Identification of Novel Therapeutic Targets in Overactive Bladder by Parallel Whole Transcriptome Analysis of Three Mouse Models
Scott R. Manson, PhD, Carlos AF Molina, MD, Paul F. Austin, MD.
Texas Children's Hospital, Houston, TX, USA.
BACKGROUND: Therapeutic options for overactive bladder (OAB) are critically lacking as it is exceedingly difficult to obtain patient tissue samples for research. While various forms of acute bladder injury mimic the OAB phenotype in mice, the relevance of these models is unclear since OAB in humans is largely idiopathic. In this study, we applied an integrative transcriptomics approach to analyze three disparate models of OAB and then differentiate molecular changes that are stimulus-specific from shared mechanisms likely to contribute to idiopathic OAB. We next used computational methods to predict downstream mechanisms and create a model of disease progression in OAB. This study revealed novel therapeutic targets and treatment strategies to evaluate in mouse models and validate in patients with OAB. METHODS: Disease progression was compared in three murine models of acute bladder injury (n=6/group): [1] mechanical injury by bladder outlet obstruction (BOO), [2] chemical injury by cyclophosphamide administration (CYP), and [3] inflammatory injury by intravesical instillation of lipopolysaccharide (LPS). Whole transcriptome analysis of 14,742 genes was performed by RNA-seq. This data was then used to conduct ingenuity pathway analysis of 204 signaling pathways, 1,219 molecular functions, and 4,935 biological processes. Findings were validated by histological methods and assessment of bladder function (micturition analysis, cystometry). RESULTS: Whole transcriptome analysis yielded high-quality sequencing data with a mean depth of 32.9M reads per sample and 90.8% reads successfully aligning to the reference genome. Each of the three OAB models exhibited significant changes in gene expression, ranging from 2.0-11.5% of all genes. Although most were stimulus-specific, we identified 97 genes that contribute to the progression of OAB in all three models. The development of an OAB phenotype was paralleled by a 38.9-fold increase in immune cells expressing the CXCR2 chemokine receptor and dramatic increases in four isoforms the CXCR2-activiating cytokines required for recruiting these cells: CXCL1 (+40.4x), CXCL2 (+38.9x), CXCL3 (+47.3x), and CXCL5 (+22.1x). Ingenuity pathway analysis showed that the OAB phenotype is paradoxically associated with a loss of smooth muscle contractility (-15.5x), development (-11.9x), and differentiation (-4.3x). This phenotypic transition was paralleled by increases in ribosome biogenesis (+15.2x), protein synthesis (+9.8x), cellular hypertrophy (+1.4x) and proliferation (+1.9x), and production of collagen (+2.4x) and cytokines (+24.9). [All results are p<0.05] CONCLUSIONS: This study reveals that the dyscoordination of bladder function in OAB involves a dramatic increase in the expression of pro-inflammatory CXCL chemokines and phenotypic switching of bladder smooth muscle cells from a differentiated ‘contractile state’ to a dedifferentiated ‘synthetic state’. This model is supported by studies which have shown that similar plasticity exists in disorders of vascular smooth muscle cells. Accordingly, therapeutic strategies designed to restore smooth muscle differentiation and suppress inflammation have a strong potential to improve outcomes in OAB.
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