Trimethylamine N-oxide Can Trigger Cardiovascular Disease by Affecting Endothelial Cell-to-Cell Junctions through ZO-1 Expression Decreasing

Background: Trimethylamine N-oxide (TMAO) is a uremic toxin that is associated with the development of cardiovascular disease in patients with chronic kidney disease (CKD). At uremic concentrations, TMAO-induced vascular damage is characterized by increased expression of inflammatory markers and vascular calcification. However, there is limited understanding regarding whether this uremic toxin adversely affects endothelial cell-cell junctions and permeability. This study aims to evaluate the effects of TMAO on the expression of Zonula occludens-1 (ZO-1), a protein component of tight junctions that plays a crucial role in regulating endothelial permeability. Methods: Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed to TMAO at normal (2.83 mg/L) and uremic concentrations (7.49 mg/L) for 24 hours. ZO-1 gene expression was assessed by RT-qPCR, while its protein levels were evaluated by western blotting and immunofluorescence. Results: ZO-1 gene expression showed no significant difference between treatments. However, we observed a significant reduction in ZO-1 protein levels in endothelial cells exposed to TMAO at uremic concentrations compared to control cells (untreated) (Figure 1A). Immunofluorescence analysis also showed a reduction in ZO-1 immunostaining in subconfluent endothelial cells after exposure to TMAO (Figure 1B). Figure's subtitle: Figure 1 - ZO-1 protein levels in endothelial cells exposed to TMAO at normal (TMAOn) and uremic (TMAOu) concentrations for 24 hours. (A) Western blotting, Kruskal-Wallis test: **P<0.01. (B) Immunofluorescence for ZO-1 (green) and nuclear staining (DAPI, blue). The scale bar indicates 50 μm, magnification 600x. Conclusion: Our data demonstrated a reduction in ZO-1 levels in endothelial cells, which may be related to increased endothelial permeability. Alterations in the endothelial barrier might contribute to the onset of cardiovascular diseases, notably atherosclerosis, which is highly prevalent among patients with CKD. Funding: Government Support – Non-U.S.