Dysfunctional LAT2 amino acid transporter is associated with cataract in mouse and humans

Cataract, the loss of ocular lens transparency, accounts for ∼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 (Slc7a8) and uniporter TAT1 (Slc16a10) are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening SLC7A8 in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects

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

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

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

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.This work has been funded by the Instituto de Salud Carlos III through the projects PI16/00267-R-FEDER and PI20/00200 to VN (Co-funded by European Regional Development Fund. ERDF, a way to build Europe), and by La Marató de TV3 through the project 202025-30 to VN and 202025-32 to FVP. Generalitat de Catalunya Grant SGR2017-191 to VN. We also thank CERCA Programme/Generalitat de Catalunya for institutional support.Peer reviewe

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

[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

El proceso de sucesión en la empresa familiar

La majoria dels experts coincideixen en definir l’empresa familiar com l’empresa a la que el capital, la gestió y el govern estan en mans de una o mes famílies que exerceixen control sobre ella. El punt mes important que diferencia l’empresa familiar de la no familiar es el propòsit de la continuïtat, que es dona en a les empreses familiars, a través de l’entrada de les successives generacions. Aquesta continuïtat es materialitza amb l’èxit del procés successori que dona lloc a la nova generació. La complexitat d’aquest tipus d’empreses deriva de la unió de les dos entitats que la formen, empresa i família. Aquesta unió suposa la combinació d’assumptes purament empresarials amb altres de caràcter més personal. La unió de tots dos interessos dins de l’empresa suposa una important font de conflictes i problemes per a l’organització. A mesura que la família es fa més gran els problemes també creixen, i les relacions entre la família i la empresa es fan més difícils i complicades. Es per això que l’empresa ha d’actuar amb temps i avançar-se a les futures dificultats que puguin presentar-se a l’organització, tractant d’evitar-les, fent servir les eines de que disposa. Per assegurar el èxit del procés successori i evitar futurs problemes interns, l’empresa, i sobretot el predecessor, han de manejar i definir de forma correcta el marc legal i el marc familiar que emmarquen les seves relacions. En primer lloc, el marc legal ha d’assegurar la bona comunicació entre la família i l’empresa, per així, evitar futurs malentesos. A través d’òrgans com el Consell d’Administració i la Junta General d’Accionistes l’empresa reforça aspectes com la unitat i el compromís, que son tan necessaris en moments de canvi generacional. Per una altra banda, el marc familiar ha de vetllar pel manteniment dels valors propis de la família que el predecessor ha inculcat al llarg dels anys, i que han servit de guia tant a l’empresa com a la família. El marc familiar el defineixen eines com el Protocol Familiar, que regula i gestiona les relacions entre la família, l’empresa i els propietaris de la organització. El òrgan encarregat de controlar el compliment d’allò pactat en el Protocol i d’encarregar-se del bon funcionament del marc familiar es el Consell de Família

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

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut de Investigació Biomèdica de Bellvitge (IDIBELL)[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

Characterization of an MLC patient carrying two MLC1 variants showing radiological improvement

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy. Approximately 75% of MLC patients have variants in MLC1, while the rest in GLIALCAM, GPRC5B and AQP4. From the GLIALCAM patients, a classical and a benign phenotype can be distinguished, in which a recessive and dominant inheritance is observed, respectively. Here, we report a new MLC patient harboring two variants in MLC1 with radiological improvement. The patient is heterozygous for the variants c.597+37C>G and c.895–1G>T affecting both mRNA splicing, and the latest causes the deletion p.Pro299_Glu353del. By analyzing mRNA and protein obtained from patient's peripheral blood leukocytes, we could demonstrate the expression of a small amount of wild-type MLC1 mRNA and protein in the patient. Thus, we suggest that the improvement of clinical and radiological abnormalities observed in all remitting MLC patients might be due to the presence of residual amounts of MLC1 protein

Efficacy of Albaconazole (UR-9825) in Treatment of Disseminated Scedosporium prolificans Infection in Rabbits

There are no effective therapeutics for treating invasive Scedosporium prolificans infections. Doses of 15, 25, and 50 mg/kg of body weight/day for the new triazole albaconazole (ABC) were evaluated in an immunocompetent rabbit model of systemic infection with this mold. Treatments were begun 1 day after challenge and given for 10 days. ABC at any dose was more effective than amphotericin B (AMB) at 0.8 mg/kg/day at clearing S. prolificans from tissue (P < 0.007). The percentages of survival at 25 mg of ABC/kg/day were similar to those obtained with AMB. Rabbits showed 100% survival when they were treated with 50 mg of ABC per kg (P < 0.0001 versus control group), and only this dosage was able to reduce tissue burden significantly in the five organs studied, i.e., spleen, kidneys, liver, lungs, and brain

Dysfunctional LAT2 Amino Acid Transporter Is Associated With Cataract in Mouse and Humans

Cataract, the loss of ocular lens transparency, accounts for ∼50% of worldwide blindness and has been associated with water and solute transport dysfunction across lens cellular barriers. We show that neutral amino acid antiporter LAT2 ) and uniporter TAT1 () are expressed on mouse ciliary epithelium and LAT2 also in lens epithelium. Correspondingly, deletion of LAT2 induced a dramatic decrease in lens essential amino acid levels that was modulated by TAT1 defect. Interestingly, the absence of LAT2 led to increased incidence of cataract in mice, in particular in older females, and a synergistic effect was observed with simultaneous lack of TAT1. Screening in patients diagnosed with congenital or age-related cataract yielded one homozygous single nucleotide deletion segregating in a family with congenital cataract. Expressed in HeLa cells, this LAT2 mutation did not support amino acid uptake. Heterozygous LAT2 variants were also found in patients with cataract some of which showed a reduced transport function when expressed in HeLa cells. Whether heterozygous LAT2 variants may contribute to the pathology of cataract needs to be further investigated. Overall, our results suggest that defects of amino acid transporter LAT2 are implicated in cataract formation, a situation that may be aggravated by TAT1 defects