Cystin is required for maintaining fibrocystin (FPC) levels and safeguarding proteome integrity in mouse renal epithelial cells A mechanistic connection between the kidney defects in cpk mice and human ARPKDRunning title: Cystin deficiency results in FPC degradation

Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice fail to express renal cystic disease. In contrast, the renal lesion in Cys1cpk/cpk (cpk) mice with loss of the cystin protein, closely phenocopy ARPKD. Recent identification of patients with CYS1-related ARPKD prompted the investigations described herein. We analyzed cystin and FPC expression in mouse models (cpk, rescued-cpk (r-cpk), Pkhd1 mutants) and cortical collecting duct (CCD) cell lines (wild type (wt), cpk). We found that cystin deficiency led to diminished FPC in both cpk kidneys and CCD cells. In r-cpk kidneys, FPC increased and siRNA of Cys1 in wt CCD cells reduced FPC. Conversely, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and the associated reduction in FPC levels impacted the architecture of the primary cilium, but not ciliogenesis. Similar Pkhd1 mRNA levels in wt, cpk kidneys and CCD cells suggested posttranslational mechanisms directed FPC loss and studies of cellular protein degradation systems revealed selective autophagy as a possible mechanism. Loss of FPC from the NEDD4 E3 ubiquitin ligase complexes caused reduced polyubiquitination and elevated levels of functional epithelial sodium channel (NEDD4 target) in cpk cells. We propose that cystin is necessary to stabilize FPC and loss of cystin leads to rapid FPC degradation. FPC removal from E3-ligase complexes alters the cellular proteome and may contribute to cystogenesis through multiple mechanisms, that include MYC transcriptional regulation.