SLC39A8介导的铁稳态对糖尿病肾小管上皮细胞铁死亡的作用及机制研究

SLC39A8 mediates iron homeostasis and regulates the ferroptosis of renal tubular epithelial cells in diabetic kidney disease

目的探索SLC39A8介导的肾脏铁稳态在糖尿病肾病肾小管上皮细胞铁死亡中的作用及可能机制。方法将16只db/db小鼠随机分为db/db组、Fe-db/db组,每组8只。另取8只db/m小鼠作为对照组。饲料铁含量不同,对照组,db/db组的铁含量约为33 ppm、Fe-db/db组的铁含量约为200 ppm。采用相关试剂盒检测尿白蛋白/肌酐比值、空腹血糖,采用生化分析仪检测肾功能(血清肌酐及尿素氮)。共聚焦免疫荧光检测肾组织SLC39A8、GPX4蛋白表达。Western blot检测SLC39A8、铁死亡相关蛋白GPX4、GSH、SLC7A11、ACSL4及TGF-β/Smad通路关键蛋白Smad3、Smad4、p-Smad3及p-Smad4蛋白表达。结果与对照组及db/db组小鼠相比,Fe-db/db组小鼠的肾功能明显降低,蛋白尿明显增加,肾组织铁水平升高。免疫荧光共聚焦及免疫电镜检测显示SLC39A8在db/db小鼠近端肾小管上皮细胞高表达,亚细胞定位于线粒体。与对照组及db/db组相比,Fe-db/db小鼠的SLC39A8蛋白表达增高、铁死亡相关因子GPX4、GSH、SLC7A11的蛋白表达降低,而ACSL4表达升高,Smad3和Smad4的磷酸化表达明显升高。结论SLC39A8介导的肾小管上皮细胞铁超载参与DKD肾小管上皮细胞的铁死亡,触发TGF-β/Smad通路活化可能是其重要机制之一。

Objective To explore the role of SLC39A8 in iron homeostasis and renal tubular epithelial cell ferroptosis in diabetic kidney disease(DKD)and the underlying mechanisms.Methods Sixteen db/db mice were randomly divided into db/db group and Fe-db/db group with 8 mice in each group.Another 8 db/m mice were used as control group.The iron content of the control group was about 33 ppm in the db/db group and about 200 ppm in the Fe-db/db group.Urinary albumin/creatinine ratio and fasting blood glucose were detected by relevant kits,and renal function(serum creatinine and urea nitrogen)was detected by biochemical analyzer.The protein expression of SLC39A8 and GPX4 in renal tissue was detected by confocal immunofluorescence.The protein expression of SLC39A8,ferroptosis-associated protein GPX4,GSH,SLC7A11,ACSL4 and TGF-β/Smad pathway key proteins Smad3,Smad4,p-Smad3 and p-Smad4 were detected by Western blot.Results Compared with the control group and db/db group,the renal function of Fe-db/db group was significantly decreased,the proteinuria was significantly increased,and the iron level in renal tissue was increased.Immunofluorescence confocal microscopy and immunoelectron microscopy showed that SLC39A8 was highly expressed in the renal proximal tubular epithelial cells of db/db mice,and its subcellular localization was in the mitochondria.Compared with the control group and db/db group,the protein expression of SLC39A8 was increased,the protein expression of ferroptosis-related factors GPX4,GSH and SLC7A11 was decreased,the protein expression of ACSL4 was increased,and the phosphorylation expression of Smad3 and Smad4 was significantly increased in Fe-db/db mice.Conclusion SLC39A8-mediated iron overload in renal tubular epithelial cells is involved in the ferroptosis of renal tubular epithelial cells in DKD,and the activation of TGF-β/Smad pathway may be one of the important mechanisms.