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Modeling Hypoxia/Reoxygenation Injury in Proximal Tubular Epithelial Cells
Published on: November 21, 2025
p66SHC-mediated mitochondrial dysfunction in renal proximal tubule cells during oxidative injury
Istvan Arany1, Amir Faisal, Jeb S Clark
1Dept. of Pediatrics, Div. of Pediatric Nephrology, Univ. of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39126, USA. iarany@ped.umsmed.edu
Abstract:
Mitochondrial dysfunction is involved in pathopysiology of ischemia-reperfusion-induced acute kidney injury (AKI). The p66shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction, which might play a role in AKI-induced renal tubular injury. Oxidative stress-mediated Serine36 phosphorylation of p66shc facilitates its transportation to the mitochondria where it oxidizes cytochrome c and generates excessive amount of reactive oxygen species (ROS). The consequence is mitochondrial depolarization and injury. Earlier we determined that p66shc plays an essential role in injury of cultured mouse renal proximal tubule cells during oxidative stress. Here, we studied the role of p66shc in ROS generation and consequent mitochondrial dysfunction during oxidative injury in renal proximal tubule cells. We employed p66shc knockdown renal proximal tubule cells and cells that overexpress wild-type, Serine phosphorylation (S36A), or cytochrome c-binding (W134F) mutants of p66shc. Inhibition of the mitochondrial electron transport chain or the mitochondrial permeability transition revealed that hydrogen peroxide-induced injury is mitochondrial ROS and consequent mitochondrial depolarization dependent. We also found that through Ser36 phosphorylation and mitochondria/cytochrome c binding, p66shc mediates those effects. We propose a similar mechanism in vivo as we demonstrated mitochondrial binding of p66shc as well as its association with cytochrome c in the postischemic kidneys of mice. Thus, manipulating p66shc might offer a new therapeutic modality to ameliorate renal ischemic injury.
Insights
The p66shc protein mediates kidney injury by causing mitochondrial dysfunction and excessive reactive oxygen species (ROS) production during oxidative stress. Targeting p66shc may offer new therapies for acute kidney injury (AKI).
Area of Science:
- Cellular Biology
- Renal Physiology
- Mitochondrial Medicine
Background:
- Mitochondrial dysfunction contributes to acute kidney injury (AKI) pathophysiology.
- The p66shc adaptor protein is implicated in mitochondrial dysfunction and potential renal tubular injury in AKI.
- Oxidative stress-induced phosphorylation of p66shc at Serine36 promotes its mitochondrial translocation, leading to cytochrome c oxidation, excessive reactive oxygen species (ROS) generation, and mitochondrial depolarization.
Purpose of the Study:
- To investigate the role of p66shc in ROS generation and mitochondrial dysfunction during oxidative injury in renal proximal tubule cells.
- To elucidate the mechanism by which p66shc mediates oxidative damage in kidney cells.
- To explore the therapeutic potential of targeting p66shc in renal ischemic injury.
Main Methods:
- Utilized p66shc knockdown renal proximal tubule cells.
- Employed cell lines overexpressing wild-type, Serine36 phosphorylation-deficient (S36A), or cytochrome c-binding deficient (W134F) p66shc mutants.
- Assessed hydrogen peroxide-induced injury, mitochondrial ROS production, and mitochondrial depolarization.
- Investigated p66shc binding to mitochondria and cytochrome c in vitro and in postischemic mouse kidneys.
Main Results:
- Hydrogen peroxide-induced renal proximal tubule cell injury is dependent on mitochondrial ROS generation and subsequent mitochondrial depolarization.
- p66shc mediates this oxidative injury through Serine36 phosphorylation and binding to mitochondria and cytochrome c.
- p66shc was found to bind to mitochondria and associate with cytochrome c in the kidneys of mice following ischemia.
Conclusions:
- p66shc plays a critical role in mediating oxidative stress-induced renal proximal tubule cell injury via ROS generation and mitochondrial dysfunction.
- The phosphorylation status at Serine36 and interaction with cytochrome c are crucial for p66shc's detrimental effects.
- Targeting p66shc represents a potential therapeutic strategy to mitigate renal ischemic injury.
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