Salt-sensitive hypertension in GR mutant rats is associated with altered plasma polyunsaturated fatty acid levels and aortic vascular reactivity.

In humans, glucocorticoid resistance is attributed to mutations in the glucocorticoid receptor (GR). Most of these mutations result in decreased ligand binding, transactivation, and/or translocation, albeit with normal protein abundances. However, there is no clear genotype‒phenotype relationship between the severity or age at disease presentation and the degree of functional loss of the receptor. Previously, we documented that a GR+/- rat line developed clinical features of glucocorticoid resistance, namely, hypercortisolemia, adrenal hyperplasia, and salt-sensitive hypertension. In this study, we analyzed the GR+/em4 rat model heterozygously mutant for the deletion of exon 3, which encompasses the second zinc finger, including the domains of DNA binding, dimerization, and nuclear localization signals. On a standard diet, mutant rats exhibited a trend toward increased corticosterone levels and a normal systolic blood pressure and heart rate but presented with adrenal hyperplasia. They exhibited increased adrenal soluble epoxide hydroxylase (sEH), favoring an increase in less active polyunsaturated fatty acids. Indeed, a significant increase in nonactive omega-3 and omega-6 polyunsaturated fatty acids, such as 5(6)-DiHETrE or 9(10)-DiHOME, was observed with advanced age (10 versus 5 weeks old) and following a switch to a high-salt diet accompanied by salt-sensitive hypertension. In thoracic aortas, a reduced soluble epoxide hydrolase (sEH) protein abundance resulted in altered vascular reactivity upon a standard diet, which was blunted upon a high-salt diet. In conclusion, mutations in the GR affecting the ligand-binding domain as well as the dimerization domain resulted in deregulated GR signaling, favoring salt-sensitive hypertension in the absence of obvious mineralocorticoid excess.

Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2.

Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic beta-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice. In beta-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low or almost undetectable levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and beta-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, beta-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the alpha- to beta-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.

Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.

Genetic evidence supports a critical role for the epithelial sodium channel (ENaC) in both clearance of fetal lung liquid at birth and total body electrolyte homeostasis. Evidence from heterologous expression systems suggests that expression of the alphaENaC subunit is essential for channel function, whereas residual channel function can be measured in the absence of beta or gamma subunits. We generated mice without gammaENaC (gammaENaC -/-) to test the role of this subunit in neonatal lung liquid clearance and total body electrolyte balance. Relative to controls, gammaENaC (-/-) pups showed low urinary [K+] and high urinary [Na+] and died between 24 and 36 h, probably from hyperkalemia (gammaENaC -/- 18.3 mEq/l, control littermates 9.7 mEq/l). Newborn gammaENaC (-/-) mice cleared lung liquid more slowly than control littermates, but lung water at 12 h (wet/dry = 5.5) was nearly normal (wet/dry = 5.3). This study suggests that gammaENaC facilitates neonatal lung liquid clearance and is critical for renal Na+ and K+ transport, and that low level Na+ transport may be sufficient for perinatal lung liquid absorption but insufficient to maintain electrolyte balance by the distal nephron. The gammaENaC (-/-) newborn exhibits a phenotype that resembles the clinical manifestations of human neonatal PHA1.

Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice.

We have isolated and characterised the promoter of the mouse Scnn1a (alpha ENaC) gene. Using transient transfections of serial deletion mutants into Scnn1a-expressing cells, we demonstrate that 1.56 kb of 5' upstream sequence is required for cell-specific expression and corticosteroid-mediated regulation. These 5' sequences are not sufficient to drive expression of a lacZ reporter gene or a rat Scnn1a cDNA in transgenic mice, where they failed to rescue Scnn1a deficiency.

Perinatal activation of a tyrosine aminotransferase fusion gene does not occur in albino lethal mice.

To investigate developmental expression of the rat tyrosine aminotransferase (TAT) gene in normal and in albino lethal mice we generated transgenic mice carrying a fusion gene of TAT 5'-sequences (11 kb) and the bacterial chloramphenicol acetyltransferase (CAT) gene. In four lines, CAT activity was found only in liver. RNA analyses on a high-expressing line showed that transgenic expression follows expression of mouse TAT mRNA: it is inducible by glucocorticoids and activated perinatally. This perinatal activation of transgene expression does not occur in lethal albino mice (c14CoS/c14CoS) which are characterized by reduced mRNA levels of several liver-specific enzymes involved in gluconeogenesis. In conclusion, the data show that the 5'-flanking region of the rat TAT gene contains elements specifying regulated expression and establish that the 5'-flanking region of the TAT gene is responsive to the enzyme deficiency characteristic of the albino lethal mice.

The ENaC channel is required for normal epidermal differentiation.

Ionic fluxes are important for critical aspects of keratinocyte differentiation, including synthesis of differentiation-specific proteins, enzymatic catalysis of protein cross-linking, post-transcriptional processing of profilaggrin, and lipid secretion. The epithelial sodium channel is expressed in epidermis and the expression of its alpha and beta subunits is enhanced as keratinocytes differentiate. In order to ascertain the role of the epithelial sodium channel in epidermal differentiation, we examined skin of mice in which the epithelial sodium channel alpha subunit had been deleted. Newborn -/- mice, in which the alpha subunit had been completely inactivated, demonstrated epithelial hyperplasia, abnormal nuclei, premature secretion of lipids, and abnormal keratohyaline granules. In addition, immunohistochemistry demonstrated that expression of the differentiation markers K1, K6, and involucrin were abnormal. These data suggest that the epithelial sodium channel modulates ionic signaling for specific aspects of epidermal differentiation, such as synthesis or processing of differentiation- specific proteins, and lipid secretion.

Implication of ENaC in salt-sensitive hypertension.

Arterial blood pressure is critically dependent on sodium balance. The kidney is the key player in maintaining sodium homeostasis. Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the highly selective, amiloride-sensitive epithelial sodium channel (ENaC). Direct evidence that dysfunction of ENaC participates in blood pressure regulation has come from the molecular analysis of two human genetic diseases, Liddle's syndrome and pseudohypoaldosteronism type 1 (PHA-1). Both, increased sodium reabsorption despite low aldosterone levels in Liddle's patients and decreased sodium reabsorption despite high aldosterone levels in PHA-1 patients, demonstrated that ENaC is an effector for aldosterone action. Gene-targeting and classical transgenic technology enable the generation of mouse models for these diseases and the analysis of the involvement of the epithelial sodium channel (ENaC) in the progress of these diseases. A first mouse model using alphaENaC transgenic knockout mice [alphaENaC(-/-)Tg] mimicked several clinical features of PHA-1, like salt-wasting, metabolic acidosis, high aldosterone levels, growth retardation and increased early mortality. Such mouse models will be necessary in testing the involvement of genetic and/or environmental factors like salt-intake in hypertension.

lacZ transgenic mice to monitor gene expression in embryo and adult.

In transgenic experiments, lacZ can be used as a reporter gene for activity of a given promoter. Its main advantage is the ease of visualization in situ, on sections or in whole mount preparations, and the availability of simple protocols. In the following, we describe our procedure for detecting promoter activity in transgenic mice, including choice of lacZ vectors, generation of the transgenic mice, and analysis of expression. We had recently used this protocol to detect tyrosinase gene promoter activity in embryonic and adult brain.

Mechano-electrical transduction in mice lacking the alpha-subunit of the epithelial sodium channel.

Sensory hair cells of the vertebrate inner ear use mechanically gated transducer channels (MET) to perceive mechanical stimuli. The molecular nature of the MET channel is not known but several findings suggested that the amiloride-sensitive epithelial Na+ channel, ENaC, might be a candidate gene for this function. In order to test this hypothesis, we examined knockout mice deficient in the alpha-subunit of ENaC, and therefore in ENaC function. First, neonatal alphaENaC(-/-) mice exhibited vestibular reflexes not different from wildtype littermates thus indicating normal vestibular function. We used organotypic cultures of cochlear outer hair cells from newborns to rescue the hair cells from the perinatal death of alphaENaC(-/-) mice. When hair bundles of cochlear outer hair cells of alphaENaC(-/-) mice were mechanically stimulated by a fluid jet in whole cell voltage clamp experiments, transducer currents were elicited that were not significantly different from those of alphaENaC(+/-) or (+/+) cochlear outer hair cells. These results suggest that the vertebrate mechano-electrical transducer apparatus does not include the alpha-subunit of the epithelial Na+ channel.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H+-ATPase.

Final urinary acidification is achieved by electrogenic vacuolar H(+)-ATPases expressed in acid-secretory intercalated cells (ICs) in the connecting tubule (CNT) and the cortical (CCD) and initial medullary collecting duct (MCD), respectively. Electrogenic Na(+) reabsorption via epithelial Na(+) channels (ENaCs) in the apical membrane of the segment-specific CNT and collecting duct cells may promote H(+)-ATPases-mediated proton secretion by creating a more lumen-negative voltage. The exact localization where this supposed functional interaction takes place is unknown. We used several mouse models performing renal clearance experiments and assessed the furosemide-induced urinary acidification. Increasing Na(+) delivery to the CNT and CCD by blocking Na(+) reabsorption in the thick ascending limb with furosemide enhanced urinary acidification and net acid excretion. This effect of furosemide was abolished with amiloride or benzamil blocking ENaC action. In mice deficient for the IC-specific B1 subunit of the vacuolar H(+)-ATPase, furosemide led to only a small urinary acidification. In contrast, in mice with a kidney-specific inactivation of the alpha subunit of ENaC in the CCD and MCD, but not in the CNT, furosemide alone and in combination with hydrochlorothiazide induced normal urinary acidification. These results suggest that the B1 vacuolar H(+)-ATPase subunit is necessary for the furosemide-induced acute urinary acidification. Loss of ENaC channels in the CCD and MCD does not affect this acidification. Thus, functional expression of ENaC channels in the CNT is sufficient for furosemide-stimulated urinary acidification and identifies the CNT as a major segment in electrogenic urinary acidification.

Reversal of convention: from man to experimental animal in elucidating the function of the renal amiloride-sensitive sodium channel.

The kidney plays a dominant role in maintaining sodium homeostasis. Despite wide variation in environmental exposure, the osmolality of the extracellular fluid that is determined by the sodium ion concentration is maintained within narrow margins. Derangement in function of proteins that transport Na+ and of those regulating the activity of these sodium-transporting proteins are likely to be responsible for a number of clinical disorders of fluid and electrolyte homeostasis. The amiloride-sensitive epithelial sodium channel (ENaC) is implicated in the control of blood pressure as demonstrated by the analysis of two genetic diseases, Liddle's syndrome and pseudohypoaldosteronism (PHA-1). Mutations have been identified in the genes coding for the alpha-, beta- or gamma-subunit of ENaC. ENaC constitutes the limiting step for sodium reabsorption in epithelial cells that line the distal nephron, distal colon, ducts of several exocrine glands and lung airways and might play an important role in pathophysiological and clinical conditions such as hypertension or lung edema.

Dysfunction of epithelial sodium transport: from human to mouse.

The highly amiloride-sensitive epithelial sodium channel (ENaC) is an apical membrane constituent of cells of many salt-absorbing epithelia. In the kidney, the functional relevance of ENaC expression has been well established. ENaC mediates the aldosterone-dependent sodium reabsorption in the distal nephron and is involved in the regulation of blood pressure. Mutations in genes encoding ENaC subunits are causative for two human inherited diseases: Liddle's syndrome, a severe form of hypertension associated with ENaC hyperfunction, and pseudohypoaldosteronism (PHA-1), a salt-wasting syndrome caused by decreased ENaC function. Transgenic mouse technologies provide a useful tool to study the role of ENaC in vivo. Different mouse lines have been established in which each of the ENaC subunits was affected. The phenotypes observed in these mice demonstrated that each subunit is essential for survival and for regulation of sodium transport in kidney and colon. Moreover, the alpha subunit plays a specific role in the control of fluid absorption in the airways at birth. Such mice can now be used to study the role of ENaC in various organs and can serve as models to understand the pathophysiology of these human diseases.

Preferential nondisjunction of specific bivalents in oocytes from Djungarian hamsters (Phodopus sungorus) following colchicine treatment.

In an attempt to study the mechanisms leading to nondisjunction during meiosis I, Djungarian hamster females were treated with colchicine (3 mg/kg), which binds specifically to tubulin. The number of ovulated oocytes per female was significantly reduced following colchicine treatment (8.2 +/- 5.3, compared to 10.6 +/- 5.9 in controls receiving saline solution only). Application of colchicine rather late during oocyte maturation (ie, 5.5 h after injection of human chorionic gonadotrophin) caused a significant increase in the number of ovulated diploid (34.5%) and hyperhaploid (11.7%) oocytes, compared to the frequencies observed in the saline-treated controls (0.8% and 3.5%, respectively). Specific bivalents (viz, the large meta- and submetacentric chromosomes of groups A, B, and C) were preferentially involved in colchicine-induced nondisjunction. The same pattern of chromosomal malsegregation was previously observed in oocytes from this hamster species following hypergonadotrophic stimulation. Preferential involvement of bivalents in the process of nondisjunction, whether induced by colchicine or hypergonadotrophic stimulation, is explained by an interference with microtubular function affecting those bivalents that are the last to segregate.

Pattern and frequency of nondisjunction in oocytes from the Djungarian hamster are determined by the stage of first meiotic spindle inhibition.

In order to study the mechanisms of nondisjunction at meiosis I in oocytes gonadotropin-stimulated Djungarian hamsters were treated at two stages [4.5 and 6 h post human chorionic gonadotropin (HCG)] during the preovulatory period with 1000 mg/kg Carbendazim (MBC). The compound, known to bind fast but reversibly to mammalian tubulin, was chosen to investigate whether the stage at which spindle function is inhibited affects the pattern of nondisjunction. Ovulated oocytes were cytologically prepared and scored for hyperhaploidy, diploidy and presegregation. Application at an early spindle phase, 4.5 h post HCG, to females stimulated with a low gonadotropin dose [3 IU pregnant mares serum (PMS); 2 IU HCG] caused a high frequency of nondisjunction (40.6%) with a more or less nonspecific pattern of malsegregated bivalents. Treatment at a late stage of spindle function (6 h post HCG) resulted in a less frequent (22.5%) but highly preferential malsegregation of those A-D group bivalents thought earlier to be late segregators. On the other hand, oocytes from females primed with a high (10 IU PMS and HCG) gonadotropin dose, a treatment assumed to delay meiosis by approximately 1.5 h, responded to MBC treatment at the late stage (6 h) with a nonspecific pattern and a high frequency (71.2%) of nondisjunction. The latter result is comparable to that in which MBC was given at the early stage (4.5 h) and after a low gonadotropin dose. The high nondisjunction response additionally indicates that spindles in hypergonadotropic stimulated oocytes are more susceptible and/or that the concentration of the inhibitor is higher in such oocytes. Only few oocytes with presegregation (3.1%; 0.0%; 1.7%) and few diploid oocytes (3.3%; 1.5%; 3.2%) with complete inhibition of meiosis I were observed. We conclude, that in Djungarian hamsters (1) the segregation of bivalents at meiosis I is asynchronous with the large A-D bivalents segregating last, (2) the phase in which spindle function is inhibited determines the pattern of nondisjunction, and (3) the resumption of meiosis I - from dictyotene to metaphase II - does not follow a rigidly timed programme but depends on the conditions of follicular maturation.

Scnn1 sodium channel gene family in genetically engineered mice.

The amiloride-sensitive epithelial sodium channel is the limiting step in salt absorption. In mice, this channel is composed of three subunits (alpha, beta, and gamma), which are encoded by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). The functions of these genes were recently investigated in transgenic (knockout) experiments, and the absence of any subunit led to perinatal lethality. More defined phenotypes have been obtained by introducing specific mutations or using transgenic rescue experiments. In this report, these approaches are summarized and a current gene-targeting strategy to obtain conditional inactivation of the channel is illustrated. This latter approach will be indispensable for the investigation of channel function in a wide variety of organ systems.

Genetic disorders of membrane transport. V. The epithelial sodium channel and its implication in human diseases.

The epithelial Na+ channel (ENaC) controls the rate-limiting step in the process of transepithelial Na+ reabsorption in the distal nephron, the distal colon, and the airways. Hereditary salt-losing syndromes have been ascribed to loss of function mutations in the alpha-, beta-, or gamma-ENaC subunit genes, whereas gain of function mutations (located in the COOH terminus of the beta- or gamma-subunit) result in hypertension due to Na+ retention (Liddle's syndrome). In mice, gene-targeting experiments have shown that, in addition to the kidney salt-wasting phenotype, ENaC was essential for lung fluid clearance in newborn mice. Disruption of the alpha-subunit resulted in a complete abolition of ENaC-mediated Na+ transport, whereas knockout of the beta- or gamma-subunit had only minor effects on fluid clearance in lung. Disruption of each of the three subunits resulted in a salt-wasting syndrome similar to that observed in humans.

Physiological and pathophysiological role of the epithelial sodium channel in the control of blood pressure.

Blood pressure regulation is an integrated physiological phenomenon known to be influenced by many biological processes and by a variety of environmental factors. Epidemiological studies nevertheless suggest that up to 30% of the variation in blood pressure could be due to genetic factors. Thus, mutations in genes that control blood pressure may be the underlying cause of essential hypertension. Arterial blood pressure is critically dependent on the sodium balance and the regulation of renal sodium excretion is one of the most important homeostatic functions of the body. The identification of genes encoding proteins that transport Na+ across cells of the kidney tubules and of those regulating the activity of these sodium-transporting proteins will therefore bring further insights into the pathophysiology of salt-sensitive hypertension.