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.

ClC‐K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC‐Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline‐tyrosine motif in the C‐terminus of barttin stimulates ClC‐K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co‐expressing ClC‐K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC‐K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock‐in mouse. Comparing wild‐type (WT) and knock‐in mice under a standard diet, we could not observe any difference in ClC‐K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high‐sodium low‐potassium diet, known to induce hyperplasia of distal convoluted tubules, knock‐in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. image Key points: It is revealed by mutagenesis and functional experiments that a previously identified proline‐tyrosine motif regulating ClC‐K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC‐K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock‐in mice with a mutation in this motif, under conditions of a high‐sodium low‐potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild‐type animals, indicating a gain of function of the channel in vivo.

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.

PSA testing plays an important role in the diagnostic workup of prostate cancer; despite this, its cancer specificity is a well-recognised limitation. Consequently, more reliable, non-invasive diagnostic tools that are capable of improving specificity while maintaining sensitivity are needed, thereby enabling better risk stratification, personalised patient management, and reduction in unnecessary invasive procedures. In this study, we characterised the small RNA profile—including miRNAs and tsRNAs—in seminal small extracellular vesicles (sEVs) using high-throughput sequencing to expand biomarker discovery for distinguishing benign from malignant prostate conditions in patients with moderately elevated PSA levels, a setting where non-invasive biomarkers are critically needed. Our analysis confirms a small number of differentially represented sncRNA transcripts contained in seminal sEVs between benign and malignant prostate disease, most of which are of low abundance. This result suggests that shared underlying molecular mechanisms are likely to occur between both prostate disease conditions, especially in patients with moderate PSA levels. Subsequent RT-qPCR analysis revealed differences in expression in PCa compared with healthy controls but not when compared with benign prostatic disease. Given the complexity of the underlying pathophysiological process and the heterogeneity in clinical phenotypes, this limitation was addressed through the application of multivariate approaches (including isomiRs or tRFs), which are proposed as fluid-based biomarkers for prostate cancer, collectively offering improved accuracy and enhancing the negative predictive value of PSA used in clinical settings.