Transepithelial water permeability in an in vitro model of renal cysts

Abstract

Renal cysts develop from microscopic tubules and may enlarge progressively several thousandfold. Sustained epithelial proliferation, intracavitary fluid accumulation, and extracellular matrix remodeling are central elements in a multistep process that leads to the formation and enlargement of cysts. MDCK cells suspended within medium-hydrated collagen gels grow to form spherical, monolayered, fluid-filled cysts that enlarge steadily. Vasopressin and other agents that increase intracellular levels of cAMP stimulate the rate of MDCK cyst growth and net fluid/solute secretion when added to defined medium in vitro. In this model, net fluid secretion is the only means by which fluid can accumulate within the cyst cavity. We used this cyst-forming line of epithelial cells to evaluate several membrane transport properties that are important in the coupled movements of solute and water in the process of secretory fluid transport. Individual cysts were microdissected from collagen gels, held by a micropipet in a thermostated chamber, and examined at a high magnification by video microscopy. Transepithelial water flow was initiated by rapidly exchanging the bath medium with hyperosmotic solutions. Net water flux, Jv, determined from the initial rate of decrease in cyst diameter, was proportionate to the transmembrane osmotic gradient of NaCI or raffinose; the reflection coefficient for NaCI was indistinguishable from 1.0. Osmotic water permeability (cm3/cm2/osm/ min × 10-6) was 739 ± 99 (N = 11) in medium augmented by an NaCI concentration of 100 mosmol/ kg. Hydraulic conductivity (Pf), estimated in control cysts, was 6.8 ± 0.9 μm/s, a value similar to that of medullary and cortical thick ascending limbs of Henle. Arginine vasopressin (10 mU/mL), methylisobutylxanthine (10-4 M), and 8-Br-cAMP (10-4 M) had no effect on water permeability. Removal of prostaglandin E1 from defined medium and addition of indomethacin did not increase water permeability in response to vasopressin. We conclude that fluid accumulates within MDCK cysts by net solute and water secretion across an epithelium of relatively low water permeability. Vasopressin stimulates solute fransport, but the hormone does not regulate water permeability in MDCK cysts. These cysts functionally resemble thick ascending limbs of Henle's loop in the coupling of salt and water transport, although the direction of net fluid movement is opposite in the two epithelia.