Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury - PubMed (original) (raw)

. 2013 Jul 16;110(29):12024-9.

doi: 10.1073/pnas.1305538110. Epub 2013 Jul 1.

Jan Hinrich Bräsen, Maurice Darding, Mi Kyung Jin, Ana B Sanz, Jan-Ole Heller, Federica De Zen, Ricardo Weinlich, Alberto Ortiz, Henning Walczak, Joel M Weinberg, Douglas R Green, Ulrich Kunzendorf, Stefan Krautwald

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Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury

Andreas Linkermann et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptor-interacting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia-reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.

Keywords: RIP1; RIP3; apoptosis; programmed necrosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Increased protection from ischemia–reperfusion damage by combined loss of RIPK3 and CypD. Mice underwent severe renal IRI. (A) Survival proportions of indicated mice following IRI. (B) Representative periodic acid–Schiff (PAS)-stained histomicrographs of mice with indicated genotype 48 h after severe IRI. White arrows point to typical necrotic changes classically observed in proximal tubules upon renal IRI. (C) Quantification by renal damage score of B. (D and E) Serum creatinine and serum urea concentrations 48 h following reperfusion or sham operation. **P < 0.01; ***P < 0.001 (n = 8–12 per group).

Fig. 2.

Fig. 2.

RIPK3 and CypD contribute to ischemia–reperfusion damage but caspase-8 does not. (A) Expression levels of RIPK1 in whole-kidney lysates taken from wt or RIPK3-ko mice during the time course of IRI in wt mice. GAPDH serves as a loading control. (B) wt, RIPK3-ko, and caspase-8 (C8)/RIPK3-dko mice underwent renal IRI 48 h before preparation of PAS-stained renal sections and its quantification using the renal damage score (C). (D and E) Corresponding serum creatinine and serum urea concentrations 48 h after reperfusion (n = 8–12 per group). (F–I) Comparison of wt, CypD-ko, and RIPK3-ko mice in a mild IRI setting (n = 7–16 per group).

Fig. 3.

Fig. 3.

Necroptosis occurs independently of the immune system. (A) Freshly isolated renal tubules were treated for 6 h after preparation with indicated agents before assessment of TUNEL positivity and quantification (B). (C and D) Representative PAS-stained kidney sections (C) and evaluation of renal damage (D) from SCID-Beige mice that underwent ischemia–reperfusion in the presence of DMSO or Nec-1. Corresponding serum concentrations of creatinine (E) and urea (F) are shown 48 h after reperfusion (n = 8 per group). (G) PI staining of freshly isolated renal tubules from wt or RIPK3-ko mice that underwent 60 min of hypoxia followed by 60 min of reperfusion. (H) LDH-release assay of the similar tubules as in G.

Fig. 4.

Fig. 4.

Combination therapy with Nec-1 and SfA provides strong protection against IRI. (A and B) RIPK3-ko and CypD-ko mice (n = 8 per group) underwent indicated treatment 15 min before onset of IRI surgery. Note that the neither the addition of Nec-1 to RIPK3-ko mice nor the application of SfA in CypD-ko mice led to any further protection compared with the DMSO-treated knockouts. (C–G) wt mice (n = 8–12 per group) underwent indicated treatment 15 min before the onset of surgery. (C) Survival proportions following IRI. (D) Representative PAS-stained histomicrographs 48 h after severe IRI are demonstrated. (E) Quantification by renal damage score of D. (F–G) Serum creatinine and serum urea concentrations 48 h following reperfusion or sham operation. Note the additive protective effect of combination therapy. *P < 0.05; **P < 0.01; n.s., not significant.

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