S. Dillman1,2, N. Heinzerling1,2, K. Fredrich1,2, J. Fawley1,2, D. M. Gourlay1,2 1Medical College Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA 2Children’s Hospital Of Wisconsin,Children’s Research Institute,Milwaukee, WI, USA
Introduction: Recent studies indicate an increase in intestinal permeability precedes necrotizing enterocolitis (NEC). Prior research has shown supplemental enteral Intestinal Alkaline Phosphatase (IAP), an endogenous protein expressed by the intestines, decreases intestinal permeability and the severity of NEC in a rodent model. The tight junction proteins, claudins, are essential in creating a functional epithelial intestinal barrier. Increased expression and altered distribuiton of Claudin-1 (Cl-1) during intestinal inflammation was associated with increased intestinal permeability leading to a breach in mucosal barrier. We hypothesize that IAP decreases intestinal permeability prior to the development NEC through modification of CL-1.
Methods: One day preterm Sprague Dawley rat pups were delivered via cesarean section and fed formula. Select pups received 4 units/kg of IAP in their formula. Pups were sacrificed on day of life 2-4 and the terminal ileum was harvested. Intestinal permeability was measured by filling loops of intestine with a fluorescein isothiocyanate-dextran (FITC-dextran) solution and measuring flux into the incubating media. Three day old dam-fed wild-type, heterozygotes, and IAP KO rat tissue was also harvested. To examine the effect of IAP on intestinal epithelial cell, in vitro experimentation involved FHs 74, a fetal epithelial cell culture. Cells were given either formula or formula plus 4 units of IAP. ZO-1, CL-1, 2, and 3 were measured by RT-PCR and Western Blot on all cells and ileal intestinal homogenates. Statistical analysis was performed using a paired t-test and a p<0.05 considered significant.
Results: Intestine loops exposed to lipopolysaccharide and FITC-dextran had a greater statistically significant increase in permeability over controls. Supplemental, enteral IAP significantly decreased intestinal permeability. Evaluation of ileal intestinal homogenates showed a significant decrease messenger RNA levels for CL-1, 2, and 3 in IAP fed rats. For Western Blot analysis, ZO-1 and CL-1 displayed a significant increase in the IAP fed group while CL-2 showed a slight decrease in protein expression. In the IAP gene knockout rats, CL-1 showed a significant increase mRNA expression compared to wild-type. In the FHs 74 cell line, Cl-1 expression was significantly decreased in the IAP group while the other claudins didn’t have noticeable expression in either group.
Conclusion: Neonatal intestinal permeability is increased by formula feeding and LPS and may increase the risk of NEC and sepsis. These data suggest IAP has a direct effect on expression and stability of CL-1 in the neonatal intestinal epithelial cell that is a novel mechanism by which IAP may function to protect newborns from NEC. .