Early expression of β- and γ-subunits of epithelial sodium channel during human airway development

2000 ◽  
Vol 278 (1) ◽  
pp. L177-L184 ◽  
Author(s):  
Dominique Gaillard ◽  
Jocelyne Hinnrasky ◽  
Sylvie Coscoy ◽  
Paul Hofman ◽  
Michael A. Matthay ◽  
...  
Keyword(s):  
Human Lung ◽  
Immunohistochemical Localization ◽  
Fluid Composition ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Membrane Protein Complex ◽  
Large Airways ◽  
Early Expression ◽  
Polyclonal Antisera ◽  
Human Airways

The amiloride-sensitive epithelial Na+channel (ENaC) is an apical membrane protein complex involved in active Na+ absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming α-, β-, and γ-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against β- and γ-ENaC, immunohistochemical localization of the channel was performed in fetal (10–35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of β- and γ-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, β- and γ-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of β- and γ-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na+ absorption might begin significantly before birth, even if secretion is still dominant.

scholarly journals Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

2020 ◽  
Author(s):  
Ameen A. Salahudeen ◽  
Shannon S. Choi ◽  
Arjun Rustagi ◽  
Junjie Zhu ◽  
Sean M. de la O ◽  
...  
Keyword(s):  
Lung Disease ◽  
Single Cell ◽  
Basal Cell ◽  
Human Lung ◽  
Basal Cells ◽  
Ciliated Cells ◽  
Club Cells ◽  
Distal Lung

ABSTRACTThe distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

Polycystin-2 expression is developmentally regulated

1999 ◽  
Vol 277 (1) ◽  
pp. F17-F25 ◽  
Author(s):  
Glen S. Markowitz ◽  
Yiqiang Cai ◽  
Li Li ◽  
Guanqing Wu ◽  
Llewellyn C. Ward ◽  
...  
Keyword(s):  
Ureteric Bud ◽  
Developmentally Regulated ◽  
Adult Kidney ◽  
Endocrine Organs ◽  
Organ Specific ◽  
Early Expression ◽  
Polyclonal Antisera ◽  
Distal Tubules ◽  
Autosomal Dominant Polycystic Kidney ◽  
High Level

PKD2 encodes a protein of unknown function that is mutated in 15% of autosomal dominant polycystic kidney disease (ADPKD) families. We used polyclonal antisera against PKD2 to examine the pattern of Pkd2 expression in staged mouse embryos. Staining for Pkd2 was documented as early as the 6th embryonic day ( day E6) in the embryonic ectoderm and endoderm. Low-intensity staining is seen in metanephric ureteric bud at day E12.5. By day E15.5, the adult pattern of expression is established with low level staining in proximal tubules and high level, basolateral staining in distal tubules. Pkd2 expression is first detected in the medullary collecting ducts at postnatal day 14. Outside of the kidney, Pkd2 expression is widely distributed in utero and more restricted postnatally. The greatest intensity of staining is seen in the fetal but not adult adrenal cortex and in red blood cell precursors. Expression also is seen in multiple endocrine organs, in cardiac, skeletal, and smooth muscle, and in multiple mesenchymal tissues. The diffuse distribution and early expression of Pkd2 suggest a fundamental developmental role. The persistent strong expression in adult kidney is consistent with a more organ-specific function in the maintenance of the mature metanephric tubule.

Distribution of ClC-2 chloride channel in rat and human epithelial tissues

2002 ◽  
Vol 282 (4) ◽  
pp. C805-C816 ◽  
Author(s):  
Joanna Lipecka ◽  
Moëz Bali ◽  
Annick Thomas ◽  
Pascale Fanen ◽  
Aleksander Edelman ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Expression Pattern ◽  
Chloride Channel ◽  
Human Intestine ◽  
Ciliated Cells ◽  
Rat Intestine ◽  
Colon Cells ◽  
Basolateral Membranes ◽  
Significant Expression ◽  
Human Airways

The ubiquitous ClC-2 Cl−channel is thought to contribute to epithelial Cl−secretion, but the distribution of the ClC-2 protein in human epithelia has not been investigated. We have studied the distribution of ClC-2 in adult human and rat intestine and airways by immunoblotting and confocal microscopy. In the rat, ClC-2 was present in the lateral membranes of villus enterocytes and was predominant at the basolateral membranes of luminal colon enterocytes. The expression pattern of ClC-2 in the human intestine differed significantly, because ClC-2 was mainly detected in a supranuclear compartment of colon cells. We found significant expression of ClC-2 at the apex of ciliated cells in both rat and human airways. These results show that the distribution of ClC-2 in airways is consistent with participation of ClC-2 channels in Cl− secretion and indicate that extrapolation of results from studies of ClC-2 function in rat intestine to human intestine is not straightforward.

Postnatal undernutrition slows development of bronchiolar epithelium in rats

1988 ◽  
Vol 255 (4) ◽  
pp. R521-R526 ◽  
Author(s):  
G. D. Massaro ◽  
L. McCoy ◽  
D. Massaro
Keyword(s):  
Small Airway ◽  
Numerical Density ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Long Term Effects ◽  
Chronic Lung Diseases ◽  
Airway Function ◽  
Childhood Environment ◽  
Bronchiolar Epithelium ◽  
Morphological Maturation

The lung's small conducting airways are sites of dysfunction early in the course of chronic lung diseases that are prevalent in humans; furthermore, there is evidence that aspects of childhood environment may adversely influence small airway function in adulthood. Because there is considerable early postnatal morphological maturation of the bronchiolar epithelium in rats, these considerations led to the present study in which we assessed the effect of early postnatal undernutrition in rats on the anatomic development of the bronchiolar epithelium. We found undernutrition, produced by increasing rat litter size shortly after birth, led to delayed development of the mitochondria and rough endoplasmic reticulum of bronchiolar Clara cells. Of particular interest, underfeeding resulted in considerably diminished mitosis by Clara cells, decreased nuclear numerical density of bronchiolar ciliated cells, evidence of diminished conversion of Clara cells to ciliated cells, and an abnormal cellular composition of the small airway epithelium that persisted well beyond the period of underfeeding. We conclude that early neonatal events can have long-term effects on the bronchiolar epithelium.

New Insight into the Development of the Respiratory Acini in Rabbits: Morphological, Electron Microscopic Studies, and TUNEL Assay

2019 ◽  
Vol 25 (3) ◽  
pp. 769-785 ◽  
Author(s):  
Doaa M. Mokhtar ◽  
Manal T. Hussein ◽  
Marwa M. Hussein ◽  
Enas A. Abd-Elhafez ◽  
Gamal Kamel
Keyword(s):  
Tunel Assay ◽  
Second Phase ◽  
Type I ◽  
Apoptotic Cells ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Blood Capillaries ◽  
Pulmonary Alveoli ◽  
Alveolar Septa ◽  
Terminal Bronchiole

AbstractThis study investigated the histomorphological features of developing rabbit respiratory acini during the postnatal period. On the 1st day of postnatal life, the epithelium of terminal bronchiole consisted of clear cells which intercalated between few ciliated and abundant non-ciliated (Clara) cells. At this age, the rabbit lung was in the alveolar stage. The terminal bronchioles branched into several alveolar ducts, which opened into atria that communicated to alveolar sacs. All primary and secondary inter-alveolar septa were thick and showed a double-capillary network (immature septa). The primitive alveoli were lined largely by type-I pneumocytes and mature type-II pneumocytes. The type-I pneumocytes displayed an intimate contact with the endothelial cells of the blood capillaries forming the blood–air barrier (0.90 ± 0.03 µm in thickness). On the 3rd day, we observed intense septation and massive formation of new secondary septa giving the alveolar sac a crenate appearance. The mean thickness of the air–blood barrier decreased to reach 0.78 ± 0.14 µm. On the 7th day, the terminal bronchiole epithelium consisted of ciliated and non-ciliated cells. The non-ciliated cells could be identified as Clara cells and serous cells. New secondary septa were formed, meanwhile the inter-alveolar septa become much thinner and the air–blood barrier thickness was 0.66 ± 0.03 µm. On the 14th day, the terminal bronchiole expanded markedly and the pulmonary alveoli were thin-walled. Inter-alveolar septa become much thinner and single capillary layers were observed. In the 1st month, the secondary septa increased in length forming mature cup-shaped alveoli. In the 2nd month, the lung tissue grew massively to involve the terminal respiratory unit. In the 3rd month, the pulmonary parenchyma appeared morphologically mature. All inter-alveolar septa showed a single-capillary layer, and primordia of new septa were also observed. The thickness of the air–blood barrier was much thinner; 0.56 ± 0.16 µm. TUNEL assay after birth revealed that the apoptotic cells were abundant and distributed in the epithelium lining of the pulmonary alveoli and the interstitium of the thick interalveolar septa. On the 7th day, and onward, the incidence of apoptotic cells decreased markedly. This study concluded that the lung development included two phases: the first phase (from birth to the 14th days) corresponds to the period of bulk alveolarization and microvascular maturation. The second phase (from the 14th days to the full maturity) corresponds to the lung growth and late alveolarization.

scholarly journals Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures

2008 ◽  
Vol 295 (1) ◽  
pp. L194-L200 ◽  
Author(s):  
Ashwini S. Sabnis ◽  
Christopher A. Reilly ◽  
John M. Veranth ◽  
Garold S. Yost
Keyword(s):  
Epithelial Cells ◽  
Human Lung ◽  
External Environment ◽  
Lung Epithelial Cells ◽  
Critical Element ◽  
Cold Temperatures ◽  
Cold Air ◽  
Lung Epithelial ◽  
Human Airways ◽  
Human Lung Epithelial Cells

Recognition of temperature is a critical element of sensory perception and allows mammals to evaluate both their external environment and internal status. The respiratory epithelium is constantly exposed to the external environment, and prolonged inhalation of cold air is detrimental to human airways. However, the mechanisms responsible for adverse effects elicited by cold air on the human airways are poorly understood. Transient receptor potential melastatin family member 8 (TRPM8) is a well-established cold- and menthol-sensing cation channel. We recently discovered a functional cold- and menthol-sensing variant of the TRPM8 ion channel in human lung epithelial cells. The present study explores the hypothesis that this TRPM8 variant mediates airway cell inflammatory responses elicited by cold air/temperatures. Here, we show that activation of the TRPM8 variant in human lung epithelial cells leads to increased expression of several cytokine and chemokine genes, including IL-1α, -1β, -4, -6, -8, and -13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α. Our results provide new insights into mechanisms that potentially control airway inflammation due to inhalation of cold air and suggest a possible role for the TRPM8 variant in the pathophysiology of asthma.

scholarly journals Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells.

1996 ◽  
Vol 44 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
T Sugiyama ◽  
M Yamamoto-Hino ◽  
K Wasano ◽  
K Mikoshiba ◽  
M Hasegawa
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Specific Cell ◽  
Airway Epithelial ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Inositol 1,4,5 Trisphosphate

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphate receptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonal antibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epithelium from trachea to distal intrapulmonary airways (bronchioles) showed positive immunoreactivity for all types of IP3R. However, cell type as well as subcellular site immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in the apical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3R Type 2 was exclusively localized to the entire cytoplasm of ciliated cells from the trachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclear cytoplasm not only of ciliated cells at all airway levels but also in Clara cells of the bronchiolar epithelium. Double fluorescent staining using combinations of KM1083 and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibody confirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Clara cells. These results indicate that the expression of three types of IP3Rs in different cell types and subcellular sites may reflect diverse physiological functions of IP3Rs within airway epithelial cells. The double staining studies suggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specific cell marker for ciliated cells of the airway epithelium.

Brief perinatal hypoxia impairs postnatal development of the bronchiolar epithelium

1989 ◽  
Vol 257 (2) ◽  
pp. L80-L85 ◽  
Author(s):  
G. D. Massaro ◽  
J. Olivier ◽  
D. Massaro
Keyword(s):  
Secretory Granules ◽  
Volume Density ◽  
Clara Cell ◽  
Lower Percentage ◽  
Numerical Density ◽  
Perinatal Hypoxia ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Pregnant Rats ◽  
Bronchiolar Epithelium

We placed pregnant rats in 10% O2 on the last day of gestation for less than or equal to 9 h plus 1-2 h (with their pups) after the onset of delivery. In the pups this brief perinatal hypoxia led to an altered cellular composition of the bronchiolar epithelium that persisted at least to age 30 days; it was characterized by a higher nuclear numerical density (Nvn) of Clara cells, a lower Nvn of ciliated cells, and a lower percentage and Nvn of Clara cells in mitoses compared with control rats. The perinatal hypoxia also led to a significantly lower volume and volume density of the secretory apparatus (rough endoplasmic reticulum and secretory granules) on day 7 in 10% O2-born rats. The data on the Nvn of Clara and ciliated cells and on Clara cell mitoses are consistent with the notion that exposure to 10% O2 impaired the differentiation of Clara cells into ciliated cells and this impairment persisted well beyond the period of exposure.

A ‘tad’ of hope in the fight against airway disease

2020 ◽  
Vol 48 (5) ◽  
pp. 2347-2357
Author(s):  
Eamon Dubaissi
Keyword(s):  
Cell Types ◽  
Airway Disease ◽  
Live Imaging ◽  
Model System ◽  
In Vivo Model ◽  
Large Airways ◽  
Human Airways ◽  
Tadpole Skin ◽  
Mucociliary Epithelia

Xenopus tadpoles have emerged as a powerful in vivo model system to study mucociliary epithelia such as those found in the human airways. The tadpole skin has mucin-secreting cells, motile multi-ciliated cells, ionocytes (control local ionic homeostasis) and basal stem cells. This cellular architecture is very similar to the large airways of the human lungs and represents an easily accessible and experimentally tractable model system to explore the molecular details of mucociliary epithelia. Each of the cell types in the tadpole skin has a human equivalent and a conserved network of genes and signalling pathways for their differentiation has been discovered. Great insight into the function of each of the cell types has been achieved using the Xenopus model and this has enhanced our understanding of airway disease. This simple model has already had a profound impact on the field but, as molecular technologies (e.g. gene editing and live imaging) continue to develop apace, its use for understanding individual cell types and their interactions will likely increase. For example, its small size and genetic tractability make it an ideal model for live imaging of a mucociliary surface especially during environmental challenges such as infection. Further potential exists for the mimicking of human genetic mutations that directly cause airway disease and for the pre-screening of drugs against novel therapeutic targets.

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