Steatosis in Liver Transplantation: Current Limitations and ... : Transplantation (original) (raw)
The prevalence of nonalcoholic fatty liver disease (NAFLD) in western countries is remarkable, it is estimated to be 10-24%,1 and strongly associates (65-80%) with obesity (defined as body mass index [BMI] >30).2 The prevalence of obesity and morbid obesity (BMI > 40) among adults in the Unitec is alarming, being 40% and 8%, respectively.3 If this trend continues, by 2030 86% of adult USA population will be overweight or obese.4 This will have an important impact on liver transplantation, by increasing the indication of liver transplantation due to cirrhosis secondary to NAFLD and also by augmenting the number of procured steatotic livers.
Nonalcoholic fatty liver disease is presently one of the principal causes of chronic liver disease in the world. Nonalcoholic fatty liver disease encompasses 2 main subtypes: nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH).5-7 Although NAFL is a somewhat a benign disorder with small probability for progression, individuals with NAFL are at increased risk of mortality when compared with general population, primarily because of cardiovascular events.8,9 In contrast, individuals with NASH are at higher risk of progression to cirrhosis, development of hepatocellular carcinoma, and liver-related complications.10 Currently, cirrhosis due to NASH is the second indication for liver transplantation in the United States.11
As of 2018, there are no approved drug treatments for NAFL or NASH.12 However, due to the improvement at understanding the pathogenesis of NAFL and NASH, there are several ongoing phase 2 and phase 3 clinical trials with drugs targeting different pathways (peroxisome proliferator-activated receptor [PPARs]), bile acid activated receptors, apoptosis, fibrosis, endocrine receptors, etc.).13-18 Although none of them is as of yet authorized for clinical use, it is highly certain that the therapeutic armament for NASH will likely grow in the following years.
Liver transplantation is the only lifesaving treatment option for patients with end-stage liver failure. The increasing gap between the demand and supply of organs has extended the interest in marginal grafts to expand the donor pool. A common type of marginal grafts is the fatty liver; such organs are more prone to preservation injury and graft dysfunction.19-21
Liver steatosis is a common finding during the procurement surgery22 and one of the major reasons to decline liver grafts for human transplantation. It is still unclear whether it is safe to use severe steatotic grafts for transplantation or not. A clear guideline for the use of fatty livers is still lacking, and the decision highly relies on the opinion of the transplant physician or surgeon. Most studies agree that macrosteatosis under 30% should not be a contraindication for transplantation.19-21 In contrast, even though there is appealing recent published data suggesting that the livers with moderate (30-60%) and severe macrosteatosis (>60%) can be safely used in low-risk donor-recipient allocation (low Model for End-Stage Liver Disease [MELD], short cold ischemia time [CIT], no retransplant or donation after circulatory death [DCD]) without compromising the outcomes,19,20 there is no consensus regarding the use of these grafts for transplantation.23-25
Extensive preclinical and clinical research on dynamic preservation with machine perfusion has opened the opportunity to assess and potentially treat fatty livers before transplantation. Although there is only limited data from use of warm and cold perfused fatty livers, the preliminary results are encouraging.26-30
This review focuses on the current and potential future therapeutic interventions for patients with NAFL and NASH. It also summarizes the present clinical practice on the use of fatty livers for transplantation and the recent advances of innovative experimental and clinically available protective strategies to improve the outcomes after transplantation with fatty livers.
CURRENT AND FUTURE TRENDS IN THE TREATMENT OF NASH
Nonalcoholic steatohepatitis has progressively become one of the principal causes of cirrhosis, hepatocellular carcinoma, and indication of liver transplantation. Apart of changes in lifestyle with diet and physical activity, there are presently no other accepted interventions for NAFL/NASH. Nevertheless, a large number of drugs have been assessed first in the in preclinical models and are currently being evaluated in phase 2 and phase 3 clinical trials.
Preclinical Studies for the Treatment of NASH
Several strategies have been evaluated in experimental models and have demonstrated outstanding results but just some of them have progressed to clinical trials. We focus this discussion specifically on those interventions that have advanced to phase 2/phase 3 clinical trials.
Pioglitazone (PPAR-γ agonist) in preclinical studies has demonstrated a reduction in the fat accumulation,31-33 additionally showing a decrease in the hepatic inflammation34,35 and fibrosis.31,35-37 Yang et al32 recently investigated through metabolomics and lipidomic analysis the effect of this drug on rats with NASH showing a significant increase in the expression of genes related to fatty acid oxidation and a downregulation of genes linked to phospholipid biosynthesis, de novo lipogenesis, fatty acid uptake, and transport in the liver32 which adds to the effects that have been shown on the increase of adiponectin and the reduction of insulin resistance in prior studies.38,39
Vitamin E has demonstrated to significantly reduce the total liver cholesterol, lipid peroxidase levels and the alpha smooth muscle actin (α-SMA)–positive areas.40 Additionally, experimental studies have found an association between the reduction of fat content and fibrosis in NASH models accompanied with downregulation of inflammatory and profibrotic genes and up-regulation of antiapoptotic genes.41
Liraglutide (glucagon-like peptide-1 [GLP-1] agonist) has shown to have a significant effect in reducing the hepatic lipid accumulation in NASH models through improving insulin sensitivity and affecting the expression of enzymes linked to the transport of lipids and β-oxydation.42 Additional investigations have demonstrated that the treatment with Liraglutide in NASH models improves the macroautophagy, outcome that has been linked to a protective effect against endoplasmic reticulum stress-induced cell death and progression of disease in NASH models.43
Elafibanor (PPAR-α/δ agonist) has demonstrated to reduce by approximately 60% the degree of macrosteatosis and significantly decrease the expression of proinflammatory genes, such as interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α), the macrophage marker, F4/80, and the fibrosis genes, TGF-β and the tissue inhibitor of metalloproteinase-2.44 In a recent article by Tølbøl et al,45 the authors demonstrated by using RNA sequencing a significant reduction in the genes associated with fibrosis and inflammation in the Elafibanor vs vehicle group.
Obeticholic acid (farnesoid X receptor [FXR] agonist) in numerous studies has demonstrated a beneficial effect in models of liver fibrosis,46-48 additionally the use of obeticholic acid in rat models has shown a significant reduction in alpha α-SMA and monocyte chemoattractant protein-1, indicative of decreased hepatic stellate cell and Kupffer cell activation, respectively.48 Of note, Goto et al49 reported in 2018, the effect of obeticholic acid to be limited to reduction of liver fibrosis and inflammation with no effect in the degree of steatosis in a mouse NASH model.
Cenicriviroc (C-C chemokine receptor [CCR2/CCR5] antagonist) was evaluated by Lefebvre et al50 who showed that this drug was able to significantly reduce the collagen deposition by 49% when compared with vehicle group in a rodent NASH model. This reduction was associated with a decrease in alanine aminotransferase measurement but no differences were found in the inflammation markers F4/80+ (macrophage marker) and C-C chemokine ligand type 2 levels. Data published in 2018 by Krenkel et al51 indicates that the effect of Cenicriviroc mainly affects the monocyte-derived macrophages (MoMF) infiltration but not Kupffer cells.
Selonsertib (apoptosis signal-regulating kinase-1 [ASK-1] inhibitor) has demonstrated a significant reduction in the liver inflammation through decreasing the levels of TNF-α, IL-6, and IL-1β.52 According to the work presented by Budas et al53 at the 2016 International Liver Congress, the use of ASK-1 inhibitor reduced the liver cholesterol content by 45% and showed a significant reduction of hepatic levels of fatty acids. In addition, Ikenaga et al54 recently showed a significant fibrosis reduction of 38% in the Selonsertib group when compared to control. Recent work by Liles et al55 at the 2017 demonstrated that the use of selonsertib ASK-1 promoted a reduction of 39% and 74% in the degree of steatosis and liver cholesterol, respectively.
Some other promising drugs are currently under development. These new agents have different targets and functions (anti-inflammatory and anti-fibrotic agents, bile acid activated receptors, PPARs, fibroblast growth factor [FGF]-19 and -21, lipid metabolism, intestinal transporters, intestinal microbiota, and endocrine receptors) (Table 1). The overall objective from all these interventions is to reduce the fat content, inflammatory response, and fibrosis.
Drugs under development for the treatment of NAFLD/NASH
Clinical Studies for the Treatment of NASH
In the context of clinical studies, the American Association for the Study of Liver Diseases (AASLD) guidelines currently recommends that only the patients that have a diagnosis of NASH proven by biopsy should be considered for medical treatment.12,56 Several agents have been evaluated in phase 2 and phase 3 clinical trials,13,14,57-62 but as of 2018, only few agents are recommended for specific situations in the treatment of patients with NASH.
Current Management and Treatments for NASH
Lifestyle modification has proven to have a beneficial effect in NAFL and NASH. A well-designed exercise regimen and weight loss is the foundation of treatment for these patients.63 Reduction of at least 7% is required to improve or resolve steatohepatitis.64,65
Prospective studies on bariatric surgery have shown to decrease the prevalence and severity and steatosis and ballooning at 1 and 5 years after the surgical intervention.66 These data are supported by a meta-analysis from 2015 where a reduction by 11.9% in the incidence of fibrosis was found with the reduction of liver weight.67
In regard to medical treatments, pioglitazone (PPAR-γ agonist) has proven in a phase 3 clinical trial (PIVENS trial-NCT00063622) and further clinical studies to have a beneficial effect in patients with NASH by reducing the degree of steatosis, inflammation, hepatocyte ballooning, NAFLD Activity Score (NAS) activity score, as well as promoting resolution of NASH and improving fibrosis13,62,68 (Table 2). The downside of this agent was the reappearance of NASH and elevation of aminotransferases after the medication was discontinued. Additionally, weight gain is an unwanted effect found with this drug.62 The 2018 AASLD guidelines for NAFL/NASH states that pioglitazone may be used to treat patients with biopsy-proven NASH after the risk and benefits have been discussed with the patient.
Results from drugs that have finished or entered in phase 3 clinical trials
Vitamin E in the phase 3 clinical study (PIVENS trial-NCT00063622) at a dose of 800 IU/d to be able to improve steatosis, inflammation, ballooning, NAS score as well as resolution of NASH13 (Table 2). These data are supported by meta-analysis that demonstrated significant histological benefits with the use of vitamin E in patients with NASH.69,70 Nevertheless, there are some studies that suggest that long-term use of vitamin E increases the incidence of hemorrhagic stroke and prostate cancer.13 At this time, the AASLD guidelines suggest that vitamin E may be used at a dose of 800 IU/d in adults without diabetes with biopsy-proven NASH after discussion of risks and benefits with the patients.
Future Potential Treatments for NASH
The following are some agents that are currently in phase 2/phase 3 clinical trials with some of them demonstrating outstanding results. The AASLD currently does not recommend the use of any of these agents.
Liraglutide (GLP-1 agonist) has demonstrated in a phase 2 study (LEAN trial-NCT01237119). a greater resolution of steatohepatitis and less progression of fibrosis when compared to control group. It also showed improvements in metabolic risk factors as weight, BMI, glucose level, and high-density lipoprotein cholesterol (HDL)18 (Table 2). Phase 3 trial is awaited to confirm these initial findings.
Obeticholic acid (FXR-agonist) demonstrated in a phase 2 study (FLINT trial-NCT01265498) to have a higher proportion of patients (62% vs 31%) with improvement in histology parameters as decrease in steatosis score and progression of liver fibrosis when compared with placebo15 (Table 2). Some of the side effects were severe pruritus in 23% of patients with discontinuation of medication in some cases and reduction in the HLD levels.15 A phase 3 clinical study has been initiated (REGERATE trial NCT02548351), and the results will determine the efficacy and safety of this drug.
Elafibranor (PPAR-α/δ agonist) demonstrated in a phase 2b study (GOLDEN trial NCT01694849) improvement in NASH resolution without fibrosis worsening. This agent additionally improved the cardiometabolic risk profile (Table 2). Nevertheless, some patients experienced elevation in creatinine level that improved after the drug was discontinued.14 The phase 3 clinical study is ongoing to determine the effect of Elafibranor on NASH resolution and long-term outcomes (RESOLVE-IT trial-NCT02704403).
Selonsertib (ASK-1 inhibitor) showed in results from the phase 2 clinical study (NCT02466516) a significant improvement in fibrosis (52%) when compared with baseline samples and simtuzumab group. In addition, selonsertib group showed beneficial effect on secondary endpoints (liver stiffness on magnetic resonance elastography, collagen content, lobular inflammation, markers of apoptosis, and necrosis) when compared with control group17 (Table 2). Based on these data, a phase 3 clinical study in individuals with stage 3 (STELLAR 3- NCT03053050) and 4 (STELLAR 4- NCT03053063) NASH has been initiated to evaluate the efficacy and safety of Selonsertib alone.
Cenicriviroc (CCR2/CCR5 receptor blocker) in a phase 2b study (CENTAUR trial NCT02217475) showed a significant decrease in fibrosis at 1 year after the start of treatment. Minor adverse effects were identified as fatigue (2.8%) and diarrhea (2.1%)16 (Table 2). This drug is currently being evaluated in a phase 3 study (AURORA trial NCT03028740) to determine its efficacy to improve fibrosis without worsening NASH.
OUTCOMES OF THE USE OF STEATOTIC LIVER GRAFTS FOR TRANSPLANTATION IN THE CURRENT PRACTICE
Mild Steatosis (<30% macrosteatosis)
It is now a generally accepted concept that the use of liver grafts with less than 30% macrosteatosis, and microsteatosis—regardless the percentage—is not associated with increased risk of primary graft nonfunction (PNF), early allograft dysfunction (EAD).19-21 Therefore, these grafts are considered safe for liver transplantation.
Moderate Steatosis (30-60% Maccrosteatosis)
In contrast to the livers with mild steatosis, the acceptance of moderate macrosteatotic grafts (30-60%) remains controversial, and it is still considered a relative contraindication. Prior studies have shown that the use of grafts with more than 30% of macrosteatosis is an independent risk factor for graft failure and EAD.19,20 Doyle et al reported in 2010 a significantly higher requirement of transfusion of packed red blood cell (8 U vs 3 U, P ≤ 0.001), fresh frozen plasma (4 U vs 2 U, P = 0.007), and cryoprecipitates in grafts with 35% or more steatosis versus those with 5% or less steatosis. Furthermore, it has been shown that those patients that receive a liver with more than 30% macrosteatosis have a longer hospital (11 days vs 5 days, P = 0.02) and intensive care unit (ICU) stay (21 days vs 11 days, P ≤ 0.001) when compared with those with 5% or less steatosis.24,71 In addition, studies have found that the recipients of these moderately steatotic livers suffer of higher postreperfusion injury and increased rate of impaired initial function (20-53%).23-25 However, some of the recent clinical series over the last decade reported favorable outcomes using liver grafts with 30% to 60% macrovesicular infiltration.23-25,71-77 In general, the development of primary graft nonfunction in those studies was reasonably low (0-4%), and the long-term prognosis was comparable to lean liver grafts.23-25,71-73,75-77 In addition, these studies found similar incidence of biliary (11% vs 14%, P = 0.88) and vascular (7.4% vs 5.1%, P = 0.65) complications in patients that received a liver graft with moderate steatosis when compared with livers with less than 30% macrosteatosis.24,25,71,72,74,76 Furthermore, in contrast to prior results, Chavin et al reported in 2013 that the length of hospital (10.3 ± 6.6 vs 10.9 ± 6.6, P = 0.92) and ICU stay (3.6 ± 2.6 vs 4.7 ± 3.8, P = 0.36) was similar for moderate fatty livers when compared with mild fatty or lean livers.23,72 These findings need to be interpreted with caution, since most studies contained small number of patients. In summary, these results suggest that grafts with moderate steatosis, in the absence of other risk factors, can be considered for transplant. However, impaired early liver function, prolonged ICU stay, and higher costs should be taken into account (Table 3).
Clinical studies and outcomes of patients transplanted with moderate steatotic livers (30-60%) in last decade
Severe Steatosis (>60% Macrosteatosis)
In the past, severe macrosteatosis had been considered to be an absolute contraindication for liver transplantation. Only a few studies have published clinical outcomes with severely macrosteatotic liver grafts often with unfavorable results.23,71-75,78,79 Investigators have found unacceptably high PNF (20-50%)73-75 and EAD (25-80%) rates.23,24,73-75 Furthermore, the incidence of early biliary complications seems to be strongly associated with the degree of graft steatosis.80 In contrast, some of the most recent studies reported excellent early and long-term outcomes. Of note, the early and long-term outcomes in those studies were similar in the rate of PNF (0% vs 0-3.8%), EAD (0% vs 0.3%), 1-year (82-94.7% vs 81-91.8%) and 3-year patient survivals (82-94.7% vs 70.5-85.8%) in the severe macrosteatosis versus no severe macrosteatosis group.72,78 The long-term outcomes likely correlate with the reduction in fatty infiltration of severe or moderate steatotic grafts that have been found after liver transplantation in prior studies.71 Some authors concluded that severely macrosteatotic grafts might be accepted for liver transplantation but only with a very careful patient selection algorithm that keeps additional risk factors to a minimum (low MELD score, donor and recipient age, short CIT, no retransplant).72,78 Again, results should be taken with caution due to the small size of the sample in these studies (Table 4).
Clinical studies and outcomes of patients transplanted with severely steatotic (>60%) livers in the last decade
DONOR AND RECIPIENT CHARACTERISTICS THAT PREDICT ADVERSE OUTCOMES WITH THE USE OF STEATOTIC LIVERS
In the clinical setting, the Donor Risk Index (DRI) is the most commonly used risk assessment score for livers from deceased donors. One drawback of this score is that DRI does not include graft macrovesicular steatosis as part of the assessment, which has demonstrated to be an important risk factor. Of note, a recent study by De Graaf et al74 have pointed out that macrosteatosis is even a more important factor than the DRI to evaluate outcomes.74 Two large-scale comprehensive studies addressed this question and evaluated the impact of graft steatosis in the context of risk assessment. Spitzer et al19 published in 2010 a study where the authors analyzed more than 5000 histology reports and the clinical outcomes after transplantation using the United Network for Organ Sharing (UNOS) database. They found that relative risk of 1-year graft loss was dramatically increased, by over 70%, when macrosteatosis in the graft was more than 30% compared to the reference group (<15%). Furthermore, even mild graft macrosteatosis (20-30%) in conjunction with long CIT (>11 hours) was also associated with a 54% higher rate of graft loss. Of note, short CIT in combination with steatotic grafts resulted in comparable outcomes to those seen in lean livers. The authors concluded in this study that livers with more than 30% of macrosteatosis can be safely used for transplantation if other risk factors (older donors, prolonged CIT, DCD grafts) are absent.19 Additional research by Dutkowski et al20 helped to develop and validate an adjusted Balance of Risk (BAR) score by using data from 17 744 liver biopsies in correlation with patient and graft outcomes. These data were obtained from the UNOS and European Liver Transplant Registry databases (Table 5).20 The adjusted BAR score includes donor and recipient age, MELD score, CIT, and the presence or absence of retransplantation. The score ranges from 0 to 27. Three risk groups were defined: a low morbidity with low mortality group (BAR score, 0-8), an increased morbidity with low mortality group (BAR score, 9-17), and a high morbidity with high mortality group (BAR score, >18)20 (Table 5). The multivariate Cox regression analysis showed that the transplant outcomes of grafts with at least moderate macrosteatosis (>30%) were only acceptable in low-risk group (BAR score, 0-9) (Table 5).20 This finding may explain the recently reported excellent outcomes using severe macrosteatotic liver graft for transplantation,72,78 where recipients were selected with a low MELD score.
Original and Adjusted BAR score
PROTECTIVE STRATEGIES FOR FATTY LIVER GRAFTS BEFORE TRANSPLANTATION
Venous systemic oxygenated persufflation (VSOP) and machine-based perfusion techniques at different temperatures (hypothermic oxygenated machine perfusion [HOPE], subnormothermic machine perfusion [SNMP], and normothermic machine perfusion [NMP]) represent the most promising protective strategies for fatty liver grafts before transplantation by improving organ viability and decrease preservation injury.27,28,30,81,82 Furthermore, its use may also provide an ideal opportunity for organ-specific pharmacologic interventions and a new modality of functional assessment before transplantation (Table 6 and 7) (Figure 1).
Preclinical animal studies with fatty liver grafts and VSOP or machine perfusion
Preclinical human studies with declined fatty livers and machine perfusion
VSOP and machine perfusion technologies for the preservation of fatty livers.
Venous Systemic Oxygenated Persufflation
Preclinical
Venous systemic oxygenated persufflation is performed by retrograde insufflation of gaseous oxygen via the hepatic vein during the cold storage time, allowing an escape route from the capillaries by creating multiply miniature pin-pricks on the surface of the liver (Figure 1). In rodent models with diet-induced fatty liver, Minor et al88,91 demonstrated the superiority of VSOP over static cold storage (SCS). Venous systemic oxygenated persufflation resulted in less parenchyma and mitochondria damage, attenuated postischemic Kupffer cell activation, improved microcirculation and functional recovery and energy recharge,91 along with reduced necrosis, and restored cellular autophagic clearance.88 Thus, overall, VSOP improved organ viability and allowed a better preservation of hepatic ultrastructure compared to SCS. The results were confirmed by other groups84,87 using fatty liver rat models. Ye et al87 used VSOP plus a high dose of glutathione (GSH) at the donor and recipient side, to attenuate consequent oxidative stress. Improved animal survival, liver function, redox status and energy charge were found in the treatment group compared to the SCS group. In 2013, Nagai et al84 published the use of a mixture of molecular oxygen and nitric oxide (NO) for VSOP with rat fatty livers, and demonstrated an improved microcirculation and portal flow, lower inflammation (IL-6 levels), elevated tissue adenosine triphosphate (ATP) levels, and improved inducible NO synthase/endothelial NO synthase balance with subsequently less histology damage over SCS84 (Table 6).
Clinical
Venous systemic oxygenated persufflation has already moved into the clinical application.27,30 The first human trial was reported in 2008, where VSOP was applied to rescue marginal liver grafts with less than 20% steatosis that were rejected by 3 transplant centers due to previous warm ischemia damage (>20 minutes) before explantation. After transplantation, all grafts performed with adequate initial function, and all the recipients survived for the minimum follow-up of 2 years without retransplantation.27 Furthermore, results from the randomized clinical trial were presented at the 2018 international congress of the transplantation society. In this study, VSOP showed a significant benefit in 5-year patient survival in the subgroups of livers with macrosteatosis, donor age is 70 years or older, and prolonged CIT96 (Table 8) (Figure 1).
Clinical studies that included fatty livers
HOPE
Preclinical
Hypothermic oxygenated machine perfusion is another strategy that has been broadly investigated for the use in marginal organs. Bessems et al90 in 2007 compared 24 h of SCS against HOPE in a model of isolated steatotic rat liver perfusion. Moderate steatosis (30-60% steatosis) was induced by methionine-choline–deficient diet. Steatotic grafts were better preserved with HOPE versus SCS, as determined by a higher ammonia clearance, increased ATP levels, and increased urea and bile production. In addition, Kron et al28 compared in a rat model of diet-induced severe macrosteatosis 1 hour of end-ischemia HOPE versus SCS. The authors found significant reduction of mitochondrial oxidative stress (8-oxo-2′-deoxyguanosine), damage-associated molecular pattern release (high-mobility group box 1 protein 1), endothelial activation (Von Willebrand factor, endothelin-1, Intercellular adhesion molecule-1), and Kupffer cell activation (Toll-like receptor-4, myeloid differentiation primary response-88 induction) in the HOPE- versus SCS-treated livers. HOPE was also associated with a reduction of proinflammatory pathways (TNF-α, IL-6). The beneficial impact of machine perfusion was dependent of the presence of oxygen in the perfusate. Without oxygen, the perfusion was no longer protective28 (Table 6).
Clinical
In 2017, Kron et al28 published the first human study using HOPE treatment for fatty liver grafts. The authors reported 6 steatotic grafts, with 5 livers being retrieved from DCD donors. The HOPE-treated steatotic livers were compared with 12 donations after brain death static cold-stored grafts that were matched for donor and recipient age and for total preservation time. The median percentage of macrosteatosis was 30% (20-40%) in the HOPE group, and 35 (20-60%) in the nonperfused group, respectively. All HOPE-treated grafts showed adequate initial graft function, with low mean peak of aspartate aminotransferase (AST) (1871 U/L), short ICU stay (3 days) and without development of PNF or need for retransplantation. Only 1 patient needed hemodialysis, and all patients survived for 1 year. In contrast, 3 of 12 recipients with SCS had PNF, 3 had retransplantation, and 75% needed hemodialysis in the control group. The mean peak AST was 3800 U/L, ICU stay was 15 days, and the 1-year patient survival was 42% in the control group. The authors concluded that HOPE is especially beneficial for fatty liver grafts28 (Table 8) (Figure 1). Results from the randomized clinical trial are awaited (NCT01317342).
Subnormothermic Machine Perfusion
Preclinical
In 2009, Vairetti et al89 investigated SNMP at 20°C for 6 hours in steatotic rat livers. The perfusate contained glucose, N-acetylcysteine, and Krebs-Henseleit solution. The authors found a rapid increase in hepatic energy content, better bile production, and reduced cellular enzyme and cytokine release in the SNMP group. Using the same rat model, Boncompagni et al85 demonstrated that SNMP reduced apoptosis and necrosis, and resulted in better preserved hepatic ultrastucture. Liu et al83 assessed the potential of SNMP for reducing the hepatic fat content by using a defatting cocktail (forskolin, hypericin, scoparone, visfatin, and GW501516), which was previously described by Nagrath et al97 and proved to upregulate lipid oxidation and export decreasing the intracellular lipid content by 50% in 3 hours by using NMP. For this model, the steatotic livers from obese Zucker rats were perfused for 6 hours at subnormothermic temperatures. The grafts that were perfused with the defatting cocktail showed a slightly higher secretion of very low-density lipoprotein and triglycerides (TG) into the perfusate without reaching significance. In contrast, no decrease of the intracellular lipid content was found when compared with not supplemented but perfused livers83 (Table 6) (Figure 1).
Clinical
To our knowledge, there are no published clinical articles showing assessment of this technique in fatty livers.
Normothermic Machine Perfusion
Preclinical
In 2009, Nagrath et al97 assessed the combination of pharmacological interventions with NMP to reduce liver steatosis. The authors developed a defatting cocktail based on the experimental results obtained from fatty hepatocyte cultures supplemented with potential active lipid metabolizing agents. The mixture contained a PPAR-α and PPAR-δ agonist, a pregnane X receptor ligand, a constitutive androstane receptor ligand, an insulin mimetic (visflatin), a glucagon mimetic and a cyclic adenosine 3′, 5′-monophosphate activator (forskolin), targeting multiple pathways involved in TG secretion and beta β-oxidation. This cocktail was added to the perfusate and tested in a NMP using liver grafts from obese Zucker rats. Within only 3 hours of perfusion, the intracellular lipid content was reduced by more than 50%.97
In 2011, Jamieson at al86 evaluated the impact of prolonged NMP (48 hours) in a porcine fatty liver model and compared to lean liver perfusion. Of note, this group showed that the TG release and urea production were greater in the fatty liver group, in contrast the glucose consumption was found lower in the same group. Of interest, the hepatic fat content decreased from 30% to 15% during perfusion as assessed by histology.86 However, according to their results the control livers progressively accumulated fat reaching approximately 20% by the end of the perfusion (Table 6).
Banan et al94 in 2016 reported a mild decrease (10%) of macrosteatosis in human discarded livers using a defatting solution that contained l-carnitine and exendin-4 during NMP (8 hours). This resulted in 8.8-fold release of TG and 2.6-fold release of low-density lipoprotein (LDL) into the perfusate compared with baseline value along with adequate synthetic function and minimal injury. In contrast, no reduction of steatosis was observed on histology when compared with control groups. The control group that was perfused without defatting agents showed no changes in the TG and LDL levels94 (Table 7).
Several studies have been published about the metabolic, functional, and hemodynamic characterization of discarded human fatty livers during prolonged normothermic perfusion29,92-95,98 (Table 7). Interestingly, the prior experimental observation by Jamieson et al86 in their fatty liver pig model seems not to be applicable for human grafts. Even prolonged normothermic perfusion has failed to result in a relevant reduction of steatosis. Nevertheless, these findings may provide further valuable knowledge on hemodynamic and functional features, also on specific metabolic patterns of perfused steatotic livers to estimate functional and viability reserve and eventually establish a novel option for the assessment using NMP technology. This may contribute to an improved organ preselection approach toward a safe way to increase the utilization of high-risk human grafts for liver transplantation.
Clinical
The first randomized clinical trial with NMP has been recently published in 2018. The trial included a total 121 NMP cases and 101 SCS cases. Results from this trial showed a significantly lower discard rate (11.7% vs 24.1%, P = 0.008), a very significant decrease in the peak of AST for DCD organs (389.7 [278.0-546.4] vs 1458.1 [944.7-2250.5], P ≤ 0.0001) and less incidence of EAD (10.1% vs 29.9%, P = 0.0001) in the NMP group versus SCS group.26 To our knowledge, there are no published clinical trials specifically dedicated to explore the effects of NMP on fatty livers. Nevertheless, the results from the clinical trial are encouraging and motivating to explore the use of NMP in marginal grafts (including fatty livers) as the next step (Table 8). Of interest, He et al29 in 2018 reported the first human case using a new technology, so-called ischemia-free organ transplantation. The authors preserved a severe steatotic liver graft (85-95% macrosteatosis) under continuous NMP condition throughout the procurement, preservation and implantation period. The recipient recovered smoothly with a functioning liver graft without postreperfusion syndrome or early biliary, vascular and immunological complications. The postreperfusion histology showed minimal hepatocyte necrosis and apoptosis Terminal deoxynucleotidyl transferase dUTP nick end labeling, minimal organ injury, minimal inflammatory cytokine release (IL-1α, IL-6, TNF-α), and sinusoidal epithelial injury (Von Willebrand factor) or activation of key inflammatory pathways (mitogen-activated protein kinase and NFκB) were absent (Figure 1).
FUTURE TRENDS IN TREATMENT OF NAFL/NASH AND THE MANAGEMENT OF STEATOTIC LIVERS BEFORE TRANSPLANTATION
The liver steatosis represents a challenge in the field of liver transplantation. As such, it needs to be addressed in an integral manner. First, the efforts should focus on public health strategies to prevent the high incidence of obesity and as a consequence the development of NASH as a cause of end-stage liver disease. Second, the effective treatment of NASH will be necessary to decrease the need of liver transplantation due to cirrhosis or hepatocellular carcinoma. Intense research in the field of NASH has increased the arsenal of drugs that could potentially be used for the treatment of this disease. There are currently several drugs under investigation in phase 2 and 3 of the clinical trials targeting different receptors (PPAR-γ, PPAR-α/δ, FXR) and pathways (GLP-1, FGF-19, FGF-20, CCR2/CCR5, ASK-1) to reduce fat content and fibrosis. Although most of the clinical trials have focused in monotherapy, it might be the combination of these interventions that will give us the best results in the future. A motivating remark is the experience from antiviral trials in the field of hepatitis C where the cure of this disease can often reverse the cirrhosis, a fact that was inconceivable years ago.99 Therefore, exploring the mechanisms of action by which the patients with hepatitis C can achieve regression of cirrhosis after cure could provide us with new tools to transform the current situation of patients with chronic liver fibrosis, including NASH.
On the other side, at the same time that the efforts aim to reduce the incidence of NASH, we should also focus on optimizing the use of those called fatty livers to decrease the mortality on the transplantation waiting list. As described before, the optimal election of organs and recipients based on scores as the adjusted BAR system in combination with technologies like machine perfusion could improve the short and long term outcomes of recipients of fatty livers. Machine perfusion in its different modalities has opened the opportunity to a new field for ex vivo interventions in an isolated environment. Although the HOPE technology has demonstrated benefits on ATP storage, decrease in PNF and hospital stay in fatty livers the NMP technology have the potential to be a tool to integrally evaluate and even target a metabolically active liver possibly with those strategies that are under investigation for the treatment of NAFL/NASH. In addition, combination of technologies (VSOP, HOPE, and NMP) with their different beneficial effects might provide additional improvement in transplantation outcomes. Randomized clinical trials will be necessary to validate the use of these technologies in steatotic livers.
CONCLUSIONS
Despite many progresses at understanding the pathogenesis of NAFL and NASH, as of today, the only available intervention to improve NAFL/NASH is the weight loss. However, the current landscape suggests that the armament of drugs available for NASH will increase in the near future and as for other multifactorial diseases, the ideal treatment will need to be tailored to each individual either as single treatment or with a combination of agents according to the disease stage.
In regard to the transplantation of fatty livers, moderate to severe macrovesicular steatosis (>30%) should be considered an independent risk factor for graft loss and EAD. Based on recent studies the increased rate of PNF and EAD with moderate steatosis can be balanced with a low-risk recipient selection (adjusted BAR score, ≤ 9). High-grade liver steatosis in the donor liver pool is expected to be a serious problem worldwide due to the pandemic of obesity, facing a new challenge in the transplantation units. New innovative preservation techniques, such as liver retrograde oxygenated persufflation, oxygenated cold machine perfusion, and subnormothermic and normothermic machine perfusion may be the solution by improving organ viability and by offering an option for pretransplant assessment. This might allow us in the future a broader and safer use of fatty livers for transplantation.
ACKNOWLEDGMENTS
I.L. expresses his gratitude to the Mexican National Council of Science and Technology (CONACyT, Mexico City, Mexico) for its support for the Graduate program at University of Toronto. We thank Uwe Mummenhoff and the Birmingham family for their generous support.
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