Kidney Week

Abstract: FR-OR065

Homozygous Variants in NOS1AP from a Patient with Steroid-Resistant Nephrotic Syndrome Cause Podocyte Polarity Dysregulation and Aberrant Glomerulogenesis in Human iPSC Kidney Organoids

Session Information

• Genetic Diseases and the Kidneys
  November 08, 2019 | Location: 144, Walter E. Washington Convention Center
  Abstract Time: 05:18 PM - 05:30 PM

Category: Genetic Diseases of the Kidneys

• 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

• Forbes, Thomas A., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Thomas A. Forbes, MBBS



Tom is a Consultant Paediatric Nephrologist at the Royal Children's Hospital (Melbourne, Australia). He completed his undergraduate medical degree with Monash University (Melbourne) in 2005 and trained in paediatrics at the Royal Children's Hospital between 2007 and 2010. During his clinical training he worked in the UK at Birmingham Children’s Hospital and Nottingham Queen’s Medical Centre where he developed an interest in rare, inherited, paediatric renal diseases. Tom returned to Melbourne in 2013 and completed a paediatric nephrology fellowship at the Royal Children's Hospital and became a Fellow of the Royal Australasian College of Physicians in 2015. His special interests are genetic renal disease and young adult transition. Tom is in the closing stages of a PhD within Prof Melissa Little's Kidney Regeneration Laboratory at the Murdoch Children's Research Institute (Melbourne). Tom's PhD is examining the use of patient iPSC derived kidney organoids as research models of inherited kidney disease. He is supported by an NHMRC Post-graduate Award (2016) and an RACP/NHMRC Jacquot Award for Excellence.

• Howden, Sara E., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Sara E. Howden,

• Majmundar, Amar J., Boston Children's Hospital, Boston, Massachusetts, United States

Amar J. Majmundar,

• Buerger, Florian, Boston Children's Hospital, Boston, Massachusetts, United States

Florian Buerger,

• Scurr, Michelle, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Michelle Scurr,

• Tan, Ker sin, Murdoch Children's Research Institute, Parkville, Victoria, Australia

Ker sin Tan,

• Starks, Lakshi T., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Lakshi T. Starks,

• Lawlor, Kynan T., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Kynan T. Lawlor,

• Little, Melissa H., Murdoch Children's Research Institute, Parkville, Victoria, Australia

Melissa H. Little,

• Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States

Friedhelm Hildebrandt,

Background

Kidney organoids generated from human induced pluripotent stem cells (hiPSC) represent an emerging disease modelling platform for the study of genetic kidney diseases. Genomic sequencing is increasing the rate of novel disease gene discovery, and over 60 genes have been identified to cause steroid resistant nephrotic syndrome (SRNS). Nitric oxide synthase 1 adaptor protein (NOS1AP) is a novel gene for SRNS, whose encoded protein regulates actin cytoskeleton remodelling by promoting CDC42 activation. CDC42 regulates PAR3-PAR6-aPKC complex maintenance of apicobasal polarity. This PAR complex colocalises with the slit diaphragm in podocytes and either CDC42 or aPKC deficiency in mice causes severe proteinuria. Here, we characterise the effect of a homozygous NOS1AP SRNS patient variant on glomerulogenesis in hiPSC kidney organoids.

Methods

A homozygous, patient-derived NOS1AP variant (c.428G>A) was gene-edited into a wild type hiPSC cell line using CRISPR-Cas9. NOS1AP homozygous and wild-type (WT) hiPSC clones were differentiated to kidney organoids in paired experiments. Organoids were examined by blinded and semi-automated analysis of histology, immunofluorescence and electron microscopy imaging.

Results

Histology sections of WT organoids demonstrated tufts of podocyte monolayers lining an established basement membrane. In contrast, NOS1AP homozygous organoids showed disorganised podocyte collections with poorly established basement membranes and pyknotic nuclear figures which were CASP-3 positive. Foot process effacement was evident on electron microscopy of NOS1AP homozygous organoids. Whole mount immunofluorescence showed disorganisation of slit diaphragm markers and reduced aPKC expression in NOS1AP homozygous glomeruli suggesting dysregulation of the PAR complex.

Conclusion

A novel, SRNS patient-derived, homozygous variant in NOS1AP causes abnormal glomerulogenesis in kidney organoids and highlights the utility of this 3D, human, in vitro, functional genomic model. We propose that pathogenic variants in NOS1AP reduce active CDC42 which impairs polarity complex expression and foot process formation in hiPSC kidney organoid glomeruli.

Funding

• Government Support - Non-U.S.