Abstract: TH-OR078

HIF Regulation of Nephron Progenitor Metabolic State Mediates Cell Fate Decisions

Session Information

Category: Developmental Biology and Inherited Kidney Diseases

  • 401 Developmental Biology

Authors

  • Murali, Anjana, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Cargill, Kasey, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Mukherjee, Elina, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Saifudeen, Zubaida R., Tulane University School of Medicine, New Orleans, Louisiana, United States
  • Sims-Lucas, Sunder, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

Hypoxia inducible factors (HIFs) are transcription factors involved in the differentiation of nephron progenitors (NP) into functional nephrons. Alterations in nephron differentiation lead to renal abnormalities. As renal oxygen increases, von Hippel-Lindau (VHL) marks HIF-1α for degradation and facilitates normal nephron differentiation. Alternatively, pathological hypoxia results in HIF-1α accumulation and initiation of processes including cellular survival and metabolism. Previous in vitro studies have also linked HIF-1α stabilization to mitochondrial pathologies suggesting that HIF-1α plays a role in cellular and mitochondrial respiration. Therefore, we hypothesize that HIF-1α alters mitochondrial function, mediating a metabolic switch to determine NP fate.

Methods

To determine the role of HIFs in the NPs we utilized VHL floxed mice bred with the Six2EGFPcre line, to generate Six2creVHLlox/lox mutant mice. RNA sequencing was conducted on E14 whole kidneys to look at differential gene expression, and validated via RT-PCR. We also performed a metabolic assessment using seahorse extracellular flux analysis, and coupled this with immunofluorescence (IF) and western blot analysis to analyze metabolic markers.

Results

RNA sequencing of Six2creVHLlox/lox mutant mice revealed metabolic gene dysregulation. Furthermore, seahorse extracellular flux analysis suggested that mutants with HIF-1α stabilization in the NPs remained in a glycolytic state, and were subsequently unable to switch to oxidative phosphorylation, which drives NP differentiation. IF staining and western blot analysis supported these results revealing fewer mature nephron structures and decreased mitochondrial content in mutant kidneys.

Conclusion

HIFs are critical in determining the metabolic state of the NPs. In the absence of VHL, HIF1α is stabilized. This stabilization maintains NPs in a state of glycolysis and in turn blocks the switch to oxidative phosphorylation causing NP differentiation defects and kidney malformations.

Funding

  • NIDDK Support