Both adenosine and NO are synthesized in vascular endoThe effects of ischemic preconditioning on rat liver thelium and are released into the surrounding vascular and integrity, as well as the implication of nitric oxide (NO) interstitial compartments during the ischemic and reperfuand adenosine in this process, has been evaluated. Presion periods. Experimental data suggest that NO is a major conditioning before ischemia-reperfusion prevented the mediator of adenosine-induced vasodilatation. 9 In addition, increases in alanine transaminase (ALT), aspartate recent evidence suggests that adenosine stimulates, by a retransaminase (AST), and lactate dehydrogenase (LDH) ceptor-mediated mechanism, the production of NO by endolevels, but did not modify blood flow. Adenosine or NO thelial cells. 10 On the other hand, it has also been reported administration previous to hepatic ischemia-reperfuthat NO could modulate the adenosine release.11 sion simulated the effect of preconditioning, whereas Ischemic preconditioning has been commonly studied in inhibition of adenosine or NO synthesis abolished the the heart, but few studies have been performed on liver preprotective effect of hepatic preconditioning. Nevertheconditioning. 12 In this study, the concerted roles of NO and less, inhibition of adenosine and simultaneous adminisadenosine in the protective effect of preconditioning against tration of NO in preconditioned animals offered similar hepatic ischemia-reperfusion-induced injury have been results to those found in the preconditioned group, indistudied. cating that, in the absence of adenosine, NO is able to maintain the preconditioning benefits. It is suggested MATERIALS AND METHODS that, in preconditioning, adenosine stimulates NO production to protect against the injury associated with Surgical Procedure. Male Wistar rats (8 in each group; 250-300 g body weight) were used. All animals (including controls) were anes- ischemia-reperfusion. (HEPATOLOGY 1997;25:934-937.)thetized with urethane (10 mg/kg intraperitoneally) and placed in a supine position on a heating pad for maintenance of body temperaIt has been reported that repetitive, short periods of is-ture between 36ЊC and 37ЊC. To induce hepatic ischemia, laparotomy chemia, separated by intermittent reperfusion, render the was performed, and the blood supply to the right lobe of the liver heart more tolerant to subsequent, longer ischemic episodes, was interrupted by placement of a bulldog clamp at the level of the hepatic artery and portal vein. Reflow was initiated by removal of and attenuate the injury observed after ischemia-reperfusion.

This study aims to determine if the protective role of adenosine in liver ischemic preconditioning is mediated by the activation of adenosine receptors and to ascertain which of these receptors is implicated in the process. Administration of adenosine A 1 and A 2 receptor antagonists to preconditioned animals indicates that hepatic preconditioning is mediated by the activation of adenosine A 2 receptors. Propentofylline (an inhibitor of adenosine transport into cells) in the preconditioned group, subjected to previous administration of an adenosine A 2 receptor antagonist, prevented the negative effect of the latter on the protection offered by preconditioning. An increase of NO production was detected just immediately after hepatic preconditioning, and the administration of an adenosine A 2 receptor antagonist to the preconditioning group prevented this increase, thus abolishing the protective effect of preconditioning. However, the administration of a NO donor to the preconditioned group subjected to previous administration of the adenosine A 2 receptor antagonist was able to maintain the preconditioning effects. In conclusion, these results indicate that, in preconditioning, the protective effect of adenosine could be a result of an increase in extracellular adenosine. This in turn would induce the activation of adenosine A 2 receptors, which, by eliciting an increase in NO generation, would protect against the injury associated with hepatic ischemia-reperfusion. (HEPATOLOGY 1999;29: 126-132.)Ischemic preconditioning, first shown in the myocardium has become a phenomenon described in the intestine, 1 brain, 2 and liver. 3 We have previously shown that ischemic preconditioning, induced by brief ischemia and reperfusion periods, renders the liver more tolerant to subsequent sustained ischemia-reperfusion. 4 We have also shown that the administration of adenosine mimics the effect of preconditioning in ischemic livers, and that the metabolization of endogenous adenosine by adenosine deaminase abolished the protective effect of preconditioning. We have further shown the beneficial effect of adenosine in inducing NO synthesis in ischemic tissue, 4 although the exact mechanism by which adenosine offered protection was not determined in that study. Recent work in heart preconditioning has shown that preconditioning-induced protection may require the activation of adenosine receptors. Thus, an adenosine receptor agonist can simulate the preconditioning whereas an adenosine receptor antagonist blocks its beneficial effect. 5 Also, recent studies in cerebral ischemia, have obtained evidence that the response to adenosine persists or is enhanced by nucleoside transport inhibitors such as propentofylline. Inhibition of adenosine uptake would increase its concentration in the extracellular space and hence potentiate its effects, particularly if adenosine binds to a specific receptor site on the external surface of the membrane. 6 Adenosine receptors have been divided into three major subclasses: A 1 , A 2 , and A 3 , 5,7 ...

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Macrophage infiltration is a common feature of the early phase of renal ischaemia/reperfusion injury. Indeed, it is generally regarded as the cause of tissue injury in this phase, although it is also clear that it can lead to tissue repair in other phases. In order to ascertain whether macrophages are directly involved in the repair/late phase, which follows the proinflammatory and injury process of renal ischaemia/reperfusion, we used two different approaches based on macrophage depletion. Firstly, we produced renal ischaemia in mice that were previously treated with clodronate liposome. Secondly, during reperfusion we re-injected RAW 264.7 to macrophage-depleted mice 24 h prior to sacrifice. The results showed that regeneration, as evaluated by stathmin and PCNA markers, was macrophagedependent: it was blocked when macrophage depletion was provoked and recovered with macrophage re-injection. The cytokine profile revealed the influence of the inflammatory environment on kidney repair: pro-inflammatory cytokines (MCP-1, MIP-1α) increased during the early stages of reperfusion, coinciding with low regeneration, and the antiinflammatory cytokine IL-10 increased during the longer periods of reperfusion when regeneration was more evident. We conclude that macrophages induce renal regeneration after ischaemia/reperfusion, depending on the inflammatory milieu.

Ischemia/reperfusion injury is a leading cause of acute renal failure triggering an inflammatory response associated with infiltrating macrophages, which determine disease outcome. To repair the inflammation we designed a procedure whereby macrophages that overexpress the anti-inflammatory agent interleukin (IL)-10 were adoptively transferred. These bone marrow-derived macrophages were able to increase their intracellular iron pool that, in turn, augmented the expression of lipocalin-2 and its receptors. Infusion of these macrophages into rats after 1 h of reperfusion resulted in localization of the cells to injured kidney tissue, caused increases in regenerative markers, and a notable reduction in both blood urea nitrogen and creatinine. Furthermore, IL-10 therapy decreased the local inflammatory profile and upregulated the expression of pro-regenerative lipocalin-2 and its receptors. IL-10-mediated protection and subsequent renal repair were dependent on the presence of iron and lipocalin-2, since the administration of a neutralizing antibody for lipocalin-2 or administration of IL-10 macrophages pretreated with the iron chelating agent deferoxamine abrogated IL-10-mediated protective effects. Thus, adoptive transfer of IL-10 macrophages to ischemic kidneys blunts acute kidney injury. These effects are mediated through the action of intracellular iron to induce lipocalin-2.

To assess the immediate and long-term effects of ischemic preconditioning (IPC) in deceased donor. liver transplantation (LT), we designed a prospective, randomized controlled trial involving 60 donors: control group (CTL, n = 30) or study group (IPC, n = 30). IPC was induced by 10-min hiliar clamping immediately before recovery of organs. Clinical data and blood and liver samples were obtained in the donor and in the recipient for measurements. IPC significantly improved biochemical markers of liver cell function such as uric acid, hyaluronic acid and Hypoxia-Induced Factor-1alpha (HIF-1a ) levels. Moreover, the degree of apoptosis was significantly lower in the IPC group. On clinical basis, IPC significantly improved the serum aspartate aminotransferase (AST) levels and reduced the need for reoperation in the postoperative period. Moreover, the incidence of primary nonfunction (PNF) was lower in the IPC group, but did not achieve statistical significance. We conclude that 10-min IPC protects against I/R injury in deceased donor LT.

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