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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

Effect of FSS on apoptosis and necrosis in 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/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments.
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pone.0131416.g004: Effect of FSS on apoptosis and necrosis in 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/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments.

Mentions: Tubular apoptosis and necrosis are exacerbated in CKD, thereby contributing to tubular atrophy [38]. In addition, our laboratory has previously shown that exposure of HK-2 cells to FSS causes hyper-secretion of TNF-α [15], known to induce apoptosis in these cells [39]. To test whether FSS-induced dedifferentiation was associated to tubular cell death, HK-2 cells exposed or not to FSS 0.5 Pa for 48h were double-labeled with annexin V and propidium iodide. Analysis was performed by flow cytometry to separate live cells (non-labeled), cells in early phase of apoptosis (annexin V-positive, negative for propidium iodide) and necrotic cells (post-apoptotic or not, double positive). As shown in Fig 4, no change in the proportions of the different cell populations was observed between FSS 0 and FSS 0.5 Pa, thereby indicating that chronic FSS does not cause apoptosis or necrosis of the 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)

Effect of FSS on apoptosis and necrosis in 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/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131416.g004: Effect of FSS on apoptosis and necrosis in 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/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments.
Mentions: Tubular apoptosis and necrosis are exacerbated in CKD, thereby contributing to tubular atrophy [38]. In addition, our laboratory has previously shown that exposure of HK-2 cells to FSS causes hyper-secretion of TNF-α [15], known to induce apoptosis in these cells [39]. To test whether FSS-induced dedifferentiation was associated to tubular cell death, HK-2 cells exposed or not to FSS 0.5 Pa for 48h were double-labeled with annexin V and propidium iodide. Analysis was performed by flow cytometry to separate live cells (non-labeled), cells in early phase of apoptosis (annexin V-positive, negative for propidium iodide) and necrotic cells (post-apoptotic or not, double positive). As shown in Fig 4, no change in the proportions of the different cell populations was observed between FSS 0 and FSS 0.5 Pa, thereby indicating that chronic FSS does not cause apoptosis or necrosis of the 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