In the present study, we demonstrated that 2 ml/kg EIso (8% vol./vol.) pretreatment alleviated renal tissue morphological damage, decreased serum Cr, BUN, CYC, TNF-α and IL-6 concentrations, reduced renal tissue MDA content, increased serum IL-10 concentration and tissue SOD activity in the I/R model of rat kidneys, as 4 ml/kg EIso pretreatment. Our results indicated that EIso may exert protective effects against renal I/R injury in rats by inhibiting systemic inflammatory response and improving antioxidation of the tissue. To our knowledge, this is the first study to show that intravenous administration of EIso could produce renal protection.
Chiari et al.  found that intravenous infusion of 6.9% EIso at a constant rate of 3.5 ml/kg per h for 30 min produces acute and delayed preconditioning against myocardial infarction in rabbits. In the same way, intravenous pretreatment of 2 or 4 ml/kg 8% EIso for 30 min affords effective protection against myocardial ischemia in rats . And these doses of EIso had no significant inhibitory effects on circulation function in pentobarbital anesthetized rats . Base on these studies, we demonstrated in the present study that EIso (2 or 4 ml/kg for 30 min) is useful for preventing renal injury after ischemia.
Measurement of serum Cr and BUN levels have been used to determine renal function in many experimental studies. Coll et al.  reported that serum concentration of CYC could reflect glomerular filtration rate very accurately, even in cases where there was only a minor reduction in glomerular filtration rate. A meta-analysis of 46 CYC-related studies to evaluate the superiority of CYC levels over serum Cr levels showed that serum CYC is a more potent marker of glomerular filtration rate than serum Cr . Serum CYC level is a more sensitive test for the early detection of renal function impairment or reduced glomerular filtration rate as compared to serum Cr . In the present study, serum Cr, BUN and CYC levels in the 2 and 4 ml/kg EIso groups were markedly decreased compared with the I/R, intralipid and 1 ml/kg EIso groups. In addition, the histological scores of renal injury were significantly decreased with 2 or 4 ml/kg EIso preconditioning. It showed that both 2 and 4 ml/kg EIso pretreatment induce protective effects against renal I/R injury in rats.
With renal I/R injury, necrotic cell death results in further activation of inflammatory cascades, resulting in more severe secondary tissue damage . The inflammatory components consist of TNF-α, IL-6 and IL-8 that cause leukocytes to accumulate in the vasa recta of the outer medulla . There are growing evidences from in vitro and in vivo models that the course of renal I/R injury is associated with intra-renal inflammation, and therefore it is now recognized that intra-renal inflammation is deeply involved in the pathogenesis of renal I/R injury . Increased levels of renal and serum IL-6 were observed in mice subjected to renal I/R . And IL-6-knockout mice suffered less renal I/R injury . The experimental hyperlipidemia induced by poloxamer 407 decreased renal I/R injury, which was mediated by reduced renal IL-6 production after the insult . Lee et al.  demonstrated that 1 minimum alveolar concentration isoflurane given during renal I/R protects against renal I/R injury in rats by reducing renal TNF-α concentrations and inhibiting nuclear factor-κB activation. Hashiguchi et al.  also showed that 1.5% isoflurane has a preconditioning effect against rat renal I/R injury when administered 20 min before ischemia, which may be related to inhibition of the protein kinases, Jun N-terminal kinase and extracellular signal-regulated kinase. Recently, pretreatment with 1.5% isoflurane was showed to ameliorate renal I/R injury in mice via upregulation of hypoxia inducible factor-1 α . In addition, several studies implicated that isoflurane activates intestinal sphingosine kinase to protect against renal I/R-induced  or bilateral nephrectomy-induced  liver and intestine injury. Other studies suggested that volatile anesthetics could protect against I/R injury in the heart  and lung  via anti-inflammatory effects. IL-10 is expressed and secreted by a variety of cell types, which prevents production of proinflammatory cytokines and chemokines by monocytes/macrophages. IL-10 is an antiinflammatory cytokine which contributes to improvement in left ventricle functional recovery after myocardial infarction in a cultured cell study . And IL-10-deficient mice revealed increased neutrophil infiltration, infarct size, and myocardial necrosis after acute myocardial infarction . In the present study, we demonstrated that both 2 and 4 ml/kg EIso preconditioning significantly reduced the serum levels of TNF-α and IL-6, increased the serum level of IL-10 as compared to the I/R, intralipid and 1 ml/kg EIso groups. Our results indicated that EIso-induced protection against renal I/R injury was probably related to preventing inflammatory response. In a recent study, Lv et al.  showed that EIso preconditioning decreased nuclear factor-κB activity, TNF-α level, myeloperoxidase activity, and intercellular adhesion molecule-1 expression in the lung, which resulted to reduce lung injury induced by hepatic I/R. Both 2 and 4 ml/kg EIso intravenously infused were also found to attenuate myocardial damage by inhibiting apoptosis after ischemia .
It is well known that oxygen free radicals contributes to the pathogenesis of I/R injury. MDA is one of the products of free radical chain reaction and lipid peroxidation. The changes in tissue MDA content reflect changes in the quantity of polyunsaturated fatty acid reductions during I/R injury. Consequently, the increase of MDA content indicates a lowering of the membrane fluidity and impairment of the normal membrane structure of the mitochondria. SOD is one of important antioxidase that scavenges oxygen free radicals and protects mitochondria against damage caused by potentially cytotoxic reactions. Usually antioxidative ability of tissue is evaluated by MDA content and SOD activity. Yurdakoc et al.  showed that isoflurane could protect against lipid peroxidation of cerebral injury in rats. Recently, a study demonstrated that EIso increased antioxidation in mitochondria to protect against liver and lung injury in the rat hemorrhagic shock model . Similarly, Xu et al. showed that 2 ml/kg EIso pretreatment could ameliorate lipopolysaccharide-induced acute lung injury in rats by decreasing pulmonary MDA level and increasing pulmonary SOD activity . Our present study showed that both 2 and 4 ml/kg EIso preconditioning resulted in a decrease of MDA content and an increase of SOD activity in the kidney tissue. These findings suggested that EIso pretreatment could enhance renal antioxidative ability, which might be one of mechanisms of renal protection of EIso with 2 or 4 ml/kg, partly protected against I/R injury.
In the present study, EIso pretreated the kidney with 2 or 4 ml/kg against I/R injury in rats. However, the lower dose of EIso (1 ml/kg) did not appear to produce protection, which is consistent with a previous report concerning a dose-dependent effect of isoflurane . There were no significant differences between the 2 and 4 ml/kg EIso groups. The similar result was found in the rat myocardial injury model . Whether formulations containing higher concentrations or different rates of infusion could be responsible for greater renal protection deserves further investigation.
EIso is composed of isoflurane and intralipid. As a vehicle, intralipid may influence the renal protection of this anaesthetic, so the effects of intralipid were also investigated in the present study. It was demonstrated that intralipid preconditioning alone did not protect renal injury. There is a conflicting report regarding the renal protective effect of intralipid during acute renal I/R . Several factors can account for these discrepant results, including differences in experimental design, as well as exposure time and drug concentration. In the present study, there were no significant differences in these variables between the intralipid and I/R groups with a same experimental protocol. Therefore, we concluded that the intralipid could be not responsible for the renal protection of EIso.
There are some limitations of this study, including the brief period of observation, and one blood and tissue sampling time point. Moreover, blood concentrations of isoflurane during infusion and before renal pedicles occlusion were not measured, so unequal doses of isoflurane may administer to rats subjected to renal ischemia.
EIso can be administrated intravenously, which makes its clinical application more practical than inhalation of isoflurane. For this reason intravenous EIso may gain more wider acceptance as a treatment option for clinical application.