Inducible Slc7a7 Knockout Mouse Model Recapitulates Lysinuric Protein Intolerance Disease

Abstract

Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y⁺LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7^-/-). The Slc7a7^-/- model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7^-/- mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7^-/- model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression.

Keywords: LPI; amino acid transporter; hyperammonemia; hypoargininemia; pulmonary alveolar proteinosis; rare disease; y+LAT1.

Figure 1

LPI mouse model and citrulline treatment. (a) Schematic representation of timeframes for tamoxifen induction and citrulline treatment. Twelve-week-old animals were fed a tamoxifen diet for 1 week. The diet was then changed to 8% protein with or without citrulline supplementation. Western blot is of total membranes from kidney and intestine against y⁺LAT1-CD98 heterodimer (135 kDa) from Slc7a7^loxP/loxP Cre⁻ (Slc7a7^−/−) and Slc7a7^loxP/loxP Cre⁺ mice. β-actin was used as a loading control; (b) Survival plot at 60 days with 13 animals per group; Slc7a7^loxP/loxP animals fed an 8% protein diet with citrulline supplementation (grey line) showed enhanced survival vs. Slc7a7^−/− animals without citrulline supplementation (dashed line). Black line represents control animals (Slc7a7 ^loxP/loxP without Cre expression treated with tamoxifen diet and 8% protein diet); (c–e) Body, epydidymal white adipose tissue weight and food intake of tamoxifen induced Slc7a7^loxP/loxP Cre⁻ (white bars) compared with control Slc7a7^loxP/loxP Cre⁺ (black bars) animals with or without citrulline supplementation as indicated on the X axis. Data correspond to the mean ± SEM of 9 animals. Statistical significance * p < 0.05, **** p < 0.0001 vs. control. ## p < 0.01 vs. citrulline treatment was analysed using a Student’s t-test.

Figure 2

Slc7a7^−/− recapitules all the main hallmarks of LPI. (a) Hyperexcretion of cationic amino acids in Slc7a7^−/−. Cationic amino acid (CAA) concentration in urine relative to creatinine concentration at 24 h and body weight of 12-month-old animals; (b) Renal clearance of cationic amino acids calculated from Supplementary Equation S1; (c) Malabsorption of lysine; (d) CAA concentration in plasma. Control (black bars or squares) and Slc7a7^−/− (white bars or squares) animals were analyzed. Data corresponds to the mean ± SEM of 6 animals per group. Statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. # p < 0.05, ## p < 0.01 vs citrulline treatment was analyzed using a Student’s t-test.

Figure 3

Slc7a7^−/− mice show hyperammonemia and brain alterations. (a) Slc7a7^−/− mice (white bars) have higher glutamine concentration in plasma; (b) hyperammonemia and (c) higher levels of orotic acid in urine compared to controls (black bars); (d) brain water content is also increased in Slc7a7^−/− mice. Citrulline treatment (+ Cit) improved all the parameters measured. Data corresponds to the mean ± SEM of 6 mice per group (a to d). Statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. ## p < 0.01 vs. citrulline treatment was analyzed using a Student’s t-test. (e) Representative images of glial fibrillary acidic protein (GFAP) immunochemistry of hippocampus sections from control and Slc7a7^−/− mice treated (2 control and 4 Slc7a7^−/− mice, lower panels) or not (9 control and 4 Slc7a7^−/− mice) with citrulline. Scale bar = 400 µm

Figure 4

Slc7a7^−/− develops pulmonary alveolar proteinosis (PAP). Sections of periodic acid–Schiff (PAS)-stained lungs reveal the classic histological features of PAP in Slc7a7^−/− animals. These features were not present in control mice. Serial sections of lung were stained with SP-B antibody and Masson’s trichrome. Lipoproteinaceous material accumulated within the alveoli was PAS and surfactant protein (SP)-B positive. Pulmonary fibrosis was observed in the Slc7a7^−/− mouse, as revealed by blue staining, which indicates collagen deposition. Scale bar = 5 µm for PAS images and scale bar = 500 µm for the rest.

Figure 5

Analysis of alveolar macrophages. (a) y⁺LAT1 mRNA expression is strongly reduced in AMs from Slc7a7^−/− mice (white bars) compared to controls ones (black bars); (b) Percentage of foamy AMs is increased in Slc7a7^−/− animals (white circles); (c) AMs from Slc7a7^−/− mice have a bigger area than those from controls (black bars) and are foamy as expected for surfactant accumulation (inset panels). Mice were fed with an 8% protein diet supplemented with citrulline for 15–55 days. Data corresponds to the mean ± SEM of 12 control and 14 Slc7a7^−/− mice. Statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001 was analyzed using a Student’s t-test. Cytological preparations of bronchoalveolar-lavage fluid were stained with panoptic. Scale bar = 50 µm.