Liver transplantation remains the only effective therapy for end-stage liver failure but is limited by a severe shortage of donor livers. Moreover, initial poor function and primary nonfunction frequently occur, especially in recipients of fatty liver grafts. Still unknown are changes that occur during cold ischemic storage that predispose to graft injury after transplantation. During the previous period of support, we developed the hypothesis that ATP depletion during cold storage inhibits the proton-pumping vacuolar ATPase (V-ATPase) in lysosomes and collapses lysosomal acidic pH gradients, which causes release of lysosomal Fe2+ into the cytosol through divalent metal transporter-1 (DMT1). Mitochondria take up this Fe2+ via the mitochondrial Ca2+ (and Fe2+) uni-porter (MCU) to predispose to Fe2+-dependent hydroxyl radical formation in mitochondria after transplantation with consequent activation of c-Jun N-terminal kinase (JNK), onset of the mitochondrial permeability transition (MPT) and cell death. Pursuing this hypothesis, we propose five specific aims.
In Aim 1, intracellular iron mobilization in hepatocytes of cold-stored mouse livers will be measured by multiphoton microscopy of calcein, an iron-indicating fluorophore. We will assess the lysosomal origin of iron and the role of MCU using the lysosomally targeted iron chelator, starch-desferal, and the MCU inhibitors, minocycline and doxycycline. Further, we will assess protection by iron chelators and MCU inhibitors against graft injury and failure after rat liver trans- plantation, including assessment of mitochondrial function after transplantation by innovative intravital mul- tiphoton microscopic techniques.
In Aim 2, a similar analysis will be performed for Kupffer cells (KCs) and sinusoidal endothelial cells (SECs) to test the hypothesis that lysosomal iron mobilization also promotes KC activation and loss of SEC viability, which are prominent early features of hepatic storage/reperfusion injury.
In Aim 3, the role of DMT1 in lysosomal iron release during liver storage and graft injury after transplantation will be assessed using pharmacological inhibitors. Using novel transgenic mice created with cre-lox technology, the effect of cell-specific DMT1 deficiency in hepatocytes, KCs and SECs on iron release and graft injury will be assessed. These experiments will identify which cell types are most important to iron translocation- dependent graft dysfunction.
For Aim 4, we hypothesize that lysosomal iron release during cold storage promotes JNK activation after reperfusion. Using iron chelators and MCU inhibitors, we will determine whether iron translocation to cytosol or mitochondria is most important for JNK activation and the relative importance of different cell types to iron-dependent JNK activation. Lastly in Aim 5, we will characterize the importance of lysosomal iron mobilization to enhanced storage/reperfusion injury in steatotic livers induced by a high-fat, high-fructose diet. We expect earlier iron mobilization by fatty livers during cold storage that promotes greater graft dysfunction and failure after transplantation. Success of this project will lead to novel translatable strategies and new drug targets against storage/reperfusion injury.

Public Health Relevance

Liver transplantation remains the only effective therapy for end-stage liver failure but is limited by a severe shortage of donor livers and the relatively frequent occurrence of poor function and primary nonfunction due to harvest, storage and reperfusion injury, especially in recipients of fatty liver grafts. This project addresses the nove hypothesis that mobilization of iron from lysosomes into mitochondria during cold storage predisposes to iron-dependent formation of reactive oxygen species after transplantation to cause mitochondrial dysfunction, cell death and liver graft failure. The information gained from the proposed experiments will be useful for improving clinical outcomes after liver transplantation and for expanding safe donation of fatty livers.

Agency
National Institute of Health (NIH)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56DK037034-28A1
Application #
8920450
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Sherker, Averell H
Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
City
Charleston
State
SC
Country
United States
Zip Code
29403
Maldonado, Eduardo N; Lemasters, John J (2014) ATP/ADP ratio, the missed connection between mitochondria and the Warburg effect. Mitochondrion 19 Pt A:78-84