Intestinal Perfusion and Permeability in Sepsis

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DESCRIPTION (provided by applicant): The parenchymal organs that are most prominently affected in the multiple organ dysfunction syndrome (MODS) are the lungs, liver, kidneys and gut. The normal functioning of these organs depends on the establishment and maintenance of compositionally distinct compartments that are lined by sheets of epithelial cells. An essential element in this process is the formation of tight junctions (TJs) between adjacent epithelial cells. The TJ acts as a regulated semi-permeable barrier that limits the passive diffusion of solutes across the paracellular pathway between adjacent cells. Thus, the barrier function of the TJ is necessary to prevent dissipation of the concentration gradients that exist between the two compartments defined by the epithelium. The histopathology of MODS in humans is remarkably bland; massive cell death, whether due to necrosis or apoptosis, is almost certainly not the cause of MODS. Rather, the final step in the development of MODS is probably the widespread dysfunction of parenchymal cells in multiple organs as a result of the deleterious effects of a poorly controlled systemic inflammatory response. Thus, a hugely under-explored area of research can be summarized by this question: How does the inflammatory response lead to parenchymal cell dysfunction? Based on our work during the previous cycle of funding, we hypothesize that MODS results, at least in part, from nitric oxide (NO)- dependent perturbations in the expression and subcellular localization of TJ proteins. To test this hypothesis, we propose to study inflammation-induced alterations in epithelial barrier function and TJ formation at levels of integration ranging from whole animals to cultured cells or subcellular fractions. In vitro, we will focus on changes in intestinal epithelial permeability using Caco-2 (human enterocyte-like) monolayers as a reductionist model system. In vivo, however, in studies using mice and rats, we will evaluate changes in epithelial barrier function not only in the gut, but also in the liver and lung as well. In a series of 6 Specific Aims, we will test these Specific Hypotheses: 1) sepsis in mice leads to alterations in TJ structure and function via mechanisms that depend on the formation of NO., reactive oxygen species (ROS), and/or ONOO-; 2) the structure of epithelial TJs is deranged in patients dying with MODS; 3) decreased transcription of the TJ protein, ZO-1, is a critical step leading to inflammation- or NO-induced alterations in epithelial barrier function; 4) cytokine- or NO-induced events impair the proper packaging and targeting of the key TJ proteins, claudin-1 and occludin, in cultured Caco-2 cells; 5) post-translational modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contributes to epithelial barrier dysfunction; 6) alterations in the function of Na+,K+-ATPase contribute to inflammation-induced derangements in epithelial barrier function.