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RIP1-Dependent Programmed Necrosis is Negatively Regulated by Caspases During Hepatic Ischemia-Reperfusion

Rosentreter, Dirk*; Funken, Dominik*†; Reifart, Jörg*†; Mende, Konstantin*†; Rentsch, Markus*; Khandoga, Andrej*

Author Information
doi: 10.1097/SHK.0000000000000371



Ischemia-reperfusion (I/R) injury causes significant morbidity and mortality and occurs during organ transplantation, resection, trauma, and septical as well ashemorrhagical shock. Hepatic I/R injury represents the main reason for early organ failure on liver transplantation. Ischemia-reperfusion–induced injury is a complex cascade of events including different mechanisms of cell death caused by oxygen deprivation and adenosine triphosphate depletion during ischemia but also by activation of cells of the innate immune system via inflammatory responses during reperfusion. The understanding of the processes leading to different forms of liver cell death is essential for the development of effective interventions to prevent hepatocellular damage and liver failure on transplantation or major surgery.

The different types of cell death on I/R include spontaneous and programmed cell death with or without initiating an inflammatory response. These types can be defined by morphological or biochemical behavior of the cell. Apoptosis is defined by its morphological criteria, including cell shrinkage, nuclear DNA fragmentation, and membrane blebbing (1). Phosphatidylserine exposure on apoptotic cells facilitates phagocytic uptake mainly by macrophages, leading to programmed cell death without causing an immune response (2). Apoptosis can be initiated by extracellular mechanisms and from any membrane-defined organelle in the cell. In contrast to apoptosis, during necrosis, the disruption of the cell membrane leads to a release of cytoplasm contents into the surrounding environment, finally resulting in inflammation (3). Programmed necrosis, also known as necroptosis, was first postulated in 1998. Necroptosis is a caspase-independent programmed cell death leading to cell swelling and membrane breakdown, resulting in morphology reminiscent of passive nonregulated necrosis, including an inflammatory response by the release of intracellular contents (4). It was shown that simultaneous caspase inhibition and tumor necrosis factor (TNF) stimulation in L929 fibrosarcoma cells undergo necrosis-like cell death (5, 6). Necroptosis is a strongly regulated process unlike necrosis and depends on the kinases receptor-interacting protein 1 (RIP1) and RIP3 (7, 8). Death receptors are able to initiate necroptosis under conditions that are insufficient to trigger apoptosis. Receptor-interacting protein 1 kinase activity and subsequent recruitment of RIP3 are necessary for the induction of necroptosis. Mixed lineage kinase domain-like protein (MLKL) was identified as a key component downstream of RIP3 in the execution of RIP1-mediated necroptosis in vitro. In contrast, activation of caspase-8 inhibits necroptosis by cleavage of RIP1. Necrostatin-1 (Nec-1) is a known specific kinase inhibitor of RIP1 and was shown to stop the initiation of necroptosis (9, 10).

Recent studies have shown a prominent role of necroptosis after cardiac, renal, and cerebral I/R (9, 11, 12). The relevance of programmed necrosis in the postischemic liver remains, however, unclear. In this study, we tested the hypothesis that, during hepatic I/R, blockade of the RIP-1–dependent pathway by using Nec-1 attenuates necrotic injury and inflammation in vivo.



For experiments, 5- to 7-week-old female C57BL/6 wild-type mice (Charles River, Sulzfeld, Germany) were used. All experiments were carried out according to the German legislation on protection of animals.

Surgical procedure and experimental protocol

The surgical procedure was described elsewhere (13). Briefly, under inhalation anesthesia with isoflurane-N2O, a polypropylene catheter was inserted into the left carotid artery in a retrograde direction for measurement of mean arterial pressure and application of fluorescence dyes, as described previously. A warm (37°C) reversible ischemia of the left liver lobe was induced for 90 min by clamping the supplying nerve vessel bundle using a microclip. Reperfusion time was 240 min in all experiments. A sham-operated group and two I/R groups were analyzed (n = 6 each): an I/R group treated with the inactive Nec-1 derivative Nec-1inactive (3.5 μg kg−1 body weight; Calbiochem, Darmstadt, Germany) and an I/R group treated with Nec-1 (3.5 μg kg−1 body weight; Sigma-Aldrich, Taufkirchen, Germany). Necrostatin-1 and Nec-1inactive were infused intra-arterially via the carotid catheter into the aortic arch 10 min before reperfusion.

Leukocyte (neutrophil)-endothelial cell interactions and sinusoidal perfusion failure

Intravital fluorescence microscopy was performed using a modified Leitz-Orthoplan microscope as described previously. Leukocytes were stained in vivo by rhodamine 6G (0.05%, 100 μL, i.a.; Sigma) and visualized in hepatic postsinusoidal venules using intravital fluorescence microscopy as described previously (14).

Thereafter, the plasma marker fluorescein isothiocyanate–conjugated Dextran (molecular weight, 150,000; 0.1 mL; 5%; Sigma) was infused, and sinusoidal perfusion was analyzed using an I2/3 filter block in sinusoids within six to nine acini. Intravital microscopy was started after 240 min of reperfusion and lasted approximately 20 min.

All videotaped images were quantitatively analyzed offline using Capimage software (Zeintl, Heidelberg, Germany). Rolling leukocytes were defined as cells crossing an imaginary perpendicular through the vessel at a velocity significantly lower than the centerline velocity in the microvessel. Their numbers are given as cells per second per vessel cross section. Leukocytes firmly attached to the endothelium for more than 20 s were counted as permanently adherent cells and were quantified as the number of cells per square millimeter of endothelial surface calculated from the diameter and length of the vessel segment observed. The sinusoidal perfusion failure was calculated as the percentage of nonperfused sinusoids of all sinusoids visible.

Liver enzymes

Blood samples were taken from the carotid artery at the end of the experiment, immediately centrifuged at 2,000g for 10 min, and stored at −80°C. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined at 37°C with an automated analyzer (Hitachi 917; Roche-Boehringer, Mannheim, Germany) using standardized test systems (HiCo GOT and HiCo GPT; Roche-Boehringer).

Caspase-3 activity

Frozen liver tissue was homogenized with 1 mL of ice-cold lysis buffer per 100 mg of liver tissue containing 25 mM HEPES, pH 7.5, 5 mM MgCl2, 0.1% CHAPS, and 0.1 mM EDTA. After filtration, the homogenates were centrifuged and the protein concentration of the supernatant was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Schwerte, Germany). Twenty micrograms of protein were used in a final volume of 200 μL in a 96-well plate with 50 μM of the selective substrate for caspase-3, caspase-7, and caspase-8 acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartic acid 7-amino-4-methylcoumarin (Ac-DEVD-AMC; PeptaNova, Sandhausen, Germany) in the presence or absence of 10 μM of the specific caspase-3, caspase-7, and caspase-8 inhibitor acetyl-l-aspartyl-l-glutaminyl-l-threonyl-l-aspart-1-al (Ac-DEVD-CHO; PeptaNova) in substrate buffer including 50 mM HEPES, pH 7.5, 1% sucrose, 0.1 % CHAPS, and 10 mM dithiothreitol. The amount of the fluorescent AMC released was measured by fluorometry (Tecan Infinite F200, Männedorf, Switzerland) with 360 nm excitation and 430 nm emission filters. Data are expressed as change in fluorescence (ΔF) * min−1 * μg−1.

Western blotting

Small pieces of liver tissue were homogenized in 1 mL of ice-cold lysis buffer (30 mM Tris/HCl, pH 7.5, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) including protease inhibitor cocktail. After 10-min incubation on ice and 10-min centrifugation (10,000g), the supernatant was collected. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific). Twenty micrograms of total lysates were run on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% (wt vol−1) milk powder in 1× PBS including 0.1% Tween, membranes were incubated with mouse-IgG2a-RIP1 (BD, Heidelberg, Germany), goat-IgG-MLKL (Santa Cruz Biotechnology, Dallas, Tex), or rabbit-IgG-GAPDH (Cell Signaling Technology, Beverly, Mass), followed by incubation with goat anti–rabbit IgG–horseradish peroxidase (Santa Cruz Biotechnology) and donkey anti–goat IgG–horseradish peroxidase (Santa Cruz Biotechnology). Signal was visualized with Wasabi software (Wasabi Systems, Norfolk, VA) and an Iluminizer (Hamamatsu Photonics, Hamamatsu, Japan) using chemiluminescence.

Statistical analysis

Analysis of variance on ranks followed by Student-Newman-Keuls methods was used for the estimation of stochastic probability in intergroup comparison (SigmaPlot 12; Jandel Scientific, Erkrath, Germany). Mean values ± SEM are given. Values of P < 0.05 were considered significant.


Leukocyte-endothelial cell interactions

Leukocyte (neutrophil)–endothelial cell interactions were analyzed in postsinusoidal venules as a sign of hepatic inflammation after hepatic I/R. Leukocytes were labeled by systemic application of rhodamine 6G. As shown in Figure 1, the numbers of rolling and firmly adherent leukocytes in postsinusoidal venules were very low in sham-operated mice, whereas they were dramatically enhanced after 90 min of ischemia followed by 240 min of reperfusion in mice treated with Nec-1inactive (21 ± 2 rolling leukocytes mm−1 * s and 215 ± 22 adherent leukocytes mm−2) . Inhibition of RIP1 using Nec-1, however, did not reduce the postischemic inflammatory reaction because leukocyte migration did not significantly differ between I/R groups with Nec-1 and Nec-1inactive.

Fig. 1
Fig. 1:
Leukocyte–endothelial cell interactions. Leukocyte rolling and adherence were quantified using intravital microscopy in postsinusoidal venules of sham-operated mice, mice after I/R (90 min/240 min) treated with Nec-1inactive, and mice after I/R treated with Nec-1. n = 6 animals per group, mean ± SEM, *P < 0.05 vs. sham-operated group.

Sinusoidal perfusion failure

Ischemia-reperfusion injury is associated with a severe deterioration of tissue perfusion leading to tissue hypoxia during reperfusion (“no-reflow phenomenon”). Sinusoidal perfusion failure was determined using in vivo microscopy as a recognized parameter of microvascular I/R injury. In the I/R group treated with Nec-1inactive as vehicle, about 20% of all sinusoids were not perfused (Fig. 2). In the Nec-1–treated I/R group, the perfusion failure was not improved compared with that in the vehicle-treated group.

Fig. 2
Fig. 2:
Sinusoidal perfusion failure. Sinusoidal perfusion failure (= percentage of nonperfused sinusoids) was measured using intravital microscopy as a parameter of microvascular hepatic injury in sham-operated mice, mice after I/R (90 min/240 min) treated with Nec-1inactive, and mice after I/R treated with Nec-1. n = 6 animals per group, mean ± SEM, *P < 0.05 vs. sham-operated group.

Liver enzyme activity

The serum activity of hepatic transaminases was measured as a marker of cell integrity quantifying the extent of hepatocellular necrotic injury. Hepatic I/R (90 min/240 min) increased dramatically the activity of AST and ALT in the vehicle-treated group (AST, 9,758 ± 2,408 U/L; ALT, 2,072 ± 657 U/L) as compared with that in the sham-operated group. In line with the data on hepatic microcirculation, no protective effect of Nec-1 on necrotic injury could be detected (Fig. 3).

Fig. 3
Fig. 3:
Liver enzyme activity. Serum activity of the liver enzymes AST (black bars) and ALT (white bars) was determined as a marker of hepatocellular necrotic injury in sham-operated mice, mice after I/R (90 min/240 min) treated with Nec-1inactive, and mice after I/R treated with Nec-1. n = 10 animals per group, mean ± SEM, *P < 0.05 vs. sham-operated group.

Detection of RIP1 expression in freshly isolated liver tissues

To investigate the in vivo presence of the molecular machinery that has been demonstrated to be required for the execution of RIP1-mediated necroptosis, Western blot analyses were performed to assess the expression of RIP1 and MLKL in lysates of homogenized liver tissue. Receptor-interacting protein 1, the key mediator of necroptosis, was expressed in livers from sham-operated mice. In both postischemic groups, however, no RIP1 expression was detectable (Fig. 4). Mixed lineage kinase domain-like protein is critically involved in the execution of RIP3/RIP1-mediated necroptosis.

Fig. 4
Fig. 4:
Liver expression of RIP1 and MLKL. Representative Western blots showing RIP1 and MLKL expression in sham-operated mice, mice after I/R (90 min/240 min) treated with Nec-1inactive, and mice after I/R treated with Nec-1. GAPDH was used as a loading control. See Figure, Supplemental Digital Content 1, at

As shown by Western blot, MLKL was slightly expressed in murine livers but did not differ between the sham-operated mice and mice treated with either NEC-1 or Nec-1inactive (Fig. 4). These results together suggest that necroptosis is not present in the liver after I/R.

Caspase-3 activity

It is well known that activated caspase-8 triggers apoptosis via cleavage of caspase-3. In addition, it might attenuate necroptosis by cleavage of RIP1. The caspase-3 activity was determined in homogenates of liver tissue from all experimental groups. The caspase-3 activity was significantly increased in both I/R groups after 240 min of reperfusion (I/R + Nec-1, 1.8 ± 0.2 ΔF * min−1 * μg−1; I/R + Nec-1inactive, 2 ± 0.3 ΔF * min−1 * μg−1) as compared with the sham-operated group. Treatment with Nec-1, however, did not affect caspase-3 activity, which remained on the same level as detected in the vehicle-treated I/R group (Fig. 5).

Fig. 5
Fig. 5:
Caspase-3 activity in liver tissue. Caspase-3 activity was determined in tissue homogenates of sham-operated mice, mice after I/R (90 min/240 min) treated with Nec-1inactive, and in mice after I/R treated with Nec-1 (closed bars). Open bars show internal controls upon incubation of tissue homogenates from all groups with caspase inhibitor Ac-DEVD-CHO. n = 6 animals per group, mean ± SEM, *P < 0.05 vs. sham-operated group, # P < 0.05 vs. internal controls.


Hepatic I/R is the most common cause of organ dysfunction and failure after liver transplantation; therefore, strategies to minimize the negative effects of ischemia are now in the forefront of clinical and experimental studies (15). Different modes of cell death play a role in hepatic I/R injury. The ultimate goal of understanding mechanisms of cell death is to prevent tissue injury in hepatic I/R. For a successive therapy of hepatic I/R injury, it is important to know which mode of cell death: apoptosis, necrosis, or a mixed form of cell death is occurring. Ischemia-reperfusion is a complex process that results in hepatic cell death via a combination of necrosis and apoptosis (16). Apoptosis is the main form of cell death on hepatic I/R (17, 18). Necrotic cell death is also a prominent feature of I/R injury in the liver and is thought to be a much more inflammatory mode of cell death as compared with apoptosis. Because of the rupture of the plasma membrane, necrosis results in the release of cellular constituents in the extracellular environment, which can elicit an inflammatory response. Recently, there has been renewed interest in necrotic cell death in the context of I/R injury because RIP1 has been implicated in programmed necrosis and is modifiable by kinase inhibition. Necrostatin-1 is a well-known inhibitor of the kinase domain of RIP1 and thus in preventing necroptosis (9, 10). In cardiac, renal, and brain I/R injury, it has already been shown that the inhibition of RIP1 by Nec-1 attenuates necrotic cell death (9, 11, 12). However, the role of necroptosis in hepatic I/R injury has remained unclear. Tumor necrosis factor-α is the best trigger described for RIP1-mediated necroptosis (4) and also plays a major role in mediating hepatic I/R. It was shown that the inhibition of TNF-α protects against hepatic I/R injury in rats (19). Here, we tested the hypothesis that Nec-1 exerts a protective effect on hepatic I/R injury and attenuates the inflammatory response in vivo.

Our results show that, unlike in other organs, Nec-1 has no protective effect on cell death induction in I/R injury in the liver. Plasma activity of the liver enzymes ALT and AST was used as a parameter of liver necrosis and strongly increased on 240 min of reperfusion compared with that in the sham group. However, treatment with Nec-1 did not alter ALT and AST levels in a significant manner and was even slightly increased.

A hallmark feature of hepatic inflammation during I/R is recruitment of various types of leukocytes to the afflicted site. Inflammatory stimuli activate endothelial cells to express adhesion molecules and chemokines that physically engage circulating leukocytes and promote their adhesion to the vessel wall. The initial interaction of neutrophils with hepatic endothelium (rolling) is mediated by selectins, whereas the firm adhesion is triggered by the interaction between beta2-integrins of leukocytes and intercellular adhesion molecule 1 on endothelial cells (20, 21). The next step is transendothelial and interstitial migration toward the stimuli from the damaged cells (22, 23). In our study, we analyzed recruitment of leukocytes in the hepatic microvasculature using intravital microscopy. We could observe a dramatic increase in the number of rolling and adherent leukocytes already after 240 min of reperfusion. However, treatment with Nec-1 did not affect leukocyte migration after 240 min. These results are in line with the data on hepatocellular injury.

Next, our data show that postischemic sinusoidal perfusion increases strongly on I/R. Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction after I/R (24). The postischemic shutdown of the hepatic microcirculation is triggered by sinusoidal narrowing caused by endothelial cell edema, (25), stellate cell–mediated vasoconstriction (26, 27), or by activated Kupffer cells. In addition, inflammation- and injury-associated adherence of leukocytes in outflow venules may alter sinusoidal perfusion because of an increase of blood viscosity (28) and, hence, vascular resistance (29). Furthermore, perfusion failure in sinusoids is thought to be caused by sluggish blood flow, intravascular hemoconcentration, and procoagulant conditions (30). Administration of Nec-1, however, does not show any effect on sinusoidal perfusion failure.

A surprising result of our study is that the RIP1 is not expressed in the liver after I/R. Also, MLKL levels did not differ between the experimental groups. Necroptotic cell death is also naturally suppressed by caspase-mediated cleavage of RIP1. In this case, apoptosis seems to be the preferred route of cell death. In our studies, RIP1 was found completely absent on I/R, which was in correlation with the measured increase in caspase activity. Our results suggest that necroptosis is prevented by caspase activity in the liver. It was already shown in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) that loss of Casp8 prevents proteolytic cleavage of RIP1 in hepatocytes (31).

Hepatic RIP1 expression was analyzed in several recent studies and can be altered by different agents and mechanisms. These studies have shown that RIP1 is upregulated during sepsis via Fas-L (32), during acetaminophen-induced liver failure via TNF-α (33), in gall bladder cancer cells (34), and is detectable during concavalin A–induced hepatitis (35). In contrast, ethanol intoxication did not affect hepatic RIP1 expression (36). Even though not in the liver, the study by Newton et al. (37) provides important information describing ubiquitination of RIP1 via the TNF receptor 1–dependent pathway within minutes after TNF-α stimulation in vitro. As is well understood, TNF-α is one of the key mediators of hepatic I/R released from Kupffer cells immediately after the onset of reperfusion. Thus, this study might give an additional explanation of the lack of RIP-1 expression in the postischemic liver.

Finally, it cannot be excluded that RIP3 alone may be able to execute programmed necrosis. However, there are no data in the literature about the role of RIP3 for necroptosis induction during hepatic I/R injury so far.

Our in vivo data show that i) RIP1-mediated necroptosis is not present in the postischemic liver and ii) I/R-induced caspase activation is associated with loss of RIP1 expression in the liver. Because caspases are able to cleave RIP1, we hypothesize that I/R-triggered caspase activation negatively regulates necroptosis and, thereby, determines apoptosis as a preferred route of cell death after hepatic I/R.


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Postischemic liver injury; cell death; necroptosis; microcirculation; inflammation; leukocyte migration; perfusion failure

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