Developing a novel 3D tubulogenesis model to investigate the role of cilia in kidney disease

Abstract

Primary cilia are a highly conserved, antenna-like organelles that protrude from the cell membrane of most eukaryotic cells. This organelle functions as a sensory hub that is critical for cellular signalling. When cilia or ciliary signalling is disrupted, it can often lead to disease. Collectively, disorders caused by ciliary dysfunction are called ciliopathies. This thesis investigates the role of cilia in two separate diseases, Autosomal dominant polycystic disease and Arthrogryposis-Renal Dysfunction-Cholestasis Syndrome. Autosomal dominant polycystic kidney disease (ADPKD) is the most common ciliopathy and is characterised by the growth of multiple fluid filled cysts in the kidneys. ADPKD kidneys present a disruption in calcium signalling, cAMP levels, and mTOR pathways, all of which drive cystogenesis. Cilia are critical in PKD because the main proteins affected in this disease, Polycystin-1 (PKD1) and Polycystin-2 (PKD2) reside in cilia. To understand this role, my research aimed to replicate the ADPKD phenotype in vitro by engineering a PKD1 knockout in a human renal cell line, and to model cystic formation in 3D using human renal cell lines. The pipeline to generate a PKD1 knockout in human renal cells was successfully tested and optimised, with current ongoing work validating knockout clones. A novel 3D tubulogenesis assay was successfully developed using human renal proximal tubule cells on a Matrigel based matrix. This assay was used to promote cyst formation, from which libraries of drugs can now be tested on to reduce cyst formation. Arthrogryposis-Renal Dysfunction-Cholestasis (ARC) Syndrome is a rare and fatal autosomal recessive and multisystem disorder, caused by mutations in vacuolar protein sorting 33 homolog B (VPS33B). Classical features of ARC syndrome include renal dysfunction. VPS33B has been shown to interact with RAB11A, which functions in early ciliogenesis, suggesting a ciliary link for VPS33B. This research aimed to replicate the ARC phenotype in human renal proximal tubule cells using a 3D Matrigel model, and to investigate whether VPS33B could affect cilia formation. Both VPS33B knockout and knockdown cells showed tubulogenesis defects and impaired ciliogenesis, which has never been reported before. In this thesis, I developed a novel 3D tubulogenesis model to understand kidney disease, and we uncovered a novel role of VPS33B in ciliogenesis. This model will be key to investigate kidney disease and to understand how this is affected by cilia.