Expression of Sphingosine-1-Phosphate Pathway in Human Overactive Bladder: A Pilot

Objectives: The pathophysiological mechanisms underlying overactive bladder (OAB) are poorly understood. Detrusor smooth muscle (DSM) contractility may play a role in symptomatology. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose effects on mammalian DSM include enhancing contractility via interaction with Rho-kinase. In rats with iatrogenic induced detrusor overactivity (DO), components of the S1P pathway are upregulated. In rabbits, S1P enhances bladder contractility. Our primary aim is to assess differences in S1P pathway expression between human OAB DSM and normal controls. Secondarily, we aim to establish the feasibility of bladder washings as a less invasive tissue collection technique to assess bladder S1P activity.

Methods: Bladder biopsies and washings were collected at routine diagnostic cystoscopy during pelvic surgery, for 11 patients with OAB (according to scores on OABQ-SF) and 11 control patients. Clinical and intraoperative data was collected from chart review. TRIzol reagent was used to extract mRNA from specimens, and after converting transcripts to cDNA, quantitative RT-PCR was performed using SYBR green dye. An OAB specimen was run alongside a control specimen in each PCR run to minimize variation. Expression of S1P receptors S1PR1, S1PR2, S1PR3, and S1P phosphorylating enzymes SPHK1 and SPHK2 was normalized to 2 housekeeping genes, GAPDH and β-actin. Delta-delta Ct method was used to determine the fold-difference in expression of gene products between OAB and control patients. Unpaired t-test and Chi-squared test were used to determine significance.

Results: 22 subjects' specimens were included in laboratory analyses. On average, subjects were 57 years old at the time of biopsy collection. 82% were white, and BMI did differ significantly between the groups (34.2 OAB vs. 26.8 control, p=0.010). As expected, UDI (72.9 vs 29.63, p=0.0002), UIQ-7 (54.76 vs 25.29, p=0.027), and OAB-Q scores (23.6 vs 11.5, p=.00015) were significantly higher in the OAB group. When normalized to GAPDH, the expression of S1PR1, S1PR2, S1PR3, SPHK1, and SPHK2 was increased in bladder biopsies of OAB patients, with respective mean relative fold-differences of 1.95, 3.60, 2.15, 1.83, and 1.90. A similar pattern remained when normalized to β-actin, with mean relative fold differences of 0.98 (S1PR1), 2.87 (S1PR2), 1.71 (S1PR3), 3.91 (SPHK1), and 4.06 (SPHK2). Gene expression was detected in bladder washings, but results were heterogeneous.

Conclusions: Our study demonstrated the feasibility of studying gene expression in extremely small samples from human bladder biopsy. A protocol was established for RNA extraction, transcription to cDNA, and relative quantitation using rtPCR. Results suggest that components of the S1P pathway are up-regulated in DSM of patients with OAB, but washings may not be reliable for this assay. Next steps include protein assays to confirm gene expression is indicative of enzyme up-regulation, up-scaling to verify findings, and inclusion of urine samples to evaluate S1P as a potential biomarker for OAB.