DOI: 10.5301/RU.2013.11500
Urologia 2013 ; 80 ( 3 ): 173- 178
REVIEW
GAGs and GAGs diseases: when pathophysiology supports the clinic Elisabetta Costantini 1, Massimo Lazzeri 2, Massimo Porena 1 Department of Medical-Surgical Specialties and Public Health, Urology and Andrology Section, University of Perugia, Perugia - Italy 2 Department of Urology, San Raffaele Turro, Vita-Salute San Raffaele University, Milan - Italy 1
Department of Medical-Surgical Specialties and Public Health, Urology and Andrology Section, University of Perugia, Perugia - Italy
Abstract: The urinary epithelium has been the subject of considerable interest and much research in recent years. What has radically changed in the last decade is the concept of what the bladder epithelium really is. It is currently no longer considered just a simple barrier and a non-specific defence against infections, and it has been recognized as a specialized tissue regulating complex bladder functions and playing a fundamental and active role in the pathogenesis of cystitis. Researchers have been focussing on the receptors and mediators that are active in the sub-epithelial layer, with the hope that understanding the role of the urothelium defect will offer the opportunity for new therapeutic strategies. On the surface of the urothelial umbrella-cells there is a thick layer of glycoproteins and proteoglycans, which together are called Glycosaminoglycans (GAGs). They constitute a hydrophilic mucosal coating and act as a barrier against solutes found in urine. In recent years they have received special attention because injury to Gags, due to different noxae, has been identified as the first step in the genesis of chronic inflammatory bladder diseases, such as recurrent urinary tract infections, chemical or radiation cystitis, interstitial cystitis and/or Bladder Pain Syndrome. Aim of this study is to define the importance of the urothelium starting from the anatomy and physiology of the bladder wall. Furthermore, we will underline the role of glycosaminoglycans, focusing both on their pathophysiological role in the principal bladder diseases and on the therapeutic aspects from the clinical point of view. Key words: Glycosaminoglycans, Bladder pain syndrome, Interstitial cystitis, Urothelium Accepted: August 20, 2013
INTRODUCTION Recently there has been a wide consensus that a primary unsolved defective urothelial lining function may be the key factor in the chronic inflammation of the bladder and dysfunctions (1). The urothelium has been recognized to be more than a simple barrier separating the luminal contents from the deeper tissues of the urinary tract. It works with the suburothelium as a functional unit, which responds to external stresses by the release of modulator agents that
regulate the activity not just of nearby afferent nerves but also of underlying smooth muscle and basal stem cell. In particular, it may act as an efficient chemo-mechanosensor (the “afferent function”), and at the same time it is able to synthesize and release into sub-urothelium layer molecules involved in the bladder storage/voiding activity (the “efferent function”). Furthermore, the urothelium may protect the basal cells from being reached by toxins or other substance capable of activating some pathological cell growth (2).
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Urothelium and GAGs The urothelium is a lining epithelium consisting of three different layers of cells. An inner line - the basal layer, of small, irregularly discoid or polyhedral cells, which may proliferate and replace the more superficial cells. This layer is in contact with intermediate, pyriform or clavate (club-shape) cells that end in the apical layer where cells are bulky, sometimes binuclear and with a polygonal contour assuming the shape of umbrella cells. The primary function of the urothelium is to guarantee an efficient system of non-permeability and to allow selective permeability. This mechanism is regulated by structural molecular barriers, which are located at the apical, lateral and basal regions. The apical barrier system consists of different classes of membrane crystalline proteins (uroplakins), assembled into hexagonal plaques coating the umbrella cells, which prevent the passage of small molecules, neutralize toxic substance, inhibit the formation of crystals and stones and avoid uropathogens adhesion (3-5). This apical coating contains a group of molecules that form a soft and flexible layer. The urothelial apical coating can be roughly split into two layers: a deep and a superficial one. The deep portion consists of carbohydrate termini of glycoproteins, proteoglycans and glycolipids. The superficial layer consists of glycosaminoglycans (GAGs), which play a fundamental role in the urothelium function and dysfunction (6). The lateral barrier consists of desmosomes, leaky and tight junctions, which prevent the intercellular percolation of urine. The basal barrier consists of a group of proteins - cadherins, claudins, laminins, which mediate the adhesion of basal cells to basal lamina and regulate the electrostatic balance of the urothelium (7). These structures confer to the urothelium the quality of being easily shaped or moulded during the micturition phases, in order to adapt itself to the tridimensional shape of any bladder volumetric changes (8). During the storage phase the club-shaped cells decrease in height and wedge themselves into basal cells, while the umbrella cells get flat and become squamous allowing an increase of apical surface (more than 20%) (9). A series of processes allow the bladder to accommodate increasing volumes of urine during filling without compromising the barrier function.
Sub-urothelium The sub-urothelial region is the interstitial tissue extending from the basal lamina, which limits the urothelium, to 174
the smooth muscle layer. It is a deformable and flexible connective matrix consisting of elastin complex and type I, III and IV collagen; the sub-urothelium layer is stabilized by fibronectin and laminin (10). The sub-urothelium layer allows blood circulation to nourish the urothelial cells; it also collects and modulates the signals generated by the urothelium and neuronal network creating a “web” able to integrate afferent and efferent activities of the bladder and establish connections of various types with terminal endings of, prevalently sensory, nerves. One of the most intriguing findings has been the discovery of the presence of a sophisticated “impulse generator”, which regulates a spontaneous autorhythmic contraction of bladder muscle cells. The bladder “impulse generator” consists of a specific class of cells, which are Ki67 and vimentin positive and are termed interstitial cells of Cajal (11). These starshaped cells respond to acetylcholine by M3 receptor and are sensitive to NO by releasing intracellular cGMP. Their main function is the regulation of the micturition reflex (12).
Pathophysiology of chronic cystitis According to a “domino-like” mechanism, different urothelial components might play a basic role in the pathophysiology of chronic cystitis. A cascade of events starting from the coating injury may lead to chronic bladder epithelial damage and chronic neurogenic inflammation (13). Given the complexity of etiological factors and different clinical conditions it is extremely difficult to define a time sequence or a priority in the steps that lead to chronic cystitis. At present there is strong evidence that different “noxae patogenae” can be responsible of urothelial damage and failure of coating function, and that the central element common to diverse bladder pathologies is the damage to bladder epithelium and in particular a damage to the bladder GAG layer. At an early stage, autoimmune diseases, chronic bacterial infections, bladder trauma, chemicals, including anticancer drugs (mitomycin or BCG exposure) or radiation exposure, can all result in coating damage. In BPS/IC a damage of superficial coating layer, consisting of GAGs, has been reported and linked to altered permeability (14). Interestingly, independently of the original cause, the inflammatory process starts and evolves in the same way. A GAG injury will lead to the loss of their “water-tight” function. As a result, not only do normal substances that are excreted in urine (i.e. H+, K+, Na+, Cl-) come into contact with the sub-epithelial layers but also abnormal substances
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such as cytotoxic drugs or toxins. This abnormal infiltration causes inflammation. Changes in apical barriers are associated with increase synthesis of intercellular adhesion molecules (ICAMs), which are capable of activating flogosis (15). ICAMs, whose expression is increased in BPS/IC patients, play a fundamental role in recruiting inflammatory cells and drive the migration of immunocompetent cells from blood to injury site. Infiltration of inflammatory cells is associated to basal lamina changes in molecular composition with a decrease of type IV collagen (16). After acute trauma, the urothelium has however the capacity to take care of its own maintenance throughout multiple repair mechanisms controlled by specific proteins. Nonresolved inflammation has been shown to block urothelium healing and GAG repair processes. It changes receptor sensitivity in peripheral organs and nervous tissue (17). Failure to control such repair seems to be a crucial step in developing chronic cystitis and pathological cell growth (18). When the coating/GAGs defect persists or its healing process fails, chronic morphological changes develop. Mastocytosis appears in the tissue and mast cells undergo activation with degranulation, leading to the release of vasoactive, inflammatory, and nociceptive mediators, such as histamine, kinins, proteases (e.g. tryptase), cytokines, leukotrienes (e.g. IL-6 and IL-8), prostaglandins, and nitric oxide (19). Consequently, primary afferent sensory C-fibers, silent in normal conditions, undergo activation and hyper-activation, resulting in visceral hypersensitivity (17). This finds tight clinical links. The neuronal hypersensitivity, the exaggerated perception to normal stimuli, leads to allodynia, the perception of nociceptive stimulation, which occurs for stimuli that would usually evoke a normal sensation (i.e. pain during bladder filling – it is a key symptom for case definition in patients with suspected BPS/IC) and to hyperalgesia: an exaggerated sensation to a stimulation, which is normally of mild intensity. Under these conditions the central nervous system receives an increased afferent barrage from peripheral nervous endings (20). This barrage, in turn, triggers central mechanisms that amplify and sustain the effect of the sensory nerve peripheral input, leading to molecular changes in the peripheral organs and in the central nervous system (19). It has been observed that changes in the density of neuropeptides in sensory nerves develop over a period of five to seven days, and that these are preceded by changes in the level of transcription factors activation. The increase of neuropeptide synthesis and their release at the level of synapses have a
clinical impact, so at bladder level it will produce chronic pain, an increase of frequency, nocturia and urgency, sustaining a neurogenic inflammation, while at the level of the central nervous system it will lead to selective expression of genes (i.e. c-Fos). A nerve sprouting will be observed in the sub-urothelium and grey matter of the spinal dorsal horn, with an increase of cranio-caudal and latero-lateral synapses resulting in hypersensibility (21). All these changes, starting with a primary damage in urothelial permeability, due to the loss of coating/GAGs layer, lead to mast cell activation, sensory nerve stimulation and activation, and result in progressive bladder injury within a self-maintaining process. Pathology is the base of clinics and it will originate poor bladder compliance, detrusor overactivity, chronic waxing and waning symptoms of frequency, urgency, urgency incontinence and pain. Finally, GAGs layer interruption may prompt abnormal cell growth of the urothelium as it exposes the deeper layers of the bladder wall and urothelial basal (stem) cell to toxic substances such as aromatic amines. Damage to the GAGs layer, resulting in a failure of the coating function, favors the loss of hydro-repellent properties. This results in toxic constituents of urine coming into direct contact with urothelium cells, which may lead to abnormal cell growth or proliferation
Clinical implication From our current understanding of chronic bladder inflammation, although there are many steps in the pathogenesis, which can be targeted during treatment selection, damage to the coating/GAG layer seems to be the primum movens leading to chronic inflammation and might represent a risk factor for pathological cell growth. On this basis, a multimodal treatment regimen with oral therapies, intravesical therapies and complementary therapies, aiming to restore the coating of the bladder urothelium, should be our current approach in order to prevent or treat the coating defect. Despite a low level of evidence, several treatments including behavioral changes (to reduce the severity of chronic inflammation, such as stress and anxiety modulation, exercise and physical therapy, bladder training, pelvic floor rehabilitation training, and dietary changes) have been shown to be useful for the treatment of patients with urinary frequency, urgency and pain (22). Drugs targeting the C-fibers, the nervous system, and pain modulators have been used over the years with unsatisfactory results.
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According to reported evidence (23) and the unmet need to treat a single target, a therapy aimed to enhancing the urothelium self-repair, through a direct action on coating for restoring the urothelial function and the overall activity of the bladder, might offer a new paradigm of how to rebuild clinical pharmacology for the treatment of “chronic cystitis”. The coating repair may activate a sequela of events leading to the functional restoration of apical and baso-lateral barriers, a stabilization of neuro-immuneendocrine components of the sub-urothelium and the avoidance of pathological cell growth. GAGs intravesical instillations, heparin, pentosan polysulfate and hyaluronic acid were shown to be able to integrate into the structure of injured coatings and block urine constituents into the sub-urothelium layer (24, 25). In this way the coating deterioration process is slowed down or stopped giving rise to a rudimentary neocoating (26). Umbrella cells re-activate the exo-endocytosis cycle restoring their selective permeability as showed by potassium test and the improvement in bladder function in patients with BPS/IC (27). Furthermore, hyaluronic acid administration may promote the restoration of the baso-lateral junction and neutralize the effects of ICAM-1 (28). Evidence supports the action of exogenous GAGs at the level of the sub-urothelium. One of the most intriguing mechanisms of action of hyaluronic acid and chondroitin sulphate is the deactivation of mast cells. Hyaluronic acid may bind to mast cells CD44 and inhibit the release of histamine and the production of pro-inflammatory mediators. Pentosan polysulfate may act in a direct and indirect way by the NF-kB in reducing the sub-urothelial inflammation (29).
CLINICAL DATA Few data are available on how GAGs act, as well as on their definitive effectiveness in the management of bladder diseases. Recent studies (30) investigated the endoscopic results after (GAGs) intravesical instillations with hyaluronic acid (HA) and chondroitin sulphate (CS) (Ialuril) in female patients affected by bladder pain syndrome (BPS)/interstitial cystitis (IC) or recurrent urinary tract infections. The authors performed a cystoscopic evaluation before and after HA-CS endovesical instillations, and demonstrated that the restoring of superficial integrity of the bladder epithelium is related to a better clinical outcome in patients with BPS/IC; when the endoscopic picture showed a nor176
malization of the bladder mucosa, fewer recurrences were present in patients with rUTIs. Some studies confirm the efficacy of GAGs combination in recurrent UTI and PBS/IC giving support to the positive results obtained in daily clinical practice. In patients with recurrent Urinary tract infections, Constantinides et al. (31) and Lipovac et al. (32) showed, in prospective cohort studies, a significant reduction of the UTI rates per patient/ year (from 4.3 to 0.3, and from 4.99 to 0.56, respectively) over a 12-month period, a prolonged median time to recurrence (from 96 to 498 days, and from 76.7 to 178.3 days) and a rate of recurrence-free at the end of the study period ranging from 65 to 70%. Furthermore, Damiano et al. (33) provided higher-level evidence by reporting a prospective, randomized, double blind, placebo-controlled study on the use of intravesical HA and CS in women with recurrent UTIs. The study showed a significant reduction of the UTI rate per patient per year versus placebo (-86.6 ± 47.6% versus -9.6 ± 24.6%). Mean time to UTI recurrence was significantly (3.5 fold) prolonged compared to placebo (185.2 ± 78.7 versus 52.7 ± 33.4 days) and 48% of patients were free from recurrence at the end of study period. Several studies were published on the use of GAGs in patients affected by Interstitial Cystitis/Bladder Pain Syndrome with promising data (34, 35). Three of them evaluated the efficacy of GAGs bladder instillation on prolonging the effects of bladder hydrodistention (36-38) demonstrating an improvement in mean bladder capacity associated with a decrease in urinary frequency and pelvic pain. Other studies assessed the reduction of symptoms score after GAGs bladder instillations in different cohorts of patients (39-44) with a 55-65% symptoms improvement or reduction in pain score. Three multicentric studies (45, 46) reported positive results on the use of chondroitin sulphate (CS) on reducing symptoms, bother and VAS pain scores, and an improvement in the mean value of bladder capacity.
CONCLUSIONS Damage to the urothelial GAGs barrier layer is thought to underlie the pathogenesis of several chronic bladder pathologies, including IC/PBS, chemical or radiation cystitis, and rUTIs. Protecting the urothelium or promptly restoring the GAG layer to prevent the cycle of inflammation and hypersensitization is the basis for the clinical use of GAGs intravesical instillations.
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GAGs therapy is becoming a cornerstone in the treatment of defective urothelium with promising clinical results. The following substances are used for intravesical GAG replenishment: chondroitin sulphate, heparin, hyaluronic acid, and pentosan polysulfate. Clinical evidence has been gained with formulations containing either chondroitin sulphate or hyaluronic acid and recently, further formulations were launched, including a combination of chondroitin sulphate and hyaluronic acid.
Disclaimers
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The authors have no proprietary interest with regard to this article. Corresponding Author: Elisabetta Costantini, MD-PhD Department of Medical-Surgical Specialties and Public Health, Urology and Andrology Section, University of Perugia Perugia - Italy [email protected]
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23. Green M, Filippou A, Sant G, Theoharides TC. Expression of intercellular adhesion molecules in the bladder of patients with interstitial cystitis. Urology. 2004;63(4):688-93. 24. Wilson CB, Leopard J, Nakamura RM, Cheresh DA, Stein PC, Parsons CL. Selective type IV collagen defects in the urothelial basement membrane in interstitial cystitis. J Urol. 1995;154(3):1222-6. 25. Sen GL, Webster DE, Barragan DI, Chang HY, Khavari PA. Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3. Genes Dev. 2008;22:1865-70. 26. Grover S, Srivastava A, Lee R, Tewari AK, Te AE. The role of inflammation in bladder function and interstitial cystitis. Adv Urol. 2011;3(1):19-33. 27. Doyle CA, Palmer JA, Munglani R, Hunt SP. Molecular consequences of noxious stimulation in molecular neurobiology of pain, progress in pain research and management. Vol 9, edited by D Borsook, IASP Press Seattle, 1997:145-170. 28. Mayer EA, Gebhart GF. Basic and clinical aspects of visceral hyperalgesia. Gastroenterology. 1994;107: 271-293. 29. Carter BD, Kaltschmidt C, Kaltschmidt B, et al. Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. Science. 1996;272:542-5. 30. Costantini E, Lazzeri M, Pistolesi D, Del Zingaro M, Frumenzio E, Boni A, Pietropaolo A, Fragalà E, Porena M. Morphological changes of bladder mucosa in patients who underwent instillation with combined sodium hyaluronic acid-chondroitin sulphate (ialuril). Urol Int. 2013;91(1):81-8. 31. Constantinides C, Manousakas T, Nikolopoulos P, Stanitsas A, Haritopoulos K, Giannopoulos A. Prevention of recurrent bacterial cystitis by intravesical administration of hyaluronic acid: a pilot study. BJU Int. 2004;93:1262-6. 32. Lipovac M, Kurz C, Reithmayr F, Verhoeven HC, Huber JC, Imhof M. Prevention of recurrent bacterial urinary tract infections by intravesical instillation of hyaluronic acid. Int J Gynaecol Obstet. 2007;96:192-5. 33. Damiano R, Quarto G, Bava I, Ucciero G, Palumbo MI, Autorino R. Prevention of recurrent urinary tract infections by intravesical administration of hyaluronic acid and chondroitin sulphate: a placebo-controlled randomised trial. Eur Urol. 2011;59:645-51. 34. Lai MC, Kuo YC, Kuo HC. Intravesical hyaluronic acid for interstitial cystitis/painful bladder syndrome: a comparative randomized assessment of different regimens. Int J Urol. 2013 Feb;20(2):203-7. 35. Lv YS, Zhou HL, Mao HP, Gao R, Wang YD, Xue XY. Intravesical hyaluronic acid and alkalinized lidocaine for the treatment of severe painful bladder syndrome/interstitial cystitis. Int Urogynecol J. 2012;23(12):1715-20.
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Urologia 2013 ; 80 ( 3 ): 173- 178
REVIEW
GAGs and GAGs diseases: when pathophysiology supports the clinic Elisabetta Costantini 1, Massimo Lazzeri 2, Massimo Porena 1 Department of Medical-Surgical Specialties and Public Health, Urology and Andrology Section, University of Perugia, Perugia - Italy 2 Department of Urology, San Raffaele Turro, Vita-Salute San Raffaele University, Milan - Italy 1
Department of Medical-Surgical Specialties and Public Health, Urology and Andrology Section, University of Perugia, Perugia - Italy
Abstract: The urinary epithelium has been the subject of considerable interest and much research in recent years. What has radically changed in the last decade is the concept of what the bladder epithelium really is. It is currently no longer considered just a simple barrier and a non-specific defence against infections, and it has been recognized as a specialized tissue regulating complex bladder functions and playing a fundamental and active role in the pathogenesis of cystitis. Researchers have been focussing on the receptors and mediators that are active in the sub-epithelial layer, with the hope that understanding the role of the urothelium defect will offer the opportunity for new therapeutic strategies. On the surface of the urothelial umbrella-cells there is a thick layer of glycoproteins and proteoglycans, which together are called Glycosaminoglycans (GAGs). They constitute a hydrophilic mucosal coating and act as a barrier against solutes found in urine. In recent years they have received special attention because injury to Gags, due to different noxae, has been identified as the first step in the genesis of chronic inflammatory bladder diseases, such as recurrent urinary tract infections, chemical or radiation cystitis, interstitial cystitis and/or Bladder Pain Syndrome. Aim of this study is to define the importance of the urothelium starting from the anatomy and physiology of the bladder wall. Furthermore, we will underline the role of glycosaminoglycans, focusing both on their pathophysiological role in the principal bladder diseases and on the therapeutic aspects from the clinical point of view. Key words: Glycosaminoglycans, Bladder pain syndrome, Interstitial cystitis, Urothelium Accepted: August 20, 2013
INTRODUCTION Recently there has been a wide consensus that a primary unsolved defective urothelial lining function may be the key factor in the chronic inflammation of the bladder and dysfunctions (1). The urothelium has been recognized to be more than a simple barrier separating the luminal contents from the deeper tissues of the urinary tract. It works with the suburothelium as a functional unit, which responds to external stresses by the release of modulator agents that
regulate the activity not just of nearby afferent nerves but also of underlying smooth muscle and basal stem cell. In particular, it may act as an efficient chemo-mechanosensor (the “afferent function”), and at the same time it is able to synthesize and release into sub-urothelium layer molecules involved in the bladder storage/voiding activity (the “efferent function”). Furthermore, the urothelium may protect the basal cells from being reached by toxins or other substance capable of activating some pathological cell growth (2).
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Urothelium and GAGs The urothelium is a lining epithelium consisting of three different layers of cells. An inner line - the basal layer, of small, irregularly discoid or polyhedral cells, which may proliferate and replace the more superficial cells. This layer is in contact with intermediate, pyriform or clavate (club-shape) cells that end in the apical layer where cells are bulky, sometimes binuclear and with a polygonal contour assuming the shape of umbrella cells. The primary function of the urothelium is to guarantee an efficient system of non-permeability and to allow selective permeability. This mechanism is regulated by structural molecular barriers, which are located at the apical, lateral and basal regions. The apical barrier system consists of different classes of membrane crystalline proteins (uroplakins), assembled into hexagonal plaques coating the umbrella cells, which prevent the passage of small molecules, neutralize toxic substance, inhibit the formation of crystals and stones and avoid uropathogens adhesion (3-5). This apical coating contains a group of molecules that form a soft and flexible layer. The urothelial apical coating can be roughly split into two layers: a deep and a superficial one. The deep portion consists of carbohydrate termini of glycoproteins, proteoglycans and glycolipids. The superficial layer consists of glycosaminoglycans (GAGs), which play a fundamental role in the urothelium function and dysfunction (6). The lateral barrier consists of desmosomes, leaky and tight junctions, which prevent the intercellular percolation of urine. The basal barrier consists of a group of proteins - cadherins, claudins, laminins, which mediate the adhesion of basal cells to basal lamina and regulate the electrostatic balance of the urothelium (7). These structures confer to the urothelium the quality of being easily shaped or moulded during the micturition phases, in order to adapt itself to the tridimensional shape of any bladder volumetric changes (8). During the storage phase the club-shaped cells decrease in height and wedge themselves into basal cells, while the umbrella cells get flat and become squamous allowing an increase of apical surface (more than 20%) (9). A series of processes allow the bladder to accommodate increasing volumes of urine during filling without compromising the barrier function.
Sub-urothelium The sub-urothelial region is the interstitial tissue extending from the basal lamina, which limits the urothelium, to 174
the smooth muscle layer. It is a deformable and flexible connective matrix consisting of elastin complex and type I, III and IV collagen; the sub-urothelium layer is stabilized by fibronectin and laminin (10). The sub-urothelium layer allows blood circulation to nourish the urothelial cells; it also collects and modulates the signals generated by the urothelium and neuronal network creating a “web” able to integrate afferent and efferent activities of the bladder and establish connections of various types with terminal endings of, prevalently sensory, nerves. One of the most intriguing findings has been the discovery of the presence of a sophisticated “impulse generator”, which regulates a spontaneous autorhythmic contraction of bladder muscle cells. The bladder “impulse generator” consists of a specific class of cells, which are Ki67 and vimentin positive and are termed interstitial cells of Cajal (11). These starshaped cells respond to acetylcholine by M3 receptor and are sensitive to NO by releasing intracellular cGMP. Their main function is the regulation of the micturition reflex (12).
Pathophysiology of chronic cystitis According to a “domino-like” mechanism, different urothelial components might play a basic role in the pathophysiology of chronic cystitis. A cascade of events starting from the coating injury may lead to chronic bladder epithelial damage and chronic neurogenic inflammation (13). Given the complexity of etiological factors and different clinical conditions it is extremely difficult to define a time sequence or a priority in the steps that lead to chronic cystitis. At present there is strong evidence that different “noxae patogenae” can be responsible of urothelial damage and failure of coating function, and that the central element common to diverse bladder pathologies is the damage to bladder epithelium and in particular a damage to the bladder GAG layer. At an early stage, autoimmune diseases, chronic bacterial infections, bladder trauma, chemicals, including anticancer drugs (mitomycin or BCG exposure) or radiation exposure, can all result in coating damage. In BPS/IC a damage of superficial coating layer, consisting of GAGs, has been reported and linked to altered permeability (14). Interestingly, independently of the original cause, the inflammatory process starts and evolves in the same way. A GAG injury will lead to the loss of their “water-tight” function. As a result, not only do normal substances that are excreted in urine (i.e. H+, K+, Na+, Cl-) come into contact with the sub-epithelial layers but also abnormal substances
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such as cytotoxic drugs or toxins. This abnormal infiltration causes inflammation. Changes in apical barriers are associated with increase synthesis of intercellular adhesion molecules (ICAMs), which are capable of activating flogosis (15). ICAMs, whose expression is increased in BPS/IC patients, play a fundamental role in recruiting inflammatory cells and drive the migration of immunocompetent cells from blood to injury site. Infiltration of inflammatory cells is associated to basal lamina changes in molecular composition with a decrease of type IV collagen (16). After acute trauma, the urothelium has however the capacity to take care of its own maintenance throughout multiple repair mechanisms controlled by specific proteins. Nonresolved inflammation has been shown to block urothelium healing and GAG repair processes. It changes receptor sensitivity in peripheral organs and nervous tissue (17). Failure to control such repair seems to be a crucial step in developing chronic cystitis and pathological cell growth (18). When the coating/GAGs defect persists or its healing process fails, chronic morphological changes develop. Mastocytosis appears in the tissue and mast cells undergo activation with degranulation, leading to the release of vasoactive, inflammatory, and nociceptive mediators, such as histamine, kinins, proteases (e.g. tryptase), cytokines, leukotrienes (e.g. IL-6 and IL-8), prostaglandins, and nitric oxide (19). Consequently, primary afferent sensory C-fibers, silent in normal conditions, undergo activation and hyper-activation, resulting in visceral hypersensitivity (17). This finds tight clinical links. The neuronal hypersensitivity, the exaggerated perception to normal stimuli, leads to allodynia, the perception of nociceptive stimulation, which occurs for stimuli that would usually evoke a normal sensation (i.e. pain during bladder filling – it is a key symptom for case definition in patients with suspected BPS/IC) and to hyperalgesia: an exaggerated sensation to a stimulation, which is normally of mild intensity. Under these conditions the central nervous system receives an increased afferent barrage from peripheral nervous endings (20). This barrage, in turn, triggers central mechanisms that amplify and sustain the effect of the sensory nerve peripheral input, leading to molecular changes in the peripheral organs and in the central nervous system (19). It has been observed that changes in the density of neuropeptides in sensory nerves develop over a period of five to seven days, and that these are preceded by changes in the level of transcription factors activation. The increase of neuropeptide synthesis and their release at the level of synapses have a
clinical impact, so at bladder level it will produce chronic pain, an increase of frequency, nocturia and urgency, sustaining a neurogenic inflammation, while at the level of the central nervous system it will lead to selective expression of genes (i.e. c-Fos). A nerve sprouting will be observed in the sub-urothelium and grey matter of the spinal dorsal horn, with an increase of cranio-caudal and latero-lateral synapses resulting in hypersensibility (21). All these changes, starting with a primary damage in urothelial permeability, due to the loss of coating/GAGs layer, lead to mast cell activation, sensory nerve stimulation and activation, and result in progressive bladder injury within a self-maintaining process. Pathology is the base of clinics and it will originate poor bladder compliance, detrusor overactivity, chronic waxing and waning symptoms of frequency, urgency, urgency incontinence and pain. Finally, GAGs layer interruption may prompt abnormal cell growth of the urothelium as it exposes the deeper layers of the bladder wall and urothelial basal (stem) cell to toxic substances such as aromatic amines. Damage to the GAGs layer, resulting in a failure of the coating function, favors the loss of hydro-repellent properties. This results in toxic constituents of urine coming into direct contact with urothelium cells, which may lead to abnormal cell growth or proliferation
Clinical implication From our current understanding of chronic bladder inflammation, although there are many steps in the pathogenesis, which can be targeted during treatment selection, damage to the coating/GAG layer seems to be the primum movens leading to chronic inflammation and might represent a risk factor for pathological cell growth. On this basis, a multimodal treatment regimen with oral therapies, intravesical therapies and complementary therapies, aiming to restore the coating of the bladder urothelium, should be our current approach in order to prevent or treat the coating defect. Despite a low level of evidence, several treatments including behavioral changes (to reduce the severity of chronic inflammation, such as stress and anxiety modulation, exercise and physical therapy, bladder training, pelvic floor rehabilitation training, and dietary changes) have been shown to be useful for the treatment of patients with urinary frequency, urgency and pain (22). Drugs targeting the C-fibers, the nervous system, and pain modulators have been used over the years with unsatisfactory results.
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According to reported evidence (23) and the unmet need to treat a single target, a therapy aimed to enhancing the urothelium self-repair, through a direct action on coating for restoring the urothelial function and the overall activity of the bladder, might offer a new paradigm of how to rebuild clinical pharmacology for the treatment of “chronic cystitis”. The coating repair may activate a sequela of events leading to the functional restoration of apical and baso-lateral barriers, a stabilization of neuro-immuneendocrine components of the sub-urothelium and the avoidance of pathological cell growth. GAGs intravesical instillations, heparin, pentosan polysulfate and hyaluronic acid were shown to be able to integrate into the structure of injured coatings and block urine constituents into the sub-urothelium layer (24, 25). In this way the coating deterioration process is slowed down or stopped giving rise to a rudimentary neocoating (26). Umbrella cells re-activate the exo-endocytosis cycle restoring their selective permeability as showed by potassium test and the improvement in bladder function in patients with BPS/IC (27). Furthermore, hyaluronic acid administration may promote the restoration of the baso-lateral junction and neutralize the effects of ICAM-1 (28). Evidence supports the action of exogenous GAGs at the level of the sub-urothelium. One of the most intriguing mechanisms of action of hyaluronic acid and chondroitin sulphate is the deactivation of mast cells. Hyaluronic acid may bind to mast cells CD44 and inhibit the release of histamine and the production of pro-inflammatory mediators. Pentosan polysulfate may act in a direct and indirect way by the NF-kB in reducing the sub-urothelial inflammation (29).
CLINICAL DATA Few data are available on how GAGs act, as well as on their definitive effectiveness in the management of bladder diseases. Recent studies (30) investigated the endoscopic results after (GAGs) intravesical instillations with hyaluronic acid (HA) and chondroitin sulphate (CS) (Ialuril) in female patients affected by bladder pain syndrome (BPS)/interstitial cystitis (IC) or recurrent urinary tract infections. The authors performed a cystoscopic evaluation before and after HA-CS endovesical instillations, and demonstrated that the restoring of superficial integrity of the bladder epithelium is related to a better clinical outcome in patients with BPS/IC; when the endoscopic picture showed a nor176
malization of the bladder mucosa, fewer recurrences were present in patients with rUTIs. Some studies confirm the efficacy of GAGs combination in recurrent UTI and PBS/IC giving support to the positive results obtained in daily clinical practice. In patients with recurrent Urinary tract infections, Constantinides et al. (31) and Lipovac et al. (32) showed, in prospective cohort studies, a significant reduction of the UTI rates per patient/ year (from 4.3 to 0.3, and from 4.99 to 0.56, respectively) over a 12-month period, a prolonged median time to recurrence (from 96 to 498 days, and from 76.7 to 178.3 days) and a rate of recurrence-free at the end of the study period ranging from 65 to 70%. Furthermore, Damiano et al. (33) provided higher-level evidence by reporting a prospective, randomized, double blind, placebo-controlled study on the use of intravesical HA and CS in women with recurrent UTIs. The study showed a significant reduction of the UTI rate per patient per year versus placebo (-86.6 ± 47.6% versus -9.6 ± 24.6%). Mean time to UTI recurrence was significantly (3.5 fold) prolonged compared to placebo (185.2 ± 78.7 versus 52.7 ± 33.4 days) and 48% of patients were free from recurrence at the end of study period. Several studies were published on the use of GAGs in patients affected by Interstitial Cystitis/Bladder Pain Syndrome with promising data (34, 35). Three of them evaluated the efficacy of GAGs bladder instillation on prolonging the effects of bladder hydrodistention (36-38) demonstrating an improvement in mean bladder capacity associated with a decrease in urinary frequency and pelvic pain. Other studies assessed the reduction of symptoms score after GAGs bladder instillations in different cohorts of patients (39-44) with a 55-65% symptoms improvement or reduction in pain score. Three multicentric studies (45, 46) reported positive results on the use of chondroitin sulphate (CS) on reducing symptoms, bother and VAS pain scores, and an improvement in the mean value of bladder capacity.
CONCLUSIONS Damage to the urothelial GAGs barrier layer is thought to underlie the pathogenesis of several chronic bladder pathologies, including IC/PBS, chemical or radiation cystitis, and rUTIs. Protecting the urothelium or promptly restoring the GAG layer to prevent the cycle of inflammation and hypersensitization is the basis for the clinical use of GAGs intravesical instillations.
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GAGs therapy is becoming a cornerstone in the treatment of defective urothelium with promising clinical results. The following substances are used for intravesical GAG replenishment: chondroitin sulphate, heparin, hyaluronic acid, and pentosan polysulfate. Clinical evidence has been gained with formulations containing either chondroitin sulphate or hyaluronic acid and recently, further formulations were launched, including a combination of chondroitin sulphate and hyaluronic acid.
Disclaimers
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The authors have no proprietary interest with regard to this article. Corresponding Author: Elisabetta Costantini, MD-PhD Department of Medical-Surgical Specialties and Public Health, Urology and Andrology Section, University of Perugia Perugia - Italy [email protected]
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