Chemical modification of cell proliferation and fluid secretion in renal cysts

Abstract

We used an in vitro model, MDCK cyst, to determine the extent to which pharmacologic compounds known to inhibit plasma membrane solute transport mechanisms could alter the enlargement of renal epithelial cysts. Solitary MDCK cells cultured within collagen gel undergo clonal growth to form true epithelial cysts in which a single layer of polarized cells (apex toward lumen) encloses a fluid-filled cavity. Repeated observations by light microscopy were used to quantitate the rate of cyst growth in diameter, and demonstrated that cyst enlargement involved an increase in cell number (proliferation) and a net increase in intracystic volume (fluid secretion). Intracyst pressure was greater than the interstitium (6.7 mm H2O +/- 3.1 SD), indicating that fluid entry was secondary to net solute accumulation. Amiloride and seven amiloride analogs that inhibited to different degrees conductive Na+ transport, Na+-dependent H+ transport and Na+-dependent Ca++ transport reversibly decreased the rate of cyst enlargement. The effectiveness of these agents to retard cyst enlargement correlated with their relative potencies to inhibit Na+-dependent Ca++ transport. Morphologic examination indicated that amiloride and amiloride analogs decreased cell proliferation and fluid secretion to the same degree. Ouabain and vanadate (Na+K,ATPase inhibitors), and L-645,695 (Na+-dependent Cl-/HCO3- inhibitor) potently slowed cyst expansion. In contrast to amiloride and amiloride analogs, these agents caused an unusual degree of cellular stratification within the cyst walls, a finding consistent with the notion that fluid secretion was inhibited to a greater extent then cellular proliferation. We conclude that chemical inhibitors of primary and secondary active solute transport can diminish or halt the enlargement of epithelial cysts in vitro by decreasing the rate of cellular proliferation and/or net fluid secretion.

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

“…When ADPKD cells are seeded within a hydrated collagen gel matrix, spherical microcysts form as the cells proliferate and transepithelial fluid secretion fills the lumen (11). The effect of glibenclamide on the forskolin-stimulated I sc suggested the possibility that the drug might slow the expansion of these microcysts.…”

“…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).…”

“…The gel was then covered with DME-F12 media containing 25 ng/ml epithelial growth factor (EGF) and 5 M forskolin. These two agents induce cyst formation and growth (11). After 10 to 12 d of incubation, the plate was placed on the stage of an inverted microscope and, using ϫ40 magnification, the number of cysts in each well was counted and the diameter of each cyst was measured using an ocular scale in which one division ϭ 25 m at ϫ40.…”

“…The methods for inducing cultured epithelial cells to form cysts have been described previously (10,11). Briefly, ADPKD cells were suspended in ice-cold type I collagen (Vitrogen; Collagen Corp, Palo Alto, CA), and 0.1 ml of the suspension, containing 1000 cells, was added to wells of a 96-well plate (Falcon; growth area of each well, 0.32 cm 2 ).…”

cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia

“…In addition to roles in regulating renal fluid and ion transport, cAMP and/or PKA modulate proliferation in cell cultures derived from throughout the kidney: human proximal tubular cells (24,28); proximal tubular epithelial cell lines MCT (71), LLC-PK1 (5,71), and PKSV-PCT (68); glomerular mesangial cells (7,33); Madin-Darby canine kidney (MDCK) cell lines (21,24,72,73); immortalized human kidney epithelial cell line IHKE-1 (3); human embryonic kidney cell line HEK293 (56); and renal cancer cells (8). Because cAMP can induce proliferation and formation of renal epithelial cysts in vitro (21), and abnormally increased principal cell proliferation is a factor in collecting duct cyst formation and expansion in a developmental renal disease, autosomal recessive polycystic kidney disease (ARPKD) (39), cAMP could play a role in cell proliferation in normal developing principal cell epithelium.…”