Skip Navigation


Human Molecular Genetics Advance Access originally published online on July 15, 2003
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
12/17/2097    most recent
ddg228v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (19)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Feliubadaló, L.
Right arrow Articles by Nunes, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feliubadaló, L.
Right arrow Articles by Nunes, V.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 17 2097-2108
DOI: 10.1093/hmg/ddg228
© 2003 Oxford University Press

Slc7a9-deficient mice develop cystinuria non-I and cystine urolithiasis

Lídia Feliubadaló1,2, María Lourdes Arbonés1,{dagger}, Sandra Mañas1, Josep Chillarón2, Joana Visa3, Margot Rodés4, Ferran Rousaud5, Antonio Zorzano2,3, Manuel Palacín2,3 and Virginia Nunes1,*

1Medical and Molecular Genetics Center, Institut de Recerca Oncològica, L'Hospitalet de Llobregat, 08907 Barcelona, Spain, 2Department of Biochemistry and Molecular Biology, Universitat de Barcelona, 08028 Barcelona, Spain, 3Parc Científic de Barcelona, 08028 Barcelona, Spain, 4Institut de Bioquímica Clínica, Corporació Sanitària Clínic, 08028 Barcelona, Spain and 5Servei de Nefrologia, Fundació Puigvert, 08025 Barcelona, Spain

Received March 28, 2003; Accepted July 5, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cystinuria is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in urolithiasis of cystine. Cystinuria is caused by defects in the amino acid transport system b0,+ (i.e. the rBAT/b0,+AT heteromeric complex). Mutations in SLC3A1, encoding rBAT, cause cystinuria type A, characterized by a silent phenotype in heterozygotes (phenotype I). Mutations in SLC7A9, encoding b0,+AT, cause cystinuria type B, in which heterozygotes in most cases hyperexcrete cystine and dibasic amino acids (phenotype non-I). To facilitate in vivo investigation of b0,+AT in cystinuria, Slc7a9 knockout mice have been generated. Expression of b0,+AT protein is completely abolished in the kidney of Slc7a9-/- mice (‘Stones’). In contrast, Stones expressed significant amounts of rBAT protein, which is covalently linked to unidentified light subunit(s). Stones mice present a dramatic hyperexcretion of cystine and dibasic amino acids, while Slc7a9+/- mice show moderate but significant hyperexcretion of these amino acids (phenotype non-I). Forty-two per cent of Stones mice develop cystine calculi in the urinary system. Calculi develop during the first month of life and grow throughout the life span of the animals. Histopathology in kidney reveals typical changes for urolithiasis (tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis). The fact that some Stones mice, generated in a mixed genetic background, develop cystine calculi from an early age, while others do not develop them in their first year of life, suggests the involvement of modifier genes in the lithiasis phenotype. Thus, Stones provide a valid model of cystinuria which can be used in the study of genetic, pharmacological and environmental factors involved in cystine urolithiasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cystinuria (OMIM 220200) is an autosomal-recessive disease of renal reabsorption and intestinal absorption of cystine and dibasic amino acids. Cystine precipitates in the urinary system to form calculi that produce obstruction, infection and, ultimately, renal insufficiency (1). Two cystinuria phenotypes are distinguished on the basis of the cystine and dibasic amino aciduria of the obligate heterozygotes: (i) normal urinary excretion in phenotype I carriers; and (ii) moderate to high excess of urinary excretion in phenotype non-I carriers. Mutations in SLC3A1, located on chromosome 2p16.3–21 and encoding rBAT, cause phenotype I cystinuria (2,3). The gene causing phenotype non-I cystinuria was assigned by linkage to chromosome 19q12–13.1 (4,5), and was identified as SLC7A9, encoding b0,+AT (6). Mutations in SLC7A9 cause phenotype non-I and also some phenotype I cases (7–9). In the cohort of patients studied by the International Cystinuria Consortium, mutations in SLC3A1 have been found in 74% of phenotype I alleles, and mutations in SLC7A9 have been found in 84% of phenotype non-I alleles and in 10% of phenotype I alleles (9). Consequently, a genotypic classification of cystinuria has been proposed: type A, due to SLC3A1 mutations, and type B, due to SLC7A9 mutations (9).

rBAT and b0,+AT form a heterodimeric complex in the brush-border membranes of the epithelial cells of the renal proximal tubule (10). Expression of the rBAT/b0,+AT heterodimeric complex in cultured cells resulted in the induction of system b0,+ amino acid transport activity (11–13). System b0,+ is an obligatory exchanger that mediates influx of dibasic amino acids and cystine, and efflux of neutral amino acids (for a review see 14). The involvement of the rBAT/b0,+AT heterodimeric complex in cystinuria indicates that system b0,+ is the main apical reabsorption system for cystine in kidney (10).

The only proven clinical manifestation of cystinuria is urolithiasis. Hexagonal crystals appear in the urine and radiopaque cystine stones develop repeatedly in most cystinuric individuals. Cystinuria is diagnosed by demonstrating selective hyperexcretion of cystine and dibasic amino acids in urine. Urolithiasis is prevented by high fluid intake and alkalinization of urine to maximize cystine solubility. Patients who persistently develop stones are sometimes treated with oral sulfhydryl agents like D-penicillamine and {alpha}-mercaptopropionylglycine (15,16). Although quite effective, these agents have multiple side-effects that often cause discontinuation of treatment (17,18).

Natural models for cystinuria have been described. Cystinuria has been found in a cat (19), in several breeds of dog (20) and in wolves (21). The Newfoundland dogs form a group with more homogeneous biochemical parameters and known etiology. All of those analyzed have a nonsense mutation in canine Slc3a1 (22). These species are, however, suboptimal for biochemical and genetic studies.

Reported here is the development of a mouse model for cystinuria type B. Homologous recombination was used to generate mice with a disruption of Slc7a9 in a mixed genetic background. All homozygous mutant mice show massive urinary hyperexcretion of cystine and dibasic amino acids, whereas heterozygotes show lower but clear hyperexcretion of these amino acids (phenotype non-I). About 40% of the homozygous mutants present cystine calculi (i.e. cystine stones) in the urinary system (bladder, renal pelvis and/or ureter). This strongly suggests that the lithiasic phenotype is affected by modifier genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted disruption of the Slc7a9 gene and generation of Slc7a9-/- mice
A targeting vector that replaces 6.1 kb of Slc7a9 genomic sequence, including exons 3–9, with a neo® cassette oriented in the opposite direction to the endogenous Slc7a9 has been constructed (Fig. 1A). Of 570 G418-gancyclovir-resistant ES cell clones analyzed, two demonstrated the appropriate recombination as confirmed by Southern blot analysis. Figure 1B shows the analysis of the three initial positives, one of which (clone 155) resulted from recombination only in the left homology arm. Clones 164 and 291 were used to generate chimeric founder mice. Heterozygotic mice from the F1 generation were identified by PCR analysis and were crossed in order to obtain b0,+AT-deficient mice. An example of the PCR used to genotype all the generations is shown in Figure 1C. Of 280 F2 mice genotyped, 59 were wild-type, 146 were heterozygous, and 75 were homozygous mutants, in accordance with the expected Mendelian ratios ({chi}2=2.34; d.f.=2).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 1. Generation of Slc7a9-knockout mice by homologous recombination in ES cells. (A) Schematic representation of the targeting strategy. The vector is represented by the second line from the top, while the wild-type and targeted Slc7a9 alleles are indicated by the top and bottom lines, respectively. The BamHI sites are indicated by B and the KpnI sites by K. Exons are represented by solid rectangles. The homology arms are represented by gray boxes. The backbone of the vector is denoted by checked boxes. In the vector and the mutant allele, the PGK-tk and PGK-neo gene cassettes are denoted by open boxes labeled accordingly. The two external probes (SE5' and SE3') are represented as solid lines at the bottom, and the PCR primers (BreF, NorR and RecR) used to genotype the mice as solid triangles. (B) Screening and confirmation of recombinant clones by Southern blot. DNA was digested with BamHI (left) or KpnI (right) and probed with SE5' and SE3' probes, respectively. The wild-type allele generates a 4.5 kb BamHI and a 7.0 kb KpnI fragment, whereas the targeted allele produces a 4.0 kb BamHI and a 10.6 kb KpnI fragment. Note that clone 155 had undergone recombination only in the 5' homology region, yielding the insertion of the whole vector. (C) Genotyping of mice by PCR from tail biopsy DNA. The combination of the three primers shown in (A) allows the discrimination between wild-type (+/+), heterozygous (+/-) and homozygous mutant (-/-) mice, containing respectively only the wild-type (410 bp), the wild-type and the targeted (336 bp) and only the targeted allele.

 
Genetic and biochemical analysis
To assess the correct splicing of the recombined alleles in target tissues, total RNA was isolated from kidney cortex and medulla, and from jejunum, of adult male Slc7a9+/+, Slc7a9+/- and Slc7a9-/- mice. Northern blot analysis (Fig. 2) showed two Slc7a9 mRNA species, the wild-type messenger of ~1.9 kb and a new messenger of ~0.9 kb generated from the recombinant allele by splicing between exons 2 and 10 (data not shown). Slc7a9+/- mice showed reduced expression of the wild-type mRNA in both tissues. Slc7a9-/- mice showed no expression of this messenger, as expected. Conversely, the recombined mRNA appeared in Slc7a9+/- mice at half the dose found in Slc7a9-/- mice.



View larger version (98K):
[in this window]
[in a new window]
  		Figure 2. Northern blot analysis of kidney and small intestine RNA from the three genotypes (top). The expected wild-type 1.9 kb transcript is detected from Slc7a9+/+ and (at a lower intensity) Slc7a9+/- mice, and absent in Slc7a9-/- mice. The 0.9 kb targeted transcript appears in Slc7a9+/- and Slc7a9-/- mice. Ethidium bromide staining (bottom) of the blot reveals equal amounts of RNA.

 
Elimination of exons 3–9 in the targeted allele (as confirmed by the sequencing of the RT–PCR product, data not shown) leads to a frameshift after amino acid residue 29 of b0,+AT, and the addition of 12 missense amino acids before the first stop codon. This would yield a truncated protein without any of the 12 putative transmembrane domains. Since b0,+AT forms a disulfide bound heterodimer with rBAT (10,13), the expression of both proteins in the presence and absence of DTT in mice from the three genotypes was studied. Western blot analysis of b0,+AT and rBAT in renal brush-border membranes is shown in Figure 3. In reducing conditions, b0,+AT appeared as two protein bands of ~40 and ~80 kDa in Slc7a9+/+ and Slc7a9+/- mice, which correspond to monomeric and homodimeric b0,+AT (10). Both protein bands showed lower expression in Slc7a9+/- mice and were completely abolished in Slc7a9-/- mice. Indeed, densitometric analysis corrected by protein loading as determined by Ponceau S staining showed that the expression of b0,+AT protein in Slc7a9+/- mice was 24% of that in Slc7a9+/+ mice (20 and 28% in two independent determinations). In addition a band of ~90 kDa of unknown nature was detected, both in the presence and absence of DTT. This band, which is not immunoprecipitated by the anti-b0,+AT serum (10), is not revealed with the pre-immune serum and its expression is not affected by the Slc7a9+/- genotype (data not shown). In the same conditions, rBAT is revealed as a protein band of ~94 kDa, which corresponds to the mature N-glycosylated monomeric form of the protein (11). The expression of rBAT was only moderately reduced in Slc7a9+/- mice (78 and 57% of that in Slc7a9+/+ mice, as described above). In contrast to b0,+AT, the rBAT protein was expressed, although clearly lower, in the renal brush-border membranes of Slc7a9-/- mice (35 and 48% of that in Slc7a9+/+ mice). In non-reducing conditions, b0,+AT and rBAT showed two bands of ~135 and ~250 kDa, as expected. The former band corresponds to the rBAT/b0,+AT heterodimer, and the latter probably represents a dimer of heterodimers (10). The nature of the b0,+AT band of ~170 kDa is unknown. The level of expression of b0,+AT and rBAT bands in non-reducing conditions reflects the situation already described for the three genotypes in reducing conditions. The most striking result is that rBAT shows the characteristic mobility of the heterodimer in the absence of b0,+AT in Slc7a9-/- mice.



View larger version (72K):
[in this window]
[in a new window]
  		Figure 3. Western blot analysis of renal membranes from the three genotypes using antibodies directed against b0,+AT (left panels) and rBAT (right panels). Fifty micrograms of protein renal brush-border membranes from adult male mice was subjected to SDS–PAGE in the absence (-DTT; bottom panels) or in the presence of 100 mM DTT (+DTT; top panels). The genotype corresponding to each lane is indicated between top and bottom panels. In reducing conditions (+DTT), b0,+AT is revealed as two protein bands: (i) b0,+AT monomer (solid arrowhead; ~40 kDa); and (ii) b0,+AT dimer (open arrowhead; ~80 kDa). In reducing conditions, rBAT appeared as a prominent unique band (open arrowhead; ~94 kDa). In non-reducing conditions, b0,+AT and rBAT appear as heterodimer bands (solid arrow; ~125 kDa) and as high molecular-weight complexes. The most conspicuous complex (open arrow; ~250 kDa) probably corresponds to a dimer of heterodimers.

 
Urinary phenotype of Slc7a9 knockout mice
The diagnostic trait of cystinuria in humans is the urinary hyperexcretion of cystine, lysine, arginine and ornithine. To assess the correspondence of this phenotype in our mice, 24 h urine from the three mouse genotypes was analyzed. Slc7a9-/- mice showed massive urine hyperexcretion of cystine and dibasic amino acids (Fig. 4). Thus, urine excretion of cystine, arginine, lysine and ornithine was between 76- and 267-fold higher in Slc7a9-/- than in Slc7a9+/+ mice (Fig. 4B and Table 1). The urine excretion of other amino acids was only slightly affected (if at all) in Slc7a9-/- mice (Fig. 4A, Table 1 and data not shown). Thus, the urine excretion of glutamine is 2-fold higher in Slc7a9-/- mice than in Slc7a9+/- and Slc7a9+/+ mice. Slc7a9+/- mice also presented hyperexcretion of cystine and dibasic amino acids, but to a much lesser extent than Slc7a9-/- mice (2- to 14-fold higher than in Slc7a9+/+ mice; Fig. 4B and Table 1). This pattern of urine hyperexcretion of amino acids is reminiscent of that reported in human classic cystinuria due to severe human SLC7A9 mutations (i.e. phenotype non-I cystinuria) (6,8,9). The urine excretion of cystine and dibasic amino acids in females showed a tendency to be higher (140–200%) than in males for the three phenotypes. This difference is significant for arginine excretion in Slc7a9+/+ and for the excretion of the three dibasic amino acids in Slc7a9-/- mice.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 4. Aminoaciduria of cystine and dibasic amino acids in homozygous (-/-) and heterozygous (+/-) mutant mice. (A) Representative amino acid chromatograms of urine of a homozygous mutant (-/-) mouse and a wild-type (+/+) mouse. The homozygous mutant (-/-) mice show large peaks for cystine, lysine, arginine and ornithine. All other amino acids showed peaks of similar size in homozygous mutant (-/-) and wild-type (+/+) mice. (B) Hyperexcretion of cystine and dibasic amino acids in the urine of homozygous mutant (-/-) and heterozygous mutant (+/-) mice. Urine was collected from 12 animals (six males and six females) in each group for 24 h and processed for amino acid level quantification by HPLC. Amino acid levels [nmol/(24 h·g body weight)] are shown on a logarithmic scale. Homozygous mutant (-/-) mice showed dramatic hyperexcretion of cystine and dibasic amino acids. Heterozygous mutant (+/-) mice showed moderate hyperexcretion of these amino acids, which was significant for arginine, lysine and cystine. A more detailed description of the urine amino acid levels of these mice is given in Table 1.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Urine and plasma amino acid levels. Amino acid level is given as [nmol/(24 h·g body weight)]. Each value is the average (±standard error of the mean) of 12 animals of each genotype for urine (top) and eight animals of each genotype for plasma (bottom). Significant differences (P<=0.05) between groups are asterisked in the ratios columns
 
Plasma amino acid levels for the three genotypes are shown in Table 1. The only clear difference between the three genotypes was found in Slc7a9-/- mice, which showed a ~30% decrease in plasmatic cystine and dibasic amino acids compared to Slc7a9+/+ and Slc7a9+/- mice. This decrease was significant for cystine and lysine. No differences were found for the other amino acids (Table 1 and data not shown). The hyperexcretion of cystine and dibasic amino acids with low to normal levels in plasma demonstrates the renal reabsorption defect for these amino acids in Slc7a9-/- and Slc7a9+/- mice.

Lithiasic phenotype of Slc7a9 knockout mice
Similarly to human classic cystinuria (for review, see 1), cystine crystalluria (Fig. 5A) was observed in ~82% of Slc7a9-/- mice (n=22) but not in Slc7a9+/+ (n=9) or Slc7a9+/- mice (n=9).



View larger version (101K):
[in this window]
[in a new window]
  		Figure 5. Cystine lithiasis in Slc7a9-/- mice. (A) Crystalluria in Slc7a9-/- mice. The picture shows cystine hexagonal crystals in the urine of an Slc7a9-/- mouse. Bar, 10 µm. (B) Radiograph of an Slc7a9-/- mouse, showing cystine calculi in the kidneys (arrowheads) and the urinary bladder (arrow). (C) Infrared spectrometry of a urinary bladder calculus from an Slc7a9-/- mouse. This spectrum matches that of pure cystine. (D) Morphological aspect of the urinary system in an Slc7a9-/- mouse compared with that of a normal individual (Slc7a9+/+). Scale in mm. (E) Radiograph of the urinary systems in (B). Arrowheads point to calculi in both kidneys. Arrows point to calculi in the urinary bladder and in the right ureter. Genotypes are indicated as in (D). (F) Cystine calculi and macroscopic changes in kidney and urinary bladder bearing a calculus removed in the necropsy of the Slc7a9-/- mouse shown in (D)–(F). The urinary bladder calculus is shown in the top right-hand corner of the figure. The renal pelvis calculus is shown at the bottom of the figure. Notice the dramatic increase in size in the urinary bladder and kidney in the Slc7a9-/- mouse compared with the wild-type. Urinary bladders (top) and kidneys (bottom) are shown excised in two halves. Genotypes are shown at the top. Scale in mm.

 
Most cystinuric patients develop cystine calculi in the urinary system (9). Slc7a9-/- mice presented lithiasis in the urinary bladder, renal pelvis and ureter (Fig. 5B–E). In contrast, lithiasis was not observed in adult Slc7a9+/+ (n=20) or Slc7a9+/- (n=24) mice. Infrared spectroscopy revealed that calculi in the Slc7a9-/- mice were composed of pure cystine (Fig. 5C). Cystine lithiasis in the urinary bladder is a fairly common phenotype in Slc7a9-/- mice. Thus, 41 out of 98 Slc7a9-/- mice analyzed (~42%) developed cystine calculi in the bladder. No sex differences were observed (lithiasis in the bladder was observed in 44 and 41% male and female, respectively; {chi}2=0.26; d.f.=1). Cystine lithiasis in the urinary bladder of Slc7a9-/- mice was observed one month after birth and the size of the calculi increased with age (Fig. 6). The proportion of animals with lithiasis was conserved throughout the life span of the animals (Fig. 6A). Cystine lithiasis in the renal pelvis, ureter and male urethra is a less common phenotype: it was observed in six mice (four females and two males), one female and two males, respectively, out of the 41 lithiasic Slc7a9-/- mice.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 6. Frequency and size of cystine lithiasis in the bladder of Slc7a9-/- mice. (A) Number of Slc7a9-/- mice with (solid bars) or without (open bars) lithiasis in the urinary bladder distributed by age in months. The 98 Slc7a9-/- mice studied correspond to F2 and F3 generations in a mixed genetic background of 129P2/OlaHsd and C57BL/6J. Mice were not radiographed during the first month of life. The proportion of mice bearing cystine calculi in the urinary bladder is 42%, which is maintained after the first month of life. (B) Estimation of the volume of the cystine calculi in the bladder of Slc7a9-/- mice distributed by age in months. The volume of the cystine calculi was estimated from dorso-ventral and latero-lateral radiographs of the mice, by multiplying their greatest diameter in both radiographs (y-axis) by their lowest diameter in each of the radiographs (x- and z- axes). Calculi tend to grow throughout the life of lithiasic Slc7a9-/- mice.

 
Crystalluria and cystine urolithiasis are not directly related in Slc7a9-/- mice. Thus, crystalluria was observed in eight out of 11 non-lithiasic Slc7a9-/- mice and in 10 out of 11 lithiasic Slc7a9-/- mice.

The presence of calculi in the urinary system can produce renal obstruction (Fig. 5D and E). Eight out of the 41 lithiasic Slc7a9-/- mice analyzed suffered hydronephrosis, hyperuremic shock or sepsis, produced by renal obstruction due to lithiasis in the urinary system between 2 and 13 months of age. Histopathological analysis of the kidneys of 11 Slc7a9-/- (with and without calculi) and three Slc7a9+/+ mice older than 4 months showed the following (Fig. 7A–H): the four lithiasic Slc7a9-/- mice presented either hydronephrosis or kidney calculi, all of them had severe tubular and pelvic dilatation, three presented tubular necrosis and chronic interstitial nephritis, and two presented tubular hyaline droplets. Of the seven non-lithiasic Slc7a9-/- mice, three presented light chronic interstitial nephritis, two moderate pelvic dilatation, and one moderate tubular dilatation. None of them presented tubular necrosis or tubular hyaline droplets. None of the three Slc7a9+/+ mice studied presented any of these lesions. Several organs from 17 mice of 5–7 weeks of age of the three genotypes were also studied histopathologically. Skin, spleen, thymus, skeletal muscle, heart, lung, stomach, intestine, liver, pancreas, brain and eye from the 17 young animals were unaltered.



View larger version (174K):
[in this window]
[in a new window]
  		Figure 7. Histopathological study of kidney sections of Slc7a9-/- and Slc7a9+/+ mice (periodic acid-Schiff stained). (A) Dilatation of the renal pelvis caused by the presence of a calculus in a Slc7a9-/- mouse (10x). (B) Tubular dilatation with flattening of renal tubular epithelial cells in the renal cortex of an Slc7a9-/- mouse (100x). (C) Tubular necrosis and tubular hyaline droplets (rounded and densely fuchsin-stained, pointed by arrowheads), both sign of atrophy of the functional tissue in the renal medulla of an Slc7a9-/- mouse (400x). (D) Chronic interstitial nephritis characterised by interstitial inflammatory infiltrate and tubular loss in the renal cortex of an Slc7a9-/- mouse. (E)–(H) Equivalent kidney sections from Slc7a9+/+ mice. pd, pelvis dilatation; m, medulla; c, cortex; t, tubule; gl, glomerulus; icn, interstitial chronic nephritis.

 
In cystine lithiasis the bladder increases its size (Fig. 5D–F). Thus, the weight of the bladder (mean±SEM; n=7) was 33±6 mg in Slc7a9+/+ mice, 42±9 mg in non-lithiasic Slc7a9-/- mice and 211±32 mg in lithiasic Slc7a9-/- mice (~6-fold higher than in the two other groups; P<0.01). Preliminary comparison of the urinary bladder of Slc7a9-/- mice with Slc7a9+/+ mice showed urothelial hyperplasia and chronic inflammation (data not shown).

Some lithiasic Slc7a9-/- mice suffered sporadic weight loss, attributable to pain induced by obstruction and friction of stones. When weight loss and abnormal behavior were detected, mice were treated with analgesics until recovery or sacrificed. However, the weight progression of Slc7a9-/- mice was not significantly different from that of Slc7a9+/- and Slc7a9+/+ mice (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Animal models of human diseases have proven their value for obtaining insight into pathogenic mechanisms and for testing therapeutic drugs and innovative treatment protocols. With both applications in mind we planned to generate a mouse model of cystinuria by targeted disruption of Slc7a9. Evidence that Stones, the knockout mouse described in this study, is a valid model of human cystinuria is based on the disruption of Slc7a9 gene, the deficiency in b0,+AT protein, impairment of the renal reabsorption of cystine and dibasic amino acids, cystine crystalluria and cystine urolithiasis.

Analysis of genomic DNA in Stones mice demonstrates the absence of the normal gene. The normal messenger is not present in any of the target tissues of Slc7a9 (i.e. kidney and small intestine). Instead, there is a smaller messenger, whose size and sequence correspond to an aberrant Slc7a9 spliced variant that would render a soluble protein of 41 amino acid residues (the first N-terminal 29 amino acid residues plus 12 missense amino acid residues). This is consistent with the finding that renal brush border membranes of Stones mice are totally deficient in b0,+AT protein.

The physiological proof of the impairment of cystine and dibasic amino acid reabsorption in the kidney of all Stones mice is the massive hyperexcretion of cystine and dibasic amino acids with subnormal levels of these amino acids in plasma. This hyperexcretion in urine may explain the low levels in plasma. Reduced levels of the four amino acids have also been reported in human cystinuria (23,24). Moreover, Stones mice present cystine crystalluria, as a result of the high urine excretion of cystine combined with its low solubility. Finally, Stones mice develop cystine calculi in the urinary system. Most calculi are located in the urinary bladder, where urine accumulates. Less commonly, calculi can also appear in the renal pelvis, ureter and male urethra. This lithiasic phenotype resembles that of human classic cystinuria. Indeed, the first cystine stone discovered was found in the urinary bladder (25), and the most common location for calculi in patients with cystinuria is pelvi-calyceal. In humans, when cystinuria is symptomatic, cystine lithiasis occurs early in life (9). Thus, most symptomatic patients have their first calculus identified before the age of 20 (83%). This corresponds well with the early appearance, in the first 3 months of life, of calculi detected by radiography in Stones mice.

Lithiasis in Stones mice produces hydronephrosis, the characteristic consequence in humans (26,27) and animals (28,29) of urinary outflow obstruction. This includes tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis. Some Stones mice died from complications from cystine lithiasis, like hyperuremic shock or sepsis.

The murine cystinuria described here strongly resembles human classic cystinuria type B with phenotype non-I (9). The urinary excretion of the affected amino acids in type B patients (i.e. those with two mutations in SLC7A9) is, compared with control individuals: 33-fold (cystine); 35-fold (lysine); 175-fold (arginine); and 74-fold (ornithine) (9). Similarly, Stones mice excrete 76-fold (cystine), 216-fold (lysine), 267-fold (arginine) and 120-fold (ornithine) that of Slc7a9+/+ mice The higher hyperexcretion in cystinuric mice might be the reflection of a higher amino acid renal load. System b0,+ is believed to be the main renal reabsorption system of cystine, whereas other transport systems should play a role in the reabsorption of dibasic amino acids, as discussed elsewhere (10). Lysine shows the largest difference in amino acid hyperexcretion between cystinuric mice and humans. This suggests a more relevant role of system b0,+ in the renal reabsorption of lysine in mice than in humans. Most type B carriers show an increase in the excretion of the four amino acids (phenotype non-I): 8-fold (cystine), 9-fold (lysine), 6-fold (ornithine) and 6-fold (arginine) that of control individuals (8). Similarly, Slc7a9+/- mice excrete 14-fold (cystine), 3-fold (lysine), 2-fold (arginine) and 2-fold (ornithine) that of Slc7a9+/+ mice. In contrast, phenotype I human carriers do not hyperexcrete these amino acids in urine (9). The fact that hyperexcretion of dibasic amino acids in Slc7a9+/- mice is lower than in human type B heterozygotes suggests that mice are gifted with an excess of system b0,+ function. Thus, 24% expression of b0,+AT results only in a 2-fold excretion of these amino acids. Stones mice provide a useful model to study the biology of the rBAT/b0,+AT heterodimeric complex. On the one hand, it has been shown that b0,+AT increases the stability of rBAT when overexpressed in cultured cells (11,13). In agreement with this, Stones mice, which are fully deficient in b0,+AT, showed reduced expression of rBAT in renal brush-border membranes. On the other hand, co-immunopurification studies showed that all b0,+AT in human and mouse renal brush-border membranes heterodimerizes with rBAT, whereas part of the rBAT protein heterodimerizes with unidentified light subunit(s) (10). Our findings in Stones mice fully support these results, because in the absence of b0,+AT the remaining rBAT protein has the electrophoretic mobility of a heterodimer in renal brush-border membranes.

Stones mice also provide a useful model to study the mechanisms underlying phenotypes I and non-I in cystinuria. Mutations in either of the two subunits that form system b0,+ (i.e. the rBAT/b0,+AT heterodimeric complex) probably explain all cases of human classic cystinuria (9). All the mutations identified in rBAT, including mild and severe mutations with large SLC3A1 deletions, cause phenotype I in cystinuria (i.e. normal excretion of cystine and dibasic amino acids in heterozygotes) (9). In contrast, most mutations of b0,+AT cause phenotype non-I (i.e. hyperexcretion of cystine and dibasic amino acids in heterozygotes), whereas mild b0,+AT mutations (i.e. with significant residual transport activity) cause phenotype I (8,9). Very recently it has been shown that b0,+AT is the ‘catalytic subunit’ of system b0,+AT (11). This indicates that severe mutations in the ‘catalytic subunit’ in heterozygosis have a greater impact on renal reabsorption than mutations in rBAT. Interestingly, patients homozygous for rBAT (type A cystinuria) or b0,+AT (type B cystinuria) show the same levels of hyperexcretion of cystine and dibasic amino acids (9). In this scenario, Slc7a9+/- mice provide a new clue: the absence of b0,+AT results in phenotype non-I. In all, these data support the following hypothesis for a mechanism of physiopathology in cystinuria: (i) the amount of b0,+AT controls the expression of the functional rBAT/b0,+AT heterodimeric complex; (ii) the rBAT protein is produced in excess in kidney, and therefore an rBAT mutation in heterozygosis does not lead to hyperexcretion of amino acids; (iii) interaction with b0,+AT stabilizes rBAT, and the excess of rBAT is degraded; and (iv) a half-dose of b0,+AT, results in half-expression of rBAT/b0,+AT heterodimer, and therefore of the system b0,+ reabsorption activity that causes hyperexcretion of cystine and dibasic amino acids.

Stones mice may show that modifier genes are involved in the development of cystine calculi. This possibility in human cystinuria is suggested by certain clues: (i) marked differences between siblings sharing the same mutations (i.e. one sibling can have very aggressive lithiasic phenotype while the other does not develop calculi) (30); (ii) in the cohort of patients of the International Cystinuria Consortium, 12 (three of them older than 40) out of 224 patients with cystinuria did not develop renal calculi (9); and (iii) even when the severity of SLC7A9 mutations can be correlated with the urinary excretion of amino acids in carriers (8), renal stone formation cannot be directly correlated with amino acid urinary excretion in patients (9). It therefore appears that, although mutations in the two known cystinuria genes are related to amino acid excretion, once a patient's urine becomes saturated with cystine, there are other factors, both environmental and genetic, that play a decisive role in calculi formation. A number of factors affecting renal calculi formation and evolution have been described for cystine and for other urolithiases (e.g. calcium oxalate lithiasis). These include the electrolyte urinary content, the presence of proteins like bikunin and other inter-alpha inhibitors (31) and osteopontin (32). Analysis of Stones mice can help us to disclose environmental and genetic factors. All Stones mice analyzed showed hyperexcretion of cystine in urine, but only 82% developed crystalluria and only 42% developed cystine lithiasis. The reason why some Stones mice produce cystine calculi while others did not is uncertain, but the mixed genetic background (strains 129P2 and C57BL/6) of the mice could be contributory. We are currently providing knockouts with a pure genetic background, by back-crossing heterozygous mice to C57BL/6J. When an appropriate generation is achieved, we will study the presence of calculi in the urinary system. A change in the frequency of lithiasis in Slc7a9-/- mice with different genetic backgrounds will confirm whether modifier genes are involved in the cystine lithiasis phenotype. Mouse genetics will help to identify these genes.

Human cystine calculi are removed by surgery and, in the case of recently formed calculi, by extracorporeal shock wave lithotripsy. Conservative treatment in patients with cystinuria combines increased oral fluid intake, reduction of sodium in the diet, moderate reduction of protein intake and urine alkalinization. Pharmacological treatment with thiols, although quite effective, is reserved for the most severe cases, due to the number and severity of their secondary effects (18,33). Lithiasic Stones mice will hopefully be useful for testing new therapeutic and metaphylaxis protocols for cystinuria.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of Slc7a9-/-mice
15.4 kb of mouse Slc7a9 genomic DNA was isolated by screening a 129/SvJ library (Stratagene) using mouse Slc7a9 cDNA as a probe. Using this genomic clone and the pSP72 vector (Promega), a replacement vector was constructed (Fig. 1A). This has a 2.8 kb left homology arm containing exons 1–2, a 1.6 kb PGK-neo cassette (in antisense orientation), a 2.9 kb right homology arm containing exons 10–11, and a 2.7 kb PGK-tk cassette. E14-1 embryonic stem (ES) cells (34) were electroporated (Biorad Gene Pulser at 500 µF, 240 V) with the PvuI-linearized targeting vector, and selected in a medium containing 170 µg/ml G418 (Life Technologies) and 2 µM gancyclovir (Syntex). Resistant ES clones were picked after 7–9 days of double selection, and cell clones were established. To screen for recombinant events, ES cell genomic DNA was digested with BamHI and analyzed by Southern blotting using a BamHI–KpnI probe upstream the left arm of homology. KpnI-digested DNA and a 3'-external probe (SE3 in Fig. 1A) were used for further analysis of positive recombinant clones. Slc7a9 heterozygous ES cells from two targeted clones were microinjected into C57BL/6J blastocysts, which were transferred into uteri of pseudopregnant CD1 females. Chimeric male progeny were crossed to C57BL/6J females (Charles River). Germline transmission of the disrupted allele was detected in the agouti progeny by PCR from tail-biopsy specimens. The same PCR was used to genotype subsequent generations. The forward primer (BreF: 5'-CTGTTCTGTTCTGACCAACTGAGGGGCA-3') is on the left homology arm. One reverse primer specific for the wild-type allele (NorR: 5'-GATACAGATGCCACTGAGAAGACCCACC-3') anneals to the deleted region, and another reverse primer specific for the recombined allele (RecR: 5'-ATTCGCAGCGCATCGCCTTCTATCGCC-3') anneals to the Neo gene. The expected sizes of wild-type and targeted alleles are shown in Figure 1C. Only clone 291 was successful in germline transmission. Analysis thus far has been carried out on the hybrid C57BL/6J-129P2/OlaHsd background. All animals were housed in the Animal Care Unit of the Institut de Recerca Oncològica (IRO; Barcelona) in accordance with animal care guidelines. All procedures were approved by the IRO Animal Use and Care Committee.

mRNA analysis
Mouse kidney cortex and medulla, and jejunum RNA were isolated from adult male mice using the Tripure Isolation Reagent (Roche), according to the manufacturer's directions. Aliquots of RNA were separated on a formaldehyde agarose gel and transferred to nylon, and Slc7a9 mRNA was detected with a 32P-labeled probe. The probe was a PCR product from the Integrated Molecular Analysis of Genomes and their Expression (IMAGE) clone 578502. A portion of Slc7a9 cDNA (NM_021291) between positions 1337 and 1811 was amplified using primers P6oli12F (5'-GCTTAAAGTGCTCT CCTACATC-3') and P6oli1R (5'-GGGCTACGAGTGAT GGACCTT-3'). RT–PCR analysis of the recombinant kidney mRNA was performed on Slc7a9-/- mice. Reverse transcription was carried out with the First-strand cDNA Synthesis Kit (Amersham), according to the manufacturer's instructions. The cDNA was amplified with several primers, all of them yielding the expected sizes. The forward primers were: P6oli16F.2 (5'-GTCTTTCTATGTACCCCAAT-3') and P6oli1F (5'-ATG AGAAATCCACCCACAGTA-3'), and the reverse primers were P6oli1R (5'-GGGCTACGAGTGATGGACCTT-3'), P6oli2R (5'-ACAATGATAGGGATGAAGAGG-3') and P6oli3R (5'-CCAGGGATGATGTAAATGATG-3'). PCR products were purified and sequenced with the same primers.

Protein analysis
Brush-border membrane vesicles of mouse kidney were prepared by the Ca2+ precipitation method (35). N-ethylmaleimide at 5 mM was present in all buffers used (except in the resuspension buffer) following Wang and Tate (36), to prevent artifactual reduction/shuffling of disulfides. The membranes were kept at -80°C until use. Brush-border membrane vesicles were obtained from the cortex and medulla of adult male mice of the three genotypes from the F2 generation. The protein content of the membrane preparations was measured by the method of Bradford (37) using {gamma}-globulin as a standard. Proteins were boiled in the presence or absence of 100 mM dithiothreitol (DTT), separated using SDS–PAGE (20 µg protein per lane), transferred and probed with a rabbit polyclonal anti-mouse b0,+AT antibody (10) and with a rabbit polyclonal anti-rabbit rBAT antibody MANR-X (38), followed by enhanced chemiluminiscence detection (Amersham) as described (10,38). As a loading control, the membranes were Ponceau S stained [0.5% (w/v) Ponceau S in 1% (v/v) acetic acid].

Urine and plasma collection and analysis
From each experimental animal, urine was collected in a mouse metabolic cage (Tecniplast) after a 2-day adaptation period. Twenty-four-hour urine from three consecutive days was collected and immediately frozen. Thawed urine was mixed with an equal part of 0.4 M homoarginine in 0.1 M HCl (as a standard for the HPLC analysis) and deproteinized by ultrafiltration through a 10 000 nominal molecular weight limit regenerated cellulose membrane (Millipore). For cystine analysis, urine samples of Slc7a9-/- mice were diluted 20-fold to solubilize possible cystine crystals prior to the addition of the homoarginine standard. Blood was collected by cardiac puncture of anesthetized animals with a heparinized syringe. After a 5 min centrifugation at 1500g, plasma was recovered, mixed with the standard and deproteinized like the urine. This was done immediately after blood collection to prevent cystine binding to proteins. Quantitative analysis of amino acids was performed by pre-column derivatization with phenylisothiocyanate (PITC) followed by reverse-phase high-performance liquid chromatography (HPLC) in a Waters apparatus with a digital computer according to the Waters PICO-TAG procedure.

Calculi detection and analysis
Calculi in the urinary system were either detected by X-ray radiography (at 28 kV, 16 mA·s) or detected and recovered during necropsy. Calculi composition was analyzed with an infrared spectrometer (Brucker), after mixing a fragment with KBr, pulverizing it in an agate mortar and pressing the mixture at 10 atm for 10 min.

Crystalluria
Crystalluria was detected in urine collected on a Petri dish after spontaneous voiding. Crystals were observed under an Olympus BX60 microscope, at a magnification of x1000.

Histology
Animals were sacrificed by CO2 and tissues were fixed with 4% paraformaldehyde O/N at 4°C. Paraffin sections of 4 µm were stained with hematoxylin and eosin or with periodic acid-Schiff, examined and photographed with a light microscope Nikon Eclipse E-800 or a lens Leica MZ125.

Statistical analysis
Data are expressed as mean±SEM. One-way ANOVA, Bonferroni and Dunnett T3 tests were used to compare values between the different genotypes. Expected and experimental frequencies were compared by chi-squared test. The level of significance was set at P<0.05.


   ACKNOWLEDGEMENTS
 
We thank Marta Oset from the Transgenic Unit Animal Research Center (Parc Científic de Barcelona) for the microinjection of the clones, and David Solanes and co-workers at the Animal Care Unit of the IRO for their support with the mice. We thank Robin Rycroft for editorial help. This study was supported in part by the Spanish Ministry of Science and Technology PM99-017-CO-01/02 (M.P., V.N.), Fundació La Marató de TV3 ref. 98/1930 (V.N.), BIOMED BMH4 CT98-3514 (V.N., M.P.), the support of the Comissionat per a Universitats i Recerca (M.P.) and grants 2001SGR00118 (A.Z., M.P.) and 2001SGR00399 (V.N.) de la Generalitat de Catalunya (Spain), and by Instituto de Salud Carlos III networks C03/07 (V.N.) and G03/054 (M.P., V.N.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Centre de Genètica Mèdica i Molecular, Institut de Recerca Oncològica, Gran Via de Les Corts Catalanes s/n km 2,7, L'Hospitalet de Llobregat, Barcelona 08907, Spain. Email: vnunes{at}iro.es Back

{dagger} Present address: Genes and Disease Program, Centre de Regulació Genòmica, 08003 Barcelona, Spain. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Palacín, M., Goodyer, P., Nunes, V. and Gasparini, P. (2002) Cystinuria. In The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York. Available at: www.genetics.accessmedicine.com.

  2. Calonge, M.J., Volpini, V., Bisceglia, L., Rousaud, F., de Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X. et al. (1995) Genetic heterogeneity in cystinuria: the SLC3A1 gene is linked to type I but not to type III cystinuria. Proc. Natl Acad. Sci. USA, 92, 9667–9671.[Abstract/Free Full Text]

  3. Calonge, M.J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F. et al. (1994) Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. Nat. Genet., 6, 420–425.[CrossRef][ISI][Medline]

  4. Bisceglia, L., Calonge, M.J., Totaro, A., Feliubadaló, L., Melchionda, S., Garcia, J., Testar, X., Gallucci, M., Ponzone, A., Zelante, L. et al. (1997) Localization, by linkage analysis, of the cystinuria type III gene to chromosome 19q13.1. Am. J. Hum. Genet., 60, 611–616.[ISI][Medline]

  5. Wartenfeld, R., Golomb, E., Katz, G., Bale, S.J., Goldman, B., Pras, M., Kastner, D.L. and Pras, E. (1997) Molecular analysis of cystinuria in Libyan Jews: exclusion of the SLC3A1 gene and mapping of a new locus on 19q. Am. J. Hum. Genet., 60, 617–624.[ISI][Medline]

  6. Feliubadaló, L., Font, M., Purroy, J., Rousaud, F., Estivill, X., Nunes, V., Golomb, E., Centola, M., Aksentijevich, I., Kreiss, Y. et al. (1999) Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (b0,+AT) of rBAT. International Cystinuria Consortium. Nat. Genet., 23, 52–57.[ISI][Medline]

  7. Leclerc, D., Boutros, M., Suh, D., Wu, Q., Palacín, M., Ellis, J.R., Goodyer, P. and Rozen, R. (2002) SLC7A9 mutations in all three cystinuria subtypes. Kidney Int., 62, 1550–1559.[CrossRef][ISI][Medline]

  8. Font, M., Feliubadaló, L., Estivill, X., Nunes, V., Golomb, E., Kreiss, Y., Pras, E., Bisceglia, L., d'Adamo, A.P., Zelante, L. et al. (2001) Functional analysis of mutations in SLC7A9, and genotype–phenotype correlation in non-type I cystinuria. Hum. Mol. Genet., 10, 305–316.[Abstract/Free Full Text]

  9. Dello Strologo, L., Pras, E., Pontesilli, C., Beccia, E., Ricci-Barbini, V., De Sanctis, L., Ponzone, A., Gallucci, M., Bisceglia, L., Zelante, L. et al. (2002) Comparison between SLC3A1 and SLC7A9 cystinuria patients and carriers: a need for a new classification. J. Am. Soc. Nephrol., 13, 2547–2553.[Abstract/Free Full Text]

  10. Fernández, E., Carrascal, M., Rousaud, F., Abián, J., Zorzano, A., Palacín, M. and Chillarón, J. (2002) rBAT-b(0,+)AT heterodimer is the main apical reabsorption system for cystine in the kidney. Am. J. Physiol. Renal Physiol., 283, F540–F548.[Abstract/Free Full Text]

  11. Reig, N., Chillarón, J., Bartoccioni, P., Fernández, E., Bendahan, A., Zorzano, A., Kanner, B., Palacín, M. and Bertran, J. (2002) The light subunit of system b(o,+) is fully functional in the absence of the heavy subunit. EMBO J., 21, 4906–4914.[CrossRef][ISI][Medline]

  12. Chairoungdua, A., Segawa, H., Kim, J.Y., Miyamoto, K., Haga, H., Fukui, Y., Mizoguchi, K., Ito, H., Takeda, E., Endou, H. et al. (1999) Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein. J. Biol. Chem., 274, 28845–28848.[Abstract/Free Full Text]

  13. Pfeiffer, R., Loffing, J., Rossier, G., Bauch, C., Meier, C., Eggermann, T., Loffing-Cueni, D., Kuhn, L.C. and Verrey, F. (1999) Luminal heterodimeric amino acid transporter defective in cystinuria. Mol. Biol. Cell, 10, 4135–4147.[Abstract/Free Full Text]

  14. Chillarón, J., Roca, R., Valencia, A., Zorzano, A. and Palacín, M. (2001) Heteromeric amino acid transporters: biochemistry, genetics, and physiology. Am. J. Physiol. Renal Physiol., 281, F995–F1018.[Abstract/Free Full Text]

  15. Chow, G.K. and Streem, S.B. (1996) Medical treatment of cystinuria: results of contemporary clinical practice. J. Urol., 156, 1576–1578.[CrossRef][ISI][Medline]

  16. Barbey, F., Joly, D., Rieu, P., Mejean, A., Daudon, M. and Jungers, P. (2000) Medical treatment of cystinuria: critical reappraisal of long-term results. J. Urol., 163, 1419–1423.[CrossRef][ISI][Medline]

  17. Crawhall, J.C. and Watts, R.W. (1968) Some complications observed in the treatment of cystinuria with D-penicillamine and N-acetyl-D-penicillamine. Postgrad. Med. J. (suppl.), 8–11.

  18. Dahlberg, P.J., van den, B., Kurtz, S.B., Wilson, D.M. and Smith, L.H. (1977) Clinical features and management of cystinuria. Mayo Clin. Proc., 52, 533–542.[ISI][Medline]

  19. DiBartola, S.P., Chew, D.J. and Horton, M.L. (1991) Cystinuria in a cat. J. Am. Vet. Med. Assoc., 198, 102–104.[ISI][Medline]

  20. Hoppe, A., Denneberg, T., Jeppsson, J.O. and Kagedal, B. (1993) Urinary excretion of amino acids in normal and cystinuric dogs. Br. Vet. J., 149, 253–268.[ISI][Medline]

  21. Bovée, K.C., Bush, M., Dietz, J., Jezyk, P. and Segal, S. (1981) Cystinuria in the maned wolf of South America. Science, 212, 919–920.[Abstract/Free Full Text]

  22. Henthorn, P.S., Liu, J., Gidalevich, T., Fang, J., Casal, M.L., Patterson, D.F. and Giger, U. (2000) Canine cystinuria: polymorphism in the canine SLC3A1 gene and identification of a nonsense mutation in cystinuric Newfoundland dogs. Hum. Genet., 107, 295–303.[CrossRef][ISI][Medline]

  23. Morin, C.L., Thompson, M.W., Jackson, S.H. and Sass-Kortsak, A. (1971) Biochemical and genetic studies in cystinuria: observations on double heterozygotes of genotype I–II. J. Clin. Invest., 50, 1961–1976.[ISI][Medline]

  24. Dent, C.E., Senior, B. and Walshe, J.M. (1954) Pathogenesis of cystinuria. II. Polarographic studies of metabolism of sulfur containing amino acids. J. Clin. Invest., 33, 1216–1226.[Medline]

  25. Wollaston, W.H. (1810) On cystic oxide, a new species of urinary calculus. Phil. Trans. Roy. Soc. London, 100, 223–230.

  26. Moller, J.C. and Skriver, E. (1985) Quantitative ultrastructure of human proximal tubules and cortical interstitium in chronic renal disease (hydronephrosis). Virchows Arch. A. Pathol. Anat. Histopathol., 406, 389–406.[CrossRef][ISI][Medline]

  27. Hill, G.S. (1989) Basic physiology and morphology of hydronephrosis. In Uropathology. Churchill Livingstone, New York, p. 467.

  28. Greaves, P. (2000) Histopathology of Preclinical Toxicity Studies. Elsevier, Oxford, pp. 545–596.

  29. Ward, J.M., Mahler, J.F., Macronpo, R.R., Sunberg, J.P. and Frederikson, R.M. (2000). Pathology of Genetically Engineered Mice. Iowa State University Press, Iowa, pp. 347–351.

  30. Dello Strologo, L., Carbonari, D., Gallucci, M., Gasparini, P., Bisceglia, L., Zelante, L., Rousaud, F., Nunes, V., Palacin, M. and Rizzoni, G. (1997) Inter and intrafamilial clinical variability in patients with cystinuria type I and identified mutation. J. Am. Soc. Nephrol., 8, 388A.

  31. Iida, S., Peck, A.B., Johnson-Tardieu, J., Moriyama, M., Glenton, P.A., Byer, K.J. and Khan, S.R. (1999) Temporal changes in mRNA expression for bikunin in the kidneys of rats during calcium oxalate nephrolithiasis. J. Am. Soc. Nephrol., 10, 986–996.[Abstract/Free Full Text]

  32. Xie, Y., Sakatsume, M., Nishi, S., Narita, I., Arakawa, M. and Gejyo, F. (2001) Expression, roles, receptors, and regulation of osteopontin in the kidney. Kidney Int., 60, 1645–1657.[CrossRef][ISI][Medline]

  33. Crawhall, J.C. (1981) Experience with penicillamine in the treatment of cystinuria. J. Rheumatol. Suppl., 7, 100–102.[Medline]

  34. Kuhn, R., Rajewsky, K. and Muller, W. (1991) Generation and analysis of interleukin-4 deficient mice. Science, 254, 707–710.[Abstract/Free Full Text]

  35. Malathi, P., Preiser, H., Fairclough, P., Mallett, P. and Crane, R.K. (1979) A rapid method for the isolation of kidney brush border membranes. Biochim. Biophys. Acta, 554, 259–263.[Medline]

  36. Wang, Y. and Tate, S.S. (1995) Oligomeric structure of a renal cystine transporter: implications in cystinuria. FEBS Lett., 368, 389–392.[CrossRef][ISI][Medline]

  37. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254.[CrossRef][ISI][Medline]

  38. Furriols, M., Chillarón, J., Mora, C., Castelló, A., Bertran, J., Camps, M., Testar, X., Vilaró, S., Zorzano, A. and Palacín, M. (1993) rBAT, related to L-cysteine transport, is localized to the microvilli of proximal straight tubules, and its expression is regulated in kidney by development. J. Biol. Chem., 268, 27060–27068.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
P. Bartoccioni, M. Rius, A. Zorzano, M. Palacin, and J. Chillaron
Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype
Hum. Mol. Genet., June 15, 2008; 17(12): 1845 - 1854.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. Font-Llitjos, L. Feliubadalo, M. Espino, R. Cleries, S. Manas, I. M. Frey, S. Puertas, G. Colell, S. Palomo, J. Aranda, et al.
Slc7a9 knockout mouse is a good cystinuria model for antilithiasic pharmacological studies
Am J Physiol Renal Physiol, September 1, 2007; 293(3): F732 - F740.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S. Porta, S. A. Serra, M. Huch, M. A. Valverde, F. Llorens, X. Estivill, M. L. Arbones, and E. Marti
RCAN1 (DSCR1) increases neuronal susceptibility to oxidative stress: a potential pathogenic process in neurodegeneration
Hum. Mol. Genet., May 1, 2007; 16(9): 1039 - 1050.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Fernandez, M. Jimenez-Vidal, M. Calvo, A. Zorzano, F. Tebar, M. Palacin, and J. Chillaron
The Structural and Functional Units of Heteromeric Amino Acid Transporters: THE HEAVY SUBUNIT rBAT DICTATES OLIGOMERIZATION OF THE HETEROMERIC AMINO ACID TRANSPORTERS
J. Biol. Chem., September 8, 2006; 281(36): 26552 - 26561.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
L. J. Van Winkle, J. K. Tesch, A. Shah, and A. L. Campione
System B0,+ amino acid transport regulates the penetration stage of blastocyst implantation with possible long-term developmental consequences through adulthood
Hum. Reprod. Update, March 1, 2006; 12(2): 145 - 157.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
M. Palacin, V. Nunes, M. Font-Llitjos, M. Jimenez-Vidal, J. Fort, E. Gasol, M. Pineda, L. Feliubadalo, J. Chillaron, and A. Zorzano
The Genetics of Heteromeric Amino Acid Transporters
Physiology, April 1, 2005; 20(2): 112 - 124.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page