Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

Abstract

Background: Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts.

Methods: Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers.

Results: Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP.

Conclusion: Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion.

Keywords: biomarker; cholangiocyte-derived microRNA; hepatocyte-derived microRNA; miR-122; miR-222; transplantation.

Figure 1

Dynamics of HDmiR‐122 and CDmiR‐222 levels in perfusate and bile during NMP. Relative values of HDmiR‐122 (A, B), CDmiR‐222 (C, D), and the HDmiR‐122/CDmiR‐222 ratio (E, F) in perfusate (A, C, E), and bile (B, D, F). In perfusate median values of both miRs peaked after 2 h of NMP and slowly declined thereafter (A, C). In bile, median relative values of HDmiR‐112 significantly increase between 0.5 and 2 h (B). The miR ratio in bile significantly increase over time during NMP. MiR levels are represented as relative values (2^−Cq). Solid lines and bars: Median ± IQR, dotted lines: relative miR value profiles for each individual sample. **P < .01, ***P < .001

Figure 2

Correlation and prediction of relative miRNA levels and miR ratio with Suzuki injury score for liver injury. Relative level for HDmiR‐122 (closed circles) and CDmiR‐222 (open circles) in perfusate at 30 min correlated with Suzuki injury scores prior to NMP (0 h NMP, A). HDmiR122/CDmiR222 ratios at 30 min were predictive for liver parenchymal injury score after 6 h of NMP (black squares, B). Spearman's correlation coefficient (r) and P‐value are indicated

Figure 3

Early relative HDmiR‐122 and CDmiR‐222 levels in perfusate are predictive for hepato‐cholangiocellular injury at end of normothermic machine perfusion (NMP). Relative level for HDmiR‐122 (closed circles) and CDmiR‐222 (open circles) in perfusate at 30 min correlated with hepato‐cholangiocellular injury markers, peak aspartate aminotransferase in perfusate (A) and peak lactate dehydrogenase in bile (B) at 6 h of NMP. Total bilirubin in bile at 6 h NMP correlated with both HDmiR‐122 and CDmiR‐222 at 30 min (C). HDmiR‐122, but not CDmiR‐222, at 30 min was associated with bicarbonate level in bile at 6 h NMP (D). Spearman's correlation coefficient (r) and P‐value are indicated

Figure 4

CDmiR‐222 levels in bile at 2 h of normothermic machine perfusion (NMP) are predictive of hepato‐cholangiocellular injury and function at end of NMP. Relative CDmiR‐222 (open circles) values, but not relative HDmiR‐122 values (closed circles), at 2 h of NMP correlated with peak aspartate aminotransferase (AST) in perfusate (A), and with lactate dehydrogenase (LDH) in bile (B) at 6 h of NMP (6 h NMP). In the case of bicarbonate in bile at 6 h NMP, also only CDmiR‐222, and not HDmiR‐122, at 2 h showed a strong, but negative, correlation (C), while both miRs were predictive of the bile/perfusate glucose ratio at 6 h (D). Spearman's correlation coefficient (r) and P‐value are indicated

Figure 5

Aspartate aminotransferase (AST) and the miR ratio are predictive for good of liver function, as measured by cumulative bile production during normothermic machine perfusion (NMP). Cumulative bile production after 6 h plotted against clinical parameter AST (A) and relative HDmiR‐122/CDmiR‐222 ratio (B) measured after 30 min of NMP. Only these two parameters showed a good correlation with the cumulative bile production after 6 h. Spearman's correlation coefficient (r) and P‐value are indicated