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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

Uninephrectomy as an animal model of increased urinary FSS.Sham- and UNx-mice were analyzed 8 months after surgery. A/ Renal function was evaluated by measuring glomerular filtration rate (GFR), single kidney GFR (skGFR) and urinary albumin/creatinine ratio (UACR). B/ Renal corpuscule surface was measured on PAS-stained kidney slices. Pictures display representative areas of staining and bars indicate 200 μm. Data represent mean ± SEM from 6 animals per group. *p<0.05, ***p<0.01 versus sham.
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pone.0131416.g006: Uninephrectomy as an animal model of increased urinary FSS.Sham- and UNx-mice were analyzed 8 months after surgery. A/ Renal function was evaluated by measuring glomerular filtration rate (GFR), single kidney GFR (skGFR) and urinary albumin/creatinine ratio (UACR). B/ Renal corpuscule surface was measured on PAS-stained kidney slices. Pictures display representative areas of staining and bars indicate 200 μm. Data represent mean ± SEM from 6 animals per group. *p<0.05, ***p<0.01 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)

Uninephrectomy as an animal model of increased urinary FSS.Sham- and UNx-mice were analyzed 8 months after surgery. A/ Renal function was evaluated by measuring glomerular filtration rate (GFR), single kidney GFR (skGFR) and urinary albumin/creatinine ratio (UACR). B/ Renal corpuscule surface was measured on PAS-stained kidney slices. Pictures display representative areas of staining and bars indicate 200 μm. Data represent mean ± SEM from 6 animals per group. *p<0.05, ***p<0.01 versus sham.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131416.g006: Uninephrectomy as an animal model of increased urinary FSS.Sham- and UNx-mice were analyzed 8 months after surgery. A/ Renal function was evaluated by measuring glomerular filtration rate (GFR), single kidney GFR (skGFR) and urinary albumin/creatinine ratio (UACR). B/ Renal corpuscule surface was measured on PAS-stained kidney slices. Pictures display representative areas of staining and bars indicate 200 μm. Data represent mean ± SEM from 6 animals per group. *p<0.05, ***p<0.01 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