Abstract: SA-PO0144
Mitochondrial Dysfunction in Hemin-Induced AKI Is Mediated by Guanine Quadruplex Dynamics
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
- Snyder, Ryan J., National Institute of Environmental Health Sciences, Durham, North Carolina, United States
- Shankar, Uma, National Institute of Environmental Health Sciences, Durham, North Carolina, United States
- Beamish, Jeffrey A., University of Michigan, Ann Arbor, Michigan, United States
- Watts, Jason A., National Institute of Environmental Health Sciences, Durham, North Carolina, United States
Background
Proximal tubule cells are dependent on energy from mitochondria for their transporter activity and transcription of mtDNA is essential for oxidative phosphorylation. Previously, we found that transcription by mitochondrial RNA polymerase (POLRMT) is regulated by the formation of guanine-quadruplexes (G4s) in nascent RNA. Hemin is a known endogenous G4-stabilizing ligand and is elevated in the proximal tubules under conditions associated with acute kidney injury (AKI) such as sepsis, rhabdomyolysis, and ischemic reperfusion (IRI). We hypothesize that impaired mitochondrial gene expression due to hemin-stabilized G4s may contribute to renal dysfunction in AKI.
Methods
We used circular dichroism analysis and an in vitro primer extension assay to measure the stability of G4s and transcriptional pausing induced by hemin. In a mouse model of kidney IRI, we measured colocalization of G4s in proximal tubules with heme oxygenase (HMOX1) expression and KIM1 injury marker from 6 hours to 4 weeks post-IRI. We also treated human proximal tubule epithelial cells (RPTECs) with non-cytotoxic doses of hemin for 1-24 hours. Gene expression in RPTECs was analyzed by QPCR and mitochondrial function was measured using Seahorse extracellular flux assay.
Results
Hemin stabilized RNA G4 structures and impaired transcription by POLRMT in vitro. Proximal tubules from IRI kidneys had higher HMOX1 expression, indicating the presence of free heme, and more G4s at 6-48 hours post-IRI, returning to contralateral control levels within 2 weeks. Human RPTECs exposed to hemin revealed a dose-dependent increase in G4 abundance which peaked at 1-6 hours. Mitochondrial-encoded OXPHOS genes were downregulated by hemin exposure while nuclear-encoded transcription factors TFAM and TEFM were unaffected, consistent with our previous work with G4-stabilizing drugs. Oxygen consumption rates in RPTECs were significantly decreased after 24 hours of hemin exposure.
Conclusion
These results show that hemin stabilizes G4s in proximal tubule cells and impairs mitochondrial gene transcription and function. We conclude that stabilization of G4 structures and dysregulation of POLRMT transcription is an additional mechanism mediating hemin-induced AKI.
Funding
- Other NIH Support