Role of urea in intestinal barrier dysfunction and disruption of epithelial tight junction in chronic kidney disease

Abstract

Background: Chronic kidney disease (CKD) impairs intestinal barrier function which leads to endotoxemia and systemic inflammation. We have found depletion of intestinal epithelial tight junction (TJ) proteins in animals with CKD. We further showed that addition of end-stage renal disease patients' plasma to the culture medium provokes a marked drop in transepithelial electrical resistance (TER) and depletion of TJ proteins in cultured human enterocytes. These effects were less severe with post- than with prehemodialysis plasma, suggesting a role of dialyzable agent(s). This study tested the hypothesis that intestinal barrier dysfunction in uremia may be due to diffusion of urea into the gut and its conversion to ammonia by microbial urease.

Methods: Human enterocytes (T84 cells) were seeded on Transwell plates and utilized when TER exceeded 1,000 mΩ·cm(2) to ensure full polarization and TJ formation. Confluent cells were then incubated for 24 h in media containing 0, 42 or 74 mg/dl urea or urea plus urease to simulate presence of microbial flora.

Results: At clinically relevant concentrations, urea caused a concentration-dependent fall in TER and the key TJ proteins claudin-1, occludin and zonula occludens 1. The effects of urea were dramatically amplified by urease causing cell detachment, dissipation of TER, and massive loss of TJ proteins.

Conclusions: Uremia-induced disruption of intestinal TJ and barrier function is, in part, mediated by urea which is generally considered to be a nontoxic retained metabolite. These findings reveal a novel mechanism for a salutary effect of urea-lowering strategies, e.g. low-protein diet and longer and more frequent dialysis regimens in advanced CKD.

Figure 1

Bar graphs depicting the trans-epithelial electrical resistance (TER) in intestinal epithelial T84 cell monolayers incubated for 24 hr in regular media and those incubated in media containing 42 or 72 mg/dl urea.

Figure 2

Representative Western blots and group data depicting protein abundance of occludin, claudin1 and ZO1 in intestinal epithelial T84 cell monolayers incubated for 24 hr in regular media and media containing 42 or 72 mg/dl urea.

Figure 3

Representative Western blots and group data depicting protein abundance of occludin, claudin1 and ZO1 in intestinal epithelial T84 cell monolayers incubated for 24 hr in media containing 42 mg/dl urea alone and those incubated in media containing 42 mg urea plus urease.

Figure 4

Representative Western blots and group data depicting protein abundance of occludin, claudin1 and ZO1 in intestinal epithelial T84 cell monolayers incubated for 24 hr in media containing 72 mg/dl urea alone and those incubated in media containing 72 mg urea plus urease.