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 Ca²⁺ 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 immunoreactivity was downregulated in CYP-injected detrusor muscle. Apamin (a SK blocker) sensitivity on spontaneous contractile activity was decreased in CYP-injected mice compared to saline-injected 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)

Keywords: Interstitial cells; cystitis; fluorescence-activated cell sorting; gene expression; ex vivo cystometry; in vitro contractility