Uremic plasma impairs barrier function and depletes the tight junction protein constituents of intestinal epithelium

Abstract

Background: Chronic kidney disease (CKD) causes intestinal barrier dysfunction which by allowing influx of endotoxin and other noxious products contributes to the CKD-associated systemic inflammation and uremic toxicity. We have recently shown that intestinal barrier dysfunction in CKD animals is due to degradation of transcellular (claudin-1 and occludin) and intracellular (ZO1) constituents of epithelial tight junction (TJ). This study determined whether CKD-associated disruption of TJ is mediated by retained uremic toxins/metabolites and, if so, whether they are removed by hemodialysis.

Methods: The TJ-forming human enterocytes (T84 cells) were seeded on the Transwell plates and utilized when transepithelial electrical resistance (TER) exceeded 1,000 mΩ/cm(2) to ensure full polarization and TJ formation. The cells were then incubated for 24 h in media containing 10% pre- or posthemodialysis plasma from end-stage renal disease (ESRD) patients or healthy individuals. TER was then measured and cells were processed for Western blot and immunohistological analyses.

Results: Compared with the control plasma, incubation in media containing predialysis plasma from ESRD patients resulted in a marked drop in TER pointing to increased epithelial permeability. This was accompanied by significant reductions in claudin-1 (85%), occludin (15%), and ZO1 (70%) abundance. The severity of TJ damage and dysfunction was significantly less in cells exposed to the postdialysis in comparison to predialysis plasma. These findings point to the presence of as-yet unidentified product(s) in the uremic plasma capable of depleting epithelial TJ.

Conclusions: Exposure to uremic milieu damages the intestinal epithelial TJ and impairs its barrier function, events which are mediated by agents which are partially removed by hemodialysis.

Figure 1

Bar graph depicting the trans-epithelial electrical resistance in intestinal epithelial T84 cell monolayers exposed to media containing plasma obtained from healthy control individuals and those containing pre-hemodialysis and post hemodialysis plasma samples from ESRD patients.

Figure 2

Representative Western blots and group data depicting protein abundance of occludin, claudin1 and ZO1 in intestinal epithelial T84 cell monolayers exposed to media containing plasma obtained from healthy control individuals and those containing pre--hemodialysis plasma samples from ESRD patients

Figure 3

Representative Western blots and group data depicting protein abundance of occludin, claudin1 and ZO1 in intestinal epithelial T84 cell monolayers exposed to media containing plasma obtained from ESRD patients immediately before (pre-HD) and after hemodialysis (post-HD).

Figure 4

Representative photomicrographs depicting claudin-1 and ZO1 immunostaining of intestinal epithelial T84 cell monolayers exposed to media containing plasma samples obtained from a healthy control individual and an ESRD patient immediately before and after hemodialysis and visualized by confocal microscopy.