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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 uninephrectomy-mediated FSS on epithelial gene expression and the density of primary cilia.Sham- and UNx-mice were analyzed 8 months after surgery. A/ The expression of ZO-1, E-cadherin and β-Catenin mRNA was quantified by real-time PCR from total RNA extracted from kidney cortex. Results are expressed as the fold induction compared to sham. B/ Immunofluorescence detection of α-acetylated Tubulin for quantification of the primary cilium. Kidney sections were counterstained with WGA and DAPI. Pictures in the left panel display representative areas of staining. Red, α-acetylated Tubulin; green, WGA-cell membranes; blue, DAPI-nuclei. Bar indicates 20 μm and white arrows show primary cilia. Graph in the right panel displays quantification of primary cilia by cortex tubular section. Data represent mean ± SEM from 6 animals per group. *p<0.05 versus sham.
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pone.0131416.g007: Effect of uninephrectomy-mediated FSS on epithelial gene expression and the density of primary cilia.Sham- and UNx-mice were analyzed 8 months after surgery. A/ The expression of ZO-1, E-cadherin and β-Catenin mRNA was quantified by real-time PCR from total RNA extracted from kidney cortex. Results are expressed as the fold induction compared to sham. B/ Immunofluorescence detection of α-acetylated Tubulin for quantification of the primary cilium. Kidney sections were counterstained with WGA and DAPI. Pictures in the left panel display representative areas of staining. Red, α-acetylated Tubulin; green, WGA-cell membranes; blue, DAPI-nuclei. Bar indicates 20 μm and white arrows show primary cilia. Graph in the right panel displays quantification of primary cilia by cortex tubular section. Data represent mean ± SEM from 6 animals per group. *p<0.05 versus sham.

Mentions: Finally, we evaluated in vivo the effect of increased urinary FSS. We used an animal model where increased FSS was induced in proximal tubule by increased urinary flow following hyperfiltration. For this, C57BL/6 mice were uninephrectomized (UNx) by removing the right kidney. The left kidney was harvested 8 months later to analyse tubular epithelial markers. As expected [22–24], total GFR was maintained within the normal range in UNx subjected animals through adaptive increased single kidney (sk) GFR (Fig 6A), thereby leading to increased urinary FSS in remnant nephrons. In addition, elevated skGFR was accompanied by a significant glomerular hypertrophy, as indicated by increase of the renal corpuscule area (Fig 6B), thereby confirming the compensatory hyperfiltration. Urine albumin excretion was not significantly modified (Fig 6A) and tubular dilatation was not detected (Fig 6B). However the mRNA level of epithelial makers ZO-1, E-Cadherin and β-Catenin was significantly decreased in UNx animals compared to sham (Fig 7A). In addition, a decreased number of primary cilia in tubular cells was detected (Fig 7B). Taking into account the observations in vitro, these data suggest that increased FSS in vivo is associated, as well, with a reduction of expression of epithelial markers.


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 uninephrectomy-mediated FSS on epithelial gene expression and the density of primary cilia.Sham- and UNx-mice were analyzed 8 months after surgery. A/ The expression of ZO-1, E-cadherin and β-Catenin mRNA was quantified by real-time PCR from total RNA extracted from kidney cortex. Results are expressed as the fold induction compared to sham. B/ Immunofluorescence detection of α-acetylated Tubulin for quantification of the primary cilium. Kidney sections were counterstained with WGA and DAPI. Pictures in the left panel display representative areas of staining. Red, α-acetylated Tubulin; green, WGA-cell membranes; blue, DAPI-nuclei. Bar indicates 20 μm and white arrows show primary cilia. Graph in the right panel displays quantification of primary cilia by cortex tubular section. Data represent mean ± SEM from 6 animals per group. *p<0.05 versus sham.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131416.g007: Effect of uninephrectomy-mediated FSS on epithelial gene expression and the density of primary cilia.Sham- and UNx-mice were analyzed 8 months after surgery. A/ The expression of ZO-1, E-cadherin and β-Catenin mRNA was quantified by real-time PCR from total RNA extracted from kidney cortex. Results are expressed as the fold induction compared to sham. B/ Immunofluorescence detection of α-acetylated Tubulin for quantification of the primary cilium. Kidney sections were counterstained with WGA and DAPI. Pictures in the left panel display representative areas of staining. Red, α-acetylated Tubulin; green, WGA-cell membranes; blue, DAPI-nuclei. Bar indicates 20 μm and white arrows show primary cilia. Graph in the right panel displays quantification of primary cilia by cortex tubular section. Data represent mean ± SEM from 6 animals per group. *p<0.05 versus sham.
Mentions: Finally, we evaluated in vivo the effect of increased urinary FSS. We used an animal model where increased FSS was induced in proximal tubule by increased urinary flow following hyperfiltration. For this, C57BL/6 mice were uninephrectomized (UNx) by removing the right kidney. The left kidney was harvested 8 months later to analyse tubular epithelial markers. As expected [22–24], total GFR was maintained within the normal range in UNx subjected animals through adaptive increased single kidney (sk) GFR (Fig 6A), thereby leading to increased urinary FSS in remnant nephrons. In addition, elevated skGFR was accompanied by a significant glomerular hypertrophy, as indicated by increase of the renal corpuscule area (Fig 6B), thereby confirming the compensatory hyperfiltration. Urine albumin excretion was not significantly modified (Fig 6A) and tubular dilatation was not detected (Fig 6B). However the mRNA level of epithelial makers ZO-1, E-Cadherin and β-Catenin was significantly decreased in UNx animals compared to sham (Fig 7A). In addition, a decreased number of primary cilia in tubular cells was detected (Fig 7B). Taking into account the observations in vitro, these data suggest that increased FSS in vivo is associated, as well, with a reduction of expression of epithelial markers.

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