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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Pediatrics, Federico II University, Naples; 2Sezione di Biologia e Genetica, Dipartimento di Scienze Biomediche e Biotecnologie, Università di Brescia, Brescia; 3Institute of Pediatrics, University of Foggia, Foggia; 4Department of Pathology, Federico II University, Naples; 5Telethon Institute of Genetics and Medicine, Naples; and 6Dipartimento di Scienze Biomediche, Università di Foggia, Foggia, Italy
Submitted 20 November 2006 ; accepted in final form 16 March 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cationic amino acid transport; hyperdibasicaminoaciduria; neonatal lethality
Clinical symptoms of LPI are variable and most frequently include vomiting, diarrhea, failure to thrive, growth retardation, hepatosplenomegaly, osteoporosis, episodes of coma, lung involvement (mainly as alveolar proteinosis), and chronic renal disease (20). Metabolic derangement in LPI is linked to impaired urea cycle function due to lower plasma levels of arginine and ornithine. This leads to hyperammonemia after protein-rich meals and increased orotic aciduria. No pathogenetic mechanisms are known for many of the main clinical findings such as hepatosplenomegaly, alveolar proteinosis, renal involvement and stunted growth. In addition, these clinical symptoms respond poorly to the common treatment of LPI, which consists of dietary restriction of proteins and citrulline supplementation.
LPI is caused by mutations in the solute carrier family 7A member 7 gene (SLC7A7) which encodes the y+LAT-1 protein. y+LAT-1 is linked by a disulfide bond to solute carrier family 3 member 2 (SLC3A2, previously named 4F2hc) and together they comprise an amino acid transporter belonging to the heterodimeric amino acid transporter (HAT) family (3, 15, 22, 23, 27). This transporter, located at the basolateral membrane of epithelial cells, induces system y+L activity, which mediates CAA transport (15). Recently, functional studies of mutant solute carrier family gene alleles have suggested that the HAT defective in LPI may have a multiheteromeric rather than heterodimeric structure (22).
The mouse Slc7a7 gene maps on chromosome 14 (syntenic region of human chromosome 14) and its predicted amino acid sequence is highly homologous to the human protein (90.4% identity; 98.6% similarity). The murine y+LAT-1 induces system y+L activity and can form disulfide-linked heterodimers with human 4F2hc (16).
To elucidate the complex and mostly unknown pathophysiology of LPI, we attempted to recapitulate LPI in a mouse model. In collaboration with Lexicon Genetics (The Woodlands, TX), a Slc7a7 mutant clone was recovered by screening of the embryonic stem (ES) cell library generated by retroviral gene trapping and used to produce a Slc7a7-deficient mouse. In the present report we describe the analysis of phenotype of the Slc7a7-deficient mouse.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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OmniBank ES cell clone OST41878 was used to generate the Slc7a7–/– mouse. All mice used for the present study were of C57BL/6J genetic background obtained by backcrossing to C57BL/6J strain mice for at least 10 generations. Mice heterozygous for the mutant allele were bred to produce Slc7a7–/– mice.
Mice were housed at 24°C on a fixed 12:12-h light-dark cycle and had free access to water and rodent chow (Mucedola, Milan, Italy). All procedures involving animals were conducted in conformity with our Institutional Animal Care and Use Committee guidelines that are in compliance with the standards outlined in the guide for the Care and Use of Laboratory Animals.
Both Slc7a7–/– mice were fed a diet contained 8% protein (special diet produced ad hoc by Mucedola) and citrulline supplementation (100 mg/ml solution; administered at 100 µg/g of body wt/diet) (Sigma-Aldrich Italy).
Genotyping of Slc7a7 fetuses and mice colony. Genomic DNA was extracted by standard protocols from tail biopsies isolated from 3-wk-old progeny of matings and from embryonic day (E) 16.5 fetuses. Fetuses and mice were genotyped by PCR using primers A and B, located in intron 2 of the Slc7a7 gene and surrounding the gene-trap insertion site, and primer LTRrev (a vector specific primer) (Fig. 1A). The sequences for primers A and B are 5'-GATGAAGTGATCCTAGCCGTAG-3' (for A) and 5'-GCAGCTCTATGTCACAGGGCG-3' and LTRrev 5'-AAATGGCGTTACTTAAGCTAGCTTGC-3' (for B). DNA (100 ng) purified from mice and fetal tail genomic was used as template for PCR in a 25 µl reaction volume. PCR conditions were: 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 39 cycles. PCR-sexing of mouse fetuses was performed as described in Ref. 12.
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Anatomical examination and insulin-like growth factor 1 immunohistochemistry. Detailed gross and histological examinations were performed on whole mount fetuses and selected tissues such as intestine, liver, spleen, lung, kidney, and brain isolated from fetuses and adult mice. Whole mount fetuses and selected tissues of each mouse (fetus and adult, respectively) were weighed and then fixed by immersion in 4% parafolmaldeyde for 24–48 h. After fixation, tissues were routinely processed and embedded in paraffin. Briefly, 4-µm-thick sections were deparaffined in xylene and rehydrated and then stained with hematoxylin and eosin for morphological assessment. Fixed liver tissues isolated from fetuses were processed for immunohistochemistry, as described in Ref. 10. Polyclonal goat antisera to mouse insulin-like growth factor 1 (Igf1) were used for the staining (Santa Cruz Biotechnology, Santa Cruz, CA). The sections were viewed with a Nikon microscope. Pictures were processed and assembled using Adobe PhotoShop. To prevent observer bias, all histological specimens were coded and examined without knowledge of animal age and genotype.
Igf1 Western blot analysis. Liver tissue extracts were isolated from fetuses and processed in lysis buffer with protease inhibitor cocktail. Ten micrograms of extracts were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membranes were incubated with polyclonal goat antisera to mouse Igf1 (Santa Cruz Biotechnology).
Quantitative real-time RT-PCR.
Real-time PCR was performed according to the recommendations supplied by Applied Biosystems (http://europe.appliedbiosystems.com/). All primers for real-time PCR were purchased from Applied Biosystems as AssaysOnDemand. All reactions were performed in triplicate. The abundance of the target mRNAs was calculated relative to a reference mRNA (
2 microglobulin). Relative expression ratios were calculated as R = 
, where R is ratio, Ct is the cycle number at the threshold, and test refers to the tested mRNA. The confidence interval was fixed at 95%.
Urine collection and analysis of amino acids and orotic acid in urine samples. Twenty-four-hour urine samples were obtained by placing the mice in specially designed minimetabolic cages. Quantitation of amino acids was performed by liquid chromatography/mass spectrometry (LC/MS). Orotic acid analysis was carried out by gas chromatography (GC)/MS.
Determination of ammonia concentration. Blood was taken from tails of Slc7a7–/– mice and wild-type controls. Blood ammonia levels were determined using the Ammonia Checker II system (Arkray), after dilution of blood samples 1:4 in water. Measurement range of the Ammonia Checker II system is 10–400N-µmol/dl (7–286 µmol/l).
Array hybridization, scanning, and data analysis. Liver and intestine were harvested from two Slc7a7–/– adult mice and two littermate controls of matching ages kept on the same diet as the mutant animals. RNAs were pooled according to the genotype. The array studies were conducted using the Operon Mouse Genome Oligo Set version 3.0 arrays, which contain 31,769 70-mer probes representing 24,878 genes and 32,829 gene transcripts. Two sets of dye-swap experiments were performed for each tissue analyzed, for a total of four replicate hybridizations.
Hybridization data were acquired using a confocal dual laser scanner and processed using GenePix Pro 4.1 (Axon Instruments). All statistical and graphical analysis were carried out in the R computing environment using the Limma package (21), which is part of the Bioconductor project (7). Within Limma, we performed local background subtraction and global Lowess normalization within each slide. After empirical Bayes analysis, genes were ranked as being differentially expressed in decreasing order of the B-statistic and in conjunction with P value obtained from moderated t-statistic. We choose those genes with B 
1, P value 
0.01 and M 1 or M –1 as "upregulated" and "downregulated" respectively. Genes were classified according to standardized Gene Ontology (GO) vocabulary using the Onto-Express (OE) software.
Accession numbers of nucleotide sequences. The accession numbers for the mouse Slc7a7 cDNA sequence is NM_011405; for the mouse Slc7a7 gene sequence, it is NT_039606.
Accession numbers of microarray data. NKI-CMF Mus musculus 32k oligo: accession number for array platform GPL3675 and accession number for expression datasets is GSE5432.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Genotyping of 606 pups generated in >200 Slc7a7+/– intercrosses revealed ratios of genotypes (28.4% wild-type, 68.6% heterozygotes, 3% Slc7a7–/– homozygotes) inconsistent with Mendelian segregation of a recessive trait. This indicated either the in utero demise of Slc7a7–/– fetuses or the death of Slc7a7–/– newborn animal shortly after birth. No difference of sex distribution was found among heterozygous mice; heterozygotes were healthy, fertile, and identical to wild-type littermates. Only 2 Slc7a7–/– mice survived, whereas 16 of them died within 24 h of birth. Eventually, the high rate of lethality allowed us to reach an almost pure C57BL/6J genetic background by backcrossings heterozygous offspring before the birth of the two surviving Slc7a7–/– mice.
The Slc7a7 lethality was further defined by genotyping fetuses at E16.5. At this developmental stage, no significant differences were found for segregation and sex ratios among the three genotypes. This confirmed the suspicion of maternal cannibalism of Slc7a7–/– pups soon after delivery. Fostering soon after birth was tried without success. Dietary supplementation of pregnant dams with either citrulline or arginine did not increase the survival rate of Slc7a7–/– animals at birth.
At E16.5 Slc7a7–/– fetuses appeared smaller than controls by gross examination (Fig. 2A) and presented a developmental delay of 2–3 days as observed by histological examination (9). At birth, Slc7a7–/– pups were smaller and appeared less vital than their littermates (Fig. 2B).
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63% of the initial weight), and died 15 days later. The second animal, a female, had several unsuccessful matings followed by a single pregnancy that ended prematurely on the day 18. All of the pups were cannibalized by the mother as evidenced by remnants of four pups. The dam displayed acute neurological symptoms (tremors, severe hypotonia, and nonresponsiveness to external stimuli) 3 h after delivery, and died shortly thereafter. Detailed gross and histological studies were performed, as described in MATERIALS AND METHODS. No significant differences were found between Slc7a7–/– and wild-type animals apart from bone developmental delay and spleen enlargement (weights of spleens: 240 mg, Slc7a7–/– male mouse; 220 mg, Slc7a7–/– female mouse; 102 mg ± 5, controls; P < 0.003).
Biochemical investigations confirm metabolic derangement of LPI in adult Slc7a7–/– mice. The biochemical hallmarks of LPI are increased renal excretion of CAA and hyperammonemia after oral protein load. Therefore, we measured CAA urinary levels in both Slc7a7–/– animals when fed a restricted diet with citrulline supplementation and, in the male mouse, when it was put on a normal diet at 25 mo. The rapid and severe clinical course of the female Slc7a7–/– animal prevented collection of urine samples. Both Slc7a7–/– adult mice showed elevated urinary excretion of arginine, ornithine, and orotic acid when kept on a low-protein diet (Table 1). Lysine excretion strongly increased when the Slc7a7–/– male mouse was put on a normal diet without citrulline supplementation (Table 1). CAA and orotic acid renal excretions were not significantly different between wild-type and heterozygous mice. Hyperammonemia was found in both Slc7a7–/– animals in blood samples taken soon after death (mean ammonia concentration ± SE in µmol/l: 135.5 ± 40.8 for Slc7a7+/+; 137.3 ± 24.7 for Slc7a7+/–, 1,050 and 1,530 for the male and the female Slc7a7–/– mice, respectively).
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Intrauterine growth restriction of fetal Slc7a7–/– mice. All of the Slc7a7–/– mice showed intrauterine growth restriction (IUGR). In addition to the determination of CAA levels in amniotic fluids, the expression of genes involved in CAA transport (Slc7a1, Slc7a2, Slc7a6, and Slc3a2) was investigated in placentas of E16.5 fetuses by quantitative real-time RT-PCR. No significant differences were observed between Slc7a7–/– animals and controls. Downregulation of Slc7a2 (0.74 fold) was found in liver of Slc7a7–/– fetuses by quantitative real-time RT-PCR.
To elucidate the pathogenesis of severe IUGR we examined the fetal liver expression of genes encoding the insulin-like growth factors 1 and 2 (Igf1-2), and their binding proteins (Igfbp1-6). Igf1 and Igf2 mRNA levels were, respectively, 3.2 and 1.7-fold lower in Slc7a7–/– animals than in wild-type animals (Fig. 3A). In the Slc7a7–/– fetus that displayed the smallest body size, liver Igf1 and Igf2 were reduced by 7.4- and 4.0-fold, respectively (Fig. 3B). These results differ from those in the adult Slc7a7–/– animals, in which Igf1 and Igf2 expression levels were unaltered relative to controls (in liver the value of Igf1 was 0.968, whereas the value of Igf2 was 0.8925). Composite results were found for Igfbps in Slc7a7–/– fetuses: downregulation of Igfb1 (2.6-fold), upregulation of Igfbp2 (3.6-fold) and Igfbp6 (3.2-fold) (Fig. 4A). In the smallest Slc7a7–/– fetus, Igfbp1 and Igfbp4 were downregulated by 48- and by 8.3-fold, respectively. The same animal showed upregulation of Igfbp2, Igfbp5, and Igfbp6 by 7.4-, 3.5-, and 31.6-fold, respectively (Fig. 4B).
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Microarray analysis was carried out on small intestine and liver harvested from the two adult Slc7a7–/– mice and from two normal littermate controls kept on the same diet and citrulline supplementation. Pooling of RNAs was performed to reduce the variability that may occur with single sample, and to obtain sufficient amount RNA for DNA array hybridizations.
To facilitate the one-by-one analysis of differentially expressed genes, we used the Expression Analysis Systematic Explorer software to automate the process of creation of descriptive annotation tables. Furthermore, to translate the data into a more meaningful biological context and to highlight sets of functionally related genes, the differentially expressed datasets were subsequently organized into GO groupings using the OE software. Table 2 shows the results of the OE analysis of differentially expressed genes relative to the GO biological process categories. These combined analysis allowed us to pinpoint among the differentially expressed genes the ones that appear relevant to the molecular and clinical features of LPI and to the phenotype observed in Slc7a7–/– mice.
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In the next two sections, we report the results (confirmed by quantitative real-time RT-PCR) concerning transcripts, identified in the entire dataset of differentially expressed genes, that may have functional implications for the pathophysiology of LPI.
Dysregulation of urea cycle and arginine metabolism in adult Slc7a7–/– mice. Liver microarray expression profiles of genes involved in the urea cycle revealed upregulation of arginase type I (EC 3.5.3.1 [EC] ), argininosuccinate lyase (EC 4.3.2.1 [EC] ) and glutamate dehydrogenase 1 (EC 1.4.1.3 [EC] ) (3.0-, 3.0-, and 2.1-fold, respectively) and 2.3-fold downregulation of ornithine transcarbamylase (EC 2.1.3.3 [EC] ). In intestine, the isoform II of arginase (EC 3.5.3.1 [EC] ) was upregulated by 5.1-fold. In contrast to isoform I, arginase type II has a mitochondrial localization and transforms arginine into ornithine and urea in an alternative metabolic pathway.
CAA transporter genes differentially expressed in adult Slc7a7–/– mice. GO analysis revealed that the most differentially expressed genes in the intestine and liver are in the biological process category termed "transport".
Within the SLC7 family, the solute carrier family 7 member 2 gene (Slc7a2) showed a 4.3-fold upregulation in liver. However, the solute carrier family 7 member 9 gene (Slc7a9), which is involved in the transport of cationic plus neutral amino acids (system b0,+), was 7.0-fold downregulated in intestine. The solute carrier family 7 member 6 gene (Slc7a6) is ubiquitously expressed and encodes y+LAT-2, the light chain of the second transporter with y+L activity (26). The expression of Slc7a6 (not present on the DNA microarray chip) was tested by quantitative real-time RT-PCR and was found to be upregulated by 5.8 fold in liver from Slc7a7–/– mice.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In collaboration with Lexicon Genetics, we generated a genetically modified mouse in which Slc7a7 gene function was disrupted. Growth retardation was an ongoing feature of the two surviving Slc7a7–/– mice compared with normal littermates kept on the same diet and with citrulline supplementation.
An acute metabolic derangement, identical to that found in human LPI, was observed after a planned withdrawal of the special diet for the male mouse, and after an accidental protein overload for the female. This metabolic derangement had a fast onset and rapidly caused the death of both animals after severe hyperammonemic neurological symptoms. Other biochemical characteristics of human LPI, such as massive urinary excretions of arginine, ornithine, and orotic acid, were present even when both animals were kept on the special dietary regimen. Hyperlysinuria appeared only in the acute stage of the disease as seen in urine collected from the Slc7a7–/– male animal. The transient normal lysine excretion can be explained by the low protein content of the diet, similar to what is observed in human patients. In fact, excretion of lysine (as well as ornithine and arginine) may be within normal values when LPI patients spontaneously restrict their protein intake before the diagnosis is established (20). Gross and histological studies did not reveal organ-specific pathology apart from splenomegaly.
The neonatal lethality of the present LPI mouse model is in contrast to the natural history of human LPI where death may occur as a consequence of either an acute metabolic derangement or a severe complication such as pulmonary alveolar proteinosis. The lethality of the murine model is due to IUGR which is not present in human LPI patients although it was reported in unaffected children born to LPI mothers (25). Determination of amniotic CAA levels in Slc7a7–/– fetuses revealed values comparable to those found in normal controls thus ruling out CAA depletion as a primary cause of IUGR. In addition, no differences were found for placental expression profiles of genes involved in CAA transport (Slc7a1, Slc7a2, Slc7a6, and Slc3a2), with the exception of Slc7a7. To unravel the mechanism causing IUGR in Slc7a7–/– fetuses, we investigated the expression of the main genes controlling intrauterine growth: insulin-like growth factors (Igfs) and their binding proteins (Igfbps) (5). The Igf1 transcript was downregulated 3.2 fold in livers pooled from Slc7a7–/– fetuses. The smallestSlc7a7–/– fetus displayed the lowest Igf1 expression (7.4-fold lower than controls).
In support of the quantitative real-time RT-PCR data, both immunohistochemical labeling and Western blot analysis showed marked reduction of Igf1 signals. Igf2 was significantly underexpressed only in the smallest Slc7a7–/– fetus. Igf1 plays a pivotal role in promoting intrauterine growth and, during this developmental stage, is independent from growth hormone control. Like Slc7a7–/– fetuses, Igf1-deficient mice exhibit a 40% decrease in birth weight and >95% of puppies die perinatally (11, 17). Therefore, marked underexpression of Igf1 in Slc7a7–/– mice is sufficient to explain the early lethality. A complex pattern of dysregulation emerged from expression profiling of the genes encoding Igf binding proteins. Igfbps comprise a family of six homologous proteins that bind Igfs and control their bioavailability. At one extreme we found downregulation of Igfbp1 and Igfbp4, and at the other a strong upregulation of Igfbp2 and Igfbp6. The degree of downregulation of Igfbp1, a binding protein specifically produced in liver, seems to correlate with that of the Igf1 transcript. In fact, both Igfbp1 and Igf1 reached the lowest levels of expression in the smallest Slc7a7–/– fetus. It is known that Igfbp1 has an inhibitory role on Igf-stimulated growth and differentiation. In addition, IUGR caused by placental insufficiency and in utero hypoxia is associated with elevated levels of Igfbp1 (28, 29). The underexpression of Igfbp1 in Slc7a7–/– fetuses may simply reflect the inverse relationship between this binding protein and Igf1 (14). Transgenic models overexpressing either Igfbp2 or Igfbp6 show reduced postnatal growth of the entire body, for the former, or of selected organs, for the latter (2, 8). In Slc7a7–/– mice, dysregulation of the Igf axis is not related to defective transport of CAA across placentas. Our data suggest that the downregulation of Igf1 in Slc7a7–/– mice has to be explained by an altered intracellular CAA homeostasis due to Slc7a7 ablation. Postnatal growth delay, a frequent clinical finding in human LPI, is not caused by the protein-restricted diet but it might be related to the alteration of the GH/IGF1 axis as recently reported (6).
Furthermore, arginine has been demonstrated to stimulate IGF1 production and collagen synthesis in osteoblast-like cells but the intimate mechanism of regulation remains unclear (4).
To further explore the diverse regulation of genes likely related to LPI pathophysiology, microarray-based gene expression profiles were obtained from the intestine and liver of the two adult Slc7a7–/– mice. The rationale underlying the choice of these organs was that Slc7a7 is normally expressed in the intestine and not in the liver, even though the liver is also involved in the pathogenesis of LPI.
Our study revealed that the absence of a functional Slc7a7 mRNA results in the dysregulation of >400 and 500 transcripts in intestine and liver, respectively. The largest enriched GO category of differentially expressed genes corresponds to those involved in transport. This is not surprising because intestine expresses a large number of transporters, molecules that play a direct role in the absorption of various compounds from the lumen. Similarly, several transporter families mediate uptake of chemicals into, and excretion of chemicals from, the liver.
In the liver, upregulation of Slc7a2 and Slc7a6 may reflect the need to restore the intracellular CAA pool since both genes are involved in CAA transport. Upregulation of Slc7a6 was found in a previous study carried out in lymphoblasts from LPI patients (19). Interestingly, Slc7a6 has the same transport activity as Slc7a7 and is ubiquitously expressed. However, its overexpression, as seen in the present animal model, does not seem to produce a compensatory effect on CAA transport. Slc7a6 probably plays a different role which is still unclear. Transcripts encoding enzymes involved in the urea cycle are all upregulated with the exception of ornithine transcarbamylase. This general upregulation may be interpreted as an attempt to compensate for the hyperammonemic condition. In fact, conditions causing increased ammonia production such as starvation or high protein intake lead to increase of all urea cycle enzymes by a transcriptional control (24). As to the downregulation of the ornithine transcarbamylase transcript, this gene responds to other stimuli (e.g., glucocorticoids) differently from all the other enzymes of the urea cycle. In contrast to what is observed in Slc7a7–/– fetuses, in the adult Slc7a7–/– animals Igfbp1 was upregulated by 71-fold, representing the most differentially expressed transcript in liver. High levels of Igfbp1 may be responsible for the severe growth inhibition and the bone developmental delay observed in these Slc7a7–/– despite normal levels of Igf1 expression. This dramatic increase of Igfbp1 might also have related to the protracted critical illness as suffered by the male Slc7a7–/– mouse (13).
In the intestine, the Slc7a9 and Slc34a2 genes both display marked downregulation. Slc7a9 encodes the light chain of b(0,+)AT, the transport system of cystine and CAA across the apical membrane of epithelial cells of intestine and kidney. The 7.0-fold downregulation of this transcript might be explained as a compensatory mechanism to reduce intracellular accumulation of CAA in intestinal epithelial cells by hampering their transport at the basolateral membrane.
Slc34a2, which encodes a Na+-Pi cotransporter responsible for intestinal phosphate absorption (30), shows a 54.8-fold downregulation. Mutations of SLC34A1, the Na+-phosphate cotransporter expressed in kidney, are associated with hypophosphatemia and osteoporosis in human subjects (18). Whether defective Slc34a2 transport activity might contribute to phosphate depletion is still unclear, although osteoporosis is an early complication commonly observed in LPI patients. The underexpression of Slc34a2in intestine of Slc7a7–/– mice suggests new studies on mechanisms linking CAA and phosphate absorption.
The Slc7a7–/– mouse model described here offers new insights into the pathophysiology of LPI and into mechanisms linking CAA transport with prenatal growth control.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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