Genetic Regulation, Tubular Processing and Clinical Relevance of Collecting Duct alpha-Defensins 1-3

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PROJECT SUMMARY/ABSTRACT Vesicoureteral reflux affects 1-2% of all children and up to one third of these patients will experience urinary tract infections (UTI). The kidney and specifically the collect duct play a critical role in the innate defense of the kidney and urinary tract. Our group has demonstrated that intercalated cells are the primary source of a wide spectrum of innate immune antimicrobial peptides. DEFA1A3 (α-defensin 1-3) and its antimicrobial protein (AMP) product, human neutrophil protein 1-3 (HNP1-3), is important in the innate immune defense of the human kidney. Just this year we have reported that a) variations in the number of DEFA1A3 DNA copies correlate with UTI susceptibility and antibiotic response in children with vesicoureteral reflux and UTIs, b) DEFA1A3 mRNA and protein product, HNP1-3 expression is not limited to leukocytes, but also present in renal collecting duct epithelial cells and c) urine HNP1-3 increase during UTIs. Our aforementioned findings correlate with the known function of HNP1-3 as an AMP that rapidly destroys microbes. These finding are important because management of VUR and UTIs is complicated by the current inability to personalize treatment according to individual risk. For instance if all patients are treated under the assumption of recurrent pyelonephritis risk, children will be exposed to unnecessary imaging related radiation and antibiotic prophylaxis to prevent UTIs. Conversely if all patients are treated conservatively, a subset will develop potentially preventable renal scarring and chronic kidney disease. Further, identification of DEFA1A3 expressed in renal collecting duct cells, raises the possibility that renal DEFA1A3 expression could be targeted for therapeutics. Because DEFA1A3 DNA copy number variation is associated with UTI and the recent discovery that renal collecting duct cells express HNP1-3, determining HNP1-3’s role in the kidney represents clinically relevant research direction. Based on prior publications and preliminary studies, we hypothesize that renal- and neutrophil-derived DEFA1A3/HNP1-3 have distinct roles in cellular physiology/ pathology and are affected by DNA copy number variations. To this end, we will identify the unique molecular mechanisms critical to storage, secretion and activation of renally- derived alpha defensins in humans and murine UTI (Aim 1). We will utilize proteomic techniques using a mouse model of bone marrow transplantation and urine in children with UTI. In Aim 2, we will use a murine model of kidney transplantation of a humanized mouse for alpha-defensins to examine the kidney’s role in innate defense against uropathogens. In Aim 3, we will utilize this murine model and in vitro studies to dissect the role of DNA copy number variations in the DEFA1A3 locus in UTI susceptibility and antibiotic synergy.