Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion

Abstract

Ischemia and reperfusion-elicited tissue injury contributes to morbidity and mortality of hepatic surgery and during liver transplantation. Previous studies implicated extracellular adenosine signaling in liver protection. Based on the notion that extracellular adenosine signaling is terminated by uptake from the extracellular towards the intracellular compartment by way of equilibrative nucleoside transporters (ENTs), we hypothesized a functional role of ENTs in liver protection from ischemia. During orthotopic liver transplantation in humans, we observed higher expressional levels of ENT1 than ENT2, in conjunction with repression of ENT1 and ENT2 transcript and protein levels following warm ischemia and reperfusion. Treatment with the pharmacologic ENT inhibitor dipyridamole revealed elevations of hepatic adenosine levels and robust liver protection in a murine model of liver ischemia and reperfusion. Studies in gene-targeted mice for Ent1 or Ent2 demonstrated selective protection from liver injury in Ent1(-/-) mice. Treatment with selective adenosine receptor antagonists indicated a contribution of Adora2b receptor signaling in ENT-dependent liver protection.

Conclusion: These findings implicate ENT1 in liver protection from ischemia and reperfusion injury and suggest ENT inhibitors may be of benefit in the prevention or treatment of ischemic liver injury.

Figure 1

ENT1 and ENT2 expression in biopsies of human liver transplants following hepatic ischemia and reperfusion. (A) First liver biopsy was taken during ischemia (I) at the conclusion of cold ischemia time (CIT) during back table preparation of the cadaveric liver allograft. A second biopsy was taken during reperfusion (R) after warm ischemia time (WIT) and reperfusion time (RT) immediately prior to closure of the abdomen following drain placement. (B) ENT1 and ENT2 transcript levels in biopsies of human liver transplants during cold ischemia (I) and after reperfusion (R); (n=3–4 independent experiments). (C) ENT1 and ENT2 protein levels (one representative blot of two is shown).

Figure 2

ENT1 and ENT2 expression in murine livers following hepatic ischemia and reperfusion. (A) The left lobe of the liver was exposed to 45min of ischemia followed by 2h of reperfusion. (B) Ent1 and Ent2 transcript levels in left livers exposed to Sham operation (0) or 45min of ischemia followed by 2h reperfusion (n=3–4 independent experiments). (C) Ent1 and Ent2 protein levels (β-actin to control for loading conditions; one representative blot of three is shown).

Figure 3

Ischemic liver injury following inhibition of adenosine transporters during hepatic ischemia and reperfusion. (A) Experimental set-up to study liver ischemia and reperfusion with (black bar) and without (white bar) dipyridamole (DIP) treatment (0.25mg/25g mouse IV). (B) Liver adenosine content measured immediately following 45 min of liver ischemia with or without DIP treatment. Wild-type mice with or without DIP treatment were exposed to (C,D) 45 min of liver ischemia and 2h of reperfusion or (E,F) prolonged reperfusion (24h) before liver function was assessed by measurement of AST, ALT, liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4); (n=4–6).

Figure 4

Ischemic liver injury in mice gene-targeted for the equilibrative nucleoside transporter 1. (A) Liver adenosine content measured immediately following 45 min of liver ischemia in Ent1^−/− mice (white bar) or littermate controls (black bar). (B–E) Ent1^−/− mice or littermate controls were exposed to 45 min of liver ischemia and 2h of reperfusion before liver function was assessed by measurement of (B) AST, ALT, (C) liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4); (n=4–6); (D) liver inflammation was assessed by measurement of IFNγ, IL-6 protein levels and neutrophil marker myeloperoxidase (MPO) in livers and (E) lung inflammation was assessed by measuring IFNγ, IL-6 and MPO in lungs. (F,G) Ent1^−/− mice or littermate controls were exposed to 45 min of liver ischemia and 24h of reperfusion before liver function was assessed by measurement of AST, ALT, liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4); (n=4–6).

Figure 5

Ischemic liver injury in mice gene-targeted for the equilibrative nucleoside transporter 2. (A) Liver adenosine content measured immediately following 45 min of liver ischemia in Ent2^−/− mice (white bar) or littermate controls (black bar). (B–E) Ent2^−/− mice or littermate controls were exposed to 45 min of liver ischemia and 2h of reperfusion before liver function was assessed by measurement of (B) AST, ALT, (C) liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4); (n=4–6); (D) liver inflammation was assessed by measurement of IFNγ, IL-6 protein levels and neutrophil marker myeloperoxidase (MPO) in livers and (E) lung inflammation was assessed by measuring IFNγ, IL-6 and MPO in lungs. (F,G) Ent2^−/− mice or littermate controls were exposed to 45 min of liver ischemia and 24h of reperfusion before liver function was assessed by measurement of AST, ALT, liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4); (n=4–6).

Figure 6

Role of adenosine signaling in Ent1^−/− mice. (A,B) Ent1^−/− mice were treated with an Adora2a specific antagonist ZM241385 (2mg/kg IV, white bar) or vehicle (black bar) and subsequently exposed to 45 min of liver ischemia and 24h of reperfusion before liver function was assessed by measurement of AST, ALT, liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4). Ent1^−/− mice were treated with an Adora2b specific antagonist PSB1115 (0.5 mg/25g mouse IV, white bar) or vehicle (black bar) and subsequently exposed to (C,D) 45 min of liver ischemia and 2h of reperfusion or (E,F) prolonged reperfusion (24h) before liver function was assessed by measurement of AST, ALT, liver histology (200-fold magnification; 1 of 3 representative slides is shown) and quantification of histologic tissue injury (Suzuki Scoring Index, 0–4).

Figure 7. Role of HIF1γ in regulating hepatic ENT and Adora receptor expression

(A) HIF1γ expression in isolated hepatocytes of Adora2b^loxP/loxP Albumin Cre+ and Albumin Cre+ control mice with and without liver ischemia. (B) Ent1 and Ent2 transcript levels in left livers of Adora2b^loxP/loxP Albumin Cre+ and Albumin Cre+ control mice exposed to Sham operation (-I) or 45min of ischemia (+I) followed by 2h reperfusion (n=3–4 independent experiments). (C) Adora1, Adora2a, Adora2b and Adora3 transcript levels in left livers of Adora2b^loxP/loxP Albumin Cre+ and Albumin Cre+ control mice exposed to Sham operation (-I) or 45min of ischemia (+I) followed by 2h reperfusion (n=3–4 independent experiments).