The antioxidant l-Ergothioneine prevents cystine lithiasis in the Slc7a9-/- mouse model of cystinuria

Abstract

The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9^-/- mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9^-/-Slc22a4^-/- mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9^-/- mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.

Keywords: Antioxidant; Cystine lithiasis; Cystinuria; Oxidative stress; Treatment; l-Ergothioneine.

Graphical abstract

Fig. 1

L-Erg treatment for 1 month increases L-Erg and S-Met-L-Erg urinary concentration without affecting metabolic parameters. Comparison of (A) mice weight, (B) water intake normalized by mice body surface area, (C) urinary excretion normalized by body surface area, and (D) urinary pH values recorded in metabolic cages before and after administration of 15 or 60 mg/L of L-Erg. (E) L-Erg and (F) S-Met-L-Erg urinary concentration in μM before and after administration of 15 mg/L or 60 mg/L of L-Erg. (G) Ratio between post and pre-treatment levels of L-Erg and S-Met-L-Erg in the urine of treated mice. E, F and G graphs are shown using a logarithmic scale for visualization purposes. (H) Calculated L-Erg mean dose during the 1-month L-Erg treatment taking into consideration L-Erg drinking water concentration, mice body weight and water intake. The dashed black line indicates the 16 mg/kg/day dose. Each dot represents an individual mouse, and the bars indicate the mean ± SEM. p-value is shown at the top if significant after Mann-Whitney-Wilcoxon test (***p < 0.001).

Fig. 2

Long-term L-Erg treatment reduces and delays stone formation in theSlc7a9^−/−mouse model. (A) Timeline of preventive L-Erg (16 mg/kg/day) treatment from weaning to seven months of age in Slc7a9^−/− mice. (B) Evolution of the rate of stone-former mice in control and treated groups (N = 40 per group). (C) Comparison of the monthly stone weight evolution in control and treated stone-former mice. The stone weight follow-up was estimated by X-ray images. (D) Post-mortem stone weight in control and treated stone-former mice after L-Erg treatment. Each dot represents an individual mouse, and the bars indicate the mean ± SEM. p-value is shown at the top if significant after Mann-Whitney-Wilcoxon test (*p < 0.05). M = Month of age, XR = X-ray.

Fig. 3

Long-term L-Erg treatment increases cystine solubility without affecting physiological parameters and cystine crystal structure. (A) Urinary excretion normalized by body surface area in control and L-Erg treated mice. (B) Urinary pH in control and L-Erg treated mice. (C) Urinary cystine concentration in μM in WT, control and L-Erg treated mice. (D)l-cystine precipitate relative to control mice after adding a supersaturated l-cystine solution (4 mM) to a urine pool from control and L-Erg treated mice. (E) Representative SEM images of the surface (I) and core (II) of a cystine stone from control mice and of the surface (III) and core (IV) of a cystine stone from treated mice, and their corresponding EDS spectra. Each dot represents an individual mouse, and the bars indicate the mean ± SEM. p-value is shown at the top if significant after Mann-Whitney-Wilcoxon test (*p < 0.05). C = Carbon, N = Nitrogen, O = Oxygen, Al = Aluminum, S =Sulphur.

Fig. 4

High-dose L-Erg treatment reduces and delays stone formation in theSlc7a9^−/−mouse model. (A) Timeline of preventive L-Erg (200 mg/kg/day) treatment from weaning to 4 months of age in Slc7a9^−/− mice. (B) Evolution of the rate of stone-former mice in control and high-dose-treated groups (N = 55 per group). (C) Comparison of the monthly stone weight evolution in control and high-dose-treated stone-former mice. The stone weight follow-up was estimated by X-ray images. (D) Post-mortem stone weight in control and high-dose-treated stone-former mice after L-Erg treatment. Urinary levels of (E) L-Erg, (F) S-Met-L-Erg and (G) their ratio in control, low-dose and high-dose-treated non-stone-former (NSF) and stone-former (SF) mice grouped by sex. Control and treated mice are plotted in different graphs for visualization purposes. Each dot represents an individual mouse, and the bars indicate the mean ± SEM. p-value is shown at the top if significant after Mann-Whitney-Wilcoxon test (*p < 0.05, **p < 0.01). M = Month of age, XR = X-ray, M = male, F = Female, Cntrl = Control, LD = Low-dose (16 mg/kg/day), HD = High-dose (200 mg/kg/day), NSF = Non-stone-former, SF = Stone-former.

Fig. 5

L-Erg metabolism is essential for treatment effectiveness. (A) Evolution of the rate of stone-former mice percentage in control and treated dKO mice (N = 22 per group). (B) Comparison of the monthly stone weight evolution in control and treated dKO stone-former mice. The stone weight follow-up was estimated by X-ray images. (C) Post-mortem stone weight in control and treated dKO stone-former mice after L-Erg treatment. (D) l-cystine precipitate relative to control mice after adding a supersaturated l-cystine solution (4 mM) to a urine pool from Slc7a9^−/− control mice (Cntrl), Slc7a9^−/− mice treated with a low-dose of L-Erg (16 mg/kg/day) (Treated LD), Slc7a9^−/− mice treated with a high dose of L-Erg (200 mg/kg/day) (Treated HD), Slc7a9^−/− control mice but in vitro supplemented with L-Erg to obtain a final urine concentration of 0.5 mM (Cntrl + L-Erg) and Slc7a9^−/− control mice but in vitro supplemented with S-Met-L-Erg to obtain a final urine concentration of 0.5 mM (Cntrl + S-Met). Each dot represents an individual mouse, and the bars indicate the mean ± SEM. p-value is shown at the top if significant after Mann-Whitney-Wilcoxon test (*p < 0.05). dKO = Slc7a9^−/−Slc22a4^−/− mice, Cntrl = Control, LD = Low dose (16 mg/kg/day), HD = High dose (200 mg/kg/day).

Fig. 6

Long-term L-Erg treatment ameliorates oxidative damage and mitochondrial function in Slc7a9−/− mice. (A) Kidney and (B) liver reduced (GSH), oxidized (GSSG) and total (GSH + GSSG) GSH content, and GSH/GSSG ratio of WT, low-dose (LD) and high-dose (HD) L-Erg treated mice, and their respective control mice. Levels were normalized by kidney or liver total protein content (grams of proteins) and plotted normalized to WT levels. (C) Ex vivo mitochondrial respiration of fresh saponin permeabilized kidney biopsies of WT, control and high-dose L-Erg treated mice at 4 (left graph) and 6 (right graph) months of age. N-Leak respiration was measured adding pyruvate and malate (PM), N-OXPHOS capacity adding ADP and glutamate (PMG), NS-OXPHOS capacity adding succinate (PMGS), NS-ETS capacity adding FCCP and S-ETS capacity adding rotenone. Oxygen flux was normalized by mg of kidney tissue. The upper p-value corresponds to the comparison between control and treated mice, and the lower p-value, to WT and control comparison. N = 5–7. (D) Kidney mitochondrial succinate dehydrogenase (CII) and cytochrome c oxidase (CIV) activity in control and high-dose L-Erg treated mice at 4 months of age. Activity levels were calculated in nmol/min per mg of protein and normalized to citrate synthase (CS) activity. Each dot represents an individual mouse, and the bars indicate the mean ± SEM. p-value is shown at the top after Mann-Whitney-Wilcoxon test. #p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Cntrl = Control, LD = Low-dose (16 mg/kg/day), HD = High-dose (200 mg/kg/day), N = NADH linked pathway, NS = NADH and succinate linked pathways, S = succinate linked pathway, OXPHOS = oxidative phosphorylation, ETS = Electron transport system, FCCP = Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone, CII = Complex II, CIV = Complex IV, CS = Citrate Synthase.