urinary bladder, cystitis and nerve/urothelial interactions

Autonomic Neuroscience: Basic and Clinical xxx (2013) xxx–xxx

AUTNEU-01611; No of Pages 6

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Autonomic Neuroscience: Basic and Clinical

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Urinary bladder, cystitis and nerve/urothelial interactions

Lori A. Birder ⁎Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United StatesDepartment Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

⁎ Departments of Medicine and Pharmacology & ChPittsburgh, School of Medicine, A 1217 Scaife Hall, PittsbTel.: +1 412 383 7368; fax: +1 412 648 7197.

E-mail address: [email protected].

1566-0702/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.autneu.2013.12.005

Please cite this article as: Birder, L.A., Urina10.1016/j.autneu.2013.12.005

a b s t r a c t
a r t i c l e i n f o
Article history:Received 4 November 2013Accepted 12 December 2013Available online xxxx

Keywords:UrotheliumAutonomic nervous systemSensory functionTransducer function

A hallmark of functional pain syndromes, such as bladder pain syndrome/interstitial cystitis (BPS/IC) is pain inthe absence of demonstrable infection or pathology of the viscera or associated nerves. There are no clear defini-tions of this syndrome, no proven etiologies and no effective treatments able to eradicate the symptoms. Thiscondition is characterized by suprapubic pain, associated with bladder filling and can also be accompanied bya persistent strong desire to void, increased frequency of urination and nocturia. Severe cases of this disorder,which affects primarily women, can have considerable impact on the quality of life of patients due to extremepain and urinary frequency, which are often difficult to treat. In addition, BPS/IC patients may also suffer co-morbid conditionswhere pain is a common symptom (such as irritable bowel syndrome, fibromyalgia). Theoriesexplaining the pathology of bladder pain syndrome are many and include an altered bladder lining and possiblecontribution of a bacterial agent.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Bladder pain syndrome/interstitial cystitis (BPS/IC) is a debilitatingchronic disease characterized by suprapubic pain related to bladder fill-ing, coupled with additional symptoms, such as increased day- andnight-time urinary frequency, without proven urinary infection orother obvious pathology. Although the symptoms presented may ap-pear similar to those of a urinary tract infection, urine culture revealsno underlying infection and there is no response to antibiotic treatment(Parsons et al., 1993; Hanno et al., 1999; Bladder Research ProgressReview Group, 2002). Between 700,000 and 1 million people in theUnited States have IC, the preponderance of who are women (BladderResearch Progress Review Group, 2002). Moreover, it has been estimat-ed that a 60% increase in the number of cases would be identified by ex-perienced clinicians who apply the strict National Institute of Diabetes,Digestive, and Kidney Diseases definition of BPS/IC (Hanno et al.,1999). While the etiology is unknown, theories explaining the patholo-gy of BPS/IC include altered barrier lining, afferent and/or CNS abnor-malities, a possible contribution of inflammatory or bacterial agentand abnormal urothelial signaling.

2. Disease process and relevant animal models

The etiology of BPS/IC is unknown; however, several causes have beenpostulated, including epithelial dysfunction (i.e., leaky urothelium),

emical Biology, University ofurgh, PA 15261, United States.

ghts reserved.

ry bladder, cystitis and nerv

infection, autoimmune response, allergic reaction, neurogenic inflam-mation, and inherited susceptibility (Bladder Research ProgressReview Group, 2002; NIH Publication No. 02-3220, 2002).

A number of animal models have been used for the study of BPS/IC,which includes administration of an irritant or immune stimulant(e.g. hydrochloric acid, turpentine, protamine sulfate, mustard oil, lipo-polysaccharide and cyclophosphamide). Studies have shown that a de-ficiency of estrogen receptor-beta in female mice develop a bladderphenotype (including alterations in the urothelium) which may sharesimilarities with human PBS/IC (Imamov et al., 2007). However, areview of such animal models discusses the potential problems inartificially inducing bladder inflammation or injury and thus may notbe considered a valid method to model the symptoms of this complexsyndrome (Westropp and Buffington, 2002; Buffington, 2008).Furthermore, the degree of bladder hyperreflexia observed in rodentsis variable and can resolve within a matter of days. This may be, inpart, due to the capacity of the damaged rodent bladder urothelium torapidly regenerate post-intravesical insult thus limiting the capacityto establish chronicity in these models reflective of the humancondition.

A naturally occurring disease occurring in cats, termed feline inter-stitial cystitis, reproduces many features of BPS/IC in humans diagnosedwith this disorder (Buffington, 2008). In addition, an experimentalautoimmune cystitis (EAC) murine model has been shown to exhibit anumber of comparable functional and histological alterations to thatin human BPS/IC (Lin et al., 2008). Also similar to BPS/IC patients,pseudorabies virus (PRV) injection in mice results in the developmentof a neurogenic cystitis associated with pelvic pain and accumulationof mast cells (Rudick et al., 2009). Stress has been shown to impair theimmune, endocrine and nervous systems and can be an important

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2 L.A. Birder / Autonomic Neuroscience: Basic and Clinical xxx (2013) xxx–xxx

factor in functional gastrointestinal (GI) and genitourinary (GU) disor-ders such as irritable bowel syndrome (IBS) and BPS/IC. For example,rats exposed to various types of stress (water avoidance, intruder stress)exhibit symptoms of bladder dysfunction including increased micturi-tion frequency as well as anxiety-like behavior (Smith et al., 2008;Wood et al., 2009). Further, an exaggerated acoustic startle responsehas been demonstrated in both cats diagnosed with feline IC as well asin BPS/IC patients (Westropp et al., 2006; Twiss et al., 2009). This re-sponse is a brainstem reflex responding to unexpected loud stimuliand parallels that of autonomic control. Even though the pathophysiol-ogy and etiology of most persistent pain syndromes are incompletely

urothelium

sacralafferentinnervation

autonomic innervation

post

A

B

Fig. 1.A)Cartoon depicting various components of the bladderwall. These include subtypes of umuscle cells; interstitial cells (in green) whose functions have not yet been defined but may pla(parasympathetic and sympathetic) innervation (red). Pre, preganglionic; post, postganglionic.urothelial cells, smooth muscle and interstitial cells. Urothelial cells can also be targets for transcell types. Urothelial cells can be activated by either autocrine (i.e. autoregulation) or paracrinesuch as bladder distension can release urothelial-acetylcholine, which then activates urothelialM3-muscarinic subtype receptor; P2R or P2X/P2Y—purinergic subtype receptors; GC—guanyprotein kinase; Ca2+-calcium; TRPV—transient receptor potential family of ion channels; NOS—the reader is referred to the web version of this article.)

Please cite this article as: Birder, L.A., Urinary bladder, cystitis and nerv10.1016/j.autneu.2013.12.005

understood, it is generally assumed that they involve changes in thetarget organ as well as alterations in both central and peripheralprocessing/modulation of nociception and pain. In addition,while alter-ations in the periphery may alter nociceptive input to the CNS, pain re-mains an emergent property of the brain. A number of recent studieshave identified structural and functional changes in the brain of patientswith chronic pain syndromes that may influence the perception of sen-sory input (May, 2008; Mayer and Bushnell, 2009).

Due to the complex nature of BPS/IC it is thus unlikely that a singleanimal model would be suitable for investigative work and thus apanel of models reflecting the key symptoms and known components

umbrella cells

intermediate cells

basal cells

suburothelialinterstitial cellssuburothelialmuscle layer

inner muscle layer

intramuscularinterstitial cells

outer muscle layer

rothelial cells (with apical or umbrella cells connected via tight junctions), layers of smoothy a role in intercellular communication, sacral afferent innervation (blue), and autonomicB)Hypotheticalmodel depicting possible interactions between afferent nervefibers (blue),mitters (such as ATP, nitric oxide—NO, acetylcholine—ACh) released from nerves or other(release from nearby nerves or other cells) mechanisms. For example, mechanical stimulicholinergic (muscarinic) receptor subtypes, resulting in release of additional transmitters.late cyclase; IP3—inositol triphosphate; PKC—protein kinase C; PKG—cGMP-dependentnitric oxide synthase. (For interpretation of the references to colors in this figure legend,


image of Fig.�1



3L.A. Birder / Autonomic Neuroscience: Basic and Clinical xxx (2013) xxx–xxx

of the condition should be utilized to investigate the potential therapeu-tic mechanisms.

2.1. Epithelial alterations

The urothelium, the epithelial lining of the urinary tract between therenal pelvis and urinary bladder, is composed of at least three cell layers(see Fig. 1A). These consist of a basal cell layer, an intermediate and asuperficial or apical layer composed of cells termed “umbrella” cells,which are interconnected by tight junctions. Apical urothelial cells func-tion as a barrier against most substances found in urine thus protectingthe underlying tissues (Negrete et al., 1996; Zeidel, 1996; Lewis, 2000;Apodaca, 2004). When this function is compromised during injury orinflammation, it can result in the passage of toxic substances into theunderlying tissue (neural/muscle layers) resulting in urgency, frequen-cy and pain during voiding. The superficial umbrella cells play a promi-nent role inmaintaining this barrier, and exhibit a number of propertiesincluding specialized membrane lipids, asymmetric unit membraneparticles and a plasmalemma with stiff plaques (Lewis, 2000; Hu et al.,2002; Apodaca, 2004).

BPS/IC has often been described as a disease of the urothelium(Graham and Chai, 2006). Ultrastructurally, an altered vascular supplyis observed in its ulcerative form with locations of moderate-to-severeredness, interspersed among a whitish discoloration. There is also evi-dence that the urothelium in BPS/IC is associated with altered synthesisof a number of proteins including those involved in cellular differentia-tion, barrier function and bacterial defense mechanisms. In this regard,studies have shown in patients diagnosed with the ulcerative form ofBPS/IC, that laser removal of damaged urothelium is associated withreduction of symptoms of bladder or pelvic pain (Rofeim et al., 2001).This treatment stimulates a rapid urothelial turnover and patientsundergoing this treatment report a prolonged period without pain(6–12 months) after therapy. In terms of barrier ‘repair’, instillation ofliposomes composed of phospholipids has been shown to support therepair of the urothelial barrier in animals following bladder irritation(Tyagi et al., 2009). Though the mechanism is not well defined, byforming a protective coating on the urothelium, liposomes may act asa mucosal protective agent and thereby decreasing the irritation of un-derlying afferent nerves. In this regard the use of intravesical liposomesfor treatment of patients with ulcerative BPS/IC has shown promise torepair and enhance the barrier function of a dysfunctional urotheliumthough further trials are needed to fully assess this type of treatment(Peters et al., 2012).

Disruption of the integrity of the urothelial barrier may bemediatedby hormonal or neural mechanisms (such as by substances released bysurrounding afferent and autonomic nerve fibers and various cell typeswithin the bladder wall such as immune or inflammatory cells). Forexample, nitric oxide (NO)has beendemonstrated to be amarker for in-flammatory bladder disorders. In addition, nitric oxide (NO)was elevat-ed in patients with interstitial cystitis as well as cats diagnosed withfeline interstitial cystitis (Hosseini et al., 2004; Birder et al., 2005).BPS/IC patients diagnosed with a Hunner lesion (areas of inflammationon the bladder wall that characterize the classic form of BPS/IC) exhib-ited high intraluminal levels of NO as well as inflammatory infiltrateswithin the urothelium and also lamina propria (Logadottir et al.,2013). Excessive NO levels in the urinary bladder can increase perme-ability of the urothelium to water/urea in addition to producing ultra-structural changes in the apical layer. The NO can be synthesized/released by a number of cell types within the bladder wall (includingthe urothelium) and, after release can act in an autocrine/paracrinemanner to alter urothelial permeability to ions and solutes (seeFig. 1B). Although the pathological mechanism is unknown, these find-ings appear to be similar to that in other epithelia where excess produc-tion of NO has been linked to changes in epithelial integrity. Disruptionof epithelial integrity may also be linked to expression of substancessuch as antiproliferative factor (APF), which has been characterized as


a frizzled-8-related sialoglycopeptide and is detected in the urine ofpatients with bladder pain syndrome (Keay et al., 2001). Abnormalitiesin urothelial growth and proliferation may also be linked to changes inexpression of trophic factors such as HB-EGF.

Changes in epithelial signaling/barrier function are not unique to theurinary bladder. For example, airway epithelia in asthmatic patients aswell as keratinocytes in certain types of skin disease also exhibit a num-ber of similar abnormalities and compromised repair processes (Bosseet al., 2008). Taken together, epithelial cells can respond to a numberof challenges (including environmental pollutants and mediators re-leased from nerves or nearby inflammatory cells) resulting in alteredexpression and/or sensitivity of various receptor/channels as well aschanges in release of mediators, which may impact function.

2.2. GAG layers

The urothelial surface is lined by surface mucin, a heterogeneous,gel-like substance composed of numerous sulfonated glycosaminogly-cans (GAG) and glycoproteins. Many clinicians believe that a defect inthe protective GAG layer of the bladder that lines the epithelium ofthe bladder is responsible for the permeability changes of the bladderin BPS/IC. However, the urothelial barrier function of the GAG layerhas been a controversial subject. Lilly and Parsons report thatintravesical treatment of the rabbit bladder with protamine sulfate in-creased urothelial permeability to water, urea and calcium bothin vivo and in vitro (Lilly and Parsons, 1990). This effect was reversedwith pentosanpolysulfate (PPS) (Lilly and Parsons, 1990). They con-cluded that the protamine sulfate affected the GAG layer and that thiswas repaired by PPS. However no microscopic evidence of the anatom-ical changeswas presented in this paper. This studywas later confirmedby Nickel et al. (1998) who compared PPS, heparin and hyaluronic acidas treatments. The authors concluded that heparin was the best of thethree agents in efficacy, but pointed out that this may be due to itsanti-inflammatory properties. Indeed the role of the GAG layer may bemore in line with an antibacterial adherence function (Hanno et al.,1981). The GAG layer may also be important for the formation and at-tachment of particulates to the urothelium and stone formation(Grases et al., 1966; Hurst, 1994).

Overall, it seems that protamine sulfate does not act at the level ofthe GAG layer but rather at the surface of the luminal cells, so called“umbrella cells” (Fig. 1A), thus enhancing urea absorption by othermechanisms. Protamine sulfate increases the permeability of the apicalmembrane (luminal surface of umbrella cells) permeability to bothmonovalent cations and anions. This effectmay be reversible dependingon the concentration of the protamine, the composition of the bathingsolution and the exposure time of the urothelium to protamine.Prolonged exposure to protamine (N15 min) is poorly reversible andis thought to be due to a decrease in paracellular resistance due to celllysis (Tzan et al., 1993, 1994).

In summary the GAG layer may have importance in bacterial anti-adherence, and prevention of urothelial damage by large macromole-cules. However, there is no definite evidence that the GAG layer actsas the primary epithelial barrier between urine and plasma.

3. Protective functions

3.1. Importance of the autonomic nervous system

Another characteristic change observed inmany types of epithelia fol-lowing injury, and in the urothelium following a spinal cord injury, is anearly degeneration followed by regeneration. In rodents, it has beenshown that 2 h after experimental transection of the spinal cord, thereis a significant disruption of the urothelial barrier with accompanyingpermeability and ultrastructural changes (Apodaca et al., 2003). Thesealterations are prevented by pretreatment with ganglionic blockers,suggesting that the autonomic (parasympathetic or sympathetic)





innervation play a role in the acute response of urothelial cells to spinalcord injury (Fig. 1A). The mechanism underlying the neuronallyinduced changes of the urothelium is unknown. There is a limited un-derstanding of tight junction assembly or turnover, however, whenthe urothelium is injured by a transection of the spinal cord orwhen ex-posed to the uropathogenic Escherichia coli, it undergoes rapid regener-ation after disruption of the urothelial barrier. Epithelial integrity ismaintained through a complex process of proliferation and differentia-tion. During a regenerative process, studies suggest that proliferationtakes precedence over differentiation.

3.2. Immune-epithelial interactions and cellular defense

Both physiological and psychological stress can result in a failure ofthe urothelial and suburothelial ‘defensive’ systems and thereby pro-mote changes in both urothelial barrier and signaling function. Epitheliaare also able to secrete a number of antimicrobial molecules such asTamm–Horsfall protein (THP), cytokines, and defensins, which aresmall peptides thought to have antimicrobial activity (Ganz, 2001;Zasloff, 2002). The defensins, which are abundant in epithelia of the in-testine, and respiratory and urinary tracts, are also thought to exhibitadditional functions including the regulation of inflammation and inthe adaptive immune response. Alterations in proteins including pro-teoglycans and bacterial defense molecules may lead to distinctivechanges in urothelial structure and play a role in bacterial adherence(Rostand and Esko, 1997). In addition, Toll-like receptors (TLRs),which are a family of membrane glycoproteins, also play an importantrole in the initiation of immune responses in various types of epitheli-um, such as the airways and both gastrointestinal and genitourinarytracts (Weichhart et al., 2008; Kumar et al., 2009; Jager et al., 2013). Al-though the urothelium maintains a tight barrier, a number of factors(e.g. mechanical or chemical trauma, infection) can modulate the barri-er function.When the barrier is compromised, the urothelium is unableto maintain the integrity of the bladder–urine interface. The result canbe changes in the function of urothelial cells and terminals of visceralafferent neurons within the bladder wall resulting in symptoms ofurgency, frequency and pain during bladder filling and voiding. Thus,a complex chemical information transfer exists between the urotheliumand cells within the bladder wall (which can include neuroendocrine,immune and autonomic nervous systems, see Fig. 1A) and disruptionin this ‘sensory web’ may be involved in bladder dysfunction.

4. “Neuron-like” properties of the urothelium

While the urothelium has been historically viewed as primarily a“barrier”, it is becoming increasingly appreciated as a responsive struc-ture capable of detecting physiological and chemical stimuli, and releas-ing a number of signaling molecules (see Fig. 1B). Data accumulatedover the last several years now indicate that urothelial cells displaya number of properties similar to sensory neurons (nociceptors/mechanoreceptors), involving diverse signal-transduction mechanismsto detect physiological stimuli. Examples of “sensor molecules” (i.e. re-ceptors/ion channels) associatedwith neurons that have been identifiedin urothelium. These include receptors for bradykinin (Chopra et al.,2005) neurotrophins (trkA and p75) (Murray et al., 2004; Birder et al.,2010), purines (P2X and P2Y) (Lee et al., 2000; Burnstock, 2001; Huet al., 2002; Birder et al., 2004; Tempest et al., 2004; Wang et al.,2005), norepinephrine (α and β) (Birder et al., 1998; Birder et al.,2002), acetylcholine (nicotinic and muscarinic) (Chess-Williams,2002; Beckel et al., 2006; Beckel and Birder, 2012), protease activatedreceptors (PARs) (D'Andrea et al., 2003), amiloride/mechanosensitiveNa+ channels (Lewis and Hanrahan, 1985; Lewis et al., 1991; Smithet al., 1998; Carattino et al., 2005) and a number of TRP channels(TRPV1, TRPV2, TRPV4, TRPM8) (Birder et al., 2001, 2002; Stein et al.,2004).


5. What is the source of pain in BPS/IC?

Chronic pain conditions such as bladder pain syndrome are associat-ed with changes in the central nervous system as well as the periphery(Berkley, 2005). While the etiology or source of pain is unknown, therelationship is likely to be complicated, involving both abnormalitiesin central pain processing as well as changes in the target organ.

The urothelium is likely to play an important role by actively com-municating with bladder nerves (in particular afferent neurons),smooth muscle cells, lamina propria interstitial cells and cells belongingto the immune and inflammatory systems. Altered expression or sensi-tivity of molecular targets such as TRPV1, acid-sensing channels andmuscarinic receptors has been reported in BPS/IC patients as well as inanimal models for the syndrome (Cruz and Dinis, 2007; Gupta et al.,2009; Ikeda and Kanai, 2009; Sanchez-Freire et al., 2011). In addition,the augmented release of transmitters, most notably ATP, from theurothelium can lead to painful sensations by excitation of purinergic re-ceptors on sensory fibers (Sun and Chai, 2006; Birder et al., 2010;Burnstock, 2012). Thus, inhibition of purinergic P2X3 receptors on affer-ent terminals (Fig. 1B) has been shown to be effective in suppressingafferent excitation in various animalmodels andmay be effective in clin-ical conditions associated with pain such as PBS/IC (Gever et al., 2010;Ford, 2012). Onabotulinum toxin A (BoNT-A) has been used in the treat-ment of lower urinary tract disorders including BPS/IC and appears tohave a positive therapeutic effect (Chancellor et al., 2008). By inhibitingSNARE-dependent exocytotic processes, BoNT-A can prevent the releaseof transmitters (such as ATP) aswell as normalize the expression of var-ious receptors, channels and trophic factors (Liu and Kuo, 2007). Studieshave shown that BoNT/A treatment normalized changes in urothelialreceptor expression and neurotransmitter levels in animals with exper-imental bladder overactivity and in patients diagnosed with detrusoroveractivity (Smith et al., 2008). These and other studies suggest thatthe urothelium may be a target for this treatment and that urothelial-released mediators may contribute to sensory urgency/pain by activat-ing or sensitizing visceral afferents innervating the urinary bladder.

6. Can we identify a ‘biomarker’?

There is a great deal of interest in identifying a ‘biomarker’ that couldbe of value in the diagnosis (and not just predictive of symptom pro-gression) for BPS/IC. A range of factors have been studied includingAPF, epidermal growth factor (EGF), insulin-like growth factor 1(IGF1), insulin-like growth factor binding protein 3 (IGFBP3) as wellas urinary chemokines and have been shown to be correlated to BPS/IC. While some reports suggest that urinary markers (e.g., APF, HB-EGF; EGF) may be useful to discriminate BPS/IC and asymptomatic con-trols (Zhang et al., 2005), these may not correlate with findings usingbladder biopsies (Erickson et al., 2008). The reason in part may be dueto variability in biopsy location, depth of sample (containing differentcell types) as well as other technical variations. There is some sugges-tion that there seems to be more of a proinflammatory state with in-creased infiltration of mast cells in BPS/IC patients as compared tocontrols. In addition, increased levels of NGF in urine and tissue havebeen linkedwith bladder pathologies including patients with idiopathicsensory urgency (urgencywithout incontinence), overactivity, and BPS/IC (Liu and Kuo, 2007; Kuo et al., 2010). Studies in cats diagnosed withfeline IC have reported increased NGF levels in bladder urothelium(Birder et al., 2010) and a major source of NGF has been shown tocome fromurothelium,whichmay contribute to increased neural excit-ability and emergence of bladder pain in BPS/IC (Micera et al., 2007).

7. Summary

In summary, these findings suggest that urothelial cells exhibit spe-cialized sensory and signaling properties that could allow them to re-spond to their chemical and physical environments and to engage in




5L.A. Birder / Autonomic Neuroscience: Basic and Clinical xxx (2013) xxx–xxx

reciprocal communication with neighboring urothelial cells as well asnerves within the bladder well. Taken together, pharmacologic inter-ventions aimed at targeting urothelial receptor/ion channel expressionor transmitter release mechanisms may provide a new strategy for theclinical management of bladder disorders such as BPS/IC.

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urinary bladder, cystitis and nerve/urothelial interactions

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