The molecular basis of cystinuria: an update

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal proximal tubule and small intestine. In 1992, a cDNA (rBAT) was isolated from kidney which induced high-affinity, sodium-independent uptake of cystine and dibasic amino acids when expressed in Xenopus oocytes. The rBAT gene was mapped to a region of chromosome 2p known to contain a cystinuria locus, and rBAT expression was demonstrated in the straight (S3) portion of renal proximal tubule and small intestine. Over 30 distinct rBAT mutations have been described in patients who inherit two fully recessive (type I) cystinuria genes. Recently, the second cystinuria gene (SLC7A9) on chromosome 19q was identified; SLC7A9 mutations were shown to cause the incompletely recessive form of cystinuria (types II and III). Patients who inherit two mutant SLC7A9 genes have recurrent nephrolithiasis comparable to those with two rBAT mutations. In some cystinuria families, patients inherit a fully recessive allele from one parent and an incompletely recessive allele from the other parent; patients with this 'mixed type' of cystinuria have somewhat milder disease. It is not yet clear whether this form of cystinuria involves rBAT as well as SLC7A9 mutations. Current evidence suggests that the transmembrane channel mediating uptake of cystine and dibasic amino acids at the luminal surface is encoded by SLC7A9; the smaller rBAT protein forms a heterodimeric complex with this channel and is critical for its targetting to the luminal membrane.     

Cystine transport activity of heterozygous rBAT mutants expressed in Xenopus oocytes

The rBAT gene encodes a transport protein for cystine and dibasic amino acids. It is a candidate gene for type I cystinuria, a genetic disorder inherited as an autosomal-recessive trait. Recently, several mutations in rBAT from Japanese patients with cystinuria have been reported from our laboratory. Some of these patients were heterozygous, which appears to be inconsistent with the previous concept that mutations in rBAT are recessive. To investigate the function of heterozygous mutants, we introduced these mutations into rBAT gene and analyzed the transport activity of cystine associated with the mutants in Xenopus oocytes. Co-injection of the mutant T1037C (L346P) and the polymorphism G1854A (M6181) into Xenopus oocytes produced a transport activity of 67.9% of the wild type. Oocytes co-injected with T2017C (C673R) and wild type had a transport activity of 70.3% of the wild type. These findings indicate that the heterozygous mutants show decreased transport activity compared to wild-type rBAT. Further, some mutants in rBAT may show decreased cystine transport activity even in heterozygous condition, which may contribute to stone-forming cystinuria.     

Human cystinuria-related transporter: localization and functional characterization

BACKGROUND: Cystinuria has been proposed to be an inherited defect of apical membrane transport systems for cystine and basic amino acids in renal proximal tubules. Although the mutations of the recently identified transporter BAT1/b(0,+)AT have been related to nontype I cystinuria, the function and localization of human BAT1 (hBAT1)/b(0,+)AT have not been well characterized. METHODS: The cDNA encoding hBAT1 was isolated from human kidney. Fluorescence in situ hybridization was performed to map the hBAT1 gene on human chromosomes. Tissue distribution and localization of expression were examined by Northern blot and immunohistochemical analyses. hBAT1 cDNA was transfected to COS-7 cells with rBAT cDNA, and the uptake and efflux of 14C-labeled amino acids were measured to determine the functional properties. The roles of protein kinase-dependent phosphorylation were investigated using inhibitors or activators of protein kinases. RESULTS: The hBAT1 gene was mapped to 19q12-13.1 on the human chromosome, which is the locus of nontype I cystinuria. hBAT1 message was expressed predominantly in kidney. hBAT1 protein was localized in the apical membrane of proximal tubules in human kidney. When expressed in COS-7 cells with a type II membrane glycoprotein rBAT (related to b(0,+)-amino acid transporter), hBAT1 exhibited the transport activity with the properties of amino acid transport system b(0,+), which transported cystine as well as basic and neutral amino acids presumably via a substrate exchange mechanism. BAT1-mediated transport was reduced by the protein kinase A activator and enhanced by the tyrosine kinase inhibitor. CONCLUSIONS: hBAT1 exhibited the properties expected for a transporter subserving the high-affinity cystine transport system in renal proximal tubules. The hBAT1 gene was mapped to the locus of nontype I cystinuria, confirming the involvement of hBAT1 in cystinuria.     

Role of rBAT Gene Products in Cystinuria

To investigate whether rBAT gene products function as a crystine transporter component or as a transport activator, we microinjected several C–terminal deletion mutants of rBAT cRNA into Xenopus oocytes, and measured transport activity for arginine, leucine and cystine in the presence and absence of sodium. Wild type rBAT significantly stimulated the uptake of all 3 amino acids 10–20 fold compared to control mutants. On the other hand, no mutant, except a Δ511–685 mutant, stimulated the uptake of these amino acids. However, the Δ511–685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Δ511–685 mutant also increased the uptake of leucine. The Δ511–685 mutant did not stimulate crystine uptake in the presence and absence of sodium. Furthermore, inhibition of L–arginine uptake by L–homoserine was seen only in the presence of sodium. These results suggest that mutant rBAT stimulates the endogenous amino acid transport system y⁺ in oocytes. Finally, rBAT gene products, as the primary cause of cystinuria, may function as activators of the amino acid transport system in renal brush border membrane.     

Cystinuria type I: identification of eight new mutations in SLC3A1

BACKGROUND: Cystinuria is a heritable disorder of amino acid transport characterized by the defective transport of cystine and the dibasic amino acids through the brush border epithelial cells of the renal tubule and intestine tract. Three types of cystinuria (I, II, and III) have been described based on the urinary excretion of cystine and dibasic amino acids in obligate heterozygotes. The SLC3A1 gene coding for an amino acid transporter named rBAT is responsible for type I cystinuria, whereas the SLC7A9 gene coding for a subunit (b0,+AT) of rBAT is involved in determining non-type I (types II and III) cystinuria. METHODS: The SLC3A1 gene sequence was investigated in a sample of seven type I/type I, three type I/non-type I, six type I/untyped, and four untyped unrelated cystinuric patients by RNA single-strand conformation polymorphism (RNA-SSCP). RESULTS: Eight new point mutations (S168X, 765+1G>T, 766-2A>G, R452Q, Y461X, S547W, L564F, and C673W) and seven previously reported mutations were detected. These new mutations increase the number of mutated alleles so far characterized in SLC3A1 to 62. CONCLUSIONS: We have found SLC3A1 mutations in 0.739 of the type I chromosomes studied. The relatively high proportion of uncharacterized type I chromosomes suggests either that there may be mutations not yet found in SLC3A1 or that many of the assigned type I chromosomes in mixed type I/non-type I patients may have mutations in SLC7A9. If the hypothesis is excluded in the future, we believe that a third gene may be involved in cystinuria.     

Renal amino acid transport: cellular and molecular events from clearance studies to frog eggs

This article reviews recent advances in the mechanisms of renal amino acid transport. Renal amino acid transport is necessary to efficiently reclaim approximately 450 mmol amino acids from the glomerular ultrafiltrate each day in man. In general, individual amino acids are transported across the epithelial membrane of the proximal tubule by a sodium (Na⁺) dependent mechanism. This cotransport process utilizes the energy of the Na⁺ gradient to enter the cell. The amino acid then exits the basolateral surface and Na⁺ is pumped out by the Na⁺–K⁺-ATPase located in the basolateral membrane. In addition to the cellular accumulation of amino acids across the luminal membrane, these compounds may be taken up by the cell from the basolateral surface. Most amino acids are transported both individually and in a series of seven group specific processes. Human disorders of amino acid transport have been described for six to the seven transport systems. The process of ontogeny of amino acid accumulation by the proximal tubule is a complex one and will be further discussed in this review. A number of factors including pH, ion dependency, electrogenicity of transport process, as well as a variety of hormonal factors, may contribute to the regulation of amino acid transport. Gene expression of several amino acid transporters has been successfully performed using the oocyte of the frogXenopus laevis. Using this system, a number of transporters have been cloned. Such a strategy will permit the cloning of virtually all transporter molecules, and thus we can anticipate the elucidation of the structure of the transporters. However, for a comprehensive understanding of cytoskeletal interactions protein phosphorylation and phospholipid domains and their linkage to the primary structure of the transporter need to be studied. The future for research in this area is indeed a bright one.     

Basolateral LAT-2 has a major role in the transepithelial flux of L-cystine in the renal proximal tubule cell line OK

During renal reabsorption, the amino acid transporters b(o,+) and y(+)L have a major role in the apical uptake of cystine and dibasic amino acids and in the basolateral efflux of dibasic amino acids, respectively. In contrast, the transporters responsible for the basolateral efflux of the apically transported cystine are unknown. This study shows the expression of system L and y(+)L transport activities in the basolateral domain of the proximal tubule-derived cell line OK and the cloning of the corresponding LAT-2 and y(+)LAT-1 cDNAs. Stable transfection with a LAT-2 antisense sequence demonstrated the specific role of LAT-2 in the basolateral system L amino acid exchange activity in OK cells. This partial reduction of LAT-2 expression decreased apical-to-basolateral trans-epithelial flux of cystine and resulted in a twofold to threefold increase in the intracellular content of cysteine. In contrast, the content of serine, threonine, and alanine showed a tendency to decrease, whereas other LAT-2 substrates were not affected. This demonstrates that LAT-2 plays a major specific role in the net basolateral efflux of cysteine and points to LAT-2 as a candidate gene to modulate cystine reabsorption.     

Cystinuria phenotyping by oral lysine and arginine loading.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acids transport that results in urolithiasis because of poor cystine solubility. Three cystinuria phenotypes, differentiated according to urinary amino acid excretion in obligate heterozygotes, were regarded as allelic variants of a monogenic disease. Two mutated amino acid transporter genes, however, have been recently identified as responsible for cystinuria. Mutations in the SLC3A1 gene. encoding for the heavy subunit of the transporter protein rBAT, were associated with type I cystinuria, whereas type II and III cystinuria were associated with mutations in the SLC7A9 gene, encoding for a light subunit of rBAT. Lysine and arginine metabolism have, therefore, been evaluated in cystinuria homozygotes and heterozygotes to better define the cystinuria phenotypes and their correlations with these emerging genotypes. PATIENTS AND METHODS: Lysine and arginine intestinal absorption and renal excretion were assessed by oral loading and compared to normal controls. Seven cystinuria homozygotes and 7 obligate heterozygotes belonging to the different types received alternately an oral dose of 0.5 mmol/kg body weight lysine or arginine. Plasma concentrations of lysine, arginine, ornithine (derived from rapid arginine conversion) were measured 0, 1, 2, and 3 hours after loading. Their urinary concentrations were measured in morning urine and in urine collected 0-6 hours after loading. RESULTS: Gut lysine absorption was deficient in type II and III, and normal in type I cystinuria homozygotes. Impaired arginine intestinal absorption, as well as massive lysine, arginine, and ornithine hyperexcretion were shared by all homozygotes, irrespective of the type. All heterozygotes shared normal lysine absorption, whereas arginine absorption was slightly impaired in type II and III heterozygotes, which also displayed high lysine, arginine, and ornithine urinary excretion after loading. CONCLUSIONS: Two cystinuria phenotypes, type I and non-type I, can be identified in both homozygous and heterozygous cystinuric subjects by oral loading tests with lysine and arginine. In agreement with recent molecular findings, non-type I cystinuria comprises mentioned type II and type III, which constitute allelic variants of a cystine and dibasic amino acid transport disorder distinct from type I cystinuria.     

Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b^0,+AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S³⁵) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P = 0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b^0,+ amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches. Gunnar Wendt-Nordahl, Sreedhar Sagi contributed equally to this work.     

Factors influencing the concentrations of the large neutral amino acids in the brain and in the CSF of dogs after portacaval anastomosis.

Portal-systemic shunting of blood is associated with hyperammonemia, an increased glutamine concentration in brain, an altered plasma neutral amino acid pattern, and high levels of several of the large neutral amino acids in brain. Since some of these amino acids are precursors for neurotransmitters and for other potentially neuroactive substances, high CNS levels of these amino acids may contribute to the development of encephalopathy. In order to determine the relative importance of changes in brain glutamine levels and changes in competition among the neutral amino acids for blood-brain transport, we measured the concentrations of the large neutral amino acids in plasma, cisternal cerebrospinal fluid and in brain tissue from various regions of dogs after end-to-side portacaval shunt. Although the changes in CSF amino acid levels correlated partially with altered amino acid plasma competitor ratios, better correlations were observed with the elevation of CSF glutamine. These results suggest a model of blood-brain amino acid transport in which a high level of glutamine in brain extracellular fluid competes with other neutral amino acids for efflux from brain, thus raising brain amino acid levels after portal-systemic shunting.     

Na+/H+ exchangers in renal regulation of acid-base balance

The kidney plays key roles in extracellular fluid pH homeostasis by reclaiming bicarbonate (HCO(3)(-)) filtered at the glomerulus and generating the consumed HCO(3)(-) by secreting protons (H(+)) into the urine (renal acidification). Sodium-proton exchangers (NHEs) are ubiquitous transmembrane proteins mediating the countertransport of Na(+) and H(+) across lipid bilayers. In mammals, NHEs participate in the regulation of cell pH, volume, and intracellular sodium concentration, as well as in transepithelial ion transport. Five of the 10 isoforms (NHE1-4 and NHE8) are expressed at the plasma membrane of renal epithelial cells. The best-studied isoform for acid-base homeostasis is NHE3, which mediates both HCO(3)(-) absorption and H(+) excretion in the renal tubule. This article reviews some important aspects of NHEs in the kidney, with special emphasis on the role of renal NHE3 in the maintenance of acid-base balance.     

Blood-brain barrier derangement in sepsis: Cause of septic encephalopathy?

Patients with sepsis often manifest disorientation, somnolence, asterixis and coma, symptoms also seen in portasystemic encephalopathy. Altered plasma concentrations of the neutral amino acids and increased blood-brain transport of these acids may play a role in portasystemic encephalopathy. Plasma amino acids and blood-brain barrier transport of neutral amino acids were investigated in a rat model of abdominal sepsis, cecal ligation and puncture. The blood-brain transport was studied by the technique of Oldendorf with carbon-14-amino acids 12 and 24 hours after the induction of sepsis. In similar groups of animals, isolation of brain capillaries was carried out by the technique of Hjelle and the capillaries were incubated with carbon-14-amino acids to study transport activity.Plasma and brain amino acids were deranged in a fashion similar to the derangements seen in portasystemic encephalopathy, with a decrease in plasma branched chain amino acids and an increase in most neutral amino acids in brain. The changes were most pronounced after 24 hours. The brain uptake of several neutral amino acids was increased in the septic rats, while the uptake of lysine, a basic amino acid, was normal. In the brain capillaries isolated from septic rats, tyrosine and leucine transport was also greater than in sham-operated animals.Elevated neutral amino acids may play a role in the encephalopathy encountered in septic patients similar to its role in patients with portasystemic encephalopathy, as similar mechanisms appear to be operating.     

Cell volume regulation: a review of cerebral adaptive mechanisms and implications for clinical treatment of osmolal disturbances. I

Control of cell size within defined limits is vital for maintenance of normal organ function. This important feature of cell physiology can be disturbed by changes in membrane transport in epithelial cells. In addition, fluctuations in the osmolality of the extracellular fluid, caused by an abnormal plasma concentration of sodium, glucose, or urea can lead to derangements in cell size. Cell volume regulation is especially important in the brain because the brain is confined within a non-compliant vault and cannot tolerate significant perturbations in cell size. Therefore, brain cells have developed a coordinated array of adaptive mechanisms designed to modulate the cytosolic content of osmotically active solutes in response to alterations in the osmolality of the extracellular fluid. This process is controlled by various hormones including arginine vasopressin, insulin, and estrogen, and is subject to changes during development. The bulk of the change in cell content of osmolytes involves inorganic electrolytes. However, excessive variation in the cytosolic ionic strength has deleterious effects on protein structure and enzyme function. Therefore, brain cells have developed the capacity to accumulate or extrude various organic osmolytes in order to adjust the cytosolic osmolality without adversely affecting cell function. These solutes are termed non-pertubing osmolytes and belong to one of three classes of molecules: amino acids, carbohydrates and polyhydric sugar alcohols, or methylamines. Cerebral cells regulate the cytosolic content of organic osmolytes primarily by altering the transmembrane flux of these solutes. There are features of the cell volume regulatory response that are shared by the brain and kidney cells. However, there are important interorgan differences including the speed of response, the exact identitiy of the organic osmolytes utilized by each organ, and the reliance upon de novo osmolyte synthesis rather than transport in modulating cytosolic content of these solutes. An improved understanding of the cerebral cell volume regulatory response has led to better treatment of clinical disorders of plasma osmolality.Part II of this survey will be published in Pediatr Nephrol (1992) Volume 6, Issue 1     

Sodium-coupled amino acid transport in renal tubule

Amino acids are reabsorbed from the tubular lumen by a saturable, carrier-mediated, concentrative transport mechanism driven by a Na+ electrochemical gradient across the luminal membrane. This process is followed by efflux mainly via carrier-mediated, Na+-independent facilitated diffusion across the basolateral membrane. Individual amino acids may have two or more Na+-dependent transport systems with different kinetic characteristics along the luminal membrane of the proximal tubule, thereby enabling very efficient amino acid reabsorption. Dual Na+-coupled transport pathways for some amino acids located in both the luminal and the peritubular membranes may operate in concert to provide the tubular epithelial cell with essential nutrients. One or more Na+ ions, H+, Cl- and in the case of acidic amino acids, K+ ion, may be involved in the translocation of the carrier complex. For most amino acids this process is electrogenic positive, favored by a negative cell interior. At least seven distinct, but largely interacting, Na+-dependent amino acid transport systems have been identified in the brush border membrane. A diet-induced adaptation in Na+-coupled taurine transport and acidosis-induced adaptive response in Na+-dependent glutamine transport are expressed at the luminal and the basolateral membrane surfaces, respectively. The aminoaciduria of early life may be related to a rapid dissipation of the Na+ electrochemical gradient necessary for amino acid reabsorption.     

Genetic variations of the SLC7A9 gene: allele distribution of 13 polymorphic sites in German cystinuria patients and controls

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. While mutations in the SLC3A1 gene cause type I cystinuria, patients with non-type I cystinuria carry mutations in the SLC7A9 gene. Both gene products form the renal amino acid transporter rBAT/b0,+AT affected in cystinuria. In the present study a total of 59 patients with different ethnic background were screened for sequence variations in SLC7A9, out of these 32 were of German origin. For determination of allele frequencies of detected polymorphisms, 58 healthy German controls were investigated. Molecular-genetic analysis was performed using single-strand conformation polymorphism analysis, restriction assays and sequencing. Allele frequencies were analyzed statistically for the detected polymorphisms. In addition to the 6 already known variants we identified 7 new polymorphisms. Statistical analyses showed a significantly different distribution of alleles between German patients and German controls in case of the polymorphisms c. 147C>T (exon 2), c.386C>T (exon 3), IVS3+22T>G, c.584C>T (exon 4), c.610T>C (exon 4), c.692C>T (exon 5), c.852C>A (exon 6) and c.872C>T (exon 6). In summary, our results show that cystinuria is a complex disease which is not only caused by mutations in SLC7A9 and SLC3A1, but also influenced by other modifying factors such as variants in SLC7A9.     

Glutathione uptake and protection against oxidative injury in isolated kidney cells

Analysis with radiotracer and high performance liquid chromatography techniques showed that glutathione (GSH) is transported intact into cells primarily of proximal tubule origin. Characteristics of GSH uptake were the same as previously reported for basal-lateral membrane vesicles, namely, uptake was Na+-dependent, inhibited by gamma-glutamylglutamate and/or probenecid, and not inhibited by cysteinylglycine or the constituent amino acids. Studies with inhibitors of gamma-glutamyltransferase (acivicin) and gamma-glutamylcysteine synthetase (buthionine sulfoximine) showed that GSH uptake, degradation and resynthesis are independent processes. The GSH uptake rate with 1 mM GSH was approximately three-fold greater than the GSH synthetic rate with 1 mM amino acids. To examine whether uptake of GSH can supplement synthesis to protect against injury, we incubated cells with a toxic concentration of t-butylhydroperoxide with or without GSH or its constituent amino acids. Although amino acids provided significant protection, GSH provided greater protection (cells with t-butylhydroperoxide plus GSH were not significantly different from cells alone). This protection by GSH was eliminated by gamma-glutamylglutamate or probenecid, indicating that GSH uptake was required for the protection seen. Protection was also eliminated when the GSSG reductase/GSH peroxidase system was inhibited by bischloronitrosourea (BCNU), indicating that GSH transport affords protection by maintaining GSH levels in the cell. Thus, intact GSH is transported into isolated proximal tubule cells by a Na+-dependent system, and this transported GSH can be used to supplement endogenous synthesis and GSSG reduction to protect cells against oxidative injury.     

Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition

Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.