Research On The Association And Mechanism Of IL-33/ST2 With Acute Kidney Injury In Children

Acute kidney injury(AKI)is one of the most common organ dysfunctions in critically ill children,and it is an independent risk factor for death[1].The high mortality rate of AKI is related to the failure to identify and establish targeted intervention therapy[2].It is of great clinical significance to explore the mechanism and biomarkers that can identify AKI early for timely treatment and thus reduce its mortality.Early and accurate identification of AKI is critical for improving patient outcomes[35].Our team is committed to the study of biomarkers of AKI in neonates and children.In the detection of factors closely related to inflammatory injury by Liqui Chip technique,we detected a significant increase of interleukin-33(IL-33)and tissue inhibitor of metalloproteinase-2(TIMP-2)in the serum and kidney of rats after renal ischemiareperfusion injury.IL-33 is a member of the interleukin-1(IL-1)family,its specific receptor is Suppression of tumorigenicity 2(ST2).As a member of the IL-1 receptor family,ST2 has transmembrane(ST2L)and soluble(sST2)isoforms.IL-33 activates the immune response by combining with ST2L protein after tissue injury.While sST2 binds to IL-33 and antagonizes its effects[6].Studies have shown that IL-33 and ST2 elevate in various renal disease models[7,8].However,there is no study on the association of IL-33/ST2 with acute kidney injury in clinical study.Therefore,through single-center and multi-center clinical studies,we intend to analyze the association of serum IL-33,sST2 with AKI and investigate the significance of serum IL-33,sST2 for the diagnosis and prognosis of AKI in critically ill children.As cell cycle arresting protein,urinary TIMP-2 is involved in renal tubular epithelial cell G1 cycle arrest in the early stage of ischemia or inflammatory injury[5].Studies in adults have shown that the combination of TIMP-2 and insulin-like growth factor binding protein-7(IGFBP-7)which is also a cell cycle arresting protein can help identifying AKI or severe AKI[9-11].However,there is a lack of evidence linking these biomarkers with AKI in neonates.Due to the influence of perinatal hypoxic asphyxia,infection and other factors,critically ill neonates are more prone to AKI than children,and the mortality rate of neonates is significantly higher than that of children.However,the diagnosis of neonatal AKI is more challenging than the diagnostic criteria used in pediatric.In this part,by establishing a prospective clinical cohort study for critically ill neonates,we analyze the association of these biomarkers with AKI or severe AKI and explored the significance of these biomarkers for the diagnosis of AKI in critically ill neonates.Through the first part of the clinical study,we found that serum sST2 is closely associated with AKI in critically ill children.While there are few studies on how IL-33/ST2 is regulated in renal injury.Renal ischemia-reperfusion injury(IRI)is the pathophysiological basis of AKI[12].A variety of cell damage and death pathways are involved,including autophagy,apoptosis,necrosis,pyroptosis and ferroptosis[13,14].Ferroptosis is an irondependent regulatory death proposed in 2012[15].Studies have shown that compared with other death modes,ferroptosis is mainly manifested by mitochondrial atrophy,membrane density aggregation and mitochondrial crest reduction,which plays an important role in kidney injury[16].However,there are few studies on the mechanism of ferroptosis in the progression of renal injury.Inflammatory response is an important part of renal ischemiareperfusion injury.Studies have confirmed that by binding to ST2L on the surface of natural killer cells,lymphocytes and macrophages,IL-33 can induce the aggregation of myeloid differentiation molecules and IL-1-related protein kinases,then activate the downstream nuclear transcription factor-KB(NF-κB)signaling pathway during injury and inflammation[6].Whether ferroptosis regulates renal injury by mediating IL-3 3/ST2 has not been reported.We hypothesized that ferroptosis during AKI may upregulate the expression of IL-33.Then by binding to its receptor ST2L,IL-33 activates NF-κB,causing inflammatory damage of renal tubular epithelial cells,and thereby aggravating renal injury.Therefore,in the second part,we investigate the expression of ferroptosis and IL-33/ST2 in acute kidney injury and related molecular mechanisms.Through the above two parts of the study,we aim to explore biomarkers of AKI and analyze the mechanisms involved,which can provide a new entry point for the diagnosis and treatment of AKI in children.Part Ⅰ Association analysis of biomarkers with acute kidney injury in childrenAssociation analysis of serum sST2 with acute kidney injury and prognosis in critically ill childrenObjective:Analysis the association between serum sST2 with AKI and prognosis through single-center and multi-center prospective clinical studies.Investigate the significance of serum sST2 for the diagnosis and prognosis of AKI in critically ill children.Methods:(1)Participating institution:Single-center clinical study:Critically ill children admitted to PICU of Children’s Hospital affiliated to Soochow University from September 2019 to February 2020 were selected as prospective subjects for this study.Multi-center experiment:Participating centers:Children’s Hospital affiliated to Soochow University,Children’s Hospital affiliated to Fudan University,Anhui Children’s Hospital,Xuzhou Children’s Hospital.Critically ill children admitted to PICU from September 2020 to February 2021 were selected as prospective subjects for this study.(2)Clinical Data Collection:Clinical information,including the pediatric risk of mortality score(PRISM Ⅲ score),AKI,sepsis,acute lung injury(ALI),shock,disseminated intravascular coagulation(DIC),multiple organ dysfunction syndrome(MODS),death and urine volume during hospitalization in PICU were collected.The use of mechanical ventilation(MV),renal replacement therapy(RRT)and drugs(diuretics and vasoactive drugs)during PICU stay,the length of PICU stay and hospital stay were collected.(3)Sample collection and analyses:After the PICU admission,daily blood samples were collected during the first 5 days of PICU admission in the single center study,and blood samples were collected on the first day of PICU admission in the multi-center study.Serum levels of IL-33 and sST2 were analyzed by ELISA.(4)Grouping:According to the occurrence of AKI during PICU stay,patients were divided into AKI group and non-AKI group.According to the occurrence of sepsis,patients were divided into sepsis group and non-sepsis group.According to the occurrence of SAAKI,patients were divided into control group(patients without sepsis and without AKI),sepsis group,AKI group and SA-AKI group.According to the occurrence of death,patients were divided into death group and survival group.(5)Analysis:Single-center study was conducted to compare the levels of serum sST2 at different time points.The association of serum sST2 with AKI,sepsis and death was analyzed.Multi-center study was conducted to further explore and verify the association of serum sST2 level on the first day of PICU admission with AKI,SA-AKI and prognosis.The association of serum sST2 with AKI,sepsis and death was analyzed by multivariate Logistic regression.Receiver operating characteristic curve(ROC)and area under the curve(AUC)were used to evaluate the discriminative value of serum sST2 for AKI and prognosis in critically ill children.Results:1.Single-center clinical study(1)Of the 159 patients,29(18.2%)developed AKI,67(42.1%)developed sepsis and 27(17.0%)died.Multivariable linear regression analysis showed that serum sST2 was associated with AKI,sepsis,and death.(2)Compared with the non-AKI group,the level of serum sST2 in the AKI group was significantly higher.Logistic regression analysis showed that after adjusting for other influencing factors,serum sST2 was still associated with AKI.There was no statistical difference in AUC values between different time points of sST2 for discriminating AKI.The first day serum sST2 achieved the highest AUC(0.812)for discriminating AKI in critically ill children.(3)The level of serum sST2 in sepsis group was significantly higher than that in nonsepsis group.Logistic regression analysis showed that serum sST2 was associated with sepsis after adjusting for other influencing factors.The efficacy of sST2 for discriminating sepsis was low.The first day serum sST2 achieved the AUC of 0.691 for discriminating sepsis in critically ill children.2.Multicenter clinical study(1)Of the 852 patients,90(10.6%)developed AKI,131(15.2%)developed sepsis,34(4.0%)developed SA-AKI and 71(8.3%)died.Multiple linear regression analysis showed that serum sST2 was associated with body weight,PRISM III,sepsis and AKI after adjusting for other factors.(2)The level of serum sST2 was higher in mild or severe AKI group than that in nonAKI group.Logistic regression analysis showed a significant association between serum sST2 and AKI after adjustment for other factors.Serum sST2 achieved an AUC of 0.811(P<0.001)and displayed a sensitivity of 82.2%and a specificity of 67.8%for discriminating AKI at the optimal cut-off value of 111.0 ng/mL.(3)The level of serum sST2 in sepsis group was significantly higher than that in nonsepsis group.Logistic regression analysis showed that there was still a significant association between serum sST2 and sepsis after adjusting for other influencing factors.Serum sST2 achieved an AUC of 0.733(P<0.001)and displayed a sensitivity of 63.4%and a specificity of 72.2%for discriminating sepsis at the optimal cut-off value of 142.7 ng/mL.(4)The level of serum sST2 in SA-AKI group were significantly higher than that in the other three groups(control group,sepsis group,AKI group).Multivariate Logistic regression analysis showed that after adjusting for other factors,serum sST2 was still significantly associated with SA-AKI.Serum sST2 achieved an AUC of 0.885(P<0.001)and displayed a sensitivity of 79.4%and a specificity of 82.9%for discriminating SA-AKI at the optimal cut-off value of 258.4 ng/mL.(5)The level of serum sST2 in death group was significantly higher than that in survival group.Binary regression analysis showed serum sST2 was associated with death,while there was no significant association between serum sST2 and death after adjusting for other factors.Conclusion:Serum sST2 is significantly associated with AKI and SA-AKI in critically ill children after adjusting for body weight and disease severity.Serum sST2 has a discriminative ability for AKI,it has a better discriminative ability for SA-AKI in critically ill children.The association of urinary TIMP-2 and IGFBP-7 with acute kidney injury in critically ill neonatesObjective:Investigate the association of urinary TIMP-2,IGFBP-7 and[TIMP2]·[IGFBP-7]with AKI or severe AKI;Analysis the value of these biomarkers for the diagnosis of AKI or severe AKI in critically ill neonates.Method:(1)Study Population:Critically ill neonates admitted to the neonatal intensive care unit(NICU)of Children’s Hospital affiliated to Soochow University from July to October 2016.(2)Clinical Data Collection:Neonatal clinical information,including the gestational age,birth weight,gender and Apgar scores were collected.Maternal information during pregnancy was also recorded,such as pregnancy complications,mode of delivery,medication applied.Clinical laboratory information,including serum creatinine(sCr)and blood urea nitrogen were recorded for each neonate.The clinical diagnosis,the use of mechanical ventilation(MV),antibiotics,steroids,caffeine,vasoactive drugs and surfactants during hospitalization and the NICU mortality were recorded.(3)Urine sample collection and biochemical analyses:After the NICU admission,the urine samples of the neonates were collected every 48(24-72)hours and immediately stored at-80℃.Urine TIMP-2 and IGFBP-7 levels were detected by ELISA.(4)Diagnosis of neonatal AKI:A diagnosis can be made if any of the following conditions are met:① The Neonatal Kidney Disease:Improving Global Outcomes(KDIGO):serum creatine(sCr)rise≥0.3 mg/dL within 48 hours or sCr rise≥1.5× reference sCr within 7 days[66].The reference sCr was defined as the lowest previous sCr value;②sCr>1.2 mg/dL[67].(5)Grouping:According to the severity of AKI,patients were divided into non-AKI group,mild AKI group and severe AKI group.KDIGO stage 1 was defined as mild AKI,KDIGO stages 2 and 3 were defined as severe AKI.(6)Statistical analysis:For patients without AKI,the first values of urinary TIMP-2,IGFBP-7 and[TIMP-2]·[IGFBP-7]were chosen for statistical analysis.For patients in AKI group,the largest values of urinary TIMP-2,IGFBP-7,or[TIMP-2]·[IGFBP-7]before the occurrence of AKI were used for analysis.Linear regression was used to analyze the correlation between biomarkers with clinical indicators.The association of biomarkers with AKI or severe AKI was analyzed by multivariate Logistic regression.ROC and AUC were used to evaluate the discriminative value of these biomarkers for AKI or severe AKI in critically ill neonates.Results:(1)Of the 237 neonates,20(8.4%)developed AKI,including 9(3.8%)patients with severe AKI.The level of urinary IGFBP-7 was higher in mild or severe AKI group than in non-AKI group.The level of urinary[TIMP-2]·[IGFBP-7]was higher in severe AKI group than in mild or non-AKI group.(2)Multivariate logistic analysis showed that urine[TIMP-2]·[IGFBP-7]was significantly associated with severe AKI.Urine[TIMP-2]·[IGFBP-7]achieved an AUC of 0.71(P=0.034)and displayed a sensitivity of 88.9%and a specificity of 50.9%for discriminating severe AKI at the optimal cut-off value of 0.045(ng/mL)2/1000.Conclusion:The combination of urinary TIMP-2 and IGFBP-7 has a discriminative ability for severe AKI in critically ill neonates.Part Ⅱ Preliminary study on the expression and mechanism of IL-33/ST2 in ischemia-reperfusion acute kidney injury in ratsObjective:Investigate the expression and the role of IL-33/ST2,ferroptosis in renal ischemia-reperfusion injury in rats.Methods:(1)Establish the model of renal IRI in rats:Sprague-Dawley(SD)rats aged 30 days were randomly divided into three groups:Control group,Sham group and IRI group.Rats in IRI group were reperfusion after 45 minutes of ischemia,and serum and kidney tissues were collected at different time points of reperfusion(IRI 1h,IRI 3,IRI 6h and IRI 24h).Serum creatinine(sCr)concentrations and histopathological changes of kidney tissues were detected.(2)Expression of cytokines during IRI:The expression of cytokines related to AKI in rat serum were detected by Liqui Chip technique.(3)Clarify the expression of IL-33 and ST2 in renal ischemia-reperfusion injury:Western blot was used to detect the expression of IL-33 and ST2 in the kidney tissues at each time point.The concentrations of IL-33 and sST2 in the serum were detected by ELISA.(4)Verify the expression of ferroptosis and lipid peroxidation in renal during IRI:The expression of long-chain acyl-CoA synthetase 4(ACSL4),Glutathione peroxidases 4(Gpx4)and 4-hydroxy-nonanoic acid(4HNE)in renal tissues at each time point were detected by Western blot.The morphology of ferroptosis in kidney was observed by electron microscopy.The levels of LPO and ATP in renal tissue were detected by LPO and ATP detection kit.(5)Observe the effects of ferroptosis inducers or inhibitors on IRI renal injury:Rats were pretreated with ferroptosis inducer(1S,3R)-RSL3(RSL3)or ferroptosis inhibitor Nacetyl-l-cysteine(NAC).The IRI 24h model was established 24 hours later.The histopathological changes of kidney tissues and ferroptosis biomarkers in rats were assessed.(6)Verify the effects of ferroptosis inducers or inhibitors on IL-33 and ST2 during IRI:The rats were pretreated with ferroptosis inducer RSL3 or ferroptosis inhibitor NAC respectively.The IRI 24h model was established 24 hours later.The expression of IL-33 and ST2 in renal tissue was detected by Western blot.The levels of IL-33 and sST2 in rat serum were detected by ELISA.(7)Investigate the effect of ferroptosis inducer RSL3 or inhibitors NAC on NF-κB protein expression in kidney:The rats were pretreated with ferroptosis inducer RSL3 or ferroptosis inhibitor NAC respectively.The IRI 24h model was established 24 hours later.The expression of phosphorylated NF-κB protein in kidney was observed.Results:(1)The rat renal ischemia-reperfusion model was established successfully.It was found that serum creatinine(sCr)increased at IRI 1h and reached the peak at IRI 24h.Compared with control group,the epithelial cells of the renal tubules became oedema and brush margin disappeared in IRI 24h group.(2)Dynamic changes of serum inflammatory factors:It was found that the levels of IL33,CXCL5,IL-12 and IL-5 in serum of rats were higher in IRI 3h group than those in control group.(3)Dynamic expressions of IL-33 and ST2:At 1h of IRI,the expression of full-length IL-33 in renal tissue increased.At 24h of IRI,the expression of cleaved IL-33 in renal tissue increased.There was no significant change in the expression of renal ST2L protein at different time points of IRI.The levels of serum IL-33 and sST2 increased at IRI 24h.(4)Dynamic changes of ferroptosis related biomarkers:The expression of 4HNE and LPO in renal tissue increased at 3h of IRI.Gpx4 expression in renal tissue decreased at IRI 6h.ACSL4 expression in renal tissue increased at IRI 24h.The mitochondria of renal tubule epithelial cells contracted and their density deepened at IRI 24h.(5)Compared with the control group,the expression of LPO and ACSL4 in renal tissue increased at IRI 24h after RSL3 pretreatment,while decreased after NAC pretreatment.HE staining showed that renal tubule epithelial cell edema increased,nuclear exfoliation became more obvious in RSL3 pretreatment group.In NAC pretreatment group,the edema of renal tubule epithelial cells reduced.(6)The expression of full-length IL-33,cleaved IL-33 and ST2L protein in renal tissue increased at IRI 24h after RSL3 pretreatment.The serum concentrations of IL-33 and sST2 in rats were significantly higher in RSL3 pretreatment group than those in control group.After NAC pretreatment,the expression of cleaved IL-33 and ST2L protein in renal tissue decreased,while the expression of full-length IL-33 increased at IRI 24h.The serum concentrations of IL-33 and sST2 in rats were significantly lower in NAC pretreatment group than those in control group.(7)The expression of phosphorylated NF-κB increased in RSL3 pretreatment group at IRI 24h,while decreased in NAC pretreatment group.Conclusion:In rats with renal ischemia-reperfusion injury,ferroptosis occurred in renal tubular epithelial cells,and the expression of IL-3 3 and NF-κB increased in renal tissues,as well as the levels of IL-33 and sST2 in serum.Ferroptosis inducer can up-regulate the expression of IL-33,ST2 and NF-κB,resulting in the aggravation of renal tissue injury.