Sp108aristolochic Acid Induces Endothelial Cell Toxicity and Vascular Dysfunction

Abstract

Introduction and Aims: Aristolochic acid nephropathy is characterized not only by marked tubular atrophy and interstitial fibrosis but also by severe peritubular capillaries rarefaction. Human umbilical vein endothelial cells (HUVECs) and rat aortic rings were then used to assess the possible vascular toxicity of aristolochic acid (AA). Methods: HUVECs were incubated during 48h with or without AA (50 or 100 µM). Cell viability was measured by MTT assay. The number of nuclei was counted by DAPI. Pecam adhesion molecule (CD31) cell expression was analyzed by immunofluorescence. We studied the endothelium-dependent relaxations to acetylcholine (ACh) in rat aortas incubated for 1h with 200 µM AA, in the presence or in the absence of superoxide dismutase (SOD). Endothelium independent relaxations to sodium nitroprusside (SNP) were also tested on preparations after endothelium denudation. Results: AA exposure resulted in a significant decrease in the total count of cells which exhibited a typical spindle shape (CD31+ cells/ total count [%] vs controls [100%]: 60.27 ± 13.50 with AA 50 µM and 53.70 ± 8.82 with AA 100 µM, respectively). The MTT assay showed a dose-dependent toxicity of AA (cell viability [%] vs controls [100%]: 70.26 ± 4.80 with AA 50 µM and 12.50 ± 3.26 with AA 100 µM, respectively). ACh-induced relaxations were significantly impaired in rat aortic rings pre-incubated with AA 200 µM: the AUC was 170.9 ± 8.9 in control conditions vs 236.0 ± 8.7 in rings incubated with AA ( p < 0.0001). This effect was reversed after washing of the aortic rings or in the presence of SOD (200 UI). The toxicity was also observed in smooth muscle cells (VSC) as the concentration-response curves to sodium nitroprusside were also modified. Conclusions: Our data show 1) a dose-dependent toxicity of AA on HUVECs 2) an impaired ACh-induced relaxation of aortic rings reversed by SOD, suggesting that superoxide anion is responsible for the AA-mediated endothelial dysfunction 3) some alterations in VSC but to a lesser extent than the endothelial function.