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      Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells

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          Abstract

          Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

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          Most cited references63

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          The tight junction: a multifunctional complex.

          Multicellular organisms are separated from the external environment by a layer of epithelial cells whose integrity is maintained by intercellular junctional complexes composed of tight junctions, adherens junctions, and desmosomes, whereas gap junctions provide for intercellular communication. The aim of this review is to present an updated overview of recent developments in the area of tight junction biology. In a relatively short time, our knowledge of the tight junction has evolved from a relatively simple view of it being a permeability barrier in the paracellular space and a fence in the plane of the plasma membrane to one of it acting as a multicomponent, multifunctional complex that is involved in regulating numerous and diverse cell functions. A group of integral membrane proteins-occludin, claudins, and junction adhesion molecules-interact with an increasingly complex array of tight junction plaque proteins not only to regulate paracellular solute and water flux but also to integrate such diverse processes as gene transcription, tumor suppression, cell proliferation, and cell polarity.
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            Adherens junction: molecular architecture and regulation.

            The adherens junction (AJ) is an element of the cell-cell junction in which cadherin receptors bridge the neighboring plasma membranes via their homophilic interactions. Cadherins associate with cytoplasmic proteins, called catenins, which in turn bind to cytoskeletal components, such as actin filaments and microtubules. These molecular complexes further interact with other proteins, including signaling molecules, rendering the AJs into highly dynamic and regulatable structures. The AJs of such nature contribute to the physical linking of cells, as well as to the regulation of cell-cell contacts, which is essential for morphogenesis and remodeling of tissues and organs. Thus, elucidating the molecular architecture of the AJs and their regulatory mechanisms are crucial for understanding how the multicellular system is organized.
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              A multi-layer microfluidic device for efficient culture and analysis of renal tubular cells.

              We have developed a simple multi-layer microfluidic device by integrating a polydimethyl siloxane (PDMS) microfluidic channel and a porous membrane substrate to culture and analyze the renal tubular cells. As a model cell type, primary rat inner medullary collecting duct (IMCD) cells were cultured inside the channel. To generate in vivo-like tubular environments for the cells, a fluidic shear stress of 1 dyn/cm(2) was applied for 5 hours, allowing for optimal fluidic conditions for the cultured cells, as verified by enhanced cell polarization, cytoskeletal reorganization, and molecular transport by hormonal stimulations. These results suggest that the microfluidic device presented here is useful for resembling an in vivo renal tubule system and has potential applications in drug screening and advanced tissue engineering.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 July 2015
                2015
                : 10
                : 7
                : e0131416
                Affiliations
                [1 ]Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Toulouse, France
                [2 ]Université Toulouse III Paul Sabatier, Institute of Metabolic and Cardiovascular Diseases - I2MC, Toulouse, France
                National Cancer Institute, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: DM RD JLB JPS BBM. Performed the experiments: DM RD MB LD SG BBM. Analyzed the data: DM RD MB JSSB BBM. Contributed reagents/materials/analysis tools: AC JB CC. Wrote the paper: JSSB JLB JPS BBM.

                Article
                PONE-D-15-00353
                10.1371/journal.pone.0131416
                4493045
                26146837
                3e067d24-82b9-4631-b2f0-ff3e0fa22c00
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 5 January 2015
                : 2 June 2015
                Page count
                Figures: 7, Tables: 1, Pages: 21
                Funding
                The authors thank the association 111 des Arts (France) for funding. This funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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