Role of p66shc in renal toxicity of oleic acid

Abstract

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.

Fig.1. Oleic acid dose-dependently increases cell injury and intracellular ROS production source of which is the NAPDH oxidase/mitochondria in RPTCs

RPTCs were treated with varying concentration of oleic acid as indicated. LDH release (A) and ROS production (B) were determined as described in Materials and Methods. N=3, *p<0.05 compared to untreated cells (C) RPTCs were pretreated with 100 µM allopurinol (Allo), 5 µM diphelinediodium (DPI), 10 µM Antimycin A (AntA) or 10 µM Rotenone (Rot) 1 hour prior to treatment with 100 µM OA. ROS production was determined as described in Materials and Methods. N=3, *p<0.05 compared to OA treatment

Fig.2. Oleic acid-induced ROS depolarizes the mitochondria, which is responsible for cell injury

(A) RPTCs were treated with 100 µM OA in the presence or absence of 100 µM NAC and mitochondrial depolarization was determined as described in Materials and Methods. N=3, *p<0.05 compared to untreated cells (B) RPTCs were pretreated with 100 µM NAC or 5 µM CsA 1 hour prior to treatment with 100 µM OA and LDH release was determined 24 hours later. N=3, *p<0.05 compared to OA-treated cells

Fig.3. Oleic acid increases expression of p66shc via stimulation of its promoter

(A) RPTCs were treated with 100 µM OA for 24 hours and expression of p66shc was determined by Western blotting. As internal control, blots were rehybridized with an anti-actin antibody. Image shown is representative of three independent experiments. (B) Densitometry of results shown in A. N=3, *p<0.05 compared to untreated (C) RPTCs were transfected with a p66shc promoter-luciferase plasmid together with a renilla plasmid as described in Materials and Methods. After 24 hours with treatment with 100 µM OA, luciferase activities were determined. Values are calculated as p66shc-Luc/renilla ratios and expressed as % of the untreated value. N=3, *p<0.05 compared to untreated

Fig.4. Adverse effects of OA require Ser36 phosphorylation and cytochrome c binding of p66shc

(A) Wild type (w.t.) or p66shc knockdown (k.d.) RPTCs or cells that were transfected with a phosphorylation deficient (S36A), a corresponding phosphomimetic mutant (S36D) or a cytochrome c binding deficient (W134F) p66shc plasmid were treated with 100 µM OA and ROS production was determined. Values are expressed as % of control (untreated) cells. N=3, *p<0.05 compared to treated w.t. values (B) Mitochondrial depolarization was determined in cells treated similar to (A). Values are expressed as % of control (untreated) cells. N=3, *p<0.05 compared to treated w.t. values (C) LDH release was determined in cells treated similar to (A). Values are expressed as % of total LDH content. N=3, *p<0.05 compared to w.t. values

Fig.5. Proposed role of p66shc in oleic acid-dependent renal toxicity

OA stimulates transcription of p66shc leading to increased expression of the p66shc protein, which in turn, is Ser36 phosphorylated by ROS generated through NADPH oxidase- or other non-p66shc-related pathways. The Ser36 phosphorylated p66shc is translocated into the mitochondria, where –after dephosphorylation- binds cytochrome c. The result is increased mitochondrial ROS production and consequently, mitochondrial depolarization as well as injury.