CFTR correctors suppress Ca²⁺-cAMP signaling and cyst growth in primary cultures of ARPKD cholangiocytes.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe ciliopathy caused by mutations in Polycystic kidney and hepatic disease 1 gene (PKHD1), resulting in progressive renal and hepatobiliary disease for which effective targeted therapies are lacking. Although reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression has been implicated in hepatic cystogenesis, whether pharmacological restoration of CFTR is associated with improvement of the underlying cellular abnormalities remains unclear. We investigated the effects of the CFTR correctors VX-809 and VX-445, and compared them with the CFTR inhibitor CFTRinh-172, in cholangiocytes isolated from Pkhd1del3-4/del3-4 mice. FPC-deficient cholangiocytes exhibited enhanced cyst growth and proliferation together with marked reductions in CFTR, polycystin-1 (PC1), and polycystin-2 (PC2) protein expression. These changes were accompanied by profound remodeling of intracellular signaling, including elevated resting cytosolic Ca²⁺, an increased thapsigargin-releasable endoplasmic reticulum Ca²⁺ pool, increased STIM1 expression, reduced IP₃ receptor expression, a marked increase in intracellular cAMP, and a switch in adenylyl cyclase isoform expression characterized by increased AC3 and reduced AC6. CFTR localization was also altered, with redistribution toward the apical membrane. Treatment with either VX-809 or VX-445 improved many of these abnormalities by increasing CFTR expression, partially restoring PC1 and PC2 expression, attenuating abnormal Ca²⁺ and cAMP signaling, reducing cholangiocyte proliferation and cyst growth, and shifting CFTR membrane distribution toward the WT pattern. In contrast, CFTRinh-172 failed to reproduce many of the same effects, indicating that inhibition of chloride conductance alone does not account for the broader cellular effects of the CFTR correctors. These findings identify coordinated remodeling of Ca²⁺- and cAMP-dependent signaling as a central feature of ARPKD cholangiocytes and show that CFTR correctors are associated with improvement of multiple disease-associated pathways. Our results support further evaluation of clinically available CFTR correctors as a potential therapeutic strategy for hepatobiliary disease in ARPKD.