FP282
FERROPTOSIS-MEDIATED CELL DEATH IS DECREASED BY CURCUMIN IN RENAL DAMAGE ASSOCIATED TO RHABDOMYOLYSIS Free

INTRODUCTION: Muscle damage (rhabdomyolysis) releases myoglobin (Mb) into the bloodstream and may induce acute renal injury (AKI). Once filtered by the kidney, Mb causes oxidative stress and tubular cell death. Several studies reported the presence of apoptosis in rhabdomyolysis, but it has been suggested that, besides apoptosis, other types of cell death may be involved in rhabdomyolysis-induced AKI. Oxidative stress plays an important role in this syndrome. Curcumin, a compound from the Curcuma longa, is a powerful antioxidant because of its inducer effect on Nrf2 (Nuclear factor (erythroid-derived 2)-like 2). The purpose of this study is to investigate the beneficial effects of curcumin on rhabdomyolysis-mediated renal damage, focusing on ferroptosis, an iron-dependent form of regulated nonapoptotic cell death.

METHODS: We performed an experimental model of AKI associated to rhabdomyolysis by the intramuscular injection of 50% glycerol (10mg/kg of weight) in 12-week-old male C57BL/6 mice and RIPK3-KO mice (RIPK3 is involved in necroptosis). Curcumin was injected intraperitoneally (1g/kg) two times: the day before and the same day of the injection of glycerol. Animals were sacrificed at 24 hours post-injection of glycerol. In other experiments, C57BL/6J mice were injected intraperitoneally with 5 mg/kg ferrostatin-1 (a ferroptosis inhibitor), 10 mg/kg zVAD (pan-caspase inhibitor), or vehicle (DMSO) 30 minutes before glycerol injection. Blood and kidney samples were collected to perform gene and protein expression studies by Real Time-PCR, western blot and immunohistochemistry. In addition, we carried out studies in murine tubular cells (MCTs) to study the molecular mechanisms involved in the protection of curcumin.

RESULTS: Rhabdomyolysis caused serum creatinine levels increase, endothelial damage, inflammatory chemokines and cytokines expression, alteration of redox balance (increased lipid peroxidation and decreased antioxidant defenses) and tubular cell death. Treatment with curcumin ameliorated all these pathological processes. While apoptosis or RIPK3-mediated necroptosis were activated in rhabdomyolysis, our results suggest a key role of ferroptosis. Thus, treatment with ferrostatin-1, a ferroptosis inhibitor, improved renal function in mice with rhabdomyolysis, whereas no beneficial effects were observed with the pan-caspase inhibitor zVAD or in RIPK3-deficient mice. In cultured renal tubular cells, Mb induced ferroptosis sensitive cell-death that was also inhibited by curcumin. In vitro studies showed that curcumin reduced Mb-mediated inflammation and oxidative stress by inhibiting the TLR4/NF-κB axis and activating the cytoprotective enzyme HO-1.

CONCLUSIONS: Our findings are the first to demonstrate the involvement of ferroptosis in rhabdomyolysis-associated renal damage, and its sensitivity to curcumin treatment. Therefore, curcumin may be a potential therapeutic approach for patients with this syndrome.