The Effects And Mechanisms Of Septin4-K174 In Regulating Oxidative Stress In Hypertensive Kidney Injury

Objective: Hypertension affects over one billion people worldwide and can have devastating effects on multiple organs throughout the body,and GBD 2017 states that the kidneys are one of the main target organs damaged by hypertension.The number of chronic kidney disease cases caused by hypertension exceeds 23.6 million worldwide,severely affecting the survival time of patients.Renal podocytes form the ultimate filtration barrier of the glomerulus and hypertension leads to damage and apoptosis of the renal podocytes,ultimately leading to glomerulosclerosis.The extent of renal podocyte injury and apoptosis is considered to be one of the major prognostic factors in kidney disease.Mitigation of apoptosis in renal podocytes has potential clinical implications for the treatment of hypertensive nephropathy.Although some progress has been made in understanding the pathogenesis of renal podocyte apoptosis,methods to inhibit renal podocyte apoptosis have not been identified.In order to develop targeted therapies for hypertensive nephropathy,detailed studies on the molecular mechanisms and pathways of renal podocyte apoptosis are needed.Septin4 belongs to the SEPTIN family and contains a highly conserved P-loop motif(GESGLGKS)located in the GTPase domain,which plays a key role in regulating Caspase activation and apoptosis and is an important marker protein of organ damage.the P-loop motif is essential for the apoptotic function of Septin4.Once Caspases are activated to induce apoptotic factor production,Septin4 promotes the release of a range of cytokines and amplifies the Caspase cascade to induce apoptosis.Although post-translational modification(PTM)plays an important role in the apoptotic function of Septin4,it is unclear whether Septin4 is regulated by acetylation.sirtuin2(SIRT2)affects intracellular metabolism by regulating the ketoacid response and the ketoacid signalling pathway.It can regulate cell survival and differentiation by deacetylating cell cycle proteins and cell survival-associated proteins.Given this key role of SIRT2 deacetylase in oxidative stress and apoptosis,it is important to determine whether Septin4 has a biological function in hypertensive kidney injury via deacetylation of SIRT2.The present work explores the key role of Septin4 in hypertensive kidney injury,as well as the post-translational modification sites and specific regulatory mechanisms by which Septin4 exercises this role.Methods: SIRT2-TG transgenic mice were constructed and Septin4 acetylation levels were assessed by Western Blot,an immunoblotting method.Eight-to ten-week-old male specific pathogen-free(SPF)mice were examined and selected in all mice.SIRT2-WT and SIRT2-TG mice were grouped in Ang II and Na Cl infused mouse models for Ang IIinduced hypertension modelling.To investigate whether the pathway of SIRT2’s deacetylation regulation of Septin4 is directly involved in the cell viability as well as morphology,cell injury and apoptotic processes induced by hypertensive nephropathy.Septin4-K174 Q transgenic mice were subsequently constructed and Septin4 acetylation levels were assessed by Western Blot,an immunoblotting method.Eight-to ten-week-old male specific pathogen-free(SPF)mice were examined and selected in all mice.In Ang II and Na Cl infused mouse models,Septin4-WT and Septin4-K174 Q mice were grouped together for Ang II-induced hypertension modelling.A series of methods such as immunoprecipitation and proteomics were then used to further investigate the acetylation of the Septin4-K174 locus on Ang II-induced hypertensive kidney injury and the mechanism.In cellular assays,the antioxidant Tempol was used to pretreat Ang II-induced hypertensive kidney injury in Septin4-WT and Septin4-K174 Q kidney foot cells and to observe cell viability as well as morphology,cell damage and apoptosis.Pretreatment of Ang II-induced hypertensive kidney injury Septin4-WT and Septin4-K174 Q kidney pedicle cells with Tempol in mice and observation of kidney tissue function as well as morphology,cell injury and apoptosis.To investigate whether Septin4 acetylation affects apoptosis and hypertensive kidney injury by influencing intracellular levels of oxidative stress.Results: 1.Western Blot assay was performed to verify the level of Septin4 acetylation in SIRT2-TG mice,and cytoplasm,nucleus and mitochondria were isolated from kidney tissues of SIRT2-TG mice to determine that overexpressed Flag-SIRT2 was predominantly in the cytoplasm.Several oxidative markers,including 3-Nitrotyrosine,8-oxo-d G,SOD1 and endogenous ROS levels,were measured in kidney tissue from SIRT2-WT and SIRT2-TG hypertensive mice.Expression of oxidative stress-related proteins and endogenous ROS levels were significantly reduced in kidney tissue from SIRT2-TG mice compared to SIRT2-WT.The amount of cleaved PARP1 and cleaved Caspase3 was significantly reduced in the SIRT2-TG group compared to the SIRT2-WT group.precursor levels of PARP1 and Caspase3 were increased with the induction of Ang II.PARP1 levels were decreased in the SIRT2-TG group compared to the SIRT2-WT group.urinary protein creatinine ratio(UPCR)was significantly lower in SIRT2-TG mice than in WT mice,while creatinine clearance was significantly higher in SIRT2-TG mice.2.Western Blot assay to verify that Septin4-K174 Q,i.e.Septin4-K174 site hyperacetylated mice,and the acetylation level of K174 site in Septin4-K174 Q mice was significantly lower than that in Septin4-WT,suggesting that Septin4-K174 Q mice were successfully modeled.Kidney tissue from Septin4-K174 Q mice showed higher levels of 3-Nitrotyrosine,8-oxo-d G,SOD1 and endogenous ROS in response to hypertensive kidney injury compared to Septin4-WT.Cleaved PARP1 and cleaved Caspase3 were significantly higher in the Septin4-K174 Q group compared to the Septin4-WT group.the UPCR was significantly higher in Septin4-K174 Q mice than in Septin4-WT mice,while creatinine clearance was significantly lower in Septin4-K174 Q mice than in Septin4-WT 3.levels of the oxidative stress markers 3-Nitrotyrosine,8-oxo-Dg,SOD1,and intracellular ROS were higher in Ang II-induced sh Septin4 after re-expression of the K174Q-Septin4 NTm and WT-Septin4 NTm plasmids than in WT-Septin4 NTm after re-expression of sh Septin4.oxidative stress damage in sh Septin4 kidney foot cells re-expressing K174 Q NTm or WT NTm was rescued after pretreatment with Tempol prior to Ang II induction,and there was no significant difference between the two groups.after K174Q-Septin4 NTm re-expression,Ang II-induced cleavage of PARP1 and cleavage of levels of Caspase3 and cytoskeletal disintegration were higher than after sh Septin4 re-expression of WT-Septin4 NTm.Apoptosis levels and cytoskeletal disintegration were rescued in sh Septin4 kidney foot cells re-expressing K174 Q NTm and WT NTm by Tempol pretreatment prior to Ang II induction,with no significant differences between the two groups.Kidney tissues from Septin4-K174 Q mice showed higher levels of 3-Nitrotyrosine,8-oxo-d G,SOD1 and endogenous ROS in response to hypertensive kidney injury compared to kidney tissues from Septin4-WT mice.Renal tissue apoptosis and deterioration of renal function in Septin4-WT and Septin4-K174 Q mice were significantly reduced by pretreatment with Tempol prior to induction with Ang II,with no significant difference between the two groups.significantly alleviated and there was no significant difference between the two groups.Conclusion: 1.Overexpression of the deacetylase SIRT2 resulted in lower levels of Septin4 acetylation and reduced Ang II-induced hypertensive kidney injury in SIRT2-TG mice compared to SIRT2-WT.2.Septin4 hyperacetylation in Septin4-K174 Q mutant mice significantly exacerbated Ang II-induced hypertensive kidney injury.In vitro and in vivo assays demonstrated that Septin4-K174Q-induced oxidative stress exacerbated renal foot cell apoptosis,cytoskeletal disintegration and impaired renal function,and that pretreatment with the antioxidant Tempol significantly rescued this process.