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Keywords:

  • ER tress;
  • PPAR-γ;
  • steatotic liver grafts;
  • TLR4;
  • transplantation;
  • TUDCA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion (I/R). We examined whether tauroursodeoxycholic acid (TUDCA), a known inhibitor of endoplasmic reticulum (ER) stress, protects steatotic and nonsteatotic liver grafts preserved during 6 h in University of Wisconsin (UW) solution and transplanted. The protective mechanisms of TUDCA were also examined. Neither unfolded protein response (UPR) induction nor ER stress was evidenced in steatotic and nonsteatotic liver grafts after 6 h in UW preservation solution. TUDCA only protected steatotic livers grafts and did so through a mechanism independent of ER stress. It reduced proliferator-activated receptor-γ (PPARγ) and damage. When PPARγ was activated, TUDCA did not reduce damage. TUDCA, which inhibited PPARγ, and the PPARγ antagonist treatment up-regulated toll-like receptor 4 (TLR4), specifically the TIR domain-containing adaptor inducing IFNβ (TRIF) pathway. TLR4 agonist treatment reduced damage in steatotic liver grafts. When TLR4 action was inhibited, PPARγ antagonists did not protect steatotic liver grafts. In conclusion, TUDCA reduced PPARγ and this in turn up-regulated the TLR4 pathway, thus protecting steatotic liver grafts. TLR4 activating-based strategies could reduce the inherent risk of steatotic liver failure after transplantation.


Abbreviations: 
ALT

alanine aminotransferase

AST

aspartate aminotransferase

ATF4

activating transcription factor 4

ATF6

activating transcription factor 6

CHOP

C/EBP homologous protein

eIF2α

eukary-otic translation initiation factor 2 subunit α

ER

endoplasmic reticulum

GRP78

78-kDa glucose-regulated/binding immunoglobulin protein

GW9662

2-chloro-5-nitro-N-phenylbenzamide

HTK

histidine tryptophan ketoglutarate

IRE1

inositol-requiring enzyme 1

I/R

ischemia-reperfusion

Ln

lean

MDA

malondialdehyde

MPL-A

monophosphoryl lipid A

MyD88

myeloid differentiation factor 88

Ob

obese

PPAR-γ

peroxisome proliferator activated receptor-γ

Rosiglitazone

(RS)-5-[4-(2-[methyl(pyridine-2-yl)-amino]ethoxy)benzyl]thiazolidine-2,4-dione

ROS

reactive oxygen species

TLR4

toll-like receptor 4

TR

transplantation

TRAF2

tumor necrosis factor-associated factor 2

TRIF

TIR domain-containing-adaptor inducing IFN-β

TUDCA

tauroursodeoxycholic acid

UPR

unfolded protein response

UW

University of Wisconsin

XBP-1

X-box-binding protein 1

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Ischemia-reperfusion (I/R) injury is a major cause of early graft dysfunction after liver transplantation. Steatosis is currently estimated to be present in up to 50% of deceased donor livers and is recognized as the key donor variable predicting posttransplant outcomes (1–3). The use of steatotic livers for transplantation is associated with increased risk of graft dysfunction or failure after surgery. In addition, many steatotic livers are discarded for transplantation, exacerbating the critical shortage of donor livers (1,4,5).

Endoplasmic reticulum (ER) stress is emerging as an important component of inflammatory responses in the liver associated with I/R processes (6,7). In response to ER stress, a signal transduction cascade termed “the unfolded protein response (UPR)” is induced (8,9). The UPR has three branches: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor (ATF6). These proteins are normally held in inactive states in ER membranes by binding to intra-ER chaperones, particularly the 78-kDa glucose-regulated/binding immunoglobulin protein (GRP78). When injury is excessive, these ER stress signal transduction pathways can also induce cell death (8,9).

Tauroursodeoxycholic acid (TUDCA), a natural hydrophilic bile salt that modulates ER stress (9,10), has been approved for clinical use as a protective agent in various liver diseases, including cholestatic liver diseases and cirrhosis (11–15). In our knowledge, only two studies evaluate ER stress in both steatotic and nonsteatotic livers subjected to I/R (6,7). In one study, TUDCA treatment protected both liver types against ER stress under warm ischemia (7). In the second study, the authors preserved liver grafts with histidine tryptophan ketoglutarate (HTK) solution for 2 h. ER stress responses occurred in both liver grafts undergoing transplantation, but were greater in the presence of steatosis (6). Under these conditions, TUDCA reduced ER stress posttransplantation, thus protecting against I/R damage. Potential action mechanisms of TUDCA other than ER stress were not evaluated in the study (6).

In this study, we investigated whether the benefits of TUDCA on ER damage in liver grafts undergoing 2 h of cold ischemia in HTK solution (6) could be also extrapolated to both steatotic and nonsteatotic liver grafts during 6 h of cold preservation in University of Wisconsin (UW) solution. UW solution has become the standard for organ preservation for transplantation (1). Furthermore, liver grafts are usually preserved from 6 to 8 h during clinical transplantation (16–19).

The molecular mechanisms involved in the therapeutic effect of TUDCA are not fully elucidated. Given the inactivation of proliferator-activated receptor-γ (PPARγ) induced by TUDCA in isolated hepatocytes (20), we investigated the effects of TUDCA on PPARγ in steatotic liver grafts undergoing transplantation. The key role of PPARγ in the vulnerability of this liver type to I/R injury associated with transplantation and the benefits of PPARγ antagonists under these conditions have been previously reported (21). In this study, we will also investigate whether the benefits of PPARγ antagonists could be explained by the regulation of toll-like receptor 4 (TLR4), a member of a highly conserved family of pattern recognition receptors. Indeed, changes in TLR4 expression induced by PPARγ regulators have been reported in nonsteatotic livers undergoing warm ischemia (22). TLR4 signaling is mediated by two distinct intracellular adaptor proteins: myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFNβ (TRIF). MyD88 and TRIF activate intracellular signaling cascades that ultimately trigger an inflammatory response (23,24). Indeed, numerous studies have reported that the TLR4 signaling pathway is responsible for the I/R damage in nonsteatotic livers under warm ischemia (25–30) and liver transplantation (31,32). In addition, a recent report indicated that disruption of TLR4 signaling ameliorated hepatic damage in steatotic livers under warm I/R (33). To date, the role of TLR4 in steatotic liver grafts under cold ischemia has not been reported.

The data presented herein indicate: ER stress is not induced when steatotic and nonsteatotic liver grafts are preserved for 6 h in UW solution; new TUDCA properties based on the modulation of PPARγ in steatotic liver transplantation; TLR4 signaling pathway induction by PPARγ antagonists and the benefits of TLR4 signaling pathway activation in steatotic liver transplantation. These results could be useful in the design of pharmacological strategies to protect steatotic livers during liver transplantation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Experimental animals

Syngeneic liver transplantation:  This study was performed using male homozygous (obese [Ob]) and heterozygous (lean [Ln]) Zucker rats aged 10 to 11 weeks.

Allogeneic liver transplantation:  Male Sprague Dawley (SD) and Wistar (WS) rats weighing 200-222 g, were either choline-deficient or standard chow diet for 10 days, respectively (34). This strain combination (SD to WS) is fully allogenic and results in acute liver transplant rejection (35).

Ob Zucker and Ob SD rats develop moderate macrovesicular and microvesicular infiltration in hepatocytes (40–60% steatosis). All procedures were performed under isoflurane inhalation anesthesia (21,36). This study conformed to European Union regulations (Directive 86/609 EEC) for animal experiments. Animals were randomly distributed into groups as described below.

Experimental design (summarized in Table 1)

Table 1.  Experimental groups, treatments and determinations of this study
  1. Abbreviations: anti-TLR4-antibody from Santa Cruz, USA; ER = endoplasmic reticulum; GW9662, 2-chloro-5-nitro-N-phenylbenzamide from Alexis Biochemical, SW; lean; MDA = malondialdehyde; MPL-A = monophosphoryl lipid A from Enzo Life Sciences, SW; Ob = obese; PPAR-γ, peroxisome proliferator activated receptor-γ; Rosiglitazone, (RS)-5-[4-(2-[methyl(pyridine-2-yl)-amino]ethoxy)benzyl]thiazolidine-2,4-dione from Alexis Biochemical SW; SD = Sprague-Dawley; TLR4 = Toll-like receptor 4; TR = transplantation; TUDCA, sodium tauroursodeoxycholate from Sigma-Aldrich, MO; UW = University of Wisconsin; WS = Wistar.

PROTOCOL 1. Syngeneic liver transplantation
Protocol 1.1. Effect of TUDCA on hepatic injury in steatotic and nonsteatotic livers in syngeneic liver transplantation
 Group 1. ShamDissection of hepatic ilium vessels from Ob and Ln Zucker rats
 Group 2. TRLivers preserved in UW solution and transplanted
 Group 2.1 TRSteatotic grafts from Ob Zucker rats with 6 h of cold ischemia and transplanted in recipient Ln Zucker rats.
 Group 2.2 TRNonsteatotic grafts from Ln Zucker rats with 6 h of cold ischemia and transplanted in recipient Ln Zucker rats.
 Group 3. TR+TUDCASame as group 2, but treated with TUDCA
 Group 3.1 TR+TUDCASame as group 2.1, but treated with TUDCA
 Group 3.2 TR+TUDCASame as group 2.2, but treated with TUDCA
Protocol 1.2. PPARγ involvement in TUDCA-induced effects on hepatic injury in steatotic livers in syngeneic liver transplantation
 Group 4. TR+PPARγ antagonistSame as group 2.1, but treated with PPARγ antagonist
 Group 5. TR+ TUDCA+PPARγ agonistSame as group 2.1, but treated with TUDCA and PPARγ agonist
Protocol 1.3. Modulation of the TLR4 pathway by PPARγ in steatotic livers in syngeneic liver transplantation
 Group 6. TR+ PPARγ antagonist +anti-TLR4Same as group 2.1, but treated with PPARγ antagonist and anti-TLR4 antibody
 Group 7. TR+TLR4 agonistSame as group 2.1, but treated with TLR4 agonist
 Group 8. TR+anti-TLR4Same as group 2.1, but treated with anti-TLR4 antibody
Tests at 4 h after reperfusion. Transaminases and damage score (Groups 1-8); ER stress markers, MDA, Nitrotyrosines and PPARγ levels (Groups 1, 2.1, 3.1); TLR4 pathway (Groups 1, 2.1, 3.1, 4, 5) and MPO, TNF, IL1 and IL6 (Groups 1, 2.1, 8).
Tests at 1, 2, 4, 12 and 18 h after reperfusion. Additional groups were subjected to intervention similar to that used for groups 2.1 and 3.1. ER stress markers, PPARγ levels and TLR4 pathway were evaluated at the mentioned reperfusion times.
Survival tests. Additional groups were subjected to intervention similar to that used for groups 2.1, 3.1, 4, 5, 6 and 7 and the survival of receptors was monitored for 14 days.
PROTOCOL 2. Allogeneic liver transplantation
Protocol 2.1. Effect of TUDCA and TLR4 on hepatic injury in steatotic and nonsteatotic livers in allogeneic liver transplantation
 Group 9. ShamDissection of hepatic ilium vessels from Ob SD and Ln WS rats
 Group 10. TRAllograftLivers preserved in UW solution and transplanted
 Group 10.1 TRAllograftSteatotic grafts from Ob SD rats with 6 h of cold ischemia and transplanted in recipient Ln WS rats.
 Group 10.2 TRAllograftNonsteatotic grafts from Ln SD rats with 6 h of cold ischemia and transplanted in recipient Ln WS rats.
 Group 11. TRAllograft+TUDCASame as group 10, but treated with TUDCA
 Group 11.1 TRAllograft+TUDCASame as group 10.1, but treated with TUDCA
 Group 11.2 TRAllograft+TUDCASame as group 10.2, but treated with TUDCA
 Group 12. TR Allograft+TLR4 agonistSame as group 10.1, but treated with TLR4 agonist
Tests at 4 h after reperfusion. Transaminases and damage score (Groups 9–12); ER stress markers, PPARγ levels and TLR4 pathway (Groups 9, 10.1, 11.1).
Survival tests. Additional groups were subjected to intervention similar to that used for groups 10.1, 11.1 and 12 and the survival of receptors was monitored for 14 days.
Drug Administration ProtocolDose and pretreatment time
 TUDCA100 mg/kg i.v., in donor rats 10 min before the surgical procedure
 PPARγ antagonist (GW9662)1 mg/kg i.p., in donor rats 1 h before the surgical procedure
 PPARγ agonist (Rosiglitazone)3 mg/kg i.p., in donor rats 1 h before the surgical procedure
 TRL4 agonist (Nontoxic MPLA)500 μg/kg i.v., in donor rats 10 min before the surgical procedure
 Anti-TLR43 mg/kg i.v., in donor rats 30 min before the surgical procedure

Protocol 1. Syngeneic liver transplantation:  1.1. Effect of TUDCA on hepatic injury in steatotic and nonsteatotic livers in syngeneic liver transplantation

Group 1. Sham. Ln and Ob Zucker animals were subjected to transverse laparotomy.

Group 2. TR. In subgroup 2.1, steatotic livers from donor rats (Ob Zucker rats) were flushed and then preserved in ice-cold UW solution for 6 h (21,36) and implanted into Ln Zucker rats, according to the Kamada cuff technique without hepatic artery reconstruction (37). In subgroup 2.2, the same surgical procedure was repeated, but with Ln Zucker rats as donor and recipients (37). The time of anhepatic phase was 17–19 minutes (38).

Group 3. TR+TUDCA. Subgroups 3.1 and 3.2: same as subgroup 2.1 and 2.2, respectively, but donor rats were treated with TUDCA (7).

1.2. PPARγ involvement in TUDCA-induced effects on hepatic injury in steatotic livers in syngeneic liver transplantation

Group 4. TR+PPARγ antagonist. Same as group 2.1, but donor rats were treated with a PPARγ antagonist (21).

Group 5. TR+TUDCA+PPARγ agonist. Same as group 2.1, but donor rats were treated with TUDCA (7) and a PPARγ agonist (21).

1.3. Modulation of the TLR4 pathway by PPARγ in steatotic livers in syngeneic liver transplantation

Group 6. TR+PPARγ antagonist+anti-TLR4. Same as group 2.1, but donor rats were treated with a PPARγ antagonist (21) and anti-TLR4 antibody (39).

Group 7. TR+TLR4 agonist. Same as group 2.1, but donor rats were treated with a TLR4 agonist (40).

Group 8. TR+anti-TLR4. Same as group 2.1, but donor rats were treated with an anti-TLR4 antibody (39).

The dose and pretreatment times for the different drugs are shown in Table 1.

Liver and plasma samples were collected from groups 1 to 8 at 4h after reperfusion. Plasma transaminases and hepatic damage score were determined in groups 1–8. Hepatic ER stress markers, MDA, nitrotyrosines and PPARγ levels were determined in groups 1, 2.1 and 3.1. Hepatic TLR4 pathway was determined in groups 1, 2.1, 3.1, 4 and 5. Hepatic MPO, TNF, IL-1 and IL-6 were also determined in groups 1, 2.1 and 8. A cold ischemia period of 6 h is long enough to induce damage after transplantation in both liver grafts and to allow high survival at 4h after reperfusion (21). This reperfusion times is included into the range in which maximum levels in the hepatic injury parameters have been shown. Therefore, these experimental conditions were appropriate to evaluate the underlying protective mechanisms of TUDCA in I/R injury associated with liver transplantation.

To evaluate the reperfusion time-dependent effect of TUDCA on ER stress, PPARγ and TLR4 pathway, animals were subjected to intervention similar to that used for groups 2.1 and 3.1 but liver samples were obtained at 1, 2, 4, 12 and 18 h after reperfusion. Hepatic ER stress parameters, PPARγ levels and TLR4 pathway were also determined.

For survival studies in isograft model, animals were subjected to intervention similar to that used for groups 2.1, 3.1, 4, 5, 6 and 7 and the survival of receptors was monitored for 14 days (36).

Protocol 2. Allogeneic liver transplantation:  2.1. Effect of TUDCA and TLR4 on hepatic injury in steatotic and nonsteatotic livers in allogeneic liver transplantation

Group 9. Sham. Ob SD and Ln WS were subjected to transverse laparotomy.

Group 10. TRAllograft. In subgroup 10.1, steatotic allografts from donor rats (Ob SD rats) were flushed and then preserved in ice-cold UW solution for 6 h (21, 36) and implanted into Ln WS rats, according to the Kamada cuff technique without hepatic artery reconstruction (37). In subgroup 10.2, the same surgical procedure was repeated, but with Ln SD rats as donors and Ln WS rats as recipients (37). All recipients were treated with cyclosporine A 3 mg/kg/d (35). In line with previous results (35), no sign of rejection (evaluated according to the Banff schema; Ref. 41) were found in all groups of protocol 2. Our preliminary data indicate that all recipients that received steatotic liver allografts with 6 h of cold ischemia and without immunosupressor died immediately within 1 h after reperfusion.

Group 11. TRAllograft+TUDCA. Subgroups 11.1 and 11.2: same as subgroups 10.1 and 10.2, respectively, but donor rats were treated with TUDCA (7).

Group 12. TRAllograft+TLR4 agonist. Same as group 10.1, but donor rats were treated with a TRL4 agonist (40).

Liver and plasma samples were collected from groups 9 to 12 at 4 h after reperfusion. Plasma trasaminases and hepatic damage score were determined in groups 9-12. Hepatic ER stress markers, PPARγ levels and TLR4 pathway were determined in groups 9, 10.1 and 11.1.

For survival studies in allograft model, animals were subjected to intervention similar to that used for groups 10.1, 11.1 and 12 and the survival of receptors was monitored for 14 days (36).

Biochemical determinations

Transaminases, malondialdehyde (MDA), nitrotyrosine levels (as an index of peroxynitrite), MPO (as an index of neutrophil accumulation), TNF, IL-1 and IL-6 were measured as described elsewhere (30,42–44).

Western blotting

This was done as described elsewhere (7) using the following antibodies: GRP78, C/EBP homologous protein-10 (CHOP), total and phospho-PERK (p-PERK), total and phospho-eukaryotic translation initiation factor 2 subunit α (p-eIF2α), activating transcription factor 4 (ATF4), ATF6α and ATF6β, X-box-binding protein 1 (sXBP-1), TLR4, MyD88 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TNF receptor-associated factor 2 (TRAF2; Cell Signaling Technology, Danvers, MA, USA) and PPARγ and TRIF (Abcam, UK). Signals were quantified with scanning densitometry.

Histology

To appraise the severity of hepatic injury, hematoxylin and eosin-stained sections were evaluated by a point-counting method on an ordinal scale (36,43).

Statistics

Data are expressed as means ± standard error and were compared statistically via one way analysis of variance (ANOVA), followed by post hoc Student–Newman–Keuls test. Survival was estimated with the Kaplan–Meier method and was compared with a long-rank test. p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Effect of TUDCA on hepatic injury in steatotic and nonsteatotic livers in syngeneic liver transplantation

Nonsteatotic grafts were not protected against hepatic injury in the TR+TUDCA group. However, TUDCA treatment protected steatotic grafts, as the biochemical and histological parameters of hepatic injury were lower than those recorded in the TR group (Figure 1).

image

Figure 1. Effect of TUDCA on hepatic injury in steatotic and nonsteatotic liver isografts 4 h after reperfusion. Transaminases (A and B) and damage score (C). The severity of hepatic injury was evaluated by a point-counting method on an ordinal scale as follows: grade 0, minimal or no evidence of injury; grade 1, mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis; grade 2, moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia and loss of intercellular borders; and grade 3, severe necrosis with disintegration of hepatic cords, hemorrhage and neutrophil infiltration. For all parts of the figure, 6 transplantations with nonsteatotic liver isografts and 6 transplantations with steatotic livers isografts for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TR.

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Next, we investigated why TUDCA protected steatotic grafts against damage. GRP78 protein levels in steatotic grafts of the TR and TR+TUDCA groups were similar to those in the sham group (Figure 2A). Given these results, we examined whether the three branches of the UPR (ATF6, IRE1 and PERK) are inactivated in steatotic grafts.

image

Figure 2. Effect of TUDCA on UPR induction, oxidative stress and PPARγ protein expression in steatotic liver isografts 4 h after reperfusion. (A) Protein levels of GRP78, (B) p-50 ATF6α, (C) XBP1(s) and TRAF2, (D) p-PERK, p-eIF2α, ATF4 and CHOP, (E) MDA and nitrotyrosine levels and (F) PPARγ protein expression. Representative western blots at the top and densitometric analysis at the bottom. The scanning values for GRP78, ATF6α and ATF6β, XBP1(s), TRAF2, ATF4, CHOP and PPARγ were divided by the scanning values for β-actin, and those of p-PERK and p-eIF2α by the total PERK and eIF2α, respectively. For all parts of the figure, six transplantations with steatotic liver isografts for each group were included in each measurement. For UPR induction: TR < versus Sham (p = NS), TR+TUDCA < versus TR (p = NS), TR+TUDCA < versus Sham (p = NS). For MDA and PPARγ, *p < 0.05 versus Sham, +p < 0.05 versus TR.

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During ER stress, ATF6 is converted from a 90-kDa protein to a 50-kDa protein (p-50 ATF6α or p-50 ATF6β; Refs. 7,45). The protein levels of p-50 ATF6α (Figure 2B) and p-50 ATF6β (data not shown) in steatotic grafts of the TR and TR+TUDCA groups were similar to those in the sham group. IRE1 activation results in the splicing of XBP1 mRNA for translation of XBP1(s; Refs. 7,8). XBP1(s) and TRAF2 protein levels in steatotic grafts of the TR and TR+TUDCA groups were similar to those in the sham group (Figure 2C). PERK phosphorylates eIF2α in response to ER stress. The p-eIF2α facilitates translation of ATF4, which induces CHOP (7,8,45). The p-PERK and p-eIF2α, ATF4 accumulation and CHOP protein levels in steatotic grafts of the TR and TR+TUDCA groups were similar to those in the sham group (Figure 2D).

TR increased MDA and nitrotyrosine levels in steatotic grafts when compared with the sham group (Figure 2E), which indicated the presence of peroxynitrite, a predominant form of reactive oxygen species in steatotic grafts (36,42). In contrast to anti-oxidant properties of TUDCA under warm hepatic ischemia (7,46), the benefits of TUDCA in steatotic liver transplantation might not be explained by a reduction in oxidative stress, because TR+TUDCA group resulted in MDA and nitrotyrosine levels in steatotic grafts similar to those in the TR group (Figure 2E). In the TR group, PPARγ protein levels were higher in steatotic grafts than in the sham group (Figure 2F). TR+TUDCA group reduced PPARγ protein levels in steatotic grafts with respect to those recorded in the TR group.

PPARγ involvement in TUDCA-induced effects on hepatic injury in steatotic livers in syngeneic liver transplantation

TR+PPARγ antagonist group resulted in lower hepatic injury parameters in steatotic grafts than in the TR group (Figures 3A–D). TR+TUDCA+PPARγ agonist group abolished the benefits of TUDCA, resulting in transaminase (Figures 3A and B) and damage score values (Figure 3C) in steatotic grafts similar to those in the TR group. This indicated that when PPARγ was activated, TUDCA did not protect steatotic grafts against damage. Steatotic grafts of the TR group showed extensive and confluent areas of coagulative necrosis with neutrophil infiltration that were reduced in number and extension in the TR+TUDCA group (Figure 3D). The hepatic lesions observed in TR+TUDCA+PPARγ agonist and TR+PPARγ antagonist groups were similar to those in the TR and TR+TUDCA groups, respectively (Figure 3D).

image

Figure 3. PPARγ involvement in TUDCA-induced effects on hepatic injury and TLR4 pathway in steatotic liver isografts 4 h after reperfusion. Transaminases (A and B) and damage score (C) as described in Figure 1 legend. Representative photographs of histological changes in steatotic livers are show in Figure 3D. (TR), widespread coagulative hepatic necrosis. (TR+TUDCA), small area of coagulative hepatic necrosis. (TR+PPARγ antagonist), hepatic lesions similar to the TR+TUDCA group. (TR+TUDCA+PPARγ agonist), hepatic lesions similar to the TR group (Bars, 1000 μm). TLR4, MyD88 and TRIF are shown in parts (E), (F) and (G), respectively. Representative western blots at the top and densitometric analysis at the bottom. The scanning values for TLR4, MyD88 and TRIF were divided by the scanning values for β-actin. For all parts of the figure, six transplantations with steatotic liver isografts for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TR, #p < 0.05 versus TR+TUDCA.

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Modulation of the TLR4 pathway by PPARγ in steatotic livers in syngeneic liver transplantation

As mentioned before, TUDCA treatment reduced PPARγ, and therefore ameliorated damage in steatotic grafts. We next evaluated whether TUDCA-induced PPARγ inhibition affected the TLR4 pathway in steatotic grafts. In the TR group, TLR4 protein levels were lower in steatotic grafts than in the sham group (Figure 3E). In the TR+TUDCA and TR+PPARγ antagonist groups, TLR4 protein levels were higher in steatotic grafts than in the TR group. TR+TUDCA+PPARγ agonist group abolished the effects of TUDCA on TLR4, because it resulted in TLR4 protein levels in steatotic grafts similar to those in the TR group (Figure 3E). Thus, TUDCA treatment reduced PPARγ and this PPARγ inhibition was associated with higher TLR4 levels than those observed in the TR group. Protein levels of MyD88 were unchanged in all groups (Figure 3F). However, a pattern similar to that observed for TLR4 protein levels were observed for TRIF protein levels in the groups evaluated (Figure 3G).

Next, we conducted experiments to evaluate whether TLR4 up-regulation consequent to PPARγ inhibition protected steatotic liver grafts. TR+PPARγ antagonist group, which increased TLR4, protected steatotic grafts (Figures 4A–C). In contrast, TR+PPARγ antagonist+anti-TLR4 group resulted in hepatic injury parameters similar to those in the TR group. Thus, TLR4 action inhibition abolished the benefits of PPARγ antagonists in steatotic liver grafts. We then activated TLR4 by administering a TLR4 agonist in the TR group (TR+TLR4 agonist group). Under these conditions steatotic liver grafts were protected (Figures 4A–C). The hepatic lesions observed in the TR+PPARγ antagonist+anti-TLR4 groups (Figure 4C) were similar to those in the TR group (Figure 3D). In the TR+TLR4 agonist group (Figure 4C), the extent and the number of necrotic areas in steatotic liver grafts was less than the TR group (Figure 3D). The recipients transplanted with steatotic grafts without any treatment (TR group) showed 30% survival at 14 days, most of the deaths occurring within 2 days (Figure 4D). The treatment with TUDCA, PPARγ antagonist and TLR4 agonist reduced lethality in recipients transplanted with steatotic grafts, and resulted in a 70% survival rate at 14 days (Figure 4D). The survival rates in TR+TUDCA+PPARγ agonist and TR+PPARγ antagonist+anti-TLR4 groups were similar to those of the TR group.

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Figure 4. Effect of TLR4 regulation on damage in steatotic liver isografts 4 h after reperfusion and survival of recipients transplanted with steatotic liver isografts at 14 days after transplantation. Trasaminases (A and B) and damage score (C) as described in Figure 1 legend. Representative photographs of histological changes in steatotic livers are shown in part (C). (TR+PPARγ antagonist+anti-TLR4), widespread coagulative hepatic necrosis with neutrophil infiltration. (TR+TLR4 agonist), small area of coagulative hepatic necrosis (Bars, 1000 μm). For all parts of the figure, 6 transplantations with steatotic livers for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TR, °p < 0.05 versus TR+PPARγ antagonist. Survival studies are shown in part (D). 10 transplantations with steatotic liver isografts for each group. TR (□), TR+TUDCA (•), TR+PPARγ antagonist (Δ), TR+TUDCA+PPARγ agonist (◊), TR+PPARγ antagonist+anti-TLR4 (▪), TR+TLR4 agonist (○).

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TLR4 blockade reduced hepatic injury, neutrophil accumulation and TNFα, IL-1 and IL-6 release from Kupffer cells in nonsteatotic livers undergoing I/R (27,30). Then, we evaluated the effects of TLR4 inhibition on hepatic damage, inflammatory cell infiltration and proinflammatory cytokine levels in steatotic grafts. In line with previous studies (44,47,48), the results presented herein indicate that hepatic damage in steatotic livers does not correspond to either the degree of inflammatory cell infiltration or proinflammatoy cytokines. Indeed, TR+anti-TLR4 group resulted in hepatic injury parameters higher than those of the TR group (Figures 5A and B). However, hepatic MPO, TNF, IL-1 and IL-6 levels observed in TR+anti-TLR4 group were similar to those in the TR group (Figures 5C–F).

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Figure 5. Effect of TLR4 inhibition on inflammatory cell infiltration and proinflammatory cytokine levels in steatotic liver isografts 4 h after reperfusion. (A and B) Transaminase levels and (C) MPO, (D) TNF, (E) IL-1 and (F) IL-6. For all parts of the figure, six transplantations with steatotic livers for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TR.

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The reperfusion time dependent effects of TUDCA on ER stress markers, PPARγ levels and TLR4 pathway in steatotic livers were measured throughout reperfusion (Figure 6). ER stress parameters were similar in Sham, TR and TR+TUDCA groups at each time (Figures 6A–D). PPARγ levels were lower in TR+TUDCA than in TR group at each time (Figure 6E). TLR4 and TRIF (but not MyD88) levels were higher in TR+TUDCA than in TR group at each time (Figures 6F–H).

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Figure 6. Reperfusion time dependent effect of TUDCA in steatotic liver isografts on UPR induction, PPARγ protein expression and TLR4 pathway. (A) Protein levels of GRP78, (B) p-50 ATF6α, (C) XBP1(s) and TRAF2, (D) p-PERK, p-eIF2α, ATF4 and CHOP, (E) PPARγ, (F) TLR4, (G) MyD88 and (H) TRIF in steatotic livers at 1, 2, 4, 12 and 18 h after reperfusion. The scanning values for GRP78, ATF6α, ATF6β, XBP1(s), TRAF2, ATF4, CHOP, PPARγ, TLR4, MyD88 and TRIF were divided by the scanning values for β-actin, and those of p-PERK and p-eIF2α by the total PERK and eIF2α, respectively. For all parts of the figure, 6 transplantations with steatotic livers for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TR.

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Effect of TUDCA and TLR4 on hepatic injury in steatotic and nonsteatotic livers in allogeneic liver transplantation

As described below, the effects of TUDCA and TLR4 on hepatic injury, ER stress parameters, oxidative stress, PPARγ levels and TLR4 pathway in allograft model followed a similar pattern to those described in isograft model. Thus, nonsteatotic allografts were not protected against hepatic injury in the TRAllograft+TUDCA group. However, TRAllograft+TUDCA group protected steatotic allografts, as the hepatic injury parameters were lower than those recorded in the TR group (Figure 7). GRP78 and the three branches of the UPR are inactivated in steatotic allografts. The protein levels of p-50 ATF6β (data not shown), GRP78, p-50 ATF6α, XBP-1(S), TRF2, p-PERK, p-eIF2α, ATF4 accumulation and CHOP (Figures 8A–D) in steatotic allografts of the TRAllograft and TRAllograft+TUDCA groups were similar to those found in the Sham group. As shown in Figure 8E, TRAllograft+TUDCA group resulted in MDA and nitrotyrosine levels in steatotic allografts similar to those in the TRAllograft group. TRAllograft+TUDCA group reduced PPARγ protein levels in steatotic allografts with respect to those in the TRAllograft group (Figure 8F). TLR4 and TRIF protein levels were lower in steatotic allografts of TRAllograft group than in the sham group (Figures 9A–C). In the TRAllograft+TUDCA group, TLR4 and TRIF protein levels were higher in steatotic allografts than in the TRAllograft group (Figures 9A and C). The protein levels of MyD88 were unchanged in all groups (Figure 9B). Recipients transplanted with steatotic allografts without any treatment (TRAllograft group) showed 30% survival at 14 days, most of the deaths occurring within 2 days (Figure 9D). The treatment with TUDCA and TLR4 agonist reduced lethality in recipients transplanted with steatotic grafts, and resulted in a 70% survival rate at 14 days (Figure 9D).

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Figure 7. Effect of TUDCA on hepatic injury in steatotic liver allografts 4 h after reperfusion. (A and B) Transaminases and (C) damage score. The severity of hepatic injury was evaluated by a point-counting method on an ordinal scale as described in Figure 1 legend. For all parts of the figure, six transplantations with steatotic livers grafts for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TRAllograft.

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Figure 8. Effect of TUDCA on UPR induction, oxidative stress and PPARγ protein expression in steatotic liver allografts 4 h after reperfusion. (A) Protein levels of GRP78, (B) p-50 ATF6α, (C) XBP1(s) and TRAF2, (D) p-PERK, p-eIF2α, ATF4 and CHOP, (E) MDA and nitrotyrosine levels and (F) PPARγ. Representative western blots at the top and densitometric analysis at the bottom. The scanning values for GRP78, ATF6α, ATF6β, XBP1(s), TRAF2, ATF4, CHOP and PPARγ were divided by the scanning values for β-actin, and those of p-PERK and p-eIF2α by the total PERK and eIF2α, respectively. For all parts of the figure, six transplantations with steatotic livers for each group were included in each measurement. For UPR induction, TRAllograft < versus Sham (p = NS), TR+TUDCA < versus TRAllograft (p = NS), TRAllograft+TUDCA < versus Sham (p = NS). For MDA and PPARγ, *p < 0.05 versus Sham, +p < 0.05 versus TR.

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Figure 9. Effect of TUDCA on TLR4 pathway in steatotic liver allografts 4 h after reperfusion and survival of recipients transplanted with steatotic liver allografts at 14 days after transplantation. (A) TLR4, (B) MyD88 and (C) TRIF. Representative western blots at the top and densitometric analysis at the bottom. The scanning values for TLR4, MyD88 and TRIF were divided by the scanning values for β-actin. For all parts of the figure, six transplantations with steatotic livers for each group were included in each measurement. *p < 0.05 versus Sham, +p < 0.05 versus TRAllograft. Survival studies are shown in part (D). Ten transplantations with steatotic livers for each group. TRAllograft (□),TRAllograft+TUDCA (•),TRAllograft+TLR4 agonist (○).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Data obtained herein suggest that TUDCA may play a beneficial role in the protection of steatotic grafts in experimental models of syngeneic and allogeneic steatotic liver transplantation. In our conditions, ER stress is not induced in steatotic and nonsteatotic liver grafts and TUDCA was only effective in steatotic grafts, through a mechanism independent of ER stress. Thus, targeting the ER with TUDCA may not be a useful therapeutic approach in clinical conditions in which steatotic liver grafts are preserved in UW solution for 6 h of cold ischemia.

In the same conditions evaluated herein, PPARγ worsened hepatic injury in steatotic livers (21). Here, we report that TUDCA reduced PPARγ over-expression in both steatotic liver isografts and allografts. We evaluated whether TUDCA-induced PPARγ inhibition affected the TLR4 pathway in steatotic grafts. The inhibition of PPARγ action with PPARγ antagonists increased TLR4 and TRIF in steatotic liver grafts. This protected them against the damage associated with transplantation. Moreover, the activation of PPARγ abolished the benefits of TUDCA on the TLR4 pathway and damage in steatotic grafts. Thus, we suggest a new mechanism, namely TLR4 pathway induction, to explain why TUDCA-induced PPARγ inhibition protects steatotic liver grafts. Our results in isograft and allograft model indicate that in steatotic liver transplantation, the reduction in TLR4 protein expression was associated with reduced TRIF protein expression, whereas no changes in MyD88 expression were observed. This suggests that TRIF (but not MyD88) is the branch of downstream signaling pathways of TLR4 in steatotic liver transplantation. Further studies (beyond the scope of this work) will be required to answer why TLR4 pathway is down-regulated in steatotic liver transplantation compared with steatotic liver without a surgical procedure (sham group). Several molecules that play key roles in the down-regulation of TLR4 signaling, such as suppressor or cytokine signaling (SOCS)-1, SOCS-3 and interleukin (IL)-1 receptor-associated kinase (IRAK)-M (49–52), should be explored as possible answers to this question. Here, we report by first time evidence of the beneficial effects of strategies to up-regulate TLR4 in steatotic liver isografts and allografts. TLR4 agonists and TUDCA (which increased TLR4) were administered only in donors, before cold ischemia period. Our results would be in line with previous studies in normotermic hepatic ischemia indicating that the induction of chemical preconditioning using TLR4 ligands protects the liver against the deleterious effects of a subsequent I/R (52). This study does not analyze why TLR4 signaling protect steatotic liver grafts against damage associated with transplantation. To answer this question, previous results obtained in isolated cells should be considered (49). These suggest that increased TLR4 signaling triggers beneficial responses; namely the induction of protective Type I IFNs in hepatocytes and antiinflammatory IL-10 in macrophages in an IRF3-dependent manner.

Strategies based on TLR4 signaling pathway regulation could improve the postoperative outcome of patients undergoing hepatic resections and increase the number of organs suitable for transplantation. These strategies may improve the outcome of patients receiving marginal grafts that would not otherwise have been transplanted, leading to new possibilities for clinical transplantation. However, before a successful therapeutic strategy based on the TLR4 signaling pathway regulation is defined, several additional points need to be addressed. The effects of TLR4 differ according to the surgical conditions. Therefore, it seems plausible that in contrast to approaches that would completely abrogate TLR4 signaling, as in warm ischemic conditions, we could activate the TLR4 pathway to protect steatotic liver grafts against damage associated with transplantation. Moreover, the response of TLR4 might vary and involve different signal transduction pathways depending on the surgical procedure. These pathways are poorly understood at present.

To sum up, TUDCA protected steatotic liver grafts preserved in UW solution for 6 h, though a mechanism independent of ER stress. Our results report new properties of TUDCA in steatotic liver transplantation, based on a relationship between TUDCA, PPARγ and TLR4. We also describe the role of the TLR4 pathway in steatotic liver transplantation. TUDCA reduced PPARγ and this up-regulated the TLR4 pathway, specifically the TRIF pathway, protecting steatotic liver grafts. The results point to new possibilities for therapeutic interventions based on TLR4 signaling activation to protect steatotic liver grafts against damage associated with transplantation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

This research was supported by the Ministerio de Ciencia e Innovación (project grant BFU2009-07410) Madrid, Spain. Jiménez-Castro M.B. is in receipt of a fellowship from the Sociedad Española de Transplante Hepatico (SETH Foundation; Barcelona, Spain). We are grateful to Michael Maudsley at the Languaje Advisory Service of the University of Barcelona for revising the English text.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References