Purinergic receptor expression is altered in extra-intestinal organs following intestinal ischemia/reperfusion injury

Abstract

Background: Intestinal ischemia/reperfusion (IIR) injury is known to initiate the systemic inflammatory response syndrome, often progressing to multiple organ failure, resulting in significant morbidity and mortality. Pro-inflammatory cytokines released following IIR injury have been implicated as mediators in the associated extra-intestinal manifestations of this disease process, specifically pulmonary and renal dysfunction. Extracellular nucleotides are known to be released in response to ischemic stress, and modulate a host of pro-inflammatory responses via interactions with purinergic receptors. In this study, we investigate the hypothesis that purinergic receptor expression is altered in clinically relevant extra-intestinal organs following IIR injury. Methods: Adult male BalbC mice (n=17; wt: 30.0 +/− 3.3 grams) were anesthetized and randomized to receive either sham laparotomy (control, n=5), or 15 minutes of superior mesenteric artery occlusion. Experimental ischemia was followed by a subsequent period of reperfusion[1 minute (n=6) or 1 hour (n=6)]. The mice were then sacrificed, and lung, kidney, and intestinal tissues were harvested for analysis. Following RNA extraction, purinergic receptor mRNA expression for P2×7, P2Y2, P2Y4, P2Y6, A2b and A3, was analyzed with real-time RT-PCR using previously optimized intron-spanning murine primers. Receptor gene patterns were normalized as a ratio of cyclophilin A expression. Results: Significant differences in tissue purinergic receptor expression were observed in both the lungs and kidneys of mice exposed to IIR injury when compared to controls. In the lung, P2Y2 receptor expression was increased in the 1 hour IIR group when compared to control (1.687 +/− 0.227 vs. 1.014 +/− 0.116; p<0.05). Pulmonary A3 receptor expression was incrementally elevated following intestinal ischemia/reperfusion injury (ctrl. 1.042 +/− 0.213 vs. IIR 1 hr. 2.093 +/− 0.142 and IIR 1 min. 1.239 +/− 0.306 vs. IIR 1 hr.; p<0.05). In the kidney, P2Y2 receptor expression was increased in the 1 hour IIR group compared to both 1 minute IIR and control (IIR 1 hr. 1.845 +/− 0.333 vs. IIR 1 min. 0.911 +/− 0.137 and vs. ctrl. 0.717 +/− 0.276; p<0.05). A3 receptor expression in the kidney was decreased in the 1 hour IIR group compared to the 1 minute IIR group (0.496 +/− 0.121 vs. 1.436 +/− 0.261; p<0.05). No significant changes were noted in the intestinal tissue purinergic receptor profiles. Conclusion: Purinergic receptor expression patterns are altered in the lung and kidney following intestinal ischemia/reperfusion injury. Intestinal purinergic receptor expression patterns are unchanged following experimental ischemia/reperfusion injury. These findings may implicate extracellular nucleotides and their receptors as possible mediators of extra-intestinal organ dysfunction associated with intestinal ischemia/reperfusion injury.