Research Paper Volume 15, Issue 20 pp 10972—10995

Figure 5. Urea activates MMP2 leading to degradation of TJPs. (A) Western blotting indicating the abundance of claudin-5 and CD31 after indoxyl sulfate (IS), trimethylamine N-oxide (TMAO), and urea treatment of bEnd.3 cells. IS and TMAO did not influence the expressions of claudin-5 and CD31, while urea suppressed both proteins dose dependently after treating bEnd.3 cells for 24 h (n = 13, 10, 9, or 12 per group, respectively). Statistical analyses among multiple groups were performed using one-way ANOVA, followed by Turkey’s post-hoc test for parametric variables or the Kruskal–Wallis test followed by Dunn’s multiple comparisons test for nonparametric variables. The Jonckheere–Terpstra trend test was used to indicate potential protein expression trends in response to a concentration gradient of uremic solutes. (B) We treated the cells with urea and a MMP inhibitor marimastat at 5 μM as the final concentration, which is the first MMP inhibitor used for clinical trials to suppress tumor growth in various cancers. Western blotting showed that the suppression of claudin-5 expression was ameliorated with marimastat, whereas CD31 expression remained unchanged (n = 13 in non-marimastat group; n = 6 in marimastat-treated group). Data are presented as mean ± standard deviation of the mean. Normality was assessed with the Shapiro–Wilk test. P < 0.05 was considered statistically significant. CKD, chronic kidney disease; MMP2, matrix metalloproteinase-2.