Establishment and characterization of a rat glomerular endothelial cell line.

BACKGROUND

Primary cell cultures have been widely used for research purposes, but kidney glomerular and vascular endothelial cells and, particularly, stable endothelial cell lines from these have been difficult to obtain.

EXPERIMENTAL DESIGN

We used electroporation and lipofectin transfection to introduce various transforming constructs, and direct infection with an oncogenic adeno 31 (Ad31) virus to immortalize cells from freshly isolated rat kidney glomeruli and characterized the cells obtained at their 20th, 40th, 60th, and 80th passage for markers of various glomerular cell types.

RESULTS

Direct infection with an oncogenic adenovirus type 31 (Ad31) resulted in a stable cell line (over 120 passages) morphologically resembling epithelial/endothelial cells line (over 120 passages) morphologically resembling epithelial/endothelial cells. These cells were constantly positive for factor VIII-related antigen, podocalyxin and for OX-43 anti-rat endothelial cell antibodies, Bandeiraea simplicifolia (BSI-B4) lectin, and expressed receptors for acetylated low density lipoprotein. Anti-Thy 1.1 and anti-desmin antibodies recognizing glomerular mesangial cells and antibodies against O-acetyl GD3 and gp330 antigens of podocytes, as well as anti-thrombospondin and anti-cytokeratin antibodies identifying parietal epithelial cells failed to bind to our cells. These rat glomerular endothelial cells (RGE cells) showed decreased serum requirements for growth, changes in morphology, loss of contact inhibition, and loose adherence to growth support. The transforming E1A gene could not be found by Southern blotting or polymerase chain reaction amplification analysis. Neither did the RGE cells produce any adenoviral proteins tested by immunoprecipitation analysis.

CONCLUSIONS

The RGE cells represent a stable cell line with various characteristics of endothelial cells. Furthermore, they remain responsive to basic FGF and grow on type IV collagen and fibronectin matrices forming capillary-like structures. Thus, we consider RGE cells a cell line of rat glomerular endothelial origin.