Miss Ana Nunez-Nescolarde, Mr Mehran Piran, Professor David J Nikolic-Paterson, Dr Alexander N Combes
1Monash University, Australia, 2Department of Nephrology, Monash Medical Centre, Australia
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.
Aim: To develop a human kidney organoid model of hypoxia-induced fibrosis to facilitate therapeutic studies.
Methods: Human iPSC-derived organoids were generated from 3 different cell lines and cultured for 18d, then subjected to hypoxia (1%O2) or normoxia (21%O2) for 48h (d20) followed by 5d recovery period in normoxia (d25). Organoids were examined by gene expression profiling, immunohistochemistry and immunofluorescence microscopy.
Results: Robust hypoxic response was evident on d20 in mRNA levels of hypoxia-inducible genes (>5-fold increase in VEGFA, HK1 p<0.001). 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. Mitochondrial dysfunction was evident in hypoxic organoids on d20 with a 50% decrease (p<0.001) of PPARGC1A levels. 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. Immunostaining at d25 for COL1A1 affirmed increased collagen I deposition and reduction in tubular density (PAX8) in hypoxia-treated organoids. 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.
Biography:
Ana has a bachelor in Microbiology from the Autonomous University of Barcelona (Spain). Awarded a Master Degree in Molecular Medicine at the Norwegian University of Science and Technology (NTNU) in 2020, Ana generated iPSC-derived brain organoids to study oxidative stress and developed a novel approach to modify human iPSC using CRISPR/Cas9. Currently, Ana is PhD student supervised by Dr Alexander Combes and Professor David Nikolic-Paterson at Monash University, with focus on fibrosis and the use of human 3D models to facilitate the discovery of new therapeutic strategies.