Protective effect of inhibiting necroptosis on gentamicin-induced nephrotoxicity

Necroptosis is defined as a novel programmed cell necrosis that is mediated by receptor interacting serine-threonine protein kinase 1 (RIPK1) and Other related signals. Necrosis, Apoptosis and inflammation are commonly considered as the leading mechanism in acute kidney injury (AKI) induced by gentamicin (GEN), which is a useful Antibiotic for treating the Infection of Gram-negative Bacterial. However, the Necroptosis in the pathogenesis of GEN-induced AKI is unknown. In this study, to investigate the process and function of Necroptosis in GEN-induced AKI, NRK-52E and HK-2 cells and SD rats were used as the models. The necroptosis-related proteins, including RIPK1, RIPK3, Mixed Lineage Kinase domain-like (MLKL) and phosphorylated MLKL (p-MLKL), were all increasing time-dependently when GEN was continuously given. By using the RIPK1 Inhibitor necrostatin-1 (NEC-1) and RIPK3 Inhibitor (CPD42), the GEN-induced toxicity of tubular cells was alleviated. Moreover, it was validated that GEN-induced cell Apoptosis and inflammation were attenuated after treating with NEC-1 or CPD42, both in vivo and in vitro. When MLKL was knocked down by siRNA, NEC-1 and CPD42 can not further protect the damage of tubular cells by GEN. Although the using of pan-caspase inhibitor Z-VAD significantly decreased GEN-induced Apoptosis, it enhanced Necroptosis and slightly promoted the decreased cell viability in GEN-treated cells, with the protective effects weaker than NEC-1 or CPD42. Finally, in vitro minimum inhibitory concentration (MIC) tests and bacteriostatic ring studies showed that NEC-1 did not interfere with the Antibiotic effects of GEN. Thus, suppressing Necroptosis can serve as a promising strategy for the prevention of GEN-induced nephrotoxicity.