Novel effects of ouabain in autosomal dominant polycystic kidney disease cystogenesis

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 or Pkd2, genes encoding for polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. ADPKD is characterized by the progressive growth of numerous fluid-filled renal cysts. Cyst formation and growth depends on proliferation of the cyst-lining epithelial cells and fluid secretion into the cyst lumen. ADPKD cystogenesis is highly influenced by non-genomic factors, many of which elicit their effects via cAMP-dependent pathways. Understanding mechanisms mediating the effects of cystogenic agents is crucial for the future development of ADPKD therapy. Previous work has shown that cells derived from the epithelial-lining of renal cysts from patients with ADPKD (ADPKD cells) have an increased affinity for the hormone ouabain. ADPKD cells respond to ouabain by an increased rate of cell proliferation. This effect depends on binding of ouabain to the Na,K-ATPase which induces activation of Src kinase, epidermal growth factor receptor (EGFR), and the extracellular regulated kinase (ERK1/2) pathway. The objective of the current study was to determine the role of ouabain in mechanisms of fluid secretion and cyst growth in ADPKD. Studies were carried out in human ADPKD cells, embryonic kidneys from the Pkd1m1Bei mouse model, and M-1 mouse cortical collecting duct cells. Results of this study show that physiologic concentrations of ouabain enhance cAMP-dependent fluid secretion and cyst growth of ADPKD cells grown in culture as monolayers or in three-dimensional structures resembling cysts. Additionally, ouabain potentiated the cAMP-dependent growth of cyst-like dilations in metanephric kidneys from the Pkd1m1Bei mouse model. These effects were mediated via activation of the Na,K-ATPase signaling apparatus, located at the basolateral domain of ADPKD cells. Intracellular mediators of ouabain's response included the EGFR-Src-ERK pathway. Ouabain alone did not increase fluid secretion and cyst growth. Rather, ouabain treatment altered the phenotype of ADPKD cells to allow enhanced responses to cAMP agonists. The potentiating effect of ouabain on cAMP-induced fluid secretion was associated with the capacity of ouabain to stimulate anion secretion via the apically located cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, ouabain increased membrane expression of the CFTR. Finally, ouabain decreased Na,K-ATPase membrane expression and ion transport at the basolateral membrane of ADPKD cells. The increased ouabain sensitivity of ADPKD cells depends on an abnormally high affinity of the Na,K-ATPase for ouabain. Increased ouabain affinity of the Na,K-ATPase was associated with abnormal expression of the C-terminus of PC-1 in M-1 cells. Altogether, the study of ouabain's effects in ADPKD have uncovered a novel role for ouabain as a physiologic agent that influences renal cyst growth in ADPKD. In addition, it has identified a new mechanism in ADPKD cystogenesis, important for the progression of the disease