Effects Of AMPKα1 Mitochondrial Location On Ferroptosis In Acute Renal Ischemia/Reperfusion Injury And The Intervention Mechanism Of Berberine

Part 1 IntroductionAcute renal injury（AKI）is a common syndrome with serious short-term and long-term complications.Early diagnosis and mechanism research are extremely important for AKI treatment.As the major cause of AKI,acute renal ischemia/reperfusion injury is usually induced by sharply reduced renal blood flow and insufficient renal oxygen and nutrition supply caused by massive hemorrhage、contrast medium、trauma、sepsis and major surgery.Acute renal ischemia/reperfusion（I/R）injury can be divided into two stages.First,lack of oxygen and nutrients during ischemia lead to kidney damage.However,after blood reperfusion,intracellular reactive oxygen species（ROS）excessive generation,and redox balance is broken,resulting in more serious renal injury than that in ischemic period.Existing studies have confirmed that AMPK not only involved in energy metabolism,but also involved in regulating physiological and pathophysiological processes such as cell growth and proliferation,inflammation,oxidative stress and apoptosis,autophagy and"ferroptosis".The paradoxical biological effects of AMPK has been concerned by researchers.Previous studies of our team confirmed that up-regulation of AMPKα1expression has protective effect on acute renal ischemia/reperfusion injury.While,the specific mechanism and whether related to ferroptosis have not been reported.Firstly,to determine ferroptosis involved in acute anoxia/reoxygenation injury of NRK-52E cells,we constructed acute anoxia/reoxygenation injury model on NRK-52E renal tubular epithelial cells pretreated with various cell death pattern inhibitors.In order to further explored the role of ferroptosis in acute anoxia/reoxygenation injury,NRK-52E renal tubular epithelial cells were pretreated with ferroptosis inhibitor Ferrostatin-1（Fer-1）,iron chelator dexrazoxane（DXZ）and mitochondrial targeted antioxidant（Mito TEMPO）and then acute anoxia/reoxygenation（A/R）injury was constructed.Ptgs2（Prostaglandin-endoperoxide synthase2）m RNA,intracellular Fe²⁺level were detected and cell ultrastructure were detected by transmission electron microscope.To verify AMPKα1 mitochondrial location protect NRK-52E cells against acute anoxia/reoxygenation injury.Overexpression AMPKα1 in cytosol/mitochondria was constructed.After acute anoxia/reoxygenation injury,AMPKα1,NOX4 and GPX4 were determined by western blotting,and oxygen consumption rate and extracelluar acidification rate were detected by Seahorse XFe²⁴Extracellular Flux analyzer.To further explore the protective mechanism of AMPKα1 mitochondrial location on NRK-52E cells damaged by acute anoxia/reoxygenation.We constructed low expression PDHA1transgenic NRK-52E cells,and measured intracellular oxidative stress and mitochondrial function.Finally,AMPKα1 mitochondrial knock-in transgenic mice and berberine（BBR）pretreated mice were constructed and bilateral acute renal ischemia/reperfusion injury model was established.Serum indexes,histomorphology and western blotting were detected to verify protection function of AMPKα1mitochondrial location,and preliminarily explored the protective effect of BBR on acute renal ischemia/reperfusion injury,so as to lay a theoretical and experimental basis for treatment of acute renal injury with similar effective traditional Chinese medicine monomer.Part 2 Ferroptosis involved in acute anoxia/reoxygenation injury of NRK-52E cellsObjective:To investigate the role of ferroptosis in acute anoxia/reoxygenation injury in NRK-52E renal tubular epithelial cells.Methods:NRK-52E cells which were pretreated with various cell death pattern inhibitors were subjected to acute anoxia/reoxygenation injury,CCK-8 and LDH activity were determined;Ptgs2 m RNA was detected by q PCR;Fe²⁺contnet,the activities of SOD、CAT and GSH-Px,GSH/GSSG ratio and MDA content were measured by spectrophotometry;intracellular ultrastructure was observed by transmission electron microscope;intracellular ROS was detected by flow cytometry;mitochondrial ROS was observed by fluorescence inverted microscope;NOX4 and GPX4 expression were determined by western blotting.Result:2μM Fer-1（ferroptosis inhibitor）、50μM Emricasan（EMR,apoptosis inhibitor）、30μM Necrostatin-1（Nec-1,necrosis inhibitor）and 300μM 3-MA（autophagy inhibitor）enhanced the cell viability and reduced LDH activity of NRK-52E cells suffered acute anoxia/reoxygenation injury.The effect of 2μM fer-1is particularly prominent.2μM Fer-1、100μM DXZ and 10μM Mito TEMPO reduced Ptgs2 m RNA、Fe²⁺level,reduced oxidative stress and inhibited excessive production of ROS in cytosol and mitochondria,increased GPX4 expression and limited NOX4 expression.Conclusion:Ferroptosis involved in acute anoxia/reoxygenation injury of NRK-52E renal tubular epithelial cells.Ferroptosis inhibitors、iron chelators and mitochondrial targeted antioxidants improved the acute anoxia/reoxygenation injury of NRK-52E cells by reducing ferroptosis.Part 3 The protective effect of AMPKα1 mitochondrial location on NRK-52 E cells damaged by acute anoxia/reoxygenationObjective: To investigate the protective effect of AMPKα1 mitochondrial location on NRK-52 E cells damaged by acute anoxia/reoxygenation.Methods: Overexpression of cytosol and mitochondria AMPKα1 transgenic cells were constructed and NRK-52 E cells which were pretreated with ferroptosis inhibitors were exposed to acute anoxia/reoxygenation injury.The cytosol and mitochondria AMPKα1 expression were detected by western blotting;CCK-8 and LDH activity were used to detect cell viability;Ptgs2 m RNA level was detected by q PCR;intracellular Fe2+level、SOD、CAT and GSH-Px activities,GSH/GSSG ratio,MDA content,mitochondrial permeability transition pore（m PTP）were measured by spectrophotometry;intracellular ROS generation and mitochondrial membrane potential（MMP）were detected by flow cytometry;mitochondrial ROS generation was observed by fluorescence inverted microscope;NOX4、GPX4、mitochondrial complexes Ⅰ and Ⅲ、AMPKα1、ACC、p-ACC and PDHA1 were detected by western blotting;enzyme linked immunosorbent assay（ELISA）was used to determine AMP/ATP ratio;oxygen consumption rate and extracelluar acidification rate were detected by Seahorse XFe24 Extracellular Flux analyzer.Results: AMPKα1 mitochondrial location reduced Ptgs2 m RNA,Fe2+ level and MDA content;enhanced the SOD 、 CAT and GSH activities,GSH/GSSG ratio;inhibited intracellular and mitochondrial ROS generation;stabilized MMP,limited m PTP opening;stabilized oxygen consumption rate;reduced extracelluar acidification rate;promoted ATP production;increased NDUFB8 and UQCRC2 expression and promoted ACC phosphorylation and PDHA1 expression.Conclusion: AMPKα1 mitochondrial location reduced oxidative stress,improved mitochondrial function,enhanced mitochondrial respiration,inhibited glycolysis,promoted energy metabolism and energy generation,resisted NRK-52 E cells ferroptosis induced by acute anoxia/reoxygenation injury.Part 4 The protective mechanism of AMPKα1 mitochondrial location on NRK-52 E cells injured by acute anoxia/reoxygenationObjective: To investigate the protective mechanism of AMPKα1 mitochondrial location on NRK-52 E cells injured by acute anoxia/reoxygenation.Methods: Low expression PDHA1 transgenic cells were constructed.After acute anoxia/reoxygenation injury,the cell viability was detected by CCK-8 and LDH activity;Ptgs2 m RNA was detected by q PCR;intracellular Fe2+ level、SOD、CAT and GSH-Px activities,GSH/GSSG ratio,MDA content and m PTP opening were measured by spectrophotometry;intracellular ROS production and MMP were detected by flow cytometry;mitochondrial ROS generation was observed by fluorescence inverted microscope;NOX4、GPX4、mitochondrial complexes Ⅰ andⅢ、AMPKα1、ACC、p-ACC and PDHA1 expression were detected by western blotting;ELISA was used to determine AMP/ATP ratio.Results: Low expression PDHA1 decreased AMPKα1 mitochondrial overexpression transgenic cells viability,increased LDH activity;inhibited the activities of SOD 、 CAT and GSH-Px,reduced GSH/GSSG ratio;increased Ptgs2 m RNA、Fe2+ level 、MDA content;increased intracellular and mitochondrial ROS production;promoted MMP loss;increased m PTP open;reduced ATP production;increased AMP/ATP ratio;reduced ACC phosphorylation and PDHA1 expression.Conclusion: AMPKα1 mitochondrial location form AMPKα1-PDHA1 axis which inhibited oxidative stress 、 stabilized mitochondrial function 、 enhanced mitochondrial respiration、inhibited glycolysis、promoted energy metabolism and ATP generation and protected NRK-52 E cells from ferroptosis induced by acute anoxia/reoxygenation injury.Low expression PDHA1 remarkably abolished the protective effect of AMPKα1 mitochondrial location on acute anoxia/reoxygenation injury in NRK-52 E cells.Part 5 AMPKα1 mitochondrial location and Berberine alleviated acute renal ischemia/reperfusion injuryObjective: To investigate the protective effect and mechanism of AMPKα1mitochondrial location and berberine on acute renal ischemia/reperfusion injury.Methods: AMPKα1 mitochondrial location transgenic C57BL/6 mice,BBR pretreated C57BL/6 mice and C57BL/6 mice co-treated with Compound C（a specific AMPK inhibitor）and BBR were constructed.Mice were subjected to bilateral acute renal ischemia/reperfusion injury.Renal cortical tissue Ptgs2 m RNA was detected by q PCR;serum creatinine（Scr）,blood urea nitrogen（BUN）,LDH activity,Fe2+ level,SOD、CAT and GSH-Px activities,GSH/GSSG ratio,MDA content and caspase3 activity were measured by spectrophotometry;renal cortical tissue ROS generation and the changes of renal cortical tissue morphology were observed by fluorescence inverted microscope;NOX4 、 GPX4 、 mitochondrial complexes Ⅰ and Ⅲ and AMPKα1、ACC、p-ACC and PDHA1 expression were detected by western blotting.ELISA was used to determine AMP/ATP ratio;the renal cortical tissue ultrastructure was observed by transmission electron microscope;TUNEL assay was used to observe renal cortical tissue apoptosis.Results: AMPKα1 mitochondrial location significantly improved ferroptosis induced by acute renal ischemia/reperfusion injury,and BBR promoted AMPKα1mitochondrial location which reduced the increased Scr and BUN level caused by acute renal ischemia/reperfusion injury;reduced renal cortical tissue Ptgs2 m RNA and Fe2+ level;increased the activity of antioxidant enzymes and weaken the level of MDA.BBR pretreatment and AMPKα1 mitochondrial location improved renal cortical tissue and mitochondria morphology;reduced renal cortical tissue caspase3 activity and apoptosis;promoted renal cortical tissue ATP production,increased the expression of renal cortical tissue mitochondrial complexes I and III、GPX4、PDHA1and ACC phosphorylation.The above effects of BBR were distinctly cancelled after co-treatment with Compound C（a specific AMPK inhibitor）and BBR.Conclusion: AMPKα1 mitochondrial location protected renal cortical tissue resisted acute renal ischemia/reperfusion injury.BBR pretreatment promoted AMPKα1 mitochondrial location,reduced oxidative stress,improved mitochondrial function,promoted mitochondrial respiratory to produce ATP,reduced renal cortical tissue ferroptosis,maintained renal function,improved renal morphology,and reduced acute renal ischemia/reperfusion injury.