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Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage.

Bejaoui M, Pantazi E, De Luca V, Panisello A, Folch-Puy E, Hotter G, Capasso C, T Supuran C, Roselló-Catafau J, Rosselló-Catafau J - PLoS ONE (2015)

Bottom Line: In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined.In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation.Interestingly, CA II supplementation was not associated with enhanced CO2 hydration.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pathology, Institute of Biomedical Research of Barcelona-Spanish National Research Council (IIBB-CSIC), IDIBAPS, Barcelona, Spain.

ABSTRACT
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.

No MeSH data available.


Related in: MedlinePlus

Hepatic injury and CA II expression and activity after cold ischemia.(A) Hepatic injury after cold ischemia in IGL-1 solution supplemented or not with CA II, measured as AST and ALT levels. The presence of CA II in IGL-1 significantly reduced AST/ALT levels; (B) CA II immunohistochemistry: CA II staining was reduced after 24h-cold storage in IGL-1 solution (IGL-1). CA II staining increased in IGL-1+CAII group compared to IGL-1 group; (C) CA II protein expression by Western blotting and densitometric analyses. A fall in CA II expression was observed after 24 h-cold ischemia in IGL-1 solution, which was slightly reversed after CA II supplementation of IGL-1 solution; (D) Quantitative CA II mRNA expression in steatotic livers preserved in IGL-1 or IGL-1+CAII. CA II mRNA levels presented similar reductions in both IGL-1 solutions compared to controls; (E) CA II activity levels in fatty livers preserved in IGL-1 or IGL-1+CAII. CA II activity decreased in livers preserved in IGL-1 solutions compared to controls; (F) ATP quantitation: ATP levels decrease significantly after cold storage. Ctr 1: liver flushed without cold preservation; IGL-1: liver preserved in IGL-1 solution; IGL-1+CAII: liver preserved in IGL-1 solution enriched with CA II. * p < 0.05 vs Ctr 1; # p < 0.05 vs IGL-1.
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pone.0134499.g001: Hepatic injury and CA II expression and activity after cold ischemia.(A) Hepatic injury after cold ischemia in IGL-1 solution supplemented or not with CA II, measured as AST and ALT levels. The presence of CA II in IGL-1 significantly reduced AST/ALT levels; (B) CA II immunohistochemistry: CA II staining was reduced after 24h-cold storage in IGL-1 solution (IGL-1). CA II staining increased in IGL-1+CAII group compared to IGL-1 group; (C) CA II protein expression by Western blotting and densitometric analyses. A fall in CA II expression was observed after 24 h-cold ischemia in IGL-1 solution, which was slightly reversed after CA II supplementation of IGL-1 solution; (D) Quantitative CA II mRNA expression in steatotic livers preserved in IGL-1 or IGL-1+CAII. CA II mRNA levels presented similar reductions in both IGL-1 solutions compared to controls; (E) CA II activity levels in fatty livers preserved in IGL-1 or IGL-1+CAII. CA II activity decreased in livers preserved in IGL-1 solutions compared to controls; (F) ATP quantitation: ATP levels decrease significantly after cold storage. Ctr 1: liver flushed without cold preservation; IGL-1: liver preserved in IGL-1 solution; IGL-1+CAII: liver preserved in IGL-1 solution enriched with CA II. * p < 0.05 vs Ctr 1; # p < 0.05 vs IGL-1.

Mentions: Transaminase levels were measured in the effluent of washout liquid (Ringer’s lactate) after cold storage. Livers preserved in IGL-1 preservation solution showed high ALT and AST levels, whereas the addition of CA II to IGL-1 solution (IGL-1+CAII) significantly reduced transaminase levels (Fig 1A). Next, we studied the CA II profiles obtained by immunohistochemistry and Western blot techniques. Livers preserved in IGL-1 solution for 24 hours showed a significant decrease in CA II staining when compared to control livers (Fig 1B). This situation was reversed by CA II addition to IGL-1 solution, as revealed by the increased CA II staining in the IGL+CAII group. These results agreed with those obtained by Western blot analysis. In fact, CA II protein expression decreased significantly after cold storage and increased after the addition of CA II to IGL-1 solution (Fig 1C). Next, we examined the mRNA levels of CA II (Fig 1D). We found that CA II mRNA expression decreased after cold storage and the addition of CA II did not affect mRNA expression. Finally, we analyzed CA II activity and observed that preservation of fatty livers in IGL-1 solution resulted in decreased CA II activity, which slightly increased after CA II addition (Fig 1E). These results were consistent with the Western blot and immunochemistry findings. Finally, we assessed ATP content in fatty liver submitted to prolonged cold storage. As expected, cold ischemia was associated with marked decrease in liver ATP content and addition of bovine CA II did not enhance ATP level.


Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage.

Bejaoui M, Pantazi E, De Luca V, Panisello A, Folch-Puy E, Hotter G, Capasso C, T Supuran C, Roselló-Catafau J, Rosselló-Catafau J - PLoS ONE (2015)

Hepatic injury and CA II expression and activity after cold ischemia.(A) Hepatic injury after cold ischemia in IGL-1 solution supplemented or not with CA II, measured as AST and ALT levels. The presence of CA II in IGL-1 significantly reduced AST/ALT levels; (B) CA II immunohistochemistry: CA II staining was reduced after 24h-cold storage in IGL-1 solution (IGL-1). CA II staining increased in IGL-1+CAII group compared to IGL-1 group; (C) CA II protein expression by Western blotting and densitometric analyses. A fall in CA II expression was observed after 24 h-cold ischemia in IGL-1 solution, which was slightly reversed after CA II supplementation of IGL-1 solution; (D) Quantitative CA II mRNA expression in steatotic livers preserved in IGL-1 or IGL-1+CAII. CA II mRNA levels presented similar reductions in both IGL-1 solutions compared to controls; (E) CA II activity levels in fatty livers preserved in IGL-1 or IGL-1+CAII. CA II activity decreased in livers preserved in IGL-1 solutions compared to controls; (F) ATP quantitation: ATP levels decrease significantly after cold storage. Ctr 1: liver flushed without cold preservation; IGL-1: liver preserved in IGL-1 solution; IGL-1+CAII: liver preserved in IGL-1 solution enriched with CA II. * p < 0.05 vs Ctr 1; # p < 0.05 vs IGL-1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520486&req=5

pone.0134499.g001: Hepatic injury and CA II expression and activity after cold ischemia.(A) Hepatic injury after cold ischemia in IGL-1 solution supplemented or not with CA II, measured as AST and ALT levels. The presence of CA II in IGL-1 significantly reduced AST/ALT levels; (B) CA II immunohistochemistry: CA II staining was reduced after 24h-cold storage in IGL-1 solution (IGL-1). CA II staining increased in IGL-1+CAII group compared to IGL-1 group; (C) CA II protein expression by Western blotting and densitometric analyses. A fall in CA II expression was observed after 24 h-cold ischemia in IGL-1 solution, which was slightly reversed after CA II supplementation of IGL-1 solution; (D) Quantitative CA II mRNA expression in steatotic livers preserved in IGL-1 or IGL-1+CAII. CA II mRNA levels presented similar reductions in both IGL-1 solutions compared to controls; (E) CA II activity levels in fatty livers preserved in IGL-1 or IGL-1+CAII. CA II activity decreased in livers preserved in IGL-1 solutions compared to controls; (F) ATP quantitation: ATP levels decrease significantly after cold storage. Ctr 1: liver flushed without cold preservation; IGL-1: liver preserved in IGL-1 solution; IGL-1+CAII: liver preserved in IGL-1 solution enriched with CA II. * p < 0.05 vs Ctr 1; # p < 0.05 vs IGL-1.
Mentions: Transaminase levels were measured in the effluent of washout liquid (Ringer’s lactate) after cold storage. Livers preserved in IGL-1 preservation solution showed high ALT and AST levels, whereas the addition of CA II to IGL-1 solution (IGL-1+CAII) significantly reduced transaminase levels (Fig 1A). Next, we studied the CA II profiles obtained by immunohistochemistry and Western blot techniques. Livers preserved in IGL-1 solution for 24 hours showed a significant decrease in CA II staining when compared to control livers (Fig 1B). This situation was reversed by CA II addition to IGL-1 solution, as revealed by the increased CA II staining in the IGL+CAII group. These results agreed with those obtained by Western blot analysis. In fact, CA II protein expression decreased significantly after cold storage and increased after the addition of CA II to IGL-1 solution (Fig 1C). Next, we examined the mRNA levels of CA II (Fig 1D). We found that CA II mRNA expression decreased after cold storage and the addition of CA II did not affect mRNA expression. Finally, we analyzed CA II activity and observed that preservation of fatty livers in IGL-1 solution resulted in decreased CA II activity, which slightly increased after CA II addition (Fig 1E). These results were consistent with the Western blot and immunochemistry findings. Finally, we assessed ATP content in fatty liver submitted to prolonged cold storage. As expected, cold ischemia was associated with marked decrease in liver ATP content and addition of bovine CA II did not enhance ATP level.

Bottom Line: In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined.In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation.Interestingly, CA II supplementation was not associated with enhanced CO2 hydration.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pathology, Institute of Biomedical Research of Barcelona-Spanish National Research Council (IIBB-CSIC), IDIBAPS, Barcelona, Spain.

ABSTRACT
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.

No MeSH data available.


Related in: MedlinePlus