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

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.

Arg tyrosine kinase modulates TGF-β1 production in human renal tubular cells under high-glucose conditions

“…So far, little is known about the in vitro EMT susceptibility of human primary tubular cell cultures. Some reports suggest that human primary tubular cells, even after TGF-β treatment, the major factor driving EMT (Carew et al, 2012), show morphological alterations but maintain a stable expression of epithelial markers like E-cadherin and its indirect inducer miR-200c (Keller et al, 2012), as also seen in the results described here. Our high-glucose-treated primary cell cultures overexpressed and secreted TGF-β1 that was able to activate fibroblasts, and the TGF-β1 overexpression was associated with a significant decrease of Arg protein and activity.…”

P2Y2R is a direct target of HIF-1α and mediates secretion-dependent cyst growth of renal cyst-forming epithelial cells

“…Next, we aimed to evaluate HIF-1α-dependent regulation of P2Y2R in renal tubular cells. To this end, we used human primary tubular cells (hPTECs) isolated from healthy parts of the kidneys of patients that were nephrectomized because of kidney cancer [15]. We first performed formaldehyde-assisted isolation of regulatory elements (FAIRE) assays in hPTECs.…”

“…Isolated cells represent a mixture of proximal and distal tubular cells with a ratio of approximately 50:50 %, as described previously [15]. Isolation of human cells from healthy parts of tumor nephrectomies was approved by the local ethics committee (Reference number 3755, EthikKommission der Medizinischen Fakultät der FriedrichAlexander Universität Erlangen-Nürnberg).…”

Cytosolic phospholipase A2α increases proliferation and de-differentiation of human renal tubular epithelial cells

“…This phenotype has been reported with immortalized HK-2 cells and is also observed in primary cultured human proximal tubular epithelial cells possibly due to lower overall E-cadherin expression than in distal tubular epithelial cells. (21, 22) In contrast, cPLA 2 α silenced HK-2 cells appeared more cuboidal with less spindle-like projections and clustered more cohesively (Figure 1E,F) than control cells.…”