors was detected by q-PCR.2.PA-treated MPC5 cells were intervened with STING inhibitor C-176,STING small interfering RNA,and TBK1 inhibitor GSK8612 to detect cGAS-STING activation and cell damage,respectively.The assay was performed as above.3.Mitochondrial function and morphology of PA-treated MPC5 cells and EtBr-intervened PA-treated MPC5 cells were detected by Seahorse and transmission electron microscopy.MPC5 cells were transfected with mtDNA to detect the activation of cGAS-STING and cell injury.4.PA-treated MPC5 cells were interfered with BAX inhibitor BAI1,BAX small interfering RNA,and mitochondrial permeability transition pore inhibitor cyclosporine A,and cGAS-STING activation and cell damage were detected,respectively.Results1.After PA stimulation,cGAS-STING was activated in MPC5 cells,the phosphorylation levels of STING downstream molecules TBK1 and NF-kB p65 were increased,IRF3 was not phosphorylated,and the protein levels of IFN-β remained unchanged.The levels of nephrin and podocin decreased,apoptosis levels increased,and the expression of the inflammatory factors IL-6 and TNF-α increased.2.C-176,STING small interfering RNA,and GSK8612 intervention reduced TBK1 and NF-kB p65 phosphorylation,nephrin and podocin protein levels were partially restored,apoptosis was reduced,and IL-6 and TNF-α levels were decreased.3.PA decreased the basal and maximal mitochondrial respiration rate of MPC5 cells and reduced ATP production,caused disruption of mitochondrial structure and matrix vacuole formation.The number of autophagic vesicles decreased with increasing PA concentration.EtBr intervention ameliorated PA-induced podocyte injury.cGAS-STING activation in mtDNA-transfected MPC5 cells induced podocyte injury and these injuries could be significantly attenuated by C-176.4.BAI1 and BAX small interfering RNA intervention inhibited cGAS-STING activation and attenuated podocyte injury.While cyclosporine A did not play this role.Conclusion1.the protective effect of C-176 or STING knockdown in lipotoxicity-associated podocyte injury is cell-autonomous,consistent with animal experiments.2.cGAS-STING pathway is activated in MPC5 cells by STING-TBK1-p65,without activating IRF3,consistent with animal experiments.3.GSK8612 inhibition of TBK1 is sufficient to produce cell-autonomous protection,indicating that TBK1 is an important effector downstream molecule of the cGAS-STING pathway in podocytes.4.Impaired autophagy in podocytes leads to the accumulation of damaged mitochondria,and mtDNA leaks into the cytoplasm through bax-mediated macropores,activating the cGAS-STING/TBK1/p65 pathway,leading to inflammatory factor production and podocyte injury.