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Abstract: PO0891

Modeling Damage-Associated Molecular Pattern Injury and Fibrosis Using Human Kidney Organoids

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Przepiorski, Aneta J., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Espiritu, Eugenel Bermudez, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Davidson, Alan J., The University of Auckland Faculty of Medical and Health Sciences, Auckland, Auckland, New Zealand
  • Hukriede, Neil A., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
Background

Recent developments in generating human kidney organoids in vitro, have provided an invaluable tool to study human renal diseases, injury, and screening new therapeutics. In order to study acute kidney injury (AKI) we have developed a human kidney organoid model of injury with the damage associated molecular pattern molecule (DAMP), hemin, which is released during hemolysis, often occurring after ischemia/reperfusion and rhabdomyolysis. To spatially and temporally characterize tubule injury in the hemin AKI model, we generated transgenic iPSC lines that carry an early apoptosis biosensor, CytochromeC-GFP. Healthy cells within organoids will localize CytochromeC to the mitochondria but, upon injury, will diffuse into the cytoplasm before activating the apoptotic pathway. This approach provides a real-time readout of injury progression in the kidney organoids.

Methods

Kidney organoids at day 14 of culture were treated for 48 hours with varying concentrations of hemin to determine the optimal dose for measurable injury at day 26. CytochromeC-GFP iPSC lines were generated using AAVS1 Safe Harbor targeting approach. CytochromeC-GFP response in the organoid was validated using menadione (mitochondrial toxin) and tested with hemin to determine the extent of injury. To test efficacy of new therapeutic compounds, hemin injured organoids were treated with varying concentrations of 4-(phenylthio)butanoic acid (PTBA) analogs for 10 days and analyzed to determine changes in fibrotic, and oxidative stress markers.

Results

We show injury in the organoids with optimal hemin dose leading to a reproducible increase in fibrotic, and oxidative stress response. CyochromeC-GFP biosensor iPSC lines allowed us to monitor organoids under hemin insult. Organoids treated with nephrotoxin or hemin exhibit cytoplasmic GFP signal in the injured cells and morphological changes of the mitochondria. Hemin injured organoids treated with PTBA analogs showed a reduction in fibrotic markers at day 26 suggesting a reduction in fibrotic scar tissue development.

Conclusion

We have developed a reliable injury model using hemin, and together with CytochromeC-GFP as a biosensor, these tools can be exploited to test nephrotoxicity, study acute kidney injury, and new therapeutic compounds in a human based in vitro model.

Funding

  • Other NIH Support