Treatment with Methylene Blue Increases Apoptosis of and Transmigration of Peripheral Blood Mononuclear Cells through Human Renal Cells

Objectives: Methylene blue (MB), an inhibitor of nitric oxide synthase and guanylate cyclase, is used to treat endothelial dysfunction in vasoplegic syndrome after cardiac surgery. As MB is suspected to contribute to renal failure, we examined its effects on human renal cells (huRC) in an in-vitro model.

Methods: HuRC were cultured and divided into groups that were exposed to 10, 30, or 60 µM MB for 30 or 120 minutes. Each of the groups was subdivided to yield 3 subgroups of which the first received no additional treatment, while inflammation was simulated in the other two. For this purpose, the second group was exposed to 100 ng/mL lipopolysaccharide B (LPS-B) before treatment with MB, and the third to 100 ng/mL LPS-B after MB. Transmigration of peripheral blood mononuclear cells (PBMC) through HuRC (transwell migration essay) and apoptosis (annexin-V detection kit) were determined.

Results: Treatment of HuRC with 10 µM MB for 30 minutes (p = 0.0587) and 120 minutes (p = 0.0157) increased apoptosis in comparison with controls. Statistical significance was also shown for controls vs.30 µM MB for 30 min (p = 0.0007), 30 µM MB for 120 minutes (p ≤ 0.0001), 60 µM MB for 30 min (p ≤ 0.0001) and 60 µM MB for 120 minutes (p ≤ 0.0001). Additionally, treatment with MB increased transmigration of PBMC through all subgroups of HuRC. A statistically significant increase in comparison with controls was demonstrated for 30 and 120 minutes of treatment with 10 µM MB (p = 0.0002 and p = 0.0010), 120 minutes of treatment with 30 µM MB (p = 0.0071) and 30 minutes of treatment with 60 µM MB (p = 0.0071). Treatment with LPS-B before as well as after exposure reduced the effect of MB on the transmigration of PBMC through HuRC.

Conclusions: Our data suggest dose and duration dependent harmful effects of MB on HuRC in that treatment with MB increases apoptosis of and transmigration of PBMC through HuRC. Longer exposure to MB as well as application of higher doses of MB increased apoptosis. The effect of MB on the transmigration of PBMC through HuRC appears to be modulated by inflammation.