Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.

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Necrosis of a cell is defined by the loss of its plasma membrane integrity. Morphologically, necrosis occurs in several forms such as coagulative necrosis, colliquative necrosis, caseating necrosis, fibrinoid necrosis, and others. Biochemically, necrosis was demonstrated to represent a number of genetically determined signalling pathways. These include (i) kinase-mediated necroptosis, which depends on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL); (ii) gasdermin-mediated necrosis downstream of inflammasomes, also referred to as pyroptosis; and (iii) an iron-catalysed mechanism of highly specific lipid peroxidation named ferroptosis. Given the molecular understanding of the nature of these pathways, specific antibodies may allow direct detection of regulated necrosis and correlation with morphological features. Necroptosis can be specifically detected by immunohistochemistry and immunofluorescence employing antibodies to phosphorylated MLKL. Likewise, it is possible to generate cleavage-specific antibodies against epitopes in gasdermin protein family members. In ferroptosis, however, specific detection requires quantification of oxidative lipids by mass spectrometry (oxylipidomics). Together with classical cell death markers, such as TUNEL staining and detection of cleaved caspase-3 in apoptotic cells, the extension of the arsenal of necrosis markers will allow pathological detection of specific molecular pathways rather than isolated morphological descriptions. These novel pieces of information will be extraordinarily helpful for clinicians as inhibitors of necroptosis (necrostatins), ferroptosis (ferrostatins), and inflammasomes have emerged in clinical trials. Anatomical pathologists should embrace these novel ancillary tests and the concepts behind them and test their impact on diagnostic precision, prognostication, and the prediction of response to the upcoming anti-necrotic therapies.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

The death of endocrine cells is involved in type 1 diabetes mellitus, autoimmunity, adrenopause and hypogonadotropism. Insights from research on basic cell death have revealed that most pathophysiologically important cell death is necrotic in nature, whereas regular metabolism is maintained by apoptosis programmes. Necrosis is defined as cell death by plasma membrane rupture, which allows the release of damage-associated molecular patterns that trigger an immune response referred to as necroinflammation. Regulated necrosis comes in different forms, such as necroptosis, pyroptosis and ferroptosis. In this Perspective, with a focus on the endocrine environment, we introduce these cell death pathways and discuss the specific consequences of regulated necrosis. Given that clinical trials of necrostatins for the treatment of autoimmune conditions have already been initiated, we highlight the therapeutic potential of such novel therapeutic approaches that, in our opinion, should be tested in endocrine disorders in the future.

Dexamethasone is widely used as an immunosuppressive therapy and recently as COVID-19 treatment. Here, we demonstrate that dexamethasone sensitizes to ferroptosis, a form of iron-catalyzed necrosis, previously suggested to contribute to diseases such as acute kidney injury, myocardial infarction, and stroke, all of which are triggered by glutathione (GSH) depletion. GSH levels were significantly decreased by dexamethasone. Mechanistically, we identified that dexamethasone up-regulated the GSH metabolism regulating protein dipeptidase-1 (DPEP1) in a glucocorticoid receptor (GR)–dependent manner. DPEP1 knockdown reversed the phenotype of dexamethasone-induced ferroptosis sensitization. Ferroptosis inhibitors, the DPEP1 inhibitor cilastatin, or genetic DPEP1 inactivation reversed the dexamethasone-induced increase in tubular necrosis in freshly isolated renal tubules. Our data indicate that dexamethasone sensitizes to ferroptosis by a GR-mediated increase in DPEP1 expression and GSH depletion. Together, we identified a previously unknown mechanism of glucocorticoid-mediated sensitization to ferroptosis bearing clinical and therapeutic implications.

Signaling pathways of regulated necrosis, such as necroptosis and ferroptosis, contribute to acute kidney injury (AKI), but the role of pyroptosis is unclear. Pyroptosis is mediated by the pore-forming protein gasdermin D (GSDMD). Here, we report a specific pattern of GSDMD-protein expression in the peritubular compartment of mice that underwent bilateral ischemia and reperfusion injury (IRI). Along similar lines, the GSDMD-protein expression in whole kidney lysates increased during the first 84 h following cisplatin-induced AKI. Importantly, unlike whole kidney lysates, no GSDMD-protein expression was detectable in isolated kidney tubules. In IRI and cisplatin-induced AKI, GSDMD-deficient mice exhibited hypersensitivity to injury as assessed by tubular damage, elevated markers of serum urea, and serum creatinine. This hypersensitivity was reversed by a combined deficiency of GSDMD and the necroptosis mediator mixed lineage kinase domain-like (MLKL). In conclusion, we demonstrate a non-cell autonomous role for GSDMD in protecting the tubular compartment from necroptosis-mediated damage in IRI.

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