Research Fellow Monash University Melbourne, Victoria, Australia
Abstract: The rise of widespread genetic sequencing has linked thousands of missense variants and variants of uncertain significance (VUS) with kidney disorders. Determining the functional impact of these variants in a physiologically relevant human model remains a significant challenge, often requiring labor-intensive, approaches for individual genes and VUS. To address this limitation, we established an iPSC-derived epithelial human kidney organoid (tubuloid) platform to facilitate high-throughput functional analysis of VUS. Tubuloids, made from iPSC-derived human kidney organoids, model the 3D structure and expression profiles of the distal nephron, loop of Henle, and proximal tubule, providing an ideal model to study a variety of VUS. UMOD is a protein coding gene predominantly expressed in the thick ascending limb of the loop of Henle. Mutations in UMOD are linked to autosomal dominant tubulointerstitial kidney disease (ADTKD-UMOD), characterized by progressive renal dysfunction and fibrosis. The pathogenicity of novel UMOD variants is challenging to predict and the mechanisms of ADTKD-UMOD remain incompletely understood. To address this, we employed lentiviral overexpression of UMOD variants including wildtype, benign, pathogenic, and novel clinical VUS in iPSC-derived tubuloids. High-content imaging, immunofluorescence, and biochemical assays were utilised to assess protein localisation, secretion, intracellular aggregation, and stress responses associated with variant expression. Wildtype and benign UMOD variants localised to the basolateral membrane of tubuloids, while known pathogenic variants and some VUS were characterised by altered protein stability and mislocalisation. Our findings provide evidence of pathogenicity for novel UMOD VUS and correlate mislocalisation patterns and associated cellular stress responses with disease severity., This tubuloid-based system offers a scalable functional genomics platform for genetic disorders, with potential to improve diagnostic outcomes for patients and foster the development of precision medicine approaches.
Funding Source: Australia Medical Research Future Fund Australian Functional Genomics Network
