Involvement of NADPH oxidases in the Na/K‑ATPase/Src/ROS oxidant amplification loop in renal fibrosis

The Na/K-ATPase/Src complex is reportedly able to affect reactive oxygen species (ROS) amplification. However, it has remained elusive whether NADPH oxidases (NOXs) are involved in this oxidant amplification loop in renal fibrosis. To test this hypothesis, interactions between oxidative features and Na/K-ATPase/Src activation were examined in a mouse model of unilateral urethral obstruction (UUO)-induced experimental renal fibrosis. Both 1-tert-butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2) and apocynin significantly attenuated the development of UUO-induced renal fibrosis. Apocynin administration attenuated the expression of NOXs and oxidative markers (e.g., nuclear factor erythroid 2-related factor 2, heme oxygenase-1,4-hydroxynonenal and 3-nitrotyrosine); it also partially restored Na/K-ATPase expression and inhibited the activation of the Src/ERK cascade. Furthermore, administration of PP2 after UUO induction partially reversed the upregulation of NOX2, NOX4 and oxidative markers, while inhibiting the activation of the Src/ERK cascade. Complementary experiments in LLC-PK1 cells corroborated the in vivo observations. Inhibition of NOX2 by RNA interference attenuated ouabain-induced oxidative stress, ERK activation and E-cadherin downregulation. Thus, it is indicated that NOXs are major contributors to ROS production in the Na/K-ATPase/Src/ROS oxidative amplification loop, which is involved in renal fibrosis. The disruption of this vicious feed-forward loop between NOXs/ROS and redox-regulated Na/K-ATPase/Src may have therapeutic applicability for renal fibrosis disorders.