More complete polarization of renal tubular epithelial cells by artificial urine

Abstract

Cell polarization using Transwell is a common method employed to study renal tubular epithelial cells. However, this conventional protocol does not precisely recapitulate renal tubular epithelial cell phenotypes. In this study, we simulated renal physiological microenvironment by replacing serum-containing culture medium in upper chamber of the Transwell with physiologic artificial urine (AU) (to mimic renal tubular fluid), whereas the lower chamber still contained serum-containing medium (to mimic plasma-enriched renal interstitium). Comparing to the conventional protocol (control), the AU-assisted protocol offered more complete polarization of MDCK renal tubular cells as indicated by higher transepithelial electrical resistance (TER) and greater levels of tight junction (TJ) proteins (ZO-1 and occludin). Transmission electron microscopy (TEM) showed greater densities of TJ and desmosome, narrower intercellular spaces, greater cell height, and longer microvilli in the AU-treated cells. Secretome analysis revealed that the AU-treated cells secreted greater proportion of the proteins matched to normal human urinary proteome via both classical and non-classical secretory pathways. Finally, modifying/omitting each component of AU (one at a time) followed by validation revealed that urea was responsible for such property of AU to improve cell polarization. These data indicate that replacing AU on the upper chamber of Transwell can improve or optimize renal cell polarization for more precise investigations of renal physiology and cell biology in vitro.

Cellular proteome datasets of human endothelial cells under physiologic state and after treatment with caffeine and epigallocatechin-3-gallate

“…In-solution tryptic digestion was performed as described previously [2], [3]. Briefly, protein samples prepared in SDT lysis buffer were reduced by heating at 95 °C for 5 min.…”

Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions

“…A key advantage of the recreation of a 2D mucosal tissue interface is that this culture format will allow the access to the apical side of the epithelium for investigating the nutrient and drug absorption, host-microbe crosstalk, or drug metabolism and toxicity testing. The 2D mucosal tissue interface using primary 3D intestinal organoids could allow modeling of intestinal physiology ex vivo or in vitro compared to currently available canine-specific immortalized cell lines [5][6][7]. The measurement of the epithelial barrier function (e.g., TEER) is convenient when investigating the physiological responses of epithelial cells following exposure to toxins, therapeutic drugs, or nutrients [38,39].…”

“…There is currently a limited number of canine-specific primary cell lines to investigate intestinal physiology ex vivo or in vitro. Well-characterized immortalized cell lines including the Madin-Darby canine kidney (MDCK) cells do not accurately model intestinal epithelial interactions in the dog due to their origin from immature kidney cells [5,6]. Recently, isolated primary canine intestinal epithelial cells have been immortalized with a temperature-sensitive mutant of the Simian Virus 40 large tumor antigen (SV40 T-Ag) [7].…”

Effect of Substrate Stiffness on Human Intestinal Enteroids Infectivity by Enteroaggregative Escherichia coli

“…Although our HIE monolayers remained generally flat during the 5 days of differentiation culture, it would be intriguing to prolong this culture duration to assess whether a similar phenomenon occurs with the HIEs. As for the height difference, greater height of epithelial cells is an indicator of greater cell polarization and differentiation [45,46], with positive correlations between cell height, robust junctional density, and cell integrity. The height of the differentiated HIEs cultured atop the hydrogels (approximately 18 µm) is closer to that of epithelial cells in tissue sections of the human small intestinal wall, which we measured as 25-27 µm.…”