Basic research in enhancement of permeability in endothelial cells for the development of a bioartificial glomerulus

Abstract

For the development of a bioartificial glomerulus, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it using an actin-microfilament polymerization inhibitor, cytochalasin B (CyB), to enlarge fenestrae diameter of the endothelial cells. CyB was added for 2h at a final concentrations of 20, 30, 40 and 50μg/mL to culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). After CyB treatment cell viability was evaluated with Cell Counting Kit-8, and fenestrae diameter of the cells was measured by scanning electron microscopy (SEM), and ultrafiltrate rate through the cell-attached semipermeable membrane was detected under 130mmHg of hydrostatic pressure at days 1, 4, 7. After exposure of platelet-rich plasma, platelet adhesions on CyB-treated and nontreated cells were determined by SEM. Fifty μg/mL of CyB was the best concentration in terms of the cell viability and enhanced ultrafiltrate rate. Scanning electron microscopy demonstrated a larger average diameter of fenestrae on CyB-treated endothelial cells at 50μg/mL of CyB, compared with non-treated cells and CyB-treated cells at 20, 30, and 40μg/mL of CyB. This phenomenon also lasted for at least 7 days. Under 130mmHg hydrostatic pressure, the CyB-treated cells at 50μg/mL of CyB produced significantly more ultrafiltration than the non-treated control group and CyB-treated group at other CyB concentrations, and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB-treated group compared with that in the non-treated control group. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These findings indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in a bioartificial glomerulus.