Kidney Week

Abstract: FR-PO356

Hypoxia Triggers a Signature of Maladaptive Repair in Human Kidney Organoids

Session Information

• Genetics, Development, Regeneration
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development‚ Stem Cells‚ and Regenerative Medicine

• 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

• Nunez Nescolarde, Ana Beatriz, Monash University, Clayton, Victoria, Australia

Ana Beatriz Nunez Nescolarde,

• Piran, Mehran, Monash University, Clayton, Victoria, Australia

Mehran Piran,

• Nikolic-Paterson, David J., Monash University, Clayton, Victoria, Australia

David J. Nikolic-Paterson,

• Combes, Alexander N., Monash University, Clayton, Victoria, Australia

Alexander N. Combes,

Background

Reduced oxygen levels during renal ischemia can induce AKI and lead to CKD through maladaptive repair. Tubulointerstitial fibrosis is a chronic and progressive feature of CKD, for which we lack effective treatments. To facilitate therapeutic studies, we develop a human kidney organoid model of hypoxia-induced renal fibrosis.

Methods

Human iPSC-derived organoids were generated from 3 different cell lines and cultured for 18 days, then subjected to hypoxia (1%O₂) or normoxia (21%O₂) for 48h (d20) followed by 5 days recovery period in normoxia (d25). Organoids were examined by gene expression profiling, immunohistochemistry and immunofluorescence microscopy.

Results

Robust hypoxic response was evident on d20 by 5 and 7-fold increases in mRNA levels of hypoxia-inducible genes VEGFA and HK1, respectively (both p<0.001, Fig 1A). Tubular damage marker KIM1/HAVCR1, stress-response gene JUN, and profibrotic signal TGFB1 were all upregulated (>2 fold p<0.001) in hypoxic organoids on d20, consistent with an AKI response (Fig 1A). Mitochondrial dysfunction was evident in hypoxic organoids on d20 with a 50% decrease (p<0.001) of PPARGC1A levels (Fig 1A). On d25, expression of hypoxia-responsive genes and AKI markers returned to control levels. However, proinflammatory cytokine CCL2 (p<0.05), extracellular matrix components COL1A1 (p<0.01) and matrix producing cells ACTA2 were significantly upregulated in hypoxia-treated organoids (Fig 1A). Immunostaining at d25 for COL1A1 affirmed increased collagen I deposition and reduction in tubular density (PAX8) in hypoxia-treated organoids (Fig 1B). We are currently profiling control and hypoxia-treated organoids from day 20 and 25 timepoints using bulk and single cell RNA-sequencing.

Conclusion

Here we show that kidney organoids transiently upregulate markers of AKI in response to hypoxia and later display signatures of maladaptive repair including elevated levels of ECM. These results suggest kidney organoids may aid in disease modelling and drug development for AKI and fibrosis.

Funding

• Government Support – Non-U.S.