The molecular pathogenesis of autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is a severe and early-onset ciliopathy, characterised by hepatorenal manifestations, and caused by variants in PKHD1. PKHD1 encodes fibrocystin, a transmembrane protein of unknown function with an extensive ectodomain. Genotype-phenotype analyses have suggested that patients with one or two missense variants have a better prognosis than those harbouring two truncating variants, who suffer from the most grievous form of the disease. Little is known about the structure of fibrocystin, limiting our understanding of how missense variants cause ARPKD and slowing the development of new treatments.
To explore a potential mechanism of disease, protein instability, a full-length fibrocystin ectodomain model was predicted using AlphaFold2 and characterised, revealing unpredicted domains and unexpected folding events. Using FoldX in combination with this model enabled protein stability predictions for a dataset of missense variants. A binary classifier trained using these scores indicated protein stability to be strongly associated with pathogenicity. Furthermore, fibrocystin destabilisation was associated with increased severity of disease in an ARPKD patient dataset. As no experimental evidence of the structure of the ectodomain exists, protein production trials were performed with the aim of generating material for structure determination. Over several trials, recombinant fibrocystin exhibited poor stability, leading to a limited yield of soluble protein. Examination of one of these constructs in mammalian cells revealed accumulation in the endoplasmic reticulum, further indicating that instability and misfolding is an important mechanism. Finally, kidney organoids harbouring the most common ARPKD-associated variant, T36M, were successfully generated from CRISPR-edited iPSCs. Characterisation of these organoids revealed insights into their differentiation which will support their development as models for ARPKD. Spontaneous cystogenesis was not observed, suggesting that modified conditions are required to recapitulate the ARPKD phenotype. Thus, this study provides new insights into fibrocystin stability and presents it as a possible mechanism underlying ARPKD.