HUMAN FIBROBLASTS IN CULTURE EXHIBIT A LOW-SENSITIVITY AGAINST CYCLOSPORINE-A TREATMENT

The nephrotoxicity of cyclosporin A (CSA) after chronic treatment is w ell known and includes in later stages tubular atrophy associated with interstitial fibrosis. In order to examine whether interstitial fibro sis due to CSA treatment in vivo is related to a hyperproliferative ac tivity of fibroblasts, the effects of CSA on the growth characteristic s of cultured human skin fibroblasts (HUSF) were investigated at CSA c oncentrations ranging from 10 ng/ml to 50 mu g/ml. We found that CSA a t concentrations higher than 75 mu g/ml inhibited cell proliferation ( p<0.05 at concentrations above 5 mu g/ml; n = 3) and cloning efficienc y (p<0.05 at concentrations above 5 mu g/ml; n = 3) in a dose-dependen t manner and caused a promotion of cell attachment at concentrations a bove 10 mu g/ml (p<0.05; n = 4), but did not influence cell spreading. At lower concentrations CSA-treated HUSF did not differ in their grow th characteristics from the corresponding controls. A 50% inhibition o f proliferation was calculated by extrapolation for a CSA concentratio n of 70 mu g/ml for HUSF The inhibition of HUSF proliferation was reve rsible even at the highest CSA concentration of 50 mu g/ml. Under the same experimental conditions, a 50% inhibition of proliferation was ob served for Madin-Darby canine kidney (MDCK) cells to be 5.5 mu g/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 inc rease in the average cell diameter from 19.2+/-0.3 mu m (control; mean +/- SEM, n = 4) to 22.2 +/-0.2 mu m for 50 mu g/ml CSA (mean +/- SEM, n = 4) and in median cell volume from 4,210+/-160 fl (control; mean /- SEM, n = 4) to 7,020+/-190 fl for 50 mu g/ml CSA (mean +/- SEM; n = 4). These alterations described above were not correlated with a cyto toxic effect as checked by a fluorescent staining for cell vitality. A lterations in the organization of cytoskeletal components such as stre ss fibers, intermediate-sized filaments and microtubules directly due to CSA treatment were not observed. In contrast, the amount of fibrone ctin present on the cell surface was considerably increased by CSA. Al though 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 fibron ectin might explain the development of interstitial fibrosis in the ki dney of humans and animals after chronic CSA treatment.