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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 induces disruption of tight junctions in renal tubular cells.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ The localization of Claudin-2 or ZO-1 was analyzed by immunofluorescence. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Red, Claudin-2; green, ZO-1; blue, DAPI-nuclei. Bars indicate 20 μm. B/ The level of mRNA encoding for Pard3, Pard6, aPKC and Cdc42 was quantified by real-time PCR and results are expressed as the fold induction compared to static condition. Data represent mean ± SEM of 4–6 experiments. *p<0.01 versus FSS 0.
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pone.0131416.g001: FSS induces disruption of tight junctions in renal tubular cells.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ The localization of Claudin-2 or ZO-1 was analyzed by immunofluorescence. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Red, Claudin-2; green, ZO-1; blue, DAPI-nuclei. Bars indicate 20 μm. B/ The level of mRNA encoding for Pard3, Pard6, aPKC and Cdc42 was quantified by real-time PCR and results are expressed as the fold induction compared to static condition. Data represent mean ± SEM of 4–6 experiments. *p<0.01 versus FSS 0.

Mentions: First, we examined the effect of FSS on tight and adherens junctions in tubular cells. For this, HK-2 tubular proximal cells were exposed to chronic and high FSS (0.5 Pa for 48h, see materials and methods) and were compared to cells maintained under static conditions. The organization of the tight junctions was evaluated by immunocytochemical analysis of the cellular distribution of two major proteins, Claudin-2 and ZO-1 (Zonula occludens-1). In static conditions (FSS 0), Claudin-2 was detected in both plasma membrane and cytoplasm (as dot-like structures) while ZO-1 was only localized at the plasma membrane majorly as a continuous belt (Fig 1A). FSS caused the disappearance of Claudin-2 and changes the staining of ZO-1, which remained at the plasma membrane but becomes punctuated and discontinuous (Fig 1A). These modifications were concomitant with downregulation of protein expression, as revealed by Western blot analysis (S1 Fig). These results indicate that FSS induces disruption of tight junctions in tubular cells. The integrity of Par complex, that plays a critical role in tight junction formation and epithelial polarization [36, 37], was also investigated by measuring expression of Pard6 (Partitioning defective-6), Pard3 (Partitioning defective-3), aPKC (atypical protein kinase C) and Cdc42 (cell division cycle 42). When HK-2 cells were subjected to FSS, no change in mRNA levels of Pard3, aPKC and Cdc42 was detected (Fig 1B). However Pard6 mRNA expression was strongly reduced (Fig 1B), suggesting that FSS induces disruption of the Par complex thereby strengthening the demonstration that FSS leads to tight junction alteration.


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 induces disruption of tight junctions in renal tubular cells.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ The localization of Claudin-2 or ZO-1 was analyzed by immunofluorescence. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Red, Claudin-2; green, ZO-1; blue, DAPI-nuclei. Bars indicate 20 μm. B/ The level of mRNA encoding for Pard3, Pard6, aPKC and Cdc42 was quantified by real-time PCR and results are expressed as the fold induction compared to static condition. Data represent mean ± SEM of 4–6 experiments. *p<0.01 versus FSS 0.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131416.g001: FSS induces disruption of tight junctions in renal tubular cells.Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ The localization of Claudin-2 or ZO-1 was analyzed by immunofluorescence. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Red, Claudin-2; green, ZO-1; blue, DAPI-nuclei. Bars indicate 20 μm. B/ The level of mRNA encoding for Pard3, Pard6, aPKC and Cdc42 was quantified by real-time PCR and results are expressed as the fold induction compared to static condition. Data represent mean ± SEM of 4–6 experiments. *p<0.01 versus FSS 0.
Mentions: First, we examined the effect of FSS on tight and adherens junctions in tubular cells. For this, HK-2 tubular proximal cells were exposed to chronic and high FSS (0.5 Pa for 48h, see materials and methods) and were compared to cells maintained under static conditions. The organization of the tight junctions was evaluated by immunocytochemical analysis of the cellular distribution of two major proteins, Claudin-2 and ZO-1 (Zonula occludens-1). In static conditions (FSS 0), Claudin-2 was detected in both plasma membrane and cytoplasm (as dot-like structures) while ZO-1 was only localized at the plasma membrane majorly as a continuous belt (Fig 1A). FSS caused the disappearance of Claudin-2 and changes the staining of ZO-1, which remained at the plasma membrane but becomes punctuated and discontinuous (Fig 1A). These modifications were concomitant with downregulation of protein expression, as revealed by Western blot analysis (S1 Fig). These results indicate that FSS induces disruption of tight junctions in tubular cells. The integrity of Par complex, that plays a critical role in tight junction formation and epithelial polarization [36, 37], was also investigated by measuring expression of Pard6 (Partitioning defective-6), Pard3 (Partitioning defective-3), aPKC (atypical protein kinase C) and Cdc42 (cell division cycle 42). When HK-2 cells were subjected to FSS, no change in mRNA levels of Pard3, aPKC and Cdc42 was detected (Fig 1B). However Pard6 mRNA expression was strongly reduced (Fig 1B), suggesting that FSS induces disruption of the Par complex thereby strengthening the demonstration that FSS leads to tight junction alteration.

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