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The established renal epithelial cell line LLC-PK1 (proximal tubule) started to form multicellular spheroids within 24 h when grown in agar overlay culture. The spheroids, average diameter 100 to 350 microns, were free-floating with a butterfly-like structure due to the formation of several hollow microspheres. The microspheres were lined with polarized epithelial cells that had an abundance of microvilli protruding into the external medium and a well developed vacuolar apparatus, including coated pits, endocytotic vacuoles, and lysosomes. The microspheres were sealed between lumen and the surrounding medium by tight junctions and fluctuated in size due to fluid being transported in an apical-to-basal direction. Vasopressin was found to stimulate this transport, whereas the addition of ouabain or HgCl2 inhibited both spheroid growth and fluctuation in size with time. Biochemical assays of brush-border and lysosomal marker enzymes demonstrated an increase in enzyme activity during spheroid formation and growth. The most dramatic changes were observed for dipeptidyl peptidase IV (two- to threefold after 1 d and 53.5-fold after 15 d), reflecting the cellular polarization and brush-border formation during spheroid formation. When the typical lysosomal enzymes were compared, the activity of peptide bond splitting enzymes increased earlier than others. In conclusion, LLC-PK1 spheroids capable of forming microspheres represent an in vitro manifestation of specialized epithelial properties maintained in cell culture, thus providing a tool for studying renal physiologic mechanisms at a cellular level.

Biochemical and ultrastructural characterization of fluid transporting LLC-PK1 microspheres.