Abstract: PUB024
PRDX6 Suppresses Ferroptosis in Renal Tubular Epithelial Cells
Session Information
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Conner, Justin Lawrence, University of California Berkeley, Berkeley, California, United States
- Vazquez-Medina, Jose P., University of California Berkeley, Berkeley, California, United States
Background
Peroxiredoxin 6 (PRDX6) contributes to cell membrane repair through its peroxidase and phospholipase activities. PRDX6 can also function as a selenium carrier, influencing GPX4 expression, the primary enzyme responsible for reducing oxidized phospholipids. PRDX6 expression increases following acute kidney injury. We studied the role of PRDX6 in handling renal lipid peroxidation using Renal Tubular Epithelial Cells (RTECs) derived from WT and PRDX6-knock-in mice carrying a single-point mutation at the PRDX6 residue required for binding oxidized phospholipids (PRDX6-H26A). We hypothesize that the PRDX6-H26A mutation sensitizes RPTCs to ferroptosis, a novel form of regulated cell death characterized by the accumulation of phospholipid hydroperoxides.
Methods
Animals
Age-matched male WT, PRDX6 KO (MMRRC_043402-JAX), and PRDX6-H26A KI (RRID:MMRRC_042293-UNC) mice, 8-10-week-old, were obtained from the Mutant Mouse Resource & Research Centers, bred and maintained in our facilities.
Tissue analyses
Renal PRDX6 and GPX4 levels were measured by Western blot. Histology was evaluated by H&E and PAS staining. Kidney ultrastructure was evaluated by transmission electron microscopy.
Cell models
Primary RTECs were isolated as previously described. PRDX6 expression was confirmed by co-immunofluorescence with EpCAM.
Induction and inhibition of ferroptosis
RTECs were treated with erastin (0.1-10 µM), which induces ferroptosis by blocking the cystine-glutamate antiporter, resulting in intracellular glutathione depletion. Ferroptosis was blocked by co-treatment with Ferrostatin-1 1.5µM , a potent radical-trapping antioxidant.
Lipid peroxidation and cell death
Lipid peroxidation was measured with the lipid peroxide-sensitive probe Liperfluo. Cell death was evaluated using bis-AAF-R11, a fluorogenic peptide substrate.
Results
GPx4 levels were comparable in the kidneys of WT and PRDX6 KO mice, which did not exhibit histological or ultrastructural alterations. PRDX6 was highly abundant in RTECs, where it co-localized with EpCAM. Both lipid peroxidation and cell death were significantly higher in RTECs derived from PRDX6-H26A than from WT mice.
Conclusion
Our results show that PRDX6 is necessary to suppress ferroptosis in RTECs. Furthermore, our results also show that PRDX6 deletion does not impact GPX4 levels in the kidney. Hence, PRDX6 likely exerts direct cytoprotective effects in the kidney by efficiently handling lipid peroxidation.
Funding
- NIDDK Support