Crocin Attenuates Nrf2/ <scp>GPX4</scp> Mediated Ferroptosis in Obstructive Nephropathy by Selectively Targeting <scp>MKK4</scp>

Renal fibrosis is a critical pathogenesis of chronic kidney disease (CKD). Crocin, an active compound derived from saffron ( Crocus sativus L.), exhibits potent antioxidant and renal protection properties. However, the effects of crocin on renal fibrosis and its underlying mechanisms remain unclear. This study aimed to investigate the potential role and molecular mechanism of crocin in protecting renal cells from ferroptosis and improving renal fibrosis. The study utilized a unilateral ureteral obstruction (UUO) rat model and erastin-induced ferroptosis model in rat renal tubular epithelial (NRK-52E) cells to evaluate the effect of crocin on ferroptosis and renal fibrosis. Phospho-specific antibody microarray analysis was conducted to profile crocin-relevant phosphate proteins among 16 signaling pathways. Surface plasmon resonance (SPR) assessments were employed to explore molecular interactions between crocin and mitogen-activated protein kinase kinase 4 (MKK4). Co-immunoprecipitation (Co-IP) experiments were used to investigate interactions between MKK4 and Nrf2. Crocin attenuated renal fibrosis as demonstrated by reducing profibrotic markers in both the UUO model and erastin-treated NRK-52E cells. It also significantly inhibited ferroptosis in vivo and in vitro. Microarray profiling and molecular docking suggested that MKK4 was a critical regulator of ferroptosis and fibrosis. SPR analysis indicated that crocin inhibited p-MKK4 by directly interacting with MKK4. Co-IP further revealed that p-MKK4 interacted with Nrf2, leading to alleviating reactive oxygen species (ROS) levels and subsequently attenuating renal ferroptosis. Crocin exerts protective effects against renal fibrosis and ferroptosis via modulation of phospho-MKK4/Nrf2/ferroptosis signaling axis. These findings suggest crocin is a promising renoprotective agent for renal fibrosis, and MKK4 represents a potential target for ferroptosis-related kidney injury.