Ammonia detoxification via ureagenesis in rat hepatocytes involves mitochondrial aquaporin-8 channels

Authors

  • Leandro R. Soria,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
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  • Julieta Marrone,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
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  • Giuseppe Calamita,

    1. Dipartimento di Bioscienze, Biotecnologie e Scienze Farmacologiche, Università degli Studi di Bari Aldo Moro, Bari, Italy
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  • Raúl A. Marinelli

    Corresponding author
    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
    • Instituto de Fisiología Experimental. Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina
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    • fax: (54)-341-4399473.


  • Potential conflict of interest: Nothing to report.

  • Supported by Grant PICT 05-0946 from Agencia Nacional de Promoción Científica y Tecnológica and Grant PIP 01563 from Consejo Nacional de Investigaciones Científicas y Técnicas (to R. A. M.).

Abstract

Hepatocyte mitochondrial ammonia detoxification via ureagenesis is critical for the prevention of hyperammonemia and hepatic encephalopathy. Aquaporin-8 (AQP8) channels facilitate the membrane transport of ammonia. Because AQP8 is expressed in hepatocyte inner mitochondrial membranes (IMMs), we studied whether mitochondrial AQP8 (mtAQP8) plays a role in ureagenesis from ammonia. Primary cultured rat hepatocytes were transfected with small interfering RNAs (siRNAs) targeting two different regions of the rat AQP8 molecule or with scrambled control siRNA. After 48 hours, the levels of mtAQP8 protein decreased by approximately 80% (P < 0.05) without affecting cell viability. mtAQP8 knockdown cells in the presence of ammonium chloride showed a decrease in ureagenesis of approximately 30% (P < 0.05). Glucagon strongly stimulated ureagenesis in control hepatocytes (+120%, P < 0.05) but induced no significant stimulation in mtAQP8 knockdown cells. Contrarily, mtAQP8 silencing induced no significant change in basal and glucagon-induced ureagenesis when glutamine or alanine was used as a source of nitrogen. Nuclear magnetic resonance studies using 15N-labeled ammonia confirmed that glucagon-induced 15N-labeled urea synthesis was markedly reduced in mtAQP8 knockdown hepatocytes (−90%, P < 0.05). In vivo studies in rats showed that under glucagon-induced ureagenesis, hepatic mtAQP8 protein expression was markedly up-regulated (+160%, P < 0.05). Moreover, transport studies in liver IMM vesicles showed that glucagon increased the diffusional permeability to the ammonia analog [14C]methylamine (+80%, P < 0.05). Conclusion: Hepatocyte mtAQP8 channels facilitate the mitochondrial uptake of ammonia and its metabolism into urea, mainly under glucagon stimulation. This mechanism may be relevant to hepatic ammonia detoxification and in turn, avoid the deleterious effects of hyperammonemia. (HEPATOLOGY 2013)

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