Supplementary Material for: Role of p66shc in Renal Toxicity of Oleic Acid

Background/Aims: Adult and childhood obesity is an independent risk factor in development of chronic kidney disease (CKD) and its progression to end-stage kidney disease. Pathologic consequences of obesity include non-esterified fatty acid-induced oxidative stress and consequent injury. Since the serine36-phosphorylated p66shc is a newly recognized mediator of oxidative stress and kidney injury, we studied its role in oleic acid (OA)-induced production of reactive oxygen species (ROS), mitochondrial depolarization and injury in cultured renal proximal tubule cells. Methods: Renal proximal tubule cells were used and treated with OA: ROS production, mitochondrial depolarization as well as injury were determined. Transcriptional effects of OA on the p66shc gene were determined in a reporter luciferase assay. The role of p66shc in adverse effects of OA was determined using knockdown, p66shc serine36 phosphorylation and cytochrome c binding-deficient cells. Results: We found that OA increased ROS production via the mitochondria - and to a less extent via the NADPH oxidase - resulting in ROS-dependent mitochondrial depolarization and consequent injury. Interestingly, OA also stimulated the promoter of p66shc. Hence, knockdown of p66shc, impairment its Ser36 phosphorylation (mutation of Ser36 residue to alanine) or cytochrome c binding (W134F mutation) significantly attenuated OA-dependent lipotoxicity. Conclusion: These results offer a novel mechanism by which obesity may lead to renal tubular injury and consequently development of CKD. Manipulation of this pathway may offer therapeutic means to ameliorate obesity-dependent renal lipotoxicity.

研究背景与目的：成人与儿童肥胖是慢性肾脏病（CKD）及其进展至终末期肾病的独立危险因素。肥胖的病理结局包括非酯化脂肪酸诱导的氧化应激及继发性损伤。鉴于丝氨酸36位点磷酸化的p66shc蛋白是新近被证实的氧化应激与肾损伤介质，本研究探讨其在油酸（OA）诱导的培养肾近端小管细胞活性氧（ROS）生成、线粒体膜电位去极化及细胞损伤中的作用。研究方法：采用肾近端小管细胞，以油酸进行干预；检测活性氧生成、线粒体膜电位去极化及细胞损伤情况。采用荧光素酶报告基因实验检测油酸对p66shc基因的转录调控效应。通过构建p66shc敲低细胞、丝氨酸36磷酸化缺陷（将Ser36残基突变为丙氨酸）及细胞色素c结合缺陷（W134F突变）的细胞模型，探究p66shc在油酸所致不良效应中的作用。研究结果：油酸可通过线粒体途径（次要途径为NADPH氧化酶途径）促进活性氧生成，进而引发活性氧依赖性的线粒体膜电位去极化及细胞损伤。值得注意的是，油酸还可激活p66shc基因的启动子。进一步实验显示，敲低p66shc、阻断其Ser36磷酸化位点或破坏其细胞色素c结合能力，均可显著减轻油酸介导的脂毒性。研究结论：本研究揭示了肥胖导致肾小管损伤进而引发慢性肾脏病的全新机制。靶向调控该通路或可为改善肥胖相关性肾脂毒性提供全新治疗策略。