Der epitheliale Natriumtransport in der postnatalen Lunge ist für einen ausgeglichenen Flüssigkeitstransport und eine gesunde Lungenfunktion unabdingbar. Eine bedeutende Rolle spielt hierbei der epitheliale Natriumkanal (ENaC). Es ist bereits bekannt, dass unter dem Einfluss von weiblichen Sexualhormonen, wie Progesteron, oder durch die Substitution von Glukokortikoiden, wie Dexamethason, die mRNA-Expression des ENaC und dessen elektrophysiologische Aktivität erhöht wird. Zur Lungenreifeinduktion werden bei Frühgeburtlichkeitsbestrebungen hohe Dosen von Glukokortikoiden verabreicht, die im fetalen Kreislauf auf hohe Progesteronkonzentrationen treffen. Die Auswirkung dieser Hormonkombination auf den epithelialen Natriumtransport ist bisher unbekannt. Um dieser Frage nachzugehen, wurden alveoläre Epithelzellen von Rattenfeten auf permeablen Membranen gezüchtet und mit unterschiedlichen Konzentrationen von Progesteron und Dexamethason inkubiert. Anschließend wurde die mRNA-Expression der drei Untereinheiten des ENaC (α, β, γ) mittels Real-Time PCR analysiert. Mit Hilfe von Ussing-Kammer Messungen wurden die Einflüsse auf den epithelialen Natriumtransport ermittelt. Durch die Experimente konnte der stimulierende Einfluss beider Hormone auf die mRNA-Expression bestätigt werden, wobei Dexamethason einen deutlich stärkeren Effekt erreichte. Durch die Kombination beider Hormone kam es zu einer signifikant geringeren mRNA-Expression und einem verminderten funktionellen Natriumtransport im Vergleich zur reinen Dexamethasoninkubation. Der Einsatz von Hormonrezeptor-Antagonisten zeigte, dass eine Blockierung des Progesteronrezeptors die mRNA-Expression erhöhte, wohingegen die Hemmung des Glukokortikoidrezeptors die mRNA-Expression der ENaCUntereinheiten verminderte. Zusammenfassend zeigen die Ergebnisse, dass Glukokortikoide und weibliche Geschlechtshormone, die einzeln zur Erhöhung der Natriumabsorption führen, durch die Kombination beider Hormone ihren Einfluss auf den Natriumtransport reduzieren.

In der vorliegenden Arbeit wurde der Einfluss von Insulin und IGF-1 auf den transepithelialen Natriumtransport über die Zellmembran von fetalen distalen Lungenepithelzellen der Ratte (fetal distal lung epithelia, FDLE) als Modell der Pneumozyten vom Typ II des späten Frühgeborenen untersucht. In Ussing-Kammer Messungen konnte eine insulinabhängige schnelle Steigerung des transepithelialen Natriumstroms gezeigt werden. Durch Western Blot-Untersuchungen sowie Inhibition spezifischer Kinasen wurden die intrazellulären Signaltransduktionsmechanismen der Insulin-induzierten Stimulation des Natriumtransports weiter charakterisiert. Es konnte eine Beteiligung der Phosphatidylinositol 3-Kinase, der Proteinkinase B, sowie von mTORC2 an den Signalwegen in den untersuchten Zellen nachgewiesen werden. Ähnliche Wirkungen auf den Natriumtransport wie Insulin hatte der Wachstumsfaktor IGF-1. Somit wurde der akute Einfluss des Insulin/IGF-1 Systems auf den epithelialen Natriumtransport in fetalen Alveolarzellen charakterisiert.

O balanço do fluoreto (F) dentro do corpo é modulado por sua ingestão, absorção e remoção, e os rins, responsáveis pela sua excreção do organismo, são particularmente vulneráveis à toxicidade do F. Os efeitos nefrotóxicos do F envolvem mudanças estruturais marcantes nos rins, além disso, estudos proteomicos têm mostrado grandes alterações no perfil de proteínas envolvidas em pontos chave na transdução de sinal. Estes efeitos podem influenciar negativamente no transporte iônico nos rins. A vista deste fato, o transporte iônico no canal de sódio epitelial (ENaC) é limitante para a taxa de reabsorção de sódio nos rins, sendo essencial para a manutenção do equilíbrio eletrolítico e homeostase do corpo. Assim, este trabalho objetivou investigar os efeitos do F, utilizando concentrações semelhantes às que podem ser encontradas no néfron durante a fluorose dentária, na viabilidade celular e expressão de transportadores de membrana (ENaC) em linhagem celular renal M-1. Para os ensaios de viabilidade das células da linhagem M-1 foi empregado os testes colorimétricos Cristal Violeta e MTT, utilizando as concentrações de tratamento com fluoreto de sódio (NaF) a 10, 40, 100, 200 e 400 M, durante períodos experimentais de 24, 48, 72 e 96 h. As mesmas concentrações e os mesmos tempos foram utilizados no tratamento com cloreto de sódio (NaCl), utilizado como controle na possível interferência de Na+ na modulação das células. Para a investigação da influência do F: 1) nos canais ENaC foi utilizada a técnica de imunofluorescência e 2) para a análise de expressão gênica das subunidades que formam o ENaC, foi utilizada a técnica RT-PCR. Em nossos resultados pudemos observar que as maiores concentrações tanto de NaF quanto de NaCl provocaram a diminuição da viabilidade celular para ambos os ensaios de viabilidade, no entanto, foi possível observar algumas diferenças na resposta do tratamento com NaF em comparação com NaCl, por meio do ensaio Cristal Violeta. Não foi observado diferenças nas imagens de imunofluorescêcia, mas outros aspectos morfológicos foram vistos nessas imagens, como o aparecimento de domes celular, sugerindo que até mesmo a maior concentração de F não foi capaz de inibir a proliferação celular. Nosso resultado mais significativo foi em relação à expressão das subunidades dos canais de ENaC, onde a concentração de 400 M foi capaz de diminuir bruscamente a expressão das três subunidades do ENaC, enquanto as concentrações de 100 e 200 M mostraram apresentar expressão igual e em alguns casos até maior que o grupo controle. Pudemos concluir que doses de F na ordem de micromolares podem modular a expressão das subunidades formadoras do ENaC, positivamente quando em baixas concentrações e negativamente quando em concentrações elevadas. / The balance of fluoride (F) within the body is modulated by its ingestion, absorption and removal, and the kidneys, responsible for their excretion of the organism, are particularly vulnerable to the toxicity of F. The nephrotoxic effects of F involve marked structural changes in the kidneys , in addition, proteomic studies have shown large changes in the profile of proteins involved in key points in signal transduction. These effects may negatively influence ion transport in the kidneys. In view of this fact, the ionic transport in the epithelial sodium channel (ENaC) is limiting to the rate of sodium reabsorption in the kidneys, being essential for the maintenance of the electrolyte balance and homeostasis of the body. Thus, the objective of this work was to investigate the effects of F, using concentrations similar to those found in the nephron during dental fluorosis, cell viability and expression of membrane transporters (ENaC) in renal cell line M-1. For the M-1 cell line viability assays, the Crystal Violet and MTT colorimetric assays were used, using the 10, 40, 100, 200 and 400 M sodium fluoride (NaF) treatment concentrations during experimental periods of 24, 48, 72 and 96 h. The same concentrations and the same times were used in the treatment with sodium chloride (NaCl), used as control in the possible interference of Na + in the modulation of the cells. For the investigation of the influence of F: 1) in the ENaC channels, the immunofluorescence technique was used and 2) for the analysis of gene expression of the subunits that form the ENaC, the RT-PCR technique was used. In our results it was observed that the higher concentrations of NaF and NaCl caused a decrease in cell viability for both viability assays, however, it was possible to observe some differences in NaF treatment response in comparison to NaCl, through test Crystal Violet. No differences were observed in immunofluorescence images, but other morphological aspects were seen in these images, such as the appearance of cellular \"domes\", suggesting that even the highest F concentration was not able to inhibit cell proliferation. Our most significant result was the expression of the subunits of the ENaC channels, where the concentration of 400 M was able to decrease expression of the three subunits of the ENaC, whereas the concentrations of 100 and 200 M showed equal expression and in some cases even higher than the control group. We can conclude that doses in the order of micromolar the F can modulate the expression of the ENaC forming subunits, positively when in low concentrations and negatively when in high concentrations.

Salty taste has evolved to maintain electrolyte homeostasis, serving as a detector for salt containing food. In rodents, salty taste involves at least two transduction mechanisms. One is sensitive to the drug amiloride and specific for Na+, involving epithelial sodium channel (ENaC). A second rodent transduction pathway, which is triggered by various cations, is amiloride insensitive and not almost understood to date. Studies in primates showed amiloride-sensitive as well as amiloride-insensitive gustatory responses to NaCl, implying a role of both salt taste transduction pathways in humans. However, sensory studies in humans point to largely amiloride-insensitive sodium taste perception. An involvement of ENaC in human sodium taste perception was not shown, so far. In this study, ENaC subunit protein and mRNA could be localized to human taste bud cells (TBC). Thus, basolateral αβγ-ENaC ion channels are likely in TBC of circumvallate papillae, possibly mediating basolateral sodium entry. Similarly, basolateral βγ-ENaC might play a role in fungiform TBC. Strikingly, δ-ENaC subunit was confined to taste bud pores of both papillae, likely mediating gustatory sodium entry in TBC, either apical or paracellular via tight junctions. However, regional separation of δ-ENaC and βγ-ENaC in fungiform and circumvallate TBC indicate the presence of unknown interaction partner necessary to assemble into functional ion channels. However, screening of a macaque taste tissue cDNA library did neither reveal polypeptides assembling into a functional cation channel by interaction with δ-ENaC or βγ-ENaC nor ENaC independent salt taste receptor candidates. Thus, ENaC subunits are likely involved in human taste transduction, while exact composition and identity of an amiloride (in)sensitive salt taste receptors remain unclear. Localization of δ-ENaC in human taste pores strongly suggests a role in human taste transduction. In contrast, δ-ENaC is classified as pseudogene Scnn1d in mouse. However, no experimental detected sequences are annotated, while evidences for parts of Scnn1d derived mRNAs exist. In order to elucidate if Scnn1d is possibly involved in rodent salt taste perception, Scnn1d was evaluated in this study to clarify if Scnn1d is a gene or a transcribed pseudogene in mice. Comparative mapping of human SCNN1D to mouse chromosome 4 revealed complete Scnn1d sequence as well as its pseudogenization by Mus specific endogenous retroviruses. Moreover, tissue specific transcription of unitary Scnn1d pseudogene was found in mouse vallate papillae, kidney and testis and led to identification of nine Scnn1d transcripts. In vitro translation experiments showed that Scnn1d transcripts are coding competent for short polypeptides, possibly present in vivo. However, no sodium channel like function or sodium channel modulating activity was evident for Scnn1d transcripts and/or derived polypeptides. Thus, an involvement of mouse δ-ENaC in sodium taste transduction is unlikely and points to species specific differences in salt taste transduction mechanisms. / Der Salzgeschmack ermöglicht elektrolytreiche Nahrungsquellen zu erkennen und ist eine essentielle Komponente für den Erhalt des Elektrolythaushalts. In Nagern sind bisher zwei Mechanismen bekannt, welche an der Vermittlung des Salzgeschmacks beteiligt sind. Ein Natrium-spezifischer, Amilorid-sensitiver Signaltransduktionsweg wird über den epithelialen Natriumkanal (ENaC) vermittelt. Ein weiterer, bisher ungeklärter Transduktionsweg, ist Amilorid-unempfindlich und wird durch verschiedene Kationen vermittelt. Studien in Primaten konnten Amilorid-sensitive als auch -insensitive gustatorische Signaltransduktionswege nachweisen, wohingegen sensorische Studien auf eine Amilorid-Unempfindlichkeit des Natrium-spezifischen humanen Salzgeschmacks hinweisen. Eine Beteiligung des ENaC bei der Vermittlung des menschlichen Salzgeschmacks wurde bislang nicht gezeigt. In dieser Arbeit konnte die mRNA als auch Proteine von ENaC Untereineiten in menschlichen Geschmacksrezeptorzellen (GRZ) lokalisiert werden. Demzufolge, sind αβγ-ENaC Ionenkanäle möglicherweise an einem basolateralen Natriumeinstrom in circumvallaten GRZ beteiligt. Die basolaterale Lokalisation von βγ-ENaC in fungiformen GRZ weißt auf eine gleichartige Funktion hin. Die außergewöhnliche Lokalisation der δ-ENaC Untereineit ausschließlich in der Porenregion von Geschmacksknospen beider Geschmackspapillen, legt eine Beteiligung dieser ENaC Untereinheit bei der Vermittlung geschmacksrelevanter apikaler bzw. transzellulärer Natriumströme nahe. Gleichwohl weist die räumliche Trennung von apikalen δ-ENaC und basolateralen βγ-ENaC auf die Existenz unbekannter Interaktionspartner hin, da beide getrennt voneinander nicht in der Lage sind effektive Natriumkanäle zu assemblieren. Die Durchmusterung einer geschmacksrelevanten cDNA Bibliothek führte weder zur Identifikation von ENaC Interaktionspartnern, noch von ENaC unabhängigen Polypeptiden, welche in der Lage sind einen Kationenkanal zu bilden. Die genaue Zusammensetzung humaner Amilorid- (in)sensitiver Salzrezeptoren bleibt daher unklar und ein spannendes Feld. Der Nachweis von ENaC in humanen GRZ und insbesondere die Poren assoziierte Lokalisation der δ-ENaC Untereinheit impliziert eine wichtige Rolle bei der gustatorischen Signaltransduktion. Erstaunlicherweise ist die orthologe δ-ENaC Untereinheit der Maus als Scnn1d Pseudogen klassifiziert. Neben dieser automatischen Annotierung sind keine experimentell ermittelten Sequenzen in Datenbanken hinterlegt obwohl Scnn1d abgeleitete mRNA nachgewiesen werden konnte. Im Rahmen dieser Arbeit wurde untersucht ob Scnn1d ein Gen oder ein transkribiertes Pseudogen ist, um eine mögliche Rolle bei der Transduktion des murinen Salzgeschmacks zu klären. Durch Sequenzabgleich mit humanen SCNN1D konnte das vollständige Scnn1d Gen auf dem Chromosom 4 der Maus identifiziert werden, wobei sich dessen Pseudogenisierung durch Mus spezifische endogene Retroviren zeigte. Darüber hinaus wurden neun gewebsspezifische Scnn1d Transkripte nachgewiesen, welche für kurze Polypeptide kodieren. Eine mögliche Funktion derselben als Ionenkanal bzw. eine modulatorische Funktion konnte nicht gezeigt werden. Eine Beteiligung des pseudogenisierten δ-ENaC an der Vermittlung des Salzgeschmacks der Maus ist daher unwahrscheinlich und deutet auf Speziesunterschiede der Salzgeschmacksvermittlung hin.

O balanço do fluoreto (F) dentro do corpo é modulado por sua ingestão, absorção e remoção, e os rins, responsáveis pela sua excreção do organismo, são particularmente vulneráveis à toxicidade do F. Os efeitos nefrotóxicos do F envolvem mudanças estruturais marcantes nos rins, além disso, estudos proteomicos têm mostrado grandes alterações no perfil de proteínas envolvidas em pontos chave na transdução de sinal. Estes efeitos podem influenciar negativamente no transporte iônico nos rins. A vista deste fato, o transporte iônico no canal de sódio epitelial (ENaC) é limitante para a taxa de reabsorção de sódio nos rins, sendo essencial para a manutenção do equilíbrio eletrolítico e homeostase do corpo. Assim, este trabalho objetivou investigar os efeitos do F, utilizando concentrações semelhantes às que podem ser encontradas no néfron durante a fluorose dentária, na viabilidade celular e expressão de transportadores de membrana (ENaC) em linhagem celular renal M-1. Para os ensaios de viabilidade das células da linhagem M-1 foi empregado os testes colorimétricos Cristal Violeta e MTT, utilizando as concentrações de tratamento com fluoreto de sódio (NaF) a 10, 40, 100, 200 e 400 M, durante períodos experimentais de 24, 48, 72 e 96 h. As mesmas concentrações e os mesmos tempos foram utilizados no tratamento com cloreto de sódio (NaCl), utilizado como controle na possível interferência de Na+ na modulação das células. Para a investigação da influência do F: 1) nos canais ENaC foi utilizada a técnica de imunofluorescência e 2) para a análise de expressão gênica das subunidades que formam o ENaC, foi utilizada a técnica RT-PCR. Em nossos resultados pudemos observar que as maiores concentrações tanto de NaF quanto de NaCl provocaram a diminuição da viabilidade celular para ambos os ensaios de viabilidade, no entanto, foi possível observar algumas diferenças na resposta do tratamento com NaF em comparação com NaCl, por meio do ensaio Cristal Violeta. Não foi observado diferenças nas imagens de imunofluorescêcia, mas outros aspectos morfológicos foram vistos nessas imagens, como o aparecimento de domes celular, sugerindo que até mesmo a maior concentração de F não foi capaz de inibir a proliferação celular. Nosso resultado mais significativo foi em relação à expressão das subunidades dos canais de ENaC, onde a concentração de 400 M foi capaz de diminuir bruscamente a expressão das três subunidades do ENaC, enquanto as concentrações de 100 e 200 M mostraram apresentar expressão igual e em alguns casos até maior que o grupo controle. Pudemos concluir que doses de F na ordem de micromolares podem modular a expressão das subunidades formadoras do ENaC, positivamente quando em baixas concentrações e negativamente quando em concentrações elevadas. / The balance of fluoride (F) within the body is modulated by its ingestion, absorption and removal, and the kidneys, responsible for their excretion of the organism, are particularly vulnerable to the toxicity of F. The nephrotoxic effects of F involve marked structural changes in the kidneys , in addition, proteomic studies have shown large changes in the profile of proteins involved in key points in signal transduction. These effects may negatively influence ion transport in the kidneys. In view of this fact, the ionic transport in the epithelial sodium channel (ENaC) is limiting to the rate of sodium reabsorption in the kidneys, being essential for the maintenance of the electrolyte balance and homeostasis of the body. Thus, the objective of this work was to investigate the effects of F, using concentrations similar to those found in the nephron during dental fluorosis, cell viability and expression of membrane transporters (ENaC) in renal cell line M-1. For the M-1 cell line viability assays, the Crystal Violet and MTT colorimetric assays were used, using the 10, 40, 100, 200 and 400 M sodium fluoride (NaF) treatment concentrations during experimental periods of 24, 48, 72 and 96 h. The same concentrations and the same times were used in the treatment with sodium chloride (NaCl), used as control in the possible interference of Na + in the modulation of the cells. For the investigation of the influence of F: 1) in the ENaC channels, the immunofluorescence technique was used and 2) for the analysis of gene expression of the subunits that form the ENaC, the RT-PCR technique was used. In our results it was observed that the higher concentrations of NaF and NaCl caused a decrease in cell viability for both viability assays, however, it was possible to observe some differences in NaF treatment response in comparison to NaCl, through test Crystal Violet. No differences were observed in immunofluorescence images, but other morphological aspects were seen in these images, such as the appearance of cellular \"domes\", suggesting that even the highest F concentration was not able to inhibit cell proliferation. Our most significant result was the expression of the subunits of the ENaC channels, where the concentration of 400 M was able to decrease expression of the three subunits of the ENaC, whereas the concentrations of 100 and 200 M showed equal expression and in some cases even higher than the control group. We can conclude that doses in the order of micromolar the F can modulate the expression of the ENaC forming subunits, positively when in low concentrations and negatively when in high concentrations.

Pregnancy-mediated sodium (Na) retention is required to provide an increase in plasma volume for the growing fetus. The mechanisms responsible for this Na retention are not clear. In the first study, we generated a renal tubule protein profile indicating α ENaC as the only sodium transport protein upregulated in mid and late pregnancy. To determine the in vivo activity of ENaC we conducted in vivo studies in late pregnant rats (day 18-20) and virgin rats to measure the natriuretic response to ENaC blockade (with benzamil). The in vivo activity of ENaC (UNaV post benzamil - UNaV post vehicle) was markedly increased in late pregnancy and this difference was abolished by pretreatment with the mineralocorticoid receptor antagonist, eplerenone. These findings demonstrate that the increased α ENaC subunit of pregnancy is associated with a mineralocorticoid-dependent increase in ENaC activity. The plasma volume increased progressively during pregnancy with the greatest plasma volume being evident in late pregnancy. ENaC inhibition abolished the difference in plasma volume status between virgin and pregnant rats. This indicates ENaC activity is a major contributor of plasma volume status in late pregnancy. Inadequate plasma volume expansion results in complicated pregnancy with growth restricted (GR) fetus and maternal/fetal death. The second study aimed to determine the importance of renal vs extrarenal ENaC in sodium retention and blood pressure regulation during pregnancy to do so we chronically blocked ENaC with either daily subcutaneous injections of benzamil (BZ) or intrarenal transfection of αENaC shRNA. Chronic ENaC blockade with benzamil prevented normal sodium retention in the pregnant rat. Prevention of sodium retention resulted in reduced maternal serum sodium concentration, blood pressure, body weight, and fetal growth restriction. However, chronic benzamil treatment had no effect on sodium retention, body weight, or BP in virgin rats. Intrarenal transfection of a shRNA targeting αENaC successfully decreased renal αENaC mRNA expression in late pregnant rats compared to controls transfected with scrambled sRNA. Intrarenal transfection of αENaC shRNA reduced sodium retention maternal, ΔBW, and pup weight. These findings suggest that renal ENaC is necessary for maintenance of sodium balance, blood pressure regulation, and progression of a healthy pregnancy. In the third study, we performed large-scale proteomic analysis on late pregnant and virgin principal collecting duct cells, isolated by laser capture microscopy. The primary aim of this project was to identify potential proteins or signaling pathways that could account for the sodium retention occurring in pregnancy. Large-scale liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed at the NIH which identified a total of 365 proteins in virgin and late pregnant collecting duct cells. We identified pregnancy associated abundance changes in six proteins related to the ubiquitin/proteasome degradation pathway. Since a major mechanism of ENaC regulation is through trafficking we focused on changes in this pathway and their implications for enhancing ENaC activity during pregnancy. The final study aimed to apply these findings to the overall theme of pregnancy as a state of arterial underfilling. We performed a Na transporter profile in kidney cortex homogenates from animals treated chronically with vasodilators (nifidipine or sodium nitrate). We found that only the abundance of transporters in the distal nephron (NCC, ENaC, AQP2) was similar to pregnancy, however differences were seen in pre-macula densa transporters (NHE3, NKCC2). The similar changes observed in the distal convoluted tubule and renal collecting duct promote Na and water retention. The changes in these transporters may explain the similar plasma volume expansions previously described in pregnant and chronically vasodilated rats, thus supporting the underfill hypothesis of pregnancy. Taken together, this project supports an important role for the collecting duct in the pregnancy mediated sodium and fluid reabsorption during pregnancy.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD.

Background: Excess plugging of small airways is associated with premature death in chronic obstructive pulmonary disease (COPD). Over-expression of beta-epithelial sodium channel (β-ENaC) in airway epithelia in mice resulted in plugging of small airways while cystic fibrosis transmembrane regulator (CFTR) negatively regulated ENaC activity in cell models. Purpose: To test the hypothesis that accumulation of mucus exudates observed with the progression of COPD is related to excess airway epithelial sodium re-absorption as a result of over-expression of β-ENaC and reduced expression of CFTR by small airway epithelia. Methods: Small airway epithelial samples from frozen lungs from patients at different levels of COPD severity were isolated by laser capture microdissection (LCM). β-ENaC, CFTR, and β-actin (control) gene expression was determined by qRT-PCR and compared to expression in entire airways and lung parenchyma surrounding these airways. β-ENaC protein as well as epithelial mucin expression and mucus plugging were localized and quantified after immunohistochemical and periodic acid Schiff staining, respectively. Results: β-ENaC mRNA expression had a strong positive correlation with that of CFTR (p<O.0001) in airway epithelia and surrounding lung parenchyma (p=O.Ol) but not whole airways. β-ENaC mRNA and protein expression were positively correlated (p=O.4O, p=O.O5) and protein expression significantly increased with GOLD stage of COPD severity. Epithelial mucin expression positively correlated with β-ENaC (p=O.38, p=O.O5) and CFTR (p=OAO, p=O.O4.) mRNA and with mucus plugging (p=O. 43 , ptO.OOO2). CFTR mRNA also correlated positively with mucus plugging (p=O. 48 , p=O.O2). Conclusions: Strong positive correlations between β-ENaC and CFTR mRNA expression that are limited to the lung parenchyma and epithelium suggest a novel mechanism of mRNA regulation. This differs from their functional relationship where an inverse relationship between CFTR expression and β-ENaC activity has been reported. Positive correlations of epithelial mucin or mucus plugging with CFTR mRNA but not β-ENaC protein expression in the small airway epithelium suggest that CFTR may regulate mucin at this site independently of β-ENaC protein. The relationship between β-ENaC mRNA andepithelial mucin expression could be due to strong correlations between β-ENaC and CFTR mRNA expression but β-ENaC’s relationship with COPD GOLD stage suggests it may nevertheless play a role in COPD. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Le canal sodique épithélial sensible à l'amiloride (ENaC) est une protéine composée de trois sous unités similaires [alpha][bêta][gamma] codées par trois gènes distincts et associées en un complexe hétérotétramérique [alpha]2[bêta][gamma]. ENaC est localisée au niveau de la membrane apicale des cellules épithéliales à jonctions serrées. C'est une protéine clé de la réabsorption du sodium au niveau du néphron distal, des voies aériennes supérieures et du colon distal. Son rôle dans la régulation du volume extracellulaire et la pression sanguine a été renforcé par la découverte de deux maladies génétiques humaines liées à la fonction du canal : le syndrome de Liddle et le pseudoaldostéronisme de type I (PHA I) due respectivement à un gain et une perte de fonction du canal. L'activité et l'expression de ce dernier sont finement régulées. Sa régulation par les sérines protéases et le sodium extracellulaire (auto-inhibition) a été mis en évidence par Chraïbi et collaborateurs en 1997, mais peu de choses sont connues sur les mécanismes et le rôle fonctionnel de la boucle extracellulaire dans ces différentes voies de régulation. Notre objectif est d'identifier les sites potentiels impliqués dans ces différentes voies de régulation. Pour ce faire, des mutations au niveau de la partie terminale de la boucle extracellulaire des sous unités [alpha], [bêta] et [gamma] du canal ont été réalisées par mutagenèse dirigée et l'effet des sérines protéases et du sodium extracellulaire a été étudié. Nos résultats, obtenus par la technique de voltage-clamp à deux électrodes, montrent que le domaine WPS (un domaine conservé dans les trois sous unités des différentes espèces et situé au niveau de la partie terminale de la boucle extracellulaire du canal) est impliqué dans la régulation d'ENaC par les sérines protéases et le sodium extracellulaire. Nous avons montré plus particulièrement que le tryptophane [alpha]W453 a un rôle fonctionnel majeur dans ces deux voies de régulation. Sa substitution par une arginine au niveau de la sous unité [alpha] rend ENaC résistant à la trypsine et abolit totalement l'inactivation du canal par le sodium extracellulaire. Par contre, la même mutation au niveau de la sous unité [gamma] ne change ni la réponse du canal à la trypsine ni sa sensibilité au sodium extracellulaire, alors que les canaux portant la mutation W/R au niveau de la sous unité [bêta] ont une sensibilité intermédiaire. L'ensemble de ces résultats montre le rôle fonctionnel de la boucle extracellulaire d'ENaC dans la régulation du canal par les sérine-protéases et le sodium extracellulaire.