Abstract: PO1994
Studying the Pathogenesis of Congenital Nephrotic Syndrome Using NPHS2 Mutant Kidney Organoids
Session Information
- Podocyte Biology
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Dorison, Aude, Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
- Howden, Sara E., Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
- Forbes, Thomas A., Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
- Ghobrial, Irene, Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
- Little, Melissa H., Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
Group or Team Name
- Kidney Development, Disease and Regeneration
Background
Nephrotic syndrome (NS) is one of the most common forms of renal disease in children. NPHS2 mutations are the most common cause of congenital NS with missense NPHS2 mutations reported to result in misfolding and mistrafficking of the encoded slit-diaphragm protein, Podocin. Such studies overexpressed mutant protein in immortalised podocyte and non-podocyte cell lines, which may not reflect the in vivo consequences of the mutant protein. Here we generated NPHS2 mutant iPSC-derived kidney organoids as a model to dissect the pathogenic process.
Methods
We have simultaneously reprogrammed and CRISPR/Cas9 gene edited a control human fibroblast line, generating 3 iPSC lines containing mutations of the endogenous NPHS2 locus as well as a control wild type (WT) line. These include the sequence variants c274G>T, c353C>T and c503G>A leading to the protein changes G92C, P118L and R168H respectively. Control and mutant lines were used to generate kidney organoids containing all nephron segments. Podocin localisation was assessed using immunofluorescence together with known markers of subcellular compartments.
Results
Podocin protein was evident in the glomeruli of all organoids, however all mutant lines revealed a marked reduction of the Podocin protein. While the G92C mutant protein co-localised with Nephrin at the plasma membrane, the R168H mutant protein displayed a punctate perinuclear staining suggesting of Golgi retention, together with peripheral co-localisation with the early endosome marker EEA1. Interestingly, the R168H mutant was previously predicted to mislocalise in the endoplasmic reticulum (ER). The P118L mutant was previously predicted to accumulate in the ER and present a transmembrane localisation in cell lines. This mutant protein was observed both in ER and cytosol in kidney organoids. Finally, all 3 mutants displayed significantly more apoptotic podocytes as evidenced by an increased cleaved-Caspase3 staining in glomeruli compared to the control WT line.
Conclusion
Discrepancies between mutant protein localisations in previous reports and our kidney organoids highlight the need for a more appropriate model to study the pathobiology of NPHS2 mutations. These organoids will allow us to explore approaches to rescue individual Podocin defects, ultimately guiding the development of therapeutic strategies for such patients.
Funding
- Government Support - Non-U.S.