Postnatal outcome of children with antenatal colonic hyperechogenicity.

OBJECTIVE: To evaluate the postnatal outcome of children with antenatal colonic hyperechogenicity, currently considered as a sign of lysinuria-cystinuria, but which may also be a sign of other disorders with a more severe prognosis. METHOD: We carried out a French multi-centric retrospective study via 15 Multidisciplinary Center for Prenatal Diagnosis from January 2011 to January 2021. We included pregnancies for which fetal colonic hyperechogenicity had been demonstrated. We collected the investigations performed during pregnancy and at birth as well as the main clinical features of the mother and the child. We then established the prevalence of pathologies such as lysinuria-cystinuria (LC), hypotonia-cystinuria syndrome (HC), or lysinuric protein intolerance (LPI). RESULTS: Among the 33 cases of colonic hyperechogenicity collected, and after exclusion of those lost to follow-up, we identified 63% of children with lysinuria-cystinuria, 8% with lysinuric rotein intolerance, and 4% with hypotonia-cystinuria syndrome. CONCLUSION: Management of prenatal hyperechoic colon should include a specialized consultation with a clinical geneticist to discuss further investigations, which could include invasive amniotic fluid sampling for molecular diagnosis. A better understanding of diagnoses and prognosis should improve medical counseling and guide parental decision making.

Disrupting Crystal Growth through Molecular Recognition: Designer Therapies for Kidney Stone Prevention.

Aberrant crystallization within the human body can lead to several disease states or adverse outcomes, yet much remains to be understood about the critical stages leading to these events, which can include crystal nucleation and growth, crystal aggregation, and the adhesion of crystals to cells. Kidney stones, which are aggregates of single crystals with physiological origins, are particularly illustrative of pathological crystallization, with 10% of the U.S. population experiencing at least one stone occurrence in their lifetimes. The human record of kidney stones is more than 2000 years old, as noted by Hippocrates in his renowned oath and much later by Robert Hooke in his treatise Micrographia. William Hyde Wollaston, who was a physician, chemist, physicist, and crystallographer, was fascinated with stones, leading him to discover an unusual stone that he described in 1810 as cystic oxide, later corrected to cystine. Despite this long history, however, a fundamental understanding of the stages of stone formation and the rational design of therapies for stone prevention have remained elusive.This Account reviews discoveries and advances from our laboratories that have unraveled the complex crystal growth mechanisms of l-cystine, which forms l-cystine kidney stones in at least 20 000 individuals in the U.S. alone. Although l-cystine stones affect fewer individuals than common calcium oxalate stones, they are usually larger, recur more frequently, and are more likely to cause chronic kidney disease. Real-time in situ atomic force microscopy (AFM) reveals that the crystal growth of hexagonal l-cystine is characterized by a complex mechanism in which six interlaced anisotropic spirals grow synchronously, emanating from a single screw dislocation to generate a micromorphology with the appearance of stacked hexagonal islands. In contrast, proximal heterochiral dislocations produce features that appear to be spirals but actually are closed loops, akin to a Frank-Read source. These unusual and aesthetic growth patterns can be explained by the coincidence of the dislocation Burgers vector and the crystallographic 61 screw axis. Inhibiting l-cystine crystal growth is key to preventing stone formation. Decades of studies of "tailor-made additives", which are imposter molecules that closely resemble the solute and bind to crystal faces through molecular recognition, have demonstrated their effects on crystal properties such as morphology and polymorphism. The ability to visualize crystal growth in real time by AFM enables quantitative measurements of step velocities and, by extension, the effect of prospective inhibitors on growth rates, which can then be used to deduce inhibition mechanisms. Investigations with a wide range of prospective inhibitors revealed the importance of precise molecular recognition for binding l-cystine imposters to crystal sites, which results in step pinning and the inhibition of step advancement as well as the growth of bulk crystals. Moreover, select inhibitors of crystal growth, measured in vitro, reduce or eliminate stone formation in knockout mouse models of cystinuria, promising a new pathway to l-cystine stone prevention. These observations have wide-ranging implications for the design of therapies based on tailor-made additives for diseases associated with aberrant crystallization, from disease-related stones to "xenostones" that form in vivo because of the crystallization of low-solubility therapeutic agents such as antiretroviral agents.

Lipidomics characterization of the lipid metabolism profiles in a cystinuria rat model: Precalculus damage in the kidney of cystinuria.

Cystinuria is a genetic disorder of cystine transport, including defective protein b0,+AT (encoded by SLC7A9), and/or rBAT (encoded by SLC3A1). Patients present hyperexcretion of cystine in the urine, recurrent cystine lithiasis, and progressive decline in kidney function. Moreover, heterodimer transport is defective. To date, little omics data are accessible regarding this metabolic disease caused by membrane proteins. Since membrane function is closely related to changes in the lipidome, we decided to explore the changes in kidney tissue of a self-established cystinuria rat model by performing lipidomic analysis by LC-MS/MS. Our results demonstrated that Slc7a9 deficiency changed the lipid profile of the renal cortex and induced vital modifications in the lipidome, including major alterations in ChE, LPA, and PA. Among those alterations, this lipidomic study highlights the lipid changes that participate in inflammatory responses during cystinuria. As a result, lipid research, perhaps has great potential, for it may lead to the identification of novel therapeutic targets for the prevention and treatment of cystinuria.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Metabolic consequences of cystinuria.

BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1.

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

Clinical, Biochemical, and Genetic Findings of Cystinuria in Chinese Children.

BACKGROUND: Cystinuria is a rare inherited renal stone disease caused by mutations in the SLC3A1 and SLC7A9 genes. The Chinese cystinuria phenotype and genotype have rarely been reported in the literature. METHODS: For this research, the clinical features and genetic etiology were analyzed in seven children, and the clinical characteristics were summarized. The blood and urine amino acids and acylcarnitine were analyzed. Additionally, the whole coding sequence and exon-intron junctions of the SLC3A1 and SLC7A9 genes were analyzed. RESULTS: These seven patients with cystinuria were from seven unrelated Chinese families, and they were diagnosed between the ages of 1 month and 16 years old. The urinary amino acids, including ornithine, arginine, and threonine, were elevated in these patients. A homozygous c.325G>A mutation in SLC7A9 was identified in two patients, and six SLC3A1 mutations were found in five patients. CONCLUSIONS: The core pedigree analysis showed that most of the parents carried mutations; however, there was no association between the clinical course and the genotype.

No evidence for point mutations in the novel renal cystine transporter AGT1/SLC7A13 contributing to the etiology of cystinuria.

BACKGROUND: Cystinuria is caused by the defective renal reabsorption of cystine and dibasic amino acids, and results in cystine stone formation. So far, mutations in two genes have been identified as causative. The SLC3A1/rBAT gene encodes the heavy subunit of the heterodimeric rBAT-b0,+AT transporter, whereas the light chain is encoded by the SLC7A9/ b0,+AT gene. In nearly 85% of patients mutations in both genes are detectable, but a significant number of patients currently remains without a molecular diagnosis. Thus, the existence of a further cystinuria gene had been suggested, and the recently identified AGT1/SLC7A13 represents the long-postulated partner of rBAT and third cystinuria candidate gene. METHODS: We screened a cohort of 17 cystinuria patients for SLC7A13 variants which were negative for SLC3A1 and SLC7A9 mutations. RESULTS: Despite strong evidences for an involvement of SLC7A13 mutations in cystinuria, we could not confirm a relevant role of SLC7A13 for the disease. CONCLUSION: With the exclusion of SLC7A13/AGT1 as the third cystinuria gene accounting for the SLC3A1 and SLC7A9 mutation negative cases, it becomes obvious that other genetic factors should be responsible for the cystinuria phenotype in nearly 15% of patients.

Associating mutations causing cystinuria with disease severity with the aim of providing precision medicine.

BACKGROUND: Cystinuria is an inherited disease that results in the formation of cystine stones in the kidney, which can have serious health complications. Two genes (SLC7A9 and SLC3A1) that form an amino acid transporter are known to be responsible for the disease. Variants that cause the disease disrupt amino acid transport across the cell membrane, leading to the build-up of relatively insoluble cystine, resulting in formation of stones. Assessing the effects of each mutation is critical in order to provide tailored treatment options for patients. We used various computational methods to assess the effects of cystinuria associated mutations, utilising information on protein function, evolutionary conservation and natural population variation of the two genes. We also analysed the ability of some methods to predict the phenotypes of individuals with cystinuria, based on their genotypes, and compared this to clinical data. RESULTS: Using a literature search, we collated a set of 94 SLC3A1 and 58 SLC7A9 point mutations known to be associated with cystinuria. There are differences in sequence location, evolutionary conservation, allele frequency, and predicted effect on protein function between these mutations and other genetic variants of the same genes that occur in a large population. Structural analysis considered how these mutations might lead to cystinuria. For SLC7A9, many mutations swap hydrophobic amino acids for charged amino acids or vice versa, while others affect known functional sites. For SLC3A1, functional information is currently insufficient to make confident predictions but mutations often result in the loss of hydrogen bonds and largely appear to affect protein stability. Finally, we showed that computational predictions of mutation severity were significantly correlated with the disease phenotypes of patients from a clinical study, despite different methods disagreeing for some of their predictions. CONCLUSIONS: The results of this study are promising and highlight the areas of research which must now be pursued to better understand how mutations in SLC3A1 and SLC7A9 cause cystinuria. The application of our approach to a larger data set is essential, but we have shown that computational methods could play an important role in designing more effective personalised treatment options for patients with cystinuria.

Spectrum of mutations in cystinuria patients presenting with prenatal hyperechoic colon.

Cystinuria is a heterogeneous, rare but important cause of inherited kidney stone disease due to mutations in 2 genes: SLC3A1 and SLC7A9. Antenatal hyperechoic colon (HEC) has been reported in some patients as a non-pathological consequence of the intestinal transport defect. We report 83 patients affected by cystinuria: 44 presented prenatally with a HEC (HEC group) and 39 with a classical postnatal form (CC group). SLC3A1 and SLC7A9 were sequenced. All patients were fully genotyped, and the relationship between the genotype and clinical features was analyzed. We identified mutations in SLC3A1 in 80% of the HEC group and in only 49% of the CC group. The SLC3A1 p.Thr216Met mutation was found in 21% of the alleles in the HEC group but was never found in the CC group. Most of the mutations found in the HEC group were considered severe mutations in contrast with the CC group. Twenty-five novel mutations were reported. This study shows a relationship between genotype and the clinical form of cystinuria, suggesting that only the patients with the most severe mutations presented with an HEC. These results emphasized the need for prenatal cystinuria screening using classical third-trimester ultrasound scan and the early management of suspected newborns.

The role of N-glycans and the C-terminal loop of the subunit rBAT in the biogenesis of the cystinuria-associated transporter.

The transport system b(0,+) mediates reabsorption of dibasic amino acids and cystine in the kidney. It is made up of two disulfide-linked membrane subunits: the carrier, b(0,+)AT and the helper, rBAT (related to b(0,+) amino acid transporter). rBAT mutations that impair biogenesis of the transporter cause type I cystinuria. It has been shown that upon assembly, b(0,+)AT prevents degradation and promotes folding of rBAT; then, rBAT traffics b(0,+)AT from the endoplasmic reticulum (ER) to the plasma membrane. The role of the N-glycans of rBAT and of its C-terminal loop, which has no homology to any other sequence, in biogenesis of system b(0,+) is unknown. In the present study, we studied these points. We first identified the five N-glycans of rBAT. Elimination of the N-glycan Asn(575), but not of the others, delayed transporter maturation, as measured by pulse chase experiments and endoglycosidase H assays. Moreover, a transporter with only the N-glycan Asn(575) displayed similar maturation compared with wild-type, suggesting that this N-glycan was necessary and sufficient to achieve the maximum rate of transporter maturation. Deletion of the rBAT C-terminal disulfide loop (residues 673-685) prevented maturation and prompted degradation of the transporter. Alanine-scanning mutagenesis uncovered loop residues important for stability and/or maturation of system b(0,+). Further, double-mutant cycle analysis showed partial additivity of the effects of the Asn(679) loop residue and the N-glycan Asn(575) on transporter maturation, indicating that they may interact during system b(0,+) biogenesis. These data highlight the important role of the N-glycan Asn(575) and the C-terminal disulfide loop of rBAT in biogenesis of the rBAT-b(0,+)AT heterodimer.

A new workflow for proteomic analysis of urinary exosomes and assessment in cystinuria patients.

Cystinuria is a purely renal, rare genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases, patients undergo nephrectomy, but given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow-up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study conducted on eight cystinuria patients versus 10 controls highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins, and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking the potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinary exosomes in different renal diseases and to search for diagnostic/prognostic markers. Data are available via ProteomeXchange with identifier PXD001430.

Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice.

Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b(0,+)AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b(0,+). No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (∼100%), but fractional excretions of lysine (∼35%), ornithine (∼16%), and cystine (∼11%) were less affected. Loss of function of b(0,+)AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.

Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine.

Cystinuria is a classic heritable aminoaciduria that involves the defective transepithelial transport of cystine and dibasic amino acids in the kidney and intestine. Six missense mutations in the human rBAT gene, which is involved in high-affinity transport of cystine and dibasic amino acids in kidney and intestine, segregate with cystinuria. These mutations account for 30% of the cystinuria chromosomes studied. Homozygosity for the most common mutation (M467T) was detected in three cystinuric siblings. Mutation M467T nearly abolished the amino acid transport activity induced by rBAT in Xenopus oocytes. These results establish rBAT as a cystinuria gene.

Antenatal Hyperechogenic Colon and Cystinuria.

A male newborn was investigated for history of antenatal hyperechogenic colon (HEC) detected at 32 weeks of gestation. In the first week of life, urinary ultrasonography showed nephrolithiasis. Urinary amino acid analysis expressed increased excretion of dibasic amino acids, and high urinary cystine levels were detected in both spot and 24-hour urine specimens. He was diagnosed as cystinuria, and genetic analysis of the patient revealed a heterozygous mutation in SLC7A9 gene. Antenatal presentation of cystinuria with HEC is rare and reported to be associated with a more severe disease course.

Clinical, biochemical and molecular characterization of cystinuria in a cohort of 12 patients.

Cystinuria is a rare autosomal inherited disorder characterized by impaired transport of cystine and dibasic aminoacids in the proximal renal tubule. Classically, cystinuria is classified as type I (silent heterozygotes) and non-type I (heterozygotes with urinary hyperexcretion of cystine). Molecularly, cystinuria is classified as type A (mutations on SLC3A1 gene) and type B (mutations on SLC7A9 gene). The goal of this study is to provide a comprehensive clinical, biochemical and molecular characterization of a cohort of 12 Portuguese patients affected with cystinuria in order to provide insight into genotype-phenotype correlations. We describe seven type I and five non-type I patients. Regarding the molecular classification, seven patients were type A and five were type B. In SLC3A1 gene, two large genomic rearrangements and 13 sequence variants, including four new variants c.611-2A>C; c.1136+44G>A; c.1597T (p.Y533N); c.*70A>G, were found. One large genomic rearrangement was found in SLC7A9 gene as well as 24 sequence variants including 3 novel variants: c.216C>T (p.C72C), c.1119G>A (p.S373S) and c.*82C>T. In our cohort the most frequent pathogenic mutations were: large rearrangements (33.3% of mutant alleles) and a missense mutation c.1400T>C (p.M467T) (11.1%). This report expands the spectrum of SLC3A1 and SLC7A9 mutations and provides guidance in the clinical implementation of molecular assays in routine genetic counseling of Portuguese patients affected with cystinuria.

Staghorn cystine stone in a 72-year-old recurrent calcium stone former.

This case deals with the first diagnosis of Type B cystinuria with cystine nephrolithiasis in a 72-year-old male. Cystinuria is an inherited disease that consists of congenital abnormalities of renal and intestinal transport of dibasic amino acids. It often leads to frequent recurrent stone formation. Cystine stones most frequently occur in the 1st through 3rd decades of life with a decreased incidence in old age. This case shows that the first diagnosis of cystinuria may be made even in the 8th decade, without any family history, and in a patient with a history of recurrent calcium stone disease. Therefore, the chance of cystinuria must be always considered, even in older calcium stone formers.

Differences in renal cortex transcriptional profiling of wild-type and novel type B cystinuria model rats.

Cystinuria is a genetic disorder of cystine transport that accounts for 1-2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b0,+AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated.

A mouse model of type B cystinuria due to spontaneous mutation in FVB/NJcl mice.

Cystinuria is an autosomal metabolic disorder caused by mutations in the SLC3A1 and SLC7A9 genes, encoding the amino acid transporter proteins rBAT and b0,+AT, respectively. Based on the causative gene, cystinuria is classified into 3 types: type A (SLC3A1), type B (SLC7A9), and type AB (SLC3A1 and SLC7A9). Patients with cystinuria exhibit hyperexcretion of cystine and dibasic amino acids in the urine and develop cystine crystals due to its low solubility in the urine, often resulting in calculus formation. In this study, we present an inbred strain FVB/NJcl mice affected with cystinuria. In the affected mouse kidney, Slc7a9 expression was completely abolished because of a large sequence deletion in the promoter region of the Slc7a9 mutant allele. Slc7a9-deficient mice with FVB/NJcl genetic background developed cystine calculi in the bladder with high penetrance, as compared to the previously reported mouse models of cystinuria. This model may be useful to understand the determinants of crystal aggregation, affecting calculus formation.

Ca2+-mediated higher-order assembly of heterodimers in amino acid transport system b0,+ biogenesis and cystinuria.

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b0,+AT, which constitute system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+ mediates higher-order assembly of system b0,+. Ca2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b0,+AT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.