Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York
Submitted 29 December 2004 ; accepted in final form 22 March 2005
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ABSTRACT |
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acute renal failure; multiorgan injury; survival
Although the pathogenesis of sepsis-induced acute organ injury and dysfunction is not completely understood, the initial hyperinflammatory process and subsequent hypoimmune phase contribute to mortality and morbidity in sepsis. The initial hyperinflammatory response seen in sepsis is associated with uncontrolled, hyperexuberant cytokine production that can be deleterious to various tissues and leads to organ injury and dysfunction. After this hyperinflammatory phase, a hypoimmune phase ensues with enhanced apoptotic cell death occurring in multiple organs including the spleen, kidney, liver, and heart (20, 21). The murine cecal ligation and double puncture model (CLP) resembles the human sepsis syndrome with respect to proinflammatory cytokine generation, apoptosis, and progression to acute sepsis-induced multiorgan injury and failure (11, 13, 33).
We previously demonstrated that endogenous as well as exogenous A1 adenosine receptor (A1AR) activation before renal ischemia and reperfusion significantly protects renal function by reducing inflammation, necrosis, and apoptosis (28). We hypothesized that endogenous A1ARs produce anti-inflammatory and anti-apoptotic effects in renal tubular cells. Therefore, in the present study, we questioned whether mice lacking A1ARs would show increased proinflammatory markers and apoptosis resulting in increased mortality and increased hepatic injury and renal dysfunction in an in vivo murine model of septic peritonitis.
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MATERIALS AND METHODS |
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The A1 knockout mice have been shown to have equivalent renal blood flow and glomerular filtration rate compared with A1 wild-type mice (16). In addition, A1 knockout mice have equivalent mRNA and protein expression of other AR subtypes compared with A1 wild-type mice. For example, our studies show that A1 knockout mice express 102, 110, and 100% of A2a, A2b, and A3 AR mRNA expression of A1 wild-type mice, respectively (n = 2 experiments), confirming the published data of Hansen et al. (16).
Induction of sepsis by cecal ligation and puncture. Mice were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg or to effect) and were allowed to spontaneously breathe room air on an electric heating pad under a warming light. CLP was performed as described previously (14). Briefly, the cecum was isolated through a small midline incision and the distal (0.5 cm) portion of the cecum below the ileocecal valve (to avoid bowel obstruction) was ligated with a 4.0 silk suture. The cecum was then punctured through and through along its antimesenteric border (double puncture) with a 20-gauge needle and a small amount of stool was extruded through the puncture site. Because the peritoneal surface is an excellent conduit for absorption, instillation of 0.5 ml of normal saline into the peritoneal cavity was performed for fluid resuscitation before closing the abdomen. Fluid resuscitation was continued during the initial 24 h following CLP, by administering subcutaneous saline (1 ml every 8 h for 24 h). Twenty-four hours after CLP, some mice were euthanized with an overdose of intraperitoneal pentobarbital sodium, and plasma and kidneys were collected.
Survival studies. To determine 7-day survival, A1 wild-type (n = 58; 26 females, 32 males), A1 heterozygous (n = 48; 22 females, 26 males), and A1 knockout (n = 45; 20 females, 25 males) littermates were subjected to CLP with a 20-gauge needle. All mice had free access to water and food and were observed by dedicated research personnel to determine 7-day survival. All severely moribund animals were euthanized with an overdose injection of anesthetic in adherence with our animal protocol. To examine whether pharmacological blockade of endogenous A1AR could exert an impact on survival, A1 wild-type (n = 23) and A1 heterozygous (n = 30) mice received a subcutaneous injection (1 mg/kg) of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; a selective A1AR antagonist) 20 min before and 6 h following CLP. To control for any confounding nonspecific effects of DPCPX that may have contributed to the observed mortality, A1 knockout mice (n = 23) also received DPCPX in the manner outlined above and were observed for 7 days.
Assessment of renal function and hepatic injury after sepsis. Renal function was assessed by measuring plasma creatinine 24 h after CLP by a colorimetric method based on the Jaffee reaction (18). Hepatic injury 24 h after CLP was assessed by measuring plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using a commercially available colorimetric method (Sigma, St. Louis, MO).
Measurement of proinflammatory cytokines.
Murine plasma TNF- and keratinocyte-derived chemokine (KC) concentrations taken 24 h after CLP were measured using commercially available ELISA kits according to the manufacturer's instructions (ALPCO, Windham NH and R&D Systems, Minneapolis, MN, respectively).
Assessment of renal inflammation.
Renal inflammation 24 h after CLP was assessed by measurement of renal corticomedullary myeloperoxidase activity (marker of leukocyte infiltration) and by immunoblotting for intercellular adhesion molecule-1 (ICAM-1) as described previously (29). In addition, renal corticomedullary expression of mRNAs encoding proinflammatory markers was also determined using semiquantitative RT-PCR as described previously (29). The proinflammatory mRNA markers studied included: KC, macrophage inflammatory protein 2 (MIP-2), ICAM-1, monocyte chemoattractant protein 1 (MCP-1), TNF-, interferon-induced protein 10 (IP-10), and regulated on activation normal T-cell expressed and secreted (RANTES). For each experiment, we also performed semiquantitative RT-PCR under conditions yielding linear results for GAPDH to confirm equal RNA input between groups. Primers were designed based on published GenBank sequences for mice (Table 1).
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EMSA was performed using the Gel Shift Assay Systems (Promega, Madison, WI). The oligonucleotides for NF-B (Promega) consensus sequences were end-labeled with 10 µCi of [
-32P]ATP (Perkin Elmer Life Technology, Wellesley, MA) and purified using a G-25 spin column (Amersham Biosciences, Piscataway, NJ). Ten micrograms of the nuclear extract were incubated with 1 µl of the labeled probe for 20 min at room temperature and electrophoresed on a 4% polyacrylamide gel (200 V at 4°C). Two micrograms of Hela cell nuclear extract (Promega) were used for a positive control and 1 µl of NF-
B p65 TransCruz polyclonal antibody (Santa Cruz Biotechnology) was coincubated with the nuclear protein and probe for a supershift reaction. One hundred-fold concentration of cold probe was coincubated as a competitor in a negative control reaction. In addition to the protein assay, we performed immunoblotting for histones to verify equal loading of nuclear fractions (data not shown). The gel was then transferred to blotting paper and exposed to film or scanned with a Phospho Imager (Molecular Dynamics, Piscataway, NJ).
Assessment of renal tubular necrosis and apoptosis. For histological preparations, explanted kidneys were bisected along the long axis and were fixed in 10% formalin solution overnight. Following automated dehydration through a graded alcohol series, transverse kidney slices were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin (H&E). Morphological assessment was performed by an experienced renal pathologist who was blinded to the treatment for each animal. A grading scale of 04, as outlined by Jablonski et al. (22), was used for the histopathological assessment of sepsis-induced damage of the proximal tubules.
We used in situ terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining to detect DNA fragmentation in apoptosis. Fixed mouse kidney sections obtained at 24 h after septic injury were deparaffinized in xylene and rehydrated through graded ethanols to water. In situ labeling of fragmented DNA was performed with TUNEL (green fluorescence) using a commercially available in situ cell death detection kit (Roche) according to the manufacturer's instruction. To visualize the total number of cells in the field, kidney sections were also stained with Hoeschst 33342 (blue fluorescence). We also performed DNA laddering on kidney samples obtained 24 h following CLP, as previously described (14).
Protein determination. Protein content was determined with the Pierce Chemical (Rockford, IL) bicinchoninic acid protein assay reagent using bovine serum albumin as a standard.
Statistical analysis. One-way ANOVA was used to compare differences in mean values between treatment groups. Survival statistics were compared with a Kaplan-Meier curve and Log Rank Test. In all cases, a probability value <0.05 was taken to indicate significance.
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RESULTS |
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A1 knockout mice show significantly increased CLP-induced organ injury and dysfunction. We examined the degree of renal dysfunction in A1 wild-type, A1 heterozygous, and A1 knockout mice by measuring plasma creatinine (Cr) and the degree of hepatic injury by measuring ALT and AST 24 h after CLP. Twenty-four hours after sham operation plasma creatinine, ALT or AST values in A1 heterozygous and A1 knockout were not different from A1 wild-type values (A1 wild-type Cr: 0.3 ± 0.1 mg/dl, n = 5; A1 wild-type ALT: 20 ± 1.9 SF U/ml, n = 8; A1 wild-type AST: 91 ± 10.6 SF U/ml, n = 8). Creatinine, ALT, and AST significantly increased at 24 h after CLP in all three groups of mice. However, A1 knockout mice showed significantly worse renal dysfunction and hepatic injury compared with A1 wild-type mice at 24 h after CLP (Table 3). A1 heterozygous mice exhibited creatinine, ALT, and AST values that were intermediate between values of A1 wild-type and A1 knockout mice at 24 h after CLP (Table 3).
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Renal myeloperoxidase activity 24 h following CLP is increased in A1 knockout mice. Myeloperoxidase (MPO) is an enzyme present in leukocytes and is an index of tissue leukocyte infiltration following injury (42). Because activated leukocyte infiltration is a hallmark of acute inflammation, we sought to determine the effect of endogenous A1AR activation on renal MPO activity 24 h following CLP-induced sepsis. Mice lacking endogenous A1ARs showed significantly higher MPO activity than both A1 wild-type and A1 heterozygous mice 24 h after the induction of sepsis (Table 3).
Renal mRNA and protein expression of proinflammatory markers following CLP is increased in A1 knockout mice.
We next examined the effects of endogenous A1AR activation on mRNA expression in renal cortices following CLP-induced sepsis. A1 wild-type, A1 heterozygous, and A1 knockout mice had similar levels of KC, RANTES, and IP-10 24 h following sham operation (data not shown). In contrast, A1 knockout mice demonstrated increased mRNA expression of KC, RANTES, and IP-10 compared with A1 wild-type mice 24 h after the induction of sepsis (Fig. 2). We observed no significant differences at the level of mRNA expression for MIP-2, ICAM-1, MCP-1, and TNF- between groups (data not shown). In a separate set of experiments, immunoblotting performed for ICAM-1 protein from dissected renal cortices taken 24 h following CLP demonstrated ICAM-1 protein expression was significantly higher in the A1 knockout group vs. the A1 wild-type group (P < 0.05; Table 3). A1 heterozygous mice showed intermediate ICAM-1 protein expression.
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DISCUSSION |
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We previously demonstrated that modulation of renal adenosine receptors can result in powerful protection against acute renal cell injury in vivo (rats and mice) and in vitro (human proximal tubule cells) (2327). We showed that A1AR activation protects against ischemia- and reperfusion-induced renal failure via mechanisms involving Gi, PKC, and ERK MAPK signaling pathways. Moreover, we demonstrated that A1 knockout mice had increased renal failure and inflammation following ischemia-reperfusion injury. This suggests that the A1AR imparts a protective benefit by reducing inflammation after ischemia-reperfusion injury. Because a hyperactive inflammatory response contributes to the development of multiorgan injury and dysfunction in sepsis, we hypothesized that mice lacking A1ARs would have worse outcomes compared with A1AR wild-type mice.
In animal models, potentiating adenosine effects (by either administration of adenosine receptor agonists or inhibiting reuptake mechanisms) have been shown to improve outcomes in sepsis (1, 12, 34, 37). The A2aAR is known to have anti-inflammatory effects and therefore has been an adenosine receptor subtype evaluated for its potential role in sepsis. Sullivan et al. (37) in an in vivo endotoxemia model of sepsis demonstrated increased survival mediated by ATL146e through the A2aAR. However, not all of adenosine's protective effects can be attributable to activation of the A2aAR. For example, Hasko et al. (17) found that adenosine is capable of inhibiting TNF- and IL-12 production in A2a knockout mice, suggesting that other adenosine receptor subtypes may also mediate anti-inflammatory effects in sepsis. To date, no study has addressed the role that endogenous A1AR activation may play in sepsis.
We adopted the CLP model to more accurately recapitulate the complex immunology seen in human sepsis. Unlike models employing endotoxin or bacteria, this model induces septic peritonitis that more closely resembles human sepsis with regard to proinflammatory cytokine generation, progression to multiorgan injury and failure, and response to certain therapeutic interventions (35). Another advantage of this septic model is the ability to manipulate the magnitude of the inflammatory response by modulating the needle gauge used for puncture. We employed a 20-gauge needle to induce in our A1 wild-type mice a level of sepsis with moderate predilection for mortality and organ injury in the absence of any interventions.
Given that our central hypothesis was to demonstrate the effect of endogenous A1AR activation on outcomes in murine sepsis, we chose not to possibly confound our results by introducing antibiotics into our studies. In fact, we were able to demonstrate differences in survival and morbidity without having to manipulate survival outcomes with antibiotic administration.
The sepsis model of CLP has two distinct phases. An early hyperdynamic stage is characterized by increased cardiac output, tissue perfusion, and decreased vascular resistance, whereas the later hypodynamic stage is characterized by decreased cardiac output and tissue perfusion (3941). At 210 h following CLP, the hyperdynamic phase predominates, which then transitions into the later hypodynamic phase (38). By 20 h after CLP, decreased tissue perfusion as well as direct inhibition of mitochondrial respiration by bacterial toxins contribute to tissue hypoxia. In this milieu, evidence exists that increased ATP breakdown and hypoxia-dependent inhibition of adenosine kinase can drive the accumulation of adenosine (10). In fact, elevated plasma and cerebrospinal fluid levels of adenosine have been reported in patients with severe sepsis (30). Adenosine has been coined as a "retaliatory molecule" due, in part, to its abundant overexpression during cellular stress and its ability to provide protection in the setting of tissue and cellular injury. Under normal conditions, extracellular adenosine levels are tightly held at 30300 nM, but during episodes of hypoxia these levels can rise to 10 µM (15, 43). In one study, severe septic patients' adenosine levels were
4 µM, and as such, are well within the binding affinity range for A1ARs. Elevated adenosine levels during sepsis may therefore serve to protect the host via activation of A1ARs.
Given the high mortality associated with sepsis (which ranges between 30 and 60%), therapeutic effects are primarily judged by their impact on survival (3, 8). Therefore, we first sought to determine whether survival differences would exist between mice lacking endogenous A1AR and their A1AR wild-type littermates in response to a septic insult. By 24 h following CLP, we observed a greater level of mortality and morbidity in the A1 knockout mice. Furthermore, log rank analysis of these 7-day survival studies demonstrated a significant survival benefit for both the A1 wild-type and A1 heterozygous vs. the A1 knockout mice.
Because compensatory physiological changes are inherent concerns with studies using knockout mice, we included a selective A1AR blocking agent to illustrate that the results we observed in our A1 knockout mice can be demonstrated in A1 wild-type mice treated with an A1AR antagonist. By using both models, we provide conclusive data that endogenous A1AR activation serves protective functions in CLP-induced sepsis. Selective blockade of A1AR activation on sepsis-induced mortality was assessed following administration of DPCPX. The effect of DPCPX in the setting of sepsis was a profound elimination of the survival advantage observed in both A1 wild-type and A1 heterozygous groups. However, A1 knockout mice administered DPCPX failed to demonstrate a change in survival, suggesting that the effect was indeed specific to A1AR blockade. These results support the hypothesis that endogenous A1AR activation has a protective role in sepsis and can lead to increased survival.
It is well known that mortality in sepsis is strongly influenced by the development of organ injury and dysfunction (6, 7, 9, 36). In concordance, our study showed that the lack of endogenous A1AR not only increased CLP-induced mortality, but it also significantly increased the magnitude of CLP-induced renal and hepatic injury.
Sepsis represents a systemic inflammatory response that initially manifests as an overproduction of stimulatory mediators including proinflammatory cytokines (i.e., TNF-) and chemokines (i.e., IL-8). In the present study, the inflammatory processes elicited during CLP-induced sepsis contributed to greater organ injury and dysfunction observed in the A1 knockout group as they do not possess the counterbalancing anti-inflammatory benefits afforded by endogenous A1AR activation. This is supported by the findings that TNF-
levels increase in the plasma by 24 h following CLP; however, TNF-
levels in A1 knockout mice were significantly elevated compared with A1 wild-type mice. This observation correlates well with studies performed by Meldrum et al. (32), which demonstrated that adenosine was capable of attenuating TNF-
levels in a cardiac ischemia-reperfusion model. Given the diminution of TNF-
levels in A1 wild-type mice compared with A1 knockout mice, our results draw into question whether this phenomenon is actually mediated via A1AR-related mechanisms. Murine KC is a proinflammatory chemokine that putatively represents the functional homolog of human IL-8. As such, KC not only serves as a potent neutrophil attractant and activator, but its overexpression has also been associated with various inflammatory conditions and is a marker for increased mortality in the CLP sepsis model (19). We also demonstrated an increase in plasma KC levels in the A1 knockout mice compared with A1 wild-type mice, providing further evidence that endogenous A1AR activation serves an anti-inflammatory and protective role during sepsis.
Further evidence that the A1AR contributes to decreased inflammation is the finding that increased mRNA encoding KC, RANTES, and IP-10 is found in the kidneys of A1 knockout mice 24 h following CLP compared with A1 wild-type mice. Similarly, protein levels of ICAM-1 and MPO were elevated in kidneys of A1 knockout mice 24 h following CLP compared with A1 wild-type mice.
Generation of cytokines and LPS during sepsis leads to toll receptor activation and propagation of the pathogenesis of sepsis. A central downstream element of toll receptor-dependent signaling is the pleiotropic transcription factor NF-B. NF-
B has been implicated in the regulation of multiple biological phenomena and disease states, including apoptosis, cell growth, stress response, innate immunity, and septic shock. Studies have demonstrated increased NF-
B expression is predictive of poor prognosis in sepsis (4). In other models of sepsis, suppression of NF-
B activation decreased acute inflammatory processes and organ dysfunction (31). Further evidence for the anti-inflammatory role of endogenous A1AR activation in sepsis is demonstrated in the present study by the significant increase in activation of NF-
B shown in A1 knockout mice compared with A1 wild-type mice in response to sepsis, which underscores the potential signaling pathways by which A1AR may be exerting its protective effects in sepsis.
In conclusion, we demonstrate that endogenous A1AR activation provides protection from CLP-induced mortality and acute organ dysfunction. Because the pathogenesis of organ dysfunction in sepsis is largely mediated by an imbalanced inflammatory response, A1AR activation improves organ function after septic insult by attenuating this hyperinflammatory process. This attenuation is global in nature and culminates in a reduction in organ injury and thereby protects from progression to multiple organ dysfunction. We speculate that endogenous A1AR activation exerts cytoprotective mechanisms that counteract inflammation and injury in CLP-induced sepsis. Given the protective benefit of the A1AR on survival and organ dysfunction, our findings may have important future therapeutic implications for patients in sepsis.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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