Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells

Human subcultures (third passage) of glomerular visceral epithelial cells (VEC) isolated from one month old kidney were successfully transfected by two recombinant plasmids containing the cloned oncogenes from the simian virus 40 large T antigen and H-ras gene. One postcrisis cell clone (56/10 A1) was selected, propagated and characterized. One hundred percent of the 56/10 A1 cells (current passage greater than 100th; doubling time 30 hrs) expressed the nuclear T-SV40 antigen assayed by IF; the cells failed to express H-ras (RNA blot analysis). Immortalized cells were morphologically and phenotypically compared to parental cell type (third passage). Phenotypic characterization of the 56/10 A1 cells was achieved using indirect immunofluorescence (IF) and immunogold silver staining coupled to bright field and epipolarization microscopy. Both parental and 56/10 A1 cells displayed positivity for cytokeratin, CALLA and PHM5, whereas von Willebrand factor was not detected in the two cell types. Since we have previously shown that human glomerular epithelial cells in culture synthetize plaminogen activator (PA) related compounds, we investigated the secretion pattern of these products in parental and transfected cells. Zymographic analysis of secreted PA related compounds revealed production of free urokinase (u-PA) and type 1 plasminogen activator inhibitor (PAI-1) complexed to tissular plasminogen activator (t-PA). Finally, in the transfected cells, increased cGMP generation under atrial natriuretic factor (ANF) stimulation agreed with previous work performed on nontransfected human VEC. In conclusion, the establishment of a human permanent cell line which retains most of the phenotypic features of parental glomerular visceral epithelial cells should represent a new tool to study human glomerular cell functions.