Novel NMP split liver model recapitulates human IRI and demonstrates ferroptosis modulators as a new therapeutic strategy.

BACKGROUND: Almost 9%of deceased donor livers are discarded as marginal donor livers (MDL) due to concern of severe ischemia reperfusion injury (IRI). Emerging data supports ferroptosis (iron regulated hepatocellular death) as an IRI driver, however lack of robust preclinical model limits therapeutic testing. In this manuscript we describe the development of a novel rigorous internal control system utilizing normothermic perfusion of split livers to test ferroptosis regulators modulating IRI. METHODS: Upon institutional approval, split human MDLs were placed on our normothermic perfusion machine, Perfusion Regulated Organ Therapeutics with Enhanced Controlled Testing (PROTECT), pumping arterial and portal blood. Experiment 1 compared right (UR) and left (UL) lobes to validate PROTECT. Experiment 2 assessed ferroptosis regulator Deferoxamine in Deferoxamine Agent Treated (DMAT) vs. No Agent Internal Control (NAIC) lobes. Liver serology, histology, and ferroptosis genes were assessed. RESULTS: Successful MDL perfusion validated PROTECT with no ALT or AST difference between UR and UL (∆ALT UR: 235, ∆ALT UL: 212; ∆AST UR: 576, ∆AST UL: 389). Liver injury markers increased in NAIC vs. DMAT (∆ALT NAIC: 586, ∆ALT DMAT: -405; ∆AST NAIC: 617, ∆AST DMAT: -380). UR and UL had similar expression of ferroptosis regulators RPL8,HO-1 and HIFα. Significantly decreased intrahepatic iron (p = .038), HO-1 and HIFα in DMAT (HO-1 NAIC: 6.93, HO-1 DMAT: 2.74; HIFαNAIC: 8.67, HIFαDMAT: 2.60)and no hepatocellular necrosis or immunohistochemical staining (Ki67/Cytokeratin-7) differences were noted. CONCLUSION: PROTECT demonstrates the therapeutic utility of a novel normothermic perfusion split liver system for drug discovery and rapid translatability of therapeutics, driving a paradigm change in organ recovery and transplant medicine. Our study using human livers, provides preliminary proof of concept for the novel role of ferroptosis regulators in driving IRI.

Lower systemic inflammation is associated with gut firmicutes dominance and reduced liver injury in a novel ambulatory model of parenteral nutrition.

BACKGROUND: Total Parenteral Nutrition (TPN) provides lifesaving nutritional support to patients unable to maintain regular enteral nutrition (EN). Unfortunately, cholestasis is a significant side effect affecting 20-40% of paediatric patients. While the aetiology of TPN-associated injury remains ill-defined, an altered enterohepatic circulation in the absence of gut luminal nutrient content during TPN results in major gut microbial clonal shifts, resulting in metabolic endotoxemia and systemic inflammation driving liver injury and cholestasis. HYPOTHESIS: To interrogate the role of gut microbiota, using our novel ambulatory TPN piglet model, we hypothesized that clonal reduction of bacteria in Firmicutes phylum (predominant in EN) and an increase in pathogenic Gram-negative bacteria during TPN correlates with an increase in serum lipopolysaccharide and systemic inflammatory cytokines, driving liver injury. METHODS: Upon institutional approval, 16 animals were allocated to receive either TPN (n = 7) or EN only (n = 9). The TPN group was subdivided into a low systemic inflammation (TPN-LSI) and high systemic inflammation (TPN-HSI) based on the level of serum lipopolysaccharide. Culture-independent identification of faecal bacterial populations was determined by 16S rRNA. RESULTS: Piglets on TPN, in the TPN-HSI group, noted a loss of enterocyte protective Firmicutes bacteria and clonal proliferation of potent inflammatory and lipopolysaccharide containing pathogens: Fusobacterium, Bacteroidetes and Campylobacter compared to EN animals. Within the TPN group, the proportion of Firmicutes phylum correlated with lower portal lipopolysaccharide levels (r = -0.89). The TPN-LSI had a significantly lower level of serum bile acids compared to the TPN-HSI group (7.3 vs. 60.4 mg/dL; p = .018), increased day 14 weight (5.67 vs. 5.07 kg; p = .017) as well as a 13.7-fold decrease in serum conjugated bilirubin. CONCLUSION: We demonstrate a novel relationship between the gut microbiota and systemic inflammation in a TPN animal model. Pertinently, the degree of gut dysbiosis correlated with the severity of systemic inflammation. This study underscores the role of gut microbiota in driving liver injury mechanisms during TPN and supports a paradigm change in therapeutic targeting of the gut microbiota to mitigate TPN-related injury. KEY MESSAGESThis study identified a differential link between gut microbiota and inflammation-the higher the dysbiosis, the worse the systemic inflammatory markers.Higher levels of Firmicutes species correlated with reduced inflammation.

Carbamazepine mitigates parenteral nutrition-associated liver disease in a novel ambulatory piglet model.

BACKGROUND: Parenteral nutrition (PN) remains a critical therapeutic option in patients who cannot tolerate enteral feeding. However, although lifesaving, PN is associated with significant side effects, including liver injury, the etiology of which is multifactorial. Carbamazepine (CBZ), an antiepileptic medication, is known to modulate hepatic fibrosis and hepatocellular injury in a variety of liver diseases. We hypothesized that CBZ could prevent PN-associated liver disease (PNALD), which we tested by using our novel ambulatory PN piglet model. METHODS: Piglets were fitted with jugular catheters and infusion pumps for PN and randomized to enteral nutrition (n = 7), PN (n = 6), or PN with parenteral CBZ (n = 6) for 2 weeks. Serum and liver tissue were analyzed via light microscopy, quantification of serum liver injury markers, Ki67 and cytokeratin-7 indexing, and real-time quantitative polymerase chain reaction. RESULTS: PN-fed piglets in our model developed manifestations of PNALD-particularly, increased serum bilirubin, gamma-glutamyltransferase, liver cholestasis, and Ki67 expression compared with that of EN-fed animals (P < 0.03). CBZ therapy in PN-fed animals led to a significant reduction in these markers of injury (P < 0.05). Investigation into the mechanism of these therapeutic effects revealed increased expression of sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor alpha (PPAR-α), and fatty acid binding protein (FABP) in PN-fed animals receiving CBZ (P < 0.03). Further investigation revealed increased LC3 expression and decreased lysosomal-associated membrane protein (LAMP1) expression with CBZ (P < 0.03). CONCLUSION: CBZ administration mitigates PNALD severity, suggesting a novel therapeutic strategy targeting PN-associated side effects, and may present a paradigm change to current treatment options.

Parenteral Nutrition and Cardiotoxicity.

Parenteral nutrition (PN) is a life-saving nutritional therapy for those situations when patients are unable to receive enteral nutrition. However, despite a multitude of benefits offered by PN, it is associated with a variety of side effects, most notably parenteral nutrition-associated liver disease (PNALD). Adverse effects of PN on other organ systems, such as brain and cardiovascular system, have been poorly studied. There have been several case reports, studies, and a recent animal study highlighting cardiotoxic effects of PN; however, much remains unclear about the underlying mechanisms causing cardiac damage. In this review, we propose a series of potential mechanisms behind PN-associated heart injury, and we provide an overview of therapeutic strategies and recent scientific advances.

Role of Bile Acids and Gut Microbiota in Parenteral Nutrition Associated Injury.

Total Parenteral Nutrition (TPN) is a life-saving therapy where all nutritional requirements are provided intravenously. While this therapy is essential for individuals unable to process their nutritional needs enterically, significant complications arise such as intestinal failure associated liver injury (IFALD). IFALD includes hepatic steatosis, cholestasis, inflammation, ultimately progressing to cirrhosis and portal hypertension and some patients may need liver transplantation. The exact mechanism underlying this condition is not well understood, but studies have recently suggested that changes in gut microbiota and intraluminal bile acid signaling are known to play a role in the development of IFALD. In enterohepatic circulation with normal enteral nutrition, gut Farnesoid X Receptor (FXR) is activated by bile acids, which triggers the release of Fibroblast Growth Factor 19 (FGF19) into portal circulation. FGF19 serves to regulate intrahepatic bile acid synthesis with enteric nutrition. This signaling pathway is impaired in TPN as studies indicate decreased serum levels of FGF19 in subjects receiving TPN. Finally, gut microbiota is severely altered in TPN due to intestinal hypomobility. The shift in gut microbiota affects our immune response and promotes endotoxins that negatively affect liver function. Targeting the pathways affecting gut microbiota and bile acid signaling has promise in treating TPN associated injuries.