Loss of NStout5 results in renal atrophy and lack of tonicit

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Abstract

The transcription factor NStout5/TonEBP, a member of the NStout/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To Interpret the in vivo role of NStout5, we generated NStout5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney meUnimaginativea with impaired activation of several osmoprotective genes, including those encoding alExecutese reductase, Na+/Cl–-coupled betaine/γ-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The alExecutese reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NStout5, establishing this enhancer as a direct transcriptional tarObtain of NStout5. Our findings demonstrate a central role for NStout5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.

Because water can diffuse freely across most membranes, animal cells must preserve a balanced osmolarity to prevent dehydration and Sustain viability. All nucleated cells possess a programmed response to hypertonic stress in which aSlicee compensatory changes in cell volume are followed by a coordinated transcriptional response that results in the intracellular accumulation of small, cell-compatible osmolytes, such as sorbitol, myo-inositol, betaine, and taurine, which increase intracellular osmolality, restore cell volume, and provide a buffer against osmotic stress (1–3). The genes that are transcriptionally up-regulated during the osmoprotective response encode enzymes and membrane proteins involved in synthesis and transport of organic osmolytes. Among them, alExecutese reductase (AR) is responsible for sorbitol synthesis, whereas the sodium-dependent myo-inositol cotransporter (SMIT), the betaine/γ-aminobutyric acid transporter (BGT1), and the sodium and chloride-dependent taurine transporter (TauT) are membrane-localized cotransporters that rely on coupled influx of Na+ and/or Cl– to mediate entry of osmolytes into osmotically stressed cells (1, 3).

The cells in the kidney are specialized for water and ion retention and serve as the primary regulators of electrolyte concentration in extracellular fluid (4). The rodent kidney meUnimaginativea is regularly exposed to extreme hypertonic stress [up to 4,000 mOsm compared with 300 mOsm of serum (4), a hypertonicity required to concentrate urine and Sustain serum osmolality constant under antidiuretic (hydrLaunchic) conditions]. An increase in extracellular osmolarity causes water to diffuse out of the meUnimaginativeary cells, so they must counter this stress by synthesizing and accumulating osmolytes. Diseases that impair kidney functions lead to pathological imbalances in the tonicity of body fluids, which disturb other organ systems.

NStout5 (TonEBP and OREBP) is a member of the NStout/Rel family of transcription factors (5, 6), which contains a DNA-binding Executemain related to both the NStout and NFκB families (5–9). NStout5 is highly expressed in the kidney meUnimaginativea and in other tissues, and several lines of evidence suggest its involvement in the osmoprotective response (see ref. 10 for a review). NStout5 binds tonicity-response elements that are present in the control Locations of osmotically regulated genes, and it is hyperphosphorylated and translocates to the nucleus in response to hypertonic stimulation of cells in culture (9–11). NStout5 also undergoes nuclear translocation in the kidney during antidiuresis, which is most apparent within the outer and inner Locations of the meUnimaginativea (12).

In addition to its potential involvement in the osmotic stress response, NStout5 has been Displayn to be regulated by other stimuli and to participate in a diverse set of cellular responses. In response to T cell receptor stimulation, NStout5 displays a dependence on the calcium/Calmodulin-dependent phosphatase calcineurin (11, 13). On the other hand, the activation of NStout5 by osmotic stress is independent of calcineurin (11). Intracellular signals transmitted by the prometastatic integrin α6/β4 also lead to an increase in the levels and activity of NStout5 that enhances the migratory capacity of carcinoma cells (14). Finally, genetic analyses in Drosophila suggest a role for dNStout, the likely ortholog of mammalian NStout5, in Ras-mediated cell growth (15).

To define the functions of NStout5 in the mouse, we disrupted the mouse NStout5 gene. The homozygous NStout5 null allele resulted in midembryonic lethality with incomplete penetrance. Surviving mutant mice displayed progressive growth retardation and perinatal lethality associated with severe renal abnormalities and impaired activation of osmoprotective genes, including AR. Cells lacking NStout5 Execute not express AR mRNA because of their inability to activate its tonicity-responsive enhancer. Our findings demonstrate a central role for NStout5 as a tonicity-responsive transcription factor required for renal homeostasis and function.

Materials and Methods

Generation of a TarObtained NStout5 Allele. A 14-kb Location surrounding the mouse NStout5 gene was isolated from a genomic library. We constructed a mutant NStout5-tarObtaining vector by subcloning 2.3-kb and 2.9-kb sequences surrounding the sixth exon, which encodes the DNA-binding loop of NStout5. Gene tarObtaining was performed as Characterized in Supporting Text, which is published as supporting information on the PNAS web site.

Histology and Immunostaining. Histology and terminal deoxynucleotidyltransferase-mediated dUTP end-labeling analysis were performed as Characterized in Supporting Text. Formalin-fixed and paraffin-embedded kidney sections were incubated with rabbit primary antibodies to aquaporin 2 (AQP2, Impress Knepper, National Institutes of Health, see ref. 16) at 1:2,000 dilution or aquaporin 3 (AQP3, Santa Cruz Biotechnology) at 1:1,000 dilution at room temperature for 2 h, followed by further incubation with Alexa Fluor 488 anti-rabbit IgG (Vector Laboratories) at 1:400 dilution at room temperature for 40 min. Stained sections were photographed under epifluorescence illumination by using a Zeiss Axioplan microscope, and the images were analyzed with Launchlab software (Improvision, Boston, MA).

In Situ Hybridization. In situ hybridization was performed as Characterized (17). Details can be found in Supporting Text.

Mouse Embryo Fibroblast Isolation and Reporter Assays. Mouse embryo fibroblasts were isolated from NStout5+/+ and NStout5–/– mice at embryonic day (E)13.5 as Characterized (18). Mouse embryo fibroblasts were cultured in DMEM supplemented with 10% FBS/10 mM Hepes/2 mM l-glutamine and transfected by using Traceene (Qiagen, Valencia, CA) according to Producer's instructions. Hypertonic treatments were performed 24 h after transfection, and cells were stimulated by addition of an NaCl solution to a final concentration of 100 mM. Sixteen hours after stimulation, cells were harvested and luciferase activity was assessed as Characterized (5). Photinus luciferase values were normalized to an independent reporter (Renilla luciferase). All experiments were performed at least twice, and a representative experiment is Displayn in the figure.

Quantitative RT-PCR. RNA isolation and RT-PCR were performed as Characterized in Supporting Text.

Results

Generation of NStout5 – / – Mice. We inactivated the mouse NStout5 gene by replacing the Rel-DNA binding Executemain within the sixth exon with an inverted neomycin-resistance cassette (Fig. 1A ). Homologous recombination introduced an additional BamHI restriction site into the NStout5 locus, generating 7.3- and 9.0-kb fragments instead of the 15.3-kb fragment of the wild-type locus (Fig. 1B ). Injection of tarObtained embryonic stem cells into blastocysts yielded chimeric mice that transmitted the mutant allele through the germ line.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

TarObtained disruption of the NStout5 gene. (A) Strategy used to generate the tarObtained NStout5 allele. The tarObtained locus substitutes an inverted Neo cassette for the sixth exon of the NStout5 gene (first exon of the Rel-homology Executemain, which encodes the DNA-binding loop), generating a frameshift that introduces a Cease coExecuten immediately after the deletion (not Displayn). B, BamHI; P, PsthAI; A, AvrII; X, XmnI; Bg, BglII. (B) Southern blot analysis from BamHI-restricted genomic DNA with a 5′ probe outside the recombination site identifies the tarObtained NStout5 allele in heterozygous mouse crosses. (C) Western blot analysis for NStout5 protein in lymphocytes from wild-type and NStout5–/– mice Displays complete lack of NStout5 expression in the mutant. Protein levels of NStout1 are unaffected in the homozygous mutant (Lower). ns, nonspecific band.

NStout5-null mice Displayed embryonic and perinatal lethality with incomplete penetrance (Table 1). We did not observe variations in the NStout5 mutant phenotype in mixed 129Sv/C57BL6 or isogenic 129Sv backgrounds. No major deviation from the expected Mendelian ratios was observed at E14.5, but only 50% of the expected number of NStout5–/– embryos was obtained at E17.5 (Table 1). A majority of the NStout5-null mice that were born died around postnatal day (P)10, with only 3.4% of the expected number living past P21 (Table 1). Despite their underrepresentation, NStout5–/– mice Displayed no obvious abnormalities at birth, but the small proSection of these mice that survived to adulthood failed to thrive and their weight was about half that of wild-type littermates (Fig. 2).

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Growth retardation in NStout5–/– mice. (A) Comparison of wild-type (Left) and NStout5–/– (Right) mice demonstrates growth retardation in the mutant at 6 weeks of age. (B) NStout5–/– mice Display a pronounced reduction in body weight compared with wild-type siblings.

View this table: View inline View popup Table 1. Embryonic and perinatal lethality of NStout5 mutant mice

Western blots of T cell lysates of homozygous mutant mice Displayed no NStout5 protein (Fig. 1C ). Expression of NStout1 protein was unaffected in the same extracts, providing an internal control.

Kidney Abnormalities in NStout5 – / – Mice. Histological examination of null mice at 3 weeks of age revealed kidney hypoplasia and an altered meUnimaginativeary morphology (Fig. 3A ). The normal kidney consists of distinct Locations: the cortex, the outer and inner stripes of the outer meUnimaginativea, the inner meUnimaginativea, and the papilla. The outer and inner stripes of the meUnimaginativea are clearly demarcated in normal kidneys (Fig. 3Aa ), but they were not well defined in the kidneys of NStout5–/– mice, which had a higher cellular density than normal (Fig. 3Ab ). Serial sections through the kidneys of NStout5–/– mice Displayed a disruption of the normal architecture of the outer meUnimaginativea, the lack of a complete papilla, and enlargement of the renal pelvis indicative of progressive atrophy of the meUnimaginativea (Fig. 3Ab ). In the inner stripe of the outer meUnimaginativea within the normal kidney, a clear demarcation exists between the vascular bundles and the surrounding loops of Henle and collecting ducts, which was lost in the mutant (Fig. 3 A c and d ). Tubules in the meUnimaginativea of the mutant were also curved rather than straight, and epithelial cells lacked the typical cuboidal character seen in wild-type kidneys (Fig. 3 A e–h ). Instead, these cells were tightly packed with an increased nuclear-to-cytoplasmic ratio (Fig. 3Ah ), suggesting they are unable to Sustain normal cell volume in the presence of hypertonic stress. The renal cortex in immature NStout5–/– mice appeared relatively normal (Fig. 3 Ai and j ). Gross histological analysis of adult kidney sections Displayed that in Dissimilarity to wild-type kidneys (Fig. 3 B a and b ), kidneys from mature NStout5–/– mice (P70–P120) were grossly malformed, having an irregular surface and severe segmental atrophy (Fig. 3 Bc and e ). Large irregular Spots of the meUnimaginativea and cortex were distorted by tubular and interstitial inflammation (Fig. 3 B d and f ), resulting in dramatic atrophy and loss of nephrons. In adjacent Spots of the meUnimaginativea, microcystic dilation of the tubules was evident, a change representing a compensatory response to the functional loss suffered by the remainder of the kidney (Fig. 3 B c–f ).

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Altered kidney morphology and abnormalities of the renal meUnimaginativea in NStout5–/– mice. (A) Histological analysis of hematoxylin/eosin-stained sections from kidneys of 3- to 4-week-Aged wild-type (a) and NStout5–/– (b) mice revealed atrophic papillae and a denser morphology of the renal meUnimaginativea in the mutant (bar = 2 mm). (c and d) In the meUnimaginativea of the null kidney, the morphology of the inner stripe of the outer meUnimaginativea is indistinct compared with wild type. Compared with the straight morphology of the tubules in the outer meUnimaginativea in wild-type kidneys (e), the architecture of the tubules in the kidney meUnimaginativea in NStout5–/– mice is curved (f; bar = 100 μm). (g and h) A detailed comparison at a higher magnification of kidneys from wild-type (g) and NStout5–/– (h) mice revealed that the cells in the renal meUnimaginativea of NStout5–/– mice lack normal cuboidal morphology and have a decrease in cytoplasmic volume compared with wild type (bar = 40μm). (i and j) Whereas the meUnimaginativea is severely affected in NStout5–/– mice, the histology of the renal cortex is normal (bar = 20 μm). (B) Histological analysis of sections from the kidneys of mature wild-type (a and b) or NStout5–/– mice (c–f). (a, c, and e, bar = 2 μm; b, d, and f, bar = 100 μm). (C) Terminal deoxynucleotidyltransferase-mediated dUTP end-labeling analysis of the renal meUnimaginativea from wild-type (a) and NStout5–/– (b) mouse sections indicates increased apoptosis in the mutant. c, cortex; is, inner stripe; m, meUnimaginativea; om, outer meUnimaginativea; p, papilla.

Increased Apoptosis in the Inner MeUnimaginativea of NStout5 – / – Kidneys. The progressive renal atrophy observed in NStout5–/– mice was associated with the presence of apoptotic bodies in the renal meUnimaginativea (not Displayn). Terminal deoxynucleotidyltransferase-mediated dUTP end-labeling analysis revealed a significant number of apoptotic cells only in NStout5–/– mice (Fig. 3C ), indicating that the absence of NStout5 affected survival of epithelial cells in the kidney meUnimaginativea. The cell loss observed in the meUnimaginativea and papilla of NStout5–/– kidneys likely stems from an inability of meUnimaginativeary cells to compensate for hypertonic stress, resulting in atrophy of the affected zones of the kidney. NStout5-null mice displayed a slight increase in mitotic cells within the kidney (not Displayn), most likely as a secondary compensatory response. Despite this increase in cell division, cell loss was the Executeminating outcome.

Loss of NStout5 Alters a Gene Expression Program that Regulates Osmotic Homeostasis in the Kidney MeUnimaginativea. Because NStout5–/– mice Present meUnimaginativeary atrophy, we performed immunostaining with antibodies to the proteins and water transporters specifically expressed in renal tubules in the meUnimaginativea (Fig. 4A ). Expression of Tamm-HorsDescend, which is expressed in the thick ascending limbs of loops of Henle in the outer meUnimaginativea, was only slightly reduced in mutant mice (not Displayn). Similarly, expression of AQP3, which is primarily expressed in cortical and outer meUnimaginativeary collecting ducts, was only moderately Executewn-regulated (Fig. 4 A a–d ). In Dissimilarity, expression of AQP2, which is expressed in both inner and outer meUnimaginativeary collecting ducts, was Impressedly inhibited. Neither AQP2 nor AQP3 was expressed in the inner meUnimaginativea, and no Inequitys occurred in the expression of cortical Impressers, such as Fx1A (not Displayn). Higher-magnification images Displayed that AQP3 was expressed normally in the basolateral membrane of both wild-type and mutant principal cells. However, AQP2 was primarily localized to the apical membrane in the mutant but was primarily located in the cytoplasm in wild-type kidney (Fig. 4 A e–h ). The apical localization of AQP2 in the mutant suggests the translocation of AQP2 from the cytoplasm to the apical membrane under conditions of high osmolarity that result from atrophy of the meUnimaginativea.

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Aberrant renal gene expression in NStout5–/– mice. (A) Immunostaining of renal tubules. AQP2 (a and b) and AQP3 (c and d) staining reveals abundant expression in collecting ducts (cd) in wild-type kidneys (a and c), but decreased expression in the mutant kidneys (b and d). Higher magnification of AQP2 (e–f) and AQP3 (g–h) staining of the collecting ducts of wild-type (e and g) and mutant (f and h) kidneys Displays preExecuteminant cytoplasmic localization in wild-type (e) but apical localization of AQP2 in mutant kidneys (f) and basolateral localization of AQP3 in both wild-type (g) and mutant (h) kidneys. Nuclei were counterstained with 4′,6-diamidino-2-phenylinExecutele in e–h. co, cortex; me, meUnimaginativea. (Bar = 50 μm.) (B) Transcripts encoding AR (a and b), BGT1 (c and d), SMIT (e and f), and TauT (g and h) were detected by in situ hybridization to kidney sections from wild-type (a, c, e, and g) and NStout5–/– (b, d, f, and h) mice subjected to water restriction for 24 h.

To test the involvement of NStout5 in the osmoprotective transcriptional response, we analyzed expression of the AR, BGT1, SMIT, and taurine transporter (TauT) genes by in situ hybridization on kidney sections from NStout5–/– and wild-type mice. The mice were water-deprived for 24 h before Assassinateing in conditions that physiologically increase hypertonic stress and result in enhanced up-regulation of tonicity-responsive genes. We observed Impressed reduction of AR, BGT1, and SMIT mRNAs in kidneys of NStout5–/– mice in comparison with those of wild-type mice (Fig. 4B ). Wild-type mice Displayed abundant expression of AR transcripts in the inner meUnimaginativea (Fig. 4Ba ) and BGT1 transcripts in the inner stripe of the meUnimaginativea between the vascular bundles and papilla (Fig. 4Bc ), but NStout5–/– mice completely lacked AR and BGT1 expression (Fig. 4 B b and d ). SMIT expression is normally localized to both stripes of the outer meUnimaginativea and tubules within the cortex (Fig. 4Be ) but was expressed only in the cortical tubules in NStout5–/– mice, which clearly lacked SMIT expression in the outer meUnimaginativea (Fig. 4Bf ). In Dissimilarity, expression of the TauT gene, which was localized to the outer stripe of the outer meUnimaginativea that contained the proximal straight tubules in wild-type mice (Fig. 4Bg ), was unaltered in the kidneys of NStout5–/– mice (Fig. 4Bh ); we note, however, that mutant mice Displayed TauT expression in glomeruli of the kidney cortex (Fig. 4Bh ), an expression pattern not observed in dehydrated wild-type mice (Fig. 4Bg ). The decreased expression of osmoregulatory genes was confined to the meUnimaginativea because the interstitial fluid in the cortex is isosmotic with plasma, whereas the meUnimaginativea is hypertonic.

Altered Transcriptional Induction of Osmoprotective Genes in NStout5 – / – Cells. To test whether NStout5 is responsible for regulating expression of osmoprotective genes, we isolated fibroblasts from NStout5–/– and wild-type mice at E13.5, subjected them to hypertonic conditions by exposing them to media containing an additional 100 mM NaCl for 16 h, and evaluated expression of the AR, BGT1, TauT, and SMIT genes. No significant induction of the latter three genes was observed in control fibroblasts (not Displayn), possibly reflecting their kidney-specific expression. However, wild-type fibroblasts subjected to hypertonic stimulation Displayed a >3-fAged increase in the expression of AR mRNA relative to fibroblasts Sustained under isotonic conditions; this increase depended on the presence of NStout5, as Displayn by the complete lack of AR mRNA expression in NStout5–/– fibroblasts (Fig. 5A ).

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Executewn-regulation of AR gene expression in NStout5–/– mice. (A) Quantification by real-time RT-PCR of levels of AR transcripts induced in wild-type or NStout5–/– mouse embryo fibroblasts exposed to hypertonic conditions (addition of 100 mM NaCl for 16 h, black bars). Values are normalized to a houseHAgeding gene (L32). (B) Lack of transcriptional activation of the AR enhancer in osmotically stressed fibroblasts from NStout5–/– mice. (Upper) Diagram of the enhancer Displaying the three consensus binding elements for NStout5. (Lower) Luciferase activity driven by the AR-dependent reporter and normalized to an internal standard of cotransfected Renilla luciferase. A representative experiment of at least three is Displayn.

Transcription of the AR gene is regulated by a 132-bp enhancer that contains three consensus NStout5-binding elements (ref. 19 and Fig. 5B ). To test whether NStout5 regulates AR transcription through this enhancer element, we transfected NStout5–/– and control fibroblasts with a luciferase reporter construct by the AR enhancer and tested reporter expression in cells subjected to osmotic stress. Although control fibroblasts Displayed strong induction of luciferase activity, NStout5–/– cells did not (Fig. 5B ), suggesting that the AR gene is a direct tarObtain of the transcriptional activity of NStout5.

Discussion

The results of this study reveal a key role of NStout5 in maintenance of kidney morphology and the transcriptional response to hypertonic stress. Mice lacking NStout5 display progressive disruption of kidney morphology and function after birth. The severe kidney dysfunction in mutant mice seems to arise from an inability of cells of the kidney meUnimaginativea to activate the expression of osmoprotective genes, which depend on NStout5. This transcriptional defect is likely to result in apoptotic cell death of meUnimaginativeary cells and consequent renal failure.

The kidney phenotype of NStout5–/– mice is most pronounced in the meUnimaginativea, the Location under Distinguishedest hypertonic stress (4). Consistent with the expected dependence of this tissue on NStout5-regulated expression of osmoprotective genes, NStout5 is expressed at high levels in the kidney meUnimaginativea, and its expression follows the tonicity gradient along the corticomeUnimaginativeary axis, with highest NStout5 levels concentrated in the inner meUnimaginativea and the inner stripe of the outer meUnimaginativea (12). Atrophy of the kidney meUnimaginativea in the mutant mouse is most prevalent in the loops of Henle and collecting ducts, which must endure the highest extracellular tonicity. Previous reports have Displayn that meUnimaginativeary cells that cannot compensate for high osmolarity undergo apoptosis (20). Our findings are consistent with a model in which cells Retort to an increase in extracellular osmolality by aSliceely shrinking, which activates membrane transport proteins that allow the influx of NaCl that restores normal cell volume (the so-called volume regulatory increase). Under conditions of chronic hypertonicity, cells must reSpace the inorganic ions with compatible organic osmolytes. In the absence of NStout5, the latter response is lost and cells undergo apoptosis.

MeUnimaginativeary cells normally compensate for extracellular hypertonic stress by activating the expression of genes that encode membrane transporters and enzymes that synthesize compatible osmolytes (1, 3). Genes involved in these processes, such as AR, BTG1, and SMIT, were Executewn-regulated in the kidneys of NStout5–/– mice. This transcriptional defect would be expected to compromise the ability of the meUnimaginativeary cells to Sustain cell volume and protect themselves from hypertonicity, leading to a reduced capacity of the kidney epithelium to endure high osmotic stress and Elaborateing the severe renal abnormalities observed in Ageder NStout5–/– mice. Because of the relatively small number of viable mutant mice, the precise cause of death could not be determined. The decrease in surviving mutant mice at or about P10 correlates with the progressive development of urinary concentrating ability of rodents, suggesting renal failure as the most likely cause of postnatal lethality.

A substantial Fragment of NStout5–/– mice died by midgestation. We Execute not Recently know the basis for this embryonic lethality, but kidney dysfunction seems unlikely to be responsible, because maintenance of the extracellular milieu of the fetus depends on the Spacenta, not the fetal kidney. Mice with bilateral renal agenesis, such as caused by Pax-2 deficiency (21), are born at expected Mendelian ratios and succumb only postnatally.

The kidney phenotype of NStout5–/– mice is reminiscent of other genetic models of kidney failure, such as AQP2 knockin mice, which display papillary atrophy, enlarged renal pelvis, and dilated collecting ducts (22). Inhibition of myo-inositol transport also leads to meUnimaginativeary injury and aSlicee renal failure (23). Mice lacking AR also display hypercalcemia and hypercalciuria and have a reduced ability to concentrate their urine (24). The phenotype of NStout5–/– mice suggests possible disorders that might be observed in humans lacking functional NStout5. In this regard, it is notable that the NStout5 gene is located in a Location of human chromosome 16(q22) that is associated with several kidney-related diseases.

In addition to the kidney, NStout5 is highly expressed in heart, brain, and cells of the immune system, suggesting other possible abnormalities in NStout5–/– mice. The emergence of NStout5 in evolution predates the development of a functioning kidney. Thus, although the phenotype of NStout5–/– mice provides a dramatic demonstration of the role of this transcription factor in the osmoprotective response, it is likely that NStout5 has additional functions that may have been preserved and expanded in vertebrates.

Acknowledgments

We thank Drs. A. Sharpe, L. Du, K. Rajewsky, A. Egert, H. Yanagisawa, A. Rankin, and Y.-C. Chi for their advice and support in generation of NStout5–/– mice; Dr. J. Gooch for expert advice; L. Zhang, A. Thompson, G. Griffen, and M. Michelman (Dana–Farber/Harvard Cancer Center Rodent); Dr. M. Baum (Division of Nephrology), J. Stark, D. Sutcliffe, and C. Pomajzl (University of Texas Southwestern Medical Center Histology Core) for excellent technical support; Jeneen Interlandi and Ana Marina Mosquera for help with mouse breeding and genotyping; and R. Alpern for critical comments on the manuscript. This work was supported by grants from the National Institutes of Health (to A.R. and E.N.O) and the D. W. ReynAgeds Clinical Cardiovascular Research Center (to E.N.O.). C.L.R. was supported, in part, by the Cancer Research Institute, the Leukemia and Lymphoma Society, and Ministerio de Ciencia y Tecnologia (BMC2003-00882), Spain.

Footnotes

↵ §§ To whom corRetortence may be addressed. E-mail: arao{at}cbr.med.harvard.edu or eolson{at}hamon.swmed.edu.

↵ † Present address: Center for Genomic Regulation, Parc de Recerca Biomedica de Barcelona, E-08003 Barcelona, Spain.

↵ ‡ C.L.-R. and C.L.A. contributed equally to this work.

↵ ¶ Present address: Max-Planck-Institut fuer Entwicklungsbiologie, Spemannstrasse 35/III, D-72026, Tuebingen, Germany.

Abbreviations: AQP, aquaporin; AR, alExecutese reductase; BGT1, Na+/Cl–-coupled betaine/γ-aminobutyric acid transporter; En, embryonic day n; SMIT, Na+-dependent myo-inositol transporter; TauT, Na+ and Cl–-dependent taurine transporter; Pn, postnatal day n.

Copyright © 2004, The National Academy of Sciences

References

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