Russell F. Husted

Glucocorticoid Induction of Epithelial Sodium Channel Expression in Lung and Renal Epithelia Occurs via trans-Activation of a Hormone Response Element in the 5′-Flanking Region of the Human Epithelial Sodium Channel α Subunit Gene

In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel α subunit (αENaC). In H441 lung cells, 100 nm dexamethasone increases amiloride-sensitive short-circuit current (3.3 μA/cm2 to 7.5 μA/cm2), correlating with a 5-fold increase in αENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of αENaC, the human αENaC 5′-flanking region was cloned and tested in H441 cells. By deletion analysis, a ∼150-base pair region 5′ to the upstream promoter was identified that, when stimulated with 100 nm dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, mαENaC expression and luciferase expression from αENaC genomic fragments were also increased by 100 nm dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated αENaC gene transcription. We conclude that glucocorticoids stimulate αENaC expression in kidney and lung via activation of a hormone response element in the 5′-flanking region of hαENaC and this response, in part, is the likely basis for the up-regulation of Na+transport in these sites.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

Mice defective in Trpm6 show embryonic mortality and neural tube defects

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.

Enhancement of electrogenic Na+ transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones.

We have investigated the effect of steroid hormones on Na+ transport by rat renal inner medullary collecting duct (IMCD) cells. These cells, grown on permeable supports in primary culture, grow to confluence and develop a transmonolayer voltage oriented such that the apical surface is negative with respect to the basal surface. The results of these experiments demonstrate that this voltage is predominantly (or exclusively) the result of electrogenic Na+ absorption. Na+ transport can be stimulated two- to fourfold by exposure to either dexamethasone or aldosterone (100 nM). Experiments using specific antagonists of the glucocorticoid and mineralocorticoid receptors indicate that activation of either receptor stimulates electrogenic Na+ transport; electroneutral Na+ transport is undetectable. Two other features of the IMCD emerge from these studies. (a) These cells appear to have the capacity to metabolize the naturally occurring glucocorticoid hormone corticosterone. (b) The capacity for K+ secretion is minimal and steroid hormones do not induce or stimulate conductive K+ secretion as they do in the cortical collecting duct.

Cellular responses to steroids in the enhancement of Na+ transport by rat collecting duct cells in culture. Differences between glucocorticoid and mineralocorticoid hormones.

It has recently been discovered that both mineralocorticoid (MC) and glucocorticoid (GC) hormones can stimulate electrogenic Na+ absorption by mammalian collecting duct cells in culture. In primary cultures of rat inner medullary collecting duct (IMCD) cells, 24-h incubation with either MC or GC agonist stimulates Na+ transport approximately threefold. We have now determined that the effects were not additive, but the time courses were different. As aldosterone is known to stimulate citrate synthase, Na+/K+ ATPase activity, and ouabain binding in cortical collecting duct principal cells, we determined the effects of steroids on these parameters in IMCD cells. MC and GC agonists both produced a small increase in citrate synthase activity. There was no increase in Na+/K+ ATPase activity but specific ouabain binding was increased more than two-fold by either agonist. To determine the role of apical Na+ entry in the steroid-induced effects, the Na+ channel inhibitor, benzamil, was used. Benzamil did not alter the stimulation of citrate synthase activity by either steroid. In contrast, GC stimulation of ouabain binding was prevented by benzamil, whereas MC stimulation was not. We conclude that there are differences in the way that MC and GC hormones produce an increased Na+ transport. Both appear to produce translocation (or activation) of pumps into the basolateral membrane. GC stimulation of pump translocation requires increased Na+ entry whereas MC stimulation does not.

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: effects of inhibitors of H+ ATPase

SummaryThe coupling between H+ transport (JH) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.JH was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2<0.5 mm Hg) and contained 10mm glucose. Under these conditions,JH was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measuredJH. The slope ofJlac onJH was 0.58±0.12 with an intercept forJlac atJH=0 of 0.55 μmol/hr. Values for δJlac/δJH were determined in groups of individual bladders whenJH was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of δJlac/δJH indicates a high degree of coupling betweenJH andJlac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the δJlac/δJH values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2 H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobicJH without change in δJlac/δJH or basalJlac and, therefore, acted primarily on the H+ pump.S addition of vanadate also inhibitedJH, but the inhibition was associated with an increase inJlac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H+-ATPase characteristics that differ from mitochondrial ATPase in that H+ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H+ and lactate, the H+/ATP stoichiometry of the pump is about 2.

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: Studies with a disulfonic stilbene

SummaryBicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO3− generated behind the H+ pump moves across this membrane into the serosal solution. This efflux of HCO3− is inhibited by SITS (4-isothiocyano-4′-acetamido-2,2′-disulfonic stilbene). When HCO3− is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl−. This secretory HCO3− flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5×10−4m SITS on HCO3− secretion and Cl− absorption were examined. The rate of H+ secretion was brought to zero by an opposing pH gradient, and 20mm HCO3− was added toS. HCO3− secretion, measured by pH stat titration, was equivalent to the increase inM→S Cl− flux after HCO3− addition. Neither theS→M flux of HCO3− nor theM→S flux of Cl− were affected by SITS. In the absence of electrochemical gradients, net Cl− absorption was observed only in the presence of HCO3− in the media; under such conditions, unidirectional and net fluxes of Cl− were not altered by serosal or mucosal SITS. H+ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO3− in series with the H+ pump and the failure of SITS to affect HCO3− secretion and Cl− absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.

Concerted actions of IL-1β inhibit Na+ absorption and stimulate anion secretion by IMCD cells

Increasing evidence indicates that factors other than adrenocorticoid hormones can influence long-term regulation of Na+ transport by inner medullary collecting duct (IMCD) cells. We now report that, of 14 interleukins tested, only interleukin-1α (IL-1α) and IL-1β inhibited Na+ transport by primary cultures of rat IMCD. IL-1β reduced both basal and mineralocorticoid (MC)-stimulated Na+ transport by 50-70%; its effect on glucocorticoid (GC)-stimulated Na+ transport was significantly less. IL-1β continued to blunt MC stimulation of Na+ transport even after it had been removed from the medium for 24 h. The onset of action to inhibit Na+ transport was within 20 min. The acute effect from the basolateral surface was greater than that from the apical surface, but the effect from each surface was additive. In addition to its inhibitory effect on Na+ transport, chronic IL-1β exposure increased both basal and cAMP-stimulated anion secretion rates. IL-1β had no acute effect on anion secretion. Monolayers chronically treated with IL-1β had an increased capacity to secrete fluid, as predicted from its effects on ion transport. Inhibitors of cyclooxygenase did not blunt the actions of IL-1β. Furthermore, IL-1β did not produce a rise in intracellular Ca2+. These results suggest novel signaling pathways induced by IL-1β regulating Na+ and Cl- transport by the IMCD.

Chloride-bicarbonate exchange in the urinary bladder of the turtle. Independence from sodium ion

The rates of Cl- absorption and HCO3- secretion were not different in turtle urinary bladders bathed in Na+-free solutions. These results in turtle bladder are inconsistent with Na+-anion cotransport but can be accounted for by a Cl-/HCO3- exchange system.