Ischemia reperfusion injury (IRI) is a primary concern in liver transplantation, especially when steatosis is present. Acetazolamide (AZ), a specific carbonic anhydrase (CA) inhibitor, has been suggested to protect against hypoxia. Here, we hypothesized that AZ administration could be efficient to protect fatty livers against cold IRI. Obese Zucker rat livers were preserved in Institut Georges Lopez-1 storage solution for 24 hours at 4°C and ex vivo perfused for 2 hours at 37°C. Alternatively, rats were also treated with intravenous injection of AZ (30 mg/kg) before liver recovery. Liver injury, hepatic function, and vascular resistance were determined. CA II protein levels and CA hydratase activity were assessed as well as other parameters involved in IRI (endothelial nitric oxide synthase, mitogen activated protein kinase family, hypoxic inducible factor 1 alpha, and erythropoietin). We demonstrated that AZ administration efficiently protects the steatotic liver against cold IRI. AZ protection was associated with better function, decreased vascular resistance, and activation of endothelial nitric oxide synthase. This was consistent with an effective mitogen activated protein kinase inactivation. Finally, no effect on the hypoxic inductible factor 1 alpha/erythropoietin pathway was observed. The present study demonstrated that AZ administration is a suitable pharmacological strategy for preserving fatty liver grafts against cold IRI.
Limited pool of donor organs for liver transplantation has led to the acceptance of marginal livers, such as steatotic ones, despite their higher risk of early allograft dysfunction and nonfunction. In fact, steatotic liver grafts are associated with an early allograft dysfunction rate of 60% compared with less than 5% for nonsteatotic grafts (Selzner et al., 2000; Selzner and Clavien, 2001; Farrell et al., 2008). This is due to their poor tolerance to ischemia reperfusion injury (IRI) (Deschenes, 2013), evidenced by exacerbated oxidative stress, mitochondrial damage, and microcirculatory alterations (Selzner and Clavien, 2001; Casillas-Ramirez et al., 2006).
Multiple mechanisms are involved in the decreased tolerance of steatotic livers to ischemic injury, and consequently, various pharmacological strategies need to be combined to effectively protect fatty livers. Particular experimental pharmacological strategies to rescue steatotic livers consisted of the improvement of hepatic microcirculation (Hakamada et al., 1997; Caraceni et al., 1999), inhibition of oxygen-free radical-mediated injury (Nakano et al., 1997; Pesonen et al., 1998; Luo et al., 2012), and inhibition of the proteasome system with bortezomib (Ramachandran et al., 2012; Zaouali et al., 2013). In clinical practice, only a few pharmacological protective strategies, consisting of bezafibrate, an activator of peroxisome proliferator-activated receptor alpha and -β/δ, were used to treat human living donors for liver transplantation (Nakamuta et al., 2005). There are other drugs that could potentially be taken by living donors, but their significant side effects limit their use (Chalasani, 2005; Liu et al., 2013). These observations point to the need for more effective and safe drugs for preventing the steatotic liver from hepatic IRI.
Carbonic anhydrases (CAs) and their inhibitors are relevant in many physiologic processes and diseases. In mammals, there are 16 isoforms with different catalytic activities, tissue concentrations, and subcellular localization, and three of them are expressed in the liver (CA II, CA VA, and CA XVI) (Pastorekova et al., 2004; Swenson, 2014). In 1956, acetazolamide (AZ) was the first nonmercurial diuretic to be used clinically. AZ inhibits CAs in the proximal tubule of the nephron, which leads to the inhibition of proton (H+) secretion and bicarbonate (HCO3−) excretion, triggering the movement of isotonic water and augmented diuresis (Supuran, 2011). AZ has been used in many hypertensive-related diseases (Supuran, 2011), and recently, it has been reported that AZ protects the kidney against IRI through NO activation (An et al., 2013).
Here, treatment with AZ before liver recovery (AZ preconditioning) has been evaluated as a useful tool to better protect fatty liver grafts against cold IRI. Moreover, the potential mechanisms were investigated.
Materials and Methods
Male homozygous obese Zucker rats, aged 9 to 10 weeks, were purchased from Charles River Laboratories (Saint-Germain-sur-l'Arbresle, France) and housed at 22°C with free access to water and standard chow. All experiments were approved by the Ethics Committees for Animal Experimentation (CEEA, Directive 697/14), University of Barcelona, and were conducted according to European Union regulations for animal experiments (Directive 86/609 CEE). All procedures were performed under isoflurane anesthesia inhalation.
The surgical technique was performed as previously described (Ben Abdennebi et al., 1998). Briefly, after cannulation of the common bile duct, livers were flushed with a chilled Institute Georges Lopez-1 (IGL-1) preservation solution (4°C) by means of catheter insertion into the aorta. After cooling, a second catheter was inserted into the portal vein to complete liver rinsing and the whole liver was excised and trimmed of surrounding tissues. Forty milliliters of preservation solution were infused through the aorta and the portal vein. Then, the livers were preserved with a further 130 ml of the same solution for 24 hours at 4°C.
Fatty livers were perfused at 37°C via the portal vein in a closed and controlled pressure circuit. Time point 0 was established when the portal catheter was satisfactorily connected to the circuit. During the first 15 minutes of perfusion (initial equilibration period), the flow was progressively increased to stabilize the portal pressure at 12 mm Hg (Pression Monitor BP-1; Pression Instruments, Sarasota, FL). The flow was controlled by a peristaltic pump (Minipuls 3; Gilson, Villiers-Le-Bel, France). The reperfusion liquid (150 ml for each perfusion) consisted of a cell culture medium (William’s medium E; BioWhittaker, Barcelona, Spain), with a Krebs-Heinseleit–like electrolyte composition enriched with 5% albumin as the oncotic supply. The medium was continuously gassed with a 95% O2 and 5% CO2 gas mixture and subsequently passed through a heat exchanger (37°C) and bubble trap prior to entering the liver. After 120 minutes of normothermic reperfusion, the effluent perfusion fluid was collected for biochemical determination and fatty livers were sampled.
AZ (A6011; Sigma Aldrich, Barcelona, Spain) was dissolved in NaOH 1 M, and then the pH was adjusted to 9.6 and distilled water was added to reach the final concentration of 60 mg/ml. AZ was injected at 30 mg/kg 10 minutes before liver procurement, according data reported by Ichikawa et al. (1998).
All animals were randomly distributed into different experimental groups, as indicated below:
Protocol 1: Effect of AZ in Liver Injury after 24 Hours of Cold Storage in IGL-1 Preservation Solution.
Control 1 (Ctr 1) (n = 4): Control livers were flushed via the portal vein with Ringer’s lactate solution immediately after laparotomy without cold storage.
IGL-1 (n = 5): Livers were preserved for 24 hours in Institute of Georges Lopez-1 (IGL-1) solution.
AZ (n = 5): Livers were pretreated with AZ intravenously at 30 mg/kg 10 minutes before liver procurement, and then preserved for 24 hours in IGL-1.
After 24 hours of cold storage in IGL-1, steatotic livers were removed from the preserved solution and flushed at room temperature with 20 ml of Ringer lactate solution. This flushed liquid was aliquoted and stored for biochemical determination [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)].
Protocol 2: Effect of AZ in Fatty Liver Injury after Cold Ischemia Reperfusion.
To examine the effect of AZ in liver injury and the underlying mechanisms, fatty livers were subjected to 2-hour normoxic reperfusion in the following groups:
Control group (Ctr 2) (n = 4): After procurement, steatotic livers were ex vivo perfused for 2 hours, as described above, without prior cold storage.
IGL-1 group (n = 6): Fatty livers were preserved in IGL-1 preservation solution for 24 hours at 4°C and then subjected to 2 hours of normothermic reperfusion at 37°C.
AZ group (n = 6): Zucker obese rats were pretreated with intravenous administration of AZ at 30 mg/kg 10 minutes before liver procurement. Then, livers were preserved for 24 hours in IGL-1 solution and finally ex vivo perfused for 2 hours at 37°C.
Liver Injury: Transaminase Assay
Hepatic injury was assessed in terms of ALT and AST levels in washout liquid or perfusate effluent with commercial kits from RAL (Barcelona, Spain). Briefly, 100 μl was added to 1 ml of the substrate provided by the commercial kit and then transaminase activity was measured at 340 nm with a UV spectrometer and calculated following the supplier’s instructions. The results were normalized using a commercial calibrator (Biocal; RAL).
Liver Function: Bile Production and Hepatic Clearance
Liver function was assessed by measuring bile production and hepatic clearance of bromosulfophthalein (BSP). Bile was collected through the cannulated common bile duct, and output was reported as microliters per gram of liver after 120 minutes of reperfusion.
BSP clearance was assessed as previously reported (Zaouali et al., 2011). Briefly, 30 minutes after the onset of perfusion, 1 ml of BSP (Sigma Aldrich) at 10 mg/ml was added to the 150 ml of the perfusate. After 120 minutes of reperfusion, the concentration of BSP in bile was measured at 580 nm with a UV-visible spectrometer and divided by the concentration of BSP in the perfusate at t = 30. The result was expressed by percentage [(t120 bile/t30 perfusate)*100].
Vascular resistance was defined as the ratio of portal venous pressure, which was maintained at 12 mm Hg during the reperfusion to flow rate, and expressed in millimeters of mercury per minute per gram of liver per ml. The perfusion flow rate was assessed continuously throughout the reperfusion period and expressed as millimeter per minute per gram of liver.
Western Blotting Technique
Liver tissue was homogenized in HEPES buffer, and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes as previously described (Bejaoui et al., 2014). Membranes were immunoblotted overnight at 4°C using the following antibodies: anticarbonic anhydrase II (ab115306; abcam, Cambridge, UK), anti stress-activated protein kinase/Jun-amino-terminal kinase (Thr183/Tyr185), anti–p-p38 mitogen activated protein kinase (MAPK) (Thr180/Tyr182, #9211), and anti extracellular signal-regulated kinase (ERK 1/2), (Thr202/Tyr204, #9101), which were all purchased from Cell Signaling (Danvers, MA); anti–endothelial nitric oxide synthase (eNOS) (610296; Transduction Laboratories, Lexington KY); and anti–b-actin (A5316; Sigma Chemical, St. Louis, MO). After washing, the bound antibody was detected after incubation for 1 hour at room temperature with the corresponding secondary antibody linked to horseradish peroxidase. Bound complexes were detected and quantified by scanning densitometry.
Quantitative Real-Time Polymerase Chain Reaction
Total liver RNA was isolated using the TRIzol reagent (Invitrogen, Barcelona, Spain). Reverse transcription was realized on a 1-µg RNA sample using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). The reaction included incubation at 25°C (5 minutes), 42°C (30 minutes), and 85°C (5 minutes), and then cDNA was stored at −80°C. Subsequent polymerase chain reaction amplification was conducted in the iCycler iQ Multi-Color real-time polymerase chain reaction system (Bio-Rad Laboratories) using SsoAdvanced Universal SYBR Green Supermix and the following rat primers for hypoxic inductible factor (HIF) 1α, forward, 5′- TCAAGTCAGCAACGTGGAAG -3′, and reverse, 3′- GTCAGCTGTGTCGGAGCTAT-5′, and its target erythropoietin (Epo), forward, 5′- CCAGCCACCAGAGAGTCTTC -3′, and reverse, 3′-GTGTCGCCTATGAAAGACGT-5′. Reactions were carried out in duplicate, and threshold cycle values were normalized to glyceraldehyde-3-phosphate dehydrogenase gene expression. The ratio of HIF-1α and Epo relative expression to glyceraldehyde-3-phosphate dehydrogenase was calculated by the ΔCt formula.
Liver samples were fixed in 10% neutral buffered formalin and embedded in Paraplast, and 5-µm sections were stained with hematoxylin and eosin according to standard procedures. Histologic evaluation was graded semiquantitatively from 0 (no damage) to 4 (severe cellular damage, such as vacuolization, cell dissociation, cell swelling, and disintegration of hepatic architecture).
Carbonic Anhydrase Activity
Each liver sample (approximately 300 mg) was homogenized in 0.05 M Tris-HCl buffer, pH 7.5. The homogenate was centrifuged twice for 30 minutes at 12,000g. The resulting supernatant was subject to protein determination and assayed for CO2 hydratase activity.
Protein concentration was determined using the Bio-Rad protein assay based on the method of Bradford (1976)
Carbonic Anhydrase Assay.
The CA activity assay was a modification of the procedure described by Chirica et al. (1997). The assay was based on monitoring of the pH variation due to the catalyzed conversion of CO2 to bicarbonate. Bromothymol blue was used as the indicator of pH variation. The assay was performed at 0°C by adding 1.0 ml of ice-cold CO2-saturated water to 1.0-ml mixtures of 25 mM Tris-SO4 buffer. The CO2-satured solution was prepared by bubbling CO2 into 100 ml of distilled water for approximately 3 hours. The CO2 solution was chilled in an ice water bath. Fifty microliters of the liver extract were added to one tube, and an equivalent amount of buffer was added to the second tube as the control. One milliliter of CO2 solution was added very quickly, and simultaneously, a stopwatch was started. The time required for the solution to change from blue to yellow was recorded (transition point of bromothymol blue is pH 6 to 7). The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution until the color transition point of the dye is reached. The time required for the color change is inversely related to the quantity of carbonic anhydrase present in the sample. Detecting the color change is somewhat subjective, but the error for triple measurements was in the range of a 0 to 1 second difference for the catalyzed reaction. Wilbur-Anderson units (WAUs) were calculated according to the following defnition: 1 WAU of activity is defined as (T0 − T)/T, where T0 (uncatalyzed reaction) and T (catalyzed reaction) are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye in a control buffer and in the presence of an enzyme, respectively.
Liver tissues were homogenized in 1/10 homobuffer [220 mM mannitol, 70 mM sucrose, 0.1 mM EDTA, 3 mM TRIS, and 0.1% bovine serum albumin (fatty acid free), pH 7.4] and then diluted four times in phosphate-buffered serum. Triglycerides, free cholesterol, and fatty free acids were determined colimetrically at the Hospital Clinic, Barcelona, Spain.
The results were analyzed using one-way analysis of variance, with three levels (control, IGL-1 solution, and AZ preconditioning). This approach allowed us to assess whether there was any significant difference in the effects among the three levels and then to explore where such differences existed based on pairwise comparisons. Adjustments for multiple comparisons were based on Tukey’s test. The assumption of normality of residuals and homogeneity of variances was checked using the Shapiro-Wilk and Bartlett tests, respectively. For all tests, a P value < 0.05 was considered statistically significant.
We determined transaminases as a tool for predicting organ damage after cold preservation (Teramoto et al., 1993; Pantazi et al., 2014). Figure 1 reveals ALT and AST releases in liver perfusate after 24 hours of liver graft storage. The higher transaminase levels released by steatotic livers after 24 hours of cold preservation in the IGL-1 solutions group confirm the high vulnerability of fatty livers to cold ischemia (14.3 ± 1.5 versus 74.5 ± 7 U/l for ALT and 18.2 ± 5.5 versus 90.8 ± 23.1 U/l for AST). A significant reduction in transaminase levels in the flushing effluent of fatty livers was observed when AZ was administered prior to graft procurement (41.8 ± 15 versus 75.5 ± 7 U/l for ALT and 45 ± 16.8 versus 90.8 ± 23.1 U/l for AST).
To investigate the possible effect of AZ preconditioning against reperfusion injury in steatotic livers, we measured AST/ALT levels in livers preserved for 24 hours in IGL-1 solution followed by 2 hours of normoxic reperfusion (37°C). We also assessed the histologic analysis. Figure 2A shows the perfusate AST/ALT levels after 2 hours of normothermic reperfusion in steatotic livers preserved in IGL-1 with or without AZ pretreatment. AST/ALT levels increased in the IGL-1 group compared with the control group (158 ± 61.7 versus 29 ± 10.9 U/l for ALT and 338.2 ± 156.5 U/l for AST). On the other hand, AZ pretreatment significantly reduced transaminase levels (62 ± 33.5 versus 158 ± 61.7 for ALT and 76 ± 39.7 versus 338 ± 156.5 for AST). This decrease in liver injury was furthermore confirmed by the histologic findings (Fig. 2B). The histologic study of control steatotic livers (Ctr 2) showed grade II to III of fatty infiltration, with hepatocyte integrity maintenance. In the IGL-1 group, fatty infiltration was conserved and severe cellular damage was seen, whereas in the AZ group, decreased cellular damage was observed when compared with the IGL-1 group.
Hepatic function was assessed by bile production and BSP clearance in bile. The prevention of liver reperfusion injury exerted by AZ was consistent with a significant improvement of liver function. As shown in Fig. 3, bile production and BSP clearance decreased in preserved steatotic livers when compared with control ones. In all cases, AZ favored a significant recovery of bile output and percentage of BSP at 2-hour reperfusion when compared with the IGL-1 group (26 ± 3.7 versus 8.7 ± 3.9 µl/g of liver per 120 minutes for bile output and 15.2 ± 6.8 versus 38 ± 2.1 of percentage of BSP).
To investigate the relationship between AZ administration and CA inhibition, we assessed CA activity (Fig. 4B). Our results confirm that AZ pretreatment at the dose of 30 mg/kg was efficient to inhibit CA hydratase activity (0.103 ± 0.034 in the IGL-1 group versus 0.020 ± 0.007 WAU/mg protein in the AZ group). Moreover, we examined the potential effect of AZ in the regulation of CA II expression, as we have previously shown that CA II addition to the IGL-1 solution protects steatotic livers against IRI (Bejaoui et al., 2015). Surprisingly, we observed that AZ induced a significant induction of CA II protein levels (Fig. 4A).
Recently, several studies have provided evidence that CA inhibitors of the sulfonamide/sulfamate type have been considered to be potential antiobesity drugs (Supuran et al., 2008). Thus, we have quantified the fat content by measuring triglycerides, free fatty acids, and free cholesterol in liver tissue and have found that AZ significantly reduced free cholesterol and moderately reduced triglycerides when compared with the IGL-1 group after 2 hours of reperfusion (Fig. 4C).
AZ is indicated in many hypertensive-related diseases, and it is also well known that enhanced liver resistance during reperfusion is associated with the poor tolerance of steatotic grafts to IRI. Our results show that AZ preconditioning induced a significant reduction in vascular resistance (Fig. 5A). We speculated that this significant reduction could be correlated with the generation of NO, a well known vasodilator mediator. For this reason, we determined eNOS expression in steatotic livers subjected to cold ischemia and reperfusion. As indicated in Fig. 5B, we observed significant eNOS activation when AZ was administered prior to cold ischemia.
During cold ischemia, MAPKs are activated and responsible for the induction of cellular damage (King et al., 2009). We found that fatty liver preservation in IGL-1 solution resulted in a marked induction of MAPKs when compared with controls, as evidenced by upregulation of phosphorylated extracellular signal-regulated kinase (pERK), p-p38, and phosphorylated Jun-amino-terminal kinase. AZ administration prior to cold ischemia reperfusion induced an important reduction of MAPKs (Fig. 6) (Fig. 7).
Finally, we investigated whether the AZ preconditioning protective effect could be related to HIF-1α upregulation. Recently, AZ has been shown to protect the kidney against IRI through HIF-1α induction (An et al., 2013). Moreover, HIF-1α upregulation was associated with better preservation of steatotic and nonsteatotic liver grafts (Zaouali et al., 2010a). Our results show that HIF-1α and its target Epo mRNA expression were downregulated after cold ischemia reperfusion. However, AZ pretreatment did not enhance HIF-1α or Epo mRNA expression (Fig. 7).
Here, we demonstrated that AZ is a promising drug to prevent the deleterious effects of IRI in steatotic livers. Its beneficial effects are reflected by a significant prevention of liver injury and improvement of hepatic function, decreasing the vulnerability of steatotic liver grafts to IRI. In our work, steatotic livers were cold preserved in IGL-1 solution that was or was not pretreated with AZ. The rationale of the proposed protocol was to induce a pharmacological preconditioning against the subsequent cold storage and reperfusion injury. This could be relevant in a clinical situation of brain-dead donors and steatotic livers, which are both risk factors in liver transplantation.
The diminished hepatic injury in rats pretreated with AZ was concomitant with a significant decrease in CA hydratase activity and an enhanced expression level of CA II. This is in line with our previous observation showing that CA II addition to the IGL-1 solution prevented IRI in steatotic livers, which was associated with an enhanced CA II expression (Bejaoui et al., 2015). Thus, the AZ protective mechanism may be related to enhanced CA II protein expression rather than CA hydratase activity inhibition.
An important factor for the susceptibility of steatotic livers to IRI is the distortion and narrowing of hepatic sinusoids due to the reduced luminal diameter (up to 50%). This fact leads to alterations of blood flow and microcirculation, hindering the suitable revascularization of the graft (Fukumori et al., 1999; Liu et al., 2013).
Clearly, our results showed that 1) AZ decreased vascular resistance through eNOS activation; and 2) the AZ vasodilator effect is correlated with CA inhibition. In the literature, the mechanism of AZ-induced vasodilatation is controversial because in some cases, it was shown to be independent of eNOS activation (Kiss et al., 1999); in others, AZ increased NO production through eNOS activation (Tuettenberg et al., 2001; An et al., 2013) and others showed that AZ-induced vasodilation is independent of CA inhibition (Hohne et al., 2007; Torring et al., 2009). Recently, Aamand et al. have shown that CA II is able to generate vasoactive NO from nitrite at high rates and with no strict requirement for low O2 (Aamand et al., 2009). Interestingly, the addition of AZ significantly increased the CA-catalyzed NO production. The CA-catalyzed NO production is significantly higher at acidic conditions similar to those found during ischemia (pH = 5.9), and the reaction also occurs under anaerobic conditions. The authors hypothesize that CA II has two active sites, one for CO2 and the other for nitrite, and that AZ may increase the affinity for the substrate nitrite by occupying nonproductive binding sites on the enzyme. Unfortunately, our experimental model does not permit corroboration of this hypothesis due to the difficulty of determining NO levels and the nitrite-reducing CA activity in liver tissue during cold ischemia and reperfusion. In our experimental model, we determined CA activity that corresponded to CO2 hydratation activity. Further determinations, such as eNOS inhibition, when AZ was used could provide more information. Also, the isolated rat aortic rings model might be more suitable to examine this hypothesis.
Recently, it has been proposed that the use of selective CA inhibitors is useful for the development of new antiobesity drugs (Supuran et al., 2008). Quantification of fat content in the liver after reperfusion showed that AZ moderately reduces steatosis. This is an interesting result because it gives new insights into the use of CA inhibitors in defatting cocktails in machine perfusion (Nagrath et al., 2009).
MAPKs consist of 1) ERK 1/2, 2) JNK 1/2, and 3) p38 MAPK and are signal transducers that transmit messages from the cell surface to the nucleus in response to oxidative and other environmental stresses. In the case of IRI, MAPKs are well known mediators of stress responses, as they have been associated with upregulation of proinflammatory and cell death pathways. MAPKs are activated during hypothermia, and several minutes after reperfusion and inhibition of their activation has been associated with ameliorated hepatic injury (Yoshinari et al., 2001; Kobayashi et al., 2002; Xu et al., 2005; Zaouali et al., 2010b). In line with this, we report that the AZ protective effect has been mediated through an overwhelming decrease in pERK, p-p38, and pJNK.
Hepatic steatosis was shown to contribute to augmented oxidative stress, leading to nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells inactivation and impaired HIF-1α induction and thereby increased susceptibility to hypoxic injury (Anavi et al., 2012). HIF-1α is a key transcriptional factor mediating cellular adaptation under stress conditions, including IRI. Indeed, it plays an important role in fatty liver protection against cold IRI, and its protective action is related to its downstream proteins, including Epo and eNOS (Zaouali et al., 2010a; Eipel et al., 2012). Moreover, HIF-1α has been implicated in the AZ beneficial effects against renal IRI (An et al., 2013) and in the case of acute mountain sickness (Xu et al., 2009). However, in our experimental conditions, results show that the AZ protective effects are not related to HIF upregulation, but future investigations for clarifying the effects on HIF are needed.
In conclusion, our results show that AZ preconditioning is a promising strategy for improving fatty liver graft viability. The beneficial effect was associated with decreased vascular resistance, activation of eNOS, and the prevention of MAPK family activation.
The authors thank Martín Rios from the Faculty of Biology of the University of Barcelona for the statistical analysis.
Participated in research design: Bejaoui, Catafau.
Conducted experiments: Bejaoui, Pantazi, De Luca, Panisello, Serafin.
Performed data analysis: Catafau, Folch-Puy, Capasso, Supuran.
Wrote or contributed to the writing of the manuscript: Bejaoui, Catafau, Capasso, Supuran.
- Received April 15, 2015.
- Accepted August 26, 2015.
This work was supported by the Ministerio de Economía y Competividad (Spain) through the “Fondo de Investigaciones Sanitarias” (FIS) [Grant PI12/00519].
This work has been presented by Bejaoui M et al. as follows: 14th Annual Congress of the French-Speaking Society of Transplantation; 2–5 Dec 2014; Caen, France and in the 13th Congress of the Catalan Society of Transplantation, 18–20 Mar 2015; Barcelona, Spain. Catalan Society of Transplantation, Barcelona, Spain.
- alanine aminotransferase
- aspartate aminotransferase
- carbonic anhydrase
- endothelial nitric oxide synthase
- extracellular signal-regulated kinase
- hypoxic inductible factor
- Institute of Georges Lopez-1
- ischemia reperfusion injury
- c-Jun N-terminal kinase
- mitogen activated protein kinase
- phosphorylated extracellular signal-regulated kinase
- Wilbur-Anderson unit
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics