Received: 27 December 2018 | Revised: 27 April 2019 | Accepted: 9 May 2019

DOI: 10.1002/nau.24050

ORIGINAL BASIC SCIENCE ARTICLE

Nicotine‐induced hypoxia in rat urothelium deteriorates

bladder storage functions

Takashi Nagai | Tetsuya Imamura | Teruyuki Ogawa | Tomonori Minagawa |

Takahisa Domen | Toshiro Suzuki | Manabu Ueno | Osamu Ishizuka

Department of Urology, Shinshu

University School of Medicine, Abstract
Matsumoto, Japan Aims: To measure the effects of nicotine on lower urinary tract symptoms
(LUTS), bladder blood flow, and the urothelial markers hypoxia‐inducible factor
Correspondence
Takashi Nagai, Department of Urology, 1α (HIF1α), uroplakin III (UPIII), and aquaporin 3 (AQP3).
Shinshu University School of Medicine, Methods: Ten‐week‐old female Sprague Dawley rats were subcutaneously
3‐1‐1 Asahi, Matsumoto 390‐8621, Japan.
injected with 2 mg/kg nicotine (n = 17) or vehicle (control, n = 18) twice daily
Email: [email protected]
for 13 days. Some nicotine‐treated rats (n = 10) were injected daily with 1 mg/kg
tadalafil for the last 6 days of nicotine treatment. One day before cystometry, the
bladders of some nicotine‐treated and control rats were instilled with 0.08%
acetic acid. Urinary frequency and volume were measured 1 day after
treatment. Blood flow in the bladder neck was measured, and the urothelia
were immunochemically assayed for HIF1α, UPIII, and AQP3.
Results: Following acetic acid treatment, both voiding interval and micturition
volume of the nicotine‐treated rats were significantly lower than controls.
Nicotine‐treated rats had lower blood flow than controls, and the urothelial
expression of HIF1α was higher than controls. Simultaneously, the expressions
of UPIII and AQP3 were decreased. Tadalafil treatment increased bladder blood
flow, and nicotine‐treated rats had increased voiding interval and micturition
volume. Further, the expression of HIF1α decreased, and both UPIII and AQP3
levels increased.
Conclusions: Nicotine‐treated rats stimulated by intravesicular acetic acid
instillation exhibited deterioration of bladder storage functions. Changes in
tissue markers in the nicotine‐treated rats were consistent with hypoxia and loss
of urothelial function. Restoration of blood flow reversed the nicotine effects.
Nicotine may induce LUTS through reduced bladder blood flow and urothelial
hypoxia.

KEYWORDS
bladder blood flow, hypoxia, lower urinary tract symptoms, nicotine, urothelium

1 | INTRODUCTION cancers.1-4 Nicotine derived from cigarette smoke also

affects hormonal balance, sympathetic nerves, blood
Cigarette smoking causes cardiovascular and respiratory flow, and produces oxidative stress within tissues and
diseases as well as lung, urothelial, renal, and other organs.5,6 Through mechanisms that are not very well
Neurourology and Urodynamics. 2019;1-11. wileyonlinelibrary.com/journal/nau © 2019 Wiley Periodicals, Inc. | 1
2 | NAGAI ET AL.

understood, cigarette smoke, including the nicotine nicotine‐treatment group, the rats were subcutaneously
component, also contributes to the induction of lower injected with 2 mg/kg body weight nicotine (0.2 mL) at
urinary tract symptoms (LUTS), especially storage 12 hours intervals for 13 days. In the nicotine‐free control
symptoms.5-9 group, 0.2 mL PSS (vehicle) was similarly injected. On the
In this study, we focused on a reduction of bladder day following the completion of nicotine or control
blood flow due to a nicotine‐induced vasoconstrictor treatment, we performed the cystometric investigations.
activity. Generally, partial ischemia within bladder
tissues, which can be caused by prostatic hypertrophy,
2.4 | Phosphodiesterase type 5 inhibitor
is associated with a deterioration of bladder storage
administration
functions. Thus, to elucidate the association of nicotine
with LUTS development, we focused on the nicotine‐ SD rats (n = 10) were subcutaneously injected with
induced hypoxic environment within the urothelium. In nicotine as described above for 13 days. From days 8 to
cystometric investigations, we determined if the nicotine‐ 13, the rats were given an intraperitoneal 0.2 mL injection
treated rats exhibited deterioration of bladder storage of 1 mg/kg‐body tadalafil simultaneously with the second
functions. We then determined if the nicotine treatments daily subcutaneous nicotine injection. On the day
decreased blood flow to the bladder and changed the following the completion of nicotine and tadalafil
expression levels of hypoxia‐inducible factor 1α (HIF1α), treatments, we performed the cystometric investigations.
an indicator of tissue hypoxia. We also assessed
concomitant changes in uroplakin III (UPIII), a urothe-
lium marker, and aquaporin 3 (AQP3), a cell membrane
2.5 | Cystometric investigation
water channel marker. Finally, we used the phospho- Six days before cystometric investigations, nicotine‐free
diesterase type 5 inhibitor tadalafil, which improves control and nicotine‐treated rats (n = 12 each) were
blood flow within the lower urinary tract,10,11 to reduce anesthetized with pentobarbital sodium solution (40 mg/kg
urothelium hypoxia and restore bladder functions and body weight; Kyoritsu Seiyaku Co, Ltd, Tokyo, Japan) and
tissue markers in the nicotine‐treated rats. inhalation of 2% sevoflurane (Pfizer Japan Co, Ltd, Tokyo,
Japan). Urinary bladder domes were exposed and incised
to allow insertion of a polyethylene catheter (PE50; Becton
2 | MATERIALS AND MET HODS
Dickinson & Company, Sparks, MD). The catheter was
fixed at that site with a 5‐0 silk thread, and the free end
2.1 | Animals
of the catheter was tunneled through into the subcutaneous
Forty‐five female Sprague Dawley (SD) rats at postnatal tissue of the back and exposed at the nape of the neck.
week 10 (190‐240 g; Japan SLC, Inc, Shizuoka, Japan) The catheterized rats were kept and treated for 6 days
were used for this study. They were treated in accordance as described above, continuing the control or nicotine
with the National Institutes of Health Animal Care injections.
Guidelines and the requirements of the animal ethics At 5 days after cannulation and 1 day before the
committee in our university. cystometric investigations, while in their respective cages,
the bladders of the conscious nicotine‐free control and
nicotine‐treated catheterized rats (n = 6 each) were instilled
2.2 | Drugs
with 0.08% acetic acid (Wako) through the bladder cannula
Nicotine hydrogen tartrate (Sigma‐Aldrich, St Louis, MO) at a rate of 10 mL/hour for 30 minutes. Similarly, the
was dissolved in a physiological salt solution (PSS). Just tadalafil‐treated conscious catheterized rats also received
before administration, the dissolved solution was diluted the acetic acid intravesicular instillation (n = 5).
with PSS to deliver 2.0 mg/kg body weight. Tadalafil, a The catheterized, unrestricted, conscious rats were
phosphodiesterase type 5 inhibitor (Toronto Research placed singly in metabolic cages. The bladder catheter
Chemicals, Toronto, Canada), was dissolved in dimethyl was connected through a T‐tube to a pressure transducer
sulfoxide (Wako, Tokyo, Japan). Just before administra- (P23 DC; Nihon Kohden, Tokyo, Japan) and syringe pump
tion, the dissolved solution was diluted with PSS to (TE‐351; Terumo, Tokyo, Japan). Room temperature saline
deliver 1.0 mg/kg body weight. was instilled through the bladder catheter at a rate of
10 mL/hour. To investigate micturition volume, a fluid
collector connected to a force‐displacement transducer
2.3 | Nicotine treatment
(Type 45196; NEC San‐ei Instruments, Tokyo, Japan) was
The SD rats were divided into two groups: nicotine‐free placed under the metabolic cage. Intravesicular pressure,
control (n = 18) and nicotine‐treatment (n = 17). In the voiding interval, and micturition volume were recorded
NAGAI ET AL. | 3

continuously on PowerLab 4/26 and LabChart (ADInstru- (61001; 1:100; mouse monoclonal; PROGEN Biotechnik
ments Pty Ltd, New South Wales, Australia). GmbH, Heidelberg Germany) for 12 hours at 4°C.
After the recording, we estimated the following Similarly, other samples were incubated with AQP3
cystometric parameters: basal pressure (cmH2O), mic- antibody (1:2000; AB3276; rabbit polyclonal; EMD
turition pressure (cmH2O), voiding interval (minute), Millipore Corporation, CA), as a marker of cell mem-
micturition volume (mL), residual volume (mL), and brane water channels, and UPIII antibody or SMA
bladder capacity (mL). Bladder capacity was calculated antibody.
by adding the micturition volume and residual volume, The sections were then rinsed with PBS and incubated
determined as the difference from the saline infusion with donkey anti‐rabbit immunoglobulin G (IgG) sec-
volume to the micturition volume. After the cystometric ondary antibody conjugated with Alexa Fluor 488 (1:250;
investigations, the rats were euthanized with an overdose Thermo Fisher Scientific KK, Foster City, CA) and
of pentobarbital sodium solution. donkey anti‐goat or ‐mouse IgG secondary antibody
conjugated with Alexa Fluor 594 (1:250; Thermo Fisher
Scientific K.K.) for 1 hour at 4°C. Finally, after
2.6 | Measurement of bladder blood
rinsing, cell nuclei were counterstained with 5 µg/mL
flow
4′, 6‐diamidino‐2‐phenylindole dihydrochloride (Thermo
Bladder blood flow in nicotine‐free control (n = 6), Fisher Scientific KK). The stained samples were observed
nicotine‐treated (n = 5), and tadalafil‐administrated with a Leica DAS Microscopethe (Leica Microsystems
(n = 5) rats that were not used in cystometric investiga- GmbH, Wetzlar, Germany).
tions was measured with a laser blood flowmeter Observers, who were not aware of the treatment
(Omegazone; Omegawave, Tokyo, Japan). The rats were status, semiquantitatively evaluated HIF1α‐, UPIII‐, and
anesthetized as above, and then their bladders were AQP3‐positive areas within the urothelium layers or the
exposed through a lower midline abdominal incision. SMA‐positive detrusors with Image‐pro Plus (Media
The bladders were injected with 0.9 mL PSS through the Cybernetics, Inc, Bethesda, MD). Fluorescently labeled
posterior wall by a 29 G needle. Bladder blood flow was areas of the HIF1α‐, UPIII‐, and AQP3‐antibodies within
recorded in arbitrary units at the bladder neck as colored the urothelium layers or detrusors were averaged from
image pixels. Software (Laser Speckle Blood Flow six observed regions (×400 power lens) in each sample
Imager‐LSI version 3.4.3: Omegawave) provided by the and expressed as a proportion of the total observed
flowmeter converted the image pixels into relative urothelium layers or detrusor areas.
numeric measurements of flow rates. After the bladder
blood flow measurements, bladders of the rats were
removed for immunohistochemical investigations. Then
2.8 | Statistical analysis
the rats were euthanized by an overdose of pentobarbital All values were expressed as means ± standard error of
sodium. the means. Two‐way repeated measures analysis of
variance and Student‐Newman‐Keuls two‐way analysis
of variance was used to analyze statistical differences in
2.7 | Immunohistochemistry
the cystometric investigations. Two‐way unpaired t tests
investigations
were used to compare the nicotine‐treated rats with the
The bladders, which were harvested from rats that were nicotine‐free control ones, or the tadalafil‐treated rats
used in blood flow measurements (nicotine‐free, n = 6; with the tadalafil‐untreated rats. Differences with P < .05
nicotine‐treated, n = 5; and tadalafil‐treated, n = 5) were were considered to be statistically significant.
fixed with 4% paraformaldehyde and embedded in
paraffin. Serial sections (5 µm) were deparaffinized and
treated with 10 mM sodium citrate (pH 6.0, 100°C,
3 | RE SU L TS
5 minutes) for antigen retrieval. The specimens were
3.1 | Effects of nicotine on bladder
then coated with 1.5% normal donkey serum (Chemicon
functions
International, Inc, Temecula, CA) and 1.5% nonfat milk
in phosphate‐buffered saline (PBS) for 1 hour at 4°C. After 13 days of nicotine treatment, the patterns of
They were incubated with HIF1α antibody (1:100; 20960‐ voiding frequency and micturition volume of the
1‐AP; rabbit polyclonal; Proteintech Inc, IL), as a tissue nicotine‐free control and nicotine‐treated rats were very
marker of hypoxia, and UPIII antibody (1:100; M‐17; sc‐ similar (Figure 1A and 1B). In nicotine‐free control rats
15186; goat polyclonal; Santa Cruz Biotechnology Inc, that received the intravesicular instillation of acetic
Santa Cruz, CA), or smooth muscle actin (SMA) antibody acid 1 day before cystometry, the voiding frequency and
4 | NAGAI ET AL.

FIGURE 1 Cystometric investigation of nicotine‐free control and nicotine‐treated rats with and without acetic acid intravesical
instillation. A and B, Micturition charts of representative nicotine‐free control (A) and nicotine‐treated (B) rats without acetic acid
intravesical instillation. C and D, Representative micturition charts of nicotine‐free control (C) and nicotine‐treated (D) rats with the acetic
acid intravesical instillation. While the nicotine‐free control rats did not show increased urinary frequency, nicotine‐treated rats had
increased urinary frequency with short voiding intervals and small micturition volumes. In each panel, the upper chart is bladder pressure;
the bottom chart is micturition volume. AA, acetic acid
NAGAI ET AL. | 5

micturition volume (Figure 1C) were similar to nicotine‐ expressed within urothelium of the control (Figure 2C)
free control rats that were not stimulated with the acetic and nicotine‐treated (Figure 2D) rats. However, expres-
acid (Figure 1A). However, the nicotine‐treated rats that sion of HIF1α within the urothelial layers in the nicotine‐
received the intravesicular instillation of acetic acid 1 day treated rats, 0.50 ± 0.01, was significantly higher than in
before cystometry exhibited increased urinary frequency control rats, 0.29 ± 0.02 (P < .01; Figure 2E). HIF1α
with shorter intervals between voidings and lower expression within the detrusors was not affected by the
micturition volume (Figure 1D) compared with the nicotine treatment (data not shown).
nicotine‐treated rats that did not receive the acetic acid Within the urothelium, expressions of the urothe-
instillation (Figure 1C). lium marker UPIII and the water channel marker
From the cystometry recordings, we calculated AQP3 were also detected in both groups (Figure 3A
micturition parameters (Table 1). There were no and 3B). However, the expression level of UPIII
differences in basal pressure, micturition pressure, in the urothelium of the nicotine‐treated rats,
or residual volume among the groups. There were also 0.10 ± 0.02, was lower than in the control rats,
no differences between the control and nicotine‐ 0.21 ± 0.01 (P < .01; Figure 3C). Similarly, expression
treated rats in voiding interval or micturition volume. of AQP3 in the urothelium of the nicotine‐treated rats,
In contrast, the voiding interval in the nicotine‐ 0.18 ± 0.01, was lower than in the controls, 0.22 ± 0.01
treated rats that received the acetic acid instillation (P < .05; Figure 3D).
was shorter than the nicotine‐free rats that were Blood flow in the bladder neck of the nicotine‐
treated similarly (P < .01). The micturition volume in treated rats that received the acetic acid instillation,
the nicotine‐treated rats that received the acetic acid 28.6 ± 0.7, was higher than in the nicotine‐treated rats
instillation was smaller than the nicotine‐free rats without acetic acid instillation, 22.7 ± 0.7 (P < .05;
that were treated similarly (P < .05). The bladder Figure S1A and S1B). However, blood flow in the
capacity in the nicotine‐treated rats that received the nicotine‐treated rats receiving acetic acid instillation
acetic acid instillation tended to decrease compared tended to be lower than in the nicotine‐free rats that
with the nicotine‐free rats that were treated similarly. did not receive the acetic acid instillation (30.7 ± 0.5;
P < .01). Consequently, the expression level of HIF1α
in the nicotine‐treated, acetic acid instilled rats,
3.2 | Effects of nicotine on bladder 0.45 ± 0.04, was the same as in the nicotine‐treated
blood flow and expression of hypoxia, rats without acetic acid instillation (Figure S1C and
urothelium, and water channel markers S1D). However, the expression level of UPIII in the
At the bladder neck, the blood flow of the nicotine‐free nicotine‐treated rats with acetic acid instillation,
control rats (Figure 2A) was 30.7 ± 0.5 (Table 2). In 0.04 ± 0.01, was lower than in the bladders of the
contrast, the blood flow of the nicotine‐treated rats nicotine‐treated rats without acetic acid instillation,
(Figure 2B) was 22.7 ± 0.7 (P < .01, Table 2). The slower 0.10 ± 0.02 (P < .01; Figure S1C‐F). The expression
flow rate suggests that the tissue could be relatively level of AQP3, 0.15 ± 0.01, in nicotine‐treated rats with
hypoxic. Therefore, we evaluated the expression levels of acetic acid instillation was similar to the nicotine‐
the hypoxic marker HIF1α, a key indicator of the treated rats without acetic acid instillation, 0.18 ± 0.01
activation of hypoxic metabolic pathways. HIF1α was (P > .05; Figure S1E and S1F).

T A B L E 1 Micturition parameters of nicotine‐free control and nicotine‐treated rats with and without acetic acid intravesicular instillation

Acetic acid intravesicular instillation

Nicotine‐free control Nicotine treatment Nicotine‐free control Nicotine treatment

Micturition parameters (n = 6) (n = 6) (n = 6) (n = 6)
Basal pressure (cmH2O) 5.25 ± 1.10 4.65 ± 1.59 2.30 ± 0.69 3.63 ± 1.13
Micturition pressure (cmH2O) 31.68 ± 2.27 29.60 ± 1.70 27.70 ± 4.91 23.26 ± 2.36
Voiding interval (min) 12.26 ± 2.02 11.91 ± 1.23 12.63 ± 1.10 7.51 ± 0.65 **
Micturition volume (mL) 2.12 ± 0.35 2.15 ± 0.23 2.39 ± 0.19 1.50 ± 0.09 *
Residual volume (mL) 0.03 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 0.02 ± 0.01
Bladder capacity (mL) 2.15 ± 0.34 2.15 ± 0.23 2.39 ± 0.19 1.61 ± 0.06
*
P < .05.
**
P < .01: compared with the nicotine‐free control rats with acetic acid intravesicular instillation.
6 | NAGAI ET AL.

FIGURE 2 Measurement of urinary

bladder blood flow at the bladder neck
and expression of HIF1α and UPIII within
the urothelium layers. A and B,
Representative images of the bladder
blood flow at the bladder neck were
analyzed. The bladder blood flows of the
nicotine‐free control rats (30.7 ± 0.5,
n = 6) were significantly higher than that
of the nicotine‐treated ones (22.7 ± 0.7;
P < .01, n = 5). Circle, the area in which
blood flow was measured. C and D,
Expression of HIF1α and UPIII. Within
the urothelium of both the control (C) and
nicotine‐treated (D) rats, HIF1α‐ (arrows,
green stain) and UPIII‐ (arrowheads,
red stain) positive areas were detected.
Blue: nuclei. E, Proportion of HIF1α
in the urothelium of the nicotine‐treated
rat was significantly higher than in
the control rats. *P < .01 compared
with the nicotine‐free control rats. HIF1α,
hypoxia‐inducible factor 1α; UPIII,
uroplakin III

3.3 | Investigations of nicotine‐induced followed 13 days of nicotine treatment. Tadalafil was

deterioration of bladder storage functions given during the last 6 days of nicotine treatment, and
with phosphodiesterase type 5 inhibitor cystometry was performed 1 day after intravesicular
instillation with 0.08% acetic acid. In rats treated with
From the above results, we inferred that nicotine‐
tadalafil, the nicotine‐dependent deterioration of blad-
induced reduction of bladder blood flow might be a
der storage functions improved (Figure 4A). The voiding
factor in the deterioration of bladder storage functions.
interval and micturition volumes were 13.84 ± 0.94 min-
Thus, the following experiments determined if tadalafil,
utes and 2.77 ± 0.15 mL, respectively, both of which
a phosphodiesterase type 5 inhibitor that increases
were significantly higher than in nicotine‐treated rats
blood flow to the lower urinary tract,10,11 could
that received the acetic acid instillation but not the
ameliorate the loss of bladder storage functions that
tadalafil injections.
NAGAI ET AL. | 7

T A B L E 2 Bladder blood flow rates at the bladder neck in (Figure 4C) decreased compared with the rats not
nicotine‐free control, nicotine‐treatment, and tadalafil‐admini- receiving it (0.50 ± 0.01; P < .01). The expression levels
strated nicotine‐treatment rats of both UPIII, 0.18 ± 0.01, and AQP3, 0.26 ± 0.02,
Nicotine‐free Nicotine‐
within the urothelium layers of nicotine‐treated rats
control Nicotine‐treatment treated with that received tadalafil increased compared with those
(n = 6) (n = 5) tadalafil (n = 5) not receiving it (0.10 ± 0.02 and 0.18 ± 0.01 respectively;
Blood flow at the 30.70 ± 0.53 22.65 ± 0.72*,† 30.32 ± 2.79 P < .01 each, Figure 4D). The values for bladder
bladder neck blood flow and marker expression levels in the
Note: Flow rates were measured in arbitrary units based on the colored pixels nicotine‐treated rats that received tadalafil were not
in the bladder neck.
*
different from the nicotine‐free control rats (P > .05 for
P < .01: compared with the nicotine‐free control rats.
†
P < .01: compared with the nicotine‐treated with tadalafil.
each comparison).

The blood flow at the bladder neck following tadalafil

administration to nicotine‐treated rats (Figure 4B) was 4 | DISCUSSION
significantly higher than in the nicotine‐treated rats not
receiving the tadalafil (P < .01, Table 2). The expression Previous reports have documented the relationship between
level of HIF1α within the urothelium layers of the cigarette smoking and LUTS.5,7,12 Cigarette‐derived nicotine
nicotine‐treated rats that received tadalafil, 0.33 ± 0.04 is a chemical mediator that induces sympathetic nervous

FIGURE 3 Expressions of UPIII and water channel marker AQP3 within the urothelium layers. A, Within the urothelium of
nicotine‐free control rats, both UPIII (arrowheads, red stain) and AQP3 (arrows, green stain) were detected. B, Similarly, nicotine‐treated
rats expressed both markers. C, Proportion of UPIII in urothelium of the nicotine‐treated rats was significantly lower than in the control rats.
D, Proportion of AQP3 in urothelium of in the nicotine‐treated rats was also lower than in the control rats. *P < .05, **P < .01 compared with
the nicotine‐free control rats. Blue: nuclei. AQP3, aquaporin 3; UPIII, uroplakin III
8 | NAGAI ET AL.

FIGURE 4 Improvements of urothelium hypoxia in the nicotine‐treated rats administered tadalafil. A, In a representative cystometric
recording following acetic acid intravesical instillation, the tadalafil‐administered rats had increased voiding intervals and micturition
volumes. B, The bladder blood flow at the bladder neck in the tadalafil‐administered rats was much higher than in similarly treated rats
without tadalafil (Compare with Figure 2B). C, Expression of both HIF1α (arrows, green stain) and UPIII (red, arrowheads) within the
urothelium of tadalafil‐administered rats were detected. Blue: nuclei. D, Expression of both UPIII (arrowheads, red stain) and AQP3 (arrows,
green stain) within the urothelium layers in the tadalafil‐administered rats were detected. HIF1α, hypoxia‐inducible factor 1α; UPIII,
uroplakin III
NAGAI ET AL. | 9

system activity and vasoconstriction. These effects enhance Urothelial umbrella cells are covered with numerous
bladder smooth muscle tone and are also associated with the plaques consisting of uroplakins, one of which is UPIII,
development of bladder storage symptoms.6,13,14 In addition, that are pivotal components of the normal urothelial
nicotine binds to nicotinic cholinergic receptors in the barrier function.21-23 Some studies reported that uropla-
urothelium and bladder afferent nerves,17,18 as well as both kin defects lead to the deficiency of urothelial perme-
central and peripheral nerves.19 However, the effects of ability barrier and abnormal voiding patterns.24-26 AQP3
nicotine on blood flow within the bladder or histological is a water channel that transports water, glycerol, and
changes in the urothelium associated with reduced blood other small solutes through the cell membrane.27-30 The
flow and possible hypoxia are unknown. Clearly, these types decreased expression of both UPIII and AQP3 within the
of changes could hinder bladder functions. urothelial layers could be responsible, at least in part, for
In this study, rats treated with nicotine for 13 days did not the reduction in bladder storage functions in the
show the cystometrically measurable deterioration of bladder nicotine‐treated rats that were stimulated with acetic
storage functions. However, the nicotine‐treated rats that acid intravesical instillation.
were stimulated with intravesicular instillation of acetic acid We sought to determine if reversal of the nicotine‐
the day before cystometric investigations exhibited significant induced reduction of blood flow to the bladder could
deterioration of bladder storage functions, including the restore the loss of storage functions and the associated
increased urinary frequency with short voiding intervals, and urothelial cell markers. Therefore, for the final 6 days of
low micturition volume. Thus, we focused on bladder blood the nicotine‐treatment, we injected tadalafil, a phospho-
flow and histological changes of urothelium. diesterase type 5 inhibitor that improves blood flow to the
In the nicotine‐treated rats, blood flow at the bladder lower urinary tract.10,11,14 The tadalafil treatment not
neck was decreased compared with the nicotine‐free only increased bladder blood flow, but it also improved
controls. In addition, expression of the hypoxia marker the bladder storage functions, increased the urothelial
HIF1α within the urothelium layers in the nicotine‐ expression of UPIII and AQP3, and decreased the
treated rats was significantly higher than in the control expression of HIF1α. We did not investigate changes in
rats; however, there was no difference in the expression cyclic guanosine monophosphate, which is increased in
of HIF1α between the detrusors of the experimental and urinary bladders of rats by tadalafil treatments; however,
control rats. These results suggest that the nicotine activation of nicotinic cholinergic receptors within the
treatments caused a reduction in bladder blood flow and urothelium could improve the bladder storage func-
induced a hypoxic environment within the urothelium. tions.15,16 In addition, we did not assess suppression of
Within the urothelial layers of the nicotine‐treated rats, bladder afferent nerve activation by the tadalafil treat-
expression of both UPIII, a urothelial marker, and AQP3, ments.31 The decreased firing of the afferent nerve in
a marker of cell membrane water channels, decreased response to tadalafil treatments might have been one of
compared with the controls. the mechanisms by which the bladder storage function
In this study, the nicotine‐treated rats were stimulated improved. At least, these results suggest that the hypoxic
with acetic acid instillation to clearly reveal the cystome- environment of the urothelium induced by the nicotine‐
trically measurable deterioration of bladder storage func- treatment contributes to the development of LUTS.
tions. Blood flow in the bladders of the nicotine‐treated This study showed that nicotine‐treated rats can
rats with acetic acid instillation increased compared with provide a useful model to investigate the development
the nicotine‐treated rats without acetic acid instillation. of LUTS associated with cigarette smoking. Never-
However, the expression levels of both HIF1α and AQP3 theless, as with all animal models, there are some
were not altered following the acetic acid instillation. In limitations. This study design is that nicotine‐treat-
contrast, the expression level of UPIII was decreased. A ment was given twice a day for only 13 days. A longer
recent study reported that the urothelium in diabetic rats period of treatment could provide additional informa-
exhibited the effects of hypoxia due to decreased and/or tion regarding the effects of nicotine on lower urinary
deteriorated bladder blood flow.20 This finding is consis- tract structure and function. In addition, we did not use
tent with the data reported here and suggests that the disease model rats, such as those with hypertension or
structure and function of the urothelium are very sensitive diabetes mellitus. In clinical practice, many patients
to the effects of reduced blood flow and the associated with LUTS have a long history of smoking. In addition,
hypoxia, regardless of the cause of the reduced blood flow. concurrent with their smoking practice, many patients
Therefore, our results also suggested that nicotine treat- have hypertension or diabetes mellitus. Therefore,
ments, which can reduce peripheral blood flow, could future studies will include long‐term nicotine
induce disorganization of the urothelium and simulta- treatment in combination with hypertensive and/or
neously enhance sensitivity to chemical stimulation. diabetes mellitus rat models.
10 | NAGAI ET AL.

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