Nitric-oxide supplementation for treatment of long-term complications in argininosuccinic aciduria

Abstract

Argininosuccinate lyase (ASL) is required for the synthesis and channeling of L-arginine to nitric oxide synthase (NOS) for nitric oxide (NO) production. Congenital ASL deficiency causes argininosuccinic aciduria (ASA), the second most common urea-cycle disorder, and leads to deficiency of both ureagenesis and NO production. Subjects with ASA have been reported to develop long-term complications such as hypertension and neurocognitive deficits despite early initiation of therapy and the absence of documented hyperammonemia. In order to distinguish the relative contributions of the hepatic urea-cycle defect from those of the NO deficiency to the phenotype, we performed liver-directed gene therapy in a mouse model of ASA. Whereas the gene therapy corrected the ureagenesis defect, the systemic hypertension in mice could be corrected by treatment with an exogenous NO source. In an ASA subject with severe hypertension refractory to antihypertensive medications, monotherapy with NO supplements resulted in the long-term control of hypertension and a decrease in cardiac hypertrophy. In addition, the NO therapy was associated with an improvement in some neuropsychological parameters pertaining to verbal memory and nonverbal problem solving. Our data show that ASA, in addition to being a classical urea-cycle disorder, is also a model of congenital human NO deficiency and that ASA subjects could potentially benefit from NO supplementation. Hence, NO supplementation should be investigated for the long-term treatment of this condition.

Figure 1

Liver-Directed Gene Transfer Corrects the Metabolic Defect in ASA Mice A total of 23 ASA mice were randomized to receive either gene therapy (GT) or a placebo. Triple therapy was withdrawn at 5 weeks of life so that the long-term effects of GT alone could be assessed. The error bars in the bar and line graphs depict standard deviation. (A) Structure of the helper-dependent adenoviral construct with the murine Asl cDNA under the regulation of the ApoE promoter. The following abbreviations are used: ITR, inverted terminal repeat; WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element; LCR, locus control region; and GhpA, growth hormone polyA. (B) A representative immunoblot of liver compares the ASL protein levels between three WT mice, three placebo-treated ASA mice, and three GT-treated ASA mice 2 weeks after GT. Densitometry quantification shows the correction of ASL protein levels. Asterisks indicate p < 0.05. The following abbreviation is used: n.s., not significant. (C) Representative ASL enzyme activity. Compared with untreated mice (n = 3), ASA GT-treated mice (n = 3) show correction of enzymatic activity in the liver. Asterisks indicate p < 0.05. The following abbreviation is used: n.s., not significant. (D) Amino acid profile of ASA mice (n = 4) versus GT-treated ASA mice (n = 4) at 8 weeks of age showing biochemical correction. Asterisks indicate p < 0.05. The following abbreviations are used: CIT, Citrulline; and ASA, argininosuccinic acid. (E) GT-treated ASA mice (n = 15) demonstrate significantly better growth than those treated with the placebo (n = 8). Asterisks indicate p < 0.05. (F) Kaplan-Meier survival curve. GT-treated ASA mice (n = 15) show 100% survival by week 16 of life, whereas all placebo-treated ASA mice (n = 8) expired by day 103. Asterisks indicate p < 0.001.

Figure 2

Tissue-Specific NO Deficiency and Hypertension Despite Correction of the Metabolic Defect in the Liver of GT-Treated ASA Mice (A) Invasive BP measurements at age 12–14 weeks (n = 3 per group) showed that GT-treated ASA mice had elevated systolic and diastolic BPs when they were compared with WT mice. Asterisks indicate p < 0.05. (B) RSNO levels measured in liver and hearts of six GT-treated ASA mice and seven WT mice showed significantly higher RSNO levels in the livers and significantly lower levels in the hearts of the GT-treated ASA mice. Asterisks indicate p < 0.05. (C) Preconstricted WT aortic rings relaxed in a concentration-dependent fashion with acetylcholine (Ach) treatment beginning at a concentration of 10 nM, wheras aortic rings from GT-treated ASA mice were unresponsive to Ach. Data points are derived from measurements from four segments of each aorta in three WT and three GT-treated mice ASA mice. The asterisk indicates p < 0.05. (D) Representative tracing of aortic-ring isometric tension in GT-treated ASA mice shows no relaxation in response to Ach or L-arginine, whereas NO donor sodium nitroprusside (SNP) leads to a significant relaxation. Three aortic rings from each mouse aorta were used for analysis. (E) In vivo vascular response to nitrite infusion in 4-month-old GT-treated ASA mice (n = 3) shows normalization of BP. Asterisks indicate p < 0.05. The following abbreviation is used: n.s., not significant. The error bars in the bar and line graphs depict standard deviation.

Figure 3

NOS Uncoupling and Increased Free-Radical Levels in ASA Mice (A) Immunoblot and densitometric quantification from the aortae of three ASA mice and three WT mice show a decrease in the active dimer form in the ASA mice, supporting NOS3 (eNOS) uncoupling. The asterisk indicates p < 0.05. (B) Isoprostane levels, a marker of free-radical production, are significantly increased in ASA mice. Asterisks indicate p < 0.05. (C) Compared to those of WT mice (n = 5), superoxide levels from the aortae of ASA mice (n = 5) are elevated. The asterisk indicates p < 0.05. (D) In vivo isotope measurement of the transfer of the guanidino-nitrogen from ¹⁵N₂-arginine to ¹⁵N-citrulline, a marker of NO flux, is decreased in the GT-treated ASA mice (n = 5 per group). The asterisk indicates p < 0.05. (E) Compared to those of WT mice, the BH₄/BH₂ ratios in the aortae of ASA hypomorphic mice and GT-treated ASA hypomorphic mice are not significantly different. The following abbreviation is used: n.s., not significant. The error bars depict standard deviation.

Figure 4

Clinical Treatment with NO Supplements Corrects Hypertension in a Subject with ASA The vertical box plots depict the 25^th and 75^th percentiles along with median blood-pressure values at various time points. The error bars depict the 5^th and 95^th percentiles. The red shaded area depicts the 50^th and 90^th blood percentiles for age and stature. Median BP values that were above the 90^th percentile prior to initiation of organic-nitrate therapy in spite of combination antihypertensive therapy (left panel) show sustained normalization with NO supplementation therapy; this sustained normalization allowed for the withdrawal of all other antihypertensive medications (middle and right panels).

Figure 5

Neuropsychological Testing Results before and after NO Supplementation (A) California Verbal Learning Test-Children's Version. Raw scores show a significant decrease in intrusions and a mild increase in the other parameters. Aside from the intrusion score, higher scores on the test indicate improved performance. (B) Children's Memory Scale: Stories Subtest Scaled Scores show improvement in scores after treatment. (C) Tower of London-Drexel University measurements show significant improvement in nonverbal problem solving with treatment (higher scores indicate poorer performance).