| Abstract: | ![]()
Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia–reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis (1–3). Necroptosis, the best-characterized pathway of regulated necrosis, involves activation of receptor-interacting protein kinase 3 (RIPK3)-mediated phosphorylation of mixed lineage kinase domain-like protein (pMLKL) and subsequent plasma-membrane rupture, which was demonstrated in several disease states, including ischemia–reperfusion injury (IRI) in all organs analyzed (2–6); however, none of these previous studies clearly investigated the mode of cell death in the primary parenchymal cells. Therefore, it remained possible that the protective effects reported upon application of the necroptosis inhibitor necrostatin-1 (Nec-1) and for RIPK3-ko mice involve vascular, nonparenchymal effects. This possibility has been ruled out in non-IRI settings by conditional tissue targeting of proteins involved in the prevention of spontaneously occurring necroptosis, such as RIPK1, and components that regulate its ubiquitinylation status [linear ubiquitinylation chain assembly complex (LUBAC), cellular inhibitors of apoptosis proteins (cIAPs)), caspase-8, and Fas-associated protein with death domain (FADD)] in the gastrointestinal tract (7, 8), the skin (9, 10), the liver (11), and immune cells (12, 13), all of which result in spontaneous RIPK3-mediated tissue necroptosis and inflammation (7–9, 11, 12, 14–17).Ferroptosis is an iron-dependent necrotic type of cell death that occurs due to lipid peroxide accumulation, which routinely is prevented by glutathione peroxidase 4 (GPX4), a glutathione-(GSH)-dependent enzyme, and therefore depends on the functionality of a glu/cys antiporter in the plasma membrane referred to as system Xc-minus (18–20). Ferroptosis has been reported to cause several diseases and may be interfered with in vitro by the small molecule ferrostatin-1 (Fer-1) (18); however, Fer-1 was suggested to have low in vivo functionality due to potential metabolic and plasma instability.In the present studies, we used inducible, conditional kidney tubule-specific genetic deletion of FADD and caspase-8, intravital microscopy, fresh isolation of primary kidney tubules, and four preclinical models of acute organ failure to further assess the relative roles of necroptosis and ferroptosis. We find that ferroptosis is of functional in vivo relevance in acute tubular necrosis and IRI, and we introduce, to our knowledge, the first ferroptosis inhibitor that is applicable for inhibition of ferroptosis in vivo. We conclude that specific combinatory therapies will be most promising for the prevention of clinically relevant IRI and that the nephron represents, to our knowledge, the first described tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. |
