Identification and characterization of cystinuria modulating genes: L-Ergothioneine as a potential treatment for preventing cystine lithiasis

Abstract

[eng] Cystinuria, with a worldwide prevalence estimated at 1:7000, is a rare inherited disease characterized by urine hyperexcretion of cystine and dibasic amino acids. Its clinical manifestation is cystine lithiasis in the urinary system due to the low solubility of cystine at physiological urine pH which causes its precipitation and, as a consequence, stone formation. Cystine stones account for 1-2 % of adult and 6-10% of pediatric urinary tract lithiasis and, the high recurrence rate of stone episodes involving repeted urologic interventions, results in chronic kidney disease in most patients. Cystinuria is caused by genetic defects in SLC3A1 and SLC7A9 genes, which encode the heavy (rBAT) and the light (b0,+AT) subunits of the renal amino acid transport system b0,+, respectively. However, a high phenotype variability is observed in cystinuric patients as even brothers with the same mutation show different onset of stone episodes, recurrence and treatment response. There is no effective treatment for cystinuria and current therapeutic approaches are conservative measures (hydration therapy, diet recommendations and urine alkalinization), and when stones appear, thiol drugs as D-penicillamine are prescribed, although present multiple side-effects. Both the lack of genotype-phenotype correlation in cystinuric patients and the absence of an effective treatment induce the search and characterization of cystinuria modulating genes to propose novel therapeutic strategies which are addressed in this thesis. First, as AGT1 (SLC7A13) was recently described as the second kidney cystine transporter, its involvement on amino acid reabsorption and cystine lithiasis was assessed in cystinuria mouse models and in cystinuric patients (Chapter I). Cystinuric mice that expressed AGT1 showed higher levels of cystine reabsorption and a lower rate of stone formation during the whole 6-month follow-up, indicating its protective effect against cystine lithiasis. However, in cystinuric patients, no cystinuria causative or modulating effect could be associated to AGT1 after screening SLC7A13 gene in 9 cystinuric patients with only one or any mutation detected in SLC3A1 and SLC7A9 genes. Then, an RNAseq analysis was performed to identify differentially expressed genes in the kidneys of the Slc7a9-/- mouse model (Chapter II). The pathway enrichment analysis of differentially expressed genes of both male and female mice unveiled an impairment of the oxidative phosphorylation system. To validate this finding, the oxidative phosphorylation system and the Tricarboxylic acid cycle function were studied in the kidneys of the Slc7a9-/- mice. A citrate intracellular depletion, a reduced NAD+/NADH ratio, a lower complex IV enzymatic activity and a decreased in the mitochondrial maximal respiration capacity was observed in the Slc7a9-/- mice, revealing a mitochondrial dysfunction in cystinuria. Finally, as in a previous group’s work Slc22a4 (encoding OCTN1) was found downregulated in Slc7a9-/- stone former mice compared to non-stone former ones, the therapeutical potential in cystine lithiasis of the main molecule transported by OCTN1, L-Ergothioneine (L-Erg), was evaluated (Chapter III). The preventive (before any stone event) and long-term administration (6 months) of L-Erg (16 mg/kg/day) induced a reduction of above 50 % in the number of stone former mice and delayed the lithiasis onset in the Slc7a9-/- treated group by increasing cystine solubility in urine. L-Erg effect in cystine lithiasis was showed to be dependent on its internalization and derived metabolism as no effect was observed when treating Slc7a9-/-Slc22a4-/- mice.