Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.     

Bronchiolar expression of aquaporin-3 (AQP3) in rat lung and its dynamics in pulmonary oedema

Aquaporins (AQPs) are water channel proteins that permit osmotically driven water movement. To determine their dynamics in pulmonary oedema, we examined the expression of mRNA and protein for AQP1, AQP3, AQP4, and AQP5 in the lungs of normal and thiourea-treated rats. In the thiourea group, lung water content increased significantly (vs. controls) with the peak at around 4 h. Semi-quantitative RT-PCR showed that AQP3 mRNA in the thiourea group rose significantly, peaking at around 4–8 h. The expression of AQP1, AQP4, AQP5, ENaC and CFTR mRNA each decreased significantly some time after the peak in lung water content. Immunoblot analysis showed that glycosylated AQP3 protein was increased 4–10 h after treatment. Expression of the other AQP proteins was not significantly altered, except for that of AQP4. Immunohistochemical examination revealed that AQP1 was expressed in endothelia, AQP3 in the basal cells of the large airways and in cuboidal cells in the bronchioles, AQP4 in the basolateral membrane of airway cells and AQP5 in type-I pneumocytes. Our results suggest that AQP3 is expressed not only in large airways, but also in bronchioles, and is related to water movement in pulmonary oedema.     

Modulation of ENaC,CFTR, and iNOS expression in bronchial epithelial cells after stimulation with Staphylococcus epidermidis (94B080) and Staphylococcus aureus (90B083)

Bacteria affect the respiratory epithelium, which is covered by airway surface liquid (ASL) and mucus. Ion concentrations in the ASL are determined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na⁺ channel (ENaC). Neonatal sepsis is a major risk factor for subsequent pulmonary disease in preterm newborns. Predominating are coagulase‐negative staphylococci (e.g., Staphylococccus epidermidis and Staphylococccus aureus). The aim of this study was to investigate modulation of CFTR, ENaC, mucins, proinflammatory cytokines, and inducible nitric oxide synthase (iNOS) in respiratory epithelial cells after S. epidermidis 94B080 and S. aureus 90B083 exposure. Bronchial epithelial cells were incubated with S. epidermidis 94B080 and S. aureus 90B083 (neonatal blood isolates) for 1–36 h. Expression of CFTR, ENaC, iNOS, and mucins was analyzed by real‐time PCR and Western blotting. Release of cytokines was analyzed by ELISA, and production of NO by the Griess assay. Expression of CFTR significantly decreased after 36 h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of β‐ and γ‐ENaC. Expression of iNOS increased, but NO was not detected. Both staphylococci caused a decrease in the intracellular Ca²⁺ concentration. S. aureus induced increased secretion of IL‐6, IL‐8, and transforming nuclear factor (TNF)‐α in a time‐dependent manner as compared with S. epidermidis. In conclusion, expression of ENaC, CFTR, and iNOS is modulated by exposure to S. aureus 90B083 and S. epidermidis 94B080. S. aureus is more potent in causing release of IL‐6, IL‐8, and TNF‐α by bronchial epithelial cells as compared with S. epidermidis. The mRNA expression for the mucus proteins MUC2, MUC5AC, and MUC5B could not be measured, neither in the presence nor in the absence of bacteria.     

Eukaryotic and prokaryotic stomatins: the proteolytic link

The 32kD membrane protein stomatin was first studied because it is deficient from the red cell membrane in two forms of the class of haemolytic anaemias known as "hereditary stomatocytosis." The hallmark of these conditions is a plasma membrane leak to the monovalent cations Na+ and K+: the protein is missing only in the most severely leaky of these conditions. No mutation has ever been found in the stomatin gene in these conditions. Stomatin-like proteins have been identified in all three domains of biology, yet their function remains enigmatic. Although the murine knock-out is without phenotype, we have identified a family showing a splicing defect in the stomatin mRNA, in which affected children showed a catastrophic multisystem disease not inconsistent with the now-known wide tissue distribution of stomatin. We report here a study of strongly homologous stomatin-like genes in prokaryotes, which reveals a close connection with a never-studied gene erroneously known as "nfed." This gene codes for a hydrophobic protein with a probable serine protease motif. It is possible that these stomatin-like genes and those which are known as"nfed" form an operon, suggesting that the two protein products are aimed at a common function. The corollary is that stomatin could be a partner protein for a membrane-bound proteolytic process, in both prokaryotes and in eukaryotes generally: this idea is consistent with experimental evidence.     

Regulation of Na+ excretion and arterial blood pressure by purinergic signalling intrinsic to the distal nephron: consequences and mechanisms

Discretionary control of Na⁺ excretion is a key component of the regulation of arterial blood pressure in mammals. Sodium excretion is fine‐tuned in the aldosterone‐sensitive distal nephron by the activity of the epithelial Na⁺ channel (ENaC). Here, ENaC functions as a final effector of the renin–angiotensin–aldosterone system (RAAS) during negative feedback control of blood pressure. Mutations affecting ENaC activity and abnormal regulation of this channel affect blood pressure through pathological changes to Na⁺ excretion. Recent evidence demonstrates that powerful signalling pathways function in parallel with the RAAS to modulate ENaC activity and blood pressure. An inclusive paradigm is emerging with respect to regulation of blood pressure where ENaC serves as a critical point of convergence for several important signalling systems that affect renal Na⁺ excretion. A robust inhibitory purinergic signalling system intrinsic to the distal nephron dynamically regulates ENaC through paracrine ATP signalling via the metabotropic P2Y₂ purinergic receptor to properly match urinary Na⁺ excretion to dietary Na⁺ intake. This enables blood pressure to be maintained within a normal range despite broad changes in dietary Na⁺ consumption. Loss of purinergic inhibition of ENaC increases blood pressure by causing inappropriate Na⁺ excretion. In contrast, stimulation of the P2Y₂ receptor promotes natriuresis and a decrease in blood pressure. Such observations identify purinergic signalling in the distal nephron as possibly causative, when dysfunctional, for certain forms of elevated blood pressure, and as a possible therapeutic target for the treatment of elevated blood pressure particularly that associated with salt sensitivity.     

Cofilin participates in regulating alpha-epithelial sodium channel by interaction with 14-3-3 isoforms

Renal epithelial sodium channel (ENaC) plays a crucial role in maintaining homeostasis and sodium absorption. While insulin participates in controlling sodium transport across the renal epithelium, the underlying molecular mechanism remain unclear. In this study, we found that insulin increased the expression and function of alpha-epithelial sodium channel (α-ENaC) as well as phosphorylation of cofilin, a family of actin-binding proteins which disassembles actin filaments, in mouse cortical collecting duct (mpkCCD_c14) cells. The wild-type (WT) cofilin and its constitutively phosphorylated form (S3D), but not its constitutively non-phosphorylable form (S3A), contributed to the elevated expression on α-ENaC. Overexpression of 14-3-3ε, β, or γ increased the expression of α-ENaC and cofilin phosphorylation, which was blunted by knockdown of 14-3-3ε, β, or γ. Moreover, it was found that insulin increased the interaction between cofilin and 14-3-3 isoforms, which indicated relevance of 14-3-3 isoforms with cofilin. Furthermore, LIMK1/SSH1 pathway was involved in regulation of cofilin and α-ENaC expression by insulin. The results from this work indicate that cofilin participates in the regulation of α-ENaC by interaction with 14-3-3 isoforms.     

Apical serine protease activity is necessary for assembly of a high-resistance renal collecting duct epithelium

Aim: We hypothesized that the serine protease prostasin is necessary for differentiation of a high-resistance renal collecting duct epithelium governed by glucocorticoid. Methods: Postnatal rat kidney and adult human kidney was used to study the expression and localization of prostasin. The murine collecting duct cell line (M-1) was cultured in Snapwell inserts to investigate the significance of prostasin for the development of transepithelial electrical resistance (TER). Results: In the cortex and medulla of rat kidney, prostasin mRNA and protein increased significantly between birth and weaning (day 21) and was detected in collecting ducts. Immunoreactive prostasin was associated with collecting ducts and loops of Henle in human kidney. In rat, adrenalectomy at day 10 had no effect on prostasin mRNA level in kidney at day 20. Cultured M-1 cells exhibited parallel increases in prostasin mRNA, protein and TER 5 days after seeding. Apical addition of the serine protease inhibitor aprotinin to M-1 cell cultures inhibited development of TER and led to aberrant localization of E-cadherin. This effect was mimicked by the protease inhibitor nafamostat. Apical addition of phospholipase C to cleave glycosylphosphatidylinositol (GPI) anchors released prostasin to the medium and attenuated development of TER with time of culture. Disruption of lipid rafts by methyl-β-cyclodextrin attenuated development of TER in M-1 cells. Omission of dexamethasone impaired development of TER in M-1 cells, while prostasin protein abundance and E-cadherin distribution did not change. Conclusion: Apical, GPI-anchored, lipid raft-associated serine protease activity, compatible with prostasin, is necessary for the development of a high-resistance collecting duct epithelium.     

Regulation of the abundance of renal sodium transporters and channels by vasopressin

Vasopressin plays a role in both salt and water balance in the kidney. Classic studies, utilizing isolated perfused tubules, have revealed that vasopressin increases sodium reabsorption in the kidney thick ascending limb and the collecting duct. Furthermore, the activity of several sodium transport proteins expressed in these segments, such as the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and the epithelial sodium channel (ENaC), have been shown to be directly increased by vasopressin. Increased protein abundance might be one means through which sodium transporter and channel activity is enhanced. We have used immunoblotting and immunohistochemistry in order to investigate the regulation of abundance of the major sodium transporters and channels expressed along the renal tubule in response to vasopressin. Chronic (7-day) studies were performed in which vasopressin levels were elevated either endogenously by water restriction of Sprague-Dawley rats or exogenously through infusion of the vasopressin V2-receptor-selective agonist, dDAVP (1-deamino-8d-arginine-vasopressin), to Brattleboro rats. We found a significant increase in protein abundance for NKCC2 and the beta- and gamma-subunits of ENaC with either water restriction or dDAVP infusion. The alpha-subunit of Na-K-ATPase was increased by water restriction, but not by dDAVP infusion, and alpha-ENaC and the thiazide-sensitive cotransporter (NCC) were increased by dDAVP infusion but not by water restriction. Acute (60-min) in vivo exposure to dDAVP led to an increase in both beta- and gamma-ENaC abundance in kidney cortex homogenates, displaying the rapid nature of some of these changes. Overall these increases in sodium transporter and channel abundances likely contribute to both the antidiuretic and antinatriuretic actions of vasopressin.