Establishment and characterization of an immortalized bovine glomerular endothelial cell line

Bovine subcultures (second passage) of glomerular endothelial cells (GEN) isolated from one-year-old kidney were successfully transfected by recombinant plasmids containing the simian virus (SV)-40 T antigen (Tag) using a lipofectin-mediated procedure. One cell clone was selected, propagated and characterized. This clone can be grown in RPMI 1640 medium supplemented with 10% fetal calf serum. The advantage of this cell line is the cultivation of bovine GEN without the addition of fibroblast growth factor or a coating of fibronectin or gelatin on the culture plate. More than 80 passages were achieved and the doubling time was 32 h. The Tag was easily identified in transfected-GEN by indirect immunofluorescence. These cells weakly expressed factor VIII-related antigen, slightly took up acetylated-low density lipoprotein and secreted a detectable amount of angiotensin-converting enzyme. Immunocytochemical staining for UAE-1 was also positive. Moreover, oncoproteins, such as Ki-67 and p53, were expressed in these cells. Cell cycle analysis by flow cytometry revealed that the percentages of G1, S, and G2/M stages in cycling transfected-GEN culture in RPMI 1640 medium supplemented with 10% fetal calf serum were 34%, 52.9%, and 13.1%, respectively. The conditioned medium from confluent transfected-GEN stimulated [3H]thymidine incorporation into glomerular mesangial cells. This cell line may provide a useful tool for examining modulators of mesangial cell growth. Thus this cell line is the first immortalized bovine GEN that retain the morphologic, phenotypic, and functional characteristics of bovine GEN.