Urea transport in MDCK cells that are stably transfected with UT-A1

2004 ◽  
Vol 286 (6) ◽  
pp. C1264-C1270 ◽  
Author(s):  
Otto Fröhlich ◽  
Janet D. Klein ◽  
Pauline M. Smith ◽  
Jeff M. Sands ◽  
Robert B. Gunn
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Arginine Vasopressin ◽  
Cell Biology ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Urea Transport ◽  
Urea Transporter ◽  
Collecting Ducts ◽  
Single Clone

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 Ω·cm2. Transepithelial [14C]urea fluxes were measured at 37°C in a HCO3−/CO2 buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [14C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts.

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

scholarly journals Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK).

1979 ◽  
Vol 81 (3) ◽  
pp. 635-648 ◽  
Author(s):  
M J Rindler ◽  
L M Chuman ◽  
L Shaffer ◽  
M H Saier
Keyword(s):  
Epithelial Cell ◽  
Adenylate Cyclase ◽  
Cell Line ◽  
Tight Junctions ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Morphological Properties ◽  
Epithelial Cell Line ◽  
Kidney Epithelial Cell ◽  
Epithelial Sheets

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.

scholarly journals Characterization of a Porcine Lung Epithelial Cell Line Suitable for Influenza Virus Studies

2001 ◽  
Vol 75 (19) ◽  
pp. 9517-9525 ◽  
Author(s):  
Sang Heui Seo ◽  
Olga Goloubeva ◽  
Richard Webby ◽  
Robert G. Webster
Keyword(s):  
Influenza Virus ◽  
Epithelial Cell ◽  
Cell Line ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Lung Epithelial Cell ◽  
Clinical Samples ◽  
Epithelial Cell Line ◽  
Porcine Endogenous Retrovirus ◽  
Lung Epithelial

ABSTRACT We established a porcine lung epithelial cell line designated St. Jude porcine lung cells (SJPL) and demonstrated that all tested influenza A and B viruses replicated in this cell line. The infectivity titers of most viruses in SJPL cells were comparable to or better than those in MDCK cells. The propagation of influenza viruses from clinical samples in SJPL cells did not lead to antigenic changes in the hemagglutinin molecule. The numbers of both Sia2-3Gal and Sia2-6Gal receptors on SJPL cells were greater than those on MDCK cells. Influenza virus infection of SJPL cells did not lead to apoptosis, as did infection of MDCK cells. No porcine endogenous retrovirus was detected in SJPL cells, and in contrast to MDCK cells, SJPL cells did not cause tumors in nude mice.

scholarly journals A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology

2016 ◽  
Vol 6 (3) ◽  
pp. 761-766 ◽  
Author(s):  
Bakiah Shaharuddin ◽  
Sajjad Ahmad ◽  
Nani Md Latar ◽  
Simi Ali ◽  
Annette Meeson
Keyword(s):  
Stem Cell ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Biology ◽  
Corneal Epithelial Cell ◽  
Epithelial Cell Line ◽  
Stem Cell Biology ◽  
Corneal Epithelial ◽  
Cell Line Model ◽  
Limbal Stem Cell

A human bronchial epithelial cell line releases arginine vasopressin: involvement of Ca2+-activated K+ channels

1998 ◽  
Vol 74 (2-3) ◽  
pp. 91-95 ◽  
Author(s):  
Jun Tamaoki ◽  
Kazuo Isono ◽  
Mitsuko Kondo ◽  
Isao Yamawaki ◽  
Etsuko Tagaya ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Arginine Vasopressin ◽  
Bronchial Epithelial Cell ◽  
Human Bronchial Epithelial Cell ◽  
Epithelial Cell Line ◽  
Bronchial Epithelial ◽  
K Channels

Gene expression alterations during HGF-induced dedifferentiation of a renal tubular epithelial cell line (MDCK) using a novel canine DNA microarray

2004 ◽  
Vol 286 (4) ◽  
pp. F702-F710 ◽  
Author(s):  
Daniel F. Balkovetz ◽  
Edward R. Gerrard ◽  
Shixiong Li ◽  
David Johnson ◽  
James Lee ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Dna Microarray ◽  
Mdck Cells ◽  
Biological Activities ◽  
Early Response ◽  
Tubular Epithelial Cell ◽  
Small Gtpases ◽  
Epithelial Cell Line ◽  
Genes Encoding

Hepatocyte growth factor (HGF) elicits a broad spectrum of biological activities, including epithelial cell dedifferentiation. One of the most widely used and best-studied polarized epithelial cell lines is the Madin-Darby canine kidney (MDCK) cell line. Here, we describe and validate the early response of polarized monolayers of MDCK cells stimulated with recombinant HGF using a novel canine DNA microarray designed to query 12,473 gene sequences. In our survey, eight genes previously implicated in the HGF signaling pathway were differentially regulated, demonstrating that the system was responsive to HGF. Also identified were 117 genes not previously known to be involved in the HGF pathway. The results were confirmed by real-time PCR or Western blot analysis for 38 genes. Of particular interest were the large number of differentially regulated genes encoding small GTPases, proteins involved in endoplasmic reticulum translation, proteins involved in the cytoskeleton, the extracellular matrix, and the hematopoietic and prostaglandin systems.

scholarly journals Characterization of a porcine intestinal epithelial cell line for influenza virus production

2012 ◽  
Vol 93 (9) ◽  
pp. 2008-2016 ◽  
Author(s):  
Zhi Sun ◽  
Victor C. Huber ◽  
Kara McCormick ◽  
Radhey S. Kaushik ◽  
Adrianus C. M. Boon ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Epithelial Cell Line ◽  
Influenza B ◽  
Specific Cell ◽  
Influenza A Viruses

We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log10 TCID50 ml−1. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin–Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 ml−1, respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.

scholarly journals Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line.

1990 ◽  
Vol 111 (4) ◽  
pp. 1351-1361 ◽  
Author(s):  
A Le Bivic ◽  
A Quaroni ◽  
B Nichols ◽  
E Rodriguez-Boulan
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cell

We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.

scholarly journals A simple epithelial cell line (MDCK) shows heterogeneity of desmoglein isoforms, one resembling pemphigus vulgaris antigen

1995 ◽  
Vol 108 (4) ◽  
pp. 1743-1750
Author(s):  
M.J. Vilela ◽  
T. Hashimoto ◽  
T. Nishikawa ◽  
A.J. North ◽  
D. Garrod
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Mdck Cells ◽  
Pemphigus Vulgaris ◽  
Epithelial Cell Line ◽  
Cell Type ◽  
Epithelial Cell Type ◽  
Blistering Disease ◽  
Circulating Autoantibodies ◽  
Canine Kidney

The epidermal blistering disease, pemphigus vulgaris (PV), is caused by circulating autoantibodies that react with a desmosomal glycoprotein desmoglein (Dsg3). This antigen is expressed only in stratified epithelial tissues. Here we show that the simple epithelial canine kidney cell line, MDCK, expresses at least two desmoglein isoforms recognised by different monoclonal antibodies. One of these isoforms is a 130 × 10(3) M(r) polypeptide that is recognised by both PV autoantisera and a monoclonal antibody reactive with a cytoplasmic domain of human Dsg3. Antibodies in PV sera bind to the surface of MDCK cells but not cause loss of intercellular adhesion. This is the first demonstration of the expression of a polypeptide related to human PV antigen by a simple epithelial cell type.

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