Impact of hypercapnia on alveolar Na+-transport : Establishing a system for ENaC-protein detection

Acute respiratory distress syndrome is a life threatening condition triggered by a variety of pulmonary and extrapulmonary causes, that is characterized by pulmonary edema and subsequently impaired gas exchange. Due to lung protective ventilation strategies, its treatment is often associated with systemic accumulation of CO2, a condition termed permissive hypercapnia. Recent studies report a negative effect of CO2 on alveolar fluid clearance, a process mediated by its two key elements the Na+,K+-ATPase and epithelial Na+-channels (ENaCs). A reduced activity of the Na+,K+-ATPase during hypercapnia has already been demonstrated, but regulation of ENaC has never been directly linked to CO2. Many molecular signaling events that are activated during hypercapnia are known to regulate ENaC function, so the present study aimed to generate and subsequently apply techniques to investigate a possible contribution of ENaC to the reduction of alveolar epithelial fluid transport upon hypercapnia. ENaC function was studied in H441 cells by Ussing chamber experiments which revealed no significant regulation during short term hypercapnia, but a clear reduction of ENaC function during sustained hypercapnia. To identify the signaling mechanism on the molecular level, epitope-tagged human ENaC constructs for the α-, β- and γ-subunit were cloned and initially expressed in A549 cells. Exposition to hypercapnia up to 4 hours did not significantly reduce cell surface expression of the ENaC-subunits, but after 24 hours, a significant decrease of β-ENaC was observed. Since the molecular sizes of α- and γ-ENaC expressed in A549 cells were differing from previously published studies, transfection of ENaC was continued in other cells. H441 cells are commonly used for ENaC studies, so their transfection was established, yielding an efficiency of about 60 %. The molecular sizes of transfected ENaC subunits matched the pattern that was expected, but expression levels were evanescent and too low for further experiments. Since ENaC detection in these two cell lines remained problematic, a novel methodology was applied. Since the primary site of ENaC expression in the lung are epithelial cells, rat primary alveolar epithelial cells type II were used as recipients for ENaC plasmids. Non-viral transfection of ATII cells has been inefficient in the past, but during the present study a protocol was generated to efficiently deliver nucleic acids to exactly this cell type. ENaC expression was largely increased in ATII cells, compared to the cell lines used, indicating that established system might be extremely useful for further studies involving ENaC turnover. Thus, a new and highly relevant, non-viral transfection technique for primary alveolar epithelial type II cells was established, providing ground-breaking opportunities for future pulmonary research.Das Atemnotsyndrom des Erwachsenen ist eine lebensbedrohliche Erkrankung, ausgelöst durch eine Reihe von Faktoren, die direkt oder indirekt auf die Lunge einwirken . Charakteristisch für dieses Syndrom sind pulmonare Ödeme und daraus resultierend ein eingeschränkter Gasaustausch. Die daher benötigte künstliche Beatmung führt im Zuge von protektiven Beatmungsstrategien oft zu einer systemischen Anreicherung von CO2 (Hyperkapnie). Einige Studien zeigen, dass erhöhte CO2-Level den Flüssigkeitstransport der Lunge einschränken. Dieser aktive Prozess wird maßgeblich durch zwei Komponenten, die Na+,K+-ATPase und epitheliale Na+-Kanäle (ENaCs), kontrolliert. Eine Beeinträchtigung der Na+,K+-ATPase durch CO2 gezeigt, für ENaCs ist dies bislang nicht bekannt. Einige bekannte Regulatoren von ENaCs werden jedoch während Hyperkapnie aktiviert. Das Ziel der vorliegenden Arbeit war, Methoden zu etablieren und anzuwenden, die einen möglichen Einfluss von CO2 auf ENaC zeigen. Funktionelle Versuche wurden an H441-Zellen mit Ussing-Kammer-Messungen durchgeführt. Während akuter Hyperkapnie konnte keine signifikante Regulation von ENaC nachgewiesen werden, jedoch war die ENaC-Funktion bei anhaltender Hyperkapnie deutlich verringert. Um die Signalwege auf molekularer Ebene zu untersuchen, wurde die α-, β- und γ- Untereinheit des humanen ENaC kloniert, genetisch modifiziert und in A549 Zellen überexprimiert. Nach bis zu vierstündiger Hyperkapnie erfolgte keine Regulation von ENaC, jedoch wurde nach 24 Stunden eine deutlich verminderte Menge β-ENaC in der Zellmembran nachgewiesen. Da die Größen von α- und γ-ENaC von den bisher publizierten abwichen, wurden weitere Versuche in H441 Zellen durchgeführt. Die Transfektion dieser Zelllinie wurde etabliert und erreichte eine Effizienz von ungefähr 60 %. Die posttranslationale Regulation der α- und γ-Untereinheiten, insbesondere die proteolytische Aktivierung funktionierten wie in der Literatur beschrieben, jedoch waren die Expressionslevel zu gering für weitere Versuche. In der Lunge werden ENaCs überwiegend in epithelialen Zellen exprimiert. Diese Zellen konnten bisher jedoch nicht effizient transfiziert werden, ohne Viren einzusetzen. In der vorliegenden Arbeit wurde jedoch eine effiziente Methode zur Transfektion von primären epithelialen Zellen der Ratte erarbeitet. Die Expression von transfizierten ENaC-Untereinheiten war in diesen Zellen deutlich erhöht, weswegen die Etablierung dieses Systems ausschlaggebend für weitere Versuche ist. Die vorliegende Arbeit beschreibt daher zum ersten Mal die nicht-virale, effiziente Transfektion von primären alveolaren Zellen und liefert damit ein bedeutendes neues Werkzeug für die Lungenforschung

Does the Bayley-III motor scale at 2 years predict motor outcome at 4 years in very preterm children?

Aim To assess the predictive validity of the Bayley Scales of Infant and Toddler Development Third Edition (Bayley-III) for later motor outcome. Method Ninety-six infants (49 males, 47 females) born at less than 30weeks' gestation admitted to two tertiary hospitals in Melbourne, Australia, were assessed with the Bayley-III Motor Scale at 2years' corrected age and were classified as suspect or definite motor impairment if they scored less than 1 or 2 standard deviations respectively, relative to the test mean. At 4years' corrected age, children completed Movement Assessment Battery for Children Second Edition (MABC-2); for the total motor score, cut-offs of not more than the 15th were used to classify motor development and cut-offs of not more than the 15th centile were classified as having a significant movement difficulty. Results Of the 96 children assessed at both ages, at 2years 9% had suspect and 4% had definite motor impairment; however, by 4years, rates had increased to 22% and 19% respectively. The specificity of the Bayley-III for motor impairments for later motor outcome was excellent (ranging from 94 to 100% for cerebral palsy [CP] and 97 to 100% for motor impairment), although the sensitivity was low (ranging from 67 to 83% for CP and 18 to 37% for motor impairment); many children with later impairment were not identified by the Bayley-III. Interpretation The Bayley-III Motor Scale at 2years underestimates later rates of motor impairment, particularly in the absence of CP at 4years on the MABC-2 total motor score in children born at less than 30weeks' gestational age

Characteristics of the study sample.

<p>Note: Two infants died prior to 2 years and are not included in this table; AIMS = Alberta Infant Motor Scale; NSMDA = Neuro-Sensory Motor Developmental Assessment; MABC-2 = Movement Assessments Battery for Children 2^nd edition</p><p>*n = 82; SD = standard deviation</p><p>Characteristics of the study sample.</p