Dataset Information

Therapeutic hyperthermia promotes lipid export and HSP70/90 during machine perfusion of human livers

ABSTRACT: Liver transplantation is the only curative option for end-stage liver disease. Donor shortages necessitate the use of higher risk donor livers, including fatty livers, which are more susceptible to ischemia reperfusion injury. Machine perfusion has improved graft utilization and is typically performed at hypothermic (8-12oC), or normothermic (35-37oC) temperatures. Here we studied the impact of mild hyperthermia (40oC) as a therapeutic intervention for fatty livers using in-depth proteomic and lipoprotein profiling of whole organ perfusion and precision cut liver slices. We observed proteomic changes with metabolic alterations over time, evidenced by a significant increase in lipid export in whole organ perfusions. Furthermore, PCLS showed significant upregulation of metabolic processes and heat shock protein response after 24 hours of hyperthermia. Machine perfusion under hyperthermic conditions may be a potential strategy to improve the utilization of fatty liver grafts, ultimately expanding the donor pool and improving transplant outcomes.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Liver, Blood

SUBMITTER: Adam Thorne

LAB HEAD: Vincent de Meijer

PROVIDER: PXD056736 | Pride | 2025-05-26

REPOSITORIES: Pride

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Therapeutic hyperthermia promotes lipid export and HSP70/90 during machine perfusion of human livers.

Thorne Adam M AM Geng Yana Y Lantinga Veerle A VA Smit Marieke M Kuivenhoven Jan Albert JA Porte Robert J RJ Kuipers Folkert F Olinga Peter P Wolters Justina C JC de Meijer Vincent E VE

Physiological reports 20250501 9

Liver transplantation is the only curative option for end-stage liver disease. Donor shortages necessitate the use of higher risk donor livers, including fatty livers, which are more susceptible to ischemia-reperfusion injury. Machine perfusion has improved graft utilization and is typically performed at hypothermic (8-12°C) or normothermic (35-37°C) temperatures. Here we studied the impact of mild hyperthermia (40°C) as a therapeutic intervention for fatty livers using in-depth proteomic and li ...[more]

PMID: 40346031

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Project description:Bile has gained increasing attention for its potential to reflect the overall health of the biliary system. Proteomic analysis of bile could deepen our understanding of the molecular pathophysiology of biliary disease and injury and provides a potential source of diagnostic biomarkers. With the emergence of normothermic machine perfusion (NMP) prior to liver transplantation, bile samples can be easily collected and analyzed. However, the unique composition of bile, including high concentrations of endogenous detergents and salts, can make application of proteomics challenging. In this study, we systematically evaluated a variety of available trypsin-digestion methods to optimize proteomics analysis of bile obtained from human NMP livers. Bile was collected from 12 human donor livers that were accepted for transplantation after NMP viability assessment. We performed tryptic digestion using 6 different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without an additional precipitation step prior to digestion. Untargeted, data-independent acquisition proteomics was performed using an Exploris 480 mass spectrometer coupled with a FAIMS device. The methods were judged for total protein IDs, variation, and specific protein characteristics to determine the most optimal method. A total of 4650 unique proteins were identified across all samples and all methods. Methods involving precipitation identified substantially more proteins (4500 vs 3815, respectively). In-gel crude was the exception, identifying a comparable number of proteins to precipitated in-gel digest. We found minimal differences in the mass, cellular component, or hydrophobicity of identified proteins between methods. Intra-method variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming other methods with considerably less variation. Age-related biological comparisons revealed significant disparities in protein abundances between methods, but younger donors consistently showed upregulation of metabolic-related processes compared to older donors. Older donors showed upregulation of immune response and cell cycle-related processes. The digestion method used for proteomic analysis of bile can heavily influence the results obtained. A method of protein purification appears to be essential for sufficient quality mass spectrometry runs and subsequent data. Sample processing speed, cost, cleanliness, and reproducibility should be carefully considered when selecting a protein digestion method for this difficult sample type.

			Action	DRS
	PCLS_Sample_10.raw	Raw
	PCLS_Sample_100.raw	Raw
	PCLS_Sample_101.raw	Raw
	PCLS_Sample_102.raw	Raw
	PCLS_Sample_103.raw	Raw