The nephrotoxicity of cyclosporin A (CSA) after chronic treatment is well known and includes in later stages tubular atrophy associated with interstitial fibrosis. In order to examine whether interstitial ñbrosis due to CSA treatment in vivo is related to a hyperproliferative activity of fibroblasts, the effects of CSA on the growth characteristics of cultured human skin fibroblasts (HUSF) were investigated at CSA concentrations ranging from 10 ng/ ml to 50 µg/ml. We found that CSA at concentrations higher than 7.5 µg/ml inhibited cell proliferation (p < 0.05 at concentrations above 5 µg/ml; n = 3) and cloning efficiency (p < 0.05 at concentrations above 5 µg/ml; n = 3) in a dose-dependent manner and caused a promotion of cell attachment at concentrations above lOµg/ml (p < 0.05; n = 4), but did not influence cell spreading. At lower concentrations CSA-treated HUSF did not differ in their growth characteristics from the corresponding controls. A 50% inhibition of proliferation was calculated by extrapolation for a CSA concentration of 70 µg/ml for HUSF. The inhibition of HUSF proliferation was reversible even at the highest CSA concentration of 50 µg/ml. Under the same experimental conditions, a 50% inhibition of proliferation was observed for Madin-Darby canine kidney (MDCK) cells to be 5.5 µg/ml, e.g. at a 15-fold lower CSA concentration. Moreover, CSA caused a dose-dependent and reversible cell elongation of HUSF and a significant increase in the average cell diameter from 19.2 ± 0.3µm (control; mean ± SEM, n = 4) to 22.2 ± 0.2 µm for 50 µg/ml CSA (mean ± SEM, n = 4) and in median cell volume from 4,21·± 160 fl (control; mean ± SEM, n = 4) to 7,020 ± 190 fl for 50 µg/ ml CSA (mean ± SEM; n = 4). These alterations described above were not correlated with a cytotoxic effect as checked by a fluorescent staining for cell vitality. Alterations in the organization of cytoskeletal components such as stress fibers, intermediate-sized filaments and microtubules directly due to CSA treatment were not observed. In contrast, the amount of fibronectin present on the cell surface was considerably increased by CSA. Although HUSF in culture do not respond to CSA treatment by an increased proliferative activity, they are much less affected by CSA than other cell types (i.e MDCK cells). Thus, the increase in cell diameter and cell volume, respectively, and the increased amounts of surface fibronectin might explain the development of interstitial fibrosis in the kidney of humans and animals after chronic CSA treatment.

Keywords:
Cyclosporin A, Fibroblasts, Cell culture
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© 1994 S. Karger AG, Basel
1994
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