There are few effective targeted strategies to reduce hepatic ischemia-reperfusion (IR) injury, a contributor to poor outcomes in liver transplantation recipients. It has been proposed that IR injury is driven by the generation of reactive oxygen species (ROS). However, recent studies implicate other mediators of the injury response, including mitochondrial metabolic dysfunction. We examined changes in global gene expression after transient hepatic ischemia and at several early reperfusion times to identify potential targets that could be used to protect against IR injury. Male Wistar rats were subjected to 30 minutes of 70% partial warm ischemia followed by 0, 0.5, 2, or 6 hours of reperfusion. RNA was extracted from the reperfused and non-ischemic lobes at each time point for microarray analysis. Identification of differentially expressed genes and pathway analysis were used to characterize IR-induced changes in the hepatic transcriptome. Changes in the reperfused lobes were specific to the various reperfusion times. We made the unexpected observation that many of these changes were also present in tissue from the paired non-ischemic lobes. However, the earliest reperfusion time, 30 minutes, showed a marked increase in the expression of a set of immediate-early genes (c-Fos, c-Jun, Atf3, Egr1) that was exclusive to the reperfused lobe. We interpreted these results as indicating that this early response represented a tissue autonomous response to reperfusion. In contrast, the changes that occurred in both the reperfused and non-ischemic lobes were interpreted as indicating a non-autonomous response resulting from hemodynamic changes and/or circulating factors. These tissue autonomous and non-autonomous responses may serve as targets to ameliorate IR injury.
Objectives
Transferrin, Tf, the protein transports iron from the blood to the tissues via endocytosis, is believed to also transport chromium(III), Cr(III). Recently, the release of Cr(III) from Tf has been postulated to be too slow for appreciable quantities of Cr(III) to be released during the lifetime of an endosome. The objective of this work was to measure the rate of Cr(III) release from human serum Tf as a function of Tf confirmation and as the transferrin-transferrin receptor (TfR) complex.
Methods
Cr(III) was added to apoTf in a buffered solution at pH 7.4 containing 25 mM bicarbonate at 37 °C. After time intervals, ultraviolet spectra were collected, or aliquots were removed and frozen for analysis by electron paramagnetic resonance (EPR) spectroscopy, which can distinguish free Cr(III) and Cr(III) bound to the two metal binding sites of Tf. To model the acidification of the endosome that triggers release of metal ions from Tf, the Cr(III)2-Tf solutions were acidified by the addition of hydrochloric acid to pH 4.5 or 5.5. At time intervals after acidification, samples were again analyzed by ultraviolet and EPR spectroscopies. Similar studies were performed in the presence of Tf receptor, which binds two equivalents of Cr(III)2-Tf.
Results
The loss of Cr(III) from the two metal-binding sites of Tf occur at different rates. Different confirmations of the Cr2-Tf complex exist depending on the conditions of Cr2-Tf formation. The conformation that forms rapidly under physiological conditions loses Cr(III) faster than conformations that form over longer periods of time. Binding of Cr(III)2-Tf to TfR facilitates the release of Cr.
Conclusions
The conformation of Cr(III)2-transferrin that forms under physiological conditions when complexed with transferrin receptor can release Cr at physiologically significant rates consistent with transferrin serving as the major Cr(III) transport agent between the blood stream and tissues.
Funding Sources
The University of Alabama College of Arts and Sciences Research Award.
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