The diverse functions of the DEG/ENaC family: linking genetic and physiological insights.

The DEG/ENaC family of ion channels was defined based on the sequence similarity between degenerins (DEG) from the nematode Caenorhabditis elegans and subunits of the mammalian epithelial sodium channel (ENaC), and also includes a diverse array of non-voltage-gated cation channels from across animal phyla, including the mammalian acid-sensing ion channels (ASICs) and Drosophila pickpockets. ENaCs and ASICs have wide ranging medical importance; for example, ENaCs play an important role in respiratory and renal function, and ASICs in ischaemia and inflammatory pain, as well as being implicated in memory and learning. Electrophysiological approaches, both in vitro and in vivo, have played an essential role in establishing the physiological properties of this diverse family, identifying an array of modulators and implicating them in an extensive range of cellular functions, including mechanosensation, acid sensation and synaptic modulation. Likewise, genetic studies in both invertebrates and vertebrates have played an important role in linking our understanding of channel properties to function at the cellular and whole animal/behavioural level. Drawing together genetic and physiological evidence is essential to furthering our understanding of the precise cellular roles of DEG/ENaC channels, with the diversity among family members allowing comparative physiological studies to dissect the molecular basis of these diverse functions.

Capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption by blocking TRPV4 channels in healthy and colitic mice.

Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (Isc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca2+ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal Isc but significantly inhibited carbachol- and caffeine-induced intestinal Isc in WT mice. Capsaicin similarly inhibited the intestinal Isc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca2+ influx via TRPV4 was required for cholinergic signaling-mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal Iscvia Na+/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic Isc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive Isc. We therefore conclude that capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action.

Dietary anions control potassium excretion: it is more than a poorly absorbable anion effect.

The urinary potassium (K+) excretion machinery is upregulated with increasing dietary K+, but the role of accompanying dietary anions remains inadequately characterized. Poorly absorbable anions, including [Formula: see text], are thought to increase K+ secretion through a transepithelial voltage effect. Here, we tested if they also influence the K+ secretion machinery. Wild-type mice, aldosterone synthase (AS) knockout (KO) mice, or pendrin KO mice were randomized to control, high-KCl, or high-KHCO3 diets. The K+ secretory capacity was assessed in balance experiments. Protein abundance, modification, and localization of K+-secretory transporters were evaluated by Western blot analysis and confocal microscopy. Feeding the high-KHCO3 diet increased urinary K+ excretion and the transtubular K+ gradient significantly more than the high-KCl diet, coincident with more pronounced upregulation of epithelial Na+ channels (ENaC) and renal outer medullary K+ (ROMK) channels and apical localization in the distal nephron. Experiments in AS KO mice revealed that the enhanced effects of [Formula: see text] were aldosterone independent. The high-KHCO3 diet also uniquely increased the large-conductance Ca2+-activated K+ (BK) channel ß4-subunit, stabilizing BKα on the apical membrane, the Cl-/[Formula: see text] exchanger, pendrin, and the apical KCl cotransporter (KCC3a), all of which are expressed specifically in pendrin-positive intercalated cells. Experiments in pendrin KO mice revealed that pendrin was required to increase K+ excretion with the high-KHCO3 diet. In summary, [Formula: see text] stimulates K+ excretion beyond a poorly absorbable anion effect, upregulating ENaC and ROMK in principal cells and BK, pendrin, and KCC3a in pendrin-positive intercalated cells. The adaptive mechanism prevents hyperkalemia and alkalosis with the consumption of alkaline ash-rich diets but may drive K+ wasting and hypokalemia in alkalosis.NEW & NOTEWORTHY Dietary anions profoundly impact K+ homeostasis. Here, we found that a K+-rich diet, containing [Formula: see text] as the counteranion, enhances the electrogenic K+ excretory machinery, epithelial Na+ channels, and renal outer medullary K+ channels, much more than a high-KCl diet. It also uniquely induces KCC3a and pendrin, in B-intercalated cells, providing an electroneutral KHCO3 secretion pathway. These findings reveal new K+ balance mechanisms that drive adaption to alkaline and K+-rich foods, which should guide new treatment strategies for K+ disorders.

Functional role of histamine receptors in the renal cortical collecting duct cells.

Histamine is an important immunomodulator, as well as a regulator of allergic inflammation, gastric acid secretion, and neurotransmission. Although substantial histamine level has been reported in the kidney, renal pathological and physiological effects of this compound have not been clearly defined. The goal of this study was to provide insight into the role of histamine-related pathways in the kidney, with emphasis on the collecting duct (CD), a distal part of the nephron important for the regulation of blood pressure. We report that all four histamine receptors (HRs) as well as enzymes responsible for histamine metabolism and synthesis are expressed in cultured mouse mpkCCDcl4 cells, and histamine evokes a dose-dependent transient increase in intracellular Ca2+ in these cells. Furthermore, we observed a dose-dependent increase in cAMP in the CD cells in response to histamine. Short-circuit current studies aimed at measuring Na+ reabsorption via ENaC (epithelial Na+ channel) demonstrated inhibition of ENaC-mediated currents by histamine after a 4-h incubation, and single-channel patch-clamp analysis revealed similar ENaC open probability before and after acute histamine application. The long-term (4 h) effect on ENaC was corroborated in immunocytochemistry and qPCR, which showed a decrease in protein and gene expression for αENaC upon histamine treatment. In summary, our data highlight the functional importance of HRs in the CD cells and suggest potential implications of histamine in inflammation-related renal conditions. Further research is required to discern the molecular pathways downstream of HRs and assess the role of specific receptors in renal pathophysiology.

Hypoxia Aggravates Inhibition of Alveolar Epithelial Na-Transport by Lipopolysaccharide-Stimulation of Alveolar Macrophages.

Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS; 1 µg/mL) and exposed to normoxia and hypoxia (1.5% O2). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISCΔamil); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.

Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC.

A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na+ channels (ENaC), but if all Na+ reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na+ over K+, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down ß- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC.

Role of epithelial ion transports in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with a complex pathogenesis. Diarrhea is a highly prevalent and often debilitating symptom of IBD patients that results, at least in part, from an intestinal hydroelectrolytic imbalance. Evidence suggests that reduced electrolyte absorption is more relevant than increased secretion to this disequilibrium. This systematic review analyses and integrates the current evidence on the roles of epithelial Na(+)-K(+)-ATPase (NKA), Na(+)/H(+) exchangers (NHEs), epithelial Na(+) channels (ENaC), and K(+) channels (KC) in IBD-associated diarrhea. NKA is the key driving force of the transepithelial ionic transport and its activity is decreased in IBD. In addition, the downregulation of apical NHE and ENaC and the upregulation of apical large-conductance KC all contribute to the IBD-associated diarrhea by lowering sodium absorption and/or increasing potassium secretion.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex.

ENaC activity and expression is decreased in the lungs of protein kinase C-α knockout mice.

We used a PKC-α knockout model to investigate the regulation of alveolar epithelial Na(+) channels (ENaC) by PKC. Primary alveolar type II (ATII) cells were subjected to cell-attached patch clamp. In the absence of PKC-α, the open probability (Po) of ENaC was decreased by half compared with wild-type mice. The channel density (N) was also reduced in the knockout mice. Using in vivo biotinylation, membrane localization of all three ENaC subunits (α, ß, and γ) was decreased in the PKC-α knockout lung, compared with the wild-type. Confocal microscopy of lung slices showed elevated levels of reactive oxygen species (ROS) in the lungs of the PKC-α knockout mice vs. the wild-type. High levels of ROS in the knockout lung can be explained by a decrease in both cytosolic and mitochondrial superoxide dismutase activity. Elevated levels of ROS in the knockout lung activates PKC-δ and leads to reduced dephosphorylation of ERK1/2 by MAP kinase phosphatase, which in turn causes increased internalization of ENaC via ubiquitination by the ubiquitin-ligase Nedd4-2. In addition, in the knockout lung, PKC-δ activates ERK, causing a decrease in ENaC density at the apical alveolar membrane. PKC-δ also phosphorylates MARCKS, leading to a decrease in ENaC Po. The effects of ROS and PKC-δ were confirmed with patch-clamp experiments on isolated ATII cells in which the ROS scavenger, Tempol, or a PKC-δ-specific inhibitor added to patches reversed the observed decrease in ENaC apical channel density and Po. These results explain the decrease in ENaC activity in PKC-α knockout lung.

Involvement of Rho GTPases and their regulators in the pathogenesis of hypertension.

Proper regulation of arterial blood pressure is essential to allow permanent adjustment of nutrient and oxygen supply to organs and tissues according to their need. This is achieved through highly coordinated regulation processes controlling vascular resistance through modulation of arterial smooth muscle contraction, cardiac output, and kidney function. Members of the Rho family of small GTPases, in particular RhoA and Rac1, have been identified as key signaling molecules playing important roles in several different steps of these regulatory processes. Here, we review the current state of knowledge regarding the involvement of Rho GTPase signaling in the control of blood pressure and the pathogenesis of hypertension. We describe how knockout models in mouse, genetic, and pharmacological studies in human have been useful to address this question.