The Role Of SIRT3 In Regulating Necrotic Apoptosis In Calcium Oxalate Kidney Stone Formation And The Intervention Of Polyethylene Glycol

Objective:The adhesion and aggregation of crystal in renal tubule epithelial cells is an important step in the formation of renal calculi,cell injury and crystal aggregation may be the key factors affecting the adhesion and deposition of crystal on cell surface.SIRT3 plays an important role in scavenging reactive oxygen species and attenuating oxidative stress injury in tissues,and regulating cell necrotic apoptosis may be an important way for SIRT3 to play its role,while polyethylene glycol has the properties of dispersant and repair cells,and may inhibit crystal aggregation.Therefore,this study intends to explore the role of SIRT3 in regulating necrotizing apoptosis in the formation of calcium oxalate kidney stones and explore the intervention of polyethylene glycol on crystal adhesion and aggregation.Methods:In the first part,SIRT3 overexpression,SIRT3 knockout and wild-type C57BL/6 male mice were used to construct mouse calcium oxalate renal crystal model by intraperitoneal injection of glyoxalic acid.The expression levels of SIRT3 and P-MLKL were detected by HE staining,SIRT3 immunohistochemistry,Von Kossa staining and protein immunoelectrophoresis in kidney samples.In the second part,mouse renal tubular epithelial cells TCMK-1 were used as the research object to construct calcium oxalate monohydrate stimulated cells as the model group.RIPK3 inhibitor GSK872 was pre-treated with calcium oxalate monohydrate stimulated cells as the inhibitor group,and the control group was added with the same amount of PBS.The crystal adhesion on the surface was observed,cell viability,cytotoxicity and oxidative stress levels were detected,and the expression levels of RIPK1,RIPK3 and PMLKL were detected by western blot.In the third part,TCMK-1 was stimulated with a variety of combinations of reactive oxygen stimulant,PEG4000 and calcium oxalate monohydrate.Cell viability,cytotoxicity and reactive oxygen levels were detected,and cell status and crystal adhesion were observed.Results:(1)Mouse renal calcium oxalate crystal deposition induces significantly increased necrotic apoptosis of renal tubular epithelial cells.Overexpression of SIRT3 can regulate necrotic apoptosis,alleviate renal crystal injury,and reduce renal crystal deposition.(2)In vitro studies showed that when the concentration of calcium oxalate monohydrate crystal was at a low level(below 400ug/ m L in a 6cm petri dish),inhibition of cell necrotic apoptosis could reduce cell damage and crystal adhesion deposition to a certain extent.When the concentration of calcium oxalate monohydrate crystals exceeded 800ug/ m L,the crystals on the cell surface gathered and formed amorphous precipitates,and the reduction of cell necrotic apoptotic cell damage could not reduce the total amount of crystal adhesion deposition.(3)The pre-use of polymer PEG4000 can keep the crystal particles suspended in the suspension system in a stable state,and can reduce cell oxidative stress damage,significantly reduce the crystal adhesion deposition on the cell surface.Conclusion:Kidney calcium oxalate crystal deposition is involved in rip K3-MLKL-mediated necrotic apoptosis,and SIRT3 can alleviate crystalline kidney injury by regulating necrotic apoptosis.In vitro studies have shown that reducing the necrotic apoptosis of renal tubular epithelial cells can reduce crystal adhesion to a certain extent,but higher crystal load can adhere to the cell surface and aggregate to form amorphous precipitation.Pre-use of polymer PEG4000 can maintain the suspension stability of crystal particles,reduce cell oxidative stress damage,and reduce cell crystal adhesion.