The Role And Mechanism Of Deacetylated P53-induced Autophagy In Sepsis-Induced Acute Kidney Injury

Sepsis is a common fatal syndrome characterized by a disorder of the host’s response to infection leading to life-threatening organ dysfunction.Acute kidney injury(AKI)is a common complication of sepsis and is associated with high morbidity and high mortality of hospitalized patients.Even after reversal,survivors not only have the potential to relapse again,but also have a high risk of turning into chronic kidney disease.Therefore,sepsis-associated acute kidney injury(SA-AKI)is an important focus and clinical burden for healthcare worldwide,and further research is needed to reduce acute and chronic adverse consequences.There are a lot of evidences that renal tubular epithelial cells are the main target of sepsis and renal function declines as sepsis progresses.In recent years,it has been proved that protecting tubular epithelial cells can improve SA-AKI.These results suggest that protecting tubular epithelial cells may be a new method for treating SA-AKI.Recently,the mechanism of autophagy in sepsis has received increasing attention.As an evolutionarily conserved intracellular self-degradation system,autophagy is involved in the establishment of intracellular homeostasis.Normally,autophagy is at a low basal level and plays an inportant role in cell metabolism and organ homeostasis through the degradation and recycling of damaged proteins,macromolecules,and organelles.However,under various stresses in vivo and in vitro(such as cell starvation,hypoxia,deprivation of nutrients and growth factors,oxidant damage,genotoxic substances,etc.)and pathological conditions,the level of intracellular autophagy will increase significantly.In sepsis,autophagy has long been considered as an adaptive protective mechanism that limits cell damage and apoptosis.Numerous studies have confirmed that autophagy has been found to play a protective role in various organs during the progress of sepsis.However,there is no consensus on the role of autophagy in SA-AKI.One study showed that autophagy activation exacerbates kidney damage in CLP-induced sepsis mice,and that inhibition of autophagy relieves kidney damage in sepsis mice.In fact,more studies tend to believe that autophagy activation has a protective role on SA-AKI.p53 is a classic pro-apoptotic protein.It is generally believed that the function of p53 is related to the regulation of cell cycle arrest and cell death,but more and more evidence indicates that the protein is a central node in the stress and nutrition response network and not only plays a role in tumor suppression,but also plays a significant role in metabolism,development,aging,neurodegeneration,and autophagy.The regulation of autophagy by p53 has a dual role due to its subcellular localization.p53 induces autophagy in the nucleus,and p53 inhibits autophagy in the cytoplasm.Therefore,we need to further explore the role of p53 in regulating autophagy.Whether p53 participates in the pathogenesis of SA-AKI through the regulation of autophagy is a question worthy of further consideration.Moreover,the key to p53 function is closely related to acetylation modification.Studies have shown that acetylation of p53 at many sites is the key to its accurate regulation in cells.This suggests that the acetylation modification of p53 may play an important role in the regulation of autophagy.Objective:This study aims to explore the role of autophagy in SA-AKI.The findings will provide new ideas for the pathogenesis of SA-AKI and provide potential targeted therapeutic methods for the treatment SA-AKI.Methods:At the animal level,C57/BL6 mice were subjected to cecal ligation and puncure(CLP)to establish a sepsis model.At the cellular level,human proximal tubular epithelial-2(HK-2)cells treated with lipopolysaccharides(LPS)were used to establish a model of sepsis.In order to explore the role of autophagy in SA-AKI,we have used many experimental techniques and methods in this study,such as pathological staining,TUNEL staining,transmission electron microscope,western blotting,laser confocal microscope observation,immunoprecipitation,siRNA interference,immunofluorescence,plasmid overexpression,and adenovirus transfection.Results:1.Observation of kidney injury and autophagy in SA-AKI.1.1 The damage of mice renal cortex tissue gradually increases in CLP-induced sepsis.1.2 Apoptosis of renal tubule epithelial cells(RTECs)in mice increased significantly at 12 h in CLP-induced sepsis.1.3 Autophagy of RTECs increased first,and then decreased after reaching the peak at 8 h in CLP-induced sepsis.1.4 Autophagy increased first,and then decreased after reaching the peak at 8 h in LPS-stimulated HK-2 cells.2.Autophagy activation improves CLP-induced kidney damage.3.Increased acetylation of p53 protein inhibits autophagy of RTECs in SA-AKI.3.1 At CLP-induced sepsis,the expression level of p53 protein in mice renal cortex is unchanged,and the acetylation level of p53 is significantly increased at 12 h.3.2 Pre-excitation of p53 attenuates autophagy of RTECs in SA-AKI,and preinhibition of p53 enhances autophagy of RTECs in SA-AKI.3.3 p53 Knockdown increased the autophagy level of HK-2 cells after LPS stimulation.3.4 The distribution of p53 protein in the cytoplasm and nucleus did not change in HK2 cells after LPS stumilation.4.Increased acetylation of p53 protein K382 site inhibits autophagy of RTECs in SAAKI.4.1 The acetylation level of p53 K382 is significantly increased at 12 h in HK-2 cells after LPS stumilation.4.2 Sirtl plasmid upregulates Sirtl protein expression in HK-2 cells,and Sirtl siRNA downregulates Sirtl protein expression in HK-2 cells.4.3 Sirtl overexpression obviously decased p53 K382 acetylation level in HK-2 cells after LPS stumilation.4.4 Sirtl overexpression obviously increased the autophagy level of HK-2 cells after LPS stimulation.4.5 Sirtl knockdown obviously increased p53 K382 acetylation level in HK-2 cells after LPS stumilation.4.6 Sirtl knockdown obviously decreased the autophagy level of HK-2 cells after LPS stimulation.4.7 Validation of Ad-p53 and Ad-p53K382R transfection efficiency in HK-2 cells.4.8 Reduced acetylation of p53 K382 enhances autophagy in HK-2 cells after LPS stumilation.Conclusions;1.This study verified that RTECs autophagy reduction is an important mechanism to promote the development of SA-AKI.Compensatory increase in autophagy of RTECs in sepsis may delay the occurrence of AKI,and increasing the autophagy level of RTECs can improve SA-AKI.2.This study found that increased acetylation of p53 protein reduces autophagy of RTECs,and K382 is a key lysine site for p53 acetylation.