These studies are designed to increase our understanding of the pathogenetic mechanisms involved in the progression of renal cystic disease. We propose to use a cell culture model of renal cyst growth (the MDCK-cyst) to investigate the cellular mechanism involved in fluid accumulation within renal cysts. Our objective is to characterize the ionic transport systems responsible for fluid accumulation in this model. Specific hypotheses to be tested are: 1) sodium and chloride transport are abnormal in cystic epithelium; and 2) altered transport is responsible for the accumulation of fluid within the cyst lumen. Microelectrodes selective for Na, K, and C1 will be used to measure intracellular and intraluminal ionic activities in the MDCK cysts. To characterize the ion transport systems of cyst forming cells, activities will be measured in the presence of furosemide, ouabain, and amiloride. The relationship between transepithelial potential difference, transepithelial resistance, short-circuit current, and ionic movements will be studied on MDCK-cyst derived monolayers in Ussing chambers. Non-cyst cell monolayers will be studied in a similar fashion to allow a comparison of transport properties between normal and cyst forming epithelia. Ouabain binding studies and ATPase enzyme assays will be used to measure the activity and pump density of the Na-K' ATPase enzyme during cyst growth. The patch clamp method will be used to study ion channels in the apical membrane of both cyst and non-cyst formings cells. Conductances, selectivities, and gating kinetics of channels found in the apical membrane of both kinds of cell will be compared. Changes in intracyst volume will be measured and correlated with specific alterations in epithelial transport induced by transport inhibitors. Our studies will determine if altered ion and fluid transport occur in the MDCK-cyst model of renal cystic disease. The relationship between altered transport and luminal fluid accumulation will also be examined. Understanding of the intracellular events that occur during cyst growth in this model may illuminate the mechanisms underlying cyst growth in renal cystic diseases.
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