Isolation and characterization of rat glomerular endothelial cells

Glomerular endothelial cells (GECs) from the kidney are in close juxtaposition to other cell types, such as mesangial cells, and may respond to as well as release different cytokines that subsequently influence the function of mesangial cells.  Weak proliferation of GECs has been described in various types of glomerulonephritis and in transplant rejection.  In addition, experimentally induced glomerulonephritis in the rabbit produced by injection of antiendothelial antibodies has been characterized by a slight proliferation of endothelial cells and the formation of subepithelial immune deposits, suggesting that endothelial cells may be an immune target in specific types of glomerulonephritis.  Vascular remodeling with the formation of a neointima is a common feature of atherosclerosis and hypertension.  Although mainly vascular smooth muscle cells contribute to the observed hyperplasia seen in these conditions, there is also evidence that the endothelium contributes to the overall proliferation.  Thus, proliferation of GECs may contribute to structural damage of glomeruli, as seen in malignant hypertension.

1. Rat glomerular endothelial cells (GECs) were isolated from Sprague-Dawley rats.

2. Kidneys from two adult female rats (100 to 120 g body weight) were pooled and glomeruli isolated by differential sieving.

3. The resulting preparation contained greater than 95% glomeruli as judged by light microscopy.

4. Glomeruli were treated with solution of primary cell isolation for 20 minutes and washed twice in primary cell culture system with 10% fetal calf serum, 2 mmol/L supplemental glutamine, 5 µg/mL insulin, 100 U/mL penicillin, and 100 µg/mL streptomycin.

5. Fifty microliters of the cell suspension was then plated into each well of a 96-well cell culture plate previously covered with fibronectin.

6. The microtiter plates were screened by phase-contrast microscopy for the presence of cells with a cobblestone appearance, which is one characteristic feature of cultured endothelial cells.

7. Cells fulfilling this criterion were subsequently cloned twice by limiting dilution, and a homogeneous cell line was obtained.

8. All experiments, cells between passages 4 and 10 were used.

9. GECs were directly photographed in cell culture flasks by phase contrast.

10. Cells were further characterized by immunofluorescence.

11. For these studies, cells were grown in glass slide chambers until they were subconfluent.

12. The cells were fixed at -20°C in acetone for 10 minutes before staining.

13. Cells were stained with the following antibodies with the use of indirect immunofluorescence: polyclonal anti–CD 31 and anti–factor VIII.

References:
1. Ballermann BJ. Regulation of bovine glomerular endothelial cell growth in vitro. Am J Physiol. 1989; 256: C182-C189.
2. Laulajainen T, Julkunen I, Haltia A, Knuutila S, Miettinen A, Holthöffer H. Establishment and characterization of a rat glomerular endothelial cell line. Lab Invest. 1993; 69: 183-192.