Generation of a novel Slc7a9G105R mutant mouse identifies new biomarkers for cystinuria

Abstract

Cystinuria is a rare inherited disease characterized by increased urinary cystine levels resulting in the formation of cystine stones in the urinary tract. Mutations in the genes encoding the cystine transporter complex, SLC3A1 and SLC7A9, are the primary drivers of the disease. Current mouse models used to study cystinuria rely on gene deficiency or spontaneous mutations in mice that do not accurately reflect the pathogenic mutations found in humans. To overcome this limitation, we generated novel Slc7a9G105R mice carrying the most common pathogenic single-point mutation in the SLC7A9 gene. Both male and female Slc7a9G105R mice developed a cystinuria phenotype by 9 weeks of age, characterized by substantial cystine stone formation and increased urinary cystine, lysine, arginine, and ornithine. Slc7a9G105R mice displayed distinct serum and urinary metabolite profiles mapped to dibasic amino acid pathways. Fecal metagenomics revealed that Slc7a9G105R mice had a heterogeneous microbiota with altered functional pathways, including increased L-cysteine biosynthesis. Depletion of the microbiota with antibiotics did not impact cystine stone burden but reduced urinary tract inflammation. Prophylactic or therapeutic dietary supplementation with alpha-lipoic acid reduced stone burden and inflammation, but it also caused damage to the urothelium. Untargeted metabolomics analysis following alpha-lipoic acid supplementation identified metabolites that can increase cystine solubility, reduce inflammation, and damage epithelial cells. Correlation analysis revealed novel serum biomarkers of stone burden, including blood urea nitrogen, 2-hydroxybutyric acid, 2-amino-2-thiazoline-4-carboxylic acid, and indole-3-acetylglycine. Collectively, the Slc7a9G105R mutant mouse model offers a precise, rapid-onset, and translational platform for investigating cystinuria pathogenesis and evaluating potential therapeutic strategies. Translational statementThe development of a novel knock-in mouse model carrying the most common pathogenic point mutation in the human SLC7A9 gene provides a clinically relevant and translationally valuable platform for investigating cystinuria pathogenesis and testing emerging therapies. This model represents the closest possible approximation of human SLC7A9-mediated cystinuria, enabling rigorous preclinical evaluation of small molecules and gene therapies. It has also facilitated the identification of candidate biomarkers for cystine stone burden and treatment response, which are urgently needed to improve disease monitoring and clinical decision-making. The next critical step is to validate these biomarkers in human cystinuria cohorts to support their clinical translation.