Disintegration of colonic epithelial tight junction in uremia: a likely cause of CKD-associated inflammation

Abstract

Background: Inflammation is a constant feature and a major mediator of the progression of chronic kidney disease (CKD) and its numerous complications. There is increasing evidence pointing to the impairment of intestinal barrier function and its contribution to the prevailing inflammation in advanced CKD. Under normal condition, the intestinal epithelium and its apical tight junction prevent entry of the luminal microorganisms, harmful microbial by-products and other noxious contents in the host's internal milieu. This study was designed to test the hypothesis that impaired intestinal barrier function in uremia must be due to disruption of the intestinal tight junction complex.

Methods: Sprague-Dawley (SD) rats were randomized to undergo 5/6 nephrectomy (CKD) or sham-operation (control) and observed for 8 weeks. In a separate experiment, SD rats were rendered uremic by addition of 0.7% adenine to their food for 2 weeks and observed for an additional 2 weeks. Rats consuming a regular diet served as controls. The animals were then euthanized and their colons were removed and processed for expression of the key constituents of the tight junction complex using real-time polymerase chain reaction, western blot analysis and immunohistological examinations.

Results: The CKD groups showed elevated plasma urea and creatinine, reduced creatinine clearance, thickened colonic wall and heavy infiltration of mononuclear leukocytes in the lamina propria. This was associated with marked reductions in protein expressions of claudin-1 (70-90%), occludin (50-70%) and ZO-1 (80-90%) in the colonic mucosa in both CKD models compared with the corresponding controls. The reduction in the abundance of the given proteins was confirmed by immunohistological examinations. In contrast, messenger RNA abundance of occludin, claudin-1 and ZO-1 was either unchanged or elevated pointing to the post-transcriptional/post-translational modification as a cause of the observed depletion of the tight junction proteins.

Conclusion: The study revealed, for the first time, that uremia results in depletion of the key protein constituents of the colonic tight junction, a phenomenon which can account for the impaired intestinal barrier function and contribute to the systemic inflammation in CKD.

Figures

Fig. 1.

Representative photomicrographs of the H&E-stained sections of colonic tissue in a rat with CKD induced by subtotal nephrectomy and a sham-operated control rat. The study revealed thickening of the wall and increased accumulation of mononuclear leukocytes in the CKD versus control rats. Original magnifications: ×10 upper panel and ×40 lower panel. Data are from at least six rats used in each group.

Fig. 2.

Representative western blots and group data depicting protein abundance of occludin, claudin 1 and ZO-1 in the ascending and descending colonic tissues of the CKD and control (CTL) groups. *P N = 6 animals in each group.

Fig. 3.

Representative photomicrographs depicting occludin, claudin-1 and ZO-1 immunostaining in the ascending and descending colon of a rat with CKD induced by subtotal nephrectomy or injection of a diet containing 0.7% adenine and a sham-operated control rat.

Fig. 4.

Representative western blots and group data depicting the abundance of phosphorylated myosin light chain (P-MLC) in the colonic tissues of the CKD and control (CTL) groups. ***P N = 6 animals in each group.

Fig. 5.

Bar graphs mRNA abundance of occludin, claudin-1 and ZO-1 in the ascending and descending colon of rats with CKD induced by subtotal nephrectomy and sham-operated control rats. Each data point represent the mean ± SE of at least three separate experiments involving at least four sets of rats. *P

Fig. 6.

Bar graphs depicting plasma MCP-1 concentration in the CKD and control (CTL) groups. *** P N = 6 animals in each groups.