Exploring the Contribution of the Transporter AGT1/rBAT in Cystinuria Progression: Insights from Mouse Models and a Retrospective Cohort Study

Abstract

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.

Keywords: SLC7A13 variants; cystine transporter; cystinuria; functional analysis; rare disease.

Figure 1

AGT1/rBAT analysis on cystinuria mouse models. (A) Schematic representation of the expression of renal cystine transporters in the cystinuria male mouse models. CssC = Cystine, Arg = Arginine, Lys = Lysine, Orn = Ornithine, Asp = Aspartate, Glu = Glutamate, and AA = amino acids. (B) Violin plot of aspartate, cystine, and glutamate urinary excretion in wild-type (WT), Slc7a9^−/−, and Slc3a1^D140G male mice grouped according to their lithiasic phenotype: non-stone former (NSF) and stone former (SF) mice. Urinary excretion was normalized by body weight (BW). (C) Monthly follow-up of the rate of stone formation in Slc7a9^−/− and Slc3a1^D140G male mice (N = 30 per condition). (D) Violin plot of AGT1/rBAT transporter expression in wild-type (WT), Slc7a9^−/− non-stone former, and Slc7a9^−/− stone former mice assessed using both AGT1 and rBAT antibodies in non-denaturing conditions. Representative membranes of AGT1/rBAT transporter protein levels and the total protein transference used to normalize data obtained. p-Values were assessed using the Mann–Whitney–Wilcoxon test.

Figure 2

In vitro characterization of AGT1 mutants. (A) Cartoon representation of the AGT1 Alphafold structural model (Q8TCU3). Helices are colored blue to red from the N-terminus. Mutated residues identified in cystinuria patients are represented by spheres. Atoms are colored as follows: O (red), N (blue), and C-atoms depending on the residue (V249: green; L330 and R380: orange; M452: red). Unwound regions of TMs 1 and 6 are also depicted and indicated. (B) Sodium-independent L-[³H]-aspartate uptake mediated by human AGT1 wild-type (WT) and mutants in transfected HeLa cells. AGT1 mutant transport activity (pmols L-aspartate · mg protein · minute) is normalized to that of wild-type AGT1. Data (mean ± SEM) correspond to quadruplicates from three independent experiments. Unpaired Student’s t-test statistical analysis of cystinuria mutants’ activity compared to that of the wild-type counterpart is represented, p-value: ** ≤0.01. (C) Representative fluorescence microscopy images of GFP-tagged wild-type (WT) and the indicated AGT1 (green) mutants overexpressed in HeLa cells, membrane staining with wheat germ agglutinin (WGA: red), and the merged images. All AGT1 mutants reached the plasma membrane.

Figure 3

Study of the impact of c.745G>A and c.988C>T SLC7A13 variants in cystinuria patients. Violin plots of (A) aspartate, (B) glutamate, and (C) cystine urinary excretion in cystinuria patients according to their cystinuria-affected gene and the carrying SLC7A13 variant. In aspartate and glutamate comparisons, as their transport is independent of the SLC7A9 gene and the number of samples was lower, heterozygous and compound heterozygous SLC7A9 patients were grouped together. (D) Comparison of the number of lithiasic events per year of each patient according to their cystinuria-affected gene and the carrying SLC7A13 variant. +/+ = no pathogenic variants, −/− = one pathogenic variant in each allele, +/− = one pathogenic variant in one allele, and ?/? = no pathogenic variants identified. Cre = creatinine.