Isorhynchophylline protects against ferroptosis in diabetic nephropathy by activating Nrf2

BACKGROUND: Diabetic nephropathy (DN) is a life-threatening complication of diabetes mellitus (DM) and the leading cause of end-stage renal disease. Ferroptosis, a novel iron-dependent mode of cell death, has been identified to participate in the pathogenesis of DN. Isorhynchophylline (IRN) is a tetracyclic indole oxide alkaloid present in Uncaria rhynchophylla (Rubiaceae), which shows protective effects against diabetic encephalopathy and acute kidney injury. Our study intends to determine whether IRN ameliorates DN progression through inhibiting ferroptosis. METHODS: The db/db diabetic mice and high glucose (HG)-stimulated human kidney tubular epithelial HK-2 cells were used to explore the potential therapeutic value of IRN in vivo and in vitro. Blood glucose levels, body weight, kidney weight, serum creatinine (SCr), blood urea nitrogen (BUN), and albumin-to-creatinine ratio (UACR) were detected to assess diabetic symptoms and renal functions in db/db mice. Hematoxylin-eosin (H&E) and periodic acid-Schiff staining (PAS) staining were performed to observe renal pathohistological changes in diabetic mice. Iron contents as well as malondialdehyde (MDA) and glutathione (GSH) in mouse tissue homogenates and HK-2 cell supernatants were examined to assess iron accumulation and oxidative stress. The levels of ferroptosis-related proteins and Nrf2/HO-1 signaling-related proteins as well as Nrf2 nuclear translocation in mouse renal tissues and HK-2 cells were detected by western blotting and immunofluorescence staining. RESULTS: IRN administration alleviated diabetic symptoms and improved renal functions in diabetic mice. IRN mitigated renal histologic damage, including glomerular hypertrophy, mesangial matrix accumulation, capillary basement membrane thickening, and thylakoid stroma expansion in diabetic mice. IRN treatment inhibited ferroptosis in both diabetic mice and HG-induced HK-2 cells by reducing iron content and MDA levels, elevating GSH levels, upregulating the protein levels of FTH-1, GPX4, and SLC7A11, and downregulating the protein levels ofTFR-1 and NCOA4. Mechanistically, IRN treatment enhanced Nrf2 and HO-1 protein levels and Nrf2 nuclear translocation in renal tissues of diabetic mice and HG-exposed HK-2 cells. CONCLUSION: IRN plays a renoprotective role in DN by suppressing ferroptosis, which might be ascribed to the Nrf2/HO-1 pathway activation, highlighting the potential therapeutic application of IRN for DN treatment.