USP9X suppresses ferroptosis in diabetic kidney disease by deubiquitinating Nrf2 <i>in vitro</i>

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 <i>in vitro</i> 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 <i>in vitro</i>.