Glycolate Oxidase Is a Safe and Efficient Target for Substrate Reduction Therapy in a Mouse Model of Primary Hyperoxaluria Type I

Abstract

Primary hyperoxaluria type 1 (PH1) is caused by deficient alanine-glyoxylate aminotransferase, the human peroxisomal enzyme that detoxifies glyoxylate. Glycolate is one of the best-known substrates leading to glyoxylate production, via peroxisomal glycolate oxidase (GO). Using genetically modified mice, we herein report GO as a safe and efficient target for substrate reduction therapy (SRT) in PH1. We first generated a GO-deficient mouse (Hao1(-/-)) that presented high urine glycolate levels but no additional phenotype. Next, we produced double KO mice (Agxt1(-/-) Hao1(-/-)) that showed low levels of oxalate excretion compared with hyperoxaluric mice model (Agxt1(-/-)). Previous studies have identified some GO inhibitors, such as 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST). We herein report that CCPST inhibits GO in Agxt1(-/-) hepatocytes and significantly reduces their oxalate production, starting at 25 µM. We also tested the ability of orally administered CCPST to reduce oxalate excretion in Agxt1(-/-) mice, showing that 30-50% reduction in urine oxalate can be achieved. In summary, we present proof-of-concept evidence for SRT in PH1. These encouraging results should be followed by a medicinal chemistry programme that might yield more potent GO inhibitors and eventually could result in a pharmacological treatment for this rare and severe inborn error of metabolism.

Figure 1

Targeted mutagenesis of the mouse Hao1 locus. (a) Design of Hao1 gene exon 3 deletion by homologous recombination in ES cells. (b) Upper Western blot of 50-µg liver protein from Hao1^+/+, Hao1^+/−, and Hao1^−/− mice probed with affinity-purified rabbit antibody raised against recombinant mouse glycolate oxidase (GO) shows lack of expression of the targeted allele and reduced levels in the heterozygous sample. Lower Reprobing of the blot with anti-glyceraldehyde-3-phosphate dehydrogenase detects even loading of the gel. (c). Left Western blot of wild-type (wt) mouse tissues (B: brain, H: heart, L: liver, K: kidney, T: testis) shows liver-specific expression of glycolate oxidase. Right No differences were found in GO expression between male and female mice. GAPDH, antiglyceraldehyde-3-phosphate dehydrogenase.

Figure 2

A 24-h urine glycolate and oxalate excretion by different mouse genotypes. Data is represented as mean ± SD (n = 6 per group). ANOVA statistical signification: ***P<0.001, NS = nonsignificative.

Figure 3

Kinetics of the mouse glycolate oxidase (GO) inhibition by 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST). (a) Cornish-Bowden plot for the inhibition of mouse glycolate oxidase by CCPST. Increased inhibitor concentrations were tested at every glycolate (substrate) concentration and represented against glycolate/velocity (v). CCPST behaves as a noncompetitive inhibitor as all lines intersect on the x axis at the point X = −K_i = −91.2 µM. (b) Dose–response curve of mouse glycolate oxidase activity against CCPST concentration. Data are represented as mean ± SD. Discontinue lines represent 95% confidence interval; nonlinear regression analysis.

Figure 4

In vitro response of mouse Agxt1^−/− primary hepatocytes. (a) Oxalate excretion in Agxt1^−/− hepatocytes treated with 5 mM glycolate compared with nontreated controls at 24, 48, and 72 hours. (b) Agxt1^−/− hepatocytes viability after treatment with increased concentrations of 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST) in presence of 5 mM glycolate, measured by methyl thiazol tetrazolium reduction assay. (c) Graphic representation of the positive relationship between CCPST added to the medium and that detected in the intracellular extract. Simple linear regression analysis (r = 0.952, P < 0.001; R² = 0.906, P < 0.001). (d) Relative amount of excreted oxalate by Agxt1^−/− hepatocytes measured at 24, 48, and 72 hours post-treatment with increased concentrations of CCPST, and compared to the corresponding nontreated control. Data are represented as mean ± SD. ANOVA statistical signification: *P < 0.05, **P < 0.01, ***P < 0.001, NS = nonsignificative, relative to control at each time point.

Figure 5

In vivo effects of 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST) treatment in PH1 mice. 24-h urine oxalate (a) and glycolate (b) excretion in Agxt1^−/− mice after daily oral administration of CCPST in complex with β-cyclodextrin during 11 days. Significant reduction in oxalate levels from the first dose compared to basal oxaluria (P < 0.05). At the 10th and 11th dose, the oxalate levels significantly decrease with respect to the first dose (P < 0.01). Mean ± SD of three independent assays of six male mice. Paired t-test statistical analysis. (c) Glycolate oxidase (GO) specific activity measured in perfused liver after the 11th oral dose of CCPST-β-cyclodextrin (CD) in Agxt1^−/− mice. Hao1-deficient mice (Hao1^−/−) was used as control of nonenzymatic GO activity. Significant reduction in GO specific activity compared to nontreated Agxt1^−/− mice (P < 0.001).