Chloride and fluid secretion by cultured human polycystic kidney cells

Chloride and fluid secretion by cultured human polycystic kidney cells. Epithelial cells cultured from the renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) secrete fluid via a process stimulated by adenosine 3′,5′-cyclic monophosphate (cAMP). We have investigated the hypothesis that fluid secretion by these cells is dependent on cAMP-mediated chloride secretion. Individual cultured ADPKD cells were suspended within a polymerized collagen matrix and stimulated to form cysts. Individual cultured cysts were placed in a chamber on the stage of an inverted microscope equipped with epifluorescent and video analysis attachments. The rate of fluid secretion, cell volume and changes in intracellular Cl - were measured. In the absence of secretagogues, fluid was absorbed from the cyst cavity (-2.36 ± 0.64 nl/min/cm 2 inner surface area). 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) plus 3-isobutyl-l-methlyxanthine (IBMX) induced a rapid reversal in the net movement of fluid to secretion (6.79 ± 1.28 nl/min/cm 2 ). Bumetanide reversibly reduced fluid secretion to 0.95 ± 0.60 nl/min/cm 2. Cell volume rapidly decreased by 7.5 ± 0.9% with the initiation of secretion and bumetanide caused an additional loss (4.2 ± 1.0%). Furosemide had a similar effect on forskolin-induced fluid secretion. Cellular chloride concentration was monitored with the use of the indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ). Removal of Cl - from the bath reduced intracellular [Cl - ] (MEQ fluorescence increased by 11.4 ± 2.3%). In cysts pretreated with furosemide to prevent Cl - entry, the application of forskolin caused a decrease in Cl - concentration (MEQ fluorescence increased by 9.3 ± 2.6%). Using monolayers of cultured ADPKD cells, grown on permeant supports, we compared the changes in short circuit current (I SC ) induced by forskolin in the presence and absence of external Cl -. Forskolin increased I SC (from 8.9 ± 2.7 to 10.6 ± 2.7 µA/cm 2 ) in the presence of Cl -, but did not significantly affect I SC in its absence. These data indicate that cultured ADPKD cells can direct fluid transport in either the absorptive or the secretory direction, and that cAMP stimulates secretion and this secretion is accompanied by a net loss of cell solute. Inhibition of secretion by bumetanide or furosemide caused an additional loss of cell solute, including Cl -. The ionic transepithelial current induced by forskolin is dependent on the presence of Cl -. These data support the thesis that chloride secretion drives fluid secretion by cultured ADPKD cells.