HOMOZYGOUS VARIANTS IN NOS1AP ARE ASSOCIATED WITH STEROID RESISTANT NEPHROTIC SYNDROME IN INFANCY AND MANIFEST PODOCYTE-SPECIFIC APICOBASAL POLARITY DISTURBANCES IN IPSC DERIVED KIDNEY ORGANOIDS

T A FORBES1,2,3, S E HOWDEN1,2, A MAJMUNDAR4, M SCURR1, K SIN TAN1, L STARKS1, K LAWLOR1, F HILDEBRANDT4, M LITTLE1,3

1Murdoch Children’s Research Institute, Parkville, Australia, 2Royal Children’s Hospital, Parkville, Australia, 3University of Melbourne, Parkville, Australia, 4Boston Children’s Hospital, Harvard Medical School, Boston, USA

Background: Kidney organoids regenerated from human induced pluripotent stem cells (hiPSC) represent an emerging disease modelling platform for validation of novel genomic variants. Using whole exome sequencing, the laboratory of Friedhelm Hildebrandt identified recessive variants in the gene Nitric oxide synthase 1 adaptor protein (NOS1AP) in two unrelated infants with steroid resistant nephrotic syndrome (SRNS). NOS1AP interacts with the actin cytoskeleton by promoting CDC42 activation. CDC42 regulates PAR3-PAR6-aPKC complex mediated maintenance of apicobasal polarity. aPKC knockout or inhibition in mouse models leads to severe proteinuria.
Aim: We aimed to characterise disturbances in apicobasal polarity of NOS1AP homozygous hiPSC-derived kidney organoids.
Methods: Using CRISPR-Cas9, we gene-edited a homozygous, patient-derived NOS1AP variant (c.428G>A) into a wild type (WT) hiPSC cell line. NOS1AP homozygous and WT hiPSC clones were differentiated to kidney organoids in paired experiments and examined using histology, immunofluorescence and electron microscopy.
Results: NOS1AP expression was confined to podocytes in kidney organoids, colocalising with NEPHRIN. Histology of WT organoids demonstrated tufts of podocyte monolayers lining an established basement membrane. In contrast, NOS1AP homozygous organoids showed poorly established basement membranes and pyknotic nuclear figures which were cleaved CASP-3 positive. Immunofluorescence demonstrated disorganisation of slit diaphragm markers and reduced aPKC and PAR3 expression in NOS1AP homozygous glomeruli suggested dysregulation of the PAR complex which is essential for podocyte polarity and foot process integrity. Putative foot process effacement was evident on electron microscopy of NOS1AP homozygous organoids.
Conclusions: Recessive NOS1AP variants are a novel genotype associated with infant-onset SRNS. NOS1AP homozygous organoids demonstrate disturbances in apicobasal polarity and progressive apoptosis specific to podocytes. In future studies, glomerular cleaved CASP-3 expression could be applied to high-throughput compound screening.


Biography:
Dr Tom Forbes is a Paediatric Nephrologist at the Royal Children’s Hospital in Melbourne, with an interest in genetic kidney diseases. He completed his clinical training at the Royal Children’s Hospital in 2015, also training at Birmingham Children’s Hospital and Nottingham University Hospital in the UK. He completed his PhD with Prof Melissa Little’s Kidney Regeneration laboratory at MCRI examining the capabilities and limitations of patient-iPSC derived kidney organoids as functional genomic models of kidney disease. He holds an MCRI Clinician Scientist Fellowship continuing his research in novel gene discovery and functional genomic disease modelling with kidney organoids.

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