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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

MAPK activation changes after enrichment of IGL-1 solution with CA II.Western blotting and densitometric analysis of p-38 (A); p-ERK (B) and p-JNK (C) in steatotic liver after 120 min of normothermic “ex vivo” perfusion. CA II addition to IGL-1 solution prevented MAPK activation. Ctr 2: Liver flushed and perfused “ex-vivo” without cold preservation; IGL-1: liver preserved in IGL-1 solution (4°C, 24 h) and subjected to 2h-normothermic “ex vivo” perfusion. IGL-1+CAII: liver preserved in IGL-1 solution (4°C, 24 h) enriched with CA II and subjected to 2h- normothermic “ex vivo” perfusion. * p < 0.05 vs Ctr 2; # p < 0.05 vs IGL-1.
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pone.0134499.g005: MAPK activation changes after enrichment of IGL-1 solution with CA II.Western blotting and densitometric analysis of p-38 (A); p-ERK (B) and p-JNK (C) in steatotic liver after 120 min of normothermic “ex vivo” perfusion. CA II addition to IGL-1 solution prevented MAPK activation. Ctr 2: Liver flushed and perfused “ex-vivo” without cold preservation; IGL-1: liver preserved in IGL-1 solution (4°C, 24 h) and subjected to 2h-normothermic “ex vivo” perfusion. IGL-1+CAII: liver preserved in IGL-1 solution (4°C, 24 h) enriched with CA II and subjected to 2h- normothermic “ex vivo” perfusion. * p < 0.05 vs Ctr 2; # p < 0.05 vs IGL-1.

Mentions: Since AMPK activation can affect ERS, a common characteristic of IRI [22–24], we further examined glucose-regulated/binding immunoglobulin protein (GRP78) which is known to be induced by ERS. We found that GRP78 was upregulated in IGL-1 group when compared to control group and that supplementation of IGL-1 with CA II resulted in attenuation of GRP78. Then, we examined signaling pathways of the unfolded protein response (UPR) involved in CA II protection. Our results show that CAII significantly decrease PKR-like ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α) and eukaryotic translation initiation factor 2 (eIF2α) activation (Fig 5B, 5C and 5D respectively). Remarkably, transcription factor 6 (ATF6) did not result in a significant activation after ischemia reperfusion (Fig 5E). It is well-known that ERS is capable to induce an apoptogenic program through activation of DNA damage-inducible gene 153/C/EBP homologous protein (GADD153/CHOP). For this reason, we assessed CHOP profile by immunohistochemistry. Interestingly, no differences between the three groups were found (Fig 5F).


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)

MAPK activation changes after enrichment of IGL-1 solution with CA II.Western blotting and densitometric analysis of p-38 (A); p-ERK (B) and p-JNK (C) in steatotic liver after 120 min of normothermic “ex vivo” perfusion. CA II addition to IGL-1 solution prevented MAPK activation. Ctr 2: Liver flushed and perfused “ex-vivo” without cold preservation; IGL-1: liver preserved in IGL-1 solution (4°C, 24 h) and subjected to 2h-normothermic “ex vivo” perfusion. IGL-1+CAII: liver preserved in IGL-1 solution (4°C, 24 h) enriched with CA II and subjected to 2h- normothermic “ex vivo” perfusion. * p < 0.05 vs Ctr 2; # p < 0.05 vs IGL-1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0134499.g005: MAPK activation changes after enrichment of IGL-1 solution with CA II.Western blotting and densitometric analysis of p-38 (A); p-ERK (B) and p-JNK (C) in steatotic liver after 120 min of normothermic “ex vivo” perfusion. CA II addition to IGL-1 solution prevented MAPK activation. Ctr 2: Liver flushed and perfused “ex-vivo” without cold preservation; IGL-1: liver preserved in IGL-1 solution (4°C, 24 h) and subjected to 2h-normothermic “ex vivo” perfusion. IGL-1+CAII: liver preserved in IGL-1 solution (4°C, 24 h) enriched with CA II and subjected to 2h- normothermic “ex vivo” perfusion. * p < 0.05 vs Ctr 2; # p < 0.05 vs IGL-1.
Mentions: Since AMPK activation can affect ERS, a common characteristic of IRI [22–24], we further examined glucose-regulated/binding immunoglobulin protein (GRP78) which is known to be induced by ERS. We found that GRP78 was upregulated in IGL-1 group when compared to control group and that supplementation of IGL-1 with CA II resulted in attenuation of GRP78. Then, we examined signaling pathways of the unfolded protein response (UPR) involved in CA II protection. Our results show that CAII significantly decrease PKR-like ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α) and eukaryotic translation initiation factor 2 (eIF2α) activation (Fig 5B, 5C and 5D respectively). Remarkably, transcription factor 6 (ATF6) did not result in a significant activation after ischemia reperfusion (Fig 5E). It is well-known that ERS is capable to induce an apoptogenic program through activation of DNA damage-inducible gene 153/C/EBP homologous protein (GADD153/CHOP). For this reason, we assessed CHOP profile by immunohistochemistry. Interestingly, no differences between the three groups were found (Fig 5F).

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