Proc Natl Acad Sci U S A 111(47): 16836-16841

PMC: PMC4250130

PMID: 25385600

Synchronized renal tubular cell death involves ferroptosis

Fritz Krombach+14 authors

Supplementary Material

^{Clinic for Nephrology and Hypertension, Christian-Albrechts-University Kiel, 24105 Kiel, Germany;}

^{Department of Biological Sciences and Department of Chemistry, University of Texas at El Paso, El Paso, TX, 79902;}

^{Department of Physiology, Christian-Albrechts-University Kiel, 24098 Kiel, Germany;}

^{Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ludwig-Maximilians-Universität München, 80366 Munich, Germany;}

^{First Department of Pediatrics, Semmelweis University, H-1083 Budapest, Hungary;}

^{Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-Universität München, 81366 Munich, Germany;}

^{Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München, 81366 Munich, Germany;}

^{Institute for Research in Biomedicine, 08028 Barcelona, Spain;}

^{Institute for Genetics, University of Cologne, 50931 Cologne, Germany;}

^{Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105-3678;}

^{Molecular Signaling and Cell Death Unit, Inflammation Research Center, Vlaams Instituut voor Biotechnologie, Ghent University, 9052 Ghent, Belgium;}

^{Methusalem Program, Ghent University, 9052 Ghent, Belgium;}

^{Division of Nephrology, Department of Internal Medicine, VA Healthcare System and University of Michigan, Ann Arbor, MI, 48109-5676;}

^{Department of Pathology, University of Hannover, 30625 Hannover, Germany;}

^{Department of Biological Sciences, Columbia University, New York, NY, 10027;}

^{Department of Chemistry, Columbia University, New York, NY, 10027;}

^{Howard Hughes Medical Institute, Columbia University, New York, NY, 10027; and}

^{Department of Systems Biology, Columbia University Medical Center, New York, NY, 10027}

^{To whom correspondence may be addressed. Email:}ed.hsku@nnamreknil.saerdna or ed.leik-inu.orhpen@dlawtuark.

Edited^* by Vishva M. Dixit, Genentech, San Francisco, CA, and approved October 10, 2014 (received for review August 12, 2014)

Author contributions: A.L., T.V.B., M.B., J.M.W., C.A.R., U.K., H.-J.A., B.R.S., D.R.G., and S.K. designed research; A.L., R.S., N.H., S.R.M., C.D., F.D.Z., A.P., G.Z., F.K., T.V.B., J.M.W., J.H.B., and S.K. performed research; A.L., R.S., P.-S.W., R.W., P.V., M.P., M.B., H.-J.A., B.R.S., D.R.G., and S.K. contributed new reagents/analytic tools; A.L., R.S., N.H., S.R.M., P.-S.W., R.W., T.V.B., P.V., M.B., J.M.W., C.A.R., J.H.B., U.K., H.-J.A., B.R.S., D.R.G., and S.K. analyzed data; and A.L. wrote the paper.

Edited^* by Vishva M. Dixit, Genentech, San Francisco, CA, and approved October 10, 2014 (received for review August 12, 2014)

Significance

Cell death by regulated necrosis causes tremendous tissue damage in a wide variety of diseases, including myocardial infarction, stroke, sepsis, and ischemia–reperfusion injury upon solid organ transplantation. Here, we demonstrate that an iron-dependent form of regulated necrosis, referred to as ferroptosis, mediates regulated necrosis and synchronized death of functional units in diverse organs upon ischemia and other stimuli, thereby triggering a detrimental immune response. We developed a novel third-generation inhibitor of ferroptosis that is the first compound in this class that is stable in plasma and liver microsomes and that demonstrates high efficacy when supplied alone or in combination therapy.

Keywords: regulated cell death, programmed cell death, ferroptosis, necroptosis, apoptosis

Significance

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.

Abstract

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 X_c-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.

Acknowledgments

We thank Katja Bruch, Maike Berger, Janina Kahl, and Monika Iversen for excellent technical assistance and Justus Cordt for expert help with mouse weight charts. A.L. received funding from the German Society for Nephrology, the Else Kröner-Fresenius Stiftung, Pfizer, and Novartis. H.-J.A. is supported by Deutsche Forschungsgemeinschaft Grants AN372/9-2, AN371/12-2, and AN372/15-1 and the Else Kröner-Fresenius Stiftung. B.R.S. is an Early Career Scientist of the Howard Hughes Medical Institute and received funding from New York State Stem Cell Science (Contract C026715 for the Chemical Probe Synthesis Facility), the US National Institutes of Health (NIH Grants R01CA097061, R01GM085081, and R01CA161061), the Whitehall Foundation, the William Randolph Hearst Foundation, and the Baby Alex Foundation. J.M.W. is supported by NIH Grant R01DK34275 and the Veterans Administration. Research in the P.V. unit is supported by Belgian grants (Interuniversity Attraction Poles Grants IAP 6/18 and IAP 7/32), Flemish grants (Research Foundation Flanders Grants FWO G.0875.11, FWO G.0973.11 N, FWO G.0A45.12 N, FWO G.0172.12, FWO G.0787.13N, G0C3114N, and FWO KAN 31528711), Ghent University grants (Multidisciplinary Research Partnership, Ghent Researchers On Unfolded Proteins in Inflammatory Disease consortium), and grants from the Flanders Institute for Biotechnology. P.V. holds Methusalem Grant BOF09/01M00709 from the Flemish Government. S.K. received grants from Pfizer, Novartis, Fresenius, and the Else Kröner-Fresenius Stiftung.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1415518111/-/DCSupplemental.

Footnotes

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Synchronized renal tubular cell death involves ferroptosis

Supplementary Material

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Significance

Abstract

Acknowledgments

Footnotes

References