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

<b><i>Background/Aims:</i></b> 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. <b><i>Methods:</i></b> 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 <i>c </i>binding-deficient cells. <b><i>Results:</i></b> 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 <i>c</i> binding (W134F mutation) significantly attenuated OA-dependent lipotoxicity. <b><i>Conclusion:</i></b> 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