Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY418)-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY418-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α2β1-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.

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A role for collecting duct epithelial cells in renal antibacterial defences

Cellular Microbiology ◽

10.1111/j.1462-5822.2011.01614.x ◽

2011 ◽

Vol 13 (8) ◽

pp. 1107-1113 ◽

Cited By ~ 20

Author(s):

Cecilia Chassin ◽

Emilie Tourneur ◽

Marcelle Bens ◽

Alain Vandewalle

Keyword(s):

Epithelial Cells ◽

Collecting Duct

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Localization of phosphatidylinositol phosphate kinase IIγ in kidney to a membrane trafficking compartment within specialized cells of the nephron

AJP Renal Physiology ◽

10.1152/ajprenal.90310.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. F1422-F1430 ◽

Cited By ~ 57

Author(s):

Jonathan H. Clarke ◽

Piers C. Emson ◽

Robin F. Irvine

Keyword(s):

Epithelial Cells ◽

Membrane Trafficking ◽

Kidney Cell ◽

Collecting Duct ◽

Thick Ascending Limb ◽

Phosphatidylinositol Phosphate ◽

The Matrix ◽

Phosphatidylinositol 4

PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kγ. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kα and PIP4Kβ, PIP4Kγ is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kγ, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kγ had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kα, bacterially expressed recombinant PIP4Kγ was completely inactive but did have the ability to associate with active PIP4Kα in vitro. Overall our data suggest that PIP4Kγ may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.

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Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ

AJP Cell Physiology ◽

10.1152/ajpcell.00250.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. C768-C778 ◽

Cited By ~ 31

Author(s):

Jaafar El Annan ◽

Dennis Brown ◽

Sylvie Breton ◽

Sylvain Bourgoin ◽

Dennis A. Ausiello ◽

...

Keyword(s):

Epithelial Cells ◽

Western Blotting ◽

Collecting Duct ◽

Small Gtpases ◽

Renal Physiology ◽

Principal Cells ◽

Arf Gtpases ◽

Kidney Epithelial Cells

ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26–180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.

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Expression of mRNA for natriuretic peptide receptor subtypes in bovine kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.2.f318 ◽

1994 ◽

Vol 267 (2) ◽

pp. F318-F324 ◽

Cited By ~ 3

Author(s):

T. Yamamoto ◽

L. Feng ◽

T. Mizuno ◽

S. Hirose ◽

K. Kawasaki ◽

...

Keyword(s):

Epithelial Cells ◽

Natriuretic Peptide ◽

Collecting Duct ◽

Biologically Active ◽

Ribonuclease Protection Assay ◽

Receptor Subtypes ◽

Total Rna ◽

Duct Cells ◽

Glomerular Epithelial Cells ◽

Collecting Duct Cells

The localization of mRNA for atrial natriuretic peptide (ANP) receptor subtypes (A, B, C) in the kidney was examined. Quantitative analysis of the ribonuclease protection assay showed that the numbers of type A receptor (ANPRA) mRNA were 6.9 x 10(7) in the glomeruli and 10.4 x 10(7) molecules/micrograms of total RNA in the inner medulla, and that of type C receptor (ANPRC) mRNA was 21.7 x 10(7) molecules/micrograms of total RNA in the glomeruli. The type B receptor (ANPRB) mRNA was present in smaller numbers (4.5-4.9 x 10(6) molecules/micrograms of total RNA) evenly throughout the kidney fractions. In situ hybridization demonstrated both ANPRA and ANPRC mRNA selectively in the glomerular epithelial cells and ANPRA mRNA in the collecting duct cells of the inner medulla. ANPRC was also localized on the foot processes of glomerular epithelial cells by immunohistochemistry using a specific antibody against the receptor. These results indicate that ANPRA is the major biologically active receptor for the ANP family of hormones in the kidney and is present selectively on the glomerular epithelial cells and inner medullary collecting duct cells. These cells are presumed to play a role in the regulation of glomerular filtration rate and sodium excretion induced by the family of ANP.

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Cell-surface translocation of annexin A2 contributes to bleomycin-induced pulmonary fibrosis by mediating inflammatory response in mice

Clinical Science ◽

10.1042/cs20180687 ◽

2019 ◽

Vol 133 (7) ◽

pp. 789-804 ◽

Cited By ~ 4

Author(s):

Yunlong Lei ◽

Kui Wang ◽

Xuefeng Li ◽

Yi Li ◽

Xuping Feng ◽

...

Keyword(s):

Epithelial Cells ◽

Pulmonary Fibrosis ◽

Inflammatory Response ◽

Cell Surface ◽

Annexin A2 ◽

Lung Epithelial Cells ◽

Cancer Drug ◽

Autophagic Flux ◽

Lung Epithelial ◽

Surface Translocation

Abstract Bleomycin, a widely used anti-cancer drug, may give rise to pulmonary fibrosis, a serious side effect which is associated with significant morbidity and mortality. Despite the intensive efforts, the precise pathogenic mechanisms of pulmonary fibrosis still remain to be clarified. Our previous study showed that bleomycin bound directly to annexin A2 (ANXA2, or p36), leading to development of pulmonary fibrosis by impeding transcription factor EB (TFEB)-induced autophagic flux. Here, we demonstrated that ANXA2 also played a critical role in bleomycin-induced inflammation, which represents another major cause of bleomycin-induced pulmonary fibrosis. We found that bleomycin could induce the cell surface translocation of ANXA2 in lung epithelial cells through exosomal secretion, associated with enhanced interaction between ANXA2 and p11. Knockdown of ANXA2 or blocking membrane ANXA2 mitigated bleomycin-induced activation of nuclear factor (NF)-κB pathway and production of pro-inflammatory cytokine IL-6 in lung epithelial cells. ANXA2-deficient (ANXA2−/−) mice treated with bleomycin exhibit reduced pulmonary fibrosis along with decreased cytokine production compared with bleomycin-challenged wild-type mice. Further, the surface ANXA2 inhibitor TM601 could ameliorate fibrotic and inflammatory response in bleomycin-treated mice. Taken together, our results indicated that, in addition to disturbing autophagic flux, ANXA2 can contribute to bleomycin-induced pulmonary fibrosis by mediating inflammatory response.

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Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

AJP Cell Physiology ◽

10.1152/ajpcell.00300.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. C27-C45 ◽

Cited By ~ 9

Author(s):

Boyang Zhao ◽

Mark A. Knepper ◽

Chung-Lin Chou ◽

Trairak Pisitkun

Keyword(s):

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Metal Ion ◽

Large Scale ◽

Collecting Duct ◽

Affinity Purification ◽

Large Fraction ◽

Proteomic Profiling ◽

Data Set ◽

Polarity Determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/ . Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions (“adherens junction,” “tight junction,” and “focal adhesion”) and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.

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