In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system.

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.

Uncompensated polyuria in a mouse model of Bartter's syndrome.

We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2-/- pups were born in expected numbers and appeared normal. However, by day 1 they showed signs of extracellular volume depletion (hematocrit 51%; wild type 37%). They subsequently failed to thrive. By day 7, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of -/- pups with indomethacin from day 1 prevented growth retardation and 10% treated for 3 weeks survived, although as adults they exhibited severe polyuria (10 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued -/- adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the -/- adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin angiotensin system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney.

The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis.

Epithelial-mesenchymal interactions are required for the development of all solid organs but few molecular mechanisms that underlie these interactions have been identified. Pod1 is a basic-helix-loop-helix (bHLH) transcription factor that is highly expressed in the mesenchyme of developing organs that include the lung, kidney, gut and heart and in glomerular visceral epithelial cells (podocytes). To determine the function of Pod1 in vivo, we have generated a lacZ-expressing null Pod1 allele. Null mutant mice are born but die in the perinatal period with severely hypoplastic lungs and kidneys that lack alveoli and mature glomeruli. Although Pod1 is exclusively expressed in the mesenchyme and podocytes, major defects are observed in the adjacent epithelia and include abnormalities in epithelial differentiation and branching morphogenesis. Pod1 therefore appears to be essential for regulating properties of the mesenchyme that are critically important for lung and kidney morphogenesis. Defects specific to later specialized cell types where Pod1 is expressed, such as the podocytes, were also observed, suggesting that this transcription factor may play multiple roles in kidney morphogenesis.

Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity.

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a lambda bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5' rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5' untranslated region. The proximal 5' flanking region was "TATA-less" but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5' flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region from -31 to -113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5' flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from -113 to -31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.

Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice.

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium.

Under the influence of sex steroids, human endometrium undergoes sequential development in preparation for implantation. Hoxa11 is essential for implantation in the mouse. Here we describe a potential role for HOXA11 in human endometrial development and implantation. Northern analysis demonstrates that HOXA11 is expressed in a menstrual cycle phase-dependent fashion in adult human endometrium. HOXA11 messenger RNA levels dramatically increase at the time of implantation and remain increased in pregnancy. In vitro, HOXA11 expression is increased in response to estrogen or progesterone. There is a dose-responsive increase over the physiologic range of progesterone concentration. Pretreatment with Cyclohexamide does not decrease the response to estrogen. Steroids are novel regulators HOX gene expression. The spatial and temporal pattern of HOXA11 expression in the human endometrium suggests a role in endometrial development, implantation, and maintenance of pregnancy.

Immunochemical characterization of Na+/H+ exchanger isoform NHE4.

Mammalian Na+/H+ exchangers (NHEs) are a family of transport proteins (NHE1-NHE5). To date, the cellular and subcellular localization of NHE4 has not been characterized using immunochemical techniques. We purified a fusion protein containing a portion of rat NHE4 (amino acids 565-675) to use as immunogen. A monoclonal antibody (11H11) was selected by ELISA. It reacted specifically with both the fusion protein and to a 60- to 65-kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65- to 70-kDa protein that was expressed most abundantly in stomach and in multiple additional epithelial and nonepithelial rat tissues including skeletal muscle, heart, kidney, uterus, and liver. Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane fractions isolated by Percoll gradient centrifugation. NHE4 was found to cofractionate with the basolateral markers NHE1 and Na+-K+-ATPase rather than the luminal marker gamma-glutamyl transferase. In stomach, NHE4 was detected by immunoperoxidase labeling on the basolateral membrane of cells at the base of the gastric gland. We conclude that NHE4 is a 65- to 70-kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the basolateral membrane.

cDNA cloning and chromosomal localization of the human and mouse isoforms of Ksp-cadherin.

Ksp-cadherin is a novel kidney-specific member of the cadherin superfamily of cell adhesion molecules. We have determined the complete cDNA coding sequences of both the human and the mouse isoforms of Ksp-cadherin. The inferred amino acid sequences of the human and mouse isoforms are 79 and 75% identical to the originally described rabbit isoform of Ksp-cadherin (Thomson et al., 1995; J. Biol. Chem. 270, 17594-17601), respectively. The relative locations of cadherin-specific sequence motifs, putative N-glycosylation sites, and characteristic protein domains are entirely conserved in all three isoforms. Multiple organ Northern analyses indicate that, as in the rabbit, both the human and the mouse Ksp-cadherin transcripts appear to have distinct kidney-specific distributions. The human Ksp-cadherin gene (CDH16) maps to chromosome 16q21-proximal 16q22. The mouse Ksp-cadherin gene (Cdh16) was localized to a highly syntenic region of distal Chromosome 8. Both the human and the mouse Ksp-cadherin genes were localized to previously identified clusters of cadherin gene sequences, consistent with the hypothesis that most cadherin family members arose by gene duplication from a single ancestral gene at a relatively early stage in the evolution of the mammalian genome.

Pod-1, a mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney.

Basic-helix-loop-helix (bHLH) proteins are transcriptional regulatory proteins that govern cell fate determination and differentiation in a variety of tissues. We have identified a novel bHLH protein, named Pod-1, that belongs to a recently-described subfamily of bHLH proteins that have essential roles in the embryonic development of mesodermal tissues. In the adult human and mouse, Pod-1 was most highly expressed in the kidney, lung and heart. In developing mouse embryos, Pod-1 was selectively expressed in mesenchymal cells at sites of epithelial-mesenchymal interaction in the kidney, lung, intestine and pancreas. Pod-1 was also expressed in visceral glomerular epithelial cells (podocytes) in the kidney, and its expression coincided with the onset of podocyte differentiation. The expression of Pod-1 in embryonic kidney explants was inhibited using antisense oligonucleotides. Inhibition of Pod-1 expression resulted in decreased mesenchymal cell condensation around the ureteric bud and a 40% decrease in ureteric branching. Pod-1 is the first tissue-restricted basic-helix-loop-helix protein that has been identified in the developing kidney where it may play a role in the regulation of morphogenetic events.

HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium.

Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.

Antisense oligonucleotides to Cux-1, a Cut-related homeobox gene, cause increased apoptosis in mouse embryonic kidney cultures.

Cux-1 is a murine homeobox gene that is highly and transiently expressed in the developing kidney. To further evaluate the role of Cux-1 in mammalian kidney development, organotypic cultures of embryonic mouse kidney were incubated with phosphorothioate-coupled antisense Cux-1 oligonucleotides (ODNs) in the presence of cationic liposomes. Inhibition of Cux-1 expression by antisense ODNs was verified by reverse transcription-PCR. Metanephroi that were incubated with antisense Cux-1 ODNs were 23% smaller than metanephroi that were incubated with sense Cux-1 ODNs. Morphologic analysis of metanephroi that were treated with antisense Cux-1 ODNs revealed that ureteric buds and induced epithelial structures were present. However, extensive areas of cell death containing shrunken cells with pyknotic nuclei were also evident. The presence of increased apoptosis was verified by ultrastructural and terminal transferase-mediated dUTP nick end labeling analyses. Two different antisense Cux-1 ODNs targeting either the translation start codon or the homeobox produced increased apoptosis. In contrast, metanephroi incubated with sense ODNs exhibited only occasional apoptotic cells. We conclude that the presence of antisense Cux-1 ODNs does not block nephron induction, but results instead in increased apoptosis. Proper regulation of Cux-1 expression may be necessary for normal kidney development.

A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes.

The mammalian female reproductive system arises from the uniform paramesonephric duct. The molecular mechanisms that establish differential development along this axis are unknown. We determined the pattern and timing of genes of the Hoxa axis in the development of the Müllerian tract. Hoxa-9, Hoxa-10, Hoxa-11, and Hoxa-13 are all expressed along the length of the paramesonephric duct in the embryonic mouse. After birth, a spatial Hox axis is established, corresponding to the postnatal differentiation of this organ system in the mouse. Hoxa-9 is expressed in the fallopian tubes, Hoxa-10 in the uterus, Hoxa-11 in the uterus and uterine cervix, and Hoxa-13 in the upper vagina. This expression pattern follows the paradigm of spatial colinearity but is a novel exception to temporal colinearity that has been considered typical of Hox genes. These genes remain expressed in the adult mouse and are expressed in the same pattern in the human. The female reproductive system undergoes dramatic structural and functional changes during the estrous cycle and in pregnancy, retaining a high degree of developmental plasticity. The late establishment of a Hox axis and persistent expression of Hox genes in the adult may play an important role in preserving this plasticity.

A unique variant of a homeobox gene related to Drosophila cut is expressed in mouse testis.

Mammalian homologues of the Drosophila cut homeobox gene encode transcriptional repressors that are involved in tissue-specific and developmental gene regulation. We examined the expression of a murine cut homologue (Cux-1) in the adult mouse. In many somatic tissues, Cux-1 was expressed as a 13-kb transcript. However, the highest expression of Cux-1 was in the testis, where a unique 2.4-kb splice variant was identified. Less abundant transcripts of 5 kb, 6.5 kb, and 8.5 kb were also detected only in the testis. The nucleotide sequence of the 2.4-kb Cux-1 transcript was identical to the 13-kb transcript in the region of overlap, but the testis-specific transcript encoded a truncated protein that contained only one Cut repeat in addition to the Cut-related homeodomain. Studies of mice homozygous for the atrichosis (at/at) mutation suggested that the 2.4-kb transcript was expressed in germ cells in the testis. In situ hybridization verified that Cux-1 was transiently expressed in post-meiotic germ cells at the round spermatid stage. Immunoblot analysis of nuclear extracts showed that the testis-specific Cux-1 transcripts encoded a 55-kDa protein. These results demonstrate that multiple products of a cut-related homeobox gene are expressed in the testis. The highly restricted pattern of expression of Cux-1 in the testis suggests that it may be involved in regulation of postmeiotic gene expression.

Primary structure, neural-specific expression, and chromosomal localization of Cux-2, a second murine homeobox gene related to Drosophila cut.

The cut locus of Drosophila encodes a diverged homeodomain-containing protein that is required for the development of external sensory (es) organs and other tissues. A homologous gene (Cux-1) that encodes a transcriptional repressor has been identified in the mouse and other mammals. We have identified a second murine homeobox-containing gene (designated Cux-2) that is structurally related to Drosophila cut. The murine Cux-2 homeobox was similar to Drosophila cut and encoded a homeodomain that contained a characteristic histidine residue at position 50. The predicted Cux-2 protein contained 1426 amino acids and included three internal 60-amino acid repeats (Cut repeats) that were previously found in Drosophila Cut and murine Cux-1. Unlike Cux-1, expression of Cux-2 was restricted to neural tissue. In the adult brain, Cux-2 was prominently expressed in neurons in the thalamus and limbic system. In embryos, Cux-2 was expressed in the developing central and peripheral nervous systems, including the telencephalon and peripheral ganglia of the trigeminal and glossopharyngeal nerves. A glutathione S-transferase fusion protein containing the carboxyl-terminal Cut repeat and homeodomain of Cux-2 exhibited sequence-specific binding to oligonucleotides derived from the promoter of the Ncam gene. Using an interspecific backcross panel, Cux-1 and Cux-2 were mapped to distinct loci that were genetically linked on distal chromosome 5. These results demonstrate that a family of homeobox genes related to Drosophila cut is located on chromosome 5 in the mouse. Cux-2 is expressed exclusively in the central and peripheral nervous systems, and the Cux-2 gene product binds to DNA in a sequence-specific manner. Cux-2 may encode a transcription factor that is involved in neural specification in mammals.

Expression of a cut-related homeobox gene in developing and polycystic mouse kidney.

Cut is a diverged homeobox gene that is essential for normal development of the Malpighian tubules in Drosophila melanogaster. Homologues of Drosophila cut that encode transcriptional repressors have been identified in several mammalian species and cell lineages. We examined the expression of a murine cut homologue (named Cux-1) in the developing mouse using Northern blot analysis and in situ hybridization. At 12.5 d.p.c. and 13.5 d.p.c., Cux-1 was highly expressed in a subset of embryonic tissues, including the developing metanephros. Within the metanephros, Cux-1 was expressed in the nephrogenic zone including both mesenchymal cells (uninduced and condensed mesenchyme) and epithelial cells (ureteric buds, renal vesicles, S-shaped bodies). During later stages of nephrogenesis, Cux-1 was down-regulated such that there was minimal expression in mature glomeruli and tubules. In addition, Cux-1 was detected in the mesonephros, mesonephric duct, and bladder. Expression of Cux-1 was also examined in polycystic kidneys from C57BL/6J-cpk/ cpk mice. At 21 days of age, Cux-1 was highly expressed in cyst epithelium of polycystic kidneys but was minimally expressed in kidneys from phenotypically normal littermates. These results demonstrate that a cut-related homeobox gene is expressed in the developing kidney and urinary tract of the mouse. Expression of Cux-1 in the kidney is inversely related to degree of cellular differentiation. Cux-1 may encode a transcriptional repressor that inhibits terminally differentiated gene expression during early stages of nephrogenesis.

Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-C1 cotransporter gene.

The murine Nkcc2/Slcl2a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)28.(C-T)28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K((NH4)+)-Cl cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.

Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.

A full-length cDNA encoding the murine renal Na-K-Cl cotransporter (NKCC2) was cloned using library screening and anchored polymerase chain reaction. The deduced protein sequence contained 1,095 amino acids and was 93.5% identical to rabbit NKCC2 and 97.6% identical to rat BSC1. Two potential sites of phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase and seven potential sites of phosphorylation by protein kinase C, which were previously identified in the rabbit and rat sequences, were phylogenetically conserved in the mouse. The expression of NKCC2 in the mouse was examined with Northern blot analysis and in situ hybridization. Expression of NKCC2 was kidney specific in both adult and embryonic mice. In the developing metanephros, NKCC2 was induced at 14.5 days post coitus and was expressed in distal limbs of immature loops of Henle but was absent from the ureteric bud, S-shaped bodies, and earlier nephrogenic structures. Similar to the rabbit, isoforms of NKCC2 that differed in the sequence of a 96-bp segment were identified in the mouse. In situ hybridization revealed that the isoforms exhibited different patterns of expression in the mature thick ascending limb of the loop of Henle as follows: isoform F was most highly expressed in the inner stripe of outer medulla, isoform A was most highly expressed in the outer stripe of the outer medulla, and isoform B was most highly expressed in the cortical thick ascending limb. To verify that the isoforms were generated by alternative splicing of mutually exclusive cassette exons, genomic clones encoding murine NKCC2 were characterized. Cassette exons were identified that corresponded to each of the three isoforms and were flanked by consensus splice donor and acceptor sequences.

Isolation and cDNA cloning of Ksp-cadherin, a novel kidney-specific member of the cadherin multigene family.

Cadherins are recognized as the principal mediators of homotypic cellular recognition and play a demonstrated role in the morphogenic direction of tissue development. We report here the identification of a structurally unique, kidney-specific member of the cadherin multigene family (Ksp-cadherin). cDNA cloning and molecular analysis of the 130-kDa protein confirmed that it was novel and indicated that it most closely resembled members of the LI-cadherin/HPT-1 cadherin subgroup. The predicted protein possesses the definitive cadherin-specific sequence motifs LDRE, DXND, and DXD in well conserved sequential arrangement, and the characteristic cysteine residues found in the last ectodomains of almost all known cadherins. Like LI-cadherin and HPT-1, Ksp-cadherin lacks the prosequence and HAV adhesion recognition sequence typical of most classical cadherins, and possesses a truncated cytoplasmic domain (18-22 amino acids). When expressed in a transient Vaccinia/T7 expression system, Ksp-cadherin displayed the classic calcium sensitivity to trypsin proteolysis that is observed in all cadherins. Immunolocalization studies and Northern analysis indicated that expression of Ksp-cadherin was kidney-specific and limited to the basolateral membranes of renal tubular epithelial cells. In summary, we have identified and cloned a novel, kidney-specific member of the cadherin multigene family that we propose be designated Ksp-cadherin.

Phylogenetically conserved sequences in the promoter of the rabbit sodium-hydrogen exchanger isoform 1 gene (NHE1/SLC9A1).

NHE1 (gene symbol SLC9A1) encodes an isoform of the amiloride-sensitive Na+-H+ exchanger that is present in many cells and the basolateral membrane of renal epithelia. Expression of NHE1 is modulated in response to chronic metabolic acidosis, growth factors, and phorbol esters. To begin examining the molecular basis for this regulation, a rabbit genomic clone that contained 6 kb of 5' flanking region, the first exon, and a portion of the first intervening sequence of NHE1 was obtained. The principal transcription start site in native rabbit tissues was located 798 bp 5' to the initiation codon. The sequence of the proximal 5' flanking region of rabbit NHE1 was similar to the human sequence and contained a TATA-box, (G + C)-boxes, homopyrimidine direct repeats, and a putative AP-1 site. When ligated to luciferase and transfected into porcine renal epithelial cells (LLC-PK1), 708 bp of proximal 5' flanking region exhibited orientation-dependent promoter activity.