Onderzoek naar Detrusoroveractiviteit. De Rol van Moleculaire Sensoren.

Het overactieve blaassyndroom (OAB) treft 12% van de volwassen westerse bevolking en heeft een plotse en moeilijk te onderdrukken aandrang om te plassen als belangrijkste symptoom, hoewel patiënten ook klagen van urineverlies, een verhoogde plasfrequentie overdag en ’s nachts. Het is momenteel niet geweten hoe OAB juist ontstaat en onderzoek naar OAB wordt bemoeilijkt door het feit dat OAB een symptoomcomplex is terwijl proefdieren natuurlijk geen symptomen kunnen meedelen. Ook zijn er belangrijke verschillen in de fysiologie van het plassen tussen menselijke patiënten en de gebruikte diermodellen. Toch kunnen diermodellen helpen bij het ophelderen van OAB. Voor dit proefschrift werd eerst een methodologisch luik uitgewerkt. De blaasfunctie kan worden beoordeeld met een urodynamisch onderzoek. We beschreven een gestandardiseerde procedure bij muizen en ratten die stapsgewijs werd gefilmd en beschreven. Vervolgens beschreven we een muismodel voor blaasdysfunctie dat veroorzaakt werd door toediening van het chemotherapeuticum cyclofosfamide. Daarna beschreven we het urologische profiel van een muismodel van multiple systeem atrofie of MSA (een neurodegeneratieve aandoening die zich presenteert met onder andere motorische stoornissen, autonome dysfunctie en plasklachten). Na het methodologische luik werden vijf projecten uitgewerkt. Anticholinergica zijn nog steeds de belangrijkste geneesmiddelen voor de behandeling van OAB, hoewel de therapietrouw bijzonder laag is. In dit proefschrift hebben we hiervoor een mogelijke verklaring gevonden. We beschrijven dat chronische toediening van anticholinergica bij ratten leidt tot een efficiëntieverlies van deze geneesmiddelen, wat veroorzaakt werd door een verschuiving van de moleculaire transmissie van de blaascontractie. De TRP (Transient Receptor Potential) superfamilie vertegenwoordigt een diverse groep van ionkanalen die fungeren als moleculaire sensoren van de cellen. In dit proefschrift werd de betrokkenheid van TRPC1 en TRPC4 in de ontwikkeling van blaasdysfunctie bij muizen die werden geïnjecteerd met cyclofosfamide onderzocht. TRPC1 en TRPC4 bleken cruciaal te zijn voor deze blaasdysfunctie door een essentiële rol te spelen in het kiemen van zenuwcellen. Een korte blootstelling van het lichaam aan een koude stimulus, zoals een koele bries of het wassen van de handen met koud water, kan een plotse drang om te plassen veroorzaken. Dit is vooral onaangenaam bij patiënten met OAB, bij wie dit vaker voorkomt en kan leiden tot ongewild urineverlies. In dit proefschrift noemen we het fenomeen acute koude geïnduceerde aandrang of Acute Cold-Induced Urgency (ACIU). Omdat plassen vaak gekoppeld is aan koudeprikkels, werd gedacht dat ACIU een soort Pavlov-reactie is. Wij toonden echter aan dat ACIU reproduceerbaar kan worden nagebootst met een eenvoudige cystometrie gebaseerde test bij verdoofde muizen en ratten. Deze resultaten bewijzen dat ACIU een evolutionair bewaarde reflex is, en dus geen Pavlov-reactie. Hierna toonden we aan dat het koude-geactiveerd ionkanaal TRPM8 een cruciale rol speelt bij ACIU. Cyclofosfamide is een chemotherapeuticum dat wordt gebruikt bij de behandeling van kanker en auto-immuunziekten. Bekende bijwerkingen zijn irritatieve plasklachten en bloederige urine. Acroleïne, een metaboliet van cyclofosfamide, zou een sleutelrol spelen in het ontstaan van deze urologische klachten door te binden aan het ionkanaal TRPA1. Andere studies wezen echter in de richting van het ionkanaal TRPV1. Wij toonden aan dat TRPV1, en niet TRPA1, een cruciale rol speelt in de ontwikkeling van blaasdysfunctie die veroorzaakt wordt door acroleine. Urineweginfecties behoren tot de meest voorkomende infectieziekten. Patiënten met een urineweginfectie (of “blaasontsteking”) hebben vaak last van een uitgesproken aandrang om te gaan plassen en van een verhoogde plasfrequentie. Urineweginfecties worden meestal veroorzaakt door Escherichia coli bacteriën. Lipopolysaccharide (LPS) is de belangrijkste component van de buitenmembraan van Escherichia coli. We toonden aan dat LPS het ionkanaal TRPV4 activeert, dat sterk tot expressie wordt gebracht in de urotheelcellen van het slijmvlies van de blaas en er fungeert als een mechano-sensor.The lower urinary tract consists out of the bladder and the urethra and acts as a dynamic reservoir that can store urine and expel it at a socially convenient time. The overactive bladder syndrome (OAB) affects an estimated 12% of the adult Western population and has urgency to urinate as key symptom, although OAB patients also complain of urgency incontinence, an increased daytime micturition frequency and nocturia. During urodynamic investigations, detrusor overactivity can be found in around half of patients with OAB symptoms. The pathophysiology of OAB remains to be elucidated but the current understanding is limited by the fact that OAB is a symptom-based diagnosis (as urgency is the key symptom) while laboratory animals obviously cannot report any symptoms. Also, due to the differences in the micturition control between human patients and animal models, experiments in laboratory animals have only limited value. Still, animal models can help in deciphering the pathophysiological mechanisms of OAB. Urodynamic investigations (i.e. cystometry) is an efficient technique to measure bladder function of small animals in vivo. For this doctoral thesis, a metholodical section was first developed. We standardized a way of performing cystometry in mice and rats. The whole procedure was filmed and explained in a “step by step” manner. Then, we described a mouse model for cystitis-induced bladder dysfunction by administrating cyclophosphamide chronically via multiple intraperitoneal injections. Creating hemorrhaghic cystitis by injecting one high dose of cyclophosphamide was already used in mice as a model of bladder dysfunction. However, the mouse model that we describe mimics the situation in the human patients better as the bladder hyperactivity is comparable with the acute model, but without the profound cystitis. Also, we described the urological phenotype of a mouse model of multiple system atrophy or MSA (which is a neurodegenerative disorder that presents with motor impairment, autonomic dysfunction and lower urinary tract symptoms) and belongs to the α-synucleinopathies which display α-synuclein positive glial cytoplasmic inclusions in the central nervous system. Transgenic mice with glial cytoplasmic inclusions have been earlier analyzed concerning neurodegeneration and motor disability but the urological phenotype has been investigated in this thesis. Our experiments showed a less efficient bladder with an increased contraction amplitude. Also, more nonvoiding contractions and a higher postmictional residual were found. After the methological section, we elaborated five research projects, in which detrusor overactivity was further explored. Anticholinergics are still the first-line pharmacotherapy for OAB, although their compliance rate is exceptionally low. The traditional view of the working mechanism of anticholinergics is that they block the muscarinic receptors on the detrusor muscle of the bladder. Normally, parasympathetic efferent neurons release acetylcholine which binds to these muscarinic receptors. However, it is known that also other signaling molecules mediate the bladder contraction. Indeed, not only acetylcholine but also ATP is released from the efferent nerve terminals. ATP couples with purinergic receptors on the detrusor muscle of the bladder. In this doctoral thesis, we have found a possible explanation for the notoriously low compliance rate of anticholinergics. We describe that a chronic administration of anticholinergics in rats induces a loss of drug efficiency, caused by a shift from muscarinic to purinergic transmission and thereby escaping the working mechanism of anticholinergics . These data imply that the poor compliance in overactive bladder patients might be due to similar mechanisms and that they may benefit from a different approach (e.g. β3 receptor agonists). The TRP (Transient Receptor Potential) superfamily represents a diverse group of ion channels that are mostly located on the cell membrane of numerous cell types. There are 28 TRP channels that are divided in seven groups: TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, TRPML. Most TRP channels are non-selectively permeable to cations such as sodium, calcium and magnesium. Some members of the TRP superfamily exhibit a high sensitivity to changes in temperature, such as the cold-activated TRPA1 and TRPM8. TRPA1 is also activated by noxious chemicals such as mustard oil and wasabi. TRPC ion channels are supposed to play a role in neuronal growth and axon guidance. TRPV1 is a heat-sensitive ion channel that is activated by capsaicin and that plays a role in the regulation of the body temperature. In this doctoral thesis, the involvement of TRPC1 and TRPC4 in the development of bladder dysfunction in mice that were chronically injected with cyclophosphamide (as described above in the methodological section) was investigated. We describe that repeated cyclophosphamide injections induce an increase in the sprouting of afferent neuronal fibers in the bladder mucosa, combined with an increased expression of TRPC1 and TRPC4 in these fibers. Genetically modified TRPC1/C4-/- double knockout mice (which do not express TRPC1 nor the TRPC4) did not display an increased sprouting of neuronal fibers. Furthermore, the urological phenotype (which was evaluated with cystometry) caused by multiple injections of cyclophosphamide normalized in the TRPC1/C4-/- double knockout mice. Therefore, we have shown that TRPC1 and TRPC4 are crucial for the neuronal sprouting in the bladder mucosa caused by cyclophosphamide injections, which appeared to be necessary for the urodynamic development of bladder dysfunction. A brief exposure of the body to a cold stimulus, such as feeling a cold breeze or washing hands with cold water, can evoke a sudden urge to void, with or without urgency incontinence. In this doctoral thesis, we propose the term acute cold-induced urgency (ACIU) for this phenomenon. Especially OAB patients frequently report ACIU but it is also observed in healthy persons, especially in children and the elderly. To this point, it was unclear whether these responses represented a form of Pavlovian conditioning or rather a conserved reflex. We demonstrated that voiding can be evoked in a simple cystometry-based assay in anesthetized mice and rats. These results prove that ACIU represents a conserved reflex, and not Pavlovian conditioning. Furthermore, we showed that ACIU can be inhibited by genetic ablation or pharmacological inhibition of the cold-activated ion channel TRPM8, and not TRPA1. Therefore, TRPM8 could be seen as a therapeutic target to relieve patients who suffer from ACIU. Cyclophosphamide is a chemotherapeutic drug that is used in the treatment of cancer (such as lymphomas and brain cancer) and autoimmune disorders (such as systemic lupus erythematosus and multiple sclerosis). A well known side effect of this treatment is bladder overactivity and hemorrhagic cystitis. Acrolein, which is a metabolite of cyclophosphamide, is proposed to play a key role in the development of these conditions by binding on TRPA1 in bladder sensory neurons. Other evidence suggests that the effects are mediated by TRPV1. Using cystometry in mice with genetic ablation and pharmacological inhibition of TRPV1 and TRPA1, it was demonstrated that TRPV1 (and not TRPA1) plays a major role in the acute irritation induced by acrolein in the mouse bladder. Furthermore, we describe that acrolein can directly activate TRPV1 by a covalent bond with residue C157. Therefore, TRPV1 could serve as a therapeutic target in the prevention of acute bladder irritation during cyclophosphamide treatment. Urinary tract infection (UTI) is one of the most common infectious diseases. Patients who suffer from a UTI do not form part of the OAB group but they share symptoms as urgency and increased frequency. UTI is most often caused by uropathogenic Escherichia coli. Lipopolysaccharide (LPS) is the major component of the outer membrane of Escherichia coli and acts as an endotoxin. The exact role LPS plays in UTI and in the development of symptoms is currently unknown, as well as the molecular mechanism behind it. We report that LPS activates TRPV4, which is highly expressed in murine and human urothelial cells and functions as a mechanosensor in the bladder. After instillation of LPS in the bladder, the voiding frequency clearly increased in wild type mice, while the effect was abolished with genetic ablation and pharmacological inhibition of TRPV4. We demonstrate that urothelial cells can recognize bacterial colonization through TRPV4 activation by LPS which could at least partially explain the urgency to urinate during cystitis.1. Introduction 1.1 The physiology of the bladder p1 1.1.1 The functional anatomy and histology of the bladder and its neural pathways p1 1.1.2 The molecular receptors of the bladder p6 1.2 The pathology of the bladder p19 1.2.1 The clinical problems p19 1.2.2 The socio-economic implications p22 1.2.3 Current treatment strategies p23 1.3 The research of the bladder p27 1.3.1 In human patients p27 1.3.2 In laboratory animals p29 2 Hypothesis and objectives 2.1 General hypothesis p33 2.2 Objectives p34 2.2.1 Methodological development p34 2.2.2 The role of molecular sensors in detrusor overactivity p34 3 Results 3.1 Methodological development:project 3.1.1-3.1.3 3.1.1 The use of cystometry in small rodents: a study of bladder chemosensation p37 3.1.2 Functional characterization of a chronic cyclophosphamide- induced overactive bladder model in mice p47 3.1.3 Bladder dysfunction in a transgenic mouse model of multiple systematrophy p57 3.2 The role of molecular sensors in detrusor overactivity: project 3.2.1 - 3.2.5 3.2.1 Chronic administration of anticholinergics in rats induces a shift from muscarinic to purinergic transmission in the bladder wall p69 3.2.2 Crucial role of TRPC1 and TRPC4 in cystitis-induced neuronal sprouting and bladder overactivity p81 3.2.3 Essential role of transient receptor potential M8 (TRPM8) in a model of acute cold-induced urinary urgency p95 3.2.4 Direct activation of TRPV1 by acrolein as trigger of acute bladder irritation p107 3.2.5 The cation channel TRPV4 mediates responses to LPS in urothelial cells p125 4 Concluding discussion and perspective 5 References 6 Summary 7 Samenvatting 8 List of publications 9 Curriculum vitaenrpages: 184status: publishe