John J. Duffy

Renal and intestinal absorptive defects in mice lacking the NHE3 Na + /H + exchanger

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3 . It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3– absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3–/–) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3– and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl–/HCO3 – exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K +-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to beta-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (+/- dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca(2+)-ATPase for Ca2+ in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the beta-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the hearts contractile responses to beta-adrenergic agonists.

Fibroblast growth factor 2 control of vascular tone

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FCF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

Phenotype Resembling Gitelman’s Syndrome in Mice Lacking the Apical Na+-Cl− Cotransporter of the Distal Convoluted Tubule

Mutations in the gene encoding the thiazide-sensitive Na+-Cl− cotransporter (NCC) of the distal convoluted tubule cause Gitelman’s syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman’s syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc −/−) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure.Ncc −/− mice retain Na+ as well as wild-type mice when fed a Na+-depleted diet; however, after 2 weeks of Na+ depletion the mean arterial blood pressure of Ncc −/− mice was significantly lower than that of wild-type mice. In addition, Ncc −/−mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg2+ and Ca2+ are altered, as observed in Gitelman’s syndrome.

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl− and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K+ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1−/− ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl− channel, were reduced in jejunum, cecum, and trachea of Nkcc1−/− mice, indicating that NKCC1 contributes to cAMP-induced Cl− secretion. In contrast, secretion of gastric acid in adult Nkcc1−/− stomachs and enterotoxin-stimulated fluid secretion in the intestine of sucklingNkcc1−/− mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K+ movements involved in generation of the K+-rich endolymph and the endocochlear potential.

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

Balance and Hearing Deficits in Mice with a Null Mutation in the Gene Encoding Plasma Membrane Ca2+-ATPase Isoform 2

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion.

Multiple isoforms of the Na+/H+ exchanger (NHE) are expressed at high levels in gastric epithelium, but the physiological role of individual isoforms is unclear. To study the function of NHE2, which is expressed in mucous, zymogenic, and parietal cells, we prepared mice with a null mutation in the NHE2 gene. Homozygous null mutants exhibit no overt disease phenotype, but the cellular composition of the oxyntic mucosa of the gastric corpus is altered, with parietal and zymogenic cells reduced markedly in number. Net acid secretion in null mutants is reduced slightly relative to wild-type levels just before weaning and is abolished in adult animals. Although mature parietal cells are observed, and appear morphologically to be engaged in active acid secretion, many of the parietal cells are in various stages of degeneration. These results indicate that NHE2 is not required for acid secretion by the parietal cell, but is essential for its long-term viability. This suggests that the unique sensitivity of NHE2 to inhibition by extracellular H+, which would allow upregulation of its activity by the increased interstitial alkalinity that accompanies acid secretion, might enable this isoform to play a specialized role in maintaining the long-term viability of the parietal cell.

Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice.

Previous studies using isolated tissues suggest that the colonic H, K-ATPase (cHKA), expressed in the colon and kidney, plays an important role in K+ conservation. To test the role of this pump in K+ homeostasis in vivo, we generated a cHKA-deficient mouse and analyzed its ability to retain K+ when fed a control or K+-free diet. When maintained on a control diet, homozygous mutant (cHKA-/-) mice exhibited no deficit in K+ homeostasis compared to wild-type (cHKA+/+ greater, similar mice. Although fecal K+ excretion in cHKA-/- mice was double that of cHKA+/+ mice, fecal K+ losses were low compared with urinary K+ excretion, which was similar in both groups. When maintained on a K+-free diet for 18 d, urinary K+ excretion dropped over 100-fold, and to similar levels, in both cHKA-/- and cHKA+/+ mice; fecal K+ excretion was reduced in both groups, but losses were fourfold greater in cHKA-/- than in cHKA+/+ mice. Because of the excess loss of K+ in the colon, cHKA-/- mice exhibited lower plasma and muscle K+ than cHKA+/+ mice. In addition, cHKA-/- mice lost twice as much body weight as cHKA+/+ mice. These results demonstrate that, during K+ deprivation, cHKA plays a critical role in the maintenance of K+ homeostasis in vivo.

Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3.

Sarco(endo)plasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) is one of two Ca2+ pumps serving intracellular Ca2+ signaling pools in non-muscle tissues; however, unlike the ubiquitous SERCA2b, it exhibits a restricted cell-type distribution. Gene targeting was used to generate a mouse with a null mutation in the SERCA3 gene. Homozygous mutant mice were viable, fertile, and did not exhibit an overt disease phenotype. Because SERCA3 is expressed in arterial endothelial cells, aortic ring preparations were analyzed to determine whether it is involved in the regulation of vascular tone. Contraction-isometric force relations in response to phenylephrine or KCl, as well as relaxation produced by exposure to a nitric oxide donor, were similar in wild-type and null mutant aortas. Acetylcholine-induced endothelium-dependent relaxation of aortas after precontraction with phenylephrine was significantly reduced in homozygous mutants (61.3 ± 5.6% in wild type, 35.4 ± 7.3% in mutants). Ca2+ imaging of cultured aortic endothelial cells demonstrated that the acetylcholine-induced intracellular Ca2+ signal is sharply diminished in SERCA3-deficient cells and also indicated that replenishment of the acetylcholine-responsive Ca2+ stores is severely impaired. These results indicate that SERCA3 plays a critical role in endothelial cell Ca2+signaling events involved in nitric oxide-mediated relaxation of vascular smooth muscle.