Abstract: SA-PO0156
Pax8 Modulates Sensitivity to Ferroptosis and Metabolic Flux in Proximal Tubule Cells
Session Information
- AKI: Mechanisms - 3
November 08, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Stahlecker, Jenna, University of Michigan, Ann Arbor, Michigan, United States
- Orban, Steven, University of Michigan, Ann Arbor, Michigan, United States
- Telang, Asha Claire, University of Michigan, Ann Arbor, Michigan, United States
- Dressler, Greg R., University of Michigan, Ann Arbor, Michigan, United States
- Snyder, Ryan J., National Institute of Environmental Health Sciences, Durham, North Carolina, United States
- Watts, Jason A., National Institute of Environmental Health Sciences, Durham, North Carolina, United States
- Beamish, Jeffrey A., University of Michigan, Ann Arbor, Michigan, United States
Background
Proximal tubule cells use a unique oxidative metabolism that fuels solute transport but that also renders them vulnerable to ferroptotic stress. The transcription factor Pax8 is upregulated in developing proximal tubules as their metabolic preferences are being established. We hypothesized that Pax8 promotes gene expression needed for proximal tubule metabolism and contributes to vulnerability to ferroptosis.
Methods
A proximal tubule epithelial cell line was derived from adult transgenic mice with conditional Pax8 and a constitutive driver of tamoxifen-dependent Cre. Cultured cells were genetically depleted of Pax8 by addition of 4-OH tamoxifen to the culture medium. Gene expression was measured using RNA sequencing. Survival after ferroptotic stress was measured after addition of the glutathione peroxidase 4 inhibitor RSL-3 or the glutathione depleting agent buthionine sulfoximine (BSO). Oxidative and glycolytic flux were measured using a Seahorse bioanalyzer. In vivo, we measured the steady-state central carbon metabolome in the outer stripe of outer medulla (OSOM) in transgenic mice lacking Pax8 in the S3 proximal tubule.
Results
Following depletion of Pax8, genes associated with oxidative phosphorylation and mTORC1 signaling were downregulated (Gene Set Enrichment Analysis, Padj < 0.002), suggesting reduced metabolic flux. Expression of specific genes that mitigate redox stress was upregulated, including Cp, which encodes ferroxidase (4.3-fold, P < 0.0001). Pax8-depleted cells had lower baseline oxygen consumption (17% reduction, P = 0.004) and extracellular acidification rate (17% reduction, P < 0.0001), but retained similar maximal respiratory capacity. Pax8 depleted cells resisted ferroptosis from both RSL-3 (109±11% vs 30±6% survival, P < 0.0001) and BSO (92±13% vs 23±18% survival, P < 0.0001). The OSOM in mice with proximal tubule depletion of Pax8 accumulates glycolytic and TCA cycle intermediates and is depleted of acetyl-CoA, ATP, and NADH, consistent with decreased central carbon flux.
Conclusion
Inactivation of Pax8 slows metabolism and confers resistance to ferroptosis. These results indicate a novel function for Pax proteins in regulating proximal tubule metabolism and redox homeostasis.
Funding
- NIDDK Support