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Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency
EURURO-6143; No. of Pages 7 EUROPEAN UROLOGY XXX (2015) XXX–XXX
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Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency Pieter Uvin a,b,c,y, Jan Franken a,b,c,y, Silvia Pinto a,c, Roma Rietjens a,b,c, Luc Grammet a,c, Yves Deruyver a,b,c, Yeranddy A. Alpizar a,c, Karel Talavera a,c, Rudi Vennekens a,c, Wouter Everaerts a,b,c, Dirk De Ridder b,c, Thomas Voets a,c,* a
Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium; b Laboratory of Experimental Urology,
Department of Development and Regeneration, KU Leuven, Leuven, Belgium; c TRP Research Platform Leuven (TRPLe), Leuven, Belgium
Article info
Abstract
Article history: Accepted March 22, 2015
Background: Acute exposure of part of the skin to cold stimuli can evoke urinary urgency, a
Keywords: Urinary urgency Cold sensing TRPM8
phenomenon termed acute cold-induced urgency (ACIU). Despite its high prevalence, particularly in patients with overactive bladder, little is known about the mechanisms that induce ACIU. Objective: To develop an animal model of ACIU and test the involvement of cold-activated ion channels transient receptor potential (TRP) M8 and TRPA1. Design, setting, and participants: Intravesical pressure and micturition were monitored in female mice (wild-type C57BL/6J, Trpa1/, Trpm8+/+, and Trpm8/) and Sprague Dawley rats. Interventions: An intravesical catheter was implanted. Localized cooling of the skin was achieved using a stream of air or topical acetone. The TRPM8 antagonist (N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide (AMTB) or vehicle was injected intraperitoneally. Outcome measurements and statistical analysis: Frequencies of bladder contractions and voids in response to sensory stimuli were compared using the Mann-Whitney or KruskalWallis test. Results and limitations: Brief, innocuously cold stimuli applied to different parts of the skin evoked rapid bladder contractions and voids in anesthetized mice and rats. These responses were strongly attenuated in Trpm8/ mice and in rats treated with AMTB. As rodent bladder physiology differs from that of humans, it is difficult to directly extrapolate our findings to human patients. Conclusions: Our findings indicate that ACIU is an evolutionarily conserved reflex rather than subconscious conditioning, and provide a useful in vivo model for further investigation of the underlying mechanisms. Pharmacological inhibition of TRPM8 may be useful for treating ACIU symptoms in patients. Patient summary: Brief cold stimuli applied to the skin can evoke a sudden desire to urinate, which can be highly bothersome in patients with overactive bladder. We developed an animal model to study this phenomenon, and found that it depends on a specific molecular cold sensor, transient receptor potential M8 (TRPM8). Pharmacological inhibition of TRPM8 may alleviate acute cold-induced urinary urgency in humans.
# 2015 European Association of Urology. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/). y These authors contributed equally. * Corresponding author. Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Herestraat 49 bus 802, B-3000 Leuven, Belgium. Tel. +32 16 330217; Fax: +32 16 345991. E-mail address: [email protected] (T. Voets).
http://dx.doi.org/10.1016/j.eururo.2015.03.037 0302-2838/# 2015 European Association of Urology. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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1.
Introduction
It is generally recognized that cold temperatures can influence bladder function in healthy individuals, and particularly in patients with lower urinary tract disorders (LUTd) [1,2]. One classic example is the ice water test, a urodynamic test in which ice-cold water is instilled into the bladder to unmask neurological pathology in LUTd patients [3]. This procedure evokes a strong detrusor contraction (the bladder cooling reflex) in children younger than 4 yr and in patients with neurogenic detrusor overactivity due to upper motor neuron lesions, but is mostly without effect in healthy adults or patients with non-neurogenic LUTd [3]. Although the exact neuronal mechanisms underlying this phenomenon are not yet fully elucidated, there is considerable evidence that the bladder cooling reflex is initiated by activation of cold-sensitive C fibers that innervate the bladder wall [3]. Under normal environmental conditions, it is highly unlikely that the temperature inside the bladder wall drops sufficiently to activate such cold-sensitive fibers. Nevertheless, low environmental temperature elicits more frequent urination in healthy individuals. Importantly, 46% of patients with overactive bladder (OAB) report that cold weather aggravates urgency symptoms [1]. Effects of whole-body cooling on LUT symptoms may be partly explained by changes in renal function in response to moderate hypothermia. Animal and human experiments have shown that hypothermia decreases antidiuretic hormone release and increases diuresis [4–7], which lead to an increase in voiding frequency and potentially to urgency. In addition, urgency symptoms can be evoked by cold stimuli that do not evoke significant central hypothermia or increased diuresis, suggesting that activation of peripheral cold sensors can directly influence bladder function. For instance, it has been shown that transfer of awake rats from room temperature to a low-temperature environment (4 8C) leads to a transient reduction in bladder capacity and increased micturition frequency [8] that correlate in time with a decrease in skin temperature and an increase in blood pressure [2]. Another widely acknowledged but poorly understood effect of external temperature on bladder function is the phenomenon whereby brief exposure of restricted parts of the body to a mildly cold stimulus can evoke urgency, with or without urgency incontinence. We term this phenomenon acute cold-induced urgency (ACIU) to distinguish it from effects of more global and prolonged exposure to cold temperatures (eg, cold weather urgency or cold stress-induced detrusor overactivity). ACIU evoked by everyday cold stimuli (eg, washing hands, entering a swimming pool, stepping on a cold floor, or experiencing a cold breeze) [9] is a frequent complaint of patients with OAB, wet or dry, but is also common in healthy individuals, especially in children and the elderly [10]. Little is known about the mechanisms underlying ACIU, particularly because of the absence of good experimental models to study the phenomenon, so specific treatments are lacking. It has been suggested that ACIU represents a
form of Pavlovian conditioning [11–13], but direct evidence supporting this view is lacking. Environmental thermal cues are conveyed to the central nervous system via sensory neurons that contain sensory endings in the skin [14]. In these endings, temperaturesensitive ion channels of the transient receptor potential (TRP) superfamily act as the main molecular thermosensors, translating environmental temperature into neuronal activity [14]. Earlier work has established that two specific TRP channels, TRPM8 and TRPA1, can act as cold-activated ion channels in sensory neurons [15,16]. In particular, Trpm8/ and Trpa1/ mice both exhibit specific deficits in sensing cold temperatures [17–20]. TRPM8 and TRPA1 have also been implicated in sensory signaling of the LUT [21–24], but their exact involvement in cold-related bladder responses remains unclear [25]. For instance, TRPM8 activity has been associated with detrusor overactivity in rats exposed to severe and prolonged cold stress [2,26,27], but this conclusion was based on the use of a relatively nonspecific antagonist (N-(4-tertiarybutylphenyl)-4-(3chloropyridin-2-yl)tetrahydropyrazine -1(2H)-carboxamide, which also inhibits other TRP channels, including TRPV1 [28]) and agonist (menthol, which also modulate various cation channels, including TRPA1 [29,30]). Here we demonstrate that bladder contraction and bladder voiding can be reproducibly evoked by brief and localized cold stimuli in anesthetized mice and rats, which thus provides an animal model that mimics the hallmarks of ACIU in humans. Moreover, using this model we demonstrate that cold-induced bladder responses are greatly diminished by genetic ablation or pharmacological inhibition of TRPM8, a cold-sensitive cation channel of the TRP superfamily [14]. Our results provide a basis for further research into the etiology of ACIU and, more importantly, suggest that TRPM8 antagonists could be used to alleviate ACIU symptoms. 2.
Materials and methods
2.1.
Animals
Experiments were conducted on 10–12-wk-old female mice and on 11–13-wk-old female Sprague Dawley rats. The mice strains that were used included wild-type (WT) C57Bl/6J mice, Trpa1/ mice backcrossed on the C57Bl/6J background for 10 generations [20], and Trpm8/ and Trpm8+/+ littermates from crosses of Trpm8+/ breeding pairs [19]. Agematched WT C57Bl/6J mice were used as controls when assessing the bladder phenotype of Trpa1/ mice [20]. Animals were housed in a conventional facility at 21 8C on a 12-h light/dark cycle with chow diet and water available ad libitum. All experiments were approved by the KU Leuven Ethics Committee for Laboratory Animals under project numbers P089 and P053/2011.
2.2.
Solutions, drugs, and anesthetics
The perfusion solution for cystometry was a 0.9% NaCl solution (Baxter). The TRPM8 inhibitor N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide (AMTB) hydrochloride salt (Tocris) was tested at a dose of 3 mg/kg. Rats were randomly divided in two groups. The first group received an intraperitoneal (IP) injection of
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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3
AMTB from a 100-mM solution in 0.5% methylcellulose; the second
or (5) rubbing the hind paw with a cotton swab. After either full
group served as controls and received only the vehicle.
termination of the cold-induced contraction or after a waiting period of
Rats and mice were anesthetized with isoflurane (Piramal Health-
60 s following the cold stimulus, the infusion was restarted.
care; 5% for induction, 2% for maintenance) during surgery. Rats were injected subcutaneously (SC) with buprenorphine (Reckitt Benckiser;
2.4.
Statistical analysis
0.03 mg/kg) every 12 h until cystometry. During cystometry, rats and mice were anesthetized with urethane (Sigma) injected SC at a dose of 1.3 g/kg.
Data were analyzed using Origin (OriginLab, Northampton, MA, USA). Group data are expressed as the mean standard error of the mean (SEM) for n animals. The Shapiro-Wilk test was used to test the normality of the
2.3.
Cystometry and sensory stimuli
data. When normality was not rejected, group comparisons were made using one-way analysis of variance with Tukey’s post hoc test. Nonpara-
Implantation of a bladder catheter for cystometry experiments was
metric data were compared using the Mann-Whitney test (comparison
performed as previously described [31]. The lower back of each animal
of two groups) or the Kruskal-Wallis test with Dunn’s post hoc test
was shaved. Anesthetized animals were placed in a supine position
(comparison of multiple groups).
under a heating lamp to maintain normal body temperature before the start of the experimental protocol.
3.
Results
During the cystometry protocol, which is outlined in detail in the Results section, perfusion was stopped and specific sensory stimuli were applied, consisting of (1) blowing air on the paw or ears during 20 s by applying a stream of carbogen (95% O2 and 5% CO2) via a 1-m-long polyethylene tube (inner diameter 1 mm) connected to a gas container at 21 8C with an exit pressure of 0.2 bar at an outflow distance of 5 cm from the animal; (2) application of 500 ml (mice) or 3 ml (rats) of acetone on the lower back using a syringe; (3) similar application of the same volume of tap water at 37 8C; (4) pinching a hind paw with a broad-tipped forceps;
To test whether ACIU can be objectively mimicked in a small-animal model, we developed a standardized assay in which cool stimuli were applied to the skin of anesthetized mice during cystometry (Fig. 1A). First, normal cystometry was performed during 30 min, which involved measurement of the intravesical pressure and visual monitoring of voids during continuous infusion of saline at a rate of
Fig. 1 – Acute cold-induced bladder responses in a mouse model of acute cold-induced urgency. (A) Cystometric trace illustrating the assay for detection of acute cold-induced bladder responses in mice. After a habituation period of 30 min (not shown), perfusion was stopped at a time point corresponding to 60% of the mean intercontractile interval (ICI) since the last normal voiding contraction. Then, after a waiting interval equivalent to twice the mean ICI, a thin stream of air was applied to one of the hind paws of the animal for 20 s. This resulted in a robust bladder contraction and a void. After termination of the contraction, perfusion was restarted and a normal cystometric pattern was restored. In this and all subsequent cystometric traces, red and green vertical lines indicate the time points at which perfusion was halted and restarted, respectively, and asterisks below the pressure trace indicate visually confirmed voids. (B,C) Same protocol as in A, showing the effect of (B) topical application of acetone on the back or (C) pinching the paw with a forceps. (D) Response frequencies (expressed as mean W standard error of the mean) to the stimuli air on paw (n = 13), paw pinching (n = 6), paw rubbing (n = 6), acetone on back (n = 14), and warm water on back (n = 6). For each animal, the response frequency was determined based on four identical stimuli. * p < 0.05, ** p < 0.01 versus both cold stimuli (Kruskal-Wallis test with Dunn’s post hoc test).
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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20 ml/min [31]. This period allowed establishment of a stable pattern of bladder filling, contraction, and voiding, from which the mean intercontractile interval (ICI) was determined [31]. After a normal void, infusion was stopped at 60% of the mean ICI, resulting in a bladder that was filled to an estimated 60% of its functional capacity. After an interval equivalent of twice the mean ICI, a brief stimulus was applied to the skin of the animal. As illustrated in Figure 1A, blowing of a stream of air over the hind paw of a WT animal during 20 s consistently evoked a bladder contraction in 80% of cases that was associated with a void in approximately 50% of cases (Fig. 1A,D). Similar response frequencies were obtained when acetone (500 ml) was applied to the skin, resulting in local evaporative cooling (Fig. 1B,D). The delay between the onset of the cold stimulus and the onset of bladder contraction was short (4.8 0.9 s), and responses that occurred >20 s after the onset of the cold stimulus were discarded as being nonspecific. In contrast to cold stimuli, vigorous pinching of a hind paw with a forceps was mostly ineffective in evoking bladder contraction or voiding (Fig. 1C,D). Similarly, two other non-noxious control stimuli (gentle rubbing of the hindpaw with a cotton swab and application of 500 ml of water at 37 8C to the back) were much less effective in evoking a bladder response (Fig. 1D). Taken together, these results show that localized, mildly cold stimuli can evoke bladder contraction and voiding in anesthetized mice, and thus mimic the hallmarks of ACIU in humans. To investigate whether the cold-activated ion channels TRPM8 and TRPA1 are involved in the cold-induced bladder responses, we applied a standardized cystometry assay to Trpm8/ and Trpa1/ mice. During the 30-min habituation period, we did not detect obvious differences in cystometric pattern among WT, Trpm8+/+, Trpm8/, and Trpa1/ mice (Fig. 2A,B), and there were no statistically significant differences in basal pressure, voiding contraction amplitude, or ICI (Fig. 2C-E). These results indicate that TRPM8 and TRPA1 are not essential for the normal voiding cycle or contractility of the mouse bladder. Importantly, we found that the occurrence of bladder responses to cold stimuli was strongly reduced in Trpm8/ mice compared to Trpm8+/+ littermates (Fig. 2A,F,G). By contrast, there was no significant difference in response frequency to cold stimuli between Trpa1/ mice and matched WT mice (Fig. 2B,F,G). These results demonstrate that acute cold-induced bladder contraction and voiding in mice depend on the cold sensor TRPM8 but not on TRPA1. As illustrated in Figure 3A, acute cold-induced bladder responses could also be reproduced in rats. Notably, using a similar stimulation scheme as in mice, response fidelity in rats was higher than in mice, with 100% voiding (nine animals; four applications per animal) in response to acetone application to the back (Fig. 3A,C,E). Earlier work has provided evidence that dichotomizing axons of lumbosacral dorsal root ganglia innervate both the skin and the bladder, and it was suggested that such dichotomizing neurons may be responsible for bladder responses to cold environmental stimuli [32]. However, we found that bladder contractions and voids could also be evoked by brief pulses of cold air applied to rat ears (Fig. 3B; void frequency
83% 17%, n = 5). This suggests that cold-induced bladder responses are not limited to stimulation of dermatomes innervated by neurons from the dorsal root ganglia that also project to the bladder (mainly L6–S1) [33]. To investigate whether the cold-induced bladder responses in rats also depend on TRPM8, we applied the most reproducible cold stimulus, acetone application to the back, in animals that were pretreated with vehicle or with the TRPM8 antagonist AMTB (3 mg/kg IP) [34]. During the first 90 min following injection of AMTB or vehicle, four separate acetone applications were applied per animal, interspaced by a normal voiding cycle (Fig. 3C,D). In vehicle-treated animals, the response rate was 100% (n = 6 animals; four voiding responses per animal; Fig. 3C,E). By contrast, within the first 60 min after injection, two thirds of the AMTB-treated animals failed to respond to the acetone stimulus (p < 0.01; Fig. 3D,E). Bladder responses to acetone application gradually recovered after 1 h (Fig. 3E), in line with the rapid clearance of AMTB (mean residence time 45 min) according to pharmacokinetic studies [34]. Taken together, these results indicate that acute cold-induced bladder responses in rats are also dependent on TRPM8. 4.
Discussion
Common daily life events, such as entering the home (latchkey incontinence), the sound or feel of running water, stepping on a cold floor, or exposure to cold wind, can evoke urinary urgency, which can be particularly bothersome in patients with OAB [10,11,35,36]. In general, it has been assumed that Pavlovian conditioning and/or subconscious automatic behavior underlies urgency symptoms elicited by such external stimuli [11–13]. In contrast to this view, we demonstrated that brief and moderately cold stimuli resulting in only modest cooling of a restricted part of the skin can consistently evoke acute bladder contractions in anesthetized mice and rats. This implies that bladder responses to acute environmental cold stimuli represent an evolutionarily conserved reflex that can be objectively measured and studied in the apparent absence of (sub)conscious processes. The key manifestation of ACIU in humans is an acute desire to urinate, with or without actual voiding, in response to a brief localized and mildly cold stimulus. Thus, ACIU can be clearly distinguished from other betterstudied effects of cold stimuli on bladder functioning, such as the bladder cooling reflex and cold stress-induced detrusor overactivity [2,26,27]. While measurement of urinary urgency in animals remains elusive [37], we believe that our assay is the closest approximation to ACIU in humans available today, and will be useful for elucidation of the underlying pathways and the development of potential treatments for ACIU. Intriguingly, although not scientifically documented to the best of our knowledge, there is extensive anecdotal evidence that immersion of the hand of a sleeping person in tepid water can evoke bedwetting in at least a subset of victims (hand-in-water prank), and our present work indicates that this trick can be quite accurately played
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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Fig. 2 – Role of TRPM8 and TRPA1 in the acute cold-induced bladder response in mice. (A,B) Cystometric traces (same protocol as in Fig. 1A) showing the effect of acute cold stimuli (acetone on back) in (A) a Trpm8S/S mouse and (B) a Trpa1S/S mouse. (C–E) Comparison of (C) mean basal pressure, (D) amplitude of a voiding contraction (D), and (E) ICI in control C57Bl/6J mice (n = 6), Trpa1S/S mice (n = 6), Trpm8+/+ mice (n = 15), and Trpm8S/S mice (n = 16). There were no significant differences in these three parameters among the four groups (one-way analysis of variance, p > 0.5). (F,G) Frequency of the occurrence of (F) bladder contraction and (G) bladder voiding on stimulation in the four genotypes. Group data are presented as mean W standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Trpm8+/+ littermates (Mann-Whitney test).
on mice and rats. On the basis of our findings, we believe that testing the bladder response of sleeping individuals to localized cold stimuli in a scientifically controlled setting will be of interest, and may provide important insight into the mechanisms of nocturnal enuresis. The acute cold stimuli used in this study inevitably had a non-noxious mechanical component, namely wetting of the skin in the case of acetone application, and air movement in the case of cold air. At present, we cannot fully exclude that mechanical stimulation of the skin is involved in the bladder response to these cold stimuli. We indeed found
that non-noxious stimuli (gentle rubbing of the paw, wetting of the skin with water at 37 8C) and noxious pinching of the paw evoked a bladder response in a small percentage of animals. Similarly, a small percentage of Trpm8/ mice exhibited an acute bladder contraction on acetone application. These data suggest that although various sensory stimuli can evoke an acute bladder response, cold stimuli acting via TRPM8 are by far the most provocative. Our results also demonstrate that acute bladder responses to cold stimuli can be strongly attenuated by
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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Fig. 3 – The TRPM8 antagonist AMTB inhibits cold-induced bladder responses in rat. (A,B) Acute cold-induced bladder responses in rats in response to (A) application of acetone (3 ml) on the back or (B) blowing of cold air on the ear. (C,D) Bladder responses to four applications of acetone (arrows) on the back of rats injected with (C) vehicle or (D) AMTB at 900 s before time 0. Perfusion was restarted between two subsequent acetone applications, resulting in one full voiding cycle. (E) Frequency (mean W standard error of the mean) of the occurrence of a voiding contraction at the indicated time points after injection with either vehicle (n = 6) or AMTB (n = 6). ** p < 0.01 versus vehicle (Mann-Whitney test).
pharmacological inhibition of TRPM8. The first results for oral administration of a TRPM8 antagonist (PF-05105679) in humans show that the drug was well tolerated at doses that were effective in a cold pressor test [38]. Therefore, there are prospects for the development of TRPM8 antagonists for specific therapies to alleviate ACIU symptoms in LUTd patients.
Author contributions: Thomas Voets had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Uvin, Franken, Pinto, Everaerts, De Ridder, Voets. Acquisition of data: Uvin, Franken, Pinto, Rietjens, Deruyver, Alpizar. Analysis and interpretation of data: Uvin, Franken, Pinto, Everaerts, De Ridder, Voets.
5.
Conclusions
Drafting of the manuscript: Uvin, Franken, Everaerts, Voets. Critical revision of the manuscript for important intellectual content: Uvin, Franken, Pinto, Deruyver, Alpizar, Talavera, Vennekens, Everaerts, De
Taken together, our findings indicate that ACIU is an evolutionarily conserved reflex rather than a subconscious conditioning response. Moreover, we presented an in vivo model that can be used to objectively measure ACIU in rodents and that could form the basis for further investigation of the mechanisms underlying ACIU. Finally, our results indicate that the cold sensor channel TRPM8 is critically involved in ACIU, which suggests that pharmacological inhibition of this druggable target may be useful for treating ACIU symptoms in patients.
Ridder, Voets. Statistical analysis: Uvin, Voets. Obtaining funding: Vennekens, Everaerts, De Ridder, Voets. Administrative, technical, or material support: Pinto, Grammet. Supervision: Talavera, Vennekens, De Ridder, Voets. Other: None. Financial disclosures: Thomas Voets certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria,
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.
[20] Karashima Y, Talavera K, Everaerts W, et al. TRPA1 acts as a cold sensor in vitro and in vivo. Proc Natl Acad Sci U S A 2009;106:1273–8. [21] Du S, Araki I, Kobayashi H, Zakoji H, Sawada N, Takeda M. Differ-
Funding/Support and role of the sponsor: This research was supported by grants from the Research Foundation Flanders (FWO), the Research Council of KU Leuven, the Astellas European Foundation, and the Belgian Science Policy IUAP P7/13. Uvin is a PhD aspirant and De Ridder is a clinician-fundamental researcher of the FWO. The sponsors played no role in the study. Acknowledgments: We thank the members of our laboratories for helpful discussions.
ential expression profile of cold (TRPA1) and cool (TRPM8) receptors in human urogenital organs. Urology 2008;72:450–5. [22] Gratzke C, Streng T, Waldkirch E, et al. Transient receptor potential A1 (TRPA1) activity in the human urethra—evidence for a functional role for TRPA1 in the outflow region. Eur Urol 2009;55:696–704. [23] Mukerji G, Yiangou Y, Corcoran SL, et al. Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol 2006;6:6.
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netic properties. J Pharmacol Exp Ther 2003;306:377–86. [29] Karashima Y, Damann N, Prenen J, et al. Bimodal action of menthol on the transient receptor potential channel TRPA1. J Neurosci 2007; 27:9874–84. [30] Mahieu F, Owsianik G, Verbert L, et al. TRPM8-independent menthol-induced Ca2+ release from endoplasmic reticulum and Golgi. J Biol Chem 2007;282:3325–36. [31] Uvin P, Everaerts W, Pinto S, et al. The use of cystometry in small rodents: a study of bladder chemosensation. J Vis Exp 2012:e3869. [32] Shibata Y, Ugawa S, Imura M, et al. TRPM8-expressing dorsal root ganglion neurons project dichotomizing axons to both skin and bladder in rats. Neuroreport 2011;22:61–7. [33] Takahashi Y, Nakajima Y. Dermatomes in the rat limbs as determined by antidromic stimulation of sensory C-fibers in spinal nerves. Pain 1996;67:197–202. [34] Lashinger ES, Steiginga MS, Hieble JP, et al. AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome. Am J Physiol Renal Physiol 2008;295: F803–10. [35] Morris CL. Urge urinary incontinence and the brain factor. Neurourol Urodyn 2013;32:441–8. [36] O’Connell KA, Torstrick A, Victor E. Cues to urinary urgency and urge incontinence: how those diagnosed with overactive bladder syndrome differ from undiagnosed persons. J Wound Ostomy Continence Nurs 2014;41:259–67. [37] Parsons BA, Drake MJ. Animal models in overactive bladder research. In: Andersson KE, Michel MC, editors.
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Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency Pieter Uvin a,b,c,y, Jan Franken a,b,c,y, Silvia Pinto a,c, Roma Rietjens a,b,c, Luc Grammet a,c, Yves Deruyver a,b,c, Yeranddy A. Alpizar a,c, Karel Talavera a,c, Rudi Vennekens a,c, Wouter Everaerts a,b,c, Dirk De Ridder b,c, Thomas Voets a,c,* a
Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium; b Laboratory of Experimental Urology,
Department of Development and Regeneration, KU Leuven, Leuven, Belgium; c TRP Research Platform Leuven (TRPLe), Leuven, Belgium
Article info
Abstract
Article history: Accepted March 22, 2015
Background: Acute exposure of part of the skin to cold stimuli can evoke urinary urgency, a
Keywords: Urinary urgency Cold sensing TRPM8
phenomenon termed acute cold-induced urgency (ACIU). Despite its high prevalence, particularly in patients with overactive bladder, little is known about the mechanisms that induce ACIU. Objective: To develop an animal model of ACIU and test the involvement of cold-activated ion channels transient receptor potential (TRP) M8 and TRPA1. Design, setting, and participants: Intravesical pressure and micturition were monitored in female mice (wild-type C57BL/6J, Trpa1/, Trpm8+/+, and Trpm8/) and Sprague Dawley rats. Interventions: An intravesical catheter was implanted. Localized cooling of the skin was achieved using a stream of air or topical acetone. The TRPM8 antagonist (N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide (AMTB) or vehicle was injected intraperitoneally. Outcome measurements and statistical analysis: Frequencies of bladder contractions and voids in response to sensory stimuli were compared using the Mann-Whitney or KruskalWallis test. Results and limitations: Brief, innocuously cold stimuli applied to different parts of the skin evoked rapid bladder contractions and voids in anesthetized mice and rats. These responses were strongly attenuated in Trpm8/ mice and in rats treated with AMTB. As rodent bladder physiology differs from that of humans, it is difficult to directly extrapolate our findings to human patients. Conclusions: Our findings indicate that ACIU is an evolutionarily conserved reflex rather than subconscious conditioning, and provide a useful in vivo model for further investigation of the underlying mechanisms. Pharmacological inhibition of TRPM8 may be useful for treating ACIU symptoms in patients. Patient summary: Brief cold stimuli applied to the skin can evoke a sudden desire to urinate, which can be highly bothersome in patients with overactive bladder. We developed an animal model to study this phenomenon, and found that it depends on a specific molecular cold sensor, transient receptor potential M8 (TRPM8). Pharmacological inhibition of TRPM8 may alleviate acute cold-induced urinary urgency in humans.
# 2015 European Association of Urology. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/). y These authors contributed equally. * Corresponding author. Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Herestraat 49 bus 802, B-3000 Leuven, Belgium. Tel. +32 16 330217; Fax: +32 16 345991. E-mail address: [email protected] (T. Voets).
http://dx.doi.org/10.1016/j.eururo.2015.03.037 0302-2838/# 2015 European Association of Urology. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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1.
Introduction
It is generally recognized that cold temperatures can influence bladder function in healthy individuals, and particularly in patients with lower urinary tract disorders (LUTd) [1,2]. One classic example is the ice water test, a urodynamic test in which ice-cold water is instilled into the bladder to unmask neurological pathology in LUTd patients [3]. This procedure evokes a strong detrusor contraction (the bladder cooling reflex) in children younger than 4 yr and in patients with neurogenic detrusor overactivity due to upper motor neuron lesions, but is mostly without effect in healthy adults or patients with non-neurogenic LUTd [3]. Although the exact neuronal mechanisms underlying this phenomenon are not yet fully elucidated, there is considerable evidence that the bladder cooling reflex is initiated by activation of cold-sensitive C fibers that innervate the bladder wall [3]. Under normal environmental conditions, it is highly unlikely that the temperature inside the bladder wall drops sufficiently to activate such cold-sensitive fibers. Nevertheless, low environmental temperature elicits more frequent urination in healthy individuals. Importantly, 46% of patients with overactive bladder (OAB) report that cold weather aggravates urgency symptoms [1]. Effects of whole-body cooling on LUT symptoms may be partly explained by changes in renal function in response to moderate hypothermia. Animal and human experiments have shown that hypothermia decreases antidiuretic hormone release and increases diuresis [4–7], which lead to an increase in voiding frequency and potentially to urgency. In addition, urgency symptoms can be evoked by cold stimuli that do not evoke significant central hypothermia or increased diuresis, suggesting that activation of peripheral cold sensors can directly influence bladder function. For instance, it has been shown that transfer of awake rats from room temperature to a low-temperature environment (4 8C) leads to a transient reduction in bladder capacity and increased micturition frequency [8] that correlate in time with a decrease in skin temperature and an increase in blood pressure [2]. Another widely acknowledged but poorly understood effect of external temperature on bladder function is the phenomenon whereby brief exposure of restricted parts of the body to a mildly cold stimulus can evoke urgency, with or without urgency incontinence. We term this phenomenon acute cold-induced urgency (ACIU) to distinguish it from effects of more global and prolonged exposure to cold temperatures (eg, cold weather urgency or cold stress-induced detrusor overactivity). ACIU evoked by everyday cold stimuli (eg, washing hands, entering a swimming pool, stepping on a cold floor, or experiencing a cold breeze) [9] is a frequent complaint of patients with OAB, wet or dry, but is also common in healthy individuals, especially in children and the elderly [10]. Little is known about the mechanisms underlying ACIU, particularly because of the absence of good experimental models to study the phenomenon, so specific treatments are lacking. It has been suggested that ACIU represents a
form of Pavlovian conditioning [11–13], but direct evidence supporting this view is lacking. Environmental thermal cues are conveyed to the central nervous system via sensory neurons that contain sensory endings in the skin [14]. In these endings, temperaturesensitive ion channels of the transient receptor potential (TRP) superfamily act as the main molecular thermosensors, translating environmental temperature into neuronal activity [14]. Earlier work has established that two specific TRP channels, TRPM8 and TRPA1, can act as cold-activated ion channels in sensory neurons [15,16]. In particular, Trpm8/ and Trpa1/ mice both exhibit specific deficits in sensing cold temperatures [17–20]. TRPM8 and TRPA1 have also been implicated in sensory signaling of the LUT [21–24], but their exact involvement in cold-related bladder responses remains unclear [25]. For instance, TRPM8 activity has been associated with detrusor overactivity in rats exposed to severe and prolonged cold stress [2,26,27], but this conclusion was based on the use of a relatively nonspecific antagonist (N-(4-tertiarybutylphenyl)-4-(3chloropyridin-2-yl)tetrahydropyrazine -1(2H)-carboxamide, which also inhibits other TRP channels, including TRPV1 [28]) and agonist (menthol, which also modulate various cation channels, including TRPA1 [29,30]). Here we demonstrate that bladder contraction and bladder voiding can be reproducibly evoked by brief and localized cold stimuli in anesthetized mice and rats, which thus provides an animal model that mimics the hallmarks of ACIU in humans. Moreover, using this model we demonstrate that cold-induced bladder responses are greatly diminished by genetic ablation or pharmacological inhibition of TRPM8, a cold-sensitive cation channel of the TRP superfamily [14]. Our results provide a basis for further research into the etiology of ACIU and, more importantly, suggest that TRPM8 antagonists could be used to alleviate ACIU symptoms. 2.
Materials and methods
2.1.
Animals
Experiments were conducted on 10–12-wk-old female mice and on 11–13-wk-old female Sprague Dawley rats. The mice strains that were used included wild-type (WT) C57Bl/6J mice, Trpa1/ mice backcrossed on the C57Bl/6J background for 10 generations [20], and Trpm8/ and Trpm8+/+ littermates from crosses of Trpm8+/ breeding pairs [19]. Agematched WT C57Bl/6J mice were used as controls when assessing the bladder phenotype of Trpa1/ mice [20]. Animals were housed in a conventional facility at 21 8C on a 12-h light/dark cycle with chow diet and water available ad libitum. All experiments were approved by the KU Leuven Ethics Committee for Laboratory Animals under project numbers P089 and P053/2011.
2.2.
Solutions, drugs, and anesthetics
The perfusion solution for cystometry was a 0.9% NaCl solution (Baxter). The TRPM8 inhibitor N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide (AMTB) hydrochloride salt (Tocris) was tested at a dose of 3 mg/kg. Rats were randomly divided in two groups. The first group received an intraperitoneal (IP) injection of
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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AMTB from a 100-mM solution in 0.5% methylcellulose; the second
or (5) rubbing the hind paw with a cotton swab. After either full
group served as controls and received only the vehicle.
termination of the cold-induced contraction or after a waiting period of
Rats and mice were anesthetized with isoflurane (Piramal Health-
60 s following the cold stimulus, the infusion was restarted.
care; 5% for induction, 2% for maintenance) during surgery. Rats were injected subcutaneously (SC) with buprenorphine (Reckitt Benckiser;
2.4.
Statistical analysis
0.03 mg/kg) every 12 h until cystometry. During cystometry, rats and mice were anesthetized with urethane (Sigma) injected SC at a dose of 1.3 g/kg.
Data were analyzed using Origin (OriginLab, Northampton, MA, USA). Group data are expressed as the mean standard error of the mean (SEM) for n animals. The Shapiro-Wilk test was used to test the normality of the
2.3.
Cystometry and sensory stimuli
data. When normality was not rejected, group comparisons were made using one-way analysis of variance with Tukey’s post hoc test. Nonpara-
Implantation of a bladder catheter for cystometry experiments was
metric data were compared using the Mann-Whitney test (comparison
performed as previously described [31]. The lower back of each animal
of two groups) or the Kruskal-Wallis test with Dunn’s post hoc test
was shaved. Anesthetized animals were placed in a supine position
(comparison of multiple groups).
under a heating lamp to maintain normal body temperature before the start of the experimental protocol.
3.
Results
During the cystometry protocol, which is outlined in detail in the Results section, perfusion was stopped and specific sensory stimuli were applied, consisting of (1) blowing air on the paw or ears during 20 s by applying a stream of carbogen (95% O2 and 5% CO2) via a 1-m-long polyethylene tube (inner diameter 1 mm) connected to a gas container at 21 8C with an exit pressure of 0.2 bar at an outflow distance of 5 cm from the animal; (2) application of 500 ml (mice) or 3 ml (rats) of acetone on the lower back using a syringe; (3) similar application of the same volume of tap water at 37 8C; (4) pinching a hind paw with a broad-tipped forceps;
To test whether ACIU can be objectively mimicked in a small-animal model, we developed a standardized assay in which cool stimuli were applied to the skin of anesthetized mice during cystometry (Fig. 1A). First, normal cystometry was performed during 30 min, which involved measurement of the intravesical pressure and visual monitoring of voids during continuous infusion of saline at a rate of
Fig. 1 – Acute cold-induced bladder responses in a mouse model of acute cold-induced urgency. (A) Cystometric trace illustrating the assay for detection of acute cold-induced bladder responses in mice. After a habituation period of 30 min (not shown), perfusion was stopped at a time point corresponding to 60% of the mean intercontractile interval (ICI) since the last normal voiding contraction. Then, after a waiting interval equivalent to twice the mean ICI, a thin stream of air was applied to one of the hind paws of the animal for 20 s. This resulted in a robust bladder contraction and a void. After termination of the contraction, perfusion was restarted and a normal cystometric pattern was restored. In this and all subsequent cystometric traces, red and green vertical lines indicate the time points at which perfusion was halted and restarted, respectively, and asterisks below the pressure trace indicate visually confirmed voids. (B,C) Same protocol as in A, showing the effect of (B) topical application of acetone on the back or (C) pinching the paw with a forceps. (D) Response frequencies (expressed as mean W standard error of the mean) to the stimuli air on paw (n = 13), paw pinching (n = 6), paw rubbing (n = 6), acetone on back (n = 14), and warm water on back (n = 6). For each animal, the response frequency was determined based on four identical stimuli. * p < 0.05, ** p < 0.01 versus both cold stimuli (Kruskal-Wallis test with Dunn’s post hoc test).
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20 ml/min [31]. This period allowed establishment of a stable pattern of bladder filling, contraction, and voiding, from which the mean intercontractile interval (ICI) was determined [31]. After a normal void, infusion was stopped at 60% of the mean ICI, resulting in a bladder that was filled to an estimated 60% of its functional capacity. After an interval equivalent of twice the mean ICI, a brief stimulus was applied to the skin of the animal. As illustrated in Figure 1A, blowing of a stream of air over the hind paw of a WT animal during 20 s consistently evoked a bladder contraction in 80% of cases that was associated with a void in approximately 50% of cases (Fig. 1A,D). Similar response frequencies were obtained when acetone (500 ml) was applied to the skin, resulting in local evaporative cooling (Fig. 1B,D). The delay between the onset of the cold stimulus and the onset of bladder contraction was short (4.8 0.9 s), and responses that occurred >20 s after the onset of the cold stimulus were discarded as being nonspecific. In contrast to cold stimuli, vigorous pinching of a hind paw with a forceps was mostly ineffective in evoking bladder contraction or voiding (Fig. 1C,D). Similarly, two other non-noxious control stimuli (gentle rubbing of the hindpaw with a cotton swab and application of 500 ml of water at 37 8C to the back) were much less effective in evoking a bladder response (Fig. 1D). Taken together, these results show that localized, mildly cold stimuli can evoke bladder contraction and voiding in anesthetized mice, and thus mimic the hallmarks of ACIU in humans. To investigate whether the cold-activated ion channels TRPM8 and TRPA1 are involved in the cold-induced bladder responses, we applied a standardized cystometry assay to Trpm8/ and Trpa1/ mice. During the 30-min habituation period, we did not detect obvious differences in cystometric pattern among WT, Trpm8+/+, Trpm8/, and Trpa1/ mice (Fig. 2A,B), and there were no statistically significant differences in basal pressure, voiding contraction amplitude, or ICI (Fig. 2C-E). These results indicate that TRPM8 and TRPA1 are not essential for the normal voiding cycle or contractility of the mouse bladder. Importantly, we found that the occurrence of bladder responses to cold stimuli was strongly reduced in Trpm8/ mice compared to Trpm8+/+ littermates (Fig. 2A,F,G). By contrast, there was no significant difference in response frequency to cold stimuli between Trpa1/ mice and matched WT mice (Fig. 2B,F,G). These results demonstrate that acute cold-induced bladder contraction and voiding in mice depend on the cold sensor TRPM8 but not on TRPA1. As illustrated in Figure 3A, acute cold-induced bladder responses could also be reproduced in rats. Notably, using a similar stimulation scheme as in mice, response fidelity in rats was higher than in mice, with 100% voiding (nine animals; four applications per animal) in response to acetone application to the back (Fig. 3A,C,E). Earlier work has provided evidence that dichotomizing axons of lumbosacral dorsal root ganglia innervate both the skin and the bladder, and it was suggested that such dichotomizing neurons may be responsible for bladder responses to cold environmental stimuli [32]. However, we found that bladder contractions and voids could also be evoked by brief pulses of cold air applied to rat ears (Fig. 3B; void frequency
83% 17%, n = 5). This suggests that cold-induced bladder responses are not limited to stimulation of dermatomes innervated by neurons from the dorsal root ganglia that also project to the bladder (mainly L6–S1) [33]. To investigate whether the cold-induced bladder responses in rats also depend on TRPM8, we applied the most reproducible cold stimulus, acetone application to the back, in animals that were pretreated with vehicle or with the TRPM8 antagonist AMTB (3 mg/kg IP) [34]. During the first 90 min following injection of AMTB or vehicle, four separate acetone applications were applied per animal, interspaced by a normal voiding cycle (Fig. 3C,D). In vehicle-treated animals, the response rate was 100% (n = 6 animals; four voiding responses per animal; Fig. 3C,E). By contrast, within the first 60 min after injection, two thirds of the AMTB-treated animals failed to respond to the acetone stimulus (p < 0.01; Fig. 3D,E). Bladder responses to acetone application gradually recovered after 1 h (Fig. 3E), in line with the rapid clearance of AMTB (mean residence time 45 min) according to pharmacokinetic studies [34]. Taken together, these results indicate that acute cold-induced bladder responses in rats are also dependent on TRPM8. 4.
Discussion
Common daily life events, such as entering the home (latchkey incontinence), the sound or feel of running water, stepping on a cold floor, or exposure to cold wind, can evoke urinary urgency, which can be particularly bothersome in patients with OAB [10,11,35,36]. In general, it has been assumed that Pavlovian conditioning and/or subconscious automatic behavior underlies urgency symptoms elicited by such external stimuli [11–13]. In contrast to this view, we demonstrated that brief and moderately cold stimuli resulting in only modest cooling of a restricted part of the skin can consistently evoke acute bladder contractions in anesthetized mice and rats. This implies that bladder responses to acute environmental cold stimuli represent an evolutionarily conserved reflex that can be objectively measured and studied in the apparent absence of (sub)conscious processes. The key manifestation of ACIU in humans is an acute desire to urinate, with or without actual voiding, in response to a brief localized and mildly cold stimulus. Thus, ACIU can be clearly distinguished from other betterstudied effects of cold stimuli on bladder functioning, such as the bladder cooling reflex and cold stress-induced detrusor overactivity [2,26,27]. While measurement of urinary urgency in animals remains elusive [37], we believe that our assay is the closest approximation to ACIU in humans available today, and will be useful for elucidation of the underlying pathways and the development of potential treatments for ACIU. Intriguingly, although not scientifically documented to the best of our knowledge, there is extensive anecdotal evidence that immersion of the hand of a sleeping person in tepid water can evoke bedwetting in at least a subset of victims (hand-in-water prank), and our present work indicates that this trick can be quite accurately played
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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Fig. 2 – Role of TRPM8 and TRPA1 in the acute cold-induced bladder response in mice. (A,B) Cystometric traces (same protocol as in Fig. 1A) showing the effect of acute cold stimuli (acetone on back) in (A) a Trpm8S/S mouse and (B) a Trpa1S/S mouse. (C–E) Comparison of (C) mean basal pressure, (D) amplitude of a voiding contraction (D), and (E) ICI in control C57Bl/6J mice (n = 6), Trpa1S/S mice (n = 6), Trpm8+/+ mice (n = 15), and Trpm8S/S mice (n = 16). There were no significant differences in these three parameters among the four groups (one-way analysis of variance, p > 0.5). (F,G) Frequency of the occurrence of (F) bladder contraction and (G) bladder voiding on stimulation in the four genotypes. Group data are presented as mean W standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Trpm8+/+ littermates (Mann-Whitney test).
on mice and rats. On the basis of our findings, we believe that testing the bladder response of sleeping individuals to localized cold stimuli in a scientifically controlled setting will be of interest, and may provide important insight into the mechanisms of nocturnal enuresis. The acute cold stimuli used in this study inevitably had a non-noxious mechanical component, namely wetting of the skin in the case of acetone application, and air movement in the case of cold air. At present, we cannot fully exclude that mechanical stimulation of the skin is involved in the bladder response to these cold stimuli. We indeed found
that non-noxious stimuli (gentle rubbing of the paw, wetting of the skin with water at 37 8C) and noxious pinching of the paw evoked a bladder response in a small percentage of animals. Similarly, a small percentage of Trpm8/ mice exhibited an acute bladder contraction on acetone application. These data suggest that although various sensory stimuli can evoke an acute bladder response, cold stimuli acting via TRPM8 are by far the most provocative. Our results also demonstrate that acute bladder responses to cold stimuli can be strongly attenuated by
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Fig. 3 – The TRPM8 antagonist AMTB inhibits cold-induced bladder responses in rat. (A,B) Acute cold-induced bladder responses in rats in response to (A) application of acetone (3 ml) on the back or (B) blowing of cold air on the ear. (C,D) Bladder responses to four applications of acetone (arrows) on the back of rats injected with (C) vehicle or (D) AMTB at 900 s before time 0. Perfusion was restarted between two subsequent acetone applications, resulting in one full voiding cycle. (E) Frequency (mean W standard error of the mean) of the occurrence of a voiding contraction at the indicated time points after injection with either vehicle (n = 6) or AMTB (n = 6). ** p < 0.01 versus vehicle (Mann-Whitney test).
pharmacological inhibition of TRPM8. The first results for oral administration of a TRPM8 antagonist (PF-05105679) in humans show that the drug was well tolerated at doses that were effective in a cold pressor test [38]. Therefore, there are prospects for the development of TRPM8 antagonists for specific therapies to alleviate ACIU symptoms in LUTd patients.
Author contributions: Thomas Voets had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Uvin, Franken, Pinto, Everaerts, De Ridder, Voets. Acquisition of data: Uvin, Franken, Pinto, Rietjens, Deruyver, Alpizar. Analysis and interpretation of data: Uvin, Franken, Pinto, Everaerts, De Ridder, Voets.
5.
Conclusions
Drafting of the manuscript: Uvin, Franken, Everaerts, Voets. Critical revision of the manuscript for important intellectual content: Uvin, Franken, Pinto, Deruyver, Alpizar, Talavera, Vennekens, Everaerts, De
Taken together, our findings indicate that ACIU is an evolutionarily conserved reflex rather than a subconscious conditioning response. Moreover, we presented an in vivo model that can be used to objectively measure ACIU in rodents and that could form the basis for further investigation of the mechanisms underlying ACIU. Finally, our results indicate that the cold sensor channel TRPM8 is critically involved in ACIU, which suggests that pharmacological inhibition of this druggable target may be useful for treating ACIU symptoms in patients.
Ridder, Voets. Statistical analysis: Uvin, Voets. Obtaining funding: Vennekens, Everaerts, De Ridder, Voets. Administrative, technical, or material support: Pinto, Grammet. Supervision: Talavera, Vennekens, De Ridder, Voets. Other: None. Financial disclosures: Thomas Voets certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria,
Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037
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stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.
[20] Karashima Y, Talavera K, Everaerts W, et al. TRPA1 acts as a cold sensor in vitro and in vivo. Proc Natl Acad Sci U S A 2009;106:1273–8. [21] Du S, Araki I, Kobayashi H, Zakoji H, Sawada N, Takeda M. Differ-
Funding/Support and role of the sponsor: This research was supported by grants from the Research Foundation Flanders (FWO), the Research Council of KU Leuven, the Astellas European Foundation, and the Belgian Science Policy IUAP P7/13. Uvin is a PhD aspirant and De Ridder is a clinician-fundamental researcher of the FWO. The sponsors played no role in the study. Acknowledgments: We thank the members of our laboratories for helpful discussions.
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Please cite this article in press as: Uvin P, et al. Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.03.037