Dynamic characterization of intestinal metaplasia in the gastric corpus mucosa of Atp4a-deficient mice.

Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H+, K+-adenosine triphosphatase (ATPase), the enzyme primarily responsible for gastric lumen acidification. Here, we describe the characterization of mice deficient in the H+, K+-ATPase α subunit (Atp4a-/-) to determine the role of this protein in the biosynthesis of this membrane system and the biology of the gastric mucosa. Atp4a-/- mice were produced by gene targeting. Wild-type (WT) and Atp4a-/- mice, paired for age, were examined at 10, 12, 14 and 16 weeks for histopathology, and the expression of mucin 2 (MUC2), α-methylacyl-CoA racemase (AMACR), Ki-67 and p53 proteins was analyzed by immunohistochemistry. For further information, phosphoinositide 3-kinase (PI3K), phosphorylated-protein kinase B (p-AKT), mechanistic target of rapamycin (mTOR), hypoxia-inducible factor 1α (HIF-1α), lactate dehydrogenase A (LDHA) and sirtuin 6 (SIRT6) were detected by Western blotting. Compared with the WT mice, hypochlorhydric Atp4a-/- mice developed parietal cell atrophy and significant antral inflammation (lymphocyte infiltration) and intestinal metaplasia (IM) with elevated MUC2 expression. Areas of dysplasia in the Atp4a-/- mouse stomach showed increased AMACR and Ki-67 expression. Consistent with elevated antral proliferation, tissue isolated from Atp4a-/- mice showed elevated p53 expression. Next, we examined the mechanism by which the deficiency of the H+, K+-ATPase α subunit has an effect on the gastric mucosa. We found that the expression of phosphorylated-PI3K, p-AKT, phosphorylated-mTOR, HIF-1α, LDHA and SIRT6 was significantly higher in tissue from the Atp4a-/- mice compared with the WT mice (P<0.05). The H+, K+-ATPase α subunit is required for acid-secretory activity of parietal cells in vivo, the normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes. Chronic achlorhydria and hypergastrinemia in aged Atp4a-/- mice produced progressive hyperplasia and mucolytic and IM, and activated the Warburg effect via PI3K/AKT/mTOR signaling.

H,K-ATPase type 2 regulates gestational extracellular compartment expansion and blood pressure in mice.

The modifications of the hemodynamic system and hydromineral metabolism are physiological features characterizing a normal gestation. Thus, the ability to expand plasma volume without increasing the level of blood pressure is necessary for the correct perfusion of the placenta. The kidney is essential in this adaptation by reabsorbing avidly sodium and fluid. In this study, we observed that the H,K-ATPase type 2 (HKA2), an ion pump expressed in kidney and colon and already involved in the control of the K+ balance during gestation, is also required for the correct plasma volume expansion and to maintain normal blood pressure. Indeed, compared with WT pregnant mice that exhibit a 1.6-fold increase of their plasma volume, pregnant HKA2-null mice (HKA2KO) only modestly expand their extracellular volume (×1.2). The renal expression of the epithelial Na channel (ENaC) α- and γ-subunits and that of the pendrin are stimulated in gravid WT mice, whereas the Na/Cl- cotransporter (NCC) expression is downregulated. These modifications are all blunted in HKA2KO mice. This impeded renal adaptation to gestation is accompanied by the development of hypotension in the pregnant HKA2KO mice. Altogether, our results showed that the absence of the HKA2 during gestation leads to an "underfilled" situation and has established this transporter as a key player of the renal control of salt and potassium metabolism during gestation.

ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl- cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

ENaC activity in the cortical collecting duct of HKα1 H+,K+-ATPase knockout mice is uncoupled from Na+ intake.

Modulation of the epithelial Na+ channel (ENaC) activity in the collecting duct (CD) is an important mechanism for normal Na+ homeostasis. ENaC activity is inversely related to dietary Na+ intake, in part due to inhibitory paracrine purinergic regulation. Evidence suggests that H+,K+-ATPase activity in the CD also influences Na+ excretion. We hypothesized that renal H+,K+-ATPases affect Na+ reabsorption by the CD by modulating ENaC activity. ENaC activity in HKα1 H+,K+-ATPase knockout (HKα1-/-) mice was uncoupled from Na+ intake. ENaC activity on a high-Na+ diet was greater in the HKα1-/- mice than in WT mice. Moreover, dietary Na+ content did not modulate ENaC activity in the HKα1-/- mice as it did in WT mice. Purinergic regulation of ENaC was abnormal in HKα1-/- mice. In contrast to WT mice, where urinary [ATP] was proportional to dietary Na+ intake, urinary [ATP] did not increase in response to a high-Na+ diet in the HKα1-/- mice and was significantly lower than in the WT mice. HKα1-/- mice fed a high-Na+ diet had greater Na+ retention than WT mice and had an impaired dipsogenic response. These results suggest an important role for the HKα1 subunit in the regulation of purinergic signaling in the CD. They are also consistent with HKα1-containing H+,K+-ATPases as important components for the proper regulation of Na+ balance and the dipsogenic response to a high-salt diet. Such observations suggest a previously unrecognized element in Na+ regulation in the CD.

Skeletal effects of a gastrin receptor antagonist in H+/K+ATPase beta subunit KO mice.

Epidemiological studies suggest an increased fracture risk in patients taking proton pump inhibitors (PPIs) for long term. The underlying mechanism, however, has been disputed. By binding to the gastric proton pump, PPIs inhibit gastric acid secretion. We have previously shown that proton pump (H(+)/K(+)ATPase beta subunit) KO mice exhibit reduced bone mineral density (BMD) and inferior bone strength compared with WT mice. Patients using PPIs as well as these KO mice exhibit gastric hypoacidity, and subsequently increased serum concentrations of the hormone gastrin. In this study, we wanted to examine whether inhibition of the gastrin/CCK2 receptor influences bone quality in these mice. KO and WT mice were given either the gastrin/CCK2 receptor antagonist netazepide dissolved in polyethylene glycol (PEG) or only PEG for 1year. We found significantly lower bone mineral content and BMD, as well as inferior bone microarchitecture in KO mice compared with WT. Biomechanical properties by three-point bending test also proved inferior in KO mice. KO mice receiving netazepide exhibited significantly higher cortical thickness, cortical area fraction, trabecular thickness and trabecular BMD by micro-CT compared with the control group. Three-point bending test also showed higher Young's modulus of elasticity in the netazepide KO group compared with control mice. In conclusion, we observed that the gastrin receptor antagonist netazepide slightly improved bone quality in this mouse model, suggesting that hypergastrinemia may contribute to deteriorated bone quality during acid inhibition.

ECL-cell carcinoids and carcinoma in patients homozygous for an inactivating mutation in the gastric H(+) K(+) ATPase alpha subunit.

A family with a missense variant of the ATP4A gene encoding the alpha subunit of the gastric proton pump (H(+) K(+) ATPase) has recently been described. Homozygous siblings were hypergastrinemic (median gastrin 486 pM) and had gastric tumours diagnosed at a median age of 33 years. In the current histopathological study, we further characterized the tumours found in the gastric corpus. The tumours had the histological appearance of carcinoids (NET G1 or G2) and were immunoreactive for the general neuroendocrine markers chromogranin A (CgA) and synaptophysin as well as the ECL-cell markers vesicular monoamine transporter 2 (VMAT2) and histidine decarbozylase (HDC). One of the tumours consisted of a NET G2 component, but also had a component with glandular growth, which morphologically was classified as an intestinal type adenocarcinoma. Many glands of the adenocarcinoma contained a large proportion of cells positive for neuroendocrine markers, especially the small vesicle marker synaptophysin and the cytoplasmic enzyme HDC. In conclusion, patients homozygous for an inactivating ATP4A mutation develop gastric ECL-cell carcinoids in their 3rd or 4th decade. The adenocarcinoma may be classified as neuroendocrine with ECL-cell differentiation.

Effects of the Histamine 1 Receptor Antagonist Cetirizine on the Osteoporotic Phenotype in H(+) /K(+) ATPase Beta Subunit KO Mice.

Epidemiological studies suggest increased fracture risk in patients using proton pump inhibitors (PPIs). We have previously shown that the H(+) /K(+) ATPase beta subunit knockout (KO) mouse, which is a model of PPI-use, have lower bone mineral density (BMD) and impaired bone quality compared to wild type (WT) mice. Like PPI users, these KO mice display elevated gastric pH and hypergastrinemia, which in turn stimulates gastric histamine release. Previous studies have suggested a negative effect of histamine on bone, thus, we wanted to study whether a histamine 1 receptor (H1R) antagonist could improve bone quality in KO mice. Female KO and WT mice aged 8 weeks received either an H1R antagonist (cetirizine) or polyethylene glycol (PEG) for 6 months. At the end of the study, KO mice displayed elevated plasma histamine levels compared to WT. As demonstrated previously, the KO mice also exhibited lower whole body BMD, reduced mechanical bone strength, and impaired bone quality assessed by µCT. No significant differences, however, were found between the KO groups receiving cetirizine or PEG for any of the measured bone parameters. In vitro gene expression analyses of histamine receptors revealed the presence of H1R and H2R both in osteoblasts and osteoclasts, and H3R in late stage osteoblasts. In conclusion, administration of the H1R antagonist cetirizine in a concentration of 3 mg/kg did not rescue the osteoporotic phenotype in H(+) /K(+) ATPase beta subunit KO mice. It can, however, not be ruled out that histamine may influence bone via other receptors. J. Cell. Biochem. 117: 2089-2096, 2016. © 2016 Wiley Periodicals, Inc.

Circadian expression of H,K-ATPase type 2 contributes to the stability of plasma K⁺ levels.

Maintenance by the kidney of stable plasma K(+) values is crucial, as plasma K(+) controls muscle and nerve activity. Since renal K(+) excretion is regulated by the circadian clock, we aimed to identify the ion transporters involved in this process. In control mice, the renal mRNA expression of H,K-ATPase type 2 (HKA2) is 25% higher during rest compared to the activity period. Conversely, under dietary K(+) restriction, HKA2 expression is ∼40% higher during the activity period. This reversal suggests that HKA2 contributes to the circadian regulation of K(+) homeostasis. Compared to their wild-type (WT) littermates, HKA2-null mice fed a normal diet have 2-fold higher K(+) renal excretion during rest. Under K(+) restriction, their urinary K(+) loss is 40% higher during the activity period. This inability to excrete K(+) "on time" is reflected in plasma K(+) values, which vary by 12% between activity and rest periods in HKA2-null mice but remain stable in WT mice. Analysis of the circadian expression of HKA2 regulators suggests that Nrf2, but not progesterone, contributes to its rhythmicity. Therefore, HKA2 acts to maintain the circadian rhythm of urinary K(+) excretion and preserve stable plasma K(+) values throughout the day.

Impaired acid secretion in cortical collecting duct intercalated cells from H-K-ATPase-deficient mice: role of HKalpha isoforms.

Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HCO(3) transport in the collecting duct (CD). At least two H-K-ATPase alpha-subunits are expressed in the CD: HKalpha(1) and HKalpha(2). Both exhibit K dependence but have different inhibitor sensitivities. The HKalpha(1) H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HKalpha(2) H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HKalpha(1) and HKalpha(2) to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH (pH(i)) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HKalpha(1), HKalpha(2), or both H-K-ATPases (HKalpha(1,2)). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from CO(2)/HCO(3)-buffered solutions to identify the membrane locations of Cl/HCO(3) exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A(1) (100 nM) and EIPA (10 microM), respectively. Here, we report 1) initial pH(i) and buffering capacity were not significantly altered in the ICs of HKalpha-deficient mice, 2) either HKalpha(1) or HKalpha(2) deficiency resulted in slower acid extrusion, and 3) A-type ICs from HKalpha(1,2)-deficient mice had significantly slower acid extrusion compared with A-type ICs from HKalpha(1)-deficient mice alone. These studies are the first nonpharmacological demonstration that both HKalpha(1) and HKalpha(2) contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet.

Prevention of autoimmune gastritis in mice requires extra-thymic T-cell deletion and suppression by regulatory T cells.

BACKGROUND AND AIMS: Autoimmune gastritis is one of the most common autoimmune diseases and is caused by a CD4(+) T-cell response to the gastric H(+)/K(+) ATPase encoded by Atp4a and Atp4b (H(+)/K(+) ATPase). Here, we have elucidated events that result in immunological tolerance to the H(+)/K(+) ATPase and thus the prevention of autoimmune gastritis. METHODS: T cells from H(+)/K(+) ATPase-deficient mice and H(+)/K(+) ATPase-specific T-cell receptor transgenic mice were purified and transferred to wild-type (WT) or H(+)/K(+) ATPase-deficient recipients to assess the impact of exposure to antigen on pathogenicity. RESULTS: The CD4(+) T-cell population from H(+)/K(+) ATPase-deficient mice was highly effective at inducing gastritis when compared with T cells from WT mice and, as a population, was comparatively resistant to the suppressive activity of regulatory T cells. Exposing T cells from H(+)/K(+) ATPase-deficient mice to H(+)/K(+) ATPase in WT mice decreased their ability to induce gastritis and resulted in a population that could be more easily suppressed by T(reg) cells. Transfer of clonotypic antigen-inexperienced H(+)/K(+) ATPase-specific T cells into WT mice resulted in extra-thymic clonal deletion. CONCLUSIONS: Prevention of autoimmune gastritis requires the extra-thymic purging of highly autoaggressive H(+)/K(+) ATPase-specific T cells to produce a T-cell repertoire that is more susceptible to the suppressive activity of regulatory T cells. Taken together with recent published data describing the role of T-cell receptor signalling in the maintenance of regulatory T-cell populations, we propose that exposure of T cells to antigen in the periphery is able to both delete autoaggressive specificities and maintain regulatory T-cell activity, establishing a balance between pathogenicity and regulation.

Gastric achlorhydria in H/K-ATPase-deficient (Atp4a(-/-)) mice causes severe hyperplasia, mucocystic metaplasia and upregulation of growth factors.

BACKGROUND: Gastric neoplasia is common in humans, yet controversy remains over contributions of chronic achlorhydria, gastrinemia and hyperplasia, to cancer risk. To study this, mice lacking the gastric H/K-ATPase (Atp4a(-/-) mice) were used to determine whether chronic loss of acid secretion, with attendant hypergastrinemia, predisposes to cancer phenotype. METHODS: Atp4a(-/-) and Atp4a(+/+) mice, paired for age and gender, were examined at 3, 8, 12 and 20 months for histopathology, and for expression of the trefoil factor family (TFF)1-3, Reg IIIbeta, gamma and delta, osteopontin, CD44, chromogranin A, Crp-ductin, and galectin, all of which are important in cell growth. RESULTS: By 8 months, the glandular stomach of the Atp4a(-/-) mice doubled in weight and thickness, and several modulators of growth were increased. Female Atp4a(-/-) mice were more hyperplastic than Atp4a(-/-) males at 12 and 20 months. By 1 year, severe mucocystic hyperplasia, incomplete intestinal metaplasia, ciliated metaplasia, a shift in mucins from neutral to acidic, and inflammation were widespread. Cells in the mucus pit zone developed a pyloric-type appearance, containing large hyaline-like, periodic acid-Schiff (PAS)-negative/alcian blue-negative inclusions. But critical characteristics of gastric neoplasia, such as nuclear atypia, invasion into the muscularis mucosa, and metastases were absent. In Atp4a(-/-) mice, chromogranin A and histidine decarboxylase, RegIIIgamma and delta, TFF3, osteopontin and CD44 were upregulated while Reg IIIbeta, and TFF1 were reduced. CONCLUSIONS: Chronic achlorhydria and hypergastrinemia in aged Atp4a(-/-) mice produced progressive hyperplasia, mucocystic and incomplete intestinal metaplasia, and the upregulation of growth factors without histological evidence of neoplasia.

Reciprocal changes in trefoil 1 and 2 expression in stomachs of mice with gastric unit hypertrophy and inflammation.

H+/K+-ATPase beta-subunit-deficient mice (129/Sv background) display numerous pathologies in the stomach. Expression of the mutation in BALB/cCrSlc mice results in the development of an aberrant 'mucus-rich' cell population. 'Mucus-rich' cells have been described in stomachs of mice with autoimmune gastritis, a disease mediated by CD4+ T cells. Other pathological features of autoimmune gastritis are similar to those in H+/K+ beta-deficient mice and include a mononuclear cell infiltrate in the gastric mucosa, non-functional or absent parietal cells, depletion of zymogenic cells, hypergastrinaemia, and gastric unit hypertrophy caused by immature cell hyperplasia. The present study investigates further the aberrant gastric 'mucus-rich' cell lineage and analyses the mRNA expression of mucus cell products TFF1 and TFF2. 'Mucus-rich' cells stained for both acidic and neutral mucins, and with a TFF2-specific antibody. Stomachs from both models expressed decreased TFF1 mRNA and reciprocally increased TFF2 mRNA. The involvement of gastrin in regulating trefoil mRNA expression was also investigated using gastrin-deficient mice. In contrast to previous findings, gastrin did not positively regulate TFF1 mRNA expression, but there was possible augmentation of TFF2. Additionally, a clear role for inflammation was established involving both polymorphonuclear and mononuclear cells in these models, and a link was found between mucosal hypertrophy and increased interleukin-11 (IL-11) expression.

Colocalization of the apical Cl-/HCO3- exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells.

The apical Cl-/HCO exchanger called the putative anion transporter (PAT1; SLC26A6) is expressed on apical membranes of villus cells in the duodenum, but its location in the stomach remains unknown. Here we examined the cell distribution and membrane location of PAT1 in mouse stomach. Immunofluorescence labeling studies with anti-PAT1 antibodies and Dolichos biflorus agglutinin indicated the exclusive expression of PAT1 in gastric parietal cells. Double immunocytochemical staining revealed colocalization of PAT1 with the gastric H-K-ATPase, consistent with expression in tubulovesicles and/or the secretory canaliculus. Radiolabeled 36Cl flux studies demonstrated the functional presence of Cl-/HCO exchange in purified tubulovesicles of parietal cells. The expression of PAT1 was significantly decreased in parietal cells of gastric H-K-ATPase-null mice, which exhibit a sharp reduction in tubulovesicle membranes. These data indicate that the Cl-/HCO exchanger PAT1 is localized on tubulovesicular membranes, and they are consistent with the hypothesis that it functions in the maintenance of intravesicular ion concentrations in the resting state and dehydration of vesicles derived from the secretory membranes following the transition from the stimulated to the resting state.

The unique ultrastructure of secretory membranes in gastric parietal cells depends upon the presence of H+, K+ -ATPase.

Ion transporters play a central role in gastric acid secretion. To determine whether some of these transporters are necessary for the normal ultrastructure of secretory membranes in gastric parietal cells, mice lacking transporters for H+, K+, Cl-, and Na+ were examined for alterations in volume density (Vd) of basolateral, apical, tubulovesicular and canalicular membranes, microvillar dimensions, membrane flexibility, and ultrastructure. In mice lacking Na+/H+ exchanger 1 (NHE1) or the Na+-K+-2Cl- cotransporter (NKCC1), the ultrastructure and Vd of secretory membranes and the secretory canalicular to tubulovesicular membrane ratio (SC/TV), a morphological correlate of secretory activity, were similar to those of wild-type mice. In mice lacking Na+/H+ exchanger 2 (NHE2) or gastric H+, K+ -ATPase alpha- or beta-subunits, the SC/TV ratio and Vd of secretory membranes were decreased, though canaliculi were often dilated. In H+, K+ -ATPase-deficient parietal cells, canalicular folds were decreased, normally abundant tubulovesicles were replaced with a few rigid round vesicles, and microvilli were sparse, stiff and short, in contrast to the long and flexible microvilli in wild-type cells. In addition, microvilli of the H+, K+ -ATPase-deficient parietal cells had centrally bundled F-actin filaments, unlike the microvilli of wild-type cells, in which actin filaments were peripherally positioned concentric to the plasmalemma. Data showed that the absence of H+, K+ -ATPase produced fundamental changes in parietal cell membrane ultrastructure, suggesting that the pump provides an essential link between the membranes and F-actin, critical to the gross architecture and suppleness of the secretory membranes.

Endogenous H/K ATPase beta-subunit promotes T cell tolerance to the immunodominant gastritogenic determinant.

A CD4(+) T cell response to the gastric H/K ATPase beta-subunit (H/Kbeta) is required for the onset of experimental autoimmune gastritis in BALB/c mice. The extent to which endogenous H/Kbeta contributes toward the tolerance of the H/Kbeta-specific T cell repertoire in normal individuals is not known. By comparison of T cell responses in H/Kbeta-deficient (o/o) and H/Kbeta-expressing BALB/c mice, in this work we show that the endogenous H/Kbeta autoantigen plays a major role in the tolerance of pathogenic H/Kbeta-specific T cells. First, T cell-dependent Ab responses to the H/Kbeta Ag were enhanced in H/K ATPase-immunized H/Kbeta-deficient mice compared with wild-type mice. Second, peptide immunization experiments indicated that immune responses to the major gastritogenic epitope of the H/K ATPase, namely H/Kbeta(253-277), were significantly more vigorous in H/Kbeta-deficient mice compared with wild-type mice. Third, unfractionated splenocytes from H/Kbeta-deficient mice, but not H/Kbeta-expressing mice, induced autoimmune gastritis after adoptive transfer to BALB/c nude mice. The enhanced responses to H/Kbeta in H/Kbeta-deficient mice were shown to be intrinsic to CD4(+)CD25(-) T cells rather than a change in status of CD4(+)CD25(+) regulatory T cells. We conclude from these studies that the H/Kbeta-specific T cells in wild-type mice represent the residue of a T cell repertoire, directed toward a single determinant, that has been subjected to partial tolerance induction.

Colonic H(+)-K(+)-ATPase in K(+) conservation and electrogenic Na(+) absorption during Na(+) restriction.

Upregulation of the colonic H(+)-K(+)- ATPase (cHKA) during hyperaldosteronism suggests that it functions in both K(+) conservation and electrogenic Na(+) absorption in the colon when Na(+)-conserving mechanisms are activated. To test this hypothesis, wild-type (cHKA(+/+)) and cHKA-deficient (cHKA(-/-)) mice were fed Na(+)-replete and Na(+)-restricted diets and their responses were analyzed. In both genotypes, Na(+) restriction led to reduced plasma Na(+) and increased serum aldosterone, and mRNAs for the epithelial Na(+) channel (ENaC) beta- and gamma-subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative to wild-type controls, cHKA(-/-) mice on a Na(+)-replete diet had elevated fecal K(+) excretion. Dietary Na(+) restriction led to increased K(+) excretion in knockout but not in wild-type mice. The amiloride-sensitive, ENaC-mediated short-circuit current in distal colon was significantly reduced in knockout mice maintained on either the Na(+)-replete or Na(+)-restricted diet. These results demonstrate that cHKA plays an important role in K(+) conservation during dietary Na(+) restriction and suggest that cHKA-mediated K(+) recycling across the apical membrane is required for maximum electrogenic Na(+) absorption.

Regulation of gastric epithelial cell development revealed in H(+)/K(+)-ATPase beta-subunit- and gastrin-deficient mice.

The gastric H(+)/K(+)-ATPase is essential for normal development of parietal cells. Here we have directly assessed the role of the H(+)/K(+)-ATPase beta-subunit (H/K-beta) on epithelial cell development by detailed quantitation of the epithelial cell types of the gastric mucosa of H/K-beta-deficient mice. H/K-beta-deficient mice had a 3.1-fold increase in the number of immature cells per gastric unit; however, the numbers of surface mucous and parietal cells were similar to those in the gastric units of wild-type mice. The effect of elevated gastrin levels in the H/K-beta-deficient mice was determined by producing mice that are also deficient in gastrin. We demonstrated that the increased production of immature cells and resulting hypertrophy is caused by the overproduction of gastrin. However, the depletion of zymogenic cells, which is another feature of H/K-beta-deficient mice, is independent of hypergastrinemia. Significantly, parietal cells of H/K-beta- and gastrin-deficient mice had abnormal secretory membranes and were devoid of resting tubulovesicular membranes. Together these data suggest a homeostatic mechanism limiting the number of immature cells that can develop into end-stage epithelial cells and indicate a direct role for H/K-beta in the development of mature parietal cells.

Distal renal tubular acidosis with severe hypokalaemia, probably caused by colonic H(+)-K(+)-ATPase deficiency.

We describe a 21 month old male infant who presented with failure to thrive associated with severe hypokalaemia and metabolic acidosis, together with hypomagnesaemia. Evaluation revealed marked renal and probable faecal potassium wasting, distal renal tubular acidosis, mild urinary magnesium wasting, and a normal gastric pH (gastric H(+)-K(+)-ATPase). Hypokalaemic forms of metabolic acidosis, such as diabetic ketoacidosis and proximal renal tubular acidosis were ruled out from the clinical picture. The hypokalaemia of distal renal tubular acidosis usually improves with alkali therapy, but this was not observed: despite correction of acidosis with 5 mmol/kg potassium citrate per day, an additional 5 mmol/kg potassium chloride was required to bring serum potassium to 3.5 mmol/l. At 3 years of age potassium was provided in the absence of potential alkali and acidosis ensued; serum bicarbonate fell to 10 mmol/l. Although a specific genetic analysis is not yet possible, the abnormalities are consistent with a novel form of distal renal tubular acidosis. The pathophysiology probably does not stem from defects in the vacuolar H(+)-ATPase but more likely from deficient activity of the colonic isoform of H(+)-K(+)-ATPase that is resident in the medullary collecting duct and mediates potassium absorption and proton secretion.

Stomachs of mice lacking the gastric H,K-ATPase alpha -subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia.

The H,K-ATPase of the gastric parietal cell is the most critical component of the ion transport system mediating acid secretion in the stomach. To study the requirement of this enzyme in the development, maintenance, and function of the gastric mucosa, we used gene targeting to prepare mice lacking the alpha-subunit. Homozygous mutant (Atp4a(-/-)) mice appeared healthy and exhibited normal systemic electrolyte and acid-base status but were achlorhydric and hypergastrinemic. Immunocytochemical, histological, and ultrastructural analyses of Atp4a(-/-) stomachs revealed the presence of chief cells, demonstrating that the lack of acid secretion does not interfere with their differentiation. Parietal cells were also present in normal numbers, and despite the absence of alpha-subunit mRNA and protein, the beta-subunit was expressed. However, Atp4a(-/-) parietal cells had dilated canaliculi and lacked typical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adult Atp4a(-/-) mice exhibited metaplasia, which included the presence of ciliated cells. We conclude that ablation of the H,K-ATPase alpha-subunit causes achlorhydria and hypergastrinemia, severe perturbations in the secretory membranes of the parietal cell, and metaplasia of the gastric mucosa; however, the absence of the pump appears not to perturb parietal cell viability or chief cell differentiation.