Loss of NFAT5 Results in Renal Atrophy and Lack of Tonicity-Responsive Gene Expression

Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression

Cristina Lo´ pez-Rodrı´guez*†‡, Christopher L. Antos‡§¶, John M. Sheltonʈ, James A. Richardson§ʈ, Fangming Lin**, Tatiana I. Novobrantseva*, Roderick T. Bronson††, Peter Igarashi‡‡, Anjana Rao*§§, and Eric N. Olson§,§§

*Department of Pathology, Harvard Medical School and Center for Blood Research, Institute for Biomedical Research, 200 Longwood Avenue, Boston MA 02115; Departments of §Molecular Biology, ʈPathology, ‡‡Internal Medicine (Nephrology), and **Pediatrics, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148; and ††Department of Pathology, Tufts University School of Veterinary Medicine, North Grafton, MA 01536

Contributed by Eric N. Olson, December 29, 2003 The transcription factor NFAT5͞TonEBP, a member of the NFAT͞Rel and in other tissues, and several lines of evidence suggest its family of transcription factors, has been implicated in diverse involvement in the osmoprotective response (see ref. 10 for a cellular responses, including the response to osmotic stress, inte- review). NFAT5 binds tonicity-response elements that are grin-dependent cell migration, T cell activation, and the Ras path- present in the control regions of osmotically regulated genes, and way in Drosophila. To clarify the in vivo role of NFAT5, we it is hyperphosphorylated and translocates to the nucleus in generated NFAT5-null mice. Homozygous mutants were geneti- response to hypertonic stimulation of cells in culture (9–11). cally underrepresented after embryonic day 14.5. Surviving mice NFAT5 also undergoes nuclear translocation in the kidney manifested a progressive and profound atrophy of the kidney during antidiuresis, which is most apparent within the outer and medulla with impaired activation of several osmoprotective genes, inner regions of the medulla (12). -including those encoding aldose reductase, Na؉͞Cl؊-coupled be taine͞␥-aminobutyric acid transporter, and the Na؉͞myo-inositol In addition to its potential involvement in the osmotic stress cotransporter. The aldose reductase gene is controlled by a tonic- response, NFAT5 has been shown to be regulated by other ity-responsive enhancer, which was refractory to hypertonic stress stimuli and to participate in a diverse set of cellular responses. in ﬁbroblasts lacking NFAT5, establishing this enhancer as a direct In response to T cell receptor stimulation, NFAT5 displays a ͞ transcriptional target of NFAT5. Our ﬁndings demonstrate a central dependence on the calcium calmodulin-dependent phosphatase role for NFAT5 as a tonicity-responsive transcription factor re- calcineurin (11, 13). On the other hand, the activation of NFAT5 quired for kidney homeostasis and function. by osmotic stress is independent of calcineurin (11). Intracellular signals transmitted by the prometastatic integrin ␣6͞␤4 also lead ecause water can diffuse freely across most membranes, to an increase in the levels and activity of NFAT5 that enhances Banimal cells must preserve a balanced osmolarity to prevent the migratory capacity of carcinoma cells (14). Finally, genetic dehydration and maintain viability. All nucleated cells possess a analyses in Drosophila suggest a role for dNFAT, the likely programmed response to hypertonic stress in which acute com- ortholog of mammalian NFAT5, in Ras-mediated cell pensatory changes in cell volume are followed by a coordinated growth (15). transcriptional response that results in the intracellular accumu- To define the functions of NFAT5 in the mouse, we disrupted lation of small, cell-compatible osmolytes, such as sorbitol, the mouse NFAT5 gene. The homozygous NFAT5 null allele myo-inositol, betaine, and taurine, which increase intracellular resulted in midembryonic lethality with incomplete penetrance. osmolality, restore cell volume, and provide a buffer against Surviving mutant mice displayed progressive growth retardation osmotic stress (1–3). The genes that are transcriptionally up- and perinatal lethality associated with severe renal abnormalities regulated during the osmoprotective response encode enzymes and impaired activation of osmoprotective genes, including AR. and membrane proteins involved in synthesis and transport of Cells lacking NFAT5 do not express AR mRNA because of their organic osmolytes. Among them, aldose reductase (AR) is inability to activate its tonicity-responsive enhancer. Our find- responsible for sorbitol synthesis, whereas the sodium- ͞␥ ings demonstrate a central role for NFAT5 as a tonicity- dependent myo-inositol cotransporter (SMIT), the betaine - responsive transcription factor required for renal homeostasis aminobutyric acid transporter (BGT1), and the sodium and and function. chloride-dependent taurine transporter (TauT) are membrane- ϩ localized cotransporters that rely on coupled influx of Na Materials and Methods and͞or ClϪ to mediate entry of osmolytes into osmotically Generation of a Targeted NFAT5 Allele. A 14-kb region surrounding stressed cells (1, 3). NFAT5 The cells in the kidney are specialized for water and ion the mouse gene was isolated from a genomic library. We retention and serve as the primary regulators of electrolyte constructed a mutant NFAT5-targeting vector by subcloning concentration in extracellular fluid (4). The rodent kidney 2.3-kb and 2.9-kb sequences surrounding the sixth exon, which medulla is regularly exposed to extreme hypertonic stress [up to encodes the DNA-binding loop of NFAT5. Gene targeting was 4,000 mOsm compared with 300 mOsm of serum (4), a hypertonicity required to concentrate urine and maintain serum Abbreviations: AQP, aquaporin; AR, aldose reductase; BGT1, Naϩ͞ClϪ-coupled betaine͞␥- osmolality constant under antidiuretic (hydropenic) conditions]. aminobutyric acid transporter; En, embryonic day n; SMIT, Naϩ-dependent myo-inositol An increase in extracellular osmolarity causes water to diffuse transporter; TauT, Naϩ and ClϪ-dependent taurine transporter; Pn, postnatal day n. out of the medullary cells, so they must counter this stress by †Present address: Center for Genomic Regulation, Parc de Recerca Biomedica de Barcelona, synthesizing and accumulating osmolytes. Diseases that impair E-08003 Barcelona, Spain. kidney functions lead to pathological imbalances in the tonicity ‡C.L.-R. and C.L.A. contributed equally to this work. of body fluids, which disturb other organ systems. ¶Present address: Max-Planck-Institut fuer Entwicklungsbiologie, Spemannstrasse 35͞III, NFAT5 (TonEBP and OREBP) is a member of the NFAT͞ D-72026, Tuebingen, Germany. Rel family of transcription factors (5, 6), which contains a §§To whom correspondence may be addressed. E-mail: [email protected] or DNA-binding domain related to both the NFAT and NF␬B [email protected]. families (5–9). NFAT5 is highly expressed in the kidney medulla © 2004 by The National Academy of Sciences of the USA

2392–2397 ͉ PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308703100 Downloaded by guest on October 7, 2021 Fig. 1. Targeted disruption of the NFAT5 gene. (A) Strategy used to generate the targeted NFAT5 allele. The targeted locus substitutes an inverted Neo cassette for the sixth exon of the NFAT5 gene (first exon of the Rel-homology domain, which encodes the DNA-binding loop), generating a frameshift that introduces a stop codon immediately after the deletion (not shown). B, BamHI; P, PsthAI; A, AvrII; X, XmnI; Bg, BglII. (B) Southern blot analysis from BamHI-restricted genomic DNA with a 5Ј probe outside the recombination site identifies the targeted NFAT5 allele in heterozygous mouse crosses. (C) Western blot analysis for NFAT5 protein in lymphocytes from wild-type and NFAT5Ϫ/Ϫ mice shows complete lack of NFAT5 expression in the mutant. Protein levels of NFAT1 are unaffected in the homozygous mutant (Lower). ns, nonspecific band.

performed as described in Supporting Text, which is published as Sixteen hours after stimulation, cells were harvested and lucif- supporting information on the PNAS web site. erase activity was assessed as described (5). Photinus luciferase values were normalized to an independent reporter (Renilla Histology and Immunostaining. Histology and terminal de- luciferase). All experiments were performed at least twice, and oxynucleotidyltransferase-mediated dUTP end-labeling analysis a representative experiment is shown in the figure. GENETICS were performed as described in Supporting Text. Formalin-fixed and paraffin-embedded kidney sections were incubated with Quantitative RT-PCR. RNA isolation and RT-PCR were per- rabbit primary antibodies to aquaporin 2 (AQP2, Mark Knepper, formed as described in Supporting Text. National Institutes of Health, see ref. 16) at 1:2,000 dilution or aquaporin 3 (AQP3, Santa Cruz Biotechnology) at 1:1,000 Results dilution at room temperature for 2 h, followed by further Generation of NFAT5؊/؊ Mice. We inactivated the mouse NFAT5 incubation with Alexa Fluor 488 anti-rabbit IgG (Vector Lab- gene by replacing the Rel-DNA binding domain within the sixth oratories) at 1:400 dilution at room temperature for 40 min. exon with an inverted neomycin-resistance cassette (Fig. 1A). Stained sections were photographed under epifluorescence il- Homologous recombination introduced an additional BamHI lumination by using a Zeiss Axioplan microscope, and the restriction site into the NFAT5 locus, generating 7.3- and 9.0-kb images were analyzed with OPENLAB software (Improvision, fragments instead of the 15.3-kb fragment of the wild-type locus Boston, MA). (Fig. 1B). Injection of targeted embryonic stem cells into blas- tocysts yielded chimeric mice that transmitted the mutant allele In Situ Hybridization. In situ hybridization was performed as through the germ line. described (17). Details can be found in Supporting Text. NFAT5-null mice showed embryonic and perinatal lethality with incomplete penetrance (Table 1). We did not observe Mouse Embryo Fibroblast Isolation and Reporter Assays. Mouse variations in the NFAT5 mutant phenotype in mixed 129Sv͞ embryo fibroblasts were isolated from NFAT5ϩ/ϩ and C57BL6 or isogenic 129Sv backgrounds. No major deviation NFAT5Ϫ/Ϫ mice at embryonic day (E)13.5 as described (18). from the expected Mendelian ratios was observed at E14.5, but Mouse embryo fibroblasts were cultured in DMEM supple- only 50% of the expected number of NFAT5Ϫ/Ϫ embryos was mented with 10% FBS͞10 mM Hepes͞2mML-glutamine and obtained at E17.5 (Table 1). A majority of the NFAT5-null mice transfected by using Effectene (Qiagen, Valencia, CA) accord- that were born died around postnatal day (P)10, with only 3.4% ing to manufacturer’s instructions. Hypertonic treatments were of the expected number living past P21 (Table 1). Despite their performed 24 h after transfection, and cells were stimulated by underrepresentation, NFAT5Ϫ/Ϫ mice showed no obvious ab- addition of an NaCl solution to a final concentration of 100 mM. normalities at birth, but the small proportion of these mice that

Lo´pez-Rodrı´guez et al. PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 ͉ 2393 Downloaded by guest on October 7, 2021 Table 1. Embryonic and perinatal lethality of NFAT5 mutant mice than straight, and epithelial cells lacked the typical cuboidal Day ϩ͞ϩϩ͞ϪϪ͞Ϫ character seen in wild-type kidneys (Fig. 3 Ae–h). Instead, these cells were tightly packed with an increased nuclear-to- E14.5 11 (11) 28 (22) 8 (11) cytoplasmic ratio (Fig. 3Ah), suggesting they are unable to E17.5 10 (10) 12 (20) 5 (10) maintain normal cell volume in the presence of hypertonic stress. P18–21 205 (205) 305 (410) 7 (205) The renal cortex in immature NFAT5Ϫ/Ϫ mice appeared rela- The numbers of mice of the indicated genotypes obtained from heterozy- tively normal (Fig. 3 Aiand j). Gross histological analysis of adult gous intercrosses are shown. Numbers in parentheses indicate the expected kidney sections showed that in contrast to wild-type kidneys (Fig. Ϫ/Ϫ number based on predicted Mendelian inheritance. At least three litters were 3 Baand b), kidneys from mature NFAT5 mice (P70–P120) analyzed for each time point. were grossly malformed, having an irregular surface and severe segmental atrophy (Fig. 3 Bcand e). Large irregular areas of the medulla and cortex were distorted by tubular and interstitial survived to adulthood failed to thrive and their weight was about inflammation (Fig. 3 Bdand f), resulting in dramatic atrophy half that of wild-type littermates (Fig. 2). and loss of nephrons. In adjacent areas of the medulla, micro- Western blots of T cell lysates of homozygous mutant mice cystic dilation of the tubules was evident, a change representing showed no NFAT5 protein (Fig. 1C). Expression of NFAT1 a compensatory response to the functional loss suffered by the protein was unaffected in the same extracts, providing an remainder of the kidney (Fig. 3 Bc–f). internal control. Increased Apoptosis in the Inner Medulla of NFAT5؊/؊ Kidneys. The Ϫ Ϫ Kidney Abnormalities in NFAT5؊/؊ Mice. Histological examination progressive renal atrophy observed in NFAT5 / mice was of null mice at 3 weeks of age revealed kidney hypoplasia and an associated with the presence of apoptotic bodies in the renal altered medullary morphology (Fig. 3A). The normal kidney medulla (not shown). Terminal deoxynucleotidyltransferase- consists of distinct regions: the cortex, the outer and inner stripes mediated dUTP end-labeling analysis revealed a significant Ϫ/Ϫ of the outer medulla, the inner medulla, and the papilla. The number of apoptotic cells only in NFAT5 mice (Fig. 3C), outer and inner stripes of the medulla are clearly demarcated in indicating that the absence of NFAT5 affected survival of epithelial cells in the kidney medulla. The cell loss observed in normal kidneys (Fig. 3Aa), but they were not well defined in the Ϫ/Ϫ kidneys of NFAT5Ϫ/Ϫ mice, which had a higher cellular density the medulla and papilla of NFAT5 kidneys likely stems from than normal (Fig. 3Ab). Serial sections through the kidneys of an inability of medullary cells to compensate for hypertonic NFAT5Ϫ/Ϫ mice showed a disruption of the normal architecture stress, resulting in atrophy of the affected zones of the kidney. NFAT5-null mice displayed a slight increase in mitotic cells of the outer medulla, the lack of a complete papilla, and within the kidney (not shown), most likely as a secondary enlargement of the renal pelvis indicative of progressive atrophy compensatory response. Despite this increase in cell division, of the medulla (Fig. 3Ab). In the inner stripe of the outer medulla cell loss was the dominating outcome. within the normal kidney, a clear demarcation exists between the vascular bundles and the surrounding loops of Henle and Loss of NFAT5 Alters a Gene Expression Program that Regulates collecting ducts, which was lost in the mutant (Fig. 3 Acand d). Osmotic Homeostasis in the Kidney Medulla. Because NFAT5Ϫ/Ϫ Tubules in the medulla of the mutant were also curved rather mice exhibit medullary atrophy, we performed immunostaining with antibodies to the proteins and water transporters specifi- cally expressed in renal tubules in the medulla (Fig. 4A). Expression of Tamm-Horsfall, which is expressed in the thick ascending limbs of loops of Henle in the outer medulla, was only slightly reduced in mutant mice (not shown). Similarly, expression of AQP3, which is primarily expressed in cortical and outer medullary collecting ducts, was only moderately down-regulated (Fig. 4 Aa–d). In contrast, expression of AQP2, which is expressed in both inner and outer medullary collecting ducts, was markedly inhibited. Neither AQP2 nor AQP3 was expressed in the inner medulla, and no differences occurred in the expression of cortical markers, such as Fx1A (not shown). Higher- magnification images showed 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 Ae–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 medulla. To test the involvement of NFAT5 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 NFAT5Ϫ/Ϫ and wild-type mice. The mice were water-deprived for 24 h before killing in conditions that physiologically increase hypertonic stress and Fig. 2. Growth retardation in NFAT5Ϫ/Ϫ mice. (A) Comparison of wild-type result in enhanced up-regulation of tonicity-responsive genes. (Left) and NFAT5Ϫ/Ϫ (Right) mice demonstrates growth retardation in the We observed marked reduction of AR, BGT1, and SMIT mRNAs Ϫ Ϫ mutant at 6 weeks of age. (B) NFAT5Ϫ/Ϫ mice show a pronounced reduction in in kidneys of NFAT5 / mice in comparison with those of body weight compared with wild-type siblings. wild-type mice (Fig. 4B). Wild-type mice showed abundant

2394 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308703100 Lo´ pez-Rodrı´guez et al. Downloaded by guest on October 7, 2021 Fig. 3. Altered kidney morphology and abnormalities of the renal medulla in NFAT5Ϫ/Ϫ mice. (A) Histological analysis of hematoxylin͞eosin-stained sections from kidneys of 3- to 4-week-old wild-type (a) and NFAT5Ϫ/Ϫ (b) mice revealed atrophic papillae and a denser morphology of the renal medulla in the mutant (bar ϭ 2 mm). (c and d) In the medulla of the null kidney, the morphology of the inner stripe of the outer medulla is indistinct compared with wild type. Compared with the straight morphology of the tubules in the outer medulla in wild-type kidneys (e), the architecture of the tubules in the kidney medulla in NFAT5Ϫ/Ϫ mice is curved (f; bar ϭ 100 ␮m). (g and h) A detailed comparison at a higher magniﬁcation of kidneys from wild-type (g) and NFAT5Ϫ/Ϫ (h) mice revealed that the cells in the renal medulla of NFAT5Ϫ/Ϫ mice lack normal cuboidal morphology and have a decrease in cytoplasmic volume compared with wild type (bar ϭ 40 ␮m). (i and j) Whereas the medulla is severely affected in NFAT5Ϫ/Ϫ mice, the histology of the renal cortex is normal (bar ϭ 20 ␮m). (B) Histological analysis of sections from the kidneys of mature wild-type (a and b) or NFAT5Ϫ/Ϫ mice (c–f). (a, c, and e, bar ϭ 2 ␮m; b, d, and f, bar ϭ 100 ␮m). (C) Terminal deoxynucleotidyltransferase-mediated dUTP end-labeling analysis of the renal medulla from wild-type (a) and NFAT5Ϫ/Ϫ (b) mouse sections indicates increased apoptosis in the mutant. c, cortex; is, inner stripe; m, medulla; om, outer medulla; p, papilla.

expression of AR transcripts in the inner medulla (Fig. 4Ba) and shown), possibly reflecting their kidney-specific expression.

BGT1 transcripts in the inner stripe of the medulla between the However, wild-type fibroblasts subjected to hypertonic stimula- GENETICS Ϫ Ϫ vascular bundles and papilla (Fig. 4Bc), but NFAT5 / mice tion showed a Ͼ3-fold increase in the expression of AR mRNA completely lacked AR and BGT1 expression (Fig. 4 Bband d). relative to fibroblasts maintained under isotonic conditions; this SMIT expression is normally localized to both stripes of the outer increase depended on the presence of NFAT5, as shown by the medulla and tubules within the cortex (Fig. 4Be) but was complete lack of AR mRNA expression in NFAT5Ϫ/Ϫ fibroblasts Ϫ/Ϫ expressed only in the cortical tubules in NFAT5 mice, which (Fig. 5A). clearly lacked SMIT expression in the outer medulla (Fig. 4Bf). Transcription of the AR gene is regulated by a 132-bp en- In contrast, expression of the TauT gene, which was localized to hancer that contains three consensus NFAT5-binding elements the outer stripe of the outer medulla that contained the proximal (ref. 19 and Fig. 5B). To test whether NFAT5 regulates AR straight tubules in wild-type mice (Fig. 4Bg), was unaltered in the Ϫ Ϫ transcription through this enhancer element, we transfected / Bh Ϫ Ϫ kidneys of NFAT5 mice (Fig. 4 ); we note, however, that NFAT5 / and control fibroblasts with a luciferase reporter mutant mice showed TauT expression in glomeruli of the kidney construct by the AR enhancer and tested reporter expression in cortex (Fig. 4Bh), an expression pattern not observed in dehy- cells subjected to osmotic stress. Although control fibroblasts drated wild-type mice (Fig. 4Bg). The decreased expression of Ϫ/Ϫ osmoregulatory genes was confined to the medulla because the showed strong induction of luciferase activity, NFAT5 cells interstitial fluid in the cortex is isosmotic with plasma, whereas did not (Fig. 5B), suggesting that the AR gene is a direct target the medulla is hypertonic. of the transcriptional activity of NFAT5. Discussion Altered Transcriptional Induction of Osmoprotective Genes in -NFAT5؊/؊ Cells. To test whether NFAT5 is responsible for regu- The results of this study reveal a key role of NFAT5 in main lating expression of osmoprotective genes, we isolated fibroblasts tenance of kidney morphology and the transcriptional response from NFAT5Ϫ/Ϫ and wild-type mice at E13.5, subjected them to to hypertonic stress. Mice lacking NFAT5 display progressive hypertonic conditions by exposing them to media containing an disruption of kidney morphology and function after birth. The additional 100 mM NaCl for 16 h, and evaluated expression of severe kidney dysfunction in mutant mice seems to arise from an the AR, BGT1, TauT, and SMIT genes. No significant induction inability of cells of the kidney medulla to activate the expression of the latter three genes was observed in control fibroblasts (not of osmoprotective genes, which depend on NFAT5. This tran-

Lo´ pez-Rodrı´guez et al. PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 ͉ 2395 Downloaded by guest on October 7, 2021 Fig. 5. Down-regulation of AR gene expression in NFAT5Ϫ/Ϫ mice. (A) Quantification by real-time RT-PCR of levels of AR transcripts induced in wild-type or NFAT5Ϫ/Ϫ mouse embryo fibroblasts exposed to hypertonic conditions (addition of 100 mM NaCl for 16 h, black bars). Values are normalized to a housekeeping gene (L32). (B) Lack of transcriptional activation of the AR enhancer in osmotically stressed fibroblasts from NFAT5Ϫ/Ϫ mice. (Upper) Diagram of the enhancer showing the three consensus binding elements for NFAT5. (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 shown.

Fig. 4. Aberrant renal gene expression in NFAT5Ϫ/Ϫ mice. (A) Immunostain- is expressed at high levels in the kidney medulla, and its ing of renal tubules. AQP2 (a and b) and AQP3 (c and d) staining reveals expression follows the tonicity gradient along the corticomed- abundant expression in collecting ducts (cd) in wild-type kidneys (a and c), but ullary axis, with highest NFAT5 levels concentrated in the inner decreased expression in the mutant kidneys (b and d). Higher magniﬁcation of medulla and the inner stripe of the outer medulla (12). Atrophy AQP2 (e–f) and AQP3 (g–h) staining of the collecting ducts of wild-type (e and of the kidney medulla in the mutant mouse is most prevalent in g) and mutant (f and h) kidneys shows predominant cytoplasmic localization the loops of Henle and collecting ducts, which must endure the in wild-type (e) but apical localization of AQP2 in mutant kidneys (f) and highest extracellular tonicity. Previous reports have shown that basolateral localization of AQP3 in both wild-type (g) and mutant (h) kidneys. medullary cells that cannot compensate for high osmolarity Nuclei were counterstained with 4Ј,6-diamidino-2-phenylindole in e–h. co, cortex; me, medulla. (Bar ϭ 50 ␮m.) (B) Transcripts encoding AR (a and b), BGT1 undergo apoptosis (20). Our findings are consistent with a model (c and d), SMIT (e and f), and TauT (g and h) were detected by in situ in which cells respond to an increase in extracellular osmolality hybridization to kidney sections from wild-type (a, c, e, and g) and NFAT5Ϫ/Ϫ by acutely shrinking, which activates membrane transport pro- (b, d, f, and h) mice subjected to water restriction for 24 h. teins that allow the influx of NaCl that restores normal cell volume (the so-called volume regulatory increase). Under conditions of chronic hypertonicity, cells must replace the inorganic scriptional defect is likely to result in apoptotic cell death of ions with compatible organic osmolytes. In the absence of medullary cells and consequent renal failure. NFAT5, the latter response is lost and cells undergo apoptosis. Ϫ Ϫ The kidney phenotype of NFAT5 / mice is most pronounced Medullary cells normally compensate for extracellular hyper- in the medulla, the region under greatest hypertonic stress (4). tonic stress by activating the expression of genes that encode Consistent with the expected dependence of this tissue on membrane transporters and enzymes that synthesize compatible NFAT5-regulated expression of osmoprotective genes, NFAT5 osmolytes (1, 3). Genes involved in these processes, such as AR,

2396 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308703100 Lo´ pez-Rodrı´guez et al. Downloaded by guest on October 7, 2021 BTG1, and SMIT, were down-regulated in the kidneys of might be observed in humans lacking functional NFAT5. In this NFAT5Ϫ/Ϫ mice. This transcriptional defect would be expected regard, it is notable that the NFAT5 gene is located in a region to compromise the ability of the medullary cells to maintain cell of human chromosome 16(q22) that is associated with several volume and protect themselves from hypertonicity, leading to a kidney-related diseases. reduced capacity of the kidney epithelium to endure high In addition to the kidney, NFAT5 is highly expressed in heart, osmotic stress and explaining the severe renal abnormalities brain, and cells of the immune system, suggesting other possible Ϫ Ϫ observed in older NFAT5 / mice. Because of the relatively abnormalities in NFAT5Ϫ/Ϫ mice. The emergence of NFAT5 in small number of viable mutant mice, the precise cause of death evolution predates the development of a functioning kidney. could not be determined. The decrease in surviving mutant mice Thus, although the phenotype of NFAT5Ϫ/Ϫ mice provides a at or about P10 correlates with the progressive development of dramatic demonstration of the role of this transcription factor in urinary concentrating ability of rodents, suggesting renal failure the osmoprotective response, it is likely that NFAT5 has addi- as the most likely cause of postnatal lethality. Ϫ Ϫ tional functions that may have been preserved and expanded in A substantial fraction of NFAT5 / mice died by midgesta- vertebrates. tion. We do not currently know the basis for this embryonic lethality, but kidney dysfunction seems unlikely to be responsi- We thank Drs. A. Sharpe, L. Du, K. Rajewsky, A. Egert, H. Yanagisawa, ble, because maintenance of the extracellular milieu of the fetus A. Rankin, and Y.-C. Chi for their advice and support in generation of depends on the placenta, not the fetal kidney. Mice with bilateral NFAT5Ϫ/Ϫ mice; Dr. J. Gooch for expert advice; L. Zhang, A. Thomp- renal agenesis, such as caused by Pax-2 deficiency (21), are born son, G. Griffen, and M. Michelman (Dana–Farber͞Harvard Cancer at expected Mendelian ratios and succumb only postnatally. Center Rodent); Dr. M. Baum (Division of Nephrology), J. Stark, D. The kidney phenotype of NFAT5Ϫ/Ϫ mice is reminiscent of Sutcliffe, and C. Pomajzl (University of Texas Southwestern Medical other genetic models of kidney failure, such as AQP2 knockin Center Histology Core) for excellent technical support; Jeneen Inter- landi and Ana Marina Mosquera for help with mouse breeding and mice, which display papillary atrophy, enlarged renal pelvis, and genotyping; and R. Alpern for critical comments on the manuscript. This dilated collecting ducts (22). Inhibition of myo-inositol transport work was supported by grants from the National Institutes of Health (to also leads to medullary injury and acute renal failure (23). Mice A.R. and E.N.O) and the D. W. Reynolds Clinical Cardiovascular lacking AR also display hypercalcemia and hypercalciuria and Research Center (to E.N.O.). C.L.R. was supported, in part, by the have a reduced ability to concentrate their urine (24). The Cancer Research Institute, the Leukemia and Lymphoma Society, and Ϫ Ϫ phenotype of NFAT5 / mice suggests possible disorders that Ministerio de Ciencia y Tecnologia (BMC2003-00882), Spain.

1. Burg, M., Kwon, E. & Kultz, D. (1997) Annu. Rev. Physiol. 59, 437–455. 14. Jauliac, S., Lo´pez-Rodrı´guez, C., Shaw, L. M., Brown, L. F., Rao, A. & Toker, 2. Haussinger, D. (1996) Biochem. J. 313, 697–710. A. (2002) Nat. Cell. Biol. 4, 540–544. 3. Kwon, H. M. & Handler, J. S. (1995) Curr. Opin. Cell Biol. 7, 465–471. 15. Huang, A. M. & Rubin, G. M. (2000) Genetics 156, 1219–1230. 4. Guyton, A. C. & Hall, J. E. (1996) in Textbook of Medical Physiology (Saunders, 16. Fernandez-Llama, P., Andrews, P., Ecelbarger, C. A., Nielsen, S. & Knepper, Philadelphia), pp. 349–366. M. (1998) Kidney Int. 54, 170–179. 5. Lo´pez-Rodrı´guez, C., Aramburu, J., Rakeman, A. S. & Rao, A. (1999) Proc. 17. Shelton, J. M., Lee, M. H., Richardson, J. A. & Patel, S. B. (2000) J. Lipid Res. Natl. Acad. Sci. USA 96, 7214–7219. 41, 532–537. 6. Graef, I. A., Gastier, J. M., Francke, U. & Crabtree, G. R. (2001) Proc. Natl. 18. Conner, D. A. (2000) in Current Protocols in Molecular Biology, eds. Ausubel, Acad. Sci. USA 98, 5740–5745. F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J., Smith, J. A. & 7. Lo´pez-Rodrı´guez, C., Aramburu, J., Rakeman, A. S., Copeland, N. G., Gilbert, Struhl, K. (Wiley, Hoboken, N.J.), pp. 23.2.1–23.2.7. D. J., Thomas, S., Disteche, C., Jenkins, N. A. & Rao, A. (1999) Cold Spring 19. Ko, B. C. B., Ruepp, B., Bohren, K. M., Gabbay, K. H. & Chung, S. S. M. (1997) Harbor Symp. Quant. Biol. 64, 517–526. J. Biol. Chem. 272, 16431–16437. 8. Stroud, J. C., Lo´pez-Rodrı´guez, C., Rao, A. & Chen, L. (2002) Nat. Struct. Biol. 20. Michea, L., Ferguson, D. R., Peters, E. M., Andrews, P. M., Kirby, M. R. & 9, 90–94. Burg, M. B. (2000) Am. J. Physiol. 278, F209–F218. 9. Miyakawa, H., Woo, S. K., Dahl, S. C., Handler, J. S. & Kwon, H. M. (1999) 21. Dressler, G. R., Wilkinson, J. E., Rothenpieler, U. W., Patterson, L. T., Proc. Natl. Acad. Sci. USA. 96, 2538–2542. Williams-Simons, L. & Westphal, H. (1993) Nature 362, 65–67. 10. Woo, S. K., Lee, S. D. & Kwon H. M. (2002) Pflu¨gers Arch. Eur. J. Physiol. 444, 22. Yang, B., Gillespie, A., Carlson, E. J., Epstein, C. J. & Verkman, A. S. (2001) GENETICS 579–585. J. Biol. Chem. 276, 2775–2779. 11. Lopez-Rodriguez, C., Aramburu, J., Jin, L., Rakeman, A. S., Michino, M. & 23. Kitamura, H., Yamauchi, A., Sugiura, T., Matsuoka, Y., Horio, M., To- Rao, A. (2001) Immunity 15, 47–58. hyama, M., Shimada, S., Imari, E. & Hori, M. (1998) Kidney Int. 53, 12. Cha, J. H., Woo, S. K., Han, K. H., Kim, Y. H., Handler, J. S., Kim, J. & Kwon, 146–153. H. M. (2001) J. Am. Soc. Nephrol. 12, 2221–2230. 24. Aida, K., Ikegishi, Y., Chen, J., Tawata, M., Ito, S., Maeda, S. & Onaya, T. 13. Trama, J., Lu, Q., Hawley, R. G. & Ho, S. N. (2000) J. Immunol. 165, 4884–4894. (2000) Biochem. Biophys. Res. Commun. 277, 281–286.

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