RESULTS: LRCs were found both within cytokeratin 5 (CK5)-positive basal cells and CK5-negative cells of the urothelium. A subsetof these cells proliferated after UPEC-induced urothelial injury. In theadult model of UPEC-induced urothelial injury, there was reproducibleloss of a functional superficial cell layer, resulting in increased urothelialpermeability, bladder weight, and stromal edema. Urothelial repairresulted in resolution of edema and normalization of bladder weight.Both CK5-positive and CK5-negative cells contributed to regenerativerepair of the urothelium.

CONCLUSIONS: Proliferation within both the basal cell layerand the intermediate cell layer plays a central role in superficial cellregeneration. Each layer contains a subset of slow-cycling urothelialcells that are recruited to proliferate in response to diffuse urothelialinjury.

Source of Funding: This work was supported in part by theNIH National Center for Research Resources (NCRR), grantnumber T32 RR023916 [07/2008-07/2011], the AmericanUrological Association Foundation Research Scholars Program[07/2011-06/2012], and the Clinical and Translational ScienceAward (CTSA) program of the National Center for AdvancingTranslational Sciences, grant number 9U54TR000021 [06/2012-present].

1146CHRONIC INFLAMMATION-INDUCED HEMATURIA INVOLVESMOLECULAR MODULATION OF THE UROTHELIAL BARRIERAND BLADDER VASCULATURE

Kim Thai, Portland, OR; Mohammad Khan, Mark Nicolls, Stanford,CA; Debalina Ray, Tyrrell Nelson, San Francisco, CA; Chi-Ling Fu,Shailja Patel, Diana Gong, Justin Odegaard, Stanford, CA;Giselle Knudsen, San Francisco, CA; Michael Hsieh*, Stanford, CA

INTRODUCTION AND OBJECTIVES: Bladder surgery-, che-motherapy-, and infection-induced inflammation can breach the urothe-lial barrier and cause hematuria. Defining the mechanisms of urothelialcompromise and hematuria in inflammation models may reveal sharedpathways suitable as therapeutic targets.

METHODS: We expanded on our mouse model of urogenitalschistosomiasis (Fu et al., PLOS Pathogens 2012) to study howchronic inflammation breaches the urothelial barrier and causes hema-turia. In this model, Schistosoma haematobium eggs are injected intothe bladder walls of mice, which recapitulates key features of humaninfection, i.e. urothelial hyperplasia and hematuria.

RESULTS: Microarray analysis demonstrated that despite theonset of urothelial hyperplasia, transcription of junctional adhesionmolecule, claudin and uroplakin genes, key components of the urothe-lial barrier, were all decreased compared to controls after bladderexposure to eggs (�2-fold, p�0.05). Luminex liquid microbead immu-noassays demonstrated higher levels of bladder vascular endothelialgrowth factor (VEGF) compared to controls after egg injection(p�0.05). Correspondingly, microarray and DAVID pathways analysisrevealed more transcription of VEGF pathway-related genes in egg- vs.control-injected bladders. Perfusion of egg-injected mice with FITC-lectin and Red Fluoromax microspheres revealed widespread bladderneovascularization and more microvascular permeability relative tocontrols (Figure). Finally, mass spectrometry of urine from egg-injectedmice demonstrated the presence of multiple blood proteins not ob-served in control-injected mice.

CONCLUSIONS: These unexpected findings suggest thatchronic inflammation-induced hematuria is associated with intricatemodulation of the bladder vasculature and urothelial barrier on thecellular and molecular level. Future work will define how VEGF andother factors mediate these processes, which may lead to new man-agement approaches for hematuria.

Source of Funding: NIH T32AI007290 (CF), the Society ofPediatric Urology (MH), NIH K08-DK087895 (MH), LucilePackard Foundation for Children’s Health (LPFCH) [MH], theStanford NIH CTSA (UL1 RR025744) [MH], the StanfordConsortium for Innovation, Design, Evaluation and Action(C-IDEA) [NIH RC4TW008781] [MH], a Stinehart/Reed Awardfrom the Institute for Stem Cell Biology and RegenerativeMedicine at Stanford(MH), the UBS Optimus Foundation(www.ubs.com/optimus) [MH], and NIH/NCRR UCSF-CTSIGrant Number UL1 RR024131

1147APPLICATION OF STATE-OF-THE-ART METHODS TO SEARCHFOR MICROBIAL CONTRIBUTIONS TO THE ETIOLOGY OFUROLOGICAL CHRONIC PELVIC PAIN SYNDROME (UCPPS)

J. Curtis Nickel*, Kingston, Canada; Alisa Stephens, Philadelphia,PA; Jun Chen, Boston, MA; Rachael Melton-Kreft, Tracy Spirk,Josh Earl, Mary O’Toole, J. William Costerton, Pittsburgh, PA;Adrie vanBokhoven, Denver, CO; Chris Mullins, Bethesda, MD;Garth Ehrlich, Pittsburgh, PA

INTRODUCTION AND OBJECTIVES: The presence of micro-organisms or an imbalance of the microbial ecology of the lower urinary

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tract has been implicated in UCPPS. We used culture-independentmolecular methods to identify the microbiota of the lower urinary tract inmen and women with UCPPS.

METHODS: Uniformly collected urine specimens were ob-tained in UCPPS and age matched healthy controls (including positivecontrols with CFS, FM, IBS). Specimens were tested by Ibis T-5000Universal Biosensor using BAC plate assay. Mass spectometric tech-nology measures unique molecular weights of multiple DNA ampliconsgenerated by PCR to detect the presence and identify the species of allbacteria. Differences in species composition for cases versus controlswere assessed using “distance measure” methods from microbial ecol-ogy. PERMANOVA and Cochran-Mantel-Haenszel methods were usedto test differences by cohort type in the presence and/or richness ofspecies. Testing, adjusted for sex, was completed for differences inoverall composition at the species, genus, and gram-stain level.

RESULTS: Baseline urine specimens were obtained from 257cases (161 female; 96 male) and 261 controls (164 female; 97 male).A total of 136 species (57 genera) were detected in VB1; VB2 con-tained 109 species (52 genera). Mean VB2 species count per personwas 2.50 and 2.29 among female UCPPS patients and controls respec-tively; 1.33 and 1.08 for males respectively. Trends were similar forVB1. Overall species composition was not significantly associated withcohort type at any level (p�0.198, 0.612 species level, p�0.313, 0.085genus level, p�0.279, 0.086 Gram-stain level in VB1 and VB2, respec-tively). At the species level in VB1, Lactobacillus gasseri and Strepto-coccus pneumoniae were more prevalent among cases than controls,while Staphylococcus capitis/caprae was underrepresented among cases(OR�3.74, p�0.007 L. gasseri, OR�0.263, p�0.011 S. capitis/caprae,OR�3.45,p�0.007 S. Pneumoniae). VB1 samples among cases werealso more likely to have gram-negative organisms than controls(OR�1.88, p�0.008), For VB2 the only statistical difference observed wasat the genus level for Corynebacterium (lower prevalence among UCPPSpatients compared to controls OR�0.34, p�0.011).

CONCLUSIONS: Despite observing some provocative trends,we did not find significant differences in the microbiome detected inlower urinary tract urine specimens from men and women with UCPPScompared to controls using state-of-the-art detection methods.

Source of Funding: NIH/NIDDK

1148THE ROLE OF MACROPHAGES IN CHRONIC BLADDERINFLAMMATION-ASSOCIATED GRANULOMA FORMATION,FIBROSIS, AND SEPSIS

Chi-Ling Fu, Stanford, CA; Kim Thai, Portland, OR; Justin Odegaard,Michael Hsieh*, Stanford, CA

INTRODUCTION AND OBJECTIVES: Chronic bladder inflam-mation leading to granuloma formation and fibrosis can be caused byvarious stimuli, including suture material, BCG, and urogenital schisto-somiasis (infection by Schistosoma haematobium worms). Althoughmacrophages are the defining feature of the granuloma and associatedfibrosis, the importance of these cells in bladder granuloma formationand fibrosis is poorly understood. We used the first tractable animal(mouse) model of urogenital schistosomiasis (published by us in PLOSPathogens) as a starting point for defining the role of macrophages inbladder granuloma formation and fibrosis.

METHODS: Mice underwent bladder wall injection with S.haematobium eggs or vehicle controls. Next, mice were administeredsystemic followed by intravesical liposomal clodronate (which selec-tively depletes macrophages) or control vehicle liposomes. Mice weresacrificed at various time points and their bladders harvested andsubjected to either flow cytometry or biochemical assays for fibrosis.

RESULTS: Serial microultrasonography revealed zones of de-creased echogenicity in the periphery of egg granulomas in mac-rophage-depleted versus -replete mice, suggestive of relative hypocel-lularity. This was confirmed by histology, biochemical assays forfibrosis, and flow cytometry, which revealed hypocellular peripheralzones in macrophage-depleted granulomas, less fibrosis (Figure 1A-B),

and fewer infiltrating leukocytes (Figure 1C). None of the controlvehicle-treated mice receiving eggs died, whereas 60% of macro-phage-depleted mice receiving high doses of eggs died by day 11post-egg injection, indicating an unexpected yet crucial role for macro-phages in prevention of bladder granuloma-associated sepsis (Figure 2).

CONCLUSIONS: Our results confirm that macrophages arecritical to bladder granuloma pathogenesis, even in the setting of asingle exposure to an inciting agent. This suggests that macrophagesmay be a suitable therapeutic target for bladder granuloma-associateddiseases.

Source of Funding: NIH T32AI007290 (CF), the Society ofPediatric Urology (MH), NIH K08-DK087895 (MH), LucilePackard Foundation for Children’s Health (LPFCH) [MH], theStanford NIH CTSA (UL1 RR025744) [MH], the StanfordConsortium for Innovation, Design, Evaluation and Action(C-IDEA) [NIH RC4TW008781] [MH], a Stinehart/Reed Awardfrom the Institute for Stem Cell Biology and RegenerativeMedicine at Stanford(MH), the UBS Optimus Foundation [MH],and NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131

1149BRILLIANT BLUE G, A P2X7 ANTAGONIST, ATTENUATESRENAL INFLAMMATION AND FIBROSIS IN UNILATERALURETERAL OBSTRUCTION IN RATS

José Monteiro Sad Pereira*, André Luis Barreira,Alberto Schanaider, Christina Maeda Takiya, Maurilo Leite Jr,Luis Carlos Miranda, Rio de Janeiro, Brazil

INTRODUCTION AND OBJECTIVES: Purinergic receptorshave been involved in inflammation and immunological response.These receptors expression have been demonstrated in granulocytes,monocytes/macrophages, dendritic cells and T and B lymphocytes.The present study investigated the effect of brilliant blue G (BBG), themost potent and selective irreversible antagonist, on rat kidneys afterunilateral ureteral obstruction (UUO).

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