DIFFERENTIATION OF RENAL EPITHELIAL CELLS IN CULTURE

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The LLC-PK1 cell line, which develops properties of renal proximal tubule in culture and the MDCK cell line, which exhibits renal distal tubule characteristics are longterm polarized epithelial cell lines of renal origin. Expression of an array of differentiated functions develops at cell confluence and can be accelerated by chemicals such as hexamethylene bisacetamide, known as differentiation inducers. We will characterize the regulation of microvillar hydrolases as markers of differentiation in LLC-PK1 cells in response to cell confluence and chemical inducers, with particular emphasis on the two most potent inducers, hexamethylene bisacetamide and 5-azacytidine, and the regulation of trehalase and maltase activities, which we have shown to be inducible by hexamethylene bisacetamide. Antisera directed against purified trehalase will be used in biosynthetic labelling and immunoprecipitation studies, immunoelectroblot analysis and fluorescence histochemistry studies in order to probe the basis for induced expression of trehalase activity. Physiological variables such as cell confluence, culture substratum, hormones, cyclic nucleotide levels and nutrients will be tested for effects on expression of differentiated markers. Our variant clonal sublines which are altered in response to inducers will be characterized and new variant cell lines isolated. Additional inducer -responsive differentiation markers will be identified in MDCK and LLC-PK1 cells in order to determine if differentiated properties are co-ordinately or individually regulated. The fundamental goal of this work is to define and reproduce normal, programmed epithelial developmental and morphogenic sequences of events in cell culture. These studies are relevant to our understanding of the cellular and developmental biology of the kidney, as well as the phenomenon of aging, which can be viewed as a programmed and intrinsic part of normal development, modulated by stochastic environmental influences.