Kupffer cell-independent acute hepatocellular oxidative stress and decreased bile formation in post-cold-ischemic rat liver

Abstract

The purpose of this study was to examine distribution and time history of oxidative stress during the hyperacute period of reperfusion in the liver grafts undergoing cold ischemia and to investigate roles of Kupffer cells as a potential oxidant source. Rat livers were harvested at 4ЊC in University of Wisconsin solution and followed by reperfusion with Krebs-Henseleit buffer under monitoring bile excretion. To investigate oxidative changes, laser-confocal microfluorography was performed in reperfused livers preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescence precursor sensing intracellular hydroperoxide generation. Livers undergoing the 16-hour cold storage displayed an impaired recovery of bile aciddependent bile output concurrent with a marked increase in hydroperoxide generation in hepatocytes, which occurred as early as 5 minutes after the onset of reperfusion, whereas the status of lobular perfusion was well maintained. Pretreatment with liposome-encapsulated dichloromethylene diphosphonate, a Kupffer cell-depleting reagent, did neither alter the reperfusion-induced periportal oxidative changes nor improve the recovery of bile output in the graft. On the other hand, EPCK, a hepatotropic antioxidant composed of vitamin E phosphate ester bound to vitamin C, not only diminished the oxidative changes but also improved the reduction of bile acid-dependent bile output. Furthermore, the reagent was capable of inhibiting H 2 O 2 -induced oxidative stress in cultured hepatocytes. These results suggest that hepatocytes constitute a major site of the oxidative insult triggered through Kupffer cell-independent mechanisms and serve as an important cellular component to be protected by antioxidant therapeutics.

Kupffer cell-induced oxidant stress during hepatic ischemia-reperfusion: Does the controversy continue?

“…Recently, vascular reactive oxygen formation has been examined as a cause of intracellular oxidant stress in hepatocytes. The study by Kumamoto et al 1 in the present issue of HEPATOLOGY emphasizes again the relevance of intracellular oxidant stress in the pathophysiology of reperfusion injury. Does this mean that we have gone full circle, that the early studies were correct all along?…”

“…The conclusion is that, in an isolated perfused liver, where complement factors are absent, the Kupffer cell-induced ROS formation is limited, and consequently its detrimental effects are considerably reduced relative to in vivo conditions. Thus, the results of Kumamoto et al 1 do not exclude Kupffer cellinduced oxidant stress in a postischemic liver but, rather, demonstrate that in a buffer-perfused liver, without the stimulating factors usually present in vivo, ROS generated by Kupffer cells do not produce detectable intracellular oxidant stress in hepatocytes during the first 30 minutes of reperfusion.A novel aspect of the report by Kumamoto et al is that it provides direct evidence for an intracellular oxidant stress in hepatocytes during reperfusion. Moreover, the protective effect of an antioxidant under these conditions suggests that this ROS formation contributes to the functional impairment of the liver during reperfusion.…”

“…Again, the report by Kumamoto et al adds an important new aspect by showing that a pathophysiologically relevant intracellular oxidant stress can occur in hepatocytes without previous gross cell injury. 1 The fact that the mitochondrial membrane potential (Rh123 fluorescence) was not fully restored during reperfusion suggests mitochondrial dysfunction in these hepatocytes. Therefore, it is likely that mitochondria are the primary source of the postischemic oxidant stress.…”

“…14 Despite a large number of follow-up studies supporting the concept of an inflammatory injury during reperfusion, individual reports, including the present report by Kumamoto et al, questioned the pathophysiological role of Kupffer cells. 1,15 It was shown that hypoxia and reoxygenation could directly activate isolated Kupffer cells. 16 Also, chemiluminescence measurements supported the concept that reactive oxygen is being generated even in the isolated perfused liver during reperfusion, 17 although this was detectable mainly after prolonged storage (Ͼ16 hours) and was relatively short lived.…”

“…Moreover, the protective effect of an antioxidant under these conditions suggests that this ROS formation contributes to the functional impairment of the liver during reperfusion. Although it is generally assumed that sinusoidal endothelial cells are the main cell type injured during cold storage of the liver, 20,21 data from Kumamoto et al indicate that hepatocytes are also affected during cold ischemia, even after moderate storage times, 1 pointing to previously unrecognized events in hepatocytes that underlie functional impairment during reperfusion.…”