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Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice
Article first published online: 6 MAR 2008
Copyright © 2008 American Association for the Study of Liver Diseases
Volume 14, Issue 6, pages 845–854, June 2008
How to Cite
Kim, J., Kim, M., Song, J. H. and Lee, H. T. (2008), Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice. Liver Transpl, 14: 845–854. doi: 10.1002/lt.21432
- Issue published online: 28 MAY 2008
- Article first published online: 6 MAR 2008
- Manuscript Accepted: 6 DEC 2007
- Manuscript Received: 11 SEP 2007
- Department of Anesthesiology at the National Cancer Center (Goyang-Si, Gyeonggi-Do, Republic of Korea). Grant Number: R01-DK058547
Hepatic ischemia reperfusion (IR) injury is a major clinical problem during the perioperative period and occurs frequently after major hepatic resection or liver transplantation. Exogenous and endogenous A1 adenosine receptor (A1AR) activation protects against renal IR injury. In this study, we questioned whether exogenous and endogenous A1AR activation protects against hepatic IR injury in vivo. A1AR wild-type (WT) or knockout mice were subjected to 60 minutes of partial hepatic IR. Some animals were treated with a selective A1AR agonist, 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1 mg/kg), or a selective A1AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.4 mg/kg), 15 minutes before hepatic ischemia. Twenty-four hours after hepatic IR, the A1 knockout mice and DPCPX-treated A1 wild-type (A1WT) mice developed significantly worse liver injury (alanine aminotransferase, liver necrosis, neutrophil infiltration, and apoptosis) compared to A1AR WT mice. However, the selective A1AR agonist CCPA failed to protect against hepatic IR injury in A1WT mice. Our results show that the endogenous A1ARs protect against hepatic IR injury in vivo by primarily reducing apoptosis and necrosis with subsequent reductions in proinflammatory neutrophil infiltration. However, in contrast to the kidneys, in which exogenous A1AR activation protected against IR injury, exogenous A1AR activation failed to protect against liver injury after IR. We conclude that endogenous A1AR activation prevents worsened murine liver IR injury primarily by reducing necrotic and apoptotic cell death. Harnessing the mechanisms of cytoprotection with endogenous A1AR activation may lead to new therapies for perioperative hepatic IR injury. Liver Transpl, 2008. © 2008 AASLD.
Hepatic ischemia reperfusion (IR) injury is a major clinical problem during the perioperative period and frequently follows major hepatic resection, liver transplantation, or septic shock.1-3 Hepatic IR injury not only causes liver dysfunction but frequently results in injury of extrahepatic organs, including the lung, kidney, and heart.4-6 Therefore, a better understanding of the pathophysiology of hepatic IR and identification of therapeutic methods to attenuate this injury have important clinical implications.
Activation of cell surface adenosine receptors (ARs) modulates cytoprotection (antiapoptotic, anti-inflammatory, and antinecrotic) in many organs including the heart, kidney, and liver.7-12 Of 4 subtypes of ARs (A1, A2a, A2b, and A3), preischemic A1 adenosine receptor (A1AR) activation has been shown to protect against IR injury in many organs including the heart, kidney, and brain.8, 13-16 Our laboratory previously demonstrated that exogenous and endogenous A1AR activation protects against renal IR injury.8, 16 Moreover, endogenous activation of A1ARs protects against renal and hepatic injury in septic shock induced with cecal ligation and puncture in mice.17
In contrast, other investigators have reported that the administration of a selective A1AR antagonist, KW3902 [8-(noradamantan-3yl)-1,3-dipropylxanthine], before ischemia attenuated hepatic IR injury in dogs.18 Therefore, we sought to further substantiate the role of A1ARs in hepatic IR injury using a mouse model of partial hepatic IR. In this study, we questioned whether exogenous and endogenous A1AR activation protects against hepatic IR injury in vivo. We used A1AR knockout (KO) mice in addition to pharmacological manipulation of A1ARs with a selective agonist and a selective antagonist in A1AR wild-type (WT) mice. We tested the hypothesis that A1 knockout (A1KO) mice would have worsened hepatic dysfunction after IR injury compared to A1 wild-type (A1WT) mice. We further hypothesized that preischemic activation or inhibition of A1ARs would protect against or worsen, respectively, hepatic IR injury in A1AR WT mice. Finally, we questioned whether A1AR-mediated protection against hepatic IR injury was associated with reduced necrosis, apoptosis, and markers of inflammation.
MATERIALS AND METHODS
Breeder pairs of noncongenic A1AR heterozygous mice were donated by Dr. J. Schnermann (National Institutes of Health). The generation and initial characterization of the A1AR KO mice with a C57BL/6 background have been described previously.16, 19 The A1AR heterozygous mice were crossed in our laboratory with C57BL/6 mice (Harlan Laboratories, Indianapolis, IN) for 12 generations to create a congenic line of A1AR KO mice. Subsequently, all of the A1AR WT male mice were obtained from Harlan Laboratories.
Murine Model of Hepatic IR
After Columbia University Institutional Animal Care and Use Committee approval, male A1AR WT or KO mice (25-30 g) were anesthetized with intraperitoneal pentobarbital (50 mg/kg or to effect). Mice were placed under a heating lamp and on a 37°C heating pad. After a midline laparotomy and intraperitoneal application of 500 U of heparin and vehicle [50% dimethyl sulfoxide (DMSO) in saline; discussed later], left lateral and median lobes of the liver were subjected to ischemia with a microaneurysm clip occluding the hepatic triad above the bifurcation. This method of partial hepatic ischemia results in a segmental (∼70%) hepatic ischemia but spares the right lobe of the liver and prevents mesenteric venous congestion by allowing portal decompression throughout the right and caudate lobes of the liver.9, 12 The liver was then repositioned in the peritoneal cavity in its original location for 60 minutes. The liver was kept moist with gauze soaked in 0.9% normal saline. The body temperature was monitored by an infrared temperature sensor (Linear Laboratories, Fremont, CA) and maintained at 37°C with a heating lamp and a heating pad. After 60 minutes, the liver was reperfused, and the wound was closed. To determine the role of exogenous manipulations of A1ARs in hepatic IR injury, some mice were treated with a single dose of a selective A1AR agonist, 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1 mg/kg intraperitoneally), or a selective A1AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.4 mg/kg intraperitoneally), 15 minutes before hepatic ischemia. CCPA and DPCPX were dissolved first in DMSO and then further diluted in saline for the final DMSO concentration of 50%. Sham-operated mice were treated with vehicle and were subjected to laparotomy and identical liver manipulations without vascular occlusion. Twenty-four hours after reperfusion, plasma was collected for the measurement of alanine aminotransferase (ALT). Three, 6, and 24 hours after reperfusion, the liver tissue subjected to IR was collected to measure the percentage of liver necrosis, neutrophil infiltration (with immunohistochemistry), apoptosis (with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining), and inflammation by reverse-transcription polymerase chain reaction (RT-PCR) for proinflammatory messenger RNAs (mRNAs) and immunoblotting for intercellular cell adhesion molecule 1 (ICAM-1).
Plasma ALT Activity
Plasma ALT was measured by the use of a aminotransferase kit according to the manufacturer's protocol (Thermo Electron, PA) with a BioTek (Winooski, Vermont) microplate reader with KC Junior software. In brief, a 200-μL aliquot of a prewarmed (37°C) mixture of L-alanine and α-ketoglutaric acid was added to 20 μL of plasma in a 96-well plate. The plate was read at 340 nm at 60-second intervals, and the rate of change in absorbance was used to calculate ALT in units per liter.
Histology and Quantification of Hepatic Necrosis After IR Injury
For histological preparations, explanted murine livers were fixed in a 10% formalin solution overnight. After automated dehydration through a graded alcohol series, transverse liver slices were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin. To quantify the degree of hepatic necrosis, hematoxylin-eosin stains were digitally photographed, and the percentage of necrotic area was quantified with NIH Image software (Image-J, version 1.37) by a person who was unaware of the treatment that each sample received. Twenty random sections were investigated per slide to determine the percentage of necrotic area.
Immunohistochemistry for Neutrophils, Macrophages, and Lymphocytes
Paraffin-embedded mouse liver sections were deparaffinized in xylene and rehydrated through graded ethanol series to water. Endogenous peroxidase activity was inhibited with 0.3% H2O2. After blocking with 10% normal rabbit serum/phosphate-buffered saline solution, the slides were incubated overnight with primary antibody (all from Serotec, Raleigh, NC) for neutrophils (clone 7/4), macrophages (F4/80), and lymphocytes (CD3) at 4°C in a humidified chamber, and this was followed by horseradish peroxidase–conjugated rabbit anti-mouse immunoglobulin G (1:100 dilution, Vector BA4001) for 30 minutes and diaminobenzidine reagent (Vector Laboratories, Burlingame, CA) for 10 minutes. Control serum immunoglobulin G was used for negative isotype control experiments. The sections were evaluated blindly through the counting of the labeled cells (100× fields).
Semiquantitative RT-PCR Assays of Proinflammatory mRNAs for Tumor Necrosis Factor α (TNF-α), Keratinocyte Derived Cytokine, Monocyte Chemoattractant Protein 1 (MCP-1), Macrophage Inflammatory Protein 2 (MIP-2), and ICAM-1
Three, 6, or 24 hours after liver ischemic injury, liver tissues subjected to IR were dissected, and total RNA was extracted with Trizol reagent according to the instructions provided by the manufacturer (Invitrogen, Carlsbad, CA), as described previously by us.8, 16, 20 RNA concentrations were determined on the basis of spectrophotometric absorbance at 260 nm, and aliquots were subjected to electrophoresis on agarose gels for verification of equal loading and RNA quality. Semiquantitative RT-PCR was performed to analyze the expression of proinflammatory genes (TNF-α, KC, MCP-1, MIP-2, and ICAM-1). The polymerase chain reaction (PCR) cycle number for each primer pair was first optimized to yield linear increases in the densitometric measurements for resulting bands with increasing PCR cycles (15-28 cycles). The starting amount of RNA was also optimized to yield linear increases in the densitometric measurements for resulting bands with the established number of PCR cycles. For each experiment, we also performed semiquantitative RT-PCR under conditions that yielded linear results for glyceraldehyde-3-phosphate dehydrogenase to confirm equal RNA input. On the basis of these preliminary experiments, 0.5 to 1.0 μg of total RNA was used as the template for all RT-PCR assays. Primers were designed on the basis of published GenBank sequences for mice. Primer pairs were chosen to yield expected PCR products of 200 to 450 base pairs and to amplify genomic regions spanning 1 or 2 introns to eliminate the confounding effect of amplification of contaminating genomic DNA as described previously.8, 16, 20 Primers were purchased from Sigma Genosys (The Woodlands, TX). RT-PCR was performed with the Access RT-PCR system (Promega, Madison, WI), which is designed for a single-tube reaction for first-strand complementary DNA synthesis (48°C for 45 minutes) with avian myeloblastosis virus reverse transcriptase and subsequent PCR with Tfl DNA polymerase. PCR cycles included denaturation at 94°C for 30 seconds, annealing at an optimized temperature for 1 minute,8, 16, 20 and extension at 68°C for 1 minute. All PCR reactions were completed with a 7-minute incubation at 68°C to allow for enzymatic completion of incomplete complementary DNAs. The products were resolved on a 6% polyacrylamide gel and stained with Syber green (Roche, Indianapolis, IN), and the band intensities were quantified with a UVP gel imaging system (Bio-Rad, Hercules, CA).
The A1WT or A1KO mouse liver tissues were obtained 24 hours after sham operation or hepatic IR, placed in ice-cold radioimmunoprecipitation buffer (150 mM NaCl, 50 mM trishydroxymethylaminomethane-HCl, 1 mM ethylenediamine tetraacetic acid, 1% Triton-X, pH = 7.4), and homogenized for 10 seconds. The samples were then centrifuged for 10 minutes at 1000g, and the resulting supernatant was collected, quantified (for protein concentration), and mixed to a 1× final concentration with Laemmli's loading buffer (50 mM trishydroxymethylaminomethane-HCl, 1% 2-mercaptoethanol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol). Equal amounts of protein (30 μg) were subjected to electrophoresis through a 7.5% polyacrylamide gel and transferred to polyvinylidene difluoride membranes. ICAM-1 expression was subsequently detected by immunoblotting using monoclonal antibody (sc-8439, Santa Cruz Biotechnologies, Santa Cruz, CA) diluted 1:500 as described previously.21
Detection of Apoptosis with In Situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL) Assay
We used in situ TUNEL staining to detect DNA fragmentation in apoptosis. Fixed mouse liver sections obtained at 24 hours after hepatic injury were deparaffinized in xylene and rehydrated through graded ethanols to water. In situ labeling of fragmented DNA was performed with TUNEL (green fluorescence) with a commercially available in situ cell death detection kit (Roche, Nutley, NJ) according to the manufacturer's instructions. To visualize the total number of cells in the field, liver sections were also stained with propidium iodide (red fluorescence). The sections were evaluated blindly through the counting of the labeled cells in 100× magnified fields.
Protein contents were determined with a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard.
The data were analyzed with the Student t test to compare means between 2 groups or with 1-way analysis of variance plus Tukey's post hoc multiple comparison test to compare mean values across multiple treatment groups. In all cases, a probability statistic of <0.05 was taken to indicate significance. All data are expressed throughout the text as mean ± standard error.
Unless otherwise specified, all reagents were purchased from Sigma (St. Louis, MO).
Endogenous Deletion or Exogenous Blockade of A1ARs Worsens Hepatic Function After IR Injury
Our model of hepatic IR resulted in severe liver dysfunction 24 hours after reperfusion indicated by large increases in plasma ALT levels. A1WT and A1KO mice that underwent sham operations had similar baseline hepatic function (ALT = 61 ± 16 U/L for A1WT, n = 6, and ALT = 62 ± 12 U/L for A1KO, n = 5; Fig. 1). However, 24 hours after hepatic ischemic injury, A1KO mice had significantly higher plasma ALT (672 ± 79 U/L, n = 11) compared to the A1WT mice (311 ± 65 U/L, n = 14, P < 0.002). A1WT mice pretreated with DPCPX before hepatic ischemia also had significantly higher ALT at 24 hours (769 ± 103 U/L, n = 8, P < 0.01). However, in contrast to the kidney in which the exogenous A1AR activation protected against renal IR injury,8 CCPA treatment failed to protect the A1AR WT mice against hepatic IR injury (400 ± 101 U/L, n = 8). We also subjected the A1KO mice to IR injury after treating them with DPCPX or with CCPA. We determined that DPCPX-treated A1KO mice subjected to IR had ALT (627 ± 56 U/L, n = 5) similar to that of A1KO mice subjected to IR. Moreover, CCPA failed to protect the A1KO mice against IR injury (615 ± 102 U/L, n = 6). Therefore, we can confirm the in vivo selectivity of these drugs for A1AR. Injection of CCPA or DPCPX alone without hepatic IR (CCPA sham or DPCPX sham) had no effect on liver function (Fig. 1).
Endogenous Deletion or Exogenous Blockade of A1ARs Worsens Hepatic Necrosis After IR Injury
Our model of 60-minute partial hepatic IR injury produced time-dependent increases in the percentage of necrotic areas of liver after reperfusion (Fig. 2A). Correlating with significantly worsened hepatic function, significantly increased liver necrosis was observed in A1KO mice subjected to hepatic IR injury compared to the A1WT mice (Fig. 2A). Representative histological slides from liver tissues from A1WT mice, DPCPX-treated A1WT mice, CCPA-treated A1WT mice, or A1KO mice subjected to 60-minute ischemia and 24-hour reperfusion and A1WT mice or A1KO mice subjected to the sham operation are shown in Fig. 2B. The quantifications of the percentage of necrotic area are shown in Fig. 2C. We failed to detect necrosis in liver sections from sham-operated mice (A1WT sham, n = 3; A1KO sham, n = 3). The A1WT mice subjected to hepatic IR resulted in moderate necrosis 24 hours after hepatic IR (% necrosis = 35 ± 8, n = 11, P < 0.01 versus sham). A significantly higher percentage of hepatic necrosis developed in the A1KO mice (% necrosis = 68 ± 8, n = 12, P < 0.01) or in DPCPX-pretreated A1WT mice (% necrosis = 74 ± 5, n = 9, P < 0.01) compared to A1WT mice subjected to IR. A1WT mice pretreated with the A1AR agonist before IR injury failed to show an improvement in hepatic necrosis (% necrosis = 45 ± 12, n = 8). Injection of CCPA or DPCPX alone (without hepatic IR) had no effect on liver histology.
Endogenous Deletion or Exogenous Blockade of A1ARs Selectively Increases Hepatic MCP-1 Expression After IR Injury
With RT-PCR, we measured the expression of hepatic proinflammatory cytokine mRNAs 3, 6, or 24 hours after IR. Hepatic IR injury was associated with significantly increased proinflammatory mRNA expression (ICAM-1, TNF-α, KC, MCP-1, and MIP-2) at all time points. However, only the expression of MCP-1 was selectively higher for the A1KO mice or DPCPX-treated A1WT mice 3 hours after hepatic IR (Fig. 3). There were no differences between A1WT mice, A1KO mice, or DPCPX-treated A1WT mice for the expression of ICAM-1, TNF-α, KC, and MIP-2 3 hours after hepatic IR. CCPA pretreatment failed to suppress an increase in MCP-1 mRNA expression 3 hours after hepatic IR injury. Moreover, proinflammatory mRNA expression did not differ between A1WT mice, A1KO mice, DPCPX-treated A1WT mice, or mice treated with CCPA 6 or 24 hours after hepatic IR (data not shown).
Endogenous Deletion of A1ARs Does Not Accentuate the Increase in Liver ICAM-1 Expression After Hepatic IR in Mice
Hepatic IR injury was associated with significantly increased proinflammatory ICAM-1 protein expression in both A1WT and A1KO mice. However, there were no differences in the degree of ICAM-1 up-regulation between the 2 groups 24 hours after hepatic IR (data not shown).
Endogenous Deletion or Exogenous Blockade of A1ARs Increases Hepatic Neutrophil Infiltration 24 Hours After IR Injury
Sixty minutes of hepatic ischemia resulted in rapid and early recruitment of neutrophils (Fig. 4A). Neutrophil infiltration 3 or 6 hours after hepatic ischemia did not differ between the A1WT and A1KO mice (Fig. 4B). However, neutrophil infiltration in A1KO mice showed a significant increase 24 hours after hepatic ischemia, whereas the A1WT mice showed a decrease. Figure 4A shows representative images of neutrophil immunohistochemistry of liver sections from sham-operated A1WT or A1KO mice, A1WT or A1KO mice subjected to IR, or A1WT mice subjected to IR after CCPA or DPCPX pretreatment. In sham A1WT mice, we detected 19 ± 2 neutrophils/field (100× magnification, n = 3), which increased to 96 ± 26 neutrophils/field (P < 0.05, n = 5) 24 hours after IR injury. A1KO mice subjected to IR injury had significantly higher neutrophil counts (895 ± 253 neutrophils/field, n = 7, P < 0.05) 24 hours after hepatic IR compared with A1KO sham-operated mice (23 ± 8 neutrophils/field, n = 3, P < 0.01) or A1WT mice subjected to IR (P < 0.05; Fig. 4B). The A1WT mice pretreated with DPCPX before IR injury had increased neutrophilic infiltration (881 ± 103 neutrophils/field, n = 5, P < 0.05) compared to the A1WT mice subjected to IR alone 24 hours after partial hepatic IR. The A1WT mice pretreated with the A1AR agonist (CCPA) and subjected to IR injury failed to show decreased neutrophil infiltration (149 ± 100 neutrophils/field, n = 5; Fig. 4C). Unlike the observation with neutrophils, macrophage and lymphocyte infiltration did not increase after hepatic IR when compared to the sham-operated animals (data not shown).
Endogenous Deletion or Exogenous Blockade of A1ARs Increases Hepatic Apoptosis After IR Injury
Apoptosis in the ischemic liver was detected with the TUNEL assay (Fig. 5). Sixty minutes of hepatic ischemia resulted in rapid apoptotic hepatic cell death in both A1WT and A1KO mice. However, the degree of apoptosis in A1KO mice was significantly greater at 3 and 24 hours after reperfusion (Fig. 5B). Sham-operated mice demonstrated few TUNEL-positive cells 24 hours after hepatic ischemia. Twenty-four hours after 60 minutes of hepatic ischemia, a significantly increased number of TUNEL-positive cells were present in the livers of A1KO mice (236 ± 51 cells per 100× field, n = 7, P < 0.05) compared to A1WT mice (85 ± 44, n = 6; Fig. 5C). Moreover, DPCPX-pretreated A1WT mice showed a significantly higher number of TUNEL positive liver cells compared to A1WT mice (290 ± 89, n = 5, P < 0.05), whereas the A1WT mice pretreated with the A1AR agonist (CCPA) and subjected to IR injury did not show decreased apoptosis (102 ± 70, n = 5).
The major findings of this study are that (1) mice deletionally lacking the A1ARs (A1KO mice) demonstrate greater hepatic injury after IR compared to the WT mice (A1WT mice); (2) preischemic pharmacological blockade of A1ARs worsened hepatic IR injury in A1WT mice; (3) exogenous activation of A1ARs before hepatic ischemia failed to protect against hepatic IR injury in A1WT mice; (4) exacerbation of hepatic function in A1KO or DPCPX-pretreated A1WT mice was associated with increased necrosis, apoptosis, and late (24-hour) neutrophil infiltration, and (5) there was a selective increase in MCP-1 mRNA expression after hepatic IR injury in A1KO or DPCPX-pretreated A1WT mice.
The release of adenosine after stress (for example, IR or septic shock) with subsequent activation of ARs serves to protect against cell death in many organ systems as well as multiple cell types, including the heart, kidney, and liver.7, 9, 22 Of the 4 AR subtypes, the activation of A1ARs and A2a adenosine receptors (A2aARs) are well known to produce cytoprotection after IR injury. In fact, increased release of adenosine is proposed to mediate the organ-protective effects of ischemic preconditioning in the liver23 and heart.24, 25 In the liver, previous studies have suggested that activation of A2aARs mediates the hepatoprotective effects of ischemic preconditioning.23 However, the hepatoprotective effects of A1AR activation in the liver have not been examined fully.
In this study, we demonstrated for the first time that blockade of endogenous A1ARs with a selective antagonist (DPCPX) or deletion of A1ARs exacerbated hepatic dysfunction after IR injury in mice, suggesting that endogenous A1ARs serve as cytoprotective receptors in the liver. However, we failed to show protection with exogenous application of a selective A1AR agonist (CCPA), indicating that further stimulation of A1ARs does not provide additional cytoprotective benefit. These findings are in contrast to the kidney or the heart, in which exogenous and endogenous A1AR activation produces protection after IR injury.8, 16 Therefore, although endogenous A1AR activation prevents the worsened injury seen in KO mice (or mice treated with DPCPX), A1AR does not in and of itself reduce liver IR injury to baseline levels but rather dampens the greater injury seen in A1AR KO mice.
Endogenous modulation of A1AR strongly reduced necrosis of the hepatic parenchyma after IR. Necrosis after IR is a key component of organ failure.1, 26 Necrotic cell death occurs directly by total breakdown of cellular homeostatic machinery due to massive depletion of adenosine triphosphate (ATP) during and after the ischemic period or indirectly during reperfusion where uncontrolled delivery of free radicals and proinflammatory hematopoetic cells cause further cellular derangements. Because early neutrophil infiltration did not differ between A1WT and A1KO mice after hepatic IR, we propose that the reduction of hepatic necrosis with endogenous A1AR activation occurs by a direct reduction in necrotic cell death of hepatocytes. Activation of A1ARs in many cell types produces cytoprotection by initiation of multiple beneficial pathways, including extracellular signal-regulated kinase (ERK), Akt, and protein kinase C (PKC), and induction of heat shock proteins.8, 16, 27-29 We have demonstrated that activation of A1ARs in the kidney, for example, produces powerful cytoprotection via ERK-dependent and PKC-dependent pathways.8, 22, 28, 30 Similarly, other investigators have demonstrated that A1ARs in the heart and brain produce cytoprotection by activation of K channels, ERK and/or PKC.31-33
We also demonstrated in this study that apoptotic cell death after hepatic IR is significantly higher in mice lacking A1ARs or in mice after pharmacological blockade of A1ARs. Hepatic apoptosis is an important contributor in the development of hepatic failure after IR injury.1-3 Apoptotic cell death represents the execution of an ATP-dependent death program often initiated by death ligand/death receptor interactions, such as Fas ligand with Fas, which leads to a caspase activation cascade and cleavage of nuclear materials such as DNA and poly(ADP-ribose) polymerase.27, 34 Our study shows that endogenous A1ARs play an important role in attenuating apoptotic liver damage after IR.
The importance of neutrophils in the development of IR injury in the liver is well established.1, 2, 35 Activated neutrophils release substances to produce further tissue injury such as products of arachidonic acid metabolism, oxygen free radicals, and neutrophil elastase.35 We showed in this study that neutrophil infiltration into the liver increases immediately after reperfusion of the ischemic liver in both A1WT and A1KO mice. However, although the A1WT livers show a reduction of infiltrating neutrophils at 24 hours after ischemia, A1KO mice and A1WT mice treated with DPCPX show a further and more accentuated increase in neutrophil infiltration after hepatic IR. Therefore, it appears that activation of endogenous A1ARs plays a role in reducing the number of neutrophils infiltrating liver tissue 24 hours after IR. Interestingly, we failed to show any reductions in neutrophil infiltration during earlier time points (3 and 6 hours) after hepatic IR in A1WT mice. These findings indicate that endogenous A1ARs do not modulate the early infiltration of neutrophils after IR. Rather, they reduce the secondary wave of infiltrating neutrophils 24 hours after IR.
Because compensatory physiological changes are inherent concerns with studies using KO mice, we included a selective A1AR antagonist (DPCPX) to demonstrate that the results we observed in our A1KO mice can be demonstrated in A1WT mice treated with an A1AR antagonist. Blockade of endogenous A1ARs with DPCPX worsens necrosis, apoptosis, and liver dysfunction after hepatic IR injury in mice. Our study is in contrast to the study by Magata et al.,18 in which the antagonism of A1ARs provided protection after hepatic IR. The reason for this discrepancy is not clear. One reason could be due to the species differences. In the study by Magata et al., they used female dogs, whereas we used mice in our study. Moreover, in the study by Magata et al., they infused a selective A1AR antagonist, KW3902, for 60 minutes at 1 μg/kg/minute. Selectivity, specificity, or tachyphylactic ability of an antagonist at a given receptor subtype (for example, A1AR versus A2aAR) is always of concern, especially when it is given as an infusion. In our study, we used both a highly selective A1AR antagonist and mice lacking A1ARs to verify the hepatoprotective role of the endogenous A1AR activation.
The production of several proinflammatory cytokines and adhesion molecules after hepatic IR is critically important in the pathophysiology of liver IR injury.2, 5, 35-37 By RT-PCR, we examined whether the expression of hepatic proinflammatory mRNAs (TNF-α, KC, MCP-1, MIP-2, and ICAM-1) is up-regulated after IR and whether deletion of A1AR modulates the proinflammatory mRNA expression. We demonstrated that, as expected, all of the proinflammatory mRNAs examined show enhanced expression after hepatic IR. However, it is surprising to find that of the proinflammatory mRNAs examined, only the MCP-1 expression was significantly increased by A1AR deletion. MCP-1 is a member of the C-C chemokine family and is up-regulated after IR injury in many organ systems. MCP-1, originally described as the JE gene, is a 148 amino acid chemotactic cytokine and plays an important role in the recruitment, accumulation, and activation of cells of monocyte/macrophage lineage.1, 2, 5, 38 Therefore, it is surprising to show that the degree of macrophage and lymphocyte infiltration after hepatic IR was not different between A1KO mice and A1WT mice. The significance of selective enhancement of MCP-1 after hepatic IR in A1KO mice is not clear and remains a topic for future studies. MCP-1 induces dose-dependent expression of tissue factors to initiate the clotting cascade.5,38 Thus, MCP-1 may play an important role in the pathogenesis of hepatic IR not only by recruiting inflammatory cells into the liver after IR but also by affecting the microcirculation.
In summary, we demonstrate in this study that endogenous A1AR activation provides protection against hepatic IR injury by reducing necrosis, apoptosis, and neutrophil infiltration 24 hours after IR. Unlike the heart and the kidney, in which exogenous A1AR activation is protective against IR injury, activation of A1ARs in the liver failed to attenuate liver dysfunction after IR injury. We speculate that endogenous A1AR activation exerts cytoprotective mechanisms that counteract necrosis and apoptosis in liver IR injury. Given the protective benefit of endogenous A1AR activation against hepatic IR and the fact that hepatic IR is common in patients after liver surgery, liver transplantation, or sepsis, our findings may have important future therapeutic implications.