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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

Alteration of adherens junctions in response to FSS.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Immunofluorescence detection of β-Catenin. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Green, β-Catenin; blue, DAPI-nuclei. Bar indicates 20 μm. B/ Real-time PCR and Western blot analysis of E-Cadherin mRNA and protein, respectively. C/ Real-time PCR was used for evaluation of mRNA levels encoding Snail1 or Snail2. In B and C, results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4–7 experiments. *p<0.05, **p<0.01 versus FSS 0.
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pone.0131416.g002: Alteration of adherens junctions in response to FSS.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Immunofluorescence detection of β-Catenin. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Green, β-Catenin; blue, DAPI-nuclei. Bar indicates 20 μm. B/ Real-time PCR and Western blot analysis of E-Cadherin mRNA and protein, respectively. C/ Real-time PCR was used for evaluation of mRNA levels encoding Snail1 or Snail2. In B and C, results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4–7 experiments. *p<0.05, **p<0.01 versus FSS 0.

Mentions: The effect of FSS on adherens complexes was studied by analyzing by immunocytochemistry the localization of β-Catenin and by quantifying the expression of E-Cadherin as well as two of its repressor transcription factors, Snail1 and Snail2. In response to FSS, although staining is still seen predominantly in the periphery of the cell, β-catenin distribution was disrupted, with a more jagged and discontinuous pattern (Fig 2A). This effect was not accompanied with significant change in β-Catenin protein expression (S1 Fig). In parallel, mRNA and protein E-Cadherin expression was significantly reduced (Fig 2B) whereas the level of mRNA encoding Snail1 and Snail2 was significantly increased (Fig 2C). These results indicate that FSS disturbs adherens junctions of tubular cells.


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)

Alteration of adherens junctions in response to FSS.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Immunofluorescence detection of β-Catenin. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Green, β-Catenin; blue, DAPI-nuclei. Bar indicates 20 μm. B/ Real-time PCR and Western blot analysis of E-Cadherin mRNA and protein, respectively. C/ Real-time PCR was used for evaluation of mRNA levels encoding Snail1 or Snail2. In B and C, results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4–7 experiments. *p<0.05, **p<0.01 versus FSS 0.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493045&req=5

pone.0131416.g002: Alteration of adherens junctions in response to FSS.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Immunofluorescence detection of β-Catenin. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Green, β-Catenin; blue, DAPI-nuclei. Bar indicates 20 μm. B/ Real-time PCR and Western blot analysis of E-Cadherin mRNA and protein, respectively. C/ Real-time PCR was used for evaluation of mRNA levels encoding Snail1 or Snail2. In B and C, results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4–7 experiments. *p<0.05, **p<0.01 versus FSS 0.
Mentions: The effect of FSS on adherens complexes was studied by analyzing by immunocytochemistry the localization of β-Catenin and by quantifying the expression of E-Cadherin as well as two of its repressor transcription factors, Snail1 and Snail2. In response to FSS, although staining is still seen predominantly in the periphery of the cell, β-catenin distribution was disrupted, with a more jagged and discontinuous pattern (Fig 2A). This effect was not accompanied with significant change in β-Catenin protein expression (S1 Fig). In parallel, mRNA and protein E-Cadherin expression was significantly reduced (Fig 2B) whereas the level of mRNA encoding Snail1 and Snail2 was significantly increased (Fig 2C). These results indicate that FSS disturbs adherens junctions of tubular cells.

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