Distinct Mesenchymal Alterations in N-Cadherin and E-Cadherin Positive Primary Renal Epithelial Cells

<div><h3>Background</h3><p>Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations.</p> <h3>Results</h3><p>We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-β for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-β, even though TGF-β signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-β. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-β-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells.</p> <h3>Conclusions</h3><p>Interference with Rho-kinase signaling provides a target to counteract TGF-β-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability which included constant expression of E-cadherin. Our cell culture system of primary epithelial cells is thus suitable to understand and modulate cellular remodeling processes of distinct tubular cells relevant for human renal disease.</p> </div

Isoform-specific changes in F-actin fiber formation by ROCK1 and ROCK2 siRNA.

<p>(A) HKC-8 cells were transfected with siRNAs as indicated. After 48 h the cells were stimulated with LPA (10 µM) for 2 h. Cells were stained for F-actin (red) and paxillin (green). Scale bar: 50 µm. (B) HKC-8 cells were treated as in A. N-cadherin was visualized by immunocytochemistry. Scale bar: 20 µm. (C) HKC-8 cells or hPTEC were transfected with siRNAs against ROCK1 (R1), ROCK2 (R2) or GFP as indicated. After 72 h, expression of N-cadherin, E-cadherin, and tubulin was detected by Western blotting. (D) hPTECs were treated as in A. Cells were stained for F-actin and for N-cadherin to distinguish between proximal and distal cells.</p

Regulation of transcription factors and miRNAs potentially involved in E-cadherin regulation.

<p>(A) hPTECs or HKC-8 cells were incubated with TGF-β (2 ng/ml) for 6 h. Snail (Sn) and Slug (Sl) mRNA expression was quantified by RT-PCR. Expression of Snail or Slug mRNA was set to 1 in each experiment. Data are means +/− SD of 4 preparations. ***p<0.001, *p<0.05, one way ANOVA with Dunnett’s post hoc test. (B): Cells were treated as in A. ZEB1 (Z1) and ZEB2 (Z2) was detected by RT-PCR. Data are means +/− SD of 6 different preparations of primary cells and 3 experiments with HKC-8 cells. *p<0.05, calculated as in 4A. (C) miRNA levels of miR200b, miR200c and miR141 were detected in 3 different preparations of hPTECs and compared to HKC-8 cells. In each set of experiments, expression of miR200b was set to 1. Error bars reflect variability of duplicate determinations. Error bars of miR200c and miR141 in HKC cells were smaller than the line thickness of the graph. (D) Cells were treated with TGF-β for 24 and 72 h (hPTECs) and 24 h (HKC-8). miRNA expression was determined by RT-PCR. Data are means +/− SD of 3 different preparations of primary cells and 2 experiments with HKC-8 cells analyzed in duplicate. Means of control cells (C) were set to 1 for each miRNA; T: TGF-β-treated cells.</p

Differential morphological plasticity of proximal and distal hPTECs upon treatment with TGF-β.

<p>(A) hPTECs were seeded at passage 1 at low (25 000 cells/cm²) or high density (75 000 cells/cm²) at day 1. Cells were treated with TGF-β (2 ng/ml) at day 2. Phase contrast images were taken at day 5. Scale bar: 200 µm. (B) Cells were treated as in A. N-Cadherin-positive proximal tubular cells (red) and E-cadherin-positive cells (green) were detected by immunocytochemistry. Scale bar: 50 µm. (C) Cells depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043584#pone-0043584-g001" target="_blank">Fig. 1B</a> (high density, TGF-β) were analyzed by epifluorescence with ApoTome technique to present x–y and x–z projections, respectively.</p

Polarization of primary epithelial cells.

<p>(A) hPTECs were cultured in permeable filter inserts for 8 days and then further incubated with TGF-β for 7 days. Cilia were detected by staining with antibodies against acetylated tubulin. Merged images show an overlay of N-cadherin (red), DAPI (blue) and acetylated tubulin (green). X–y and x–z orientation is presented as indicated. Scale bar: 10 µm. (B) hPTECs were treated as described in A. Proximal and distal cells were distinguished by staining for N-cadherin and E-cadherin respectively. Scale bar: 20 µm. (C) Polarized hPTECs were treated with TGF-β for 2 h. Cells were stained with antibodies against E-cadherin and Smad 2/3. Cells not stained with E-cadherin were considered to be proximal hPTECs (right panel). Scale bar: 10 µm.</p

Inhibition of Rho kinases interferes with TGF-β-induced morphological alterations.

<p>(A) hPTECs cultured as control cells or were treated with H1152 (0.75 µM), TGF-β (2 ng/ml) or TGF-β plus H1152 as indicated for 72 h. Phase contrast images of cells seeded at low or high density are depicted. Scale bar: 100 µm: (B) N-cadherin was detected by immunocytochemistry in cells treated with TGF-β or TGF-β plus H1152 for 72 h. F-actin was visualized by rhodamine-phalloidin staining. Arrow heads indicate stress fibers and cortical F-actin, respectively. Scale bar: 50 µm. Right hand panels show N-cadherin structures at higher magnification. Scale bar: 10 µM. (C) hPTECs were treated with TGF-β or TGF-β plus 10 µM Y27632 (Y) or 0.75 µM H1152 (H) for 24 h and 72 h. E-cadherin and N-cadherin protein expression was detected by Western blotting. Expression of N-cadherin (means +/− SD of 3–4 independent preparations) was summarized with N-cadherin expression in TGF-β-treated cells set to 1 in each experiment. ***p<0.001, **p<0.01, one way ANOVA with Dunnett’s post hoc test. (D) E-cadherin and F-actin were detected in cells treated as in B.</p

Regulation of N-and E-cadherin expression by TGF-β.

<p>(A) hPTECs cultured in plastic dishes were treated with TGF-β (2 ng/ml) for 24 and 72 h. N-and E-cadherin protein expression was detected in cellular homogenates by Western blot analysis. Expression of the respective protein in control cells after 24 or 72 h was set to 1. Data are means +/− SD of 3–5 independent preparations. **p<0.01, one way ANOVA with Dunnett’s post hoc test. (B) Cells were treated as in A. mRNA expression of N-and E-cadherin was determined by quantitative RT-PCR. Expression of N-cadherin or E-cadherin in control cells was set to 1 in each experiment. Data are means of 2–4 independent preparations. (C) Polarized hPTECs were treated with TGF-β for 72 h. E-cadherin and N-cadherin were detected in cellular homogenates by Western blotting. Vinculin served as control. (D) HKC-8 cells were treated with TGF-β for 24 and 72 h. To summarize E-cadherin protein and mRNA of different experiments, expression of the respective control cells was set to 1. Data are means of 4–8 experiments. ***p<0.001, one way ANOVA with Dunnett’s post hoc test.</p

Structural alterations in polarized hPTECs.

<p>(A) hPTECs were incubated in permeable filter inserts for 8 days to achieve polarization. They were further incubated with TGF-β (2 ng/ml) and H1152 (0.75 µM) for 7 days. Cells were stained for F-actin and E-cadherin. N-cadherin proximal tubular cells are visualized by F-actin only and are marked by the dotted lines (left panels). Scale bar: 50 µm. (B) Higher magnification of F-actin fibers in polarized cells treated with TGF-β or with TGF-β plus H1152 as described in 7A. Scale bar: 20 µm. (C) Cells were treated as in 7A. Proximal and distal cells were detected by antibodies directed against N-and E-cadherin, respectively. Scale bar: 50 µm.</p