USP9X suppresses ferroptosis in diabetic kidney disease by deubiquitinating Nrf2 in vitro.

Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates many critical genes associated with iron storage and transportation, the activity of which is influenced by E3 ligase-mediated ubiquitination. We wondered whether there is a deubiquitinase that mediates the deubiquitination of Nrf2 to stabilize Nrf2 expression and further prevent diabetic kidney disease (DKD). High glucose (HG) was applied to induce an in vitro model of DKD. The effects of HG on HK-2 cell viability, apoptosis, Fe²⁺ level, Nrf2, and ubiquitin-specific protease 9X (USP9X) were assessed by cell counting kit-8 (CCK-8) assay, flow cytometry, iron assay, and Western blot. The direct interaction between Nrf2 and USP9X was analyzed using co-immunoprecipitation and ubiquitination assay. After transfection and ferrostatin-1 (Fer-1) intervention, Nrf2 and USP9X levels, cell viability, apoptosis, and Fe²⁺ level were tested again. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) contents, and ferroptosis-related markers were assessed by ROS assay kit, ELISA, and Western blot. HG reduced cell viability and levels of USP9X and Nrf2, while elevating apoptosis and Fe²⁺ level. An interaction between USP9X and Nrf2 has been verified and USP9X deubiquitinated Nrf2. Nrf2 up-regulation augmented the viability, GSH content, and ferroptosis-related protein expressions, while suppressing the apoptosis, Fe²⁺ level, MDA, and ROS content in HG-mediated HK-2 cells, which was reversed by USP9X silencing. Fer-1 offset the combined modulation of Nrf2 and siUSP9X on HG-induced HK-2 cells. USP9X mediates Nrf2 deubiquitinase to hamper the ferroptosis in DKD in vitro.