ATRIAL NATRIURETIC PEPTIDE REDUCES PRESERVATION AND REPERFUSION INJURY IN RAT LIVER: ROLE OF CGMP AND HEME OXYGENASE-1

Abstract

O255 Aims: Preconditioning with atrial natriuretic peptide (ANP) markedly reduces hepatic ischemia and reperfusion (I/R) injury via the inhibition of proinflammatory response and the induction of cytoprotective molecules. However, molecular mechanisms are as yet widely unknown. Heme oxygenase (HO)-1 is well recognized as an antioxidant and cytoprotective gene. The aim of this study is to investigate the involvement of HO-1 induced by ANP in rat liver I/R model. Methods: Lewis male rats were intravenously pretreated with saline (as control), ANP (4 mg/kg) or 8-Bromo cyclic guanosine monophosphate (8-Br-cGMP; 10 mg/kg) 6 hrs before liver harvesting. HO-1 mRNA expression, protein synthesis and enzyme activity were determined by reverse transcription polymerase chain reaction, Western immunoblotting, bilirubin formation, and immunohistochemical analyzes. In a different group, liver grafts were stored with chilled University of Wisconsin solution for 30 hrs and transplanted orthotopically. Immediately after revascularization a potent inhibitor of HO enzyme activity, zinc protoporphyrin IX (ZnPP; 1.5 mg/kg) was intravenously administered in animals which had received ANP or 8-Br-cGMP pretreated livers. Following reperfusion serum level of tumor necrosis factor (TNF)-αlpha and immunohistochemical expression of vascular cell adhesion molecule (VCAM)-1 were assessed. Propium iodide (PI)- and in situ nick-end labeling (TUNEL)-positive cells were determined by immunohistochemical methods. Animal survival was also monitored. Results: At 24 hrs after treatment, ANP or 8-Br-cGMP induced a significant increase in hepatic HO-1 gene and protein expression with high enzyme activity. HO-1 was dominantly expressed in Kupffer-like dendritic cells. Following reperfusion, the expression of TNF-αlpha mRNA and VCAM-1 protein as well as the numbers of necrotic and apoptotic cells were markedly reduced in ANP or 8-Br-cGMP pretreated livers. Higher animal survival rates were observed in both experimental groups (p<0.05, ANP; 7/10, 8-Br-cGMP; 6/10 vs. Saline; 0/10). These cytoprotective effects were abrogated in the presence of HO enzyme inhibitor, ZnPP, introduced immediately after reperfusion. Conclusions: These results suggest that prior induction of HO-1 by ANP and its second messenger analogue 8-Br-cGMP may lead to anti-inflammatory and anti-apoptotic effects on hepatic I/R injury depending on its enzyme activity. Thus, clinically used and less-toxic ANP would be potentially applicable to donor preconditioning or pharmacologic treatment for pathologic conditions that lead to oxidative stress including I/R injury.