scholarly journals A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ce Zhang ◽  
Yue Chen ◽  
Shijin Sun ◽  
Yikai Zhang ◽  
Lina Wang ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Function ◽  
Mdck Cells ◽  
Ldl Receptor ◽  
Membrane Targeting ◽  
Protein Distribution ◽  
Recognition Mechanism ◽  
Renal Epithelial Cells

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin–Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon

2005 ◽  
Vol 288 (3) ◽  
pp. C624-C632 ◽  
Author(s):  
Samuel C. Wolff ◽  
Ai-Dong Qi ◽  
T. Kendall Harden ◽  
Robert A. Nicholas
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Confocal Microscopy ◽  
Cell Function ◽  
Mdck Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Receptor Subtypes

Eight human G protein-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current ( Isc) or Ca2+ mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y1, P2Y11, P2Y12, and P2Y14 receptors reside at the basolateral membrane, P2Y2, P2Y4, and P2Y6 receptors are expressed at the apical membrane, and the P2Y13 receptor is unsorted. Biotinylation studies and Isc measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the Gq-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) in lung and colonic epithelial cells (16HBE14o− and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells.

Differential localization of human nongastric H+-K+-ATPase ATP1AL1 in polarized renal epithelial cells

2000 ◽  
Vol 279 (3) ◽  
pp. F417-F425 ◽  
Author(s):  
Jürgen Reinhardt ◽  
Alexander V. Grishin ◽  
Hans Oberleithner ◽  
Michael J. Caplan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Ion Pump ◽  
Β Subunit ◽  
Ion Pumps ◽  
Α Subunit ◽  
Renal Epithelial Cells

The human H+-K+-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K+-transporting ATPases. In concert with the structurally related gastric H+-K+-ATPase, it plays a major role in K+ reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK1 renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na+-K+-ATPase β-subunit or the gastric H+-K+-ATPase β-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the α-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na+-K+-ATPase β-subunit. Cotransfection of ATP1AL1 with the gastric H+-K+-ATPase β-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by 86Rb+uptake measurements. In contrast, cotransfected LLC-PK1cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the α-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Polycystin-1 and Gα12 regulate the cleavage of E-cadherin in kidney epithelial cells

2015 ◽  
Vol 47 (2) ◽  
pp. 24-32 ◽  
Author(s):  
Jen X. Xu ◽  
Tzong-Shi Lu ◽  
Suyan Li ◽  
Yong Wu ◽  
Lai Ding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signaling Pathway ◽  
Cell Polarity ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Renal Epithelial Cells ◽  
Extracellular Milieu ◽  
Kidney Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney ◽  
E Cadherin

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused β-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023 X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/β-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD.

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.

Metabolic inhibition-induced transient Ca2+ increase depends on mitochondria in a human proximal renal cell line

2007 ◽  
Vol 293 (2) ◽  
pp. F533-F540 ◽  
Author(s):  
Adrian Caplanusi ◽  
Andrew J. Fuller ◽  
Romer A. Gonzalez-Villalobos ◽  
Timothy G. Hammond ◽  
L. G. Navar
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Fold Increase ◽  
Recovery Phase ◽  
Metabolic Inhibition ◽  
Human Origin ◽  
Renal Epithelial Cells ◽  
Proximal Tubular ◽  
Renal Cell Line ◽  
Signaling Process

During ischemia or hypoxia an increase in intracellular cytosolic Ca2+ induces deleterious events but is also implicated in signaling processes triggered in such conditions. In MDCK cells (distal tubular origin), it was shown that mitochondria confer protection during metabolic inhibition (MI), by buffering the Ca2+ overload via mitochondrial Na+-Ca2+ exchanger (NCX). To further assess this process in cells of human origin, human cortical renal epithelial cells (proximal tubular origin) were subjected to MI and changes in cytosolic Ca2+ ([Ca2+]i), Na+, and ATP concentrations were monitored. MI was accomplished with both antimycin A and 2-deoxyglucose and induced a 3.5-fold increase in [Ca2+]i, reaching 136.5 ± 15.8 nM in the first 3.45 min. Subsequently [Ca2+]i dropped and stabilized to 62.7 ± 7.3 nM by 30 min. The first phase of the transient increase was La3+ sensitive, not influenced by diltiazem, and abolished when mitochondria were deenergized with the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The subsequent recovery phase was impaired in a Na+-free medium and weakened when the mitochondrial NCX was blocked with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Thus Ca2+ entry is likely mediated by store-operated Ca2+ channels and depends on energized mitochondria, whereas [Ca2+]i recovery relied partially on the activity of mitochondrial NCX. These results indicate a possible mitochondrial-mediated signaling process triggered by MI, support the hypothesis that mitochondrial NCX has an important role in the Ca2+ clearance, and overall suggest that mitochondria play a preponderant role in the regulation of responses to MI in human renal epithelial cells.

scholarly journals Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro

Reproduction ◽  
2001 ◽  
pp. 305-315 ◽  
Author(s):  
CE Green ◽  
J Bredl ◽  
WV Holt ◽  
PF Watson ◽  
A Fazeli
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Mammalian Species ◽  
Free Cell ◽  
Carbohydrate Recognition ◽  
Sperm Binding ◽  
Oviductal Epithelium ◽  
Species Specific

After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l(-1). A significant (P < or = 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l(-1), 50 mmol lactose l(-1) and 50 mmol mannose l(-1). These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition.

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