ESPU Congress 2018 - Abstract Book

13 11–14 APRIL, 2018, HELSINKI, FINLAND 13:39–13:42 S1-4 (PP) RAPAMYCIN REGULATION OF CORE CLOCK GENES IS ASSOCIATED WITH ALTERED DE-OBSTRUCTION SIGNATURES COORDINATE WITH PHYSIOLOGY Karen AITKEN  1 , Annette SCHRODER  2 , Jia-Xin JIANG  3 , Aliza SIEBENALLER  4 , Thenuka THANABALASINGHAM  5 , Martin SIDLER  6 and Darius BAGLI  7 1) PGCRL room 159420TUV, DSCB, Sickkids Research Institute, Toronto, CANADA - 2) Hospital for Sick Children, Urology Division3, Department of Surgery; Developmental and Stem Cell Biology, Research Institute, Toronto, CANADA - 3) Hospital for Sick Children, University of Toronto, Developmental and Stem Cell Biology, Research Institute; Department of Physiology, Faculty of Arts a, Toronto, CANADA - 4) Hospital for Sick Children, Developmental and Stem Cell Biology, Research Institute, Toronto, CANADA - 5) H, Developmental and Stem Cell Biology, Research Institute, Toronto, CANADA - 6) Hospital for Sick Children, University of Toronto, Developmental and Stem Cell Biology, Research Institute; Institute of Medical Sciences, Faculty of M, Toronto, CANADA - 7) Hospital for Sick Children, University of Toronto, Developmental and Stem Cell Biology, Research Institute; Department of Surgery, Faculty of Medicine, Toronto, CANADA PURPOSE While rapamycin improves smooth muscle phenotype and gene expression patterns during stretch, altered matrix, hypoxia and obstruction(Aitken, 2010, Am.J.Path., Schroder, JUrol, 2013, Jiang, PLOSONE, 2015), the effect of rapamycin on gene expression during de-obstruction(dOB) is unknown. We hypothesized that rapamycin-induced changes in gene expression during dOB point to new pathways with functional relevance. MATERIAL AND METHODS Sprague-Dawley female rats underwent PBO by tying a silk suture around the proximal urethra and a 0.9 mm steel rod, leaving only the suture in place. PBO animals were randomized to 6 week PBO or 6 week PBO plus de-obstruction (dOB). dOB were randomized to vehicle (saline) or rapamycin for 6 weeks. Shams were performed over 6 and 12 weeks. High-throughput QPCR was performed to identify genes dysregulated during dOB normalized with rapamycin treatment and correlated with function. Effects of gene products were examined in human SMC by immunofluorescent staining of SMC markers. We used transcription factor binding site programs (DAVID, DIRE). Clock gene dependency of target genes and SMC markers was queried by treating SMC with a reverba agonist (SR9009) and by performing QPCR and immunofluorescence. RESULTS IGFBP7, BMP2, and SOD3 correlated with functional improvements in rapamycin treated animals. We identified the E-boxes bound by circadian regulators CLOCK/BMAL or NPAS2/BMAL as a po- tential sites of transcriptional regulation amongst the three genes. We found that obstruction and dOB upregulated mRNA expression of CLOCK and NPAS2. Rapamycin downregulated mRNA of CLOCK, NPAS2, alongside the 3 physiologic genes, p<0.01. SR9009 downregulated several core clock genes alongside IGFBP7, p<0.01. Exogenous IGFBP7 increased SMC hypertrophy in vitro. CONCLUSIONS Core clock genes direct expression of the de-obstruction-induced rapamycin-responsive gene IGFBP7, concordant with bladder smooth muscle cell hypertrophy.