Role Of Sirt3 In Lipopolysaccharide-induced Mitochondrial Damage In Renal Tubular Epithelial Cells

Objective:Sepsis is a common fatal disease that can cause multiple organ dysfunction,including kidney.The incidence of sepsis-associated acute kidney injury(AKI)is relatively high.Meanwhile,studies have also shown that sepsis is the most common cause of AKI,which accounts for 47.5% of total events.Therefore,the prevention and treatment of sepsis-associated AKI are of great significance.The pathogenesis of sepsis-associated AKI is not completely clear.Recently,studies have found that the disordered energy metabolism of renal tubular epithelial cells is a vital cause of the occurrence of sepsis-associated AKI.Mitochondria are the center of cellular energy metabolism and highly dynamic organelles,maintaining dynamic balance through continuous fusion and fission,called mitochondrial dynamics,to adapt to the energy requirement of cells under different environmental conditions.Silent information regulator 3(Sirt3)is a major member of the silent information regulator-related enzyme family located in the mitochondria.Sirt3 participates in almost all mitochondrial-related physiological processes by regulating the acetylation level of key proteins.Studies have shown that up-regulating the expression of Sirt3 in renal tubular epithelial cells can reduce the production of reactive oxygen species(ROS)and improve mitochondrial dynamics homeostasis,suggesting that Sirt3 is involved in regulating the stability of mitochondrial function and dynamics.Previous study has also confirmed that Sirt3 plays a protective role in sepsis-associated AKI,but whether it is through regulating the homeostasis of mitochondrial dynamics and its specific mechanism is still rarely reported.This study intends to investigate the role of Sirt3 in regulating mitochondrial homeostasis,and its regulatory mechanism through in vivo and in vitro experiments in the lipopolysaccharide(LPS)-induced sepsis AKI model.Methods:Part 1Eighteen male wild-type C57BL/6 mice aged 8-10 weeks,weighing 20-26 g in SPF environment,were randomly divided into Control group,LPS24 h group and LPS48 h group,each with 6 animals.Mice in the LPS 24 h group and LPS48 h were injected intraperitoneally with LPS solution(10 mg/kg),and mice in the Control group were injected intraperitoneally with the same dose of normal saline.Blood samples were collected according to the grouping time points to test the renal function of the mice(serum creatinine,blood urea nitrogen),and then the mice were sacrificed and the kidney tissues were collected.The pathological changes of the kidney were observed by light microscopy,and the apoptosis level of renal tubular epithelial cells was assessed by TUNEL staining.The level of mitochondrial oxidative stress was assessed by DHE staining.The morphology of mitochondria was observed by transmission electron microscope,and the expression of Drp1 and OPA1 related to mitochondrial fission and fusion was evaluated by Western blotting.The expression of Sirt3 in renal tubular epithelial cells was evaluated by immunohistochemistry,Immunofluorescence and Western blotting.Human renal tubular epithelial cells(HK-2)were cultured in vitro and divided into Control group and LPS group.The stimulation concentration of LPS was 10 μg/m L for 24 h.After that,the cells of each group were collected.The cell apoptosis level was assessed by Hoechst-33342 staining.The expression of mitochondrial apoptosis related proteins were evaluated by Western blotting such as Cyt-c and Bax.The mitochondrial morphology changes were observed by immunofluorescence,and the expression of Drp1 and OPA1 was evaluated by Western blotting.The expression of Sirt3 was evaluated by immunofluorescence and Western blotting.Part 2Sirt3 knockout heterozygous mouse model was constructed by a commercial company.Sirt3 knockout heterozygous mice were mated and bred to obtain Sirt3 knockout homozygous mice.The genotype of the mouse was detected by PCR,and the successful construction of the Sirt3 knockout model was verified by Western blotting.8-10 weeks old Sirt3 knockout mice and wild-type mice of the same generation were used for experiments.LPS(10 mg/kg)or the same dose of normal saline were injected intraperitoneally,and the stimulation time was 24 h.The pathological changes of the kidney were observed by light microscopy,and the apoptosis level of renal tubular epithelial cells was assessed by TUNEL staining.The level of mitochondrial oxidative stress was assessed by DHE staining.The morphology of mitochondria was observed by transmission electron microscope,and the expression of Drp1 and OPA1 was evaluated by Western blotting.The pc DNA3.1-Sirt3 recombinant plasmid was transfected into HK-2 cells to construct a Sirt3 overexpression cell model,and LPS(10 μg/m L)or the same dose of normal saline was added for stimulation for 24 hours.Then,the cells of each group were collected.The apoptosis level was detected by flow cytometry,and the changes of mitochondrial membrane potential and oxidative stress level were assessed by JC-1staining and ROS staining.Mitochondria morphology was observed by immunofluorescence staining,and the expression of Drp1 and OPA1 was evaluated by Western blotting.Part 3Sirt3 gene knockout mouse and Sirt3 overexpression cell model were constructed respectively.Western blotting and immunoprecipitation were used to evaluate the influence of Sirt3 on the expression of OPA1’s upstream regulatory molecule i-AAA protease(YME1L1)and its acetylation level after LPS stimulation.YME1L1-WT,YME1L1-K237 R and YME1L1-K237 Q plasmids were constructed by a commercial company to simulate the deacetylation and acetylation status of YME1L1.After transfecting the three plasmids into HEK-293 cells,the expression of OPA1 was evaluated by immunoblotting,and the morphological changes of mitochondria were observed by immunofluorescence.Immunofluorescence was used to observe the co-localization of Sirt3 and YME1L1,and the interaction between Sirt3 and YME1L1 was detected by immunoprecipitation.Results:Part 1(1)The levels of Scr and BUN in AKI mice with sepsis induced by LPS were significantly increased.The pathological observation showed that LPS stimulation resulted in vacuolar degeneration of renal tubular epithelial cells,shedding of renal tubular epithelial cells,and dilatation of the renal tubule lumen.TUNEL staining showed that LPS induced increased apoptosis of renal tubular epithelial cells.(2)LPS increased ROS production of mitochondria.LPS increased mitochondrial fission,and decreased fusion.(3)LPS induced down-regulation of Sirt3 expression in mouse renal tubular epithelial cells.(4)LPS stimulation induced increased apoptosis and mitochondrial fission,and decreased fusion of HK-2 cells in vitro.(5)LPS induced down-regulation of Sirt3 expression in HK-2 cells in vitro.Part 2(1)The results of agarose gel electrophoresis showed that homozygous(SIRT3-/-)mice could only detect 820 bp band;Western blotting showed that Sirt3 was not expressed in Sirt3 knockout mice,which proved that Sirt3 knockout mouse model was successfully constructed.(2)Compared with the control group,Sirt3 gene knockout aggravated the LPS-induced kidney pathological damage and renal tubular epithelial cell apoptosis in mice.(3)DHE staining,transmission electron microscope and Western blotting showed that Sirt3 gene knockout aggravated the LPS-induced mitochondrial damage and abnormal dynamics of renal tubular epithelial cells.(4)Compared with the control group,overexpression of Sirt3 gene reduced the LPS-induced apoptosis of HK-2 cells.(5)JC-1 and ROS staining,immunofluorescence and Western blotting showed that overexpression of Sirt3 gene alleviated the LPS-induced mitochondrial damage and abnormal dynamics in HK-2cells.Part 3(1)Immunoprecipitation showed that the acetylation level of YME1L1 in renal tubular epithelial cells was increased after LPS stimulation,Sirt3 gene knockout further increased the acetylation level of YME1L1,and overexpression of Sirt3 decreased the acetylation level of YME1L1.(2)Deacetylation of YME1L1 increased the expression of the long form of mitochondrial fusion-related protein OPA1(L-OPA1)and mitochondrial fusion,and decreased mitochondrial fission.(3)Immunofluorescence and immunoprecipitation demonstrated that Sirt3 and YME1L1 were co-localized in space and interacted with each other.Conclusion:LPS stimulation induces renal tubular epithelial cell apoptosis and mitochondrial damage,and down-regulates the expression of Sirt3.Decreased expression of Sirt3 results in renal tubular epithelial cell apoptosis by regulating mitochondrial function and dynamics and increased expression of Sirt3 can promote OPA1-mediated mitochondrial fusion by deacetylating YME1L1 to reduce LPS-induced mitochondrial damage in renal tubular epithelial cells.