title: Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via cAMP and EGFR-Src-MEK pathways.
abstract: In autosomal-dominant polycystic kidney disease (ADPKD), renal cysts develop by aberrant epithelial cell proliferation and transepithelial fluid secretion. We previously showed that ouabain increases proliferation of cultured human ADPKD cells via stimulation of the EGF receptor (EGFR)-Src-MEK/ERK signaling pathway. We examined whether ouabain affects fluid secretion and in vitro cyst growth of human ADPKD cell monolayers, ADPKD cell microcysts cultured in a three-dimensional collagen matrix, and metanephric organ cultures from Pkd1(m1Bei) mice. Physiological concentrations of ouabain alone did not affect net transepithelial basal-to-apical fluid transport in ADPKD monolayers or growth of cultured ADPKD microcysts. In contrast, in the presence of forskolin or 8-bromo-cAMP, ouabain significantly enhanced ADPKD fluid secretion and microcyst expansion. Ouabain exerted this effect by enhancing cAMP-dependent Cl(-) secretion via the CFTR. Similarly, ouabain accelerated cAMP-dependent cyst enlargement in Pkd1(m1Bei) mice metanephroi, with a more prominent response in homozygous than heterozygous mice. Ouabain had no effect on fluid secretion and cystogenesis of normal human kidney cells and caused only slight cystic dilations in wild-type mouse kidneys. The effects of ouabain in ADPKD cells and Pkd1(m1Bei) metanephroi were prevented by inhibitors of EGFR (AG1478), Src (PP2), and MEK (U0126). Together, our results show that ouabain, used in physiological concentrations, has synergistic effects on cAMP-mediated fluid secretion and cyst growth, via activation of the EGFR-Src-MEK pathway. These data provide important evidence for the role of ouabain as an endogenous hormone that exacerbates ADPKD cyst progression.
authors.affiliation: Dept. of Molecular and Integrative Physiology, 3901 Rainbow Blvd., Kansas City, KS 66160, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Jansson
authors.forename: Anh-Nguyet T
authors.initials: AN
authors.lastname: Nguyen
authors.forename: Brenda S
authors.initials: BS
authors.lastname: Magenheimer
authors.forename: Gail A
authors.initials: GA
authors.lastname: Reif
authors.forename: Lavakumar Reddy
authors.initials: LR
authors.lastname: Aramadhaka
authors.forename: Elsa
authors.initials: E
authors.lastname: Bello-Reuss
authors.forename: Darren P
authors.initials: DP
authors.lastname: Wallace
authors.forename: James P
authors.initials: JP
authors.lastname: Calvet
authors.forename: Gustavo
authors.initials: G
authors.lastname: Blanco
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003560:Cysts / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D010042:Ouabain / Q000378:metabolism*; D016891:Polycystic Kidney, Autosomal Dominant / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D005576:Colforsin; D010042:Ouabain; D000242:Cyclic AMP; C512478:EGFR protein, human; D066246:ErbB Receptors