Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury

Summary Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated γ-glutamyltranspeptidase in >95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectinArachis hypogaea (rat proximal tubule) and negatively forLotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, γ-glutamyltranspeptidase and leucine aminopeptidase, and Na⁺-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium. This project was supported by grant 2-R01-DK15440-16A1 from the National Institutes of Health, Bethesda, MD, and by grant N0001 4-88-K-0427 from the Department of the Navy, Washington, DC.