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

Maggiorani D, Dissard R, Belloy M, Saulnier-Blache JS, Casemayou A, Ducasse L, Grès S, Bellière J, Caubet C, Bascands JL, Schanstra JP, Buffin-Meyer B - PLoS ONE (2015)

Bottom Line: Expression of Pard6 was also decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Toulouse, France; Université Toulouse III Paul Sabatier, Institute of Metabolic and Cardiovascular Diseases - I2MC, Toulouse, France.

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 48 h. 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.

No MeSH data available.


Related in: MedlinePlus

FSS-exposed cells exhibit loss of the primary cilium without marked change in actin cytoskeleton organization.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ α-acetylated Tubulin was analyzed by immunofluorescence to visualize the primary cilium. White arrows show primary cilia. B/ Phalloidin was used to stain the actin cytoskeleton (basal [left] and subapical [right]). Cells were counterstained with DAPI. Pictures display representative areas of staining from 5 independent experiments. Green, α-acetylated Tubulin; red, Phalloidin; blue, DAPI-nuclei. Bars indicate 20 μm.
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pone.0131416.g003: FSS-exposed cells exhibit loss of the primary cilium without marked change in actin cytoskeleton organization.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ α-acetylated Tubulin was analyzed by immunofluorescence to visualize the primary cilium. White arrows show primary cilia. B/ Phalloidin was used to stain the actin cytoskeleton (basal [left] and subapical [right]). Cells were counterstained with DAPI. Pictures display representative areas of staining from 5 independent experiments. Green, α-acetylated Tubulin; red, Phalloidin; blue, DAPI-nuclei. Bars indicate 20 μm.

Mentions: Next, we examined whether FSS (0.5 Pa, 48h) induces loss of the primary cilium in tubular cells. Cilia were visualized by immunostaining with anti α-acetylated Tubulin antibody. As shown in Fig 3A, cilia were observed in HK-2 cells in static conditions and FSS induced disappearance of cilia, accompanied by relocation of α-acetylated Tubulin into the cytoplasm. Since intercellular junctions and the cilium basis interact with actin cytoskeleton, we also studied the effect of FSS on the organization of the actin cytoskeleton in HK-2 cells. For this, we used Phalloidin staining, which detects filamentous (F) actin, and Apotome-imaging microscopy, to sequentially analyze basal and apical sides. In static conditions, actin was found as numerous long and thick cytosolic stress fibers spanning the entire cross sectional area of the cells at the basal side while actin microfilaments are organized as a thin circumferential network at cell-cell contacts at the subapical side (Fig 3B). Under FSS 0.5 Pa (48h), no marked change in actin microfilament organization was observed (Fig 3B), suggesting that FSS-treated cells did not display marked F-actin rearrangement. These results indicate that the perturbation of intercellular junctions and the deciliation of tubular cells generated by FSS are independent from F-actin rearrangement.


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

Maggiorani D, Dissard R, Belloy M, Saulnier-Blache JS, Casemayou A, Ducasse L, Grès S, Bellière J, Caubet C, Bascands JL, Schanstra JP, Buffin-Meyer B - PLoS ONE (2015)

FSS-exposed cells exhibit loss of the primary cilium without marked change in actin cytoskeleton organization.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ α-acetylated Tubulin was analyzed by immunofluorescence to visualize the primary cilium. White arrows show primary cilia. B/ Phalloidin was used to stain the actin cytoskeleton (basal [left] and subapical [right]). Cells were counterstained with DAPI. Pictures display representative areas of staining from 5 independent experiments. Green, α-acetylated Tubulin; red, Phalloidin; blue, DAPI-nuclei. Bars indicate 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493045&req=5

pone.0131416.g003: FSS-exposed cells exhibit loss of the primary cilium without marked change in actin cytoskeleton organization.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ α-acetylated Tubulin was analyzed by immunofluorescence to visualize the primary cilium. White arrows show primary cilia. B/ Phalloidin was used to stain the actin cytoskeleton (basal [left] and subapical [right]). Cells were counterstained with DAPI. Pictures display representative areas of staining from 5 independent experiments. Green, α-acetylated Tubulin; red, Phalloidin; blue, DAPI-nuclei. Bars indicate 20 μm.
Mentions: Next, we examined whether FSS (0.5 Pa, 48h) induces loss of the primary cilium in tubular cells. Cilia were visualized by immunostaining with anti α-acetylated Tubulin antibody. As shown in Fig 3A, cilia were observed in HK-2 cells in static conditions and FSS induced disappearance of cilia, accompanied by relocation of α-acetylated Tubulin into the cytoplasm. Since intercellular junctions and the cilium basis interact with actin cytoskeleton, we also studied the effect of FSS on the organization of the actin cytoskeleton in HK-2 cells. For this, we used Phalloidin staining, which detects filamentous (F) actin, and Apotome-imaging microscopy, to sequentially analyze basal and apical sides. In static conditions, actin was found as numerous long and thick cytosolic stress fibers spanning the entire cross sectional area of the cells at the basal side while actin microfilaments are organized as a thin circumferential network at cell-cell contacts at the subapical side (Fig 3B). Under FSS 0.5 Pa (48h), no marked change in actin microfilament organization was observed (Fig 3B), suggesting that FSS-treated cells did not display marked F-actin rearrangement. These results indicate that the perturbation of intercellular junctions and the deciliation of tubular cells generated by FSS are independent from F-actin rearrangement.

Bottom Line: Expression of Pard6 was also decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Toulouse, France; Université Toulouse III Paul Sabatier, Institute of Metabolic and Cardiovascular Diseases - I2MC, Toulouse, France.

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 48 h. 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.

No MeSH data available.


Related in: MedlinePlus