AB315. SPR-42 Cyclophosphamide-induced overactive bladder via downregulation of relaxation factors in detrusor PDGFRα+ cells

Objective: Morphology and functional role of PDGFRα cells have been recently characterized in the detrusor muscle layer. Detrusor relaxation is caused by activation of small conductance Ca activated-K (SK) channels and purinergic inhibitory responses in detrusor PDGFRα cells. Loss of PDGFRα cells or alteration of P2Y receptors and SK channels will affect detrusor excitability. Cyclophosphamide (CYP)-treated animals exhibited overactive bladder (OAB). We hypothesized that the downregulation of P2Y receptors and/or SK channels in PDGFRα cells will display the phenotype of CYP-induced OAB. Methods: CYP was injected intraperitoneally in PDGFRα/ eGFP and SMC/eGFP mice. We harvested the detrusor muscle without urothelium and disperse the cells for the fluorescence activated cell sorting (FACS). Sorted PDGFRα cells and smooth muscle cells (SMCs) were used for molecular study to compare the changes in transcripts between CYP-injected and control group. Transcripts were examined included; Pdgfrα, P2ry1, P2ry2, P2ry4, Kcnn1, Kcnn2, Kcnn3 and inflammation marker (Il-6). Immunohistochemistry, mechanical contractility and ex vivo cystometry were also performed. Results: Quantitative analysis of PCR revealed that CYP-injected detrusor muscle increased transcriptional expression of Il-6, but decreased the expression of Pdgfrα. Transcriptional changes in CYP-injected sorted PDGFRα cells from PDGFRα/eGFP mice showed Pdgfα, Kccn3 (SK3), P2ry1, P2ry2 and P2ry4 genes were decreased compared with saline-injected control. Sorted SMCs from SMC/eGFP mice did not show significant expression of those genes and no detectable changes. Immunohistochemistry showed SK3 in PDGFRα immunoreact iv i ty was downregulated in CYP-injected detrusor muscle. Apamin (a SK blocker) sensitivity on spontaneous contractile activity was decreased in CYP-injected mice compared to salineinjected mice. In ex vivo cystometry, increased spontaneous non-voiding contractions and less apamin sensitivity were observed in CYP-injected mice. Conclusions: These findings are the first report to investigate the role of PDGFRα cells in relation to OAB mechanisms. In conclusion, we found that CYP-induced OAB is resulted from down regulation of PDGFRα, P2Y receptors and SK channels in CYP-injected bladder. These results provide novel mechanisms of functional role of PDGFRα cells on OAB. Funding Source(s): NIDDK, RO1 DK098388 and Urology Care Foundation Research Scholar Award (Interstitial Cystitis Association)