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Bile proteome reveals biliary regeneration during normothermic preservation of human donor livers

ABSTRACT: Liver transplantation is the only treatment option for patients with end-stage liver disease. However, a persistent disparity remains between supply and demand for donor organs suitable for transplantation, resulting in a waiting list mortality of up to 20%. This has resulted in an increasing use of high-risk grafts from suboptimal, extended criteria donors, including livers from donation after circulatory death (DCD) donors, and donors with significant comorbidities such as advanced age, diabetes mellitus and livers with high fat content. While cold storage remains sufficient for livers considered optimal for transplantation, ECD grafts are more vulnerable to cold ischemic damage, resulting in many of these grafts being rejected. As an alternative to cold storage, ex-situ normothermic machine perfusion (NMP) has become an increasingly popular method for preservation, transportation and assessment of ECD livers prior to transplantation. This allows for functional assessment of the graft and provides the opportunity to assess key processes that can be used to determine organ viability. The use of both hepatocellular and biliary viability criteria in selection of suboptimal livers from ECD donors has led to the successful transplantation of these grafts with a low incidence of post-transplant cholangiopathies. However, the mechanisms of biliary injury, and recovery thereof, following static cold storage remains poorly understood. Insight into these mechanisms may further improve preservation and selection of ECD livers. Therefore, we performed an in-depth proteomics analysis of bile samples collected from human ECD livers during NMP to identify potential mechanisms linked to biliary injury, function and regeneration over the course of the perfusion, and to assess whether biliary protein profiles can distinguish between transplantable and non-transplantable grafts.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Bile

SUBMITTER: Adam Thorne

LAB HEAD: Vincent E

PROVIDER: PXD046355 | Pride | 2023-11-21

REPOSITORIES: Pride

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			Action	DRS
	NMP_Bile_Proteomics_02.raw	Raw
	NMP_Bile_Proteomics_03.raw	Raw
	NMP_Bile_Proteomics_04.raw	Raw
	NMP_Bile_Proteomics_05.raw	Raw
	NMP_Bile_Proteomics_06.raw	Raw

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Project description:There is a ever widening gap between the availability of donor livers suitable for transplantation and liver transplant demand of a growing waiting list. To expand the pool of liver grafts, donation after circulatory death is increasingly being used. Livers procured after circulatory death are exposed to a harmful period of warm Ischemia (WI) during the donation process, reducing post-transplant results significantly. Additionally, the conventional technique of static cold storage does not protect grafts that suffered WI. New dynamic preservation strategies by means of Normothermic Machine Perfusion (NMP) showed enhanced preservation of these type of grafts, but it is unknown how NMP influences liver cell biology during perfusion. Hepatocytes and cholnagiocytes have been shown to release Extracellular Vesicles (EVs) in the systemic circulation and bile, respectively. We hypothesized that EVs are released during liver NMP as well, in perfusate and bile. EVs have been separated from perfusate and bile during NMP of porcine livers, and the RNA cargo of the EVs has been sequenced to gain insights on liver cell biology during machine perfusion. Pigs were randomly allocated to undergo liver procurement either immediately after the start of surgery (no-WI, n=5) or after a period of WI of 60-minutes (clamping of thoracic aorta; WI-60, n=5). All livers (n=10) were cooled down and flushed out with ice-cold preservation solution, according to standard clinical practice. Porcine blood was collected and washed with a cell saver to obtain packed red blood cells for NMP. The hepatic artery, portal vein, and inferior vena cava were cannulated and after a period of about 2-hours of cold storage, NMP was started. All liver underwent NMP for 6-hours. Packed red blood-based perfusate samples were taken at 1, 3, and 6h of NMP; whereas, the bile produced the first 3-hours and the last 3-hours was pooled in 2 samples. Perfusate and bile samples were processed immediately for EVs separation with charge-based precipitation in a mixture of protamine and polyethylene glycol. The total RNA was extracted from EVs. Libraries of mRNA and of small RNA were prepared and analyzed for quality and size range, and sequenced on an Illumina HiSeq4000 instrument. Count-based differential expression analysis was done with R-based Bioconductor package DESeq2, and the following comparisons were performed: - Unpaired (between groups) comparison - perfusate samples WI-60 vs. no-WI - bile samples WI-60 vs. no-WI - Paired (withing group) comparison - no-WI - perfusate sample 1h vs. perfusate sample 3h - perfusate sample 1-3h vs. perfusate sample 6h - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples - WI-60 - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples Results were adjusted for multiple testing with the Benjamini-Hochberg procedure, to control for false discovery rate. Additionally, the function of the transcripts identified in the EVs of both groups and of the differentially expressed transcripts was investigated with gene ontology enrichment analysis for the domains \\"biological process\\" and \\"pathways\\".

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.

Project description:The fate of transplanted kidneys is substantially influenced by the graft quality as transplantation of kidneys from elderly and expanded criteria deceased donors (ECD) is associated with higher occurrence of delayed graft function, rejection and inferior long term outcome. Little is known about early molecular fingerprints of these events in different donor categories. Borderline changes represent the most frequent histological finding early after kidney transplantation. Therefore, transcriptomic profiles of early case biopsies diagnosed as borderline changes were studied in ECD, standard criteria deceased donors (SCD) and living donor (LD) kidney transplantation using RNA microarray (Agilent SurePrintG3). The increased transcripts typical for ECD as compared to LD (n=437) showed enrichment of extracellular matrix (ECM) -receptor interaction (p=0.004) and complement and coagulation pathways (p=0.004) and GO terms related to platelet activation, blood coagulation, regulation of cell cycle, acute inflammatory response, wound healing or defense response (p<0.001). Gene annotation analysis of transcripts with increased expression in ECD grafts compared to SCD (n=244) showed the highest enrichment of inflammatory response (p=0.013), response to wounding (p=0.001) and defense response (p=0.005) and ECM-receptor interaction pathway (p=0.043). Comparative gene expression overlaps of ECD, SCD and LD using Venn’s diagrams found 62 up- and 16 down-regulated genes in ECD compared to both LD and SCD. Shared increased transcripts in ECD vs. both SCD and LD included, thrombospondin 2 (THBS2), angiopoietin-like 4 (ANGPTL4), collagens (COL6A3, COL1A1), chemokines CCL13 or interleukin IL11 and most significant down-regulated transcripts included proline rich 35 (PRR35) and fibroblast growth factor 9 (FGF9). Transcriptomic profile of higher inflammation and extracellular matrix remodeling in early borderline changes in the ECD kidney allografts suggest mechanisms how ischemia/reperfusion injury aggravates alloimmune response in the presence of chronic vascular changes.

Project description:Ischemia-reperfusion injury during liver transplantation is responsible for early allograft dysfunction (EAD) and failure, both of which are associated with a high risk of morbidity and mortality in the recipient. The purpose of this study was to study major transcriptional alterations in livers procured from different types of human liver donors in order to identify genetic profiles predictive of post-implantation function. We have analyzed samples form living donors (LD), donors after cardiac death (DCD), donors after brain death, with subsequent post-implantation EAD in the recipient (DBD-EAD); and donors after brain death without EAD (DBD). Two samples were obtained from each donor: sample A was taken immediately before cold perfusion (baseline) and sample B 2h after portal reperfusion. We identified clear differences in gene expression patterns according to donor source. Both samples A and B from DBD-EAD and DCD demonstrated over-expression of pro-apoptotic and inflammatory transcripts. However, in DBD and LD, expression of these genes was low at baseline and rose only after reperfusion. DBD and LD demonstrated the greatest increase in overall genetic expression after reperfusion when sample B was contrasted with A, indicating less baseline graft injury in these two groups. Grafts from LD were characterized by activation of transcripts related to anti-ischemic and regenerative processes and fewer pro-inflammatory gene transcripts. This transcriptional events occurring in liver allografts could allow for the prediction of post-transplant function. Pro-inflammatory and ischemic transcriptional changes in the grafts are directly related to donor type and may be useful targets for the development of future therapeutic strategies. The complete database comprised the expression for samples taken from 33 liver grafts. Sample A was taken immediately before cold perfusion (baseline) and sample B 2 h after portal reperfusion. Donors were from one of four groups: living (LD); after cardiac death (DCD); after brain death, with subsequent post-implantation EAD in the recipient (DBD-EAD); and after brain death without EAD (DBD). RNA was extracted from the 66 samples and analyzed using Illumina Beadarray technology. A group of 3 samples from healthy volunteers and 3 samples form LD taken at the start of surgery are included as controls or reference samples. This dataset is part of the TransQST collection.

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Bile proteome reveals biliary regeneration during normothermic preservation of human donor livers

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