叉头盒K1缺失减轻缺血再灌注诱导的急性肾损伤

Forkhead box K1 deficiency alleviates ischemia-reperfusion-induced acute kidney injury

目的探讨叉头盒K1(forkhead box K1,FOXK1)在急性肾损伤(acute kidney injury,AKI)中的作用及机制,以期为AKI的防治提供新的思路和靶点。方法构建3种AKI模型:按随机数字表法将30只雄性8~10周龄22~24 g无特定病原体野生型C57BL/6小鼠随机分为生理盐水组(0.9%氯化钠水溶液0.1 ml/10 g,腹腔注射)、脂多糖组(脂多糖溶液10 mg/kg,腹腔注射)、顺铂组(顺铂溶液20 mg/kg,腹腔注射)、缺血再灌注(ischemia reperfusion,IR)组及假手术组,每组6只,各组小鼠均于造模24 h后处死取材。肾功能情况通过检测血肌酐、血尿素氮水平;HE染色法观察各组肾组织病理学改变;Western印迹法检测肾组织FOXK1、肾损伤分子1(kidney injury molecule 1,KIM1)以及自噬标志物p62、Beclin1和LC3蛋白表达水平,实时荧光定量PCR法检测Foxk1 mRNA表达水平。予以人肾小管上皮细胞(HK2细胞)缺氧24 h、复氧6 h构建缺氧复氧(hypoxia reoxygenation,HR)诱导AKI体外模型,检测HK2细胞上述指标表达水平。同时,在体内外构建Foxk1基因缺失的肾小管上皮细胞小鼠或HK2细胞,并诱导建立AKI模型,观察上述各指标表达变化。结果与生理盐水组相比,脂多糖组及顺铂组肾组织血肌酐、血尿素氮均较高,KIM1蛋白表达较高(均P<0.05),FOXK1蛋白和mRNA表达无明显改变(均P>0.05);与假手术组相比,IR组肾组织血肌酐、血尿素氮均较高,KIM1蛋白表达较高,FOXK1蛋白和mRNA表达均较低(均P<0.05)。与对照组比较,体外HR诱导的AKI细胞模型中FOXK1蛋白和mRNA表达均较低(均P<0.05)。体内实验中,与假手术组相比,IR组肾小管损伤较重,血肌酐、血尿素氮均较高,p62蛋白表达较低,KIM1、Beclin1和LC3蛋白表达均较高(均P<0.05),且与Foxk1flox/flox IR组相比,Foxk1cKO IR组肾小管损伤明显较轻,血肌酐、血尿素氮均较低,KIM1、p62蛋白表达均较低,Beclin1和LC3蛋白表达均较高(均P<0.05)。体外实验中,与shCtrl HR组比较,shFoxk1 HR组KIM1、p62蛋白表达均较低,Beclin1、LC3蛋白均较高(均P<0.05)。结论FOXK1在缺血性AKI模型中表达降低。Foxk1基因缺失通过介导自噬激活,减轻肾小管上皮细胞损伤,保护缺血性AKI。

Objective To investigate the effect and mechanism of forkhead box K1(FOXK1)in acute kidney injury(AKI),and to provide new ideas and targets for preventing and treating AKI.Methods Three models of AKI were established:30 male specific pathogen free wild type C57BL/6 mice aged 8-10 weeks and weighting 22-24 g were randomly divided into saline group(0.9%normal saline 0.1 ml/10 g,intraperitoneal injection),lipopolysaccharide(LPS)group(LPS solution 10 mg/kg,intraperitoneal injection),cisplatin group(cisplatin solution 20 mg/kg,intraperitoneal injection),ischemia-reperfusion(IR)group,and sham-operated group by the random number table,with 6 mice in each group.The mice in each group were sacrificed 24 hours after modeling to obtain experimental materials.The serum creatinine(Scr)and blood urea nitrogen(BUN)were tested to measure the renal function.HE staining was performed to observe histopathological changes of renal tissues.Western blotting was used to detect the protein expression of FOXK1,kidney injury molecule 1(KIM-1),autophagy markers p62,Beclin1 and LC3 in renal tissues.Quantitative real-time PCR was used to detect the mRNA expression of Foxk1.Human renal tubular epithelial cells(HK‐2 cells)were exposed to hypoxia for 24 h,followed by reoxygenation for 6 h to establish an in vitro AKI model induced by hypoxia reoxygenation(HR).The expression changes of the above indicators in HK‐2 cells were detected.Then,Foxk1 gene deletion in renal tubular epithelial cells was performed in vivo and in vitro,and AKI models were induced to observe the expression changes of the above indicators.Results Compared with the saline group,Scr,BUN and the protein expression level of KIM-1 were higher in LPS group and cisplatin group(all P<0.05),while FOXK1 protein and mRNA expression had no significant change(both P>0.05).Compared with the sham-operated group,Scr,BUN and the protein expression level of KIM-1 were higher,and the expression levels of FOXK1 protein and mRNA were significantly lower in the IR group(all P<0.05).FOXK1 protein and mRNA expression levels in the HR-induced AKI cell model group were lower than those in the control group(both P<0.05).In the in vivo experiments,compared with the sham-operation group,the renal tubular injury was more aggravated,Scr and BUN were higher,p62 protein expression was lower,and the protein expression levels of KIM-1,Beclin1 and LC3 were higher in the IR group(all P<0.05).Compared with Foxk1flox/flox IR goup,renal tubular injury was more alleviated,Scr,BUN and the protein expression levels of KIM-1 and p62 were lower,while the protein expression levels of Beclin1 and LC3 were higher in Foxk1cKO IR group(all P<0.05).Compared with shCtrl HR group,shFoxk1 HR group had lower protein expression levels of KIM‐1 and p62 and higher expression levels of Beclin1 and LC3 in vitro(all P<0.05).Conclusions The expression of FOXK1 is decreased in ischemic AKI model.Foxk1 deficiency alleviates renal tubular epithelial cell injury and protects against ischemic AKI through activating autophagy.