Synchronized renal tubular cell death involves ferroptosis

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.

Keywords: apoptosis; ferroptosis; necroptosis; programmed cell death; regulated cell death.

Fig. 1.

Conditional deletion of FADD or caspase-8 does not sensitize renal tubules to necroptosis. (A) Scheme of the doxycyclin-inducible conditional tubular knockout. (B) After 3 wk of doxycyclin-treatment, no detection of the FADD protein in freshly isolated renal tubules was possible; L929 cells serve as a positive control. (C) Periodic acid–Schiff (PAS) staining of normal renal morphology in Pax8-rtA; Tet-on.Cre × FADD fl/fl mice after 3-wk treatment with doxycyclin via the drinking water. (D–F) Similarly, caspase-8 was inducibly depleted from tubules. Note that the anti-mouse monoclonal antibody against caspase-8 cross-reacts with a nonspecific protein just below the band of caspase-8. Mouse embryonic fibroblasts (MEFs) from caspase-8/RIPK3-dko mice serve as a negative control, MEFs from cyclophilin D-deficient ppif-ko mice serve as a positive control, as do the C57BL/6 WT mice. (G) Serum creatinine levels remain in the normal range in all mice investigated as indicated. (H) Doxycyclin-induced conditional FADD-deficient or caspase-8–deficient mice react to 20 mg/kg body weight cisplatin-induced acute kidney injury similarly to nonstimulated mice. (I) Necrostatin-1 (Nec-1; 50 µM) does not influence the amount of LDH released from hypoxic renal tubules, either in the presence (Left) or absence (Right) of glycine (glc) (n = 8–10 per group).

Fig. 2.

Ferroptosis mediates synchronized tubular necrosis and contributes to immune-cell extravasation into ischemic tissue. (A) Snapshots from Movie S1 of functional freshly isolated proximal renal tubule segments undergoing rapid synchronized necrosis upon fatty-acid depletion. (B) In the presence of fatty acids, addition of erastin accelerated, whereas 16–86 (a third-generation ferrostatin; see Fig. 3) prevented, tubular necrosis. (C) During the time course of hydroxyquinoline/Fe-induced tubular necrosis, Fer-1 and the Nox-inhibitor GKT prevented LDH release whereas Nec-1 did not show protection. (D) Reflected light oblique transillumination imaging of leukocyte passages through postcapillary venules in IRI of the cremaster muscle in the presence of vehicle or Fer-1. (E) FITC dextran leakage in the same model. (F) Leukocyte rolling is affected by Fer-1 within the first hour after reperfusion. (G) Significantly reduced leukocyte transmigration in the presence of Fer-1.

Fig. 3.

Generation and in vitro testing of a ferrostatin derivative for in vivo applications. (A) Synthetic route of the most microsomal and plasma stable ferrostatin analog (SRS16-86). (B) Model structure of the two novel ferrostatin derivates SRS16-79 (inactive compound) and SRS16-86 (active compound). (C) FACS analysis for the necrotic marker 7AAD and phosphatidylserine accessibility (annexin V) in HT1080 and NIH 3T3 cells treated with either vehicle or 50 µM erastin in the presence or absence of 1 µM Fer-1, 16-86, or 16-79. (D) Absence of cleaved caspase-3 in necroptosis and ferroptosis. Western blot of cleaved caspase-3 in lysates from 100 ng/mL TNFα plus 1 μM Smac mimetics plus 25 μM zVAD-treated HT-29 cells, 50 μM erastin-treated NIH 3T3 cells, and 50 μM erastin-treated HT1080 cells for 24 h. Monoclonal anti-Fas–treated Jurkat cells (100 ng/mL) served as positive control. TSZ, TNFa/SMAC-mimetic/zVAD.

Fig. 4.

Ferroptosis significantly contributes to renal ischemia–reperfusion injury. (A and B) Representative PAS stainings and quantification of renal damage from mice treated with severe ischemic durations before reperfusion. Mice were killed 48 h after reperfusion. (C and D) Functional markers of acute kidney injury after severe IRI (as in A and B). (E and F) Histologic PAS staining and quantification of sham operation or ultrasevere IRI (50 min of ischemia before reperfusion) in WT mice treated with [Nec-1 + SfA] combination therapy together with either 16-79 or 16-86. (G and H) C57BL/6 were treated as in E, and functional markers of acute kidney injury were measured 48 h after the onset of reperfusion. n.s., not significant; *P = 0.05–0.02, **P = 0.02–0.001, ***P ≤ 0.001; n = 10 per group in all experiments).