This renewal application focuses on studying the efficacy of nitric oxide (NO) supplementation as a treatment for the vascular complications in argininosuccinic aciduria (ASA) caused by deficiency of argininosuccinate lyase (ASL), the second most common urea cycle disorder and a human model of nitric oxide (NO) deficiency. ASL serves distinct catalytic vs. structural functions that provide a structural model for understanding why ASA patients defy the arginine paradox, i.e., they are unable to generate NO efficiently in spite of supplemental arginine therapy. In a proof-of-principle case study, we successfully treated an ASA patient with severe hypertension refractory to multiple antihypertensive medications with NO supplementation. Our data imply that NO deficiency at the tissue level contributes to the complications of ASA and preventing hyperammonemia alone may not prevent long-term morbidity. To translate this discovery into a new treatment strategy, we propose to answer the following questions:
Aim 1. Can NO supplementation be used to treat endothelial dysfunction in patients with ASA? We will perform a double-blind, placebo-controlled, cross over study in patients with ASA. We hypothesize that a subset of ASA patients will demonstrate endothelial dysfunction due to lack of NOS-dependent NO production from the vascular endothelium and that supplementation with an NOS-independent NO source such as low dose sodium nitrite via the nutriceutical Neo40(R) would correct this defect.
Aim 2. Can we develop in vitro assays that predict in vivo clinical response to NO supplementation and clinical variability? We will determine the ability of ASA trial patient fibroblasts to produce NO as measured by nitrite and nitrosothiols and correlate the clinical response and phenotype in Aim 1 with in vitro production of NO in response to arginine supplementation.
Aim 3. What is the mechanistic basis of NO regulation by the ASL/NOS complex? NO synthesis may be regulated in part by the participation of NOS in both positive and negative regulatory complexes. To explore the mechanisms that regulate these structural complexes, we will test the hypothesis that NO production is controlled by a balance of negative regulatory complex involving NOS and caveolin vs. a positive, synthetic complex involving NOS and ASL. The insights from these studies may offer a paradigm for studying the diverse biological effects of NO in other disease contexts. While the pathogenesis of ASA is complex and may extend to other arginine- dependent mechanisms, our preclinical and clinical studies support the dominant contribution of NO deficiency on the endpoints tested in this study. Moreover, the approaches and mechanisms proposed here may be broadly application to dysregulation of NO in other disease states.

Public Health Relevance

This project will test whether supplementation with a nutriceutical source of nitric oxide (inorganic nitrite) can be used to treat the vascular dysfunction in patients with argininosuccinic aciduria due to deficiency of argininosuccinate lyase (ASL). We will determine whether mutations that lead to loss of ASL confers increased risk of NO deficiency and response to this treatment by studying the cells of patients. Finally we will determine how ASL controls NO production in response to high demand.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Therapeutic Approaches to Genetic Diseases (TAG)
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Leschek, Ellen W
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Baylor College of Medicine
Schools of Medicine
United States
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Kho, Jordan; Tian, Xiaoyu; Wong, Wing-Tak et al. (2018) Argininosuccinate Lyase Deficiency Causes an Endothelial-Dependent Form of Hypertension. Am J Hum Genet 103:276-287
Madan, Simran; Kron, Bettina; Jin, Zixue et al. (2018) Arginase overexpression in neurons and its effect on traumatic brain injury. Mol Genet Metab 125:112-117
Hook, Debra; Diaz, George A; Lee, Brendan et al. (2016) Protein and calorie intakes in adult and pediatric subjects with urea cycle disorders participating in clinical trials of glycerol phenylbutyrate. Mol Genet Metab Rep 6:34-40
Burrage, Lindsay C; Sun, Qin; Elsea, Sarah H et al. (2015) Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency. Hum Mol Genet 24:6417-27
Nagamani, Sandesh C S; Diaz, George A; Rhead, William et al. (2015) Self-reported treatment-associated symptoms among patients with urea cycle disorders participating in glycerol phenylbutyrate clinical trials. Mol Genet Metab 116:29-34
Lee, Brendan; Diaz, George A; Rhead, William et al. (2015) Blood ammonia and glutamine as predictors of hyperammonemic crises in patients with urea cycle disorder. Genet Med 17:561-8
Burrage, Lindsay C; Jain, Mahim; Gandolfo, Laura et al. (2014) Sodium phenylbutyrate decreases plasma branched-chain amino acids in patients with urea cycle disorders. Mol Genet Metab 113:131-5
Berry, Susan A; Lichter-Konecki, Uta; Diaz, George A et al. (2014) Glycerol phenylbutyrate treatment in children with urea cycle disorders: pooled analysis of short and long-term ammonia control and outcomes. Mol Genet Metab 112:17-24
Posey, Jennifer E; Burrage, Lindsay C; Miller, Marcus J et al. (2014) Lysinuric Protein Intolerance Presenting with Multiple Fractures. Mol Genet Metab Rep 1:176-183