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

Christof Keller,Jonathan Zuehlke,Margarete Goppelt-Struebe,Frank Kunath,Sven Kroening,Bettina Krueger,Jean-Claude Dussaule

doi:10.1371/journal.pone.0043584

Abstract

BackgroundRenal 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.ResultsWe 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.ConclusionsInterference 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.

Highlights

Epithelial cells possess the unique property to reversibly alter their phenotype and to adopt features of mesenchymal cells
Proximal tubular cells are the only epithelial cells in the human adult organism which express N-cadherin instead of E-cadherin as major cell-cell adhesion protein [3,4]. This difference is rarely acknowledged in studies investigating molecular properties of proximal epithelial cells, which may be due to the fact that cell lines derived from human proximal tubules such as HK-2 or HKC-8 express both, N-cadherin and varying levels of Ecadherin
Human primary tubular cells at passage one were seeded on collagen-IV-coated glass cover slips and were cultivated until day 5 in the absence of serum

Summary

Introduction

Epithelial cells possess the unique property to reversibly alter their phenotype and to adopt features of mesenchymal cells. During this process cells lose polarity, E-cadherin-mediated adherens junctions are dissolved, and the cells express mesenchymal markers [1]. Proximal tubular cells are the only epithelial cells in the human adult organism which express N-cadherin instead of E-cadherin as major cell-cell adhesion protein [3,4]. This difference is rarely acknowledged in studies investigating molecular properties of proximal epithelial cells, which may be due to the fact that cell lines derived from human proximal tubules such as HK-2 or HKC-8 express both, N-cadherin and varying levels of Ecadherin. We hypothesized that they differ in their capacity to undergo epithelial to mesenchymal alterations

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