The efficacy of HBOC-201 in ex situ gradual rewarming kidney perfusion in a rat model

Gradual rewarming from hypothermic to normothermic is a novel perfusion modality with superior outcome to sudden rewarming to normothermic. However, the identification of an oxygen carrier that could function at a temperature range from 4 to 7 degrees C or whether it is necessary to use oxygen carrier during kidney rewarming, remains unresolved. This study was designed to test the use of a hemoglobin-based oxygen carrier (HBOC) during gradual kidney rewarming as an alternative to simple dissolved oxygen. In this study, 10 rat kidneys were randomly divided into the control and the HBOC group. In the control group, no oxygen carrier was used during rewarming perfusion and the perfusion solution was oxygenated only by applying diffused carbogen flow. The protocol mimicked a donor after circulatory death (DCD) kidney transplantation, where after 30 minutes warm ischemia and 120 minutes cold storage in University of Wisconsin solution, the DCD kidneys underwent gradual rewarming from 10 to 37 degrees C during 90 minutes with or without HBOC. This was followed by 30 minutes of warm ischemia in room temperature to mimic the anastomosis time and 120 minutes of reperfusion at 37 degrees C to mimic the early post-transplant state of the graft. The HBOC group demonstrated superior kidney function which was highlighted by higher ultrafiltrate production, better glomerular filtration rate and improved sodium reabsorption. There was no significant difference between the 2 groups regarding the hemodynamics, tissue injury, and adenosine triphosphate levels. In conclusion, this study suggests better renal function recovery in DCD kidneys after rewarming with HBOC compared to rewarming without an oxygen carrier

Tryptophan Metabolism via the Kynurenine Pathway:Implications for Graft Optimization during Machine Perfusion

Access to liver transplantation continues to be hindered by the severe organ shortage. Extended-criteria donor livers could be used to expand the donor pool but are prone to ischemia-reperfusion injury (IRI) and post-transplant graft dysfunction. Ex situ machine perfusion may be used as a platform to rehabilitate discarded or extended-criteria livers prior to transplantation, though there is a lack of data guiding the utilization of different perfusion modalities and therapeutics. Since amino acid derivatives involved in inflammatory and antioxidant pathways are critical in IRI, we analyzed differences in amino acid metabolism in seven discarded non-steatotic human livers during normothermic- (NMP) and subnormothermic-machine perfusion (SNMP) using data from untargeted metabolomic profiling. We found notable differences in tryptophan, histamine, and glutathione metabolism. Greater tryptophan metabolism via the kynurenine pathway during NMP was indicated by significantly higher kynurenine and kynurenate tissue concentrations compared to pre-perfusion levels. Livers undergoing SNMP demonstrated impaired glutathione synthesis indicated by depletion of reduced and oxidized glutathione tissue concentrations. Notably, ATP and energy charge ratios were greater in livers during SNMP compared to NMP. Given these findings, several targeted therapeutic interventions are proposed to mitigate IRI during liver machine perfusion and optimize marginal liver grafts during SNMP and NMP

Subnormothermic Machine Perfusion of Steatotic Livers Results in Increased Energy Charge at the Cost of Anti-Oxidant Capacity Compared to Normothermic Perfusion

There continues to be significant debate regarding the most effective mode of ex situ machine perfusion of livers for transplantation. Subnormothermic (SNMP) and normothermic machine perfusion (NMP) are two methods with different benefits. We examined the metabolomic profiles of discarded steatotic human livers during three hours of subnormothermic or normothermic machine perfusion. Steatotic livers regenerate higher stores of ATP during SNMP than NMP. However, there is a significant depletion of available glutathione during SNMP, likely due to an inability to overcome the high energy threshold needed to synthesize glutathione. This highlights the increased oxidative stress apparent in steatotic livers. Rescue of discarded steatotic livers with machine perfusion may require the optimization of redox status through repletion or supplementation of reducing agents

Metabolic and lipidomic profiling of steatotic human livers during ex situ normothermic machine perfusion guides resuscitation strategies

There continues to be a significant shortage of donor livers for transplantation. One impediment is the discard rate of fatty, or steatotic, livers because of their poor post-transplant function. Steatotic livers are prone to significant ischemia-reperfusion injury (IRI) and data regarding how best to improve the quality of steatotic livers is lacking. Herein, we use normothermic (37°C) machine perfusion in combination with metabolic and lipidomic profiling to elucidate deficiencies in metabolic pathways in steatotic livers, and to inform strategies for improving their function. During perfusion, energy cofactors increased in steatotic livers to a similar extent as non-steatotic livers, but there were significant deficits in anti-oxidant capacity, efficient energy utilization, and lipid metabolism. Steatotic livers appeared to oxidize fatty acids at a higher rate but favored ketone body production rather than energy regeneration via the tricyclic acid cycle. As a result, lactate clearance was slower and transaminase levels were higher in steatotic livers. Lipidomic profiling revealed ω-3 polyunsaturated fatty acids increased in non-steatotic livers to a greater extent than in steatotic livers. The novel use of metabolic and lipidomic profiling during ex situ normothermic machine perfusion has the potential to guide the resuscitation and rehabilitation of steatotic livers for transplantation

Split-Liver Ex Situ Machine Perfusion:A Novel Technique for Studying Organ Preservation and Therapeutic Interventions

Ex situ machine perfusion is a promising technology to help improve organ viability prior to transplantation. However, preclinical studies using discarded human livers to evaluate therapeutic interventions and optimize perfusion conditions are limited by significant graft heterogeneity. In order to improve the efficacy and reproducibility of future studies, a split-liver perfusion model was developed to allow simultaneous perfusion of left and right lobes, allowing one lobe to serve as a control for the other. Eleven discarded livers were surgically split, and both lobes perfused simultaneously on separate perfusion devices for 3 h at subnormothermic temperatures. Lobar perfusion parameters were also compared with whole livers undergoing perfusion. Similar to whole-liver perfusions, each lobe in the split-liver model exhibited a progressive decrease in arterial resistance and lactate levels throughout perfusion, which were not significantly different between right and left lobes. Split liver lobes also demonstrated comparable energy charge ratios. Ex situ split-liver perfusion is a novel experimental model that allows each graft to act as its own control. This model is particularly well suited for preclinical studies by avoiding the need for large numbers of enrolled livers necessary due to the heterogenous nature of discarded human liver research

Hypothermic Oxygenated Machine Perfusion Prevents Arteriolonecrosis of the Peribiliary Plexus in Pig Livers Donated after Circulatory Death

BACKGROUND: Livers derived from donation after circulatory death (DCD) are increasingly accepted for transplantation. However, DCD livers suffer additional donor warm ischemia, leading to biliary injury and more biliary complications after transplantation. It is unknown whether oxygenated machine perfusion results in better preservation of biliary epithelium and the peribiliary vasculature. We compared oxygenated hypothermic machine perfusion (HMP) with static cold storage (SCS) in a porcine DCD model. METHODS: After 30 min of cardiac arrest, livers were perfused in situ with HTK solution (4°C) and preserved for 4 h by either SCS (n = 9) or oxygenated HMP (10°C; n = 9), using pressure-controlled arterial and portal venous perfusion. To simulate transplantation, livers were reperfused ex vivo at 37°C with oxygenated autologous blood. Bile duct injury and function were determined by biochemical and molecular markers, and a systematic histological scoring system. RESULTS: After reperfusion, arterial flow was higher in the HMP group, compared to SCS (251±28 vs 166±28 mL/min, respectively, after 1 hour of reperfusion; p = 0.003). Release of hepatocellular enzymes was significantly higher in the SCS group. Markers of biliary epithelial injury (biliary LDH, gamma-GT) and function (biliary pH and bicarbonate, and biliary transporter expression) were similar in the two groups. However, histology of bile ducts revealed significantly less arteriolonecrosis of the peribiliary vascular plexus in HMP preserved livers (>50% arteriolonecrosis was observed in 7 bile ducts of the SCS preserved livers versus only 1 bile duct of the HMP preserved livers; p = 0.024). CONCLUSIONS: Oxygenated HMP prevents arteriolonecrosis of the peribiliary vascular plexus of the bile ducts of DCD pig livers and results in higher arterial flow after reperfusion. Together this may contribute to better perfusion of the bile ducts, providing a potential advantage in the post-ischemic recovery of bile ducts

Criteria for Viability Assessment of Discarded Human Donor Livers during Ex Vivo Normothermic Machine Perfusion

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial

Association of Paraoxonase-1 Genotype and Phenotype with Angiogram Positive Coronary Artery Disease

Funding Information: This study was supported by Mashhad and Isfahan University of Medical Sciences. The authors would like to thank technicians of Sina, Sadi, Ghaem catheterization laboratory and technicians of Isfahan Alzahra genetics laboratory.Peer reviewedPublisher PD