Apical Entry Research Articles

Novel method for augmented reality guided endodontics: An in vitro study

ObjectiveThe aim of this study is to evaluate the accuracy in endodontics of a novel augmented reality (AR) method for guided access cavity preparation in 3D-printed jaws. MethodsTwo operators with different levels of experience in endodontics performed pre-planned virtually guided access cavities through a novel markerless AR system on three sets of 3D-printed jaw models (Objet Connex 350, Stratasys) mounted on a phantom. After the treatment, a post-operative high-resolution CBCT scan (NewTom VGI Evo, Cefla) was taken for each model and registered to the pre-operative model. All the access cavities were then digitally reconstructed by filling the cavity area using 3D medical software (3-Matic 15.0, materialize). For the anterior teeth and the premolars, the deviation at the coronal and apical entry points as well as the angular deviation of the access cavity were compared to the virtual plan. For the molars, the deviation at the coronal entry point was compared to the virtual plan. Additionally, the surface area of all access cavities at the entry point was measured and compared to the virtual plan. Descriptive statistics for each parameter were performed. A 95% confidence interval was calculated. ResultsA total of 90 access cavities were drilled up to a depth of 4 mm inside the tooth. The mean deviation in the frontal teeth and in the premolars at the entry point was 0.51 mm and 0.77 mm at the apical point, with a mean angular deviation of 8.5° and a mean surface overlap of 57%. The mean deviation for the molars at the entry point was 0.63 mm, with a mean surface overlap of 82%. ConclusionThe use of AR as a digital guide for endodontic access cavity drilling on different teeth showed promising results and might have potential for clinical use. However, further development and research might be needed before in vivo validation.

ClC-K Kidney Chloride Channels: From Structure to Pathology.

The molecular basis of chloride transport varies all along the nephron depending on the tubular segments especially in the apical entry of the cell. The major chloride exit pathway during reabsorption is provided by two kidney-specific ClC chloride channels ClC-Ka and ClC-Kb (encoded by CLCNKA and CLCNKB gene, respectively) corresponding to rodent ClC-K1 and ClC-K2 (encoded by Clcnk1 and Clcnk2). These channels function as dimers and their trafficking to the plasma membrane requires the ancillary protein Barttin (encoded by BSND gene). Genetic inactivating variants of the aforementioned genes lead to renal salt-losing nephropathies with or without deafness highlighting the crucial role of ClC-Ka, ClC-Kb, and Barttin in the renal and inner ear chloride handling. The purpose of this chapter is to summarize the latest knowledge on renal chloride structure peculiarity and to provide some insight on the functional expression on the segments of the nephrons and on the related pathological effects.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

The kinesin motor Klp98A mediates apical to basal Wg transport.

ABSTRACTDevelopment and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.

Computer simulations of transapical mitral valve repair with neochordae implantation: Clinical implications.

ObjectivesTransapical beating heart neochordae implantation is an innovative mitral valve repair technique that has demonstrated promising clinical results in patients with primary mitral regurgitation. However, as clinical experience continues to increase, neochordae implantation criteria have not been fully standardized. The aim of this study was to investigate the biomechanical effects of selecting an antero-lateral apical access site compared with a postero-lateral site, and suboptimal neochordae length compared with optimal suture length, on restoring physiologic left heart dynamics.MethodsTransapical neochordae implantation using 3 and 4 sutures was computer simulated under 3 posterior mitral leaflet prolapse conditions: isolated P2, multiscallop P2/P3 and multiscallop P2/P1. Physiologic, pre- and postrepair left heart dynamics were evaluated using a fluid-structure interaction modeling framework.ResultsDespite the absence of residual mitral regurgitation in all postrepair models with optimal neochordae length, selecting an antero-lateral apical entry site for the treatment of P2/P3 prolapse generated a significant increase (>80%) in neochordae tension and P2 peak stress, with respect to a postero-lateral entry site. During isolated P2 prolapse repair, although neochordae overtension by 5% led to minimal hemodynamic changes in the regurgitant volume compared with using an optimal suture length, a significant increase in systolic and diastolic neochordae tension (>300%) and posterior leaflet average stress (70%-460%) was quantified. On the other hand, neochordae undertension by 5% led to worsening of regurgitation severity.ConclusionsThis parametric computer study represents a further step toward an improved understanding of the biomechanical outcomes of transapical neochordae technologies.

Ebola Virus Uptake into Polarized Cells from the Apical Surface.

Ebola virus (EBOV) causes severe hemorrhagic fever with high mortality rates. EBOV can infect many types of cells. During severe EBOV infection, polarized epithelial and endothelial cells are damaged, which promotes vascular instability and dysregulation. However, the mechanism causing these symptoms is largely unknown. Here, we studied virus infection in polarized Vero C1008 cells grown on semipermeable Transwell by using EGFP-labeled Ebola virus-like particles (VLPs). Our results showed that Ebola VLPs preferred to enter polarized Vero cells from the apical cell surface. Furthermore, we showed that the EBOV receptors TIM-1 and Axl were distributed apically, which could be responsible for mediating efficient apical viral entry. Macropinocytosis and intracellular receptor Niemann–Pick type C1 (NPC1) had no polarized distribution, although they played roles in virus entry. This study provides a new view of EBOV uptake and cell polarization, which facilitates a further understanding of EBOV infection and pathogenesis.

The molecular mechanism of SLC34 proteins: insights from two decades of transport assays and structure-function studies.

The expression cloning some 25years ago of the first member of SLC34 solute carrier family, the renal sodium-coupled inorganic phosphate cotransporter (NaPi-IIa) from rat and human tissue, heralded a new era of research into renal phosphate handling by focussing on the carrier proteins that mediate phosphate transport. The cloning of NaPi-IIa was followed by that of the intestinal NaPi-IIb and renal NaPi-IIc isoforms. These three proteins constitute the main secondary-active Na+-driven pathways for apical entry of inorganic phosphate (Pi) across renal and intestinal epithelial, as well as other epithelial-like organs. The key role these proteins play in mammalian Pi homeostasis was revealed in the intervening decades by numerous in vitro and animal studies, including the development of knockout animals for each gene and the detection of naturally occurring mutations that can lead to Pi-handling dysfunction in humans. In addition to characterising their physiological regulation, research has also focused on understanding the underlying transport mechanism and identifying structure-function relationships. Over the past two decades, this research effort has used real-time electrophysiological and fluorometric assays together with novel computational biology strategies to develop a detailed, but still incomplete, understanding of the transport mechanism of SLC34 proteins at the molecular level. This review will focus on how our present understanding of their molecular mechanism has evolved in this period by highlighting the key experimental findings.

Differential roles of WNK4 in regulation of NCC in vivo.

The Na+-Cl- cotransporter (NCC) in distal convoluted tubule (DCT) plays important roles in renal NaCl reabsorption. The current hypothesis for the mechanism of regulation of NCC focuses on WNK4 and intracellular Cl- concentration ([Cl-]i). WNK kinases bind Cl-, and Cl- binding decreases the catalytic activity. It is believed that hypokalemia under low K+ intake decreases [Cl-]i to activate WNK4, which thereby phosphorylates and stimulates NCC through activation of SPAK. However, increased NCC activity and apical NaCl entry would mitigate the fall in [Cl-]i. Whether [Cl-]i in DCT under low-K+ diet is sufficiently low to activate WNK4 is unknown. Furthermore, increased luminal NaCl delivery also stimulates NCC and causes upregulation of the transporter. Unlike low K+ intake, increased luminal NaCl delivery would tend to increase [Cl-]i. Thus we investigated the role of WNK4 and [Cl-]i in regulating NCC. We generated Wnk4-knockout mice and examined regulation of NCC by low K+ intake and by increased luminal NaCl delivery in knockout (KO) and wild-type mice. Wnk4-KO mice have marked reduction in the abundance, phosphorylation, and functional activity of NCC vs. wild type. Low K+ intake increases NCC phosphorylation and functional activity in wild-type mice, but not in Wnk4-KO mice. Increased luminal NaCl delivery similarly upregulates NCC, which, contrary to low K+ intake, is not abolished in Wnk4-KO mice. The results reveal that modulation of WNK4 activity by [Cl-]i is not the sole mechanism for regulating NCC. Increased luminal NaCl delivery upregulates NCC via yet unknown mechanism(s) that may override inhibition of WNK4 by high [Cl-]i.

Pericardiocentesis guided by echocardiography performed in echocardiography laboratory – Safety profile of the single center prospective registry

IntroductionPericardiocentesis is an invasive procedure for treatment of large pericardial effusion or cardiac tamponade and for diagnostics of pericardial effusion of unknown origin. Fluoroscopy navigation has been the preferred method during the past decades. Nevertheless, new imaging methods such as echocardiography emerged as an alternative guiding method for pericardiocentesis. These methods may improve safety of the procedure. MethodsAll consecutive pericardiocenteses performed in noninvasive cardiology department of a tertiary cardiovascular center during the period between 1998 and 2012 were prospectively recorded. We focused on the procedural safety and procedural success rate. ResultsDuring a 15-year period, 253 pericardiocenteses were performed in 185 patients. Most of the procedures (240 cases) were performed under echocardiographic control in our noninvasive cardiology laboratory under strictly sterile conditions and with equipment for cardiopulmonary resuscitation on site. Etiology of effusion was heart transplantation in 38 patients (25%), postoperative in 20 patients (14%), infective pericarditis in 25 patients (16%), malignancy in 18 patients (12%), and invasive procedures in 19 patients (8%). Apical approach was the most frequent in 218 patients (92%), parasternal in 13 patients (5%) and subxiphoideal in 7 patients (3%). The procedural success rate was 97% overall, with a total complication rate of 3% (2 major complications (0.3%); 7 minor complications (2.7%)). Minor complications included 2 cases of small pneumothorax, 2 cases of pericardial fluid drainage into pleural space, 2 cases of transient right chamber entries, and in 1 case the procedure was complicated by hemopericardium without the need for surgical management. Major complications included 2 cases due to ventricular perforation, one with left ventricule wall laceration in a loculated effusion and one complication due to right ventricular laceration, both resulting in hemopericardium and requiring emergency surgical repair. ConclusionEchocardiography-guided pericardiocentesis performed by echocardiologists in noninvasive cardiology department under strictly sterile conditions and with equipment for cardiopulmonary resuscitation is a safe procedure with infrequent complications. Apical entry site is safe and the dominant approach for pericardiocentesis under echocardiographic navigation.

Glucocorticoids and the diurnal rhythm of the thiazide‐sensitive NaCl cotransporter

Reabsorbing ~7% of the sodium load, the distal convoluted tubule plays key roles in blood pressure (BP) homeostasis. Here, Na‐Cl co‐transport (NCC) is the major route for apical sodium entry making thiazide diuretics, NCC inhibitors, a mainstay hypertension treatment. Predictive adaptions of sodium excretory rhythms are supported by an intrinsic renal clock, which regulates transporter activity according to physiological need. Peripheral clocks can be influenced by glucocorticoids, which also have a circadian rhythm and we therefore hypothesized that the diurnal rhythm of NCC was regulated by glucocorticoids. C57BL6 mice were kept on a 12h light cycle with subjective day starting at 7am local time. Urine was collected in 12h periods and kidneys harvested at 1am (night) and 1pm (day). Slc12a3 (NCC encoding gene) mRNA and NCC protein abundance were similar between night and day but phosphorylation at threonine 53 was significantly higher at night compared to the day. Both plasma and urinary corticosterone levels were elevated at night. Glucocorticoid inducible leucine zipper (GILZ) and serum and glucocorticoid inducible kinase (SGK1) transcripts were upregulated at 1am compared to 1pm. Chronic corticosterone infusion flattened the corticosterone rhythm at intermediate levels. The diurnal rhythm of T53 phosphorylation was dampened in these mice but not in vehicle‐treated mice. These data indicate that glucocorticoids contribute to the regulation of a diurnal rhythm of NCC phosphorylation. This may be clinically relevant particularly in the pathogenesis of hypertension in conditions associated with elevated glucocorticoid levels such as metabolic syndrome and chronic stress.

Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity.

Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.

Exposure to contaminated sediments induces alterations in the gill epithelia in juvenile Solea senegalensis: a comparative in situ and ex situ study

Exposure to contaminated sediments induces alterations in the gill epithelia in juvenile Solea senegalensis: a comparative in situ and ex situ study

Basolateral Mg2+ Extrusion via CNNM4 Mediates Transcellular Mg2+ Transport across Epithelia: A Mouse Model

Transcellular Mg2+ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

ObjectiveRegulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.Methods and ResultsWe report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\\TRPV4 channel mediated increase in intracellular calcium.ConclusionWe conclude that in the absence of cilia, an EGF activated TRPP2\\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

The Amiloride-Sensitive Endothelial Sodium Channel and Vascular Tone

See related article, pp 1053–1059 In this issue of Hypertension , Jeggle et al1 provide important new insights into the characteristics and functional significance of an amiloride-sensitive sodium channel that is expressed in vascular endothelial cells. This work extends the previous analyses by this group,2,3 and using atomic force microscopy and state-of-the art animal and cell models, provides provocative yet conclusive evidence for the central role of this channel in regulation vascular reactivity and stiffness. The Figure depicts the heteromeric structure of the amiloride-sensitive sodium channel. Although the overall topology of the channel is well described,4 there is no general agreement about the subunit composition even within epithelial cells, much less in other tissues like vascular endothelial cells that are the subject of the current report. The bulk of the subunits’ structures are expressed in the intertwined disulfide-rich extracellular domains, the role of which has not been fully explained. The subunits of the epithelial sodium channel (ENaC) were originally defined by now-classical expression cloning studies by Canessa and Rossier.5 The human genomic clones and the critical insight into the gain of function effect that characterizes Liddle syndrome6 was based on analogy to defects in the mechanosensor structures of degenerins in Caenorhabditis elegans .7 Although longitudinal flow is certainly a feature of many epithelial tubule systems, a direct connection among the putative mechanical transduction function, the extracellular domains, and regulation of apical sodium entry via ENaC is not obvious. In contrast, there are pulsatile pressures and flows in the systemic vascular system. Local regulation of vascular tone in response to changes in pressure and shear stress, an important feature of the microvascular circulation, lending credance to the idea that the extensive extracellular of the vascular endothelial …

Tissue kallikrein activation of the epithelial Na channel

Epithelial Na Channels (ENaC) are responsible for the apical entry of Na(+) in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K(+) or low-Na(+) diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (I(Na)) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity.

The epithelial sodium channel δ-subunit: new notes for an old song

Amiloride-sensitive epithelial Na(+) channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na(+) reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na(+) channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity of different ENaC subunit combinations provides an opportunity to gain insight into the regulation of the channel. In this review, we examine the evolution of ENaC genes, channel subunit composition, the distinct functional and pharmacological features that δ confers to ENaC, and how this can be exploited to better understand this ion channel. Finally, we briefly consider possible functional roles of the ENaC δ-subunit.

The epithelial sodium/proton exchanger, NHE3, is necessary for renal and intestinal calcium (re)absorption

Passive paracellular proximal tubular (PT) and intestinal calcium (Ca(2+)) fluxes have been linked to active sodium (re)absorption. Although the epithelial sodium/proton exchanger, NHE3, mediates apical sodium entry at both these sites, its role in Ca(2+) homeostasis remains unclear. We, therefore, set out to determine whether NHE3 is necessary for Ca(2+) (re)absorption from these epithelia by comparing Ca(2+) handling between wild-type and NHE3(-/-) mice. Serum Ca(2+) and plasma parathyroid hormone levels were not different between groups. However, NHE3(-/-) mice had increased serum 1,25-dihydroxyvitamin D(3). The fractional excretion of Ca(2+) was also elevated in NHE3(-/-) mice. Paracellular Ca(2+) flux across confluent monolayers of a PT cell culture model was increased by an osmotic gradient equivalent to that generated by NHE3 across the PT in vivo and by overexpression of NHE3.( 45)Ca(2+) uptake after oral gavage and flux studies in Ussing chambers across duodenum of wild-type and NHE3(-/-) mice confirmed decreased Ca(2+) absorption in NHE3(-/-) mice compared with wild-type mice. Consistent with this, intestinal calbindin-D(9K), claudin-2, and claudin-15 mRNA expression was decreased. Microcomputed tomography analysis revealed a perturbation in bone mineralization. NHE3(-/-) mice had both decreased cortical bone mineral density and trabecular bone mass. Our results demonstrate significant alterations of Ca(2+) homeostasis in NHE3(-/-) mice and provide a molecular link between Na(+) and Ca(2+) (re)absorption.