Diabetes mellitus (DM) is often associated with lower urinary tract dysfunction (LUTD). Although streptozotocin (STZ)-induced DM rats have been used as a rodent model of the LUTD, this is an acutely-induced, severe DM model with high glucose levels, and may not be suitable for studying the physiological condition encountered in DM patients, especially those with type II DM that is induced insufficient insulin production with insulin resistance. Thus, this study first sought to develop a more physiological rat model of DM by using low-dose insulin administration to maintain the moderately-high blood glucose level, thereby minimizing the initial diuretic effects. Also, DM is known to impair nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-dependent urethral relaxation mechanisms during micturition, resulting in inefficient voiding together with DM cystopathy [1-3]. Thus, secondly, this study examined the effects of soluble guanylate cyclase (sGC), which can increase cGMP production, independent of NO, inside the cell, on bladder and urethral dysfunctions in STZ-induced DM rats.

Female Sprague-Dawley rats (11-12 weeks) were used, and DM was induced using a single intraperitoneal injection of STZ (65 mg/kg). We divided rats into four groups: (1) non-DM (N) group, (2) DM with low-dose insulin (DI) group, (3) non-insulin DM with vehicle (D) group, and (4) non-insulin DM with sGC (GC) group. In insulin-treated DM rats (Group DI), slow-releasing insulin pellets (2 units/24 hours) were implanted 3 days after inducing DM. In Group GC, a sGC activator (BAY 60-2770,1 mg/kg/day) was orally administered from 6 to 8 weeks after inducing DM. First, in Groups DI and D, we performed 24-h voiding assay at 2, 4, and 8 weeks, cystometry and urethral perfusion pressure (UPP) recordings at 8 weeks with or without low-dose insulin treatment, and compared the data with those in Group N. In cystometry, we measured opening pressure (OP, pressure at which the urethra opens and urine flow starts), intercontraction intervals (ICI), the number of non-voiding contractions (NVCs) per minute, postvoid residual (PVR), bladder capacity, bladder compliance, and voiding efficiency [1]. NVC was defined as an increase in intravesical pressure of more than 8 cmH2O above the baseline. In UPP recordings, we measured urethral pressures (UP) at which urethra started to relax (UPUR), UP nadir which is the lowest pressure during urethral relaxation, UP reduction which is difference between UPUR and UP nadir, and high-frequency oscillation (HFO) amplitude of UP during voiding [2]. Secondly, we evaluated the effects of sGC treatment on the cystometric and UPP parameters at 8 weeks of DM in Groups D and GC. Thirdly, in a separate group of animals, the urethra and the bladder were harvested at 8 weeks of DM to evaluate the mRNA levels various markers using real-time PCR, which included NO-related markers such as phosphodiesterase type 5 (PDE5), multidrug resistance protein 5 (MRP5) and Ca2+ channels, ischemia markers such as hypoxia-inducible factor 1 alpha (HIF-1α), and inflammatory markers such as transforming growth factor beta 1 (TGF-β1) and tumor necrosis factor alpha (TNFα). All values are expressed as means ± standard deviations. We used the Kruskal-Wallis one-way analysis of variance to analyze statistical differences and Dunn’s post hoc test between in Groups N, DI, and D, and the Mann Whitney U test to evaluate statistical differences between in Groups D and GC. A P-value < 0.05 was considered statistically significant.

Moderately-high levels of blood glucose (292.3 ± 59.5 mg/dL) were maintained in Group DI vs. Group D (495.6 ± 7.6 mg/dL). In voiding assay, 24-h voided volume was significantly higher in Group D than in Group N at 2, 4, and 8 weeks (115.0 ± 24.2 vs 6.4 ± 3.2 mL, 88.3 ± 28.7 vs 5.7 ± 2.0 mL, and 82.8 ± 17.0 vs 9.8 ± 3.5 mL, respectively). In cystometry, OP, NVCs per minute, and PVR were significantly higher in Group D than in Groups N and GC (OP: 43.2 ± 11.0 vs 24.3 ± 2.7 and 30.4 ± 8.2 cmH2O, NVCs: 0.2 ± 0.1 vs 0.0 ± 0.0 and 0.0 ± 0.0 number/min, PVR: 0.2 ± 0.1 vs 0.0 ± 0.0 and 0.1 ± 0.0 mL) (Fig. 1A, B). Voiding efficiency was significantly lower in Group D than in Groups N, DI, and GC (91.6 ± 3.5 vs 99.4 ±1.1, 99.2 ± 0.8, and 97.3 ± 0.7 %). In UPP recordings, UPUR was significantly lower in Group D than in Groups DI and GC (21.6 ± 3.0 vs 32.5 ± 5.9 and 33.5 ± 9.2 cmH2O). UP reduction and HFO amplitude were significantly lower in Group D than in Groups N and GC (UP reduction: 6.7 ± 4.1 vs 15.5 ± 2.3 and 15.5 ± 5.1 cmH2O, HFO a: 1.1 ± 0.4 vs 4.0 ± 2.0 and 3.5 ± 1.8 cmH2O) (Fig. 1C, D). In addition, mRNA expression levels of Ca2+ channels and PDE5 in the urethra were significantly higher in Group D than in Groups N and GC. MRP5 in the urethra was significantly lower in Group GC than in Group D (Fig. 2A). mRNA expression levels of HIF-1α, TGF-β1, and TNFα in the bladder were significantly higher in Group D than in Groups N, DI, and GC (Fig. 2B).

This is the first report to examine the effects of sGC on DM-induced LUTD in diabetic rats. Previous studies in rodent diabetes models demonstrated that diabetic urethral dysfunction was associated with impaired NO-mediated urethral relaxation mechanisms including reduced NO synthase activity in rats [3]. In this study, the sGC treatment, which can directly increase cGMP production independent of NO, improved bladder overactivity evident as reduced NVCs as well as urethral relaxation during bladder contraction, as evidenced by an increase in UP reduction in 8-weeks DM rats. Molecular studies also showed that the sGC treatment reduced mRNA expression of PDE5 (a cGMP degrading enzyme), MRP5 (a multidrug resistance protein to transport cGMP out of the cell) and Ca2+ channels, suggesting that sGC activation increases cGMP accumulation and reduces muscle contractility. Also, HFO activity of the urethra, which represents the pumping activity of external urethral sphincter during voiding, was increased after sGC treatment in 8-weeks DM rats, suggesting the further improvement of bladder emptying during micturition. Moreover, in this study, the sGC treatment seems to be effective in reducing ischemia and inflammatory changes in the bladder, evident as decreased expressions of HIF-1α, TGF-β1, and TNFα after the treatment. STZ-induced DM rats have been used as a DM model with LUTD. However, diuresis-induced bladder overdistention due to high blood glucose levels significantly contributes to LUTD, especially bladder overactivity in the early phase in STZ-DM rats. The results of this study revealed that the low-dose insulin treatment can control the blood glucose concentration at a moderately-high level, and significantly reduced urine overproduction and prevented the progression of bladder overactivity, urethral dysfunction and inefficient voiding during 8 weeks of DM.

Low-dose insulin-treated DM rats with a lesser degree of diuresis-induced bladder overdistention would be a useful model for studying the natural progression of DM-induced LUTD. Activation of sGC, which can increase cGMP levels inside the cell, would be an effective option for the treatment of DM-induced LUTD including bladder overactivity and inefficient voiding due to impaired urethral relaxation.

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