ROLE OF NAK-ATPASE AND POLCARITY DEFECTS IN ADPKD CYST F

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A defined culture system for the analysis of human autosomal dominant polycystic kidney disease (ADPKD) epithelia has been devised and characterized by this investigator during the last grant period. Comparisons made of monolayer cultures derived from individually microdissected ADPKD cysts and age-matched normal proximal (PST) and distal (CCT) tubules showed several abnormalities of growth, basement membrane synthesis, hormone response, and enzyme activities in ADPKD cells. An alteration of major consequence for cystic enlargement was increased activity of NaK-ATPase and mislocation to the apical cell surfaces in ADPKD cells. This could be responsible for reversed vectorial transport of sodium into cystic tubule lumens and lead to osmotic fluid accumulation. This possibility was confirmed experimentally in ADPKD monolayers grown on permeable membrane supports separating apical and basal compartments since 80% of 22Na was transported from the basal to apical media. The present proposal will focus on delineating the precise and detailed nature of these NaK-ATPase and membrane polarity defects. The hypothesis that ADPKD the NaK-ATPase expressed by ADPKD epithelia is abnormal will be tested by examining the kinetics of catalytic activity; molecular subunit composition; rates of subunit synthesis, cellular processing and degradation; messenger RNA levels and translational activity in vitro. To examine the full extent of membrane polarity defects: the distribution and activity levels of apical and basolateral enzymes and NHS-biotin labelling patterns will be determined. The hypothesis that altered location of NaK- ATPase to apical membranes is caused by defective sorting mechanisms in ADPKD will be tested by examination of the putative peptide signal sequence; and by examination of patterns of fatty acid (3H-myristate, 3H- palmitate and 14C-ethanolamine) labelling. To determine to what extent apical membrane alterations are due to defects in fatty acid acylation, susceptibility to specific cleavage of thioester-palmitate, thioester-amide and glycosyl phosphatidylinositol bonds will be tested. Whole tissues, cultured normal and ADPKD epithelial cells, apical and basolateral membrane vesicles will be used and subjected to pulse chase and NHS-biotin labelling protein extraction, characterization by SDS-PAGE, Western blotting and immunoprecipitation, enzyme assays, electron immuno-and cytochemical localization; RNA extraction; Northern analysis; and in vitro translation.