Effect Of Aerobic Glycolysis On Sepsis-Induced Acute Kidney Injury And Its Mechanism

Background and Objective:The immune response dysfunction in patients with sepsis is characterized by uncontrolled inflammatory response and immunosuppression.These dysfunctional immune responses lead to cell dysfunction and then organ failure.Acute kidney injury(AKI)is one of the most common and serious complications in sepsis.The main pathogenesis of acute kidney injury in sepsis is cell hypoxia,which is caused by extensive microvascular blood flow disturbance around renal tubules and glomeruli;and cell metabolism,energy synthesis and mitochondrial dysfunction caused by various inflammatory mediators released.Hypoxia and metabolic disturbance lead to many biological processes’ disturbance.Many cell death pathways,such as necrosis,apoptosis,autophagy,programmed necrosis,netosis and pyrosis,are directly activated by uncontrolled inflammatory response or pathogens in sepsis.Autophagy is an evolutionarily conserved multi-step degradation pathway mediated by lysosomal hydrolases,which degrades damaged organelles,proteins and other macromolecules in cells.It is mainly mediated by positive or negative regulatory factors such as AMP activated protein kinase(AMPK),sirtuins(sirts)and mammalian target of rapamycin,MTOR and others.Many studies have shown that autophagy is involved in the occurrence and development of acute kidney injury in sepsis,and has a protective effect on acute kidney injury.The mechanism of autophagy regulation in septic acute kidney injury still remains unclear.Aerobic glycolysis is that when oxygen is enough,the glycolysis pathway in cells is activated,and the lactate is obviously increased,plays an important role in the development of sepsis.The measurement of serum lactate level is usually included in the clinical management of critical diseases,and is often used to evaluate the disease activity,treatment response and prognosis of sepsis patients.The hyperlactation of sepsis mainly comes from activated immune cells,regulates the immune function of innate immune cells and adaptive immune cells.The intervention of 2-deoxyglucose(2-DG)as aerobic glycolysis inhibitor can significantly reduce the serum lactoic acid level,improve the function of multiple organs and improve survival rate.Our previous studies have shown that 2-DG inhibits aerobic glycolysis and protects multiple organ damage in sepsis mice,including acute renal injury.Why inhibition of aerobic glycolysis can protect acute renal injury in sepsis needs further study.It is not clear whether aerobic glycolysis is involved in the occurrence of acute renal injury in sepsis.It is of great significance to explore whether the glucose metabolism of kidney tissue transverse to aerobic glycolysis and how the change affects acute renal injury in sepsis.In this study,the sepsis mouse model was established by cecal ligation and puncture(CLP),and the changes of renal function,renal pathological damage,apoptosis,the expression of autophagy markers and autophagy regulatory factors SIRT3 and AMPK in mice by intervention of 2-deoxyglucose or 3-MA was determined in order to explore the effect and mechanism of aerobic glycolysis on sepsis and acute kidney injury.Renal tubular epithelial cells were stimulated by lipopolysaccharide(LPS)to establish sepsis cell model,and 2-DG or3-MA intervention was performed to observe apoptosis,the expression of autophagy markers and autophagy regulatory factors SIRT3 and AMPK,and to study the effect of aerobic glycolysis on renal tubular epithelial cell apoptosis at the cellular level and explore whether 2-DG interfere with autophagy via SIRT3/AMPK pathway in sepsis-induced acute kidney injury.Methods:(1)sepsis mouse model was established by CLP.The levels of serum lactic acid,serum urea nitrogen(BUN),creatinine and kidney injury molecule-1(KIM-1)were detected by commercial kit.The pathological changes of renal tissue were observed by H & E staining.TUNEL staining was used to detect the apoptosis of renal cells.Realtime-PCR was used to detect the m RNA expression levels of glycolysis related genes pyruvate dehydrogenase kinase 1(PDK1),lactate dehydrogenase A(LDHA)and pyruvate kinase M2(PKM2)in the kidney of septic mice.The levels of autophagy markers LC3-I,LC3-II and p62 and autophagy regulators SIRT3,AMPK and p-AMPK were detected by Western blot or immunofluorescence.(2)2 g / kg 2-DG was injected intraperitoneally 3 hours before CLP,and the levels of serum lactic acid,serum urea nitrogen,creatinine and KIM-1 were detected by commercial kits.The pathological changes of renal tissue were observed by H & E staining.TUNEL staining was used to detect the apoptosis of renal tissue.The m RNA levels of PDK1,LDHA and PKM2 were detected by real-time PCR.Western blot was used to detect the levels of autophagy markers LC3-I,LC3-II and p62,as well as autophagy regulatory factors SIRT3,AMPK and p-AMPK.The effects of inhibition of aerobic glycolysis on renal function,renal pathological damage,apoptosis,autophagy markers and autophagy regulatory factors in septic mice were observed.(3)The HK-2 cells are stimulated by 1μg/ml LPS and the apoptosis of HK-2 cells was detected by flow cytometry.The m RNA expression levels of PDK1,LDHA and PKM2 were detected by real-time PCR.Western blot was used to detect the levels of autophagy markers LC3-I,LC3-II and p62,and the expression of autophagy regulators SIRT3,AMPK and p-AMPK.(4)The septic mice or HK-2 cells stimulated by LPS were pretreated with autophagy inhibitor 3-MA(30 mg / kg)in order to observe wherther 3-MA regulate the effects of 2-DG on renal function and pathological damage,apoptosis,the expression of autophagy markers and autophagy regulatory factors.(5)The HK-2 cells stimulated by LPS were pretreated with lactic acid.The levels of autophagy markers LC3-I,LC3-II and p62 and autophagy regulators SIRT3,AMPK and p-AMPK were detected by Western blot.Results:(1)Aerobic glycolysis was increased in the renal tissue of septic mice,and 2-DG pretreatment reduced the acute renal injury of septic mice.The levels of lactic acid,bun,creatinine and KIM-1 in serum of sepsis mice were significantly increased at 24 hours after CLP,and the m RNA expression levels of glycolysis related genes PDK1,LDHA and PKM2 in renal tissue were increased.Aerobic glycolysis inhibitor 2-DG reduced the levels of serum BUN,creatinine and KIM-1 in septic mice,and alleviated the pathological damage of renal tissue.(2)Autophagy was enhanced in renal tissue of septic mice,and 2-DG pretreatment further enhanced autophagy in renal tissue.The expression of autophagy marker LC3-I was decreased,but the expression of LC3-II was significantly increased,the ratio of LC3-II / I was significantly increased,and the expression of p62 was decreased after CLP;After 2-DG pretreatment,the ratio of LC3-II / I increased and the expression of p62 decreased more significantly,and the apoptosis of renal tubular epithelial cells decreased significantly.(3)Aerobic glycolysis and autophagy were increased in human renal tubular epithelial cells HK-2 stimulated by LPS.2-DG pretreatment further enhanced the autophagy of HK-2 induced by LPS,reduced the levels of SIRT3 and p-AMPK induced by LPS,and inhibited the apoptosis of HK-2.When HK-2 cells were stimulated with LPS,the levels of lactate,LDHA and PKM2 m RNA,LC3-II / I ratio,p62 expression and apoptosis of HK-2 cells were significantly increased.However,2-DG treatment decreased the levels of LDHA and PKM2 m RNA and the levels of lactate in the supernatant induced by LPS,and further increased LC3-II /I ratio,decreased the expression of p62 and reduced the apoptosis of renal tubular epithelial cells.When HK-2 cells were stimulated by LPS,the levels of SIRT3 and p-AMPK in HK-2 cells were significantly increased,while 2-DG treatment decreased the increase of SIRT3 and p-AMPK induced by LPS.(4)Pretreatment with autophagy inhibitor 3-m A attenuated the protective effect of 2-DG on septic mice and HK-2 under stimulation with LPS.After pretreatment with autophagy inhibitor 3-MA,the inhibition of2-DG on serum BUN,SCR and KIM-1 levels,renal cell apoptosis and LPS induced HK-2 cell apoptosis in septic mice were attenuated signifiacantly.(5)Lactate pretreatment inhibited LPS-induced autophagy and reversed the enhancement effect of 2-DG on autophagy.Lactic acid pretreatment decreased LPS-induced the upregulation of LC3-II / I ratio,SIRT3 and p-AMPK,increased p62 expression,and partially eliminated the effect of 2-DG on the increase of LC3-II / I ratio,SIRT3 and p-AMPK and the decrease of p62 expression,thus finally reversed the inhibition of 2-DG on apoptosis.