In vitro effect of uremic plasma and use of probiotics on the barrier function, apoptosis and inflammation in human enterocytes

Abstract

The gastrointestinal tract functions as a barrier between the external environment and the internal milieu of the body. The epithelial lining of the gastrointestinal tract forms a regulated, selectively permeable barrier permitting the passive entry of luminal nutrients, ions, and water while restricting pathogen access to underlying tissue compartments. In addition, intestinal epithelial cells (IECs) express numerous receptors and pro inflammatory mediators that allow IECs to communicate with the immune system. It is thought that exposure to certain microbial products in the intestinal lumen has an important role to limit local inflammation and consequently to contribute to the colonic homeostasis. The intestinal microbiota consists of various microorganisms with a predominance of bacteria. Among the various bacterial genera that compose the microbiota, there are those which are beneficial to the host, mainly Lactobacillus and Bifidobacterium, as well as those which can be harmful. In vitro studies with human colonic epithelial cells have shown that administration of specific probiotics is able to inhibit the secretion of pro-inflammatory cytokines and to enhance the barrier function of these cells. In Chronic Kidney Disease (CKD) it has been suggested that alterations of the gut are associated with inflammatory state and uremic toxicity. Studies suggest that uremia may impair the intestinal barrier function, by promoting increased intestinal permeability, which allows the entry of antigens or pro-inflammatory products from the lumen, leading to activation of macrophages, T-lymphocytes, release of pro inflammatory cytokines and recruitment of circulating inflammatory cells, favoring the inflammatory state. In addition, when compared with healthy subjects, patients on hemodialysis have marked changes in the composition of the intestinal microbiota. These changes may be, at least in part, a cause or consequence of the intestinal barrier dysfunction and the associated inflammation. Objective: To evaluate the in vitro effect of uremic plasma on the transepithelial electrical resistance, inflammation and apoptosis in the intestinal epithelial cells and the in vitro effect of a specific probiotics on the cells insulted by uremic plasma, that is, to assess whether the probiotic is able to minimize cellular damages caused by uremic plasma. Methods: Pre and post dialysis blood samples from 30 patients on hemodialysis (HD) will be collected to form a pool of plasma. A pool of plasma of 15 healthy subjects will serve as a control. T84 cells will be utilized for the different incubation conditions. Initially cells will be incubated with fetal bovine serum, healthy plasma, predialysis plasma and postdialysis plasma, the incubation time for each condition will be determined according a time-response curve. In a second step, using the same incubation protocol mentioned before, the T84 cells will be incubated with two probiotic bacterias: Lactobacillus rhamnosus GG or Bifidobacterium breve Reuter. Time and probiotic concentration will be determined according to a time and dose-response curve. After the incubation period, transepithelial electrical resistance will be measured, and the cells will be processed and the following parameters will be determined: expression of claudin 1, occludin 1, ZO-1 and NFk-B p65 (immunofluorescence); expression of TLR-4 and TLR-9, IL-6 and TNF intracellular, apoptosis and production of reactive oxygen species (flow cytometry) and detection of TNF and IL-6 in the culture supernatant and NFk-B (p65) in the nucleus(ELISA). (AU)