PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats

Takafumi Kabuto; So Inamura; Hisato Kobayashi; Xinmin Zha; Keiko Nagase; Minekatsu Taga; Masaya Seki; Nobuki Tanaka; Yoshinaga Okumura; Osamu Yokoyama; Naoki Terada

doi:10.1371/journal.pone.0301883

. 2024 Sep 18;19(9):e0301883. doi: 10.1371/journal.pone.0301883

PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats

Takafumi Kabuto ¹, So Inamura ¹, Hisato Kobayashi ¹, Xinmin Zha ¹, Keiko Nagase ¹, Minekatsu Taga ¹, Masaya Seki ¹, Nobuki Tanaka ¹, Yoshinaga Okumura ¹, Osamu Yokoyama ¹, Naoki Terada ^1,^*

Editor: Yung-Hsiang Chen²

PMCID: PMC11410213 PMID: 39292699

Abstract

Purpose

Bladder dysfunction associated with type 2 diabetes mellitus (T2DM) includes urine storage and voiding disorders. We examined pathological conditions of the bladder wall in a rat T2DM model and evaluated the effects of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil.

Materials and methods

Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats were used as the T2DM and control groups, respectively. Tadalafil was orally administered for 12 weeks. Micturition behavior was monitored using metabolic cages, and bladder function was evaluated by cystometry. Bladder blood flow was evaluated by laser speckle imaging, and an organ bath bladder distention test was used to measure adenosine triphosphate (ATP) release from the bladder urothelium. The expression levels of vesicular nucleotide transporter (VNUT), hypoxia markers, pro-inflammatory cytokines and growth factors in the bladder wall were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Bladder wall contractions in response to KCl and carbachol were monitored using bladder-strip tests.

Results

With aging, OLETF rats had higher micturition frequency and greater urine volume than LETO rats. Although bladder capacity was not significantly different, non-voiding bladder contraction occurred more frequently in OLETF rats than in LETO rats. Bladder blood flow was decreased and ATP release was increased with higher VNUT expression in OLETF rats than in LETO rats. These effects were suppressed by tadalafil administration, with accompanying decreased HIF-1α, 8-OHdG, IL-6, TNF-α, IGF-1, and bFGF expression. The impaired contractile responses of bladder strips to KCl and carbachol in OLETF rats with aging were restored by tadalafil administration.

Conclusions

The T2DM rats had polyuria, increased ATP release induced by decreased bladder blood flow and impaired contractile function. PDE5 inhibition improved these changes and may prevent T2DM-associated urinary frequency and bladder storage and voiding dysfunctions.

Introduction

Many epidemiological studies have shown that non-urological disorders such as hypertension, type 2 diabetes mellitus (T2DM), and dyslipidemia are associated with lower urinary tract symptoms in both men and women [1]. Lifestyle factors, and especially the presence of T2DM, can affect lower urinary tract function. In T2DM, lower urinary tract dysfunction has been evaluated using urodynamic studies, revealing detrusor overactivity (DO) in 55%–61% of individuals with T2DM, detrusor underactivity (DU) in 9%–23%, and areflexia in 9%–23% [2, 3]. Moreover, DO and DU are mixed in females with T2DM [4]. According to a review by Daneshgari et al., a mixture of lower urinary tract disorders can be present in T2DM, often with storage disorders in the early stages and voiding disorders in the later stages [5].

Clinical and basic studies suggest that atherosclerosis in both men and women induces decreased bladder blood flow, leading to chronic ischemia of the bladder [6]. Chronic bladder ischemia causes oxidative stress, resulting in bladder denervation and the two in the bladder wall, and leading to DO that progresses to DU. However, the mechanisms underlying how chronic bladder ischemia causes the development of DO have not yet been elucidated. In our earlier study of salt-sensitive hypertensive rats, hypertensive-related bladder ischemia resulted in a decrease in bladder capacity and an increased release of adenosine triphosphate (ATP) from the bladder urothelium [7]. We thus ask the question: does the same mechanism underlie the development of DO in other T2DM models? Most of the research on animal models of T2DM has been conducted using rabbits and rats in which arteriosclerosis was artificially created; few basic studies using T2DM models have been published. We have used Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a pathological model of T2DM and Long-Evans Tokushima Otsuka (LETO) rats as a control. The OLETF rat model develops insulin resistance by 12 weeks of age, hyperinsulinemia by 25 weeks of age, and subsequently has decreased insulin levels [8]. Hyperglycemia is maintained throughout the disease course. The plasma insulin and glucose levels of 36- and 48-week-old OLETF and LETO rats are described in our previous report [9]. The features of this pathological model closely resemble the natural history of human T2DM.

Using this rat model of T2DM, we have previously reported that T2DM-induced chronic ischemia leads to oxidative stress, resulting in prostate enlargement through the upregulation of several cytokines. Treatment with the phosphodiesterase-5 (PDE5) inhibitor tadalafil improves prostate ischemia and might prevent its enlargement via the suppression of cytokines and growth factors [10]. Bladder ischemia-induced chronic inflammation may also be associated with the development of DO and DU [11]. In the present study, we therefore aimed to elucidate (i) the mechanisms of DO and DU development in T2DM, and (ii) whether DO and DU can be improved by long-term treatment with tadalafil.

The bladder urothelium has an important role in mechanosensory transduction [12, 13]. In response to mechanical stimuli, ATP is released from epithelial cells and activates purinergic receptors on submucosal afferent fibers, thus facilitating bladder sensory signaling [14]. It has been reported that ATP release is mainly regulated by vesicular nucleotide transporter (VNUT) in the bladder urothelium [15]. Furthermore, although bladder ischemia results in an increase in pro-inflammatory cytokines and growth factors, it remains unknown how this phenomenon is involved in bladder function. In the current study, we therefore evaluated bladder blood flow; ATP release from the bladder urothelium; bladder expression levels of VNUT, hypoxia markers, and pro-inflammatory cytokines and growth fators; and bladder contraction using the T2DM rat model with tadalafil treatment.

Materials and methods

Animal preparation

Four-week-old male OLETF and LETO rats were purchased from SLC Inc. (Shizuoka, Japan). LETO rats are a control strain that do not have characteristics of T2DM. The animals were housed at the University of Fukui Animal Center at a constant temperature of 23°C and 50%–60% humidity with a normal 12-hr light/dark schedule. Tap water and standard rat chow were freely ingested. All animal experiments were conducted in accordance with the guidelines established by the Fukui University Committee for Animal Experimentation (Permission no: R01056).

Oral tadalafil (100 μg/kg/day) was administered to the OLETF and LETO rats for 12 consecutive weeks, beginning when the rats were 36 weeks old; the sham group received only the vehicle for 12 weeks. The experimental dose (100 μg/kg/day) was set based on the standard treatment dose of tadalafil in Japanese patients (5 mg/day) and the average body weight in Japan (60 kg), leading to a calculated standard dose of 83 μg/kg.

In total, 54 rats were divided into the following nine groups (6 rats/group), as depicted in Fig 1A: one group each of L-36 (36-week-old LETO rats), L-48 (48-week-old LETO rats treated with vehicle for 12 weeks), and L-48(t) (48-week-old LETO rats treated with tadalafil for 12 weeks), and two groups each of O-36 (36-week-old OLETF rats), O-48 (48-week-old OLETF rats treated with vehicle for 12 weeks), and O-48(t) (48-week-old OLETF rats treated with tadalafil for 12 weeks).

Micturition behavior

A metabolic cage was used to measure the voiding parameters of the rats. Rat urine was collected through a urine collection funnel and weighed on an electronic balance. The cumulative weight of the collected urine was recorded every 10 min. The rats were kept in the metabolic cages for approximately 60 hr to acclimate to the cage, and the values recorded in the last 24 hr of that period were used. Each monitoring period started at 18:00. The data collected were used to calculate the mean voided volume and the number of micturitions per 24 hr. Residual urine volume was examined by ultrasonography.

Cystometry in conscious rats

Cystometry procedures in conscious rats were performed as our previous reports [16]. Prior to cystometry, a catheter was surgically implanted into the bladder of each rat. The catheter was made of polyethylene tubing and was inserted via a lower abdominal incision. After surgery, sufficient time was provided for the rat to recover from anesthesia. Next, he bladder catheter was connected to a pressure transducer via a T-tube. The pressure transducer was used to convert changes in bladder pressure into electrical signals that were recorded. Cystometry was done with physiological saline at room temperature at 0.04 ml per minute. Bladder capacity and bladder contraction pressure were measured.

Laser speckle blood flow imaging system

The blood flow of the bladders of LETO and OLETF rats was evaluated using a laser speckle blood flow imaging system, the Omegazone™ (OZ-2, Omegawave Inc., Tokyo, Japan), as described by Forrester et al. [17]. Each rat was anesthetized using halothane. The bladder was exposed and a catheter was then inserted through the bladder dome. The catheter was connected to the infusion pump, and saline was infused into the bladder until the pressure rose to 10 cm H₂O. The bladder surface was diffusely irradiated with a 780-nm semiconductor laser, and the scattered light was treated with a hybrid filter and detected using a charge-coupled device camera. A single blood flow image was generated by averaging the numbers obtained from 20 consecutive raw speckle images. The total blood flow of the entire bladder was then calculated by summing the values on one side and those on the other side of the surface of the bladder.

Organ bath bladder distention test

After rats were sacrificed by decapitation at 48 weeks of age, the bladder and urethra were removed and weighed. The amount of ATP released from the stretched bladder urothelium was then measured according to the method of Tanaka et al. [18] with slight modifications. Briefly, one end of the infusion catheter was inserted through the urethra into the bladder, and the bladder neck was secured with surgical sutures. The bladder urothelium was rinsed three times with 0.3 mL of Krebs solution before the catheter was connected to the infusion pump and pressure transducer. The bladder was then fixed vertically in a 10-mL organ bath in which 0.3 mL of Krebs solution was gasified with 5% CO₂ and 95% O₂ at 37°C. Next, 1.5 mL of Krebs solution was infused into the bladder at a rate of 0.04 mL/s. The solution in the bladder was then collected on ice using gravity. ATP was measured using the ATPlite™ luciferin-luciferase assay with a Fusion luminometer (Perkin Elmer, Waltham, MA, USA) according to the manufacturer’s instructions. The amount of ATP released was converted to the concentration per mg of tissue.

Real-time polymerase chain reaction (PCR)

The bladders of OLETF rats (O-36, O-48, and O-48(t)) were used to measure mRNA expression. After the rats were sacrificed by decapitation, bladder tissue was cut into small pieces and ground into powder using a mortar and pestle under liquid nitrogen. Total RNA was isolated from the tissue using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For single-strand complementary DNA synthesis, 2 μg of total RNA was used with the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, in a final volume of 20 μL. The reverse transcription step was at 37°C for 60 min, followed by an inactivation step at 95°C for 5 min. The mRNA expression was quantitatively analyzed using the SYBR green fluorescence method with an ABI 7300 Real-Time PCR System (Applied Biosystems), with glyceraldehyde 3-phosphate dehydrogenase as an internal control. Differences in the mRNA expression of VNUT, hypoxia-inducible factor-1 alpha (HIF-1α), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF) in the bladder were determined. Each fold change was calculated and used for the statistical analysis. The forward and reverse primers are listed in S1 Table.

Enzyme-linked immunosorbent assay (ELISA)

Bladder tissue (15 mg) was cut into small pieces and ground into powder using a mortar and pestle under liquid nitrogen. The soluble and insoluble protein fractions were extracted using the CelLytic™ MEM Protein Extraction Kit (Sigma-Aldrich Japan, Tokyo, Japan) and the CelLytic™ NuCLEAR™ Extraction Kit (Sigma-Aldrich Japan), respectively, according to the manufacturer’s instructions. IL-6, TNF-α, IGF-1, and bFGF were measured using ELISA with the Rat IGF-1 ELISA Kit (ab213902, Abcam, Cambridge, UK), Rat IL-6 ELISA Kit (ab100772, Abcam), Rat TNF alpha ELISA Kit (ab100785, Abcam), Rat bFGF ELISA Kit (Invitrogen, Waltham, MA, USA), and AssayMax™ Dihydrotestosterone ELISA Kit (Assaypro, St. Charles, MO, USA), respectively. Protein concentrations were calculated as picograms of protein per milligram of tissue wet weight.

8-hydroxy-2’-deoxyguanosine (8-OHdG) measurement

For the measurement of 8-OHdG, bladder tissue was homogenized with 0.1 M phosphate buffer containing 1 mM ethylenediaminetetraacetic acid. After centrifugation for 10 min, the supernatant was purified using DNeasy Blood & Tissue Kits (Qiagen) according to the manufacturer’s instructions; this was followed by DNA digestion using Nuclease P1 (Wako, Tokyo, Japan). After the addition of 1 unit of alkaline phosphatase (Wako) per 100 μg of DNA and incubation at 37°C for 30 min, the samples were boiled for 10 min and kept on ice until use. The levels of 8-OHdG were determined using an 8-OHdG ELISA kit (ab201734, Abcam).

In vitro bladder-strip experiments

For the in vitro bladder-strip experiments, OLETF rats (i.e., O-36, O-48, and O-48(t) rats) were used; these were different from the rats used for the investigation of ATP release from the bladder urothelium. After the rats were sacrificed by decapitation, full-thickness longitudinal bladder strips (7 mm long × 3 mm wide) were excised and mounted in a 10-mL organ bath containing Krebs solution at 37°C and continuously bubbled with 95% O₂ and 5% CO₂. The strips were equilibrated under a resting tension of 1 g for 45 min, and the Krebs solution was changed every 15 min. Following equilibration, the strips were exposed to a solution containing high K⁺ (62 mM KCl) to normalize the contractile force. After washout of the KCl solution, the isometric contractions of the strips caused by the cumulative application of carbachol (3×10⁻⁷, 10⁻⁶, 3×10⁻⁶, 10⁻⁵, and 3×10⁻⁵ M) were recorded via force transducers (Nihon Kohden, Tokyo, Japan) and Labchart & Scope software (ADInstruments, Colorado Springs, CO, USA). After the experiment, the bladder strips were weighed to normalize the contractile force.

Data analysis

The results are presented as the mean ± standard error of the mean (SEM). All data were analyzed with analysis of variance (ANOVA) and independent t-tests using SPSS for Windows, Version 16.0 (SPSS Inc., Chicago, IL, USA). For all analyses, p<0.05 was considered significant.

Results

Body weights, bladder wet weights, and micturition characteristics of LETO and OLETF rats

The respective body weights of the LETO and OLETF rats (n = 6, each) were 546 ± 28 g and 671 ± 31 g (p<0.01) at 36 weeks, and 557 ± 27 g and 658 ± 32 g (p<0.01) at 48 weeks; they were significantly higher in the OLETF rats. The respective bladder wet weights of the LETO and OLETF rats were 86 ± 3 mg and 175 ± 7 mg (p<0.01) at 36 weeks, and 103 ± 5 mg and 196 ± 14 mg at 48 weeks (p<0.01); they were significantly higher in the OLETF rats. In both groups, tadalafil treatment did not change the body weight or bladder wet weight of the rats. The mean voided urine volume per body weight was not significantly different between the LETO and OLETF rats (2.7 ± 0.2 mL and 2.8 ± 0.6 mL, respectively; p = 0.678) and did not change with age or tadalafil treatment.

Cystometry in conscious rats was performed in the LETO and OLETF rats (n = 2, each) at 36 weeks. The bladder capacity (0.7 ± 0.1 mL and 0.7 ± 0.2 mL, p = 0.37), and the bladder contraction pressure (39.3 ± 10.7 cmH₂O and 33.4 ± 10.3 cmH₂O, p = 0.16) were not significantly different between LETO and OLETF rats. Non-voiding contractions appeared only in OLETF rats and not appeared in LETO rats (Fig 1B). There was no residual urine in OLETF or LETO rats during the experiments.

The total urine volume per 24 hr was significantly higher in the OLETF rats than in the LETO rats (n = 6, each) at both 36 (10.4 ± 1.8 mL and 4.8 ± 2.3 mL, p<0.01) and 48 (18.5 ± 3.8 mL and 4.2 ± 0.7 mL, p<0.01) weeks (Fig 1C). The frequency of micturition per 24 hr was also significantly higher in the OLETF rats than in the LETO rats at both 36 (5.9 ± 1.7 and 3.3 ± 1.6, p<0.01) and 48 (10.9 ± 2.4 and 2.9 ± 0.4, p<0.01) weeks (Fig 1D).

The total urine volume and frequency of micturition in the OLETF rats were significantly higher at 48 weeks than at 36 weeks (p<0.01). Furthermore, tadalafil treatment significantly decreased the total urine volume (from 18.5 ± 3.8 mL to 10.7 ± 2.0 mL, p<0.05) and frequency of micturition (from 10.9 ± 2.4 to 6.0 ± 1.2, p<0.05) at 48 weeks in the OLETF rats. By contrast, the total urine volume and frequency of micturition in the LETO rats were not significantly different between 36 and 48 weeks, and were unchanged by tadalafil treatment (Fig 1C, 1D).

ATP production and VNUT expression in the bladder

To clarify the mechanisms underlying the differences in bladder contraction between OLETF and LETO rats, we measured the ATP concentrations released from the bladder urothelium using the organ bath bladder distention test (n = 3, each) (Fig 2A). The ATP concentrations of the OLETF rats were significantly higher than those of the LETO rats (p<0.01), and ATP concentrations were significantly decreased by tadalafil treatment in both LETO (p<0.05) and OLETF (p<0.01) rats (Fig 2B). These results indicate that ATP production from the bladder urothelium is associated with frequent bladder contraction in OLETF rats, and is improved by tadalafil treatment.

Fig 2 — A: Organ bath bladder distention test. A whole bladder was set in an organ bath and injected with saline, which was collected using gravity after the syringe was removed. Republished from ref. No. 17 under a CC BY license, with permission from Osamu Yokoyama, original copyright in 2011. B: Changes in ATP levels released from the stretched bladder urothelium of LETO and OLETF rats at 48 weeks under distention, with 1.5 mL of Krebs solution at approximately 0.04 mL/s. C: Bladder VNUT mRNA levels relative to those at 36 weeks in LETO rats. Data are shown as the mean ± SEM. *p<0.05, **p<0.01.

To clarify the mechanisms associated with the changes in ATP production, we evaluated the bladder expression of VNUT. At 36 weeks, VNUT expression was significantly higher in the OLETF rats than in the LETO rats (p<0.05) (Fig 2B); at 48 weeks, VNUT expression was significantly increased in both LETO (p<0.05) and OLETF (p<0.01) rats. Moreover, VNUT expression was significantly decreased by tadalafil treatment in both LETO (p<0.01) and OLETF (p<0.01) rats. These results suggest that changes in bladder VNUT expression might be associated—at least in part—with ATP production in the bladder urothelium.

Changes in bladder blood flow measured by laser speckle blood flow imaging

Blood flow to the bladder was measured using the Omegazone laser speckle blood flow imaging system just before the bladder was extracted from each rat (n = 3, each) (Fig 3A). The blood flow is displayed as changes in laser frequency in the speckle imaging. The Omegazone uses pixels of different colors: blue indicates areas with poor blood flow and yellow indicates areas with high blood flow (Fig 3B). The imaging results revealed that blood flow was significantly lower at 48 weeks than at 36 weeks in both LETO (p<0.01) and OLETF (p<0.005) rats. At 48 weeks, blood flow was significantly lower in the OLETF rats than in the LETO rats (p<0.01). Tadalafil treatment produced significant increases in blood flow to the bladder in both the LETO (p<0.01) and OLETF (p<0.05) rats (Fig 3C). These results indicate that, in older rats, blood flow to the bladder is lower in OLETF rats than in LETO rats, and is increased by tadalafil treatment.

Fig 3 — A: Image of a rat bladder dissected and exposed under anesthesia. B: Laser speckle image of a rat bladder. C: Comparisons of bladder blood flow among the six rat groups. Blood flow is expressed as a percentage; the value of the L-36 rats was considered 100% (i.e., the levels are expressed relative to the levels at 36 weeks in LETO rats). Six samples were included in each group. Data are shown as the mean ± SEM. *p<0.05, **p<0.01.

Expression levels of HIF-1α and 8-OHdG in the bladders of OLETF rats

To evaluate bladder hypoxia induced by changes in blood flow, we evaluated HIF-1α expression levels in the bladders of OLETF rats (n = 3, each); HIF-1α expression was significantly higher at 48 weeks than at 36 weeks (p<0.01). Although HIF-1α expression tended to be decreased by tadalafil treatment, this difference was not significant (Fig 4A). 8-OHdG is an oxidative stress marker that is made when DNA is damaged by reactive oxygen species. In the bladders of OLETF rats, 8-OHdG levels were significantly higher at 48 weeks than at 36 weeks (p<0.01) and were significantly decreased by tadalafil treatment (p<0.01) (Fig 4B). These results indicate that hypoxia is induced by decreased blood flow in the bladders of older OLETF rats.

Fig 4 — Changes in HIF-1α **(A)** and 8-OHdG **(B)** mRNA levels in the bladders of OLETF rats at 36 weeks and at 48 weeks with and without tadalafil. Six samples were included in each group, and two replicates were performed for each sample. Data are shown as the mean ± SEM. *p<0.05, **p<0.01.

mRNA and protein expression of pro-inflammatory cytokines and growth factors in the bladders of OLETF rats

In OLETF rats (n = 3, each), we evaluated changes in pro-inflammatory cytokines (IL-6 and TNF-α) and growth factors (IGF-1 and bFGF) that are associated with bladder inflammation. The mRNA expression levels of IL-6, TNF-α, IGF-1, and bFGF were significantly higher at 48 weeks than at 36 weeks (p<0.01) and tended to be decreased by tadalafil treatment, although these apparent decreases were only significant for TNF-α (p<0.01) (Fig 5A). The protein expression levels of IL-6, TNF-α, IGF-1, and bFGF were also significantly higher at 48 weeks than at 36 weeks (p<0.01), and tended to be decreased by tadalafil treatment; these differences were significant for IL-6, TNF-α, and IGF-1 (p<0.05 or p<0.01). Together, these results indicate the altered expression levels of pro-inflammatory cytokines and growth factors in OLETF rats at an advanced age and with tadalafil treatment.

Fig 5 — mRNA expression (relative to glyceraldehyde 3-phosphate dehydrogenase) **(A)** and protein concentrations **(B)** of pro-inflammatory cytokines (IL-6 and TNF-α) and growth factors (IGF-1 and bFGF) in the bladders of OLETF rats at 36 weeks and at 48 weeks with and without tadalafil, using real-time PCR and ELISA. Six samples were included in each group, and two replicates were performed for each sample. Data are shown as the mean ± SEM. *p<0.05, **p<0.01.

Contractile responses of bladder strips to KCl, carbachol, and atropine in OLETF rats

We used bladder strips from OLETF rats (n = 3, each) to evaluate the bladder contraction responses to KCl and carbachol. Bladder contraction responses to KCl administration were significantly lower at 48 weeks than at 36 weeks (p<0.05). Furthermore, tadalafil treatment significantly increased bladder contractions (p<0.01) (Fig 6A). The dose–response curves of bladder contractions with a dose of carbachol revealed that contractions were increased by carbachol administration. Furthermore, sensitivity to carbachol was lower in the bladder at 48 weeks than at 36 weeks; this sensitivity in the bladder at 48 weeks was restored by tadalafil treatment (Fig 6B). Together, these results indicate that the bladder contractile responses of OLETF rats decrease with aging and are recovered by tadalafil treatment.

Fig 6 — Contractile responses of bladder strips to 62 mM KCl **(A)**, carbachol (3×10⁻⁷, 10⁻⁶, 3×10⁻⁶, 10⁻⁵, or 3×10⁻⁵ M) **(B)** from OLETF rats at 36 weeks and at 48 weeks with and without tadalafil. Six samples were included in each group, and two replicates were performed for each sample. Data are shown as the mean ± SEM. *p<0.05, **p<0.01.

Discussion

The results of the present study demonstrated that in a rat model of T2DM, the expression levels of HIF-1α, 8-OHdG, various pro-inflammatory cytokines and growth factors were increased as a result of bladder perfusion disorder. Moreover, our findings indicate that increased pro-inflammatory cytokines and growth factors may enhance the bladder urothelial release of ATP via VNUT upregulation. The bladder contractile response to KCl and muscarinic stimulation decreased with aging in the rats. Long-term (12-week) administration of tadalafil improved blood flow and oxidative stress, resulting in decreased ATP release from the urothelium and improved bladder storage and voiding function. The current study is the first to suggest that urothelium-derived ATP may be associated with urine storage dysfunction in T2DM. It is also the first to indicate that tadalafil can inhibit ATP release and improve bladder contractions.

Diabetes can cause lower urinary tract dysfunction, including DO or DU [5, 19]. There are many reports of bladder function or morphology in animal models of streptozotocin-induced type 1 diabetes mellitus, most of which are characterized by the presence of bladder hypertrophy [20]. Two hereditary models of T2DM (namely, Zucker diabetic fatty rats and db/db mice) with blood glucose levels that are similar to those of the type 1 diabetes mellitus model show the same degree of, or only slight, hypertrophy [21]. By contrast, the Goto-Kakizaki rat—a hereditary model of T2DM—shows mild-to-moderate bladder hypertrophy [22]. Our T2DM model (i.e., OLETF rats) had an increased bladder wet weight with age, and bladder hypertrophy was observed compared with LETO rats. Moreover, the daily urine volume was significantly larger in OLETF rats than in LETO rats. Similarly, polyuria, caused by renal dysfunction, commonly appears in T2DM patients with aging. Diabetic polyuria induces the stimulation of DNA synthesis in the bladder, resulting in increased protein synthesis and hyperplasia of the smooth muscle and epithelial layers [23].

Although overactive bladder is observed from an early stage of T2DM in humans, the presence of DO has not yet been reported in a cystometric evaluation of a T2DM animal model [24]. In rats fed a fructose-rich diet, no change in bladder capacity was observed but increased non-voiding contractions during the storage phase were identified by cystometric recordings [25]; however, quantitative data of non-voiding contractions were not provided. In our OLETF model, the mean voided volume did not differ between LETO and OLETF rats, although non-voiding contractions were observed before the micturition reflex on cystometric evaluation. Total urine volume was associated with frequent micturition in OLETF rats. Moreover, our findings indicate that tadalafil might be able to suppress urine overproduction caused by T2DM. It has been reported that tadalafil treatment reduces glucose levels and has anti-inflammatory cardioprotective effects in leptin receptor-null diabetic mice [26]. Additionally, several preclinical and clinical studies have demonstrated the beneficial metabolic effects of PDE5 inhibitors for manifestations of metabolic syndrome [27, 28]. A randomized control study also showed the effects of high-dose tadalafil on decreasing hemoglobin A1c levels in patients with well-controlled T2DM [29]. However, no previous reports have shown the effects of PDE5 inhibitors on renal dysfunction caused by T2DM. Further experiments are therefore required to elucidate the association between PDE5 and renal function.

The present study is the first to demonstrate increased ATP release from the bladder urothelium in an animal T2DM model. Given that the PDE5 inhibitor improved bladder blood flow and decreased ATP release in the rats, it can be speculated that—at least in the present model—hypoxia triggers ATP release from the urothelium. Nephropathy, retinopathy, and neuropathy (the three major complications of T2DM) develop when vascular endothelial cells are exposed to hyperglycemia, subsequently causing microcirculatory disturbance. This condition develops into macrovascular complications, including coronary artery and cerebrovascular complications [30]. It has been reported that OLETF rats develop spontaneous persistent hyperglycemia with increased atherogenesis in arteries throughout the body before the onset of T2DM [31]. These atherosclerotic changes in the internal iliac and bladder feeding artery lead to bladder ischemia, resulting in increased levels of HIF-1α, 8-OHdG, and various pro-inflammatory cytokines and growth factors in the bladder wall. Several mechanisms have been postulated to underlie the release of ATP from the bladder urothelium during bladder distension, including vesicular exocytosis and connexin/pannexin channels [32–35]. It has also been suggested that ATP release is regulated mainly by VNUT (gene name: Slc17A9) in the bladder urothelium [15]. ATP accumulates and is stored in vesicles inside epithelial cells. The molecular machine VNUT is an active transporter that concentrates ATP into secretory vesicles [36]. The mild stretching of bladder urothelium harvested from VNUT-knockout mice reduces ATP release, suggesting that VNUT-mediated ATP release is involved in the urine storage mechanism that promotes bladder relaxation during the early stages of filling [37]. ATP release from the bladder urothelium is elevated in various human pathological conditions, such as overactive bladder, benign prostate hyperplasia, spinal cord injury, and interstitial cystitis [38–40]. We have also demonstrated that urothelium-derived ATP is increased in salt-sensitive hypertensive rats, with a corresponding decrease in the mean voided volume [7]. In the present study, bladder distention-evoked ATP release was significantly higher in OLETF rats than in LETO rats. In addition, VNUT expression was markedly elevated in the bladders of OLETF rats. There have been few reports of the enhancement of ATP release by inflammation. Mice with conditional hepatic double-knockout of Irs1 and Irs2 show characteristics of T2DM and overactive bladder [41]. In this animal model, when TNF-α-mediated signaling (which was increased in the serum and bladder) was inhibited, overactive bladder was reversed without affecting hyperglycemia. In the present study, the expression levels of IL-6, TNF-α, IGF-1, and bFGF in the bladders of OLETF rats increased with age and decreased with tadalafil administration. Further investigations are therefore necessary to elucidate the association between inflammation and ATP release in the bladder urothelium.

Although the nitric oxide/cyclic guanosine monophosphate (cGMP) pathway is an endothelium-derived signaling pathway that induces vascular smooth muscle relaxation, it is suggested that this pathway is also involved in bladder relaxation. PDE5 inhibitor, sildenafil, increases cGMP levels and inhibits the distension-induced release of ATP from the bladder urothelium in mice [42]. Another study indicated that sildenafil inhibits ATP release from mucosa isolated from the detrusor muscle [43]. Together, these findings suggest that the nitric oxide/cGMP pathway may suppress epithelial ATP release. A possible mechanism by which PDE5 inhibitors might suppress ATP release involves the attenuation of Ca²⁺ influx via transient receptor potential vanilloid 2 and 4 [44]. In the present study, 12 weeks of tadalafil administration markedly decreased ATP release from the bladder urothelium. It is unclear whether this was an acute effect via the nitric oxide/cGMP pathway or a chronic effect associated with decreased pro-inflammatory cytokines as a result of improved blood flow. However, given that the effect occurred >2 days after the completion of the 12-week tadalafil treatment, and because the insides of the bladders were rinsed three times with 0.5 mL Krebs solution, we speculate that the effect may have been the result of improved bladder blood flow caused by the 12-week tadalafil regimen.

We also observed that the contractile responses to KCl and carbachol were significantly lower in bladder strips from 48-week-old OLETF rats than in those from 36-week-old OLETF rats, suggesting that muscarinic receptor hyposensitivity and/or decreased contractile factors associated with increased collagen deposition in bladder smooth muscle occurs during the late stage of T2DM. Although we did not conduct a histological examination, the bladder is known to become less responsive to muscarinic stimulation, which is accompanied by bladder hypertrophy. One of the later stages of diabetic bladder dysfunction has been described as voiding dysfunction arising from an underactive bladder [5]. This is presumed to be caused by the accumulation of oxidative stress products as the result of long-lasting hyperglycemia [45]. In rats, chronic bladder ischemia produced by endothelial injury of the iliac arteries decreases the contractile responses of bladder strips to KCl, electrical field stimulation, and carbachol, with collagen deposition in the smooth muscle layer. In this previous study, when tadalafil was administered for 8 weeks concurrently with the endothelial injury, both the decline in bladder contractility and collagen deposition were prevented. Similarly, our present findings in rats indicate that tadalafil administration starting at 36 weeks can prevent T2DM-associated bladder contraction disorders. PDE5 inhibitors may thus be useful therapeutic tools for voiding dysfunction associated with T2DM.

Limitations

The difference in the voiding frequency in LETO and OLETF rats might be mainly caused by the difference in total urine volume. Based on the results that tadalafil treatment decreased the urine volume in OLETF rats, it is suggested that tadalafil is effective for polyuria caused by T2DM. The changes in the total urine volume might be associated with the renal deficiency caused by T2DM. However, the mechanisms for them were not evaluated in this study. We are planning to make experiments in the future study.

To explore bladder function, we conducted in vitro contraction experiments of the detrusor muscle but did not perform histological examinations of the bladder wall. It is therefore not possible to say with certainty why the contractions in response to carbachol were reduced. Furthermore, an important question remains: why does T2DM cause increased ATP release from the bladder urothelium? Although pro-inflammatory cytokines have been suggested as the responsible candidates, cytokine-specific inhibitors need to be used to test this possibility.

Conclusions

Using OLETF rats, a series of pathological conditions associated with T2DM were reproduced, including polyuria, bladder wall ischemia, increases in pro-inflammatory cytokines and growth factors, epithelial-mediated ATP release, and impaired bladder contractility. Tadalafil, a PDE5 inhibitor, improved polyuria, bladder ischemia, reduced the levels of pro-inflammatory cytokines and growth factors, inhibited ATP release from the bladder urothelium, and improved bladder contractility. This pathological model will continue to help to elucidate the pathology of renal and bladder dysfunction associated with T2DM and contribute to the design of new therapeutic strategies.

Supporting information

S1 Table. Primers sequences of the genes examined by real-time PCR.

(TIF)

pone.0301883.s001.tif^{(850KB, tif)}

Acknowledgments

We thank all of the project members. We also thank Mark Cleasby, PhD, and Bronwen Gardner, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Data Availability

Data are available from the University of Fukui Institutional Data Access. Data contain potentially identifying or sensitive patient information. A Research Ethics Committee in University of Fukui has imposed them. E-mail address: rinsho-rinri@ml.u-fukui.ac.jp.

Funding Statement

The study was conducted with financial support from Nippon Shinyaku. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0301883.r001

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PONE-D-24-11736Phosphodiesterase-5 inhibition inhibits epithelial ATP release and restores detrusor contractility in rats with type 2 diabetes via an increase in bladder blood flowPLOS ONE

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Reviewer #1: The manuscript entitled " Phosphodiesterase-5 inhibition inhibits epithelial ATP release and restores detrusor contractility in rats with type 2 diabetes via an increase in bladder blood flow” describes the impairment of bladder activity that is associated T2DM and age. The authors also investigated the possible therapeutic effect of long term tadalafil treatment on this condition. I think the presentation of data and the language of manuscript is not suitable and not fully meet the quality standards for the publishing Plos One. Here are some points;

1. The manuscript needs a review of grammar, syntax and language in general by a native speaker since some sentences do not correspond to a scientific language

2. The abstract section does not reflect the whole article.

3. It's unclear why they wanted to determine the effect of tadalafil?

4. How the authors have chosed the dose of tadalafil?

5. In Fig.1, there is no statistical data showing that tadalafil treatment significantly reduces micturition frequency in OLETF rats.

6. In bladder contractility studies, authors said that they also found that the bladder showed a dose dependent response to carbachol, but the sensitivity of the bladder to carbachol was lower in rats of 48 weeks of age than in those of 36 weeks of age. But there is no statistical differences between groups in figure 6B and 6C.

Reviewer #2: The authors demonstrated that OLETF rats, a model of T2DM, display, 1) impaired bladder blood flow, 2) enhanced urothelial ATP release, 3) upregulation of hypoxic and inflammatory factors, 4) diminished detrusor muscle contractility, associated with frequent urination. They also showed that those symptoms/changes were ameliorated by oral administration of tadalafil, and concluded that PEE5 inhibitors have therapeutic potential in treating T2DM associated bladder dysfunction.

I am afraid that causal relationship amongst the findings was not sufficiently proven to draw their conclusion.

My major concerns are as follows

1) The development of T2DM phenotype in OLETF rats should be confirmed by checking their blood glucose and insulin levels, total urine volume etc. Similarly, bladder function should also be examined in more detail with cystometry to find if overactive or underactive bladder phenotype is developed. The frequent urination in OLETF rats may also result from residual urine due to diminished detrusor contractility.

2) The authors suggested that the increased urothelial ATP release is due to hypoxia and/or inflammation, presumably in the urothelium, subsequent to bladder hypoperfusion. To determine the site of hypoxia and/or inflammation, real-time PCR and ELISA should be performed using isolated bladder mucosa and mucosa-denuded detrusor muscle preparations along with corresponding immunohistochemistry.

3) As it was pointed out by the authors, the lack of histological examinations is a substantial drawback of this study. Atherosclerotic changes in internal iliac/bladder feeding artery should be examined. In addition, morphological examination of microvascular architecture/density in the bladder, particularly the mucosa, is strongly encouraged to carry out.

Minor

1. Please confirm if financial disclosure is correct (cover page vs ln 432).

2. Any reason for using the term ‘epithelium’ but not ‘urothelium’?

3. For ATP measurements, maintaining the bladder at 20 cmH2O for 10 min is certainly unphysiological.

4. Blood pressure of OLETF and LETO rats with or without tadalafil administration should be provided.

5. Why are comparisons of detrusor muscle contractility between OLETF and LETO rats lacking?

6. Did KCl or CCh develop sustained contractions? The complete inhibition of KCl-induced contractions with atropine is hard to believe. KCl-induced depolarisation would stimulate the release of neural ACh but also directly contact detrusor muscle by activating voltage-dependent calcium channels. EFS-induced detrusor contractions should be evaluated.

7. In general, the discussion is often quite speculative.

Reviewer #3: The authors focused on the bladder of a well-characterized rat model of type 2 diabetes, evaluated physiochemical changes, and showed that these changes were reversed by tadalafil. I also found that the authors conducted validation with a lot of data using a multifaceted approach. On the other hand, some of the data interpretation was difficult to understand, and there were some areas where we would like to see more careful explanations.

Major revision

I found it very interesting that there was a clear difference in wet weight of the bladder itself between LETO and OLETF rats, almost 2-fold, but no effect of tadalafil administration, while there was no difference in urine volume between the groups and no effect of tadalafil administration (line 210- 215). In contrast, the daily micturition frequency, the focus of the authors in this study, clearly shows an increase in frequency in OLETF rats and a suppression of that increase with tadalafil administration, as shown in Figure 1B (line 216-222). I understood that these results show that the size of the bladder itself, i.e., the amount of urine stored, does not change between the group, but OLETF rats urinate more frequently and in short bursts, as in frequent urination.

1) These fact may give various suggestions, so the wet weight of the bladder or the total daily urine volume should be shown in Fig. 1.

2) Next, the authors need to present an argument that the phenomenon of increased frequency of urination despite no change in total urine output is due to the physiochemical effects of the bladder itself, which will be argued later in the result.

3) What the authors do not show in their treatment of the data is that they measure urine output every 10 minutes as a urinary behavior, but do not show temporal changes within 24 hours. The authors should present data for both groups on their temporal changes of urine output, so that we could discuss about how the high frequency of urination is not due to behavioral factors such as differences in circadian rhythms, etc.

Minor revision

1) The authors need to provide the appropriate references at lines 63-65.

2) The authors specify in line 100 that the number of animals was six in each of the six groups, but it is unclear how many animals were used in total. The different experiments include "Laser speckle blood flow imaging," "Organ bath bladder distention test," "PCR, ELISA," and "In vitro bladder-strip experiments," but it is impossible for all of them to be the same test animals. The authors should indicate how the number of animals was set up.

3) The results are shown for ATP release in line 226, but I did not know how to interpret this result. Some of what is written in lines 324 through 331 of the Discussion should be presented in the Introduction.

4) Fig2 in lines 242 and 245 seems to be a mistake for Fig3.

5) Is it not a mistake that the difference in urination between groups appears for the first time (not in the results) in lines 312 to 313 of Discussion? This contradicts the statement in lines 213 to 214 of the results.

6) Some of the content from lines 344 to 348 of the discussion should also be written in Introduction.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Susumu Urakawa

Prof, Graduate School of Biomedical and Health Sciences, Musculoskeletal Functional research and regeneration, Hiroshima Univ

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PLoS One. 2024 Sep 18;19(9):e0301883. doi: 10.1371/journal.pone.0301883.r002

Author response to Decision Letter 0

8 Jul 2024

Reviewer #1:

The manuscript entitled " Phosphodiesterase-5 inhibition inhibits epithelial ATP release and restores detrusor contractility in rats with type 2 diabetes via an increase in bladder blood flow” describes the impairment of bladder activity that is associated T2DM and age. The authors also investigated the possible therapeutic effect of long term tadalafil treatment on this condition. I think the presentation of data and the language of manuscript is not suitable and not fully meet the quality standards for the publishing Plos One. Here are some points;

1. The manuscript needs a review of grammar, syntax and language in general by a native speaker since some sentences do not correspond to a scientific language

Response: Thank you for your suggestion. Although the original manuscript received proofreading from a native speaker, the revised manuscript has again been edited by a native speaker to ensure that appropriate scientific language is used throughout.

2. The abstract section does not reflect the whole article.

Response: Thank you for your comment. We have made changes to the Abstract (primarily to the Results and Conclusions sections) to reflect the manuscript’s contents more clearly.

3. It's unclear why they wanted to determine the effect of tadalafil?

Response: Tadalafil administration is a standard treatment for benign prostatic hyperplasia. As described in the Introduction section, we have previously reported that T2DM-induced chronic ischemia leads to oxidative stress, thus resulting in prostate enlargement through the upregulation of several cytokines and growth factors, using a rat model of T2DM. Together, these findings indicate that tadalafil treatment improves prostate ischemia and might prevent its enlargement via the suppression of cytokines and growth factors (Kobayashi et al. Life Science 2022). We therefore wanted to explore the effects of tadalafil on bladder epithelium in the present study, and to evaluate its association with lower urinary tract symptoms. To better explain the rationale for our study, we have added sentences to the revised manuscript (page 5, line 81).

4. How the authors have chosed the dose of tadalafil?

Response: The standard treatment dose of tadalafil for Japanese patients is 5 mg per day. Given that the average body weight in Japan is 60 kg, the calculated standard dose was 83 µg/kg. We therefore set the experimental dose of tadalafil as 100 µg/kg per day in this study. We have added text to this effect in the revised manuscript (page 7, line 107).

5. In Fig.1, there is no statistical data showing that tadalafil treatment significantly reduces micturition frequency in OLETF rats.

Response: Thank you for noticing our mistake. When we checked the data, we noted that there were indeed significant differences in micturition frequency between L-36 vs. L-48, O-36 vs. O-48, and O-48 vs. O-48(t). We have corrected Fig. 1B (now Fig. 1D) in the revised manuscript.

Response: Again, thank you for noticing our mistake. There were indeed significant differences in bladder contraction with carbachol and atropine between 48 and 36 weeks of age. In accordance with your comment, we have corrected Fig. 6B and C in the revised manuscript.

Reviewer #2:

The authors demonstrated that OLETF rats, a model of T2DM, display, 1) impaired bladder blood flow, 2) enhanced urothelial ATP release, 3) upregulation of hypoxic and inflammatory factors, 4) diminished detrusor muscle contractility, associated with frequent urination. They also showed that those symptoms/changes were ameliorated by oral administration of tadalafil, and concluded that PEE5 inhibitors have therapeutic potential in treating T2DM associated bladder dysfunction.

I am afraid that causal relationship amongst the findings was not sufficiently proven to draw their conclusion.

My major concerns are as follows

Response: Thank you for your comments. In response to your first point, as noted in the revised Introduction (page 5, line 74), we have previously evaluated the blood glucose and insulin levels of OLETF rats; they are indeed significantly different from those of control (LETO) rats (Itoga et al. BMJ Open Diabetes Res Care 2020).

As you have noted, total urine volume is associated with frequent urination in OLETF rats. The urine volume per 24 hr was significantly higher in OLETF rats than in LETO rats at both 36 (10.4 ± 1.8 mL and 4.8 ± 2.3 mL, p<0.01) and 48 (18.5 ± 3.8 mL and 4.2 ± 0.7 mL, p<0.01) weeks. The frequency of micturition per 24 hr was also significantly higher in OLETF rats than in LETO rats at both 36 (5.9 ± 1.7 and 3.3 ± 1.6, p<0.01) and 48 (10.9 ± 2.4 and 2.9 ± 0.4, p<0.01) weeks. Furthermore, the urine volume and frequency of micturition in the OLETF rats was significantly higher at 48 weeks than at 36 weeks (p<0.01). Tadalafil treatment significantly decreased the urine volume (from 18.5 ± 3.8 mL to 10.7 ± 2.0 mL, p<0.05) and frequency of micturition (from 10.9 ± 2.4 to 6.0 ± 1.2, p<0.05) at 48 weeks in the OLETF rats. The urine volume and frequency of micturition in LETO rats were not significantly different between 36 and 48 weeks, and were unchanged by tadalafil treatment. These results suggest that tadalafil might be able to suppress urine overproduction caused by T2DM. The mechanism behind the decrease in urine volume is possibly due to the decrease in blood glucose, furthermore, due to the improvement of renal function caused by tadalafil. Further experiments are needed to elucidate the association between PDE5 and renal function. These data are shown in the revised Fig. 1C, and a summary of this information was added to the revised manuscript (page 14, line 234).

Cystometry is an important procedure for evaluating bladder function. In the present study, awake cystometry was performed in both the LETO and OLETF rats at 36 weeks. Although non-voiding contractions appeared more frequently in OLETF rats than in LETO rats, there were no significant differences in functional bladder capacities. Furthermore, no residual urine was detected using ultrasonography in the OLETF or LETO rats during the experiments. The data are shown in the revised Fig. 1B, and associated text was added to the manuscript (page 13, line 230).

Response: Thank you for your important suggestions. It would indeed be a good idea to perform real-time PCR and ELISA using isolated bladder mucosa and mucosa-denuded detrusor muscle, to observe the inflammatory lesions. Unfortunately, we have already used all rat bladder tissue in this study (in the organ bath bladder distention test or the in vitro bladder-strip experiments). We therefore have no tissue remaining for additional experiments. These questions will therefore be addressed in future studies.

Response: As you have noted, the lack of histological examinations is a substantial limitation of our study. Unfortunately, all of the rats used in the study have been sacrificed, and it is therefore difficult to obtain the bladder and surrounding tissue for further experiments. However, it has previously been reported that atherosclerotic changes in arteries appear throughout the body of OLETF rats (Tamura et al. Atherosclerosis 2000). This report was therefore cited as reference 25, and associated text was added to the revised manuscript (page 21, line 360).

Minor

1. Please confirm if financial disclosure is correct (cover page vs ln 432).

Response: This study was funded and supported by Nippon Shinyaku. However, the authors have no conflicts of interest to disclose. We have also corrected the Acknowledgements section.

2. Any reason for using the term ‘epithelium’ but not ‘urothelium’?

Response: Thank you for this suggestion. The term “epithelium” was changed to “urothelium” throughout the revised manuscript.

3. For ATP measurements, maintaining the bladder at 20 cmH2O for 10 min is certainly unphysiological.

Response: We used the same methods as in our previous report (Tanaka et al. J Urol. 2011;185(1):341-6). You are correct in noting that the stated method (of maintaining the bladder at 20 cmH2O) was incorrect; the distention with 1.5 mL of Kreb solution at approximately 0.04 mL/s is correct. We have made the appropriate changes to the revised Materials and Methods (page 9, line 153) and Figure legends (page 27, line 470).

4. Blood pressure of OLETF and LETO rats with or without tadalafil administration should be provided. 　

Response: The blood pressure of OLETF and LETO rats has been examined in a previous report (Itoga et al. BMJ Open Diabetes Res Care. 2020); the clinical data suggested that blood pressure might not be changed by treatment with tadalafil. We therefore did not examine blood pressure in the current study.

5. Why are comparisons of detrusor muscle contractility between OLETF and LETO rats lacking?

Response: The objective of the detrusor muscle contractility experiments was to evaluate the efficacy of tadalafil for bladder contractions. Because the results of micturition characteristics indicated that tadalafil was not effective in LETO rats, we investigated the effects of tadalafil on detrusor muscle contractility in OLETF rats only.

Response: Thank you for your suggestions. The EFS-induced detrusor contractions will be evaluated in a future study.

7. In general, the discussion is often quite speculative.

Response: Thank you; we have simplified the revised Discussion by removing any speculative sentences.

Reviewer #3:

The authors focused on the bladder of a well-characterized rat model of type 2 diabetes, evaluated physiochemical changes, and showed that these changes were reversed by tadalafil. I also found that the authors conducted validation with a lot of data using a multifaceted approach. On the other hand, some of the data interpretation was difficult to understand, and there were some areas where we would like to see more careful explanations.

Major revision

1) These facts may give various suggestions, so the wet weight of the bladder or the total daily urine volume should be shown in Fig. 1.

Response: As you have noted, it is interesting that bladder weight was higher in OLETF rats than in LETO rats; however, bladder weight was not decreased by tadalafil treatment. As described in our response to Reviewer 2’s major concern #1, cystometry did not show any differences in functional bladder capacity. Furthermore, in our micturition behavior analyses, the voided urine volume per body weight did not significantly differ between OLETF and LETO rats. As you have suggested, we have shown the total daily urine volume in both types of rats in the revised Fig. 1C and the Results section. The total urine volume per 24 hr was significantly higher in OLETF rats than in LETO rats at both 36 (10.4 ± 1.8 mL and 4.8 ± 2.3 mL, p<0.01) and 48 (18.5 ± 3.8 mL and 4.2 ± 0.7 mL, p<0.01) weeks. Tadalafil treatment significantly decreased the urine volume (from 18.5 ± 3.8 mL to 10.7 ± 2.0 mL, p<0.05) and frequency of micturition (from 10.9 ± 2.4 to 6.0 ± 1.2, p<0.05) at 48 weeks in the OLETF rats. The urine volume and frequency of micturition in LETO rats were not significantly different between 36 and 48 weeks, and were unchanged by tadalafil treatment. Text describing these results has been added to the revised manuscript (page 14, line 234).

2）Next, the authors need to present an argument that the phenomenon of increased frequency of urination despite no change in total urine output is due to the physiochemical effects of the bladder itself, which will be argued later in the result.

Response: Total urine volume was associated with frequent micturition in OLETF rats. Moreover, our findings indicate that tadalafil might be able to suppress urine overproduction caused by T2DM. It has been reported that tadalafil treatment reduces glucose levels and has anti-inflammatory cardioprotective effects in leptin receptor-null diabetic mice. Additionally, several preclinical and clinical studies have demonstrated the beneficial metabolic effects of PDE5 inhibitors for manifestations of metabolic syndrome. A randomized control study also showed the effects of high-dose tadalafil on decreasing hemoglobin A1c levels in patients with well-controlled T2DM. However, no previous reports have shown the effects of PDE5 inhibitors on renal dysfunction caused by T2DM. Further experiments are therefore required to elucidate the association between PDE5 and renal function. Text to this effect has been added to the revised manuscript (page 20, line 342).

Response: As you have noted, a circadian rhythm disorder of urine production might be associated with urinary frequency in this T2DM model rats. However, it seems difficult to evaluate the association between urinary frequency and circadian rhythms of urine production because the total urine volume was different between OLETF rats and LETO rats.

Minor revision

1) The authors need to provide the appropriate references at lines 63-65.

Response: Thank you; we have added the appropriate references to these lines.

PLoS One. doi: 10.1371/journal.pone.0301883.r003

Decision Letter 1

Yung-Hsiang Chen

25 Jul 2024

PONE-D-24-11736R1PDE5 inhibitor potentially inhibits epithelial ATP release and recovers detrusor contractility in type 2 diabetic rats via increased bladder blood flowPLOS ONE

Dear Dr. Terada,

Please submit your revised manuscript by Sep 08 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Yung-Hsiang Chen, Ph.D.

Academic Editor

PLOS ONE

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: No

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: No

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #2: The authors have now provided key research data, namely the total urine volume and bladder capacity.

These data are very helpful to explore mechanisms underlying the voiding phenotype of T2DM model (OLETF) rats, but raised a serious concern about the authors’ interpretation/conclusion. Thus, increased voiding frequency could be simply due to polyuria but not bladder dysfunction.

OLETF rats had x2.2 total urine volume and x1.8 voiding frequency at 36 weeks, x4.4 total urine volume and x3.9 voiding frequency at 48 weeks compared with control (LETO) rats. Tadalafil treatment reduced the total urine volume from x4.4 to x1.7 and the voiding frequency from x3.9 to x1.8 of control values.

Morphological and functional changes in the bladder of OLETF rats could be a consequence of polyuria rather than the cause of increased voiding frequency. After all, the manuscript including the title should be largely rewritten.

The results of cystometry lack statistical analysis, e.g., bladder capacity, number of non-voiding contractions or peak voiding pressure. The number of experiments (animals) should be provided along with p values throughout the text.

Cystometric traces lack scale bars. In Fig1B, L-36 and O-36 traces appeared to be shown in different time scales (see the difference in the duration of voiding contractions). Please provide clearer images.

The authors did not answer why atropine completely diminished KCL-induced contractions of DSM strips. This should not be happened if experiments were carried out properly (see my comment to the original submission).

Run out of animals is not a good excuse as both OLETF and LETO rats seem to be commercially available (http://www.hoshino-lab-animals.co.jp/English/products/OLETF_ENG..html).

Yet, additional experiments are desirable but not mandatory in the present study.

Please check the accuracy of references (for example No.39).

Reviewer #3: In response to Reviewer #3, Major revision 3, I don't understand why it is difficult to evaluate the association. At least, it would be possible to divide the data into light and dark periods and show that the frequency ratio is almost the same between the groups (more in the light period).

In Fig. 1B, the units of the vertical and horizontal axes of the graph must be indicated, and is this a typical example?

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

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Attachment

Submitted filename: PONE-D-24-11736R1.docx

pone.0301883.s002.docx^{(15.4KB, docx)}

PLoS One. 2024 Sep 18;19(9):e0301883. doi: 10.1371/journal.pone.0301883.r004

Author response to Decision Letter 1

21 Aug 2024

1. The authors have now provided key research data, namely the total urine volume and bladder capacity. These data are very helpful to explore mechanisms underlying the voiding phenotype of T2DM model (OLETF) rats, but raised a serious concern about the authors’ interpretation/conclusion. Thus, increased voiding frequency could be simply due to polyuria but not bladder dysfunction. OLETF rats had x2.2 total urine volume and x1.8 voiding frequency at 36 weeks, x4.4 total urine volume and x3.9 voiding frequency at 48 weeks compared with control (LETO) rats. Tadalafil treatment reduced the total urine volume from x4.4 to x1.7 and the voiding frequency from x3.9 to x1.8 of control values.

Response: Thank you for your comments. As the reviewer mentioned, the difference in the voiding frequency in LETO and OLETF rats might be mainly caused by the difference in total urine volume. Based on the results that tadalafil treatment decreased the urine volume in OLETF rats, it is suggested that tadalafil is effective for polyuria caused by T2DM. The changes in the total urine volume might be associated with the renal deficiency caused by T2DM. However, the mechanisms for them were not evaluated in this study. We are planning to make experiments in the future study. They were added in the Limitation section (Page 24, line 423) and the words “polyuria” and “renal” were added in the Conclusions section (Page 25 line438, 440, 443)

-Page 1, line 1. The title was changed from “PDE5 inhibitor potentially inhibits epithelial ATP release and recovers detrusor contractility in type 2 diabetic rats via increased bladder blood flow” inoto “PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats”.

-Page 3, line 42. The conclusions of the abstract were changed as follows; “The T2DM rats had polyuria, increased ATP release induced by decreased bladder blood flow and impaired contractile function. PDE5 inhibition improved these changes and may prevent T2DM-associated urinary frequency and bladder storage and voiding dysfunctions.”

-Page 24, line 423. Several sentences were added.

-Page 25, line 438, 440 and 443. “Polyuria” and “renal” were added.

2. The results of cystometry lack statistical analysis, e.g., bladder capacity, number of non-voiding contractions or peak voiding pressure.

Response: Thank you for your suggestion. As the results of cystometry, the bladder capacity, the bladder contraction pressure and the number of non-voiding contractions were shown. Moreover, several corrections were made in the methods of cystometry in conscious rats with an additional reference.

-Page 14, line 231. Sentences were changed as follows; “Cystometry in conscious rats was performed in the LETO and OLETF rats (n=2, each) at 36 weeks. The bladder capacity (0.7 ± 0.1 mL and 0.7 ± 0.2 mL, p=0.37), and the bladder contraction pressure (39.3 ± 10.7 cmH2O and 33.4 ± 10.3 cmH2O, p=0.16) were not significantly different between LETO and OLETF rats. Non-voiding contractions appeared only in OLETF rats and not appeared in LETO rats”

-Page 8, line 126. A sentence was changed as follows; “Cystometry procedures in conscious rats were performed as our previous reports(16)”.

-Page 8, line 133. A sentence “Cystometry was done with physiological saline at room temperature at 0.04 ml per minute. Bladder capacity and bladder contraction pressure were measured” was added.

3.The number of experiments (animals) should be provided along with p values throughout the text.

Response: Thank you for your suggestion. The number of animals used in each experiments were shown in the Results section.

-The number of rats were added as follows; Page 13, line 223 (n=6, each), Page 14, kine 239 (n=6, each), Page 15, line 253(n=3, each), Page 16, line 269 (n=3, each), Page 16, line 281 (n=3, each), Page 17, line 291 (n=3, each) and Page 18, line 303 (n=3, each).

4. Cystometric traces lack scale bars. In Fig1B, L-36 and O-36 traces appeared to be shown in different time scales (see the difference in the duration of voiding contractions). Please provide clearer images.

Response: Thank you for your suggestion. All the figures of cystometric traces were shown in a same scale. Scale bars were added in figure 1B. Then, the figures were made clearer.

-Figure 1 was changed.

5. The authors did not answer why atropine completely diminished KCL-induced contractions of DSM strips. This should not be happened if experiments were carried out properly (see my comment to the original submission).

Response: As the reviewer pointed out, the results of the effect of atropine for KCL-induced contraction is complicated. To prevent misunderstandings, the results were deleted in this manuscript. The figure 6C was deleted accordingly.

-Figure 6 was changed.

6. Run out of animals is not a good excuse as both OLETF and LETO rats seem to be commercially available (http://www.hoshino-lab-animals.co.jp/English/products/OLETF_ENG..html). Yet, additional experiments are desirable but not mandatory in the present study.

Response: Thank you for your comments. As the reviewer mentioned, the OLETF an LETO rats are commercially available. Additional experiments to elucidate the mechanisms of polyuria or the pathological changes in the bladder wall will be performed and make another paper.

Please check the accuracy of references (for example No.39).

Response: The references (42 and 43) were corrected and the sentenced in Page 22, line 390 were also corrected.

PLoS One. doi: 10.1371/journal.pone.0301883.r005

Decision Letter 2

Yung-Hsiang Chen

2 Sep 2024

PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats

PONE-D-24-11736R2

Dear Dr. Terada,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

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PLoS One. doi: 10.1371/journal.pone.0301883.r006

Acceptance letter

Yung-Hsiang Chen

6 Sep 2024

PONE-D-24-11736R2

PLOS ONE

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Supplementary Materials

S1 Table. Primers sequences of the genes examined by real-time PCR.

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Data Availability Statement

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PERMALINK

PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats

Takafumi Kabuto

So Inamura

Hisato Kobayashi

Xinmin Zha

Keiko Nagase

Minekatsu Taga

Masaya Seki

Nobuki Tanaka

Yoshinaga Okumura

Osamu Yokoyama

Naoki Terada

Roles

Abstract

Purpose

Materials and methods

Results

Conclusions

Introduction

Materials and methods

Animal preparation

Fig 1. Measurement of micturition behavior in the rats.

Micturition behavior

Cystometry in conscious rats

Laser speckle blood flow imaging system

Organ bath bladder distention test

Real-time polymerase chain reaction (PCR)

Enzyme-linked immunosorbent assay (ELISA)

8-hydroxy-2’-deoxyguanosine (8-OHdG) measurement

In vitro bladder-strip experiments

Data analysis

Results

Body weights, bladder wet weights, and micturition characteristics of LETO and OLETF rats

ATP production and VNUT expression in the bladder

Fig 2. Measurements of the amount of ATP released by bladder distention and VNUT expression in the bladder.

Changes in bladder blood flow measured by laser speckle blood flow imaging

Fig 3. Determination of bladder blood flow in LETO and OLETF rats.

Expression levels of HIF-1α and 8-OHdG in the bladders of OLETF rats

Fig 4.

mRNA and protein expression of pro-inflammatory cytokines and growth factors in the bladders of OLETF rats

Fig 5.

Contractile responses of bladder strips to KCl, carbachol, and atropine in OLETF rats

Fig 6.

Discussion

Limitations

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Yung-Hsiang Chen

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Acceptance letter

Yung-Hsiang Chen

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Associated Data

Supplementary Materials

Data Availability Statement

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