Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Nov 1.
Published in final edited form as: Liver Transpl. 2010 Nov;16(11):1267–1277. doi: 10.1002/lt.22148

Inhibition of Inducible Nitric Oxide Synthase Prevents Graft Injury after Transplantation of Livers from Rats after Cardiac Death

Yanjun Shi 1, Hasibur Rehman 1, Gary L Wright 1, Zhi Zhong 1
PMCID: PMC2967449  NIHMSID: NIHMS224840  PMID: 21031542

Abstract

This study investigated the roles of inducible nitric oxide synthase (iNOS) in the failure of rat liver grafts from cardiac death donors (GCDD). Livers were explanted after 30-min aorta clamping and implanted after 4-hour storage in UW solution. iNOS expression increased slightly in grafts from non-cardiac death donors (GNCDD) but markedly in GCDD. Serum nitrite and nitrate and hepatic 3-nitrotyrosine adducts, indicators of NO and peroxynitrite production, respectively, were substantially higher after transplantation of GCDD than GNCDD. Production of reactive nitrogen species (RNS) was largely blocked by 1400W (5 μM), a specific iNOS inhibitor. Alanine aminotransferase (ALT) release, bilirubin, necrosis and apoptosis were 6.4-, 6.5-, 2.3- and 2.7-fold higher after transplantation of GCDD than GNCDD. 1400W effectively blocked these alterations. 1400W also increased survival of GCDD from 33% to 80%. Increased RNS production and failure of GCDD were associated with activation of c-Jun-N-terminal kinase (JNK), an effect that was blocked by inhibition of iNOS. Inhibition of JNK also improved the outcome after transplantation of GCDD. Together, the data indicate that iNOS increases substantially in GCDD, leading to RNS over-production, JNK activation, and more severe graft injury. Inhibitors of iNOS are suggested as effective therapies to improve the outcome after transplantation of GCDD.

Keywords: 1400W, c-Jun N-terminal kinase, reactive nitrogen species, non-heart-beating donors, primary non-function

INTRODUCTION

Severe shortage of donor livers strictly limits the use of orthotopic liver transplantation (1,2). Xenotransplantation, isolated hepatocyte transplantation, extracorporeal liver perfusion and liver assist devices provide bridges to transplantation for severely ill patients, but they have yet to make a significant impact on waiting list mortality (1–3). Since liver donation is currently mostly from brain-dead donors, the number of brain-dead donors before cardiac death suitable for liver donation remains a key limiting factor for utilizing liver transplantation as therapy for end-stage liver diseases. Use of marginal donor livers such as fatty livers and livers from cardiac death donors (CDD) provides an important solution to alleviate the critical shortage of donor organs. It was estimated that recovery of livers from CDD for transplantation could significantly expand the organ donation pool (4–7). However, hepatic warm ischemia after cardiac arrest compromises graft viability and increases the necessity of retransplantation (4–6,8,9).

CDD includes uncontrolled and controlled CDD. Uncontrolled CDD are those who are dead on arrival or die after unsuccessful resuscitation (4,6,7). These donors are often patients who are sent to the emergency department and could be considered for organ donation after declaration of death. CDD also includes the circumstances where cardiac arrest occurs in a heart-beating donor before organs are recovered. Typically, organs recovered from uncontrolled donors suffer a long period of warm ischemia. Controlled CDD are those awaiting cardiac arrest, usually terminally ill patients after planned withdrawal of life support at the request of the family (4,7). Warm ischemic time of these donor organs is usually shorter as there are opportunities to obtain family consent and organize the organ recovery procedures prior to withdrawal of support. Current published reports of successful cardiac death liver transplantation are mostly from controlled CDD (4,7). Organs from controlled CDD experience at least 10 min of warm ischemia prior to cold preservation.

The mechanism of primary non-function of grafts from CDD (GCDD) remains unclear. Previous studies showed that antioxidants protect against injury of these grafts, suggesting that reactive oxygen species (ROS) play a role in failure of GCDD (10–12). However, the impact of reactive nitrogen species (RNS) on the outcome of CDD liver transplantation remains unclear. The effects of RNS in hepatic ischemia/reperfusion (IR) and liver transplantation are controversial (13). RNS could cause cell injury by inhibiting a variety of biological important processes such as mitochondrial respiration, protein synthesis and gluconeogenesis (14,15). Many studies showed that inhibition of iNOS decreases hepatic IR injury (13,16–18). On the other hand, a NO precursor has been observed to decrease liver injury after transplantation of GNCDD (13). Whether RNS are protective or detrimental may depend on the roles of different subtype of NOS under various circumstances. It is generally accepted that eNOS is cell protective by mediating vasodilatation whereas iNOS mediates cytotoxicity (14). Despite previous studies on RNS in IR and liver transplantation, the role of iNOS in the outcome of grafts from CDD remains unclear. Accordingly, the purpose of this study was to test the effects of 1400W, a specific iNOS inhibitor, on graft injury after transplantation of livers from CDD.

METHODS

Animals and Liver Transplantation

Male Lewis rats (250–300 g) were used in liver transplantation experiments. To simulate liver donation after cardiac death, heparin (400 IU) in 0.5 ml of lactated Ringer's solution was injected intravenously into the donor rat, and the thorax was opened 5 min later under isoflurane anesthesia. Previous studies of rat liver donation after cardiac arrest predominantly used 30 min of ischemic time (10,19–24). Therefore, the descending aorta was clamped for 30 min with a mini-bulldog vascular clamp, followed immediately by recovery of the liver (GCDD). Some rats were anesthetized with isoflurane and livers were recovered 30 min later without thorax opening and aorta clamping as GNCDD controls. Some livers were harvested immediately after the abdomen is opened without thorax opening and aorta clamping as the standard harvesting controls (SHG).

Liver transplantation was performed using the arterialized two-cuff technique described elsewhere (25). Each graft was stored in UW solution at 0–1°C for 4 h. In some experiments, 1400W, a specific inhibitor of iNOS (26), and SP600125, a specific inhibitor for c-Jun N-terminal kinase (JNK), were added to the storage solution at a concentration of 5 μM and 20 μM, respectively. For implantation, the liver of the recipient was removed after clamping the suprahepatic and subhepatic vena cava and portal vein. The donor explants were rinsed with 5 mL of lactated Ringer's solution with or without 1400W, and implanted by connecting the suprahepatic vena cava with a running suture, then inserting cuffs into the appropriate vessels and securing them with 6-0 silk suture. The hepatic artery and the bile duct were anastomosed with intraluminal splints. Portal vein clamping time was for 18 to 20 minutes, and implantation surgery required less than 50 minutes in total. For sham operation, ligaments around the liver were dissected. Fifty minutes later, the abdominal wall was closed with running suture without transplantation. Rats were observed 7 days for survival after transplantation. All animals were given humane care in compliance with institutional guidelines using protocols approved by the Institutional Animal Care and Use Committee.

Measurement of Serum Alanine Aminotransferase (ALT), Total Bilirubin and Nitrite/Nitrate

To measure ALT release and accumulation of bilirubin due to liver injury, blood samples were collected from the inferior vena cava at 18 h and 7 days after implantation. Serum ALT and total bilirubin were determined by analytical kits from Pointe Scientific (Uncoln Park, MI). To assess NO production, serum nitrite and nitrate levels were detected using an analytic kit from Cayman Chem. (Ann Arbor, MI).

Histology

Livers were collected immediately after 4 h-cold storage or at 18 h after sham or implantation surgery. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.), livers were harvested, and slides were prepared as described elsewhere (27). In sections stained with hematoxylin and eosin (H+E), ten random fields per slide were captured in a blinded manner using a Universal Imaging Image-1/AT image acquisition and analysis system (West Chester, PA) with an Axioskop 50 microscope (Carl Zeiss, Inc., Thornwood, NY) and a 10× objective lens. Necrotic areas were quantified by computerized image analysis using an IP Lab 3.7v software (BD Biosciences, Rockville, MD) and dividing necrotic areas by total cellular area of the images.

Immunohistochemical Staining for Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) and 3-Nitrotyrosine Adducts

Apoptosis was assessed by TUNEL staining using an In Situ Cell Death Detection Kit (27). TUNEL-positive and negative cells were counted in a blinded manner in 10 randomly selected fields using a 40× objective lens.

To detect 3-nitrotyrosine adducts, a marker of peroxynitrite formation, sections were deparaffinized with xylene (Mallinckrodt Baker Inc. Paris, Kentucky) and tissue was rehydrated by taken through a graded series of alcohol/water mixtures. Hydrated sections were heated in 10 mM citrate acid (pH 6) in microwave for antigen retrieval and then exposed to rabbit anti-nitrotyrosine polyclonal antibodies (Millipore, Billerica, MA) at a concentration of 1:150 in 0.1 M phosphate buffer with 0.5% Tween 20 for 30 min at room temperature. Peroxidase-conjugated anti-rabbit IgG1 antibody (DAKO Corp., Carpinteria, CA) was then applied, and 3,3'-diaminobenzidine chromagen was added as the peroxidase substrate. After the immunostaining procedure, a light counterstain of Meyer's hematoxylin was performed so that 3-nitrotyrosine-labeled cells could be identified easily.

Western Blotting

Immunoblotting of proteins was performed as described (28) with primary antibodies specific for iNOS (BD Biosciences, San Jose, CA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), phosphorylated JNK1/2, JNK1/2, phosphorylated c-Jun, c-Jun, phosphorylated ERK1/2, ERK1/2, phosphorylated p38 MAPK, and p38 MAPK at concentrations of 1:100 to 1000 (Santa Cruz Biotech., Santa Cruz, CA), and actin (ICN, Costa Mesa, CA) at a concentration of 1:3000 at 4°C over night, respectively. Horseradish peroxidase-conjugated secondary antibodies were applied, and detection was by chemiluminescence (Pierce Biotec., Rockford, IL).

Statistical Analysis

Groups were compared using Kaplan-Meier test, ANOVA, Kruskal-Wallis test, or Student's t-test as appropriate. Data shown are means ± S.E.M or 25-percentile, median and 75-percentile, as indicated in figure legends. Numbers of animals were 8 to 12 per group in the survival experiment and 5 per group for all other parameters. Differences were considered significant at p<0.05.

RESULTS

iNOS is Upregulated in GCDD after Liver Transplantation

To investigate whether iNOS increases after transplantation of GCDD, expression of iNOS was detected by Western blotting (Fig. 1A). iNOS is barely detectable in livers from sham-operated rats and cold-stored, untransplanted livers from CDD. After transplantation, iNOS increased slightly to a similar extent in both SHG and GNCDD (Fig. 1A). By contrast, after transplantation of GCDD, expression of iNOS increased markedly compared to SHG and GNCDD (Fig. 1A). iNOS expression peaked at 6 h after transplantation and maintained at high levers until 18 h (Fig. 1A). Although an enzyme inhibitor does not necessarily inhibit the expression of its target, 1400W partially blunted upregulation of iNOS after transplantation of GCDD at 6–18 h (Fig. 1A).

Fig. 1. Upregulation of Inducible Nitric Oxide Synthase and Increased Reactive Nitrogen Species after Transplantation of Liver Grafts from CDD.

Fig. 1

Open in a new tab

Rat livers of standard harvest (SH) or from non-cardiac death donors (NCDD) and cardiac death donors (CDD) were stored in UW solution (Storage) with or without 1400W (5 μM). Livers were collected after cold storage, 18 h after sham-operation (Sham) or 6 – 18 h after transplantation (Tx). A shows the representative images of iNOS detected by Western blotting. 3-Nitrotyrosine adducts were detected immunohistochemically (B). Panels are: upper left, liver from sham-operated rat; upper right, cold-stored, unimplanted liver from CDD; middle left, implanted graft of SH; middle right, implanted graft from NCDD; lower left, implanted graft from CDD; lower right, 1400W-pretreated, implanted liver graft from CDD. Bar is 50 μm. Blood was collected 18 h after sham-operation or liver transplantation. Serum nitrite and nitrate values are shown in C. a, p<0.05 vs sham-operation; b, p<0.05 vs grafts of SH or from NCDD; c, p<0.05 vs grafts from CDD (mean ± S.E.M.). Cold-stored, unimplanted livers were not statistically different from sham operation, and grafts from NCDD were not statistically different from grafts of SH.

Increases of RNS Formation after Transplantation of GCDD: Prevention by 1400W

Since iNOS increased substantially after transplantation of GCDD, we further investigated whether RNS formation increased after liver transplantation. 3-Nitrotyrosine adducts, an indicator of peroxynitrite formation, was barely detectable in livers from sham-operated rats and cold-stored explants from CDD (Fig. 1B. upper panels). 3-Nitrotyrosine adducts increased slightly after transplantation of SHG and GNCDD (Fig. 1B. middle panels). By contrast, after transplantation of GCDD, 3-nitrotyrosine adducts increased markedly (Fig. 1B. lower left). Formation of 3-nitrotyrosine adducts in GCDD was largely blocked by inhibition of iNOS with 1400W (Fig. 1B. lower right).

Production of nitric oxide was also assessed by nitrite and nitrate levels in sera. Basal levels of nitrite and nitrate were 25 μM in sera of sham-operated rats (Fig. 1C). Transplantation of SHG or GNCDD did not increase serum nitrite and nitrate significantly (Fig. 1C). By contrast, serum nitrite and nitrate increased to 65 μM after transplantation of GCDD (Fig. 1C). Increases of nitrite and nitrate after transplantation of GCDD were totally blocked by 1400W (Fig. 1C). Taken together, these data indicate that the production of RNS increases substantially after transplantation of GCDD due to increased expression of iNOS. We also find that 1400W is effective at suppressing the excessive production of these RNS.

Inhibition of iNOS Mitigates Hepatic Cell Death after Transplantation of GCDD

To investigate the role of iNOS in injury of GCDD, we evaluated the effects of 1400W on pathological changes after liver transplantation. No pathological changes were observed in livers at 18 h after sham operation (Fig. 2, 1st row left). In cold-stored, untransplanted livers from CDD, although slight cell swelling was observed, no necrosis occurred (Fig. 2, 2nd row left and Fig. 3A). After transplantation of SHG, necrotic areas increased slightly to 5.1% (Fig. 3A). After transplantation of GNCDD, necrotic areas increased to a similar extent as SHG (7.1%, p>0.05 vs SHG, Fig. 2, 3rd row left and Fig. 3A). By contrast, after transplantation of GCDD, necrotic areas increased dramatically to 16% (Fig. 2, 4th row left and Fig. 3A). Inhibition of iNOS with 1400W decreased necrotic areas to 7.1% in implanted GCDD (Fig. 2, 5th row left and Fig. 3A).

Fig. 2. Hepatic Necrosis and Apoptosis after Transplantation of Livers from CDD: Mitigation by 1400W.

Fig. 2

Open in a new tab

Conditions were as in Fig. 1. Livers were collected after cold storage or 18 h after sham-operation (Sham) or transplantation (Tx). Columns are: left, hematoxylin and eosin (H + E) staining; right, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Rows are: 1st, sham-operation; 2nd, cold-stored, unimplanted liver from CDD; 3rd, implanted liver graft from NCDD; 4th, implanted liver graft from CDD; 5th, 1400W-pretreated, implanted liver graft from CDD. Shown are representative images. Arrows identify necrotic areas. White bar is 100 μm. Black bar is 50 μm.

Fig. 3. 1400W Prevents Necrosis and Apoptosis in Liver Grafts from CDD.

Fig. 3

Open in a new tab

Conditions were as in Fig. 2. Necrosis (A) was quantified by image analysis of 10 randomly selected fields per liver in a blinded manner. TUNEL-positive cells (B) were counted in 10 randomly selected fields using a 40× objective lens. Values are 25-percentile, median and 75-percentile. a, p<0.05 vs sham-operation; b, p<0.05 vs grafts of SH or from NCDD; c, p<0.05 vs grafts from CDD. Cold-stored, unimplanted livers were not statistically different from sham operation, and grafts from NCDD were not statistically different from SHG in both necrosis and apoptosis. Cleaved caspase-3 and actin were detected by immunoblotting (C). Shown are representative images.

Apoptosis was detected by TUNEL staining. TUNEL-positive cells were rare in livers from sham-operated rats (Fig. 2, 1st row right and Fig. 3B) and cold-stored, untransplanted livers from CDD (Fig. 2, 2nd row right and Fig. 3B). Apoptosis increased slightly to 0.8% in implanted SHG (Fig. 3B) and 1.1% in GNCDD (Fig. 2, 3rd row right and Fig. 3B, p>0.05 vs SHG) but increased to 3% in GCDD (Fig. 2, 4th row right and Fig. 3B). Treatment with 1400W decreased apoptosis to 1.1% in GCDD (Fig. 2, 5th row right and 3B).

Apoptosis was further confirmed by activation of caspase-3. Cleaved caspase-3 was barely detectable in livers from sham-operated rats and in cold-stored, untransplanted livers from CDD (Fig. 3C). Cleaved caspase-3 increased slightly after transplantation of SHG and GNCDD (Fig. 3C). By contrast, cleaved caspase-3 increased markedly in transplanted GCDD (Fig. 3C), confirming onset of apoptosis. The increase in cleaved caspase-3 was largely blunted by 1400W (Fig. 3C).

In sum, although both necrosis and apoptosis increased in livers from CDD after transplantation, necrosis is the predominant form of cell death in GCDD. Inhibition of iNOS prevents both necrosis and apoptosis after transplantation of GCDD.

Inhibition of iNOS Improves Function and Survival of Liver Grafts from CDD

Graft injury was also assessed by serum ALT activity at 18 h after sham-operation or liver transplantation. Serum ALT levels were 69 U/L in rats that received the sham operation (Fig. 4A). ALT increased to ~680 U/L after transplantation of SHG and to ~750 U/L after transplantation of GNCDD (p>0.05 vs SHG, Fig. 4A). After implantation of GCDD, ALT increased to ~4,800 U/L (Fig. 4A), indicating more severe liver injury. 1400W decreased serum ALT in rats received GCDD to ~1,600 U/L, which is not significantly different from those receiving GNCDD (Fig. 4A).

Fig. 4. 1400W Protects Against ALT Release and Accumulation of Total Bilirubin after Transplantation of Liver Grafts from CDD.

Fig. 4

Open in a new tab

Conditions were as in Fig. 1. Blood was collected 18 h after sham operation (sham) or transplantation (Tx) of liver grafts from rats with or without exposure to aorta clamping (CDD & NCDD). ALT (A) and total bilirubin (B) in sera were measured. Values are 25-percentile, median and 75-percentile. a, p< 0.05 vs sham-operation; b, p< 0.05 vs SHG or grafts from NCDD; c, p< 0.05 vs grafts from CDD. Grafts from NCDD were not statistically different from grafts of SH in both ALT and bilirubin.

Total bilirubin was 0.17 mg/dL in rats subjected to the sham operation. Bilirubin was not increased after transplantation of SHG or GNCDD (Fig. 4B). In rats received GCDD, bilirubin increased to 1.3 mg/dL at 18 h (Fig. 4B), indicating poorer function of GCDD. 1400W decreased bilirubin to 0.29 mg/dL in rats that received GCDD (Fig. 4B).

All rats survived after sham operation (data not shown) or transplantation of SHG and GNCDD (Fig. 5). Survival decreased substantially to 33% after transplantation of GCDD (Fig. 5). Death occurred mainly in the first 3 days after transplantation. Importantly, in rats that received GCDD pretreated with 1400W, survival increased to 80% (Fig. 5), indicating that inhibition of iNOS effectively increases survival of GCDD.

Fig. 5. 1400W Improves Survival after Transplantation of Liver Grafts from CDD.

Fig. 5

Open in a new tab

Conditions were as in Fig. 1. Rats were observed 7 days for survival after transplantation (Tx) of liver grafts from rats with or without exposure to aorta clamping (CDD & NCDD). Difference is statistically significant (p< 0.05) between grafts from CDD to NCDD and between grafts from CDD with 1400W treatment to grafts from CDD without 1400W treatment by Kaplan-Meier test (n = 8 – 12 per group). Survival of grafts from NCDD was the same as grafts of SH.

One of the major concerns of GCDD is biliary complications after transplantation. To investigate the effects of iNOS inhibition on biliary systems of GCDD, serum bilirubin in survivors at 7 days after transplantation was detected. Serum bilirubin further increased to 2.2 ± 0.6 mg/dL (mean ± SEM) in the recipients of GCDD at 7 days given that serum ALT has decreased to 290 ± 14 U/L (~6% of the value at 18 h). Serum bilirubin at 7 days after transplantation was decreased to 0.4 ± 0.06 mg/dL by 1400W (p = 0.03 by Student's t-test vs. no 1400W treatment). Although biliary complications are possibly still mild in 7 days, these data suggest that liver injury in the very early stage after transplantation of GCDD contributes to biliary complications in later stages. Inhibition of iNOS could decrease early injury thus minimizing biliary complications later. Studies will be performed in the future to investigate the effects of iNOS inhibition on biliary complications of GCDD in the late stages after transplantation.

Activation of JNK after Transplantation of GCDD: Prevention by 1400W

Activation of JNK mediates hepatic IR injury and graft failure after transplantation of liver grafts after long-term cold storage (29). Therefore, we investigated whether iNOS expression in GCDD increases JNK activation. Phosphorylated JNK2 (54 kDa) was barely detectable in livers from sham-operated rats, in cold-stored, untransplanted livers from CDD (Fig. 6, upper panel). Phosphorylated JNK2 increased slightly after transplantation of SHG and GNCDD after liver transplantation (Fig. 6A and B). Phosphorylated JNK2 was 64-fold higher in implanted GCDD compared to livers from sham-operated rats (Fig. 6A and B), indicating substantial activation of JNK2. Increases of phosphorylated JNK2 were blunted by 1400W (Fig. 6A and B). Phosphorylated JNK1 (45 kDa) was not detectable in all groups except for a small increase in implanted GCDD (Fig. 6A). JNK1 and 2 expressions were not altered in any of the groups (Fig. 6A).

Fig. 6. 1400W Inhibites Activation of c-Jun N-Terminal Kinase after Transplantation of Grafts from CDD.

Fig. 6

Open in a new tab

Conditions were as in Fig. 1. Phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK), JNK1/2, phosphorylated c-Jun (p-c-Jun), c-Jun, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated p38 MAPK (p-p38), p38 MAPK (p38) and actin were detected by Western blotting. Panel A shows representative gels. Panel B plots mean p-JNK2/actin ratios and Panel C plots mean p-c-Jun/actin ratios. Values are 25-percentile, median and 75-percentile. a, p< 0.05 vs sham-operation; b, p< 0.05 vs implanted grafts of SH and grafts from NCDD; c, p< 0.05 vs implanted grafts from CDD. Cold-stored, unimplanted livers were not statistically different from sham operation, and grafts from NCDD were not statistically different from grafts of SH in both p-JNK2 and p-c-Jun.

Activation of JNK was further confirmed by c-Jun phosphorylation. Phosphorylatd c-Jun was barely detectable in livers from sham-operated rats and in cold-stored, untransplanted livers from CDD (Fig. 6A). Phosphorylatd c-Jun was not significantly increased in SHG or GNCDD after liver transplantation (Fig. 6A and C). Phosphorylated c-Jun was 92-fold higher in GCDD after transplantation compared to livers from sham-operated rats (Fig. 6A and C), consistent with substantial activation of JNK. Increases of phosphorylated c-Jun were blunted by 1400W. By contrast, expression of c-Jun was not altered in any groups studied (Fig. 6A).

Activation of ERK1/2 and p38 MAPK was also investigated. Phosphorylatd ERK1/2 was barely detectable in livers from sham-operated rats and in cold-stored, untransplanted livers from CDD (Fig. 6A). Phosphorylated ERK1/2 increased slightly in SHG or GNCDD after liver transplantation and increased more in GCDD after transplantation (Fig. 6A), consistent with activation of ERK1/2. However, increases of phosphorylated ERK1/2 GCDD were not decreased by 1400W. Expression of ERK1/2 was not altered in any groups studied (Fig. 6A). A similar trend was also observed for p38 MAPK activation.

Inhibition of JNK Improves the Outcome of Transplantation of GCDD

Since iNOS inhibition prevented JNK activation and attenuated liver injury after transplantation of GCDD, we further investigated whether inhibition of JNK by SP600125 affects the outcome of transplantation of GCDD. SP600125 decreased necrotic area in grafts from CDD by 68% (Fig. 7A), apoptosis by 66% (Fig. 7B), ALT release by 75% (Fig. 7C) and hyperbilirubinemia by 75% (Fig. 7D). These data indicate that JNK indeed mediates the injurious effects in CDD grafts.

Fig. 7. Inhibition of c-Jun N-Terminal Kinase Improves the Outcome of Transplantation of Grafts from CDD.

Fig. 7

Open in a new tab

Conditions were as in Fig. 3 and 4 except that SP600125, a JNK inhibitor, was added to the UW solution at a final concentration of 20 μM. Necrotic area (A), TUNEL-positive cells (B), serum ALT (C) and bilirubin (D) were analyzed. Values are mean ± SEM. **. P<0.01 vs vehicle treatment by Student's t-test (n = 4 per group).

DISCUSSION

Inhibition of iNOS Attenuates Injury of GCDD

Severe donor organ shortage requires us to seek additional usable donor livers for transplantation. Livers from CDD provide a potential source which could significantly expand the organ donation pool (4–6). However, previous studies show that survival of GCDD is substantially lower after transplantation as compared to brain-dead donors (4–6,8,9,30). Therefore, it is important to develop effective therapies to improve survival of these grafts. Normothermic and hypothermic machine perfusion improves function and survival of GCDD compared to simple cold storage (23,24,31,32) and represents promising therapies. However, the methodology of machine preservation is complex, which could limit its clinical application. Inhibition of lipid peroxidation, elimination of Kupffer cells, inhibition of TNFα, phosphodiesterase, and endothelin improved survival of GCDD (5,33–36). However, many of these drugs require pretreatment of donors, which are sometimes difficult in clinical settings. Other treatments have proved ineffective at improving the performance of GCDD. For example, N-acetylcysteine, an antioxidant, had no effect on the survival and lipid peroxidation following transplantation of GCDD in pigs (37).

In this study, we investigated the effects of iNOS inhibition on the outcome of CDD liver transplantation. Although ROS are known to be involved in primary non-function of GCDD, it is unclear whether RNS also play a role. Expression of iNOS is up-regulated in liver cells after IR (38–40). GCDD are exposed to longer warm ischemia compared to GNCDD and therefore could lead to more profound upregulation of iNOS. Consistent with this hypothesis, iNOS expression and RNS production increased slightly after transplantation of SHG and GNCDD but dramatically in GCDD (Fig. 1), indicating a substantially higher nitrative stress in GCDD.

NO can be protective or injurious, depending on the physiological or pathological process, timing, location and the amount of NO production. NO exerts many of its physiological effects by activation of guanylyl cyclase. The availability of other reactive intermediates with which NO may interact also determines its toxicity. Because of its radical character, NO reacts with superoxide radicals (O2−.) at a near diffusion-limited rate to produce another highly reactive species peroxynitrite (ONOO−) which causes oxidative/nitrative modification to biomolecules, thus modulating physiological and pathophysiological processes (41–44). In addition, peroxynitrite can be converted to highly reactive hydroxyl-like radicals (45). In cultured hepatocytes, RNS inhibit protein synthesis, gluconeogenesis, glycogenolysis, cytochrome P450, membrane sodium/potassium ATPase activity, and mitochondrial respiration (15,41,42). Inhibition of iNOS decreased hepatic IR injury (13,16–18), suggesting the essential role of iNOS in IR injury. By contrast, other reports showed that NO precursor (e.g. L-arginine) supplementation, blockade of the L-arginine/NO pathway with non-specific NOS inhibitor L-NAME, endothelial nitric oxide synthase (eNOS)-deficiency and adenoviral delivery of iNOS gene decreased graft injury and improved outcome after transplantation of livers from NCDD (13,46,47). Inhalation of NO during liver transplantation accelerates recovery of liver function following surgery (48). It is unclear, however, whether iNOS is beneficial or detrimental for the outcome of CDD liver transplantation. Discrepancies between studies could be due to differences in models, degree and mechanisms of injury as well as selectivity, dose and timing of administration of NOS inhibitors. NO donors, on one hand, produce NO, but on the other hand, inhibit iNOS expression (49). Therefore, interpretation of their effects should also be cautious.

In this study, substantial upregulation of iNOS and increased production of RNS in GCDD were associated with more severe graft injury (necrosis, apoptosis and ALT release), poorer graft function (bilirubin) and decreased survival (Figs. 1 to 5). Inhibition of iNOS with 1400W blocked excessive RNS production (Fig. 1) and improved the outcome of transplantation of GCDD significantly (Figs. 2 to 5). 1400W has substantially higher selectivity to iNOS than nNOS and eNOS (5000:25:1) compared to other iNOS inhibitors. Therefore, it provides a reliable tool to investigate the role of iNOS in injury of GCDD. Together, these data support the hypothesis that upregulation of iNOS in GCDD leads to excessive RNS production which causes graft injury and failure.

iNOS Increases Injury of GCDD by Causing JNK Activation

JNK is a protein kinase which is activated in response to a variety of physiological and pathophysiological stresses including IR, hypoxia, ultraviolet radiation, hyperthermia, cholestasis, lipotoxicity, drugs, toxic cytokines, metals and pathogen-derived antigens (50–56). JNK plays an important role in the mitochondrial intrinsic apoptotic pathways, as well as in death receptor-initiated extrinsic apoptotic pathways (57). JNK activates apoptotic signaling by upregulating pro-apoptotic genes, phosphorylating proapoptotic Bcl2 family proteins, affecting recruitments of caspase 8 to death-inducing signaling complex (DISC), modulating mitochondrial pro-apoptotic proteins, and causing permeabilization of mitochondria which leads to release of cytochrome c and activation of Apaf-1/caspase 9 complexes (57–59).

Sustained JNK activation plays an important role in toxic cytokine and ROS-induced necrotic cell death (60). Activation of JNK was observed after transplantation of livers from NCDD and inhibition of JNK with CC-401 decreased injury and increased survival of liver grafts after prolong preservation (29). Mitochondrial dysfunction via the mitochondrial permeability transition (MPT) leads to failure of ATP production and necrotic cell death after warm IR and liver cold preservation/transplantation (27,61–63). Since IR activates JNK and GCDD are exposed to extended warm ischemia prior to cold storage, we examined whether JNK activation occurred to a greater extend after transplantation of liver grafts from CDD. Indeed, phosphorylated JNK and c-Jun increased substantially after transplantation of GCDD compared to SHG and GNCDD, whereas JNK1/2 and c-Jun expression was similar in all groups before and after transplantation (Fig 6). These results indicate a much higher activation of JNK in GCDD than SHG and GNCDD (Fig. 6). Importantly, this JNK activation is associated with more severe necrosis and apoptosis of hepatocytes, poorer liver function, and higher mortality after transplantation of GCDD (Figs. 2 – 5). Inhibition of JNK by SP600125 significantly improved the outcome after transplantation of GCDD (Fig. 7), further confirming the important role of JNK activation in injury of GCDD.

Two isoforms of JNK are expressed in the liver, namely JNK1 and JNK2. Recent work shows that activation of JNK2, but not JNK1, after liver warm IR and transplantation promotes MPT-dependent liver injury (62,64). In this study, JNK1 and JNK2 were expressed equally in GNCDD and CDD before and after liver transplantation (Fig. 6). By contrast, phosphorylated JNK2 increased substantially in GCDD compared to GNCDD whereas phosphorylated JNK1 was increased only slightly in GCDD (Fig. 6). These data are consistent with the hypothesis that JNK2 activation plays an important role in the injury and failure of GCDD.

The relationship between JNK activation and NO production is complex. JNK activation in conjunction with increases of iNOS promotes cardiotoxin-induced myocyte apoptosis by provoking Bcl-2 inactivation and subsequent activation of the intrinsic apoptotic pathway signaling (65). Some studies showed that JNK activation mediates upregulation of iNOS expression caused by toxins and cytokines (66–68). By contrast, activation of JNK after IR is attenuated in iNOS knockout mice (69), indicating iNOS controls JNK activation. Peroxynitrite generator SIN-1 results in protein nitration and tyrosine kinase and Rac activation, leading to increased JNK and p38 MAPK activities and cell apoptosis (70). Therefore, iNOS could effect upstream or downstream elements of the JNK pathway. In this study, activation of JNK in GCDD was inhibited by a specific iNOS inhibitor 1400W (Fig. 6), suggesting that excessive RNS production from iNOS leads to JNK activation in GCDD.

Taken together, our data indicate that transplantation of GCDD results in substantial upregulation of iNOS and excessive RNS formation, which leads to JNK2 activation and more severe cell death and failure of these grafts. Thus, a strategy is suggested where treatment of marginal liver grafts from CDD with specific iNOS inhibitors might allow for more frequent and successful use of these grafts in clinical transplantation.

Acknowledgments

Supported, in part, by Grant DK70844 and DK037034 from the National Institute of Health.

Abbreviations used

ALT

alanine aminotransferase

ATP

adenosine 5'-triphosphate

CDD

cardiac death donors

eNOS

endothelial nitric oxide synthase

ERK

extracellular signal-regulated kinase

GCDD

grafts from cardiac death donors

GNCDD

grafts from non-cardiac death donors

H+E

hematoxylin and eosin staining

hpf

high power field

iNOS

inducible nitric oxide synthase

IR

ischemia/reperfusion

JNK

c-Jun N-terminal kinase

MPT

mitochondria permeability transition

NCDD

non-cardiac death donors

NO

nitric oxide

3-NT

3-nitrotyrosine adducts

p38 MAPK

p38 mitogen-activated protein kinase

RNS

reactive nitrogen species

ROS

reactive oxygen species

SH

standard harvest

SHG

standard-harvested grafts

Sham

sham operation

TNFα

tumor necrosis factor-α

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

Tx

transplantation

UW solution

University of Wisconsin cold storage solution

1400W

N-(1-naphtyl)ethylendiamine dihydrochloride

REFERENCES

  • 1.Friend PJ, Imber CJ. Current status of liver transplantation. Methods Mol Biol. 2006;333:29–46. doi: 10.1385/1-59745-049-9:29. [DOI] [PubMed] [Google Scholar]
  • 2.Said A, Einstein M, Lucey MR. Liver transplantation: an update 2007. Curr Opin Gastroenterol. 2007;23:292–298. doi: 10.1097/MOG.0b013e3280f9df41. [DOI] [PubMed] [Google Scholar]
  • 3.Alexander JW, Vaughn WK. The use of “marginal” donors for organ transplantation. Transplantation. 1991;51:135–141. doi: 10.1097/00007890-199101000-00021. [DOI] [PubMed] [Google Scholar]
  • 4.Abt PL, Desai NM, Crawford MD, et al. Survival following liver transplantation from non-heart-beating donors. Ann Surg. 2004;239:87–92. doi: 10.1097/01.sla.0000103063.82181.2c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Reddy S, Zilvetti M, Brockmann J, McLaren A, Friend P. Liver transplantation from non-heart-beating donors: current status and future prospects. Liver Transpl. 2004;10:1223–1232. doi: 10.1002/lt.20268. [DOI] [PubMed] [Google Scholar]
  • 6.Abt PL, Fisher CA, Singhal AK. Donation after cardiac death in the US: history and use. J Am Coll Surg. 2006;203:208–225. doi: 10.1016/j.jamcollsurg.2006.03.014. [DOI] [PubMed] [Google Scholar]
  • 7.Deshpande R, Heaton N. Can non-heart-beating donors replace cadaveric heart-beating liver donors? J Hepatol. 2006;45:499–503. doi: 10.1016/j.jhep.2006.07.018. [DOI] [PubMed] [Google Scholar]
  • 8.Casavilla A, Ramirez C, Shapiro R, et al. Experience with liver and kidney allografts from non-heart-beating donors. Transplantation. 1995;59:197–203. doi: 10.1097/00007890-199501000-00008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.D'Alessandro AM, Fernandez LA, Chin LT, et al. Donation after cardiac death: the University of Wisconsin experience. Ann Transplant. 2004;9:68–71. [PubMed] [Google Scholar]
  • 10.Miyagi S, Iwane T, Akamatsu Y, Nakamura A, Sato A, Satomi S. The significance of preserving the energy status and microcirculation in liver grafts from non-heart-beating donor. Cell Transplant. 2008;17:173–178. doi: 10.3727/000000008783906874. [DOI] [PubMed] [Google Scholar]
  • 11.Yagi T, Sadamori H, Nakagawa H, et al. Hepatropic soluble vitamin E derivative EPC-K1 prevents warm ischemia/reperfusion injury of non-heart-beating donor liver transplantation in pigs. Transplant Proc. 1997;29:1390–1393. doi: 10.1016/s0041-1345(96)00607-0. [DOI] [PubMed] [Google Scholar]
  • 12.Lauschke H, Kotting M, Akbar S, Minor T. Use of taurine as antioxidant in resuscitating livers from non-heart-beating donors by gaseous oxygen persufflation. J Invest Surg. 2003;16:7–11. [PubMed] [Google Scholar]
  • 13.Shah V, Kamath PS. Nitric oxide in liver transplantation: pathobiology and clinical implications. Liver Transpl. 2003;9:1–11. doi: 10.1053/jlts.2003.36244. [DOI] [PubMed] [Google Scholar]
  • 14.Anggard E. Nitric oxide: mediator, murderer, and medicine. Lancet. 1994;343:1199–1206. doi: 10.1016/s0140-6736(94)92405-8. [DOI] [PubMed] [Google Scholar]
  • 15.Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med. 2003;3:519–526. doi: 10.2174/1566524033479582. [DOI] [PubMed] [Google Scholar]
  • 16.Isobe M, Katsuramaki T, Hirata K, Kimura H, Nagayama M, Matsuno T. Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver. Transplantation. 1999;68:803–813. doi: 10.1097/00007890-199909270-00013. [DOI] [PubMed] [Google Scholar]
  • 17.Urakami H, Abe Y, Grisham MB. Role of reactive metabolites of oxygen and nitrogen in partial liver transplantation: lessons learned from reduced-size liver ischaemia and reperfusion injury. Clin Exp Pharmacol Physiol. 2007;34:912–919. doi: 10.1111/j.1440-1681.2007.04640.x. [DOI] [PubMed] [Google Scholar]
  • 18.He S, Rehman H, Wright GL, Zhong Z. Inhibition of inducible nitric oxide synthase prevents mitochondrial damage and improves survival of steatotic partial liver grafts. Transplantation. 2010;89:291–298. doi: 10.1097/TP.0b013e3181c99185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Nakamura A, Akamatsu Y, Miyagi S, Fukumori T, Sekiguchi S, Satomi S. A free radical scavenger, edaravone, prevents ischemia-reperfusion injury in liver grafts from non-heart-beating donors. Transplant Proc. 2008;40:2171–2174. doi: 10.1016/j.transproceed.2008.06.049. [DOI] [PubMed] [Google Scholar]
  • 20.Iwane T, Akamatsu Y, Narita T, Nakamura A, Satomi S. The effect of perfusion prior to cold preservation and addition of biliverdin on the liver graft from non-heart-beating donors. Transplant Proc. 2006;38:3358–3361. doi: 10.1016/j.transproceed.2006.11.002. [DOI] [PubMed] [Google Scholar]
  • 21.Stegemann J, Hirner A, Rauen U, Minor T. Use of a new modified HTK solution for machine preservation of marginal liver grafts. J Surg Res. 2010;160:155–162. doi: 10.1016/j.jss.2008.10.021. [DOI] [PubMed] [Google Scholar]
  • 22.Bessems M, Doorschodt BM, van Vliet AK, van Gulik TM. Improved rat liver preservation by hypothermic continuous machine perfusion using polysol, a new, enriched preservation solution. Liver Transpl. 2005;11:539–546. doi: 10.1002/lt.20388. [DOI] [PubMed] [Google Scholar]
  • 23.Lee CY, Jain S, Duncan HM, et al. Survival transplantation of preserved non-heart-beating donor rat livers: preservation by hypothermic machine perfusion. Transplantation. 2003;76:1432–1436. doi: 10.1097/01.TP.0000088674.23805.0F. [DOI] [PubMed] [Google Scholar]
  • 24.Lee CY, Zhang JX, Jones JW, Jr., Southard JH, Clemens MG. Functional recovery of preserved livers following warm ischemia: improvement by machine perfusion preservation. Transplantation. 2002;74:944–951. doi: 10.1097/00007890-200210150-00008. [DOI] [PubMed] [Google Scholar]
  • 25.Gao W, Lemasters JJ, Thurman RG. Development of a new method for hepatic rearterialization in rat orthotopic liver transplantation: reduction of liver injury and improvement of surgical outcome by arterialization. Transplantation. 1993;56:19–24. doi: 10.1097/00007890-199307000-00004. [DOI] [PubMed] [Google Scholar]
  • 26.Garvey EP, Oplinger JA, Furfine ES, et al. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997;272:4959–4963. doi: 10.1074/jbc.272.8.4959. [DOI] [PubMed] [Google Scholar]
  • 27.Zhong Z, Theruvath TP, Currin RT, Waldmeier PC, Lemasters JJ. NIM811, a Mitochondrial Permeability Transition Inhibitor, Prevents Mitochondrial Depolarization in Small-for-Size Rat Liver Grafts. Am J Transplant. 2007;7:1103–1111. doi: 10.1111/j.1600-6143.2007.01770.x. [DOI] [PubMed] [Google Scholar]
  • 28.Rehman H, Connor HD, Ramshesh VK, et al. Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts. Transplantation. 2008;85:1322–1331. doi: 10.1097/TP.0b013e31816de302. [DOI] [PubMed] [Google Scholar]
  • 29.Uehara T, Xi P, X, Bennett B, et al. c-Jun N-terminal kinase mediates hepatic injury after rat liver transplantation. Transplantation. 2004;78:324–332. doi: 10.1097/01.tp.0000128859.42696.28. [DOI] [PubMed] [Google Scholar]
  • 30.Otero A, Gomez-Gutierrez M, Suarez F, et al. Liver transplantation from Maastricht category 2 non-heart-beating donors. Transplantation. 2003;76:1068–1073. doi: 10.1097/01.TP.0000085043.78445.53. [DOI] [PubMed] [Google Scholar]
  • 31.Schon MR, Kollmar O, Wolf S, et al. Liver transplantation after organ preservation with normothermic extracorporeal perfusion. Ann Surg. 2001;233:114–123. doi: 10.1097/00000658-200101000-00017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Dutkowski P, Furrer K, Tian Y, Graf R, Clavien PA. Novel short-term hypothermic oxygenated perfusion (HOPE) system prevents injury in rat liver graft from non-heart beating donor. Ann Surg. 2006;244:968–976. doi: 10.1097/01.sla.0000247056.85590.6b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ikegami T, Nishizaki T, Hiroshige S, Ohta R, Yanaga K, Sugimachi K. Experimental study of a type 3 phosphodiesterase inhibitor on liver graft function. Br J Surg. 2001;88:59–64. doi: 10.1046/j.1365-2168.2001.01621.x. [DOI] [PubMed] [Google Scholar]
  • 34.Xu HS, Stevenson WC, Pruett TL, Jones RS. Donor lazaroid pretreatment improves viability of livers harvested from non-heart-beating rats. Am J Surg. 1996;171:113–116. doi: 10.1016/S0002-9610(99)80084-1. [DOI] [PubMed] [Google Scholar]
  • 35.Astarcioglu H, Karademir S, Unek T, et al. Beneficial effects of pentoxifylline pretreatment in non-heart-beating donors in rats. Transplantation. 2000;69:93–98. doi: 10.1097/00007890-200001150-00017. [DOI] [PubMed] [Google Scholar]
  • 36.Gu M, Takada Y, Fukunaga K, et al. Pharmacologic graft protection without donor pretreatment in liver transplantation from non-heart-beating donors. Transplantation. 2000;70:1021–1025. doi: 10.1097/00007890-200010150-00006. [DOI] [PubMed] [Google Scholar]
  • 37.Manika A, Trinh T, Lagace G, et al. N-acetylcysteine in pig liver transplantation from non-heart-beating donors. Transplantation. 1999;68:327–330. doi: 10.1097/00007890-199908150-00002. [DOI] [PubMed] [Google Scholar]
  • 38.Lanteri R, Acquaviva R, Di GC, et al. Rutin in rat liver ischemia/reperfusion injury: effect on DDAH/NOS pathway. Microsurgery. 2007;27:245–251. doi: 10.1002/micr.20345. [DOI] [PubMed] [Google Scholar]
  • 39.Yanagida H, Kaibori M, Yoshida H, et al. Hepatic ischemia/reperfusion upregulates the susceptibility of hepatocytes to confer the induction of inducible nitric oxide synthase gene expression. Shock. 2006;26:162–168. doi: 10.1097/01.shk.0000223130.87382.73. [DOI] [PubMed] [Google Scholar]
  • 40.Koeppel TA, Mihaljevic N, Kraenzlin B, et al. Enhanced iNOS gene expression in the steatotic rat liver after normothermic ischemia. Eur Surg Res. 2007;39:303–311. doi: 10.1159/000104401. [DOI] [PubMed] [Google Scholar]
  • 41.Miranda KM, Espey MG, Jourd'heuil D, et al. The chemical biology of nitric oxide. In: Ignarro LJ, editor. Nitric Oxide: Biology and Pathobiology. Academic Press; San Diego: 2000. pp. 41–55. [Google Scholar]
  • 42.Radi R, Denicola A, Alvarez B, Ferrer-Sueta G, Rubbo H. The biological chemistry of peroxynitrite. In: Ignarro LJ, editor. Nitric oxide:biology and pathobiology. Academic Press; San Diego, CA: 2000. pp. 57–82. [Google Scholar]
  • 43.Poderoso JJ. The formation of peroxynitrite in the applied physiology of mitochondrial nitric oxide. Arch Biochem Biophys. 2009 doi: 10.1016/j.abb.2008.12.020. [DOI] [PubMed] [Google Scholar]
  • 44.Peluffo G, Radi R. Biochemistry of protein tyrosine nitration in cardiovascular pathology. Cardiovasc Res. 2007;75:291–302. doi: 10.1016/j.cardiores.2007.04.024. [DOI] [PubMed] [Google Scholar]
  • 45.Molin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000;20:1430–1442. doi: 10.1161/01.atv.20.6.1430. [DOI] [PubMed] [Google Scholar]
  • 46.Theruvath TP, Zhong Z, Currin RT, Ramshesh VK, Lemasters JJ. Endothelial nitric oxide synthase protects transplanted mouse livers against storage/reperfusion injury: Role of vasodilatory and innate immunity pathways. Transplant Proc. 2006;38:3351–3357. doi: 10.1016/j.transproceed.2006.10.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kaizu T, Ikeda A, Nakao A, et al. Donor graft adenoviral iNOS gene transfer ameliorates rat liver transplant preservation injury and improves survival. Hepatology. 2006;43:464–473. doi: 10.1002/hep.21067. [DOI] [PubMed] [Google Scholar]
  • 48.Lang JD, Jr., Teng X, Chumley P, et al. Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation. J Clin Invest. 2007;117:2583–2591. doi: 10.1172/JCI31892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Xia ZY, Wang XY, Chen X, Xia Z. Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats. Injury. 2007;38:53–59. doi: 10.1016/j.injury.2006.09.021. [DOI] [PubMed] [Google Scholar]
  • 50.Malhi H, Gores GJ. Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease. Semin Liver Dis. 2008;28:360–369. doi: 10.1055/s-0028-1091980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Rosette C, Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science. 1996;274:1194–1197. doi: 10.1126/science.274.5290.1194. [DOI] [PubMed] [Google Scholar]
  • 52.Bradham CA, Stachlewitz RF, Gao W, et al. Reperfusion after liver transplantation in rats differentially activates the mitogen activated protein kinases. Hepatology. 1997;25:1128–1135. doi: 10.1002/hep.510250514. [DOI] [PubMed] [Google Scholar]
  • 53.Kon K, Kim JS, Jaeschke H, Lemasters JJ. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology. 2004;40:1170–1179. doi: 10.1002/hep.20437. [DOI] [PubMed] [Google Scholar]
  • 54.Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ. Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem. 2006;281:15258–15267. doi: 10.1074/jbc.M512953200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Gunawan BK, Liu ZX, Han D, Hanawa N, Gaarde WA, Kaplowitz N. c-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity. Gastroenterology. 2006;131:165–178. doi: 10.1053/j.gastro.2006.03.045. [DOI] [PubMed] [Google Scholar]
  • 56.Yang JI, Yoon JH, Myung SJ, et al. Bile acid-induced TGR5-dependent c-Jun-N terminal kinase activation leads to enhanced caspase 8 activation in hepatocytes. Biochem Biophys Res Commun. 2007;361:156–161. doi: 10.1016/j.bbrc.2007.07.001. [DOI] [PubMed] [Google Scholar]
  • 57.Dhanasekaran DN, Reddy EP. JNK signaling in apoptosis. Oncogene. 2008;27:6245–6251. doi: 10.1038/onc.2008.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc Natl Acad Sci U S A. 2003;100:2432–2437. doi: 10.1073/pnas.0438011100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Tournier C, Hess P, Yang DD, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science. 2000;288:870–874. doi: 10.1126/science.288.5467.870. [DOI] [PubMed] [Google Scholar]
  • 60.Morgan MJ, Kim YS, Liu ZG. TNFalpha and reactive oxygen species in necrotic cell death. Cell Res. 2008;18:343–349. doi: 10.1038/cr.2008.31. [DOI] [PubMed] [Google Scholar]
  • 61.Zhong Z, Ramshesh VK, Rehman H, et al. Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol. 2008;295:G823–G832. doi: 10.1152/ajpgi.90287.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Theruvath TP, Snoddy MC, Zhong Z, Lemasters JJ. Mitochondrial Permeability Transition in Liver Ischemia and Reperfusion: Role of c-Jun N-Terminal Kinase 2. Transplantation. 2008;85:1500–1504. doi: 10.1097/TP.0b013e31816fefb5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Theruvath TP, Zhong Z, Currin RT, Pediaditakis P, Lemasters JJ. Minocycline mitigates storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition. Hepatology. 2008;47:235–246. doi: 10.1002/hep.21912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Theruvath TP, Czerny C, Ramshesh VK, Zhong Z, Chavin KD, Lemasters JJ. C-Jun N-terminal kinase 2 promotes graft injury via the mitochondrial permeability transition after mouse liver transplantation. Am J Transplant. 2008;8:1819–1828. doi: 10.1111/j.1600-6143.2008.02336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Sinha-Hikim I, Braga M, Shen R, Sinha Hikim AP. Involvement of c-Jun NH2-terminal kinase and nitric oxide-mediated mitochondria-dependent intrinsic pathway signaling in cardiotoxin-induced muscle cell death: role of testosterone. Apoptosis. 2007;12:1965–1978. doi: 10.1007/s10495-007-0120-6. [DOI] [PubMed] [Google Scholar]
  • 66.Chen CC, Tsai PC, Wei BL, Chiou WF. 8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages. Eur J Pharmacol. 2008;590:430–436. doi: 10.1016/j.ejphar.2008.05.018. [DOI] [PubMed] [Google Scholar]
  • 67.Chan ED, Morris KR, Belisle JT, et al. Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways. Infect Immun. 2001;69:2001–2010. doi: 10.1128/IAI.69.4.2001-2010.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Chakravortty D, Kato Y, Sugiyama T, et al. Inhibition of caspase 3 abrogates lipopolysaccharide-induced nitric oxide production by preventing activation of NF-kappaB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells. Infect Immun. 2001;69:1315–1321. doi: 10.1128/IAI.69.3.1315-1321.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Zingarelli B, Hake PW, Yang Z, O'Connor M, Denenberg A, Wong HR. Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-kappaB and AP-1 activation and enhances myocardial damage. FASEB J. 2002;16:327–342. doi: 10.1096/fj.01-0533com. [DOI] [PubMed] [Google Scholar]
  • 70.Natal C, Modol T, Oses-Prieto JA, Lopez-Moratalla N, Iraburu MJ, Lopez-Zabalza MJ. Specific protein nitration in nitric oxide-induced apoptosis of human monocytes. Apoptosis. 2008;13:1356–1367. doi: 10.1007/s10495-008-0263-0. [DOI] [PubMed] [Google Scholar]

RESOURCES