Orthotopic mouse liver transplantation to study liver biology and allograft tolerance.

Orthotopic liver transplantation in the mouse is a powerful research tool that has led to important mechanistic insights into the regulation of hepatic injury, liver immunopathology, and transplant tolerance. However, it is a technically demanding surgical procedure. Setup of the orthotopic liver transplantation model comprises three main stages: surgery on the donor mouse; back-table preparation of the liver graft; and transplant of the liver into the recipient mouse. In this protocol, we describe our procedure in stepwise detail to allow efficient completion of both the donor and recipient operations. The protocol can result in consistently high technical success rates when performed by personnel experienced in the protocol. The technique can be completed in ∼2-3 h when performed by an individual who is well practiced in performing mouse transplantation in accordance with this protocol. We have achieved a perioperative survival rate close to 100%.

Contribution of alloantigens to hepatic ischemia/reperfusion injury: Roles of natural killer cells and innate immune recognition of nonself.

Hepatic ischemia/reperfusion injury (IRI) remains a major clinical problem and involves the innate immune system's recognition of "nonself." Considering the efficient nonself recognition by natural killer (NK) cells, we hypothesize in this study that hepatic IRI associated with liver transplantation (LT) could be augmented in allogeneic rather than in syngeneic (Syn) grafts due to alloantigen recognition by innate immune cells, especially by NK cells. Using green fluorescent protein (GFP)/Sprague-Dawley rats, we tested our hypothesis in a rat LT model with 18 hours of cold storage in University of Wisconsin solution. Hepatic IRI was significantly augmented in allografts with higher alanine transaminase levels, increased necrosis, and vigorous proinflammatory mediator up-regulation compared to Syn grafts. Injury increased in allografts associated with augmented GFP+ host leukocyte infiltration due to significantly increased host CD11b/c+ and RP-1(+) neutrophil recruitment. A large number of liver-resident (donor) mature CD11b/c+ NK cells quickly diminished from allografts, but not from Syn grafts. Depletion of mature NK cells from liver grafts with anti-asialo monosialotetrahexosylganglioside significantly improved hepatic IRI and reduced neutrophil infiltration and proinflammatory mediators. In conclusion, early innate immune responses were more significantly enhanced in allografts than in Syn grafts during hepatic IRI, in part through NK cell recognition of "missing self."

Plasmacytoid dendritic cell-derived IFN-α promotes murine liver ischemia/reperfusion injury by induction of hepatocyte IRF-1.

UNLABELLED: Plasmacytoid dendritic cells (pDC) constitute the body's principal source of type I interferon (IFN) and are comparatively abundant in the liver. Among various cytokines implicated in liver ischemia and reperfusion (I/R) injury, type I IFNs have been described recently as playing an essential role in its pathogenesis. Moreover, type I IFNs have been shown to up-regulate hepatocyte expression of IFN regulatory factor 1 (IRF-1), a key transcription factor that regulates apoptosis and induces liver damage after I/R. Our aim was to ascertain the capacity of IFN-α released by liver pDC to induce liver damage through hepatic IRF-1 up-regulation after I/R injury. Our findings show that liver pDC mature and produce IFN-α in response to liver I/R. Liver pDC isolated after I/R induced elevated levels of IRF-1 production by hepatocytes compared with liver pDC isolated from sham-operated mice. Notably, hepatic IRF-1 expression was reduced significantly by neutralizing IFN-α. In vivo, IFN-α neutralization protected the liver from I/R injury by reducing hepatocyte apoptosis. This was associated with impaired expression of IRF-1 and proapoptotic molecules such as Fas ligand, its receptor (Fas) and death receptor 5, which are regulated by IRF-1. Furthermore, pDC-depleted mice failed to up-regulate hepatic IFN-α and displayed less liver injury associated with reduced levels of hepatic interleukin (IL)-6, tumor necrosis factor-α, and hepatocyte apoptosis after I/R compared with controls. CONCLUSION: these data support the hypothesis that IFN-α derived from liver pDC plays a key role in the pathogenesis of liver I/R injury by enhancing apoptosis as a consequence of induction of hepatocyte IRF-1 expression.

Recipient hyperbilirubinaemia protects cardiac graft in rat heterotopic heart transplantation.

OBJECTIVES: Since bilirubin is a known powerful antioxidant, this study examined whether recipient hyperbilirubinaemia protected heart grafts from ischaemia/reperfusion (I/R) injury and chronic rejection associated with rat cardiac transplantation. METHODS: Heterotopic heart transplantation (HTx) was performed using congenitally hyperbilirubinaemic GUNN (j/j) and normobilirubinaemic GUNN (+/+) rats. Syngenic grafts from +/+ rats were transplanted into +/+ or j/j rats with 6 or 18 h cold storage in University of Wisconsin solution to study I/R injury. To evaluate the effect on chronic rejection, Brown Norway rat heart grafts were transplanted into +/+ or j/j rats under short-course tacrolimus immunosuppression. RESULTS: The +/+ grafts in j/j rats demonstrated significantly lower serum creatine phosphokinase and higher left ventricular developed pressures and had smaller infarct areas than +/+ rats at 3 h after reperfusion. Graft survival with 18 h cold storage increased from 0% in +/+ rats to 41.7% in j/j rats. Malondialdehyde (a marker of lipid peroxidation), mRNA of the inflammatory mediators and phosphorylation of ERK1/2 were significantly decreased in the grafts transplanted into j/j rats compared with those transplanted into +/+ rats 1-3 h after reperfusion. The mean allograft survival in j/j recipients was prolonged to a median survival of 150 days from 84 days in +/+ recipients and was associated with less macrophage infiltrates and less intragraft inflammatory cytokine mRNA at d60. In vitro T-cell proliferation was significantly inhibited in the presence of bilirubin. CONCLUSIONS: Recipient hyperbilirubinaemia ameliorated cardiac I/R injury, as well as chronic allograft rejection following HTx via regulation of inflammatory responses or T-cell proliferation.

A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) that express glial fibrillary acidic protein (GFAP) are located between the sinusoidal endothelial cells and hepatocytes. HSCs are activated during liver injury and cause hepatic fibrosis by producing excessive extracellular matrix. HSCs also produce many growth factors, chemokines and cytokines, and thus may play an important role in acute liver injury. However, this function has not been clarified due to unavailability of a model, in which HSCs are depleted from the normal liver. METHODS: We treated mice expressing HSV-thymidine kinase under the GFAP promoter (GFAP-Tg) with 3 consecutive (3 days apart) CCl4 (0.16 µl/g; ip) injections to stimulate HSCs to enter the cell cycle and proliferate. This was followed by 10-day ganciclovir (40 µg/g/day; ip) treatment, which is expected to eliminate actively proliferating HSCs. Mice were then subjected to hepatic ischemia/reperfusion (I/R) or endotoxin treatment. RESULTS: CCl4/ganciclovir treatment caused depletion of the majority of HSCs (about 64-72%), while the liver recovered from the initial CCl4-induced injury (confirmed by histology, serum ALT and neutrophil infiltration). The magnitude of hepatic injury due to I/R or endotoxemia (determined by histopathology and serum ALT) was lower in HSC-depleted mice. Their hepatic expression of TNF-α, neutrophil chemoattractant CXCL1 and endothelin-A receptor also was significantly lower than the control mice. CONCLUSIONS: HSCs play an important role both in I/R- and endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects.

CD39 expression by hepatic myeloid dendritic cells attenuates inflammation in liver transplant ischemia-reperfusion injury in mice.

UNLABELLED: Hepatic innate immune cells, in particular, interstitial dendritic cells (DCs), regulate inflammatory responses and may promote inherent liver tolerogenicity. After tissue injury, adenosine triphosphate (ATP) is released and acts as a damage-associated molecular pattern that activates innate immune cells by pattern recognition receptors. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) rapidly hydrolyzes extracellular ATP to maintain physiological levels. We hypothesized that CD39 expression on liver DCs might contribute to regulation of their innate immune functions. Mouse liver conventional myeloid DCs (mDCs) were hyporesponsive to ATP, compared with their splenic counterparts. This disparity was ascribed to more efficient hydrolysis of ATP by higher expression of CD39 on liver mDCs. Human liver mDCs expressed greater levels of CD39 than those from peripheral blood. The comparatively high expression of CD39 on liver mDCs correlated strongly with both ATP hydrolysis and adenosine production. Notably, CD39(-/-) mouse liver mDCs exhibited a more mature phenotype, greater responsiveness to Toll-like receptor 4 ligation, and stronger proinflammatory and immunostimulatory activity than wild-type (WT) liver mDCs. To investigate the role of CD39 on liver mDCs in vivo, we performed orthotopic liver transplantation with extended cold preservation using CD39(-/-) or WT donor mouse livers. Compared to WT liver grafts, CD39(-/-) grafts exhibited enhanced interstitial DC activation, elevated proinflammatory cytokine levels, and more-severe tissue injury. Moreover, portal venous delivery of WT, but not CD39(-/-) liver mDCs, to donor livers immediately post-transplant exerted a protective effect against graft injury in CD39(-/-) to CD39(-/-) liver transplantation. CONCLUSIONS: These data reveal that CD39 expression on conventional liver mDCs limits their proinflammatory activity and confers protective properties on these important innate immune cells against liver transplant ischemia/reperfusion injury.

Augmenter of liver regeneration (ALR) is a novel biomarker of hepatocellular stress/inflammation: in vitro, in vivo and in silico studies.

The liver is a central organ involved in inflammatory processes, including the elaboration of acute-phase proteins. Augmenter of liver regeneration (ALR) protein, expressed and secreted by hepatocytes, promotes liver regeneration and maintains viability of hepatocytes. ALR also stimulates secretion of inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and nitric oxide from Kupffer cells. We hypothesized that ALR may be involved in modulating inflammation induced by various stimuli. We found that hepatic ALR levels are elevated at 24 h, before or about the same time as an increase in the mRNA expression of TNF-α and IL-6, after portacaval shunt surgery in rats. Serum ALR also increased, but significantly only on d 4 when pathological changes in the liver become apparent. In rats, serum ALR was elevated after intraperitoneal administration of lipopolysaccharide alone and in a model of gram-negative sepsis. Serum ALR increased before alanine aminotransferase (ALT) in endotoxemia and in the same general time frame as TNF-α and IL-6 in the bacterial sepsis model. Furthermore, mathematical prediction of tissue damage correlated strongly with alterations in serum ALR in a mouse model of hemorrhagic shock. In vitro, monomethyl sulfonate, TNF-α, actinomycin D and lipopolysaccharide all caused increased release of ALR from rat hepatocytes, which preceded the loss of cell viability and/or inhibition of DNA synthesis. ALR may thus serve as a potential diagnostic marker of hepatocellular stress and/or acute inflammatory conditions.

Gut bacteria drive Kupffer cell expansion via MAMP-mediated ICAM-1 induction on sinusoidal endothelium and influence preservation-reperfusion injury after orthotopic liver transplantation.

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.

Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury.

UNLABELLED: Dendritic cells (DCs) induce and regulate both innate and adaptive immune responses; however, their in vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver-resident DC and locally recruited blood-borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by using DC-deficient, fms-like tyrosine kinase 3 ligand (Flt3L) knockout (KO) mice in hepatic warm (70% partial clamping for 60 minutes) and cold IR injury (liver transplant [LTx] with 24-hour cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c+ F4/80- DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in wildtype (WT) mice with lower alanine aminotransferase (ALT) levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic messenger RNA (mRNA) and protein levels for inflammatory cytokines (tumor necrosis factor alpha [TNFα], interleukin [IL]-6) and chemokines (CCL2, CXCL2) were also significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver-resident and blood-borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3L KO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver-resident DC. CONCLUSION: Using both warm and cold hepatic IR models, this study suggests differential roles of liver-resident versus blood-borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury.

Long-term survival, nutritional autonomy, and quality of life after intestinal and multivisceral transplantation.

OBJECTIVE: To assess long-term survival, graft function, and health-related quality of life (QOL) after visceral transplantation. BACKGROUND: Despite continual improvement in early survival, the long-term therapeutic efficacy of visceral transplantation has yet to be defined. METHODS: A prospective cross-sectional study was performed on 227 visceral allograft recipients who survived beyond the 5-year milestone. Clinical data were used to assess outcome including graft function and long-term survival predictors. The socioeconomic milestones and QOL measures were assessed by clinical evaluation, professional consultation, and validated QOL inventory. RESULTS: Of 376 recipients, 227 survived beyond 5 years, with conditional survival of 75% at 10 years and 61% at 15 years. With a mean follow-up of 10 ± 4 years, 177 (92 adults, 85 children) are alive, with 118 (67%) recipients 18 years or older. Nonfunctional social support and noninclusion of the liver in the visceral allograft are the most significant survival risk factors. Nutritional autonomy was achievable in 160 (90%) survivors, with current serum albumin level of 3.7 ± 0.5 gm/dL and body mass index of 25 ± 6 kg/m(2). Despite coexistence or development of neuropsychiatric disorders, most survivors were reintegrated to society with self-sustained socioeconomic status. In parallel, most of the psychological, emotional, and social QOL measures significantly (P < 0.05) improved after transplantation. Current morbidities with potential impact on global health included dysmotility (59%), hypertension (37%), osteoporosis (22%), and diabetes (11%), with significantly (P < 0.05) higher incidence among adult recipients. CONCLUSIONS: With new tactics to further improve long-term survival including social support measures, visceral transplantation has achieved excellent nutritional autonomy and good QOL.

Costimulation blockade in pig artery patch xenotransplantation - a simple model to monitor the adaptive immune response in nonhuman primates.

BACKGROUND: CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade. METHODS: Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration. RESULTS: Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration. CONCLUSIONS: (i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.

Interferon regulatory factor-2 is protective against hepatic ischemia-reperfusion injury.

Interferon regulatory factor (IRF)-1 is a nuclear transcription factor that induces inflammatory cytokine mediators and contributes to hepatic ischemia-reperfusion (I/R) injury. No strategies to mitigate IRF1-mediated liver damage exist. IRF2 is a structurally similar endogenous protein that competes with IRF1 for DNA binding sites in IRF-responsive target genes and acts as a competitive inhibitor. However, the role of IRF2 in hepatic injury during hypoxic or inflammatory conditions is unknown. We hypothesize that IRF2 overexpression may mitigate IRF1-mediated I/R damage. Endogenous IRF2 is basally expressed in normal livers and is mildly increased by ischemia alone. Overexpression of IRF2 protects against hepatic warm I/R injury. Furthermore, we demonstrate that IRF2 overexpression limits production of IRF1-dependent proinflammatory genes, such as IL-12, IFNß, and inducible nitric oxide synthase, even in the presence of IRF1 induction. Additionally, isograft liver transplantation with IRF2 heterozygote knockout (IRF2(+/-)) donor grafts that have reduced endogenous IRF2 levels results in worse injury following cold I/R during murine orthotopic liver transplantation. These findings indicate that endogenous intrahepatic IRF2 protein is protective, because the IRF2-deficient liver donor grafts exhibited increased liver damage compared with the wild-type donor grafts. In summary, IRF2 overexpression protects against I/R injury by decreasing IRF1-dependent injury and may represent a novel therapeutic strategy.

Selective expansion of allogeneic regulatory T cells by hepatic stellate cells: role of endotoxin and implications for allograft tolerance.

Hepatic stellate cells (HSCs) may play an important role in hepatic immune regulation by producing numerous cytokines/chemokines and expressing Ag-presenting and T cell coregulatory molecules. Due to disruption of the endothelial barrier during cold-ischemic storage and reperfusion of liver grafts, HSCs can interact directly with cells of the immune system. Endotoxin (LPS), levels of which increase in liver diseases and transplantation, stimulates the synthesis of many mediators by HSCs. We hypothesized that LPS-stimulated HSCs might promote hepatic tolerogenicity by influencing naturally occurring immunosuppressive CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Following their portal venous infusion, allogeneic CD4(+) T cells, including Tregs, were found closely associated with HSCs, and this association increased in LPS-treated livers. In vitro, both unstimulated and LPS-stimulated HSCs upregulated Fas (CD95) expression on conventional CD4(+) T cells and induced their apoptosis in a Fas/Fas ligand-dependent manner. By contrast, HSCs induced Treg proliferation, which required cell-cell contact and was MHC class II-dependent. This effect was augmented when HSCs were pretreated with LPS. LPS increased the expression of MHC class II, CD80, and CD86 and stimulated the production of IL-1α, IL-1ß, IL-6, IL-10 and TNF-α by HSCs. Interestingly, production of IL-1α, IL-1ß, IL-6, and TNF-α was strongly inhibited, but that of IL-10 enhanced in LPS-pretreated HSC/Treg cocultures. Adoptively transferred allogeneic HSCs migrated to the secondary lymphoid tissues and induced Treg expansion in lymph nodes. These data implicate endotoxin-stimulated HSCs as important immune regulators in liver transplantation by inducing selective expansion of tolerance-promoting Tregs and reducing inflammation and alloimmunity.

T-cell-based immunosuppressive therapy inhibits the development of natural antibodies in infant baboons.

BACKGROUND: We set out to determine whether B-cell tolerance to A/B-incompatible alloantigens and pig xenoantigens could be achieved in infant baboons. METHODS: Artery patch grafts were implanted in the abdominal aorta in 3-month-old baboons using A/B-incompatible (AB-I) allografts or wild-type pig xenografts (pig). Group 1 (Gp1) (controls, n=6) received no immunosuppressive therapy (IS) and no graft. Gp2 (n=2) received an AB-I or pig graft but no IS. Gp3 received AB-I grafts+IS (Gp3A: n=2) or pig grafts+IS (Gp3B: n=2). IS consisted of ATG, anti-CD154mAb, and mycophenolate mofetil until age 8 to 12 months. Gp4 (n=2) received IS only but no graft. RESULTS: In Gp1, anti-A/B and cytotoxic anti-pig immunoglobulin-M increased steadily during the first year. Gp2 became sensitized to donor-specific AB-I or pig antigens within 2 weeks. Gp3 and Gp4 infants that received anti-CD154mAb made no or minimal anti-A/B and anti-pig antibodies while receiving IS. DISCUSSION: The production of natural anti-A/B and anti-pig antibodies was inhibited by IS with anti-CD154mAb, even in the absence of an allograft or xenograft, suggesting that natural antibodies may not be entirely T-cell independent. These data are in contrast to clinical experience with AB-I allotransplantation in infants, who cease producing only donor-specific antibodies.

The loss of renal dendritic cells and activation of host adaptive immunity are long-term effects of ischemia/reperfusion injury following syngeneic kidney transplantation.

Ischemia/reperfusion injury associated with kidney transplantation induces profound acute injury, influences early graft function, and affects long-term graft outcomes. To determine whether renal dendritic cells play any role during initial innate ischemia/reperfusion injury and the subsequent development of adaptive immune responses, we studied the behavior and function of renal graft and host infiltrating dendritic cells during early and late phases of renal ischemia/reperfusion injury. Wild type to green fluorescent protein (GFP) transgenic rat kidney transplantation was performed with and without 24-h cold storage. Ischemia/reperfusion injury in cold-stored grafts resulted in histopathological changes of interstitial fibrosis and tubular atrophy by 10 weeks, accompanied by upregulation of mRNAs of mediators of interstitial fibrosis and inflammation. In normal rat kidneys, we identified two populations of renal dendritic cells, predominant CD103(-)CD11b/c(+) and minor CD103(+)CD11b/c(+) cells. After transplantation without cold storage, grafts maintained CD103(-) but not CD103(+) GFP-negative renal dendritic cells for 10 weeks. In contrast, both cell subsets disappeared from cold-stored grafts, which associated with a significant GFP-expressing host CD11b/c(+) cell infiltration that included CD103(+) dendritic cells with a TNF-α-producing phenotype. These changes in graft/host dendritic cell populations were associated with progressive infiltration of host CD4(+) T cells with effector/effector-memory phenotypes and IFN-γ secretion. Thus, renal graft ischemia/reperfusion injury caused graft dendritic cell loss and was associated with progressive host dendritic cell and T-cell recruitment. Renal-resident dendritic cells might function as a protective regulatory network.

Carbon monoxide inhibits apoptosis during cold storage and protects kidney grafts donated after cardiac death.

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia-induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO-UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase-3 and PARP expression. In contrast, grafts in CO-UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO-UW with less TUNEL(+) cells and reduced mRNA upregulation for proinflammatory mediators (IL-6, TNF-α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO-UW for 24 h, graft PARP expression, TUNEL(+) cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia-induced apoptosis and significantly improved kidney graft function and outcomes of KTx.

Use of carbon monoxide in minimizing ischemia/reperfusion injury in transplantation.

Although carbon monoxide (CO) is known to be toxic because of its ability to interfere with oxygen delivery at high concentrations, mammalian cells endogenously generate CO primarily via the catalysis of heme by heme oxygenases. Recent findings have indicated that heme oxygenases and generation of CO serve as a key mechanism to maintain the integrity of the physiological function of organs and supported the development of a new paradigm that CO, at low concentrations, functions as a signaling molecule in the body and exerts significant cytoprotection. Consequently, exogenously delivered CO has been shown to mediate potent protection in various injury models through its anti-inflammatory, vasodilating, and antiapoptotic functions. Ischemia/reperfusion (I/R) injury associated with organ transplantation is one of the major deleterious factors limiting the success of transplantation. Ischemia/reperfusion injury is a complex cascade of interconnected events involving cell damage, apoptosis, vigorous inflammatory responses, microcirculation disturbance, and thrombogenesis. Carbon monoxide has a great potential in minimizing I/R injury. This review will provide an overview of the basic physiology of CO, preclinical studies examining efficacy of CO in I/R injury models, and possible protective mechanisms. Carbon monoxide could be developed to be a valuable therapeutic molecule in minimizing I/R injury in transplantation.

Treprostinil Improves Hepatic Cytochrome P450 Activity during Rat Liver Transplantation.

BACKGROUND: Cytochrome P450 (CYP450) activity is an important indicator of liver graft function. CYP450 activity is altered by pro-inflammatory cytokines, which are associated with ischemia-reperfusion (I/R) injury during orthotopic liver transplantation (OLT). Treprostinil, an FDA-approved prostacyclin analog, ameliorated cold I/R injury during rat OLT. We hypothesized that treprostinil would improve CYP450 activity in liver graft during cold I/R injury post-OLT. METHODS: OLT was performed in syngeneic male Lewis rats with 18 h graft preservation in cold UW solution. Donor and recipients received treprostinil (100 ng/kg/min) or matching placebo for 24 h before and up to 48 h post-OLT. Liver graft mRNA and protein expression of CYP450 isoforms were analyzed by qRT-PCR and Western blot analysis, respectively. The formation rates of 1-hydroxymidazolam and 6ß-hydroxytestosterone, 6-hydroxychlorzoxazone, 2α- and 16α-hydroxytestosterone in liver graft microsomes served as markers for CYP3A, CYP2E1, and CYP2C11 activity, respectively, and were measured by LC-MS. RESULTS: Treprostinil significantly decreased serum ALT and AST levels at 6-48 h after OLT, compared to placebo. The expressions of TNFα and IFNγ mRNA in the liver graft were significantly inhibited in the treprostinil-treated group at 1 h post-reperfusion. Treprostinil restored CYP2E1 protein expression to that of normal liver and significantly improved CYP3A activity to more than two-fold of placebo early post-OLT. CONCLUSIONS: Treprostinil significantly ameliorated hepatic injury, reduced expression of pro-inflammatory cytokines, and improved CYP450 activity in liver graft early post-OLT. These findings suggest that treprostinil has the potential to serve as a therapeutic option to protect liver graft function against I/R injury during clinical OLT.

Carbon monoxide induces hypothermia tolerance in Kupffer cells and attenuates liver ischemia/reperfusion injury in rats.

Ischemia/reperfusion (I/R) injury in liver grafts, which is initiated by cold preservation and is augmented by reperfusion, is a major problem that complicates graft quality, posttransplant patient care, and outcomes of liver transplantation (LT). Kupffer cells (KCs) play important roles in I/R injury; however, little is known about their changes during cold preservation. We examined whether a pretreatment with carbon monoxide (CO), a cytoprotective product of heme degradation, could influence KC activity during cold storage and protect liver grafts against LT-induced I/R injury. In vitro, primary rat KCs were stimulated for 24 hours under hypothermic conditions (4°C, 20% O(2)), with lipopolysaccharide, or under hypoxic conditions (37°C, 5% O(2)) with or without a CO pretreatment. When rat KCs were exposed to hypothermic conditions, they produced reactive oxygen species (ROS), but they did not produce tumor necrosis factor α (TNF-α) or nitric oxide. The preincubation of KCs with CO up-regulated heat shock protein 70 (HSP70) and inhibited ROS generation. When liver grafts from donor rats exposed to CO (250 ppm) for 24 hours were transplanted after 18 hours of cold preservation in University of Wisconsin solution, HSP70 expression increased in these grafts versus control grafts, and serum aspartate aminotransferase and alanine aminotransferase levels as well as necrotic areas and inflammatory infiltrates were significantly reduced after LT. CO-pretreated liver grafts showed less up-regulation of TNF-α and inducible nitric oxide synthase messenger RNA (mRNA) and reduced expression of proapoptotic B cell lymphoma 2-associated X protein mRNA, cleaved caspase-3, and poly(adenosine diphosphate ribose) polymerase. In conclusion, the pretreatment of donors with CO ameliorates LT-associated I/R injury with increased hepatic HSP70 expression, particularly in the KC population.