Abstract: TH-PO402
Hypoxic Injury Triggers Maladaptive Repair in Human Kidney Organoids
Session Information
- Development, Organoids, Injury, and Regeneration
October 24, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 600 Development, Stem Cells, and Regenerative Medicine
Authors
- Nunez Nescolarde, Ana Beatriz, Monash University, Melbourne, Victoria, Australia
- Piran, Mehran, Monash University, Melbourne, Victoria, Australia
- Cheng, Zhengqi, The University of Melbourne, Melbourne, Victoria, Australia
- Wells, Christine A., The University of Melbourne, Melbourne, Victoria, Australia
- Nikolic-Paterson, David J., Monash Medical Centre, Clayton, Victoria, Australia
- Combes, Alexander N., Monash University, Melbourne, Victoria, Australia
Background
Hypoxia is an important driver of AKI. Individuals who survive AKI often develop CKD through a maladaptive repair process characterised by failed epithelial regeneration, fibrosis, inflammation and metabolic dysregulation. Despite detailed knowledge built in rodent models, translational challenges underscore a need for human models.
Methods
Kidney organoids were generated from three independent human induced pluripotent stem cell lines and cultured for 48h in hypoxic (1% O2) or normoxic conditions from day (d) 18 to 20 of differentiation. Organoids were collected at d20 to assess immediate injury, or at d25, after 5d of culture in normoxic conditions, to assess repair. The response was analysed using transcriptomics, proteomics and metabolomics analyses. In addition, the role of the hypoxia regulator, HIF1A, was evaluated by exposing to hypoxia CRISPR edited HIF1A-/- organoids. Finally, direct co-culture with human induced macrophages was performed and their location and expression signatures were interrogated using immunoassays and spatial transcriptomics.
Results
Kidney organoids exposed to hypoxia upregulated hypoxic response genes (VEGFA, HK1), tubular injury markers (JUN, TGFB1, GDF15), and transcriptional signatures of AKI-associated cell stress and death pathways (apoptosis, ferroptosis). Deletion of HIF1A abrogated most of the hypoxic transcriptional response. However, we found the expression of GDF15 to be HIF1A-independent. Instead, GDF15 expression seemed to be mediated by the increase in DDIT3 due to endoplasmic reticulum stress. At d25, injured organoids had increased ceramide production, elevated levels of maladaptive repair markers (S100A8, S100A9), and genes associated with collagen production, inflammation and fibrotic mediators. Dysregulation of iron metabolism (HMOX1, FTL) and lipid peroxidation were also lasting effects of the hypoxic injury. Single-cell RNAseq localised expression of maladaptive repair genes, and activation of TNF, NF-κB and JAK-STAT signalling pathways to tubular epithelial cells. Furthermore, co-cultured with macrophages modified the hypoxic response by enhancing TNF-α signalling via NF-kB and TGF-ß signalling pathways.
Conclusion
This multi-omic analysis provides compelling evidence supporting the use of organoids as in vitro models of ischemic AKI and maladaptive repair.
Funding
- Government Support – Non-U.S.