cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells

Abstract

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Ataxia Telangiectasia Mutated (ATM) Inhibition Transforms Human Mammary Gland Epithelial Cells

“…354230, BD Biosciences) as described (19) and grown for 7 or 14 days. Transwell Assay-Transwells (8.0-m membrane pores; polycarbonate; diameter, 6.5 mm; catalog no.…”

Spatially restricted hyaluronan production by Has2 drives epithelial tubulogenesis in vitro

“…The finding that Has2 was induced a few hours after addition of either TGF-␤1 or HGF suggests that this enzyme is involved in the initiation of the tubulogenic process. In this respect, it is relevant that collecting duct mCCD-N21 cells, which spontaneously form branched tubules in a 3-D matrix environment (30), constitutively express Has2 (data not shown).…”

“…Cells were grown in DMEM with Glutamax supplemented with 5% FCS (Life Sciences). mCCD-N21 mouse kidney cells (30) were grown in a 1:1 mixture of DMEM and F12 medium with Glutamax (DMEM/F12; Life Sciences) supplemented with 2% FCS, 5 g/ml insulin (Sigma), 5 g/ml holo-transferrin (Sigma), 10 ng/ml epidermal growth factor (EGF; Pepro Tech, London, UK), 50 nM dexa-methasone (Sigma), 60 nM sodium selenite (Sigma), and 1 nM 3,3=,5-triiodo-L-thyronine (Sigma).…”

“…To assess whether HA is necessary not only for the initial sprouting of epithelial tubules but also for their subsequent elongation, we studied the effect of 4-MU on mCCD-N21 cells, a mouse renal collecting duct cell line that displays the ability to spontaneously form tubular structures in collagen/ Matrigel gels (30). mCCD-N21 cells were treated with 0.25 mM 4-MU, a concentration that we previously found does not inhibit cell growth on plastic.…”

“…However, it has not been established whether endogenously produced HA is actually necessary for tubule formation. To address this issue, we took advantage of three different in vitro assays of epithelial morphogenesis developed in our laboratory: 1) tube formation by mammary gland-derived J3B1A cells in response to low concentrations of transforming growth factor-␤1 (TGF-␤1) (29); 2) tube formation by kidney-derived MDCK cells in response to addition of HGF (31); and 3) spontaneous tube formation by collecting duct mCCD-N21 cells (30). Using these experimental models, we show that Has2 expression and HA produced at the tips of epithelial cells play a critical role in driving tubulogenesis and branching in vitro.…”