Phenytoin inhibits necroptosis

Abstract

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Necroptosis in Acute Kidney Injury

“…Screening FDA-approved drugs also identified also phenytoin as necroptosis inhibitor acting on RIPK1 kinase domain in vitro and in vivo [15]. Accordingly, phenytoin attenuated post-ischemic AKI in mice [15]. Wogonin displayed similar efficacy on RIPK1 modulation in vitro and attenuated cisplatin-induced AKI in mice [16].…”

“…Indeed, in vivo sorafenib tosylate inhibited TNF-induced systemic inflammation as well as post-ischemic AKI in mice [14]. Screening FDA-approved drugs also identified also phenytoin as necroptosis inhibitor acting on RIPK1 kinase domain in vitro and in vivo [15]. Accordingly, phenytoin attenuated post-ischemic AKI in mice [15].…”

“…Therefore, Mlkl- deficient mice are currently the ultimate experimental tool to generate undisputable evidence for an involvement of necroptosis in AKI. Mlkl- deficient mice display a consistent attenuation in the following experimental AKI models: post-ischemic AKI (Table 1) [3, 15], cisplatin-induced AKI [8], oxalate nephropathy [10], and MPO-ANCA-induced glomerular injury [18]. The fact that lack of MLKL had a much lesser effect on survival upon post-ischemic AKI as compared to ­ Ripk1 KD and Ripk3 –/– mice suggests that the contribution of necroptosis in post-ischemic AKI may indeed be overestimated by using less specific tools such as Ripk3- deficient mice or Nec-1 [3].…”

Cell Death Pathways Drive Necroinflammation during Acute Kidney Injury

“…pMLKL, by unknown means that involve the ESCRT-III complex [3], results in plasma membrane rupture. With respect to AKI, RIPK3-deficient mice [4] and MLKL-deficient mice [5] are protected from renal ischemia-reperfusion injury. Inhibition of necroptosis, therefore, appears to be a promising therapeutic target, but highly specific kinase inhibitors of RIPK1 (Nec-1s, ponatinib) did not protect from IRI in our hands (A.L., unpublished data).…”

The clinical relevance of necroinflammation—highlighting the importance of acute kidney injury and the adrenal glands

“…Several pathways of regulated necrosis contribute to the pathogenesis of SIRS and sepsis. This has been demonstrated for necroptosis [112,118,[142][143][144][145][146][147] and pyroptosis [148][149][150][151].…”