Chloride and fluid secretion by cultured human polycystic kidney cells

Abstract

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/cm2 inner surface area). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) plus 3-isobutyl-1-methlyxanthine (IBMX) induced a rapid reversal in the net movement of fluid to secretion (6.79 +/- 1.28 nl/min/cm2). Bumetanide reversibly reduced fluid secretion to 0.95 +/- 0.60 nl/min/cm2. 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 (ISC) induced by forskolin in the presence and absence of external Cl-. Forskolin increased ISC (from 8.9 +/- 2.7 to 10.6 +/- 2.7 microA/cm2) in the presence of Cl-, but did not significantly affect ISC 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.

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

“…When the cells were grown to confluence on filter supports, cell polarity of the cultures was confirmed by the achievement of a transepithelial electrical resistance by the cell monolayer, measured with an EVOM volt ohmmeter (World Precision Instruments, Sarasota, FL) as described previously (31). In culture, both ADPKD and NHK cells are able to establish a monolayer, form tight junctional complexes, and develop a transepithelial electrical potential difference, and they carry out an active transepithelial transport of salt and fluid (27,30,32). As previously shown, these cells are epithelial in nature, and the majority of the cells stain positive for specific lectin markers for the collecting duct and distal nephron, such as Arachis hypogaea and Dolichos biflorus agglutinin (26).…”

“…convoluted and collecting tubules, on the basis of lectin markers and the existence of transport mechanisms that were expressed in those nephron segments (26,30,32) (Figure 1). The similar origin of the cells suggests that the changes in ouabain response that were observed are a consequence of the cellular phenotype of normal renal epithelial cells versus mural cystic cells and not due to differences in the progenitor cell type.…”

“…A protocol for the use of discarded human kidney tissues was approved by the institutional review board at KUMC. Primary cultures were prepared as described previously (30). Cells were seeded and grown in DMEM/F12 supplemented with 5% heatinactivated FBS, 100 IU/ml penicillin G and 0.1 mg/ml streptomycin, 5 g/ml insulin, 5 g/ml transferrin, and 5 ng/ml sodium selenite (ITS).…”

Sulfonylurea-Sensitive K+ Transport is Involved in Cl− Secretion and Cyst Growth by Cultured ADPKD Cells

“…This procedure has been described in detail elsewhere (6,7). Steps were taken to reduce the contamination of fibroblasts during the dissection period and during the separation of the epithelial cells from the connective tissue (8). ADPKD cells were maintained in a 1:1 mixture of Dulbecco modified Eagle's medium and Ham F12 (DME/F12; JRH Biosciences, Lenexa, KS) supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT), 110 IU/ml penicillin G, 0.1 mg/ml streptomycin (P/S; Sigma Chemical, St. Louis MO), and 5 g/ml insulin, 5 g/ml transferrin, and 5 ng/ml sodium selenite (ITS; Biomedical Products, Bedford, MA) on plastic until they were harvested by trypsinization.…”

Cyst formation following disruption of intracellular calcium signaling

“…Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon shortterm culture of Madin-Darby canine kidney (MDCK) cells (6)(7)(8)(9)(10)(11)(12) or cells from patients (13)(14)(15)(16)(17)(18) or PC1-null mice (19,20; for review, see ref. 21).…”