Protective effect of inhibiting necroptosis on gentamicin‐induced nephrotoxicity

Abstract 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.