Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells

<div><p>Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent <i>in vitro</i> 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 <i>in vitro</i> and <i>in vivo</i> 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 48h. 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 <i>in vivo</i> 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 <i>in vitro</i> and <i>in vivo</i>. 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.</p></div

Effect of uninephrectomy-mediated FSS on epithelial gene expression and the density of primary cilia.

<p>Sham- and UNx-mice were analyzed 8 months after surgery. <b>A</b>/ 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>B</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 <i>versus</i> sham.</p

Alteration of adherens junctions in response to FSS.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A/</b> 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>B/</b> Real-time PCR and Western blot analysis of E-Cadherin mRNA and protein, respectively. <b>C/</b> 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 <i>versus</i> FSS 0.</p

<i>In vitro</i> studies assessing the effects of FSS on epithelial characteristics of renal cells.

<p>FSS was tested on renal cells and changes in organization of actin cytoskeleton, adherens junctions (AJ), tight junctions (TJ) or primary cilium were determined by comparison with no FSS condition, where cells remained in static media durig the same time than flow tests.</p><p>Lateral, at the periphery of the cells; ECM, extracellular matrix; <b>↑</b>, increase in; ↓, decrease in; ≠, change in; → no change in.</p><p>¹The population of tubular cells is heterogeneous because composed of cells derived from all tubular segments.</p><p>^2,3The supramolecular polymers used were self-assembled into nano-meter scale fibers by electro-spinning to mimic basement membrane; bioactivity was introduced into these nano-fibers by intercalation of different ECM peptides, in order to ameliorate synthetic basement membrane.</p><p>⁴FSS induction was performed through flow of medium culture at a rate of 1 mL/h. However, without knowing the size of perfusion chamber, it is impossible to give the intensity of FSS.</p><p>^5,6Topographically patterned substrate mimics micro- and nano-scale topographic structures contained in the basement membrane of the kidney tubule; blank substrates lacked of geometric structures.</p><p><i>In vitro</i> studies assessing the effects of FSS on epithelial characteristics of renal cells.</p

FSS does not induce a mesenchymal phenotype in tubular cells.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A</b>/ Transcript level of mesenchymal markers such as Vimentin and αSMA (cytoskeleton), Fibronectin and Collagen I (ECM), N-Cadherin (intercellular junction) was quantified by real-time PCR. Results are expressed as the fold induction compared to static condition. <b>B</b>/ Protein level of Vimentin, αSMA and Fibronectin was measured by Western blot. Results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4 experiments. *p<0.05, **p<0.01 <i>versus</i> FSS 0.</p

Effect of FSS on apoptosis and necrosis in tubular cells.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A/</b> 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>B</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.</p

FSS-exposed cells exhibit loss of the primary cilium without marked change in actin cytoskeleton organization.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A/</b> α-acetylated Tubulin was analyzed by immunofluorescence to visualize the primary cilium. White arrows show primary cilia. <b>B/</b> Phalloidin was used to stain the actin cytoskeleton (basal [left] and subapical [right]). Cells were counterstained with DAPI. Pictures display representative areas of staining from 5 independent experiments. Green, α-acetylated Tubulin; red, Phalloidin; blue, DAPI-nuclei. Bars indicate 20 μm.</p

Uninephrectomy as an animal model of increased urinary FSS.

<p>Sham- and UNx-mice were analyzed 8 months after surgery. <b>A</b>/ Renal function was evaluated by measuring glomerular filtration rate (GFR), single kidney GFR (skGFR) and urinary albumin/creatinine ratio (UACR). <b>B</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 <i>versus</i> sham.</p

Changes in body weight (A), energy intake (B), food intake (C), water intake (D), fat mass (E) and lean mass (F) in control fed mice (■) and HFFD fed mice (▲).

<p>Data are means ± SEM for 36 mice per group at 1, 2 and 3 months, 24 mice per group at 6 months and 12 mice per group at 8 months. *P≤0.05, **P≤0.01, ***P≤0.001.</p

Glomerular hyperfiltration (A), urinary albumin to creatinine ratio (ACR) (B) and arterial blood pressure (C) in HFFD fed mice (■) compared to control fed mice (▲)

<p>Data are means ± SEM for 9 mice per group; *p=0.0429; **p=0.004.</p