P0009 PP AN IN VITRO CELLULAR MODEL OF PROTEIN-LOSING ENTEROPATHY IMPLICATES HEPARAN SULFATE, INFLAMMATORY CYTOKINES, AND HYDROSTATIC PRESSURE AS CRITICAL PLAYERS

Abstract

Introduction: Protein-losing enteropathy (PLE), the excessive loss of plasma proteins through the intestine, occurs in seemingly unrelated disorders, e.g. Crohn’s disease, Congenital Disorders of Glycosylation (CDG), or after Fontan surgery to correct congenital heart malformations. However, PLE in these diverse diseases shares several common features. It is episodic and heparan sulfate proteoglycans (HSPG) on the basolateral surface of intestinal epithelial cells are absent or mislocalized during these episodes. The importance of HSPGs for PLE is supported by prior evidence that the electrostatic charge provided by the highly anionic HSPGs prevents albumin loss through the glomerulus, and HSPGs are absent in patients with nephrotic syndrome. In addition, the onset of episodic PLE is often associated with a pro-inflammatory state, suggesting multiple factors may combine to exceed critical thresholds. However, mechanisms that link PLE with a loss of HSPG and an inflammatory response are yet unknown. We investigated 1.) whether loss of HS directly causes protein leakage, 2.) whether the pro-inflammatory cytokine TNF-alpha influences this process, and 3.) whether increased pressure (analogous to venous hypertension in post-Fontan PLE patients) exacerbates protein leakage. Methods: We measured protein leakage as the flux of FITC-conjugated albumin through a monolayer of intestinal HT29 cells grown on transwell inserts. The amount of cell-associated glycosaminoglycan (GAG) chains was determined with 35S-labeling. The integrity of the monolayer was monitored according to transepithelial electrical resistance (TEER). Results: Heparanase treatment (0.6 mU/mL) reduced cell-associated GAG chains to 35%, decreased TEER to 65%, and increased albumin flux 1.6 fold. These effects were concentration dependent and were competed by soluble HS but not chondroitin sulfate. TNF-alpha (20 ng/mL) enhanced albumin flux 5.4 fold and reduced TEER to 15%. The effects of heparanase and TNF-alpha were synergistic with an 8.7 fold increase in albumin flux and a complete loss of TEER. Increasing hydrostatic pressure also increased albumin flux and suggested additional synergy with heparanase treatment. Conclusion: Our findings imply that PLE can result from multiple independent or interdependent processes, some of which may involve HSPG.