Distinct mesenchymal alterations in N-cadherin and E-cadherin positive primary renal epithelial cells.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Erlangen, Germany.
Christof
C
Keller
Sven
S
Kroening
Jonathan
J
Zuehlke
Frank
F
Kunath
Bettina
B
Krueger
Margarete
M
Goppelt-Struebe
D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007686:Kidney Tubules, Distal / Q000166:cytology; D007687:Kidney Tubules, Proximal / Q000166:cytology; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D000071799:Zinc Finger E-box-Binding Homeobox 1; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D015820:Cadherins; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D035683:MicroRNAs; D051899:Smad2 Protein; D051900:Smad3 Protein; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; C515851:ROCK1 protein, human; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases