Anti-MLKL monoclonal antibody Made to order

Paraquat is a highly toxic prooxidant that triggers oxidative stress and multi-organ failure including that of the heart. To date, effective treatment of paraquat toxicity is still not established. Necroptosis, a newly discovered form of programmed cell death, was recently shown to be strongly associated with cardiovascular disease. Receptor interaction proteins 1 (RIP1), receptor interaction proteins 3 (RIP3), and mixed lineage kinase domain like (MLKL) are key proteins in the necroptosis pathway. Necrostatin-1 (Nec-1) is a specific inhibitor of necroptosis which acts by blocking the interaction between RIP1 and RIP3. In the present study, we studied the effect of Nec-1 on paraquat-induced cardiac contractile dysfunction and reactive oxygen species (ROS) production in the heart tissues using a mouse model. Our results revealed impaired contractile function, deranged intracellular Ca2+ handling and echocardiographic abnormalities in mice challenged with paraquat. We further found enhanced expressions of RIP1, RIP3, and MLKL along with overproduction of ROS in mice heart tissues. Nec-1 pre-treatment prevented cardiac contractile dysfunction in paraquat-challenged mice. Furthermore, Nec-1 reduced RIP1-RIP3 interaction, down-regulated the RIP1-RIP3-MLKL signal pathway, and dramatically inhibited the production of ROS. Collectively, these findings suggest that Nec-1 alleviated paraquat-induced myocardial contractile dysfunction through inhibition of necroptosis, an effect which was likely mediated via the RIP1-RIP3-MLKL signaling cascade. Further, ROS appeared to play an important role in this process. Thus, this process may represent a novel therapeutic strategy for the treatment of paraquat-induced cardiac contractile dysfunction.

Background Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. Methods Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. Results Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk32/2 mice significantly attenuated gentamicin-induced AKI. Conclusions A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.