This proposal entails a comprehensive examination of the mechanism(s) controlling renal amino acid metabolism, ammoniagenesis and gluconeogenesis in various acid base states. The work will address five primary themes: (1) the metabolic signals initiating augmented ammoniagenesis in acidosis; (2) the rate limiting metabolic pathway(s) for ammonia production; (3) the role of effectors and/or inhibitors of ammoniagenesis on the underlying biochemistry; (4) the putative relationship between renal ammonia and glucose formation; (5) the biochemical adaptation occurring both during the induction of and recovery from acidosis and alkalosis. The core hypothesis of the work is that diminished flux through the citrate synthetase pathway is an initial step in a coordinated sequence of events leading to an increased rate of ammoniagenesis in acidosis. A corollary hypothesis is that the diminution of the alpha-ketoglutarate pool secondary to reduced flux through citrate synthetase is accompanied by enhanced flux through glutamate dehydrogenase toward oxidative deamination of glutamate with the release of NH3. We also will explore the possibility that the augmentation of ammoniagenesis and gluconeogenesis in acidosis are (or are not) inter-dependent phenomenon subject to common metabolic signals and controls, with the increase of glucose production providing an outlet for glutamate carbon. The significance of diminished flux through the glutamine synthetase reaction in abetting HN3 production will be studied as well. These topics will examined by incubating isolated rat renal tubules with 15N and/or 13C labelled precursors in the presence and absence of metabolic modulators and/or inhibitors. Subsequent determination of important precursor-product relationships and flux rates will be made using gas chromatography-mass spectrometry and/or nuclear magnetic resonance spectroscopy. The data so obtained will be of scientific import by deepening our understanding of renal ammoniagenesis in response to perturbations of hydrogen ion homeostasis. The project will evaluate such adaptations in terms of the stage of acidosis or alkalosis, i.e., during the induction of or recovery from either state.
