The Protective Effect Of Nrf2 Regulating Mitochondrial Homeostasis In Sepsis Associated Acute Kidney Injury And Analysis Of The Relationship Between CRRT Practice Changes And Outcome In Pediatric Intensive Care Unit

Chapter One:The protective effect of Nrf2 regulating mitochondrial homeostasis in sepsis-induced acute kidney injuryBACKGROUNDSepsis is a life-threatening organ dysfunction caused by uncontrolled inflammatory response to infection.The clinical morbidity and mortality of sepsis associate acute kidney injury(SA-AKI)is extremely high,and some surviving patients progress to chronic kidney disease after discharge.Previous studies have confirmed that mitochondrial damage play a key role in the pathogenesis of SA-AKI.As the main regulator of cell redox homeostasis,Nrf2 promotes cell adaptive responses in the presence of stress.In addition,Nrf2 is involved maintenance of mitochondrial homeostasis which related to the regulation of mitochondrial function,mitochondrial biogenesis and mtophagy.However,the relationship between Nrf2 and the regulation of mitochondrial related pathways in SA-AKI is not clear.Therefore,this study mainly focuses on functional role of Nrf2 in SA-AKI and its possible protective mechanism.OBJECTIVETo explore the relationship between Nrf2 and acute kidney injury in sepsis,and the regulatory mechanism of Nrf2 on mitochondrial homeostasis.METHODS1)Cecal ligation and perforation(CLP)model was used to establish a rat model of sepsis.The kidney tissue and serum specimens were collected,the levels of urea and creatinine were used to detect renal function,western blot and immunohistochemistry were used to detect the expression of Nrf2 in the kidney tissue,and morphological changes of kidney were observed by HE sections.2)LPS stimulated NRK-52E cells to establish vitro model of SA-AKI.First,whether Nrf2 is activated is verified.Subsequently,lentiviral virus were used to to up-regulate Nrf2 in NRK-52E cells.The following indicators are detected:cell injury(cell viability,apoptosis-related proteins and TUNEL apoptosis staining),oxidative stress(ROS,MDA and SOD),inflammation(TNF-α,IL-6 and inflammation pathway related proteins),mitochondrial function(mtROS,MMP and ATP),mitochondrial biogenesis-related proteins(PGC-la,TFAM),mitophagy-related proteins(Parkin,PINK1,LC3)changes,mitochondrial morphology changes,immunofluorescence co-localization(COXIV and LC3).3)Nrf2 agonist TBHQ or inhibitor ML385 pretreated experimental animals.After CLP treatment,kidney tissue and blood samples were collected,renal function indexes,oxidative stress indicators(MDA and SOD),inflammation indicators(TNF-α,IL-6),morphological changes of kidney tissue,mitochondrial biogenesis-related proteins(PGC-1α,TFAM),mitophagy-related proteins(Parkin,PINK1,LC3)changes and mitochondrial morphology changes were detected.RESULTS1)After the establishment of the CLP model,the serum urea and creatinine levels of rats increased,HE staining confirmed the pathological changes of the kidney,and the expression of Nrf2 in the kidney tissue was up-regulated.2)After LPS stimulation,Nrf2 activation in NRK-52E cells.Over-expression of Nrf2 in vitro attenuated oxidative stress levels,apoptosis,and the inflammatory response;enhanced mitophagy and mitochondrial biogenesis;and mitigated mitochondrial damage.3)In CLP model,the NRF2 agonist TBHQ alleviated the renal damage,however inhibition by ML385 aggravated these damage.In vivo,Nrf2 activation enhanced mitophagy and mitochondrial biogenesis;restored ATP production function;and mitigated mitochondrial structure damage.Conclusion:1)Nrf2 activation plays an endogenous protective role in SA-AKI.2)The protective mechanism of Nrf2 may be through maintaining mitochondrial homeostasis(promoting mitophagy and restoring mitochondrial biogenesis),alleviating oxidative stress and mitochondrial dysfunction,thus mitigating the cell injury;targeting Nrf2 may be an important therapeutic target for SA-AKI.Chapter Two:Analysis of the relationship between CRRT practice changes and outcome in pediatric intensive care unitOBJECTIVERetrospective analysis of the main practice changes of continuous renal replacement therapy in the pediatric intensive care unit during the 10 years period in our center and the impact on the survival.METHODSThe data of critically ill children who received continuous renal replacement therapy(CRRT)in the our hospital from January 2010 to December 2019 were collected.Data including demographics,clinical information and CRRT parameters were obtained through medical records.The treatment interval was divided into 2010-2014(former period)and 2015-2019(latter period).The differences of survival rate and CRRT practice between the two periods were compared.Finally,the influence of main CRRT practice changes on outcome was analyzed.RESULTSA total of 289 children were included in our study.Of the two study periods,2010-2014 and 2015-2019,the proportion of CRRT initiation time>24 h was significantly reduced(32.73%vs 60.60%,P<0.001),the percentage of fluid overload(FO%)at CRRT initiation was lower(3.8%vs 12.1%,P<0.001),the percentage of regional citrate anti-coagulation protocol was increased(100%vs 22.7%,P<0.001),and the ICU survival rate was significantly improved(36.4%vs 58.7%,P=0.001)in the latter period compared to the former.In addition,subgroup analysis found that survival were higher in patients wtih CRRT initiation time<24h,regional citrate anti-coagulation protocol and FO<10%.Conclusion:The survival rate of patients received CRRT treatment in our center has improved over past 10 years,and some changes have taken place during these periods.Among them,early initiation of CRRT,lower FO,and regional citrate anti-coagulation method seem to be related to the improvement of outcome.