HUMAN AQUAPORIN-8 (hAQP8) TRANSDUCTION STIMULATES AMMONIA DETOXIFICATION IN PRIMARY HEPATOCYTE CULTURE

MARRONE, JULIETA; DANIELLI MAURO; MARINELLI, RAÚL A.; CAPIGLIONI ALEJO; ALVAREZ MARIA DE LUJAN

Buenos Aires

Congreso; Congreso Sociedades Biociencias; 2017

Sociedad Argentina de Investigación Clínica (SAIC), Sociedad Argentina de Fisiología (SAFIS), SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR, SOCIEDAD ARGENTINA DE INMUNOLOGÍA

The ammonia detoxification is crucial to prevent the development of pathological states as hyperammonemia and encephalopathy. In previous gene silencing studies, our laboratory has demonstrated the critical role that hepatocyte mitochondrial AQP8 plays in the urea cycle, specifically by promoting ammonia uptake. Aim: to study the effect of hAQP8 transduction in hepatocytes on ammonia detoxification via ureagenesis. Methods and Results: We made use of the adenovector AdhAQP8 which was designed by us encoding hAQP8 and containing EGFP as reporter gene. An adenovector encoding for EGFP alone was used as control. Primary culture rat hepatocytes were exposed to AdhAQP8 or control vector. After 48 h, hAQP8 was properly expressed in inner mitochondrial membranes as assessed by subcellular fractionation and immunoblotting. The transduction process did not alter LDH leakage or cell morphology. When urea synthesis was augmented in transduced hepatocytes by incubation with increasing concentrations of NH4Cl (0, 0.5, 1.0, and 1.5 mM), it was observed that ureagenesis from ammonia significantly increased by about 50% (P < 0.02) in hAQP8-transduced cells. For these studies, urea was assessed by a non-enzymatic spectrophotometric assay (Abnova urea assay kit). Nuclear magnetic resonance studies using 15N-labeled ammonia confirmed that 15N-labeled urea synthesis was markedly increased in hAQP8-transduced hepatocytes (+180%, P < 0.001). Conclusion: The mitochondrial expression of hAQP8 in rat hepatocytes significantly increased ammonia detoxification by ureagenesis. This finding might have potential therapeutic implications for hyperammonemia disorders.