P66shc-mediated Mitochondrial Dynamic Alterations Contribute To Tubular Oxidative Injury In Human Diabetic Nephropathy

Background:Diabetic nephropathy (DN) is a common etiology of secondary chronic kidney disease, and a main cause of ESRD. And cellular oxidative injury and apoptosis in renal tubules is one of the early pathological characteristics of DN. Recent evidence suggest that mitochondrial dysfunction, especially mitochondrial dynamic alterations and the overproduction of mitochondrial reactive oxygen species (ROS), play central roles in the pathogenesis of diabetic complications, including DN, but the relations between them is unclear. Adaptor protein p66Shc is known as a master regulator of cellular oxidative stress and apoptosis, as well as the signalling pathways of cell cycle. Under hyperglycemia stimulation, p66Shc is activated and translocated to mitochondria, which induce mitochondrial ROS overproduction and lead to tubular cell oxidative stress and apoptosis. Whereas under diabetic ambience, the evidence of mitochondrial fragmentation in patients with DN is still lacking, the causal relations between mitochondrial dynamics and mitochondrial ROS production under hyperglycemia condition is undefined, and whether p66shc is the link between them that results in hyperglycemia-induced tubular cell oxidative injury and death need to be clarified. Therefore, the aim of this study is to elucinate these problems and underlying mechanisms. Objective:In this study, we investigate the role of mitochondrial dynamic changes and ROS overproduction, as well as the expression of the adaptor protein p66shc and mitochndrial shaping proteins in the renal specimen of DN patients and control non-DN group, to explore the cause and mechanism of tubular injury in diabetic state. In vitro, we investigate the causal relations between mitochondrial dynamics and ROS production under hyperglycemia condition and analyze whether p66shc is the link between these two mitochondrial dysfunctions that results in hyperglycemia-induced tubular cell oxidative injury and death.Methods:Renal biopsy specimens from10patients with DN and10with minimal change disease as control, as well as human tubular epithelial cells (HK-2) were studied. Renal pathological lesions were evaluated with HE, PAS and PASM staining. Mitochondrial dynamics was measured by electron microscopy in vivo, and by confocal microscopy in vitro with Mito Red staining in live cells. The expression of mitochondrial pro-fission protein Drpl, pro-fusion protein Mfn1and p66shc were evaluated by immunohistochemistry, immunoflurescence and western blot assays. Cellular oxidative stress and mitochondrial ROS levels was measured by DHE and Mito Sox staining, respectively. The pharmacological Drpl inhibitor mdivi-1was used to analyze the causal relations between mitochondrial dynamics and ROS production under diabetic ambience. In addition, wide type-p66shc or siRNA plasmid was transfected in vitro to elucidate the effect of p66shc on Drp1expression, mitochondrial dynamics and ROS production.Results:In patients with DN, renal tubular sclerosis and atrophy was shown in renal biopsy specimen, besides glomerular lesions. Compared with the non-DN control group, filamentous mitochondria became fragmented mainly in diabetic renal tubules, whereas Drp1was significantly increased and Mfnl decreased. These changes were accompanied by the elevation of p66shc expression and cellular ROS overproduction. In HK-2cells, the percentage of cells with mitochondrial fragmentation and Drp1expression were both significantly elevated under high-glucose treatment and in a time-and dose-dependent manner. However, the pharmacological Drp1inhibitor mdivi-1, attenuated mitochondrial fragmentation and ROS levels. Notably, siRNA knockdown of p66shc down-regulated hyperglycemia-induced Drpl over-expression, alleviated mitochondrial fragmentation and ROS production. However, over-expression of p66shc under low glucose ambience up-regulated Drp1expression, induced mitochondrial fragmentation and ROS overproduction, whereas adding mdivi-1in the above condition could reverse these changes.Conclusions:This study provides first evidence of mitochondrial fragmentation in human diabetic nephropathy, mitochondrial dynamic alterations is mediated by p66shc through its interaction with Drp1, which may serve as an upstream trigger of mitochondrial ROS overproduction and results in tubular oxidative injury under hyperglycemia conditions. Our findings provide insights into a novel mechanism contributing to mitochondrial dysfunction in the pathogenesis of DN.