Morphological examination of the effects of defibrotide on experimentally induced bladder injury and its relation to interstitial cystitis

This morphological study aims to investigate the effects of defibrotide, a deoxyribonucleic acid derivative drug with cytoprotective, immunosuppressive and vasorelaxant effects, on protamine sulfate induced bladder injury. Wistar albino female rats were catheterized and intravesically infused with phosphate buffered solution (control group) or, either protamine sulfate (bladder injury group) or protamine sulfate+defibrotide (bladder injury+defibrotide group) dissolved in phosphate buffered solution. The morphology of the urinary bladder was investigated using light and electron microscopy. The number of mast cells in the mucosa, mucosal alterations, intercellular junctions, surface topography and the glycosaminoglycan (GAG) layer as well as microvillus formation on the luminal surface were evaluated. In the bladder injury group, ulcerated areas, irregularity of the GAG layer, increased number of mast cells, vacuole formation, dilated perinuclear cistern, formation of pleomorphic and uniform microvilli and dilatations in the intercellular spaces in the urothelium were observed. In the bladder injury+defibrotide group a relatively normal urothelial topography, GAG layer and a few mast cells in the mucosa, some dilatations between the intercellular areas, less uniform microvilli, regular perinuclear cistern and tight junctions were observed. These results show that defibrotide can inhibit PS induced bladder damage.     

The effect of pentosanpolysulphate and carbenoxolone on bacterial adherence to the injured urothelium

The effect of the glycosaminoglycan (GAG) layer on the adherence of Escherichia coli to the bladder urothelium of rats has been studied. The study was performed by destroying the GAG layer and the changes were observed using the electron microscope. Bacterial adherence to the bladder with a destroyed GAG layer was much higher than to the normal bladder. Following the destruction of the GAG layer, the instillation of sodium pentosanpolysulphate significantly reduced the adhesion of bacteria. Prophylactic intramuscular administration of carbenoxolone increased the speed of regeneration of the destroyed GAG layer.     

Glycosaminoglycans in urothelial carcinomas

Summary The glycosaminoglycan (GAG) content in human urothelial carcinomas was biochemically determined and compared to that of normal urothelium and bladder wall of the calf. The total GAG content was elevated in urothelial carcinomas, and the distribution pattern differed from that of normal urothelium and bladder wall. Whereas urothelial carcinomas contained heparan sulphate, dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate, only heparan sulphate could be detected in the normal urothelium. The GAG determination was based on hexosamine analysis and thin layer chromatography after elution on Dowex 1 X 2 columns.Abbreviations ABC chondrointin ABC lyase - AC chondroitin AC lyase - CH4-S chondroitin 4-sulfate - CH6-2 chondroitin 6-sulfate - DS dermatan sulfate - GAG glycosaminoglycan (s) - Gal-N galactosamine - Glc-N glucosamine - HS heparan sulphate - KS keratan sulphate - TLC thin layer chromatography     

Inactivation of antiadherence effect of bladder surface glycosaminoglycans as possible mechanism for carcinogenesis

We have shown that the urinary bladder secretes and binds to its surface a glycosaminoglycan layer whose nonspecific antiadherence effect protects the bladder from infection and perhaps from stone formation. If bladder cancer is caused by agents present in the urine, as is widely believed, this mechanism may also protect against carcinogenesis. We performed the current study to determine whether suspected carcinogens or cocarcinogens in the urine gain access to the transitional cells by impairing or inactivating the surface antiadherence effect. Using an in vivo method to quantitate bacterial adherence to the rabbit bladder, we compared adherence in control and glycosaminoglycan-deficient bladders to adherence in bladders treated with one of several suspected urinary carcinogens. There were statistically significant differences between adherence in control bladders and adherence in bladders treated with the tryptophan metabolites 3-hydroxykynurenine and 3-hydroxyanthranilic acid, sodium cyclamate, and sodium saccharin. These data indicate that perhaps certain suspected urinary bladder carcinogens inactivate the anti-adherence effect of the glycosaminoglycan layer at the bladder surface and thereby penetrate to the transitional cells to exert their tumorigenic effects; or they may serve as cocarcinogens that inactivate the glycosaminoglycan barrier and permit other urinary carcinogens to transform the transitional cells.     

Nitrofurantoin not surface active agent in rabbit urinary bladder

It has been recently suggested that nitrofurantoin may induce symptoms of interstitial cystitis by acting as a surface active agent that destroys glycosaminoglycan (GAG) on the bladder surface. Evidence accumulated over the past decade has demonstrated that the bladder surface GAG prevents bacterial adherence. In this experiment, exposure of the bladder lumen to nitrofurantoin at more than twice the therapeutic concentration did not destroy the bladder GAG layer (as evidenced by periodic acid-Schiff histochemistry) nor increase bacterial adherence as did a true surface active agent (Triton X-100). Acid treatment as well as all tested organic solvents except 50% dimethyl sulfoxide (DMSO) also removed the bladder GAG layer and increased bacterial adherence. These results indicate that neither nitrofurantoin nor 50% DMSO has adverse effects on the bladder surface and thus is unlikely to initiate the interstitial cystitis symptom complex by means of surfactant activity.     

Study of the early stages of renal stone formation: experimental model using urothelium of pig urinary bladder

An experimental model that enables the close simulation of conditions prevailing in the kidney during the formation of stones, using living tissue of pig urinary bladder, was developed. The results obtained clearly confirmed the importance of the antiadherent glycosaminoglycan (GAG) layer in preventing the development of solid concretions on the urothelium. Of importance was the capacity of the necrosed urothelium to act as a heterogeneous nucleant of calcium oxalate monohydrate (COM) and dihydrate (COD) crystals, demonstrating the major urolithiasic risk factor that alterations of the healthy epithelium covering the renal papilla may pose in humans. The significant increase in brushite and hydroxyapatite crystals detected on the urothelium when the pH of the artificial urine was 6.5, and the protective GAG layer was reduced or the tissue was necrosed, was also notable. The crystallization inhibitory effects caused by citrate and phytate were also studied. It was found that whereas citrate, at normal urinary concentrations, caused a slight reduction in crystallization, with phytate there was total elimination of crystallization-when it was present at very low concentrations such as 1.0 g/ml.     

Glycosaminoglycan content of human bladders. a method of analysis using cold-cup biopsies

A glycocalyx layer composed of glycosaminoglycans (GAGs) and other molecules lines the transitional epithelium of the urinary bladder. This layer forms a barrier between the transitional cells and urinary bladder environment and is believed to help prevent the adherence of bacteria, minerals and carcinogens. Investigators postulate that quantitative and/or qualitative defects in the GAG component may be responsible for a spectrum of acute and chronic disease processes ranging from urinary tract infections to cancer. While the presence of epithelium GAGs has been confirmed biochemically and histochemically, few rigorous characterizations have been performed. This study establishes the methodology and feasibility of using routine cold-cup biopsies from cadaveric human bladders for GAG analysis and establishes baseline contents of the sulfated and non-sulfated GAGs in the urinary bladder glycocalyx. Using detergent extraction, the GAGs from cold-cup biopsies (n = 34) from four cadaveric bladders were isolated. The isolates were subjected to two colorimetric assays to quantify both sulfated and non-sulfated GAGs. The nonsulfated GAG content of the bladder epithelium ranged from 2.15 x 10(-4) to 5.50 x 10(-4) mmol/kg of dry, defatted bladder. The sulfated GAG content ranged from 2.00 x 10(-1) to 7.40 x 10(-1) mmol/kg of dry, defatted bladder. These values are consistent with reports found in the literature using electrophoresis on full-thickness human bladder specimens. The GAG content of human bladder epithelium can be readily and accurately characterized from cold-cup biopsy samples. Our future plans involve using this routinely used technique to analyze samples from live control and disease-state bladders thereby demonstrating any quantitative and/or qualitative differences in GAG constituents.     

Influence of the glycosaminoglycan layer on the permeation of hypericin in rat bladders in vivo

OBJECTIVE: To investigate the influence of a glycosaminoglycan (GAG) layer on the specific location of hypericin in superficial urothelial carcinoma lesions of the bladder after intravesical instillation. MATERIALS AND METHODS: Fisher rat bladders were incubated with 15 or 30 microm hypericin for 2 h. To examine the influence of the GAG layer on the permeation of hypericin, bladders were pre-treated with chondroitinase ABC, n-dodecyl-beta-d-maltoside (DDM) or sodium dodecyl sulphate (SDS) to disrupt, or protamine to neutralise the GAG layer before incubating with hypericin. After incubation, the photosensitizer permeation was examined quantitatively in cryostat sections of the bladders, using fluorescence microscopy and image analysis. RESULTS: Disrupting or neutralising the GAG layer in the bladder had no influence on the permeation of hypericin. Pre-treatment of the bladder with chondroitinase, DDM or SDS resulted in a significantly lower accumulation of hypericin, whereas neutralising the GAG layer in rats with protamine had no significant effect on the biodistribution of hypericin. CONCLUSION: The GAG matrix causes no obstacle to the permeation of hypericin in the urothelium of the bladder, and modification of this GAG layer cannot explain the enhanced accumulation of hypericin in superficial bladder tumours.     

Inhibition of sodium urate crystal adherence to bladder surface by polysaccharide

Bladder surface glycosaminoglycan (polysaccharide) has been shown to act in a nonspecific manner as an antiadherence factor and may potentially be important in the pathogenesis (and prevention) of renal calculi. It has been shown that when present, bladder surface GAG will reduce the adherence of both calcium and calcium oxalate crystals. This study was conducted to see if a similar effect would be found with uric acid crystals, in that the presence of the normal bladder mucus with its contained GAG and exogenously supplied polysaccharide, pentosanpolysulfate, would be inhibitory to crystal adherence. Radioactively labeled uric acid crystals were made by dissolving 14C labeled sodium urate to a point of saturation at pH 7.5 and then rapidly lowering the pH to 5.5. These crystals were placed into four groups of bladders: 1) normal bladders, 2) bladders rendered mucus deficient with hydrochloric acid, 3) mucus deficient bladders which were pre-treated with sodium pentosanpolysulfate prior to crystal addition and 4) mucus deficient bladders to which were added crystals pretreated with pentosanpolysulfate. There was a significant rise in crystal adherence after acid treatment of the bladder, a 2.3-fold increase, which was blocked with prior treatment of the crystals with pentosanpolysulfate and to a lesser degree by pretreating mucus deficient bladders with pentosanpolysulfate.     

Urinary glycosaminoglycan excretion during the menstrual cycle in normal young women

PURPOSE: We investigated whether the menstrual cycle affects urinary glycosaminoglycan (GAG) excretion in normal young women. MATERIALS AND METHODS: Urine samples from 10 healthy women 19 to 21 years old were collected daily during the whole menstrual cycle. Concentration of total urinary GAG was assessed as mug hexuronic acid per mg creatinine. Proportions of sulfated GAG species were determined by agarose gel electrophoresis. RESULTS: Mean excretion values +/- SD for period days 4 to 13 and 15 to 28 of the cycle were significantly different (0.445 +/- 0.041 vs 0.356 +/- 0.035 microg/mg, p <0.001). Correlation between values for the first and second halves of the cycle showed that this difference was consistent irrespective of individual variations in GAG excretion (r = 0.9757, p <0.001). Proportions of urinary sulfated GAG did not change during the cycle. CONCLUSIONS: Excretion of total urinary GAG during the normal menstrual cycle of young women has a biphasic pattern with significantly higher values occurring in the first half of the cycle. This variation implies modulation by estrogens and consequently it should be considered when comparing the GAG concentration in urine samples from women of childbearing age.     

A Deficit of Proteoglycans on the Bladder Uroepithelium in Interstitial Cystitis

Physical and immunohistochemical data confirm that the luminal surface of the bladder is richly endowed with proteoglycans. Their likely function is to make the bladder nonadherent and produce a water layer to exclude salts and urinary proteins from the surface. IC patients are shown to have a deficit of chondroitin sulphate from the luminal surface as determined by immunohistochemistry. These studies strongly support previous hypotheses suggesting an etiologic role for loss of barrier function in IC and a rationale for replacement therapy with chondroitin sulphate.     

Effect of drugs on crystal adhesion to injured urothelium.

The normal urothelium is covered by a glycosaminoglycan (GAG) layer which acts as a barrier to the adhesion of crystals. Destruction of the GAG layer increases the number of adhered crystals, and it is therefore assumed that it promotes crystal growth and stone formation. Intravesical instillation of pentosanpolysulfate, an exogenous glycosaminoglycan, after destruction of this layer reduces the adhesion of crystals to the urothelium. Intramuscular administration of carbenoxolone sodium following the experimental destruction of the GAG layer increases the rate of healing of the layer and reduces the number of adhered crystals.     

Structure,function, and pathology of proteoglycans and glycosaminoglycans in the urinary tract

Summary The roles of glycosaminoglycans and proteoglycans in the physiology of the urinary tract are reviewed. The structures of proteoglycans and glycosaminoglycans are reviewed together with their role in control of epithelial differentiation through stromal-epithelial interactions and as modulators of cytokines. Heparan sulfate proteoglycans appear to be important in maintaining selectivity of the kidney tubular basement membrane, and the majority of the glycosaminoglycan found in the urine appears to come from the upper tract. Evidence suggesting that a dense layer of glycosaminoglycans on the urothelial surface is important to maintaining urothelial impermeability is reviewed and new data showing a high density of proteoglycans on the lumenal surface of the urothelium is presented. The role of this layer in maintaining antibacterial adherence and impermeability was discussed together with data suggesting that failure of this layer is an etiologic factor in interstitial cystitis. A model of the bladder surface is also presented to illustrate the role of proteoglycans and exogenous glycosaminoglycans in the defenses of normal bladder lumen and the failure of these defenses in the interstitial cystitis bladder.This work was supported in part by a grant from the Presbyterian Health Foundation (to R. E. H.).     

Prevention of urinary tract infection by the exogenous glycosaminoglycan sodium pentosanpolysulfate

The transitional cells at the surface of the urinary bladder secrete and bind to their surfaces 1 or more glycosaminoglycans whose presence prevents bacterial adherence to the mucosa. Because it is believed that adherence is prerequisite to infection, we did the current studies to determine whether the antiadherence effect of natural and synthetic glycosaminoglycans prevents infection. We exposed intact rabbit bladders, mucin deficient rabbit bladders, and rabbit bladders treated with sodium pentosanpolysulfate, a similar but synthetic substitute for the surface glycosaminoglycan(s), to bacteria in vivo. We measured infection rates 48 hours after exposure. The infection rate was significantly higher in mucin deficient bladders than in controls (p less than 0.02). There was no significant difference between infection rates in controls and infection rates in bladders treated with sodium pentosanpolysulfate. These results support our impression that a bladder with an intact mucin layer is better able to resist infection than is a mucin deficient bladder. The natural surface glycosaminoglycan(s) and the synthetic substitutes that reproduce their antiadherence effect appear to be protecting factors.     

Urinary glycosaminoglycan excretion in metastatic prostate cancer

Summary Urinary glycosaminoglycan (GAG) excretion is increased in many disseminated cancers. We have postulated that patients with metastatic prostatic cancer may have an increased urinary GAG excretion and that this parameter may have potential as a tumor marker during the management of these patients. Urinary GAGs were precipitated with cetylpiridinium chloride, separated by cellulose acetate electrophoresis and stained with alcian blue. Heparan sulphate and chondroitin sulphate were identified in all, and hyaluronic acid in most patients by cellulose acetate electrophoresis and specific enzyme digestion. A fourth alcianophilic, non-GAG band could be identified in the urinary extract and is thought to be a urinary glycoprotein. In patients with localised prostatic cancer, total urinary GAG excretion was elevated in 2 of 18 patients, while in patients with metastatic prostatic cancer not in remission after hormonal therapy, 21 of 31 patients had elevated total urinary GAG levels. Separating and quantifying the urinary heparan sulphate and chondroitin sulphate content increased the sensitivity to 22 of 31 patients. Urinary glycosaminoglycan excretion may have value as a tumor marker in the follow-up of patients with prostatic cancer.     

The association of elevated urinary total to sulfated glycosaminoglycan ratio and high molecular mass hyaluronic acid with interstitial cystitis

PURPOSE: A decrease in the glycosaminoglycan (GAG) layer on the urothelium is believed to be one of the possible causes of interstitial cystitis. Consequently, GAG-like substances and hyaluronic acid (HA) have been prescribed for treating this condition. To delineate the possible role of GAG and HA in the interstitial cystitis disease process, we compared the urinary levels of total GAGs (sulfated + non-sulfated), sulfated GAGs and HA in interstitial cystitis patients and normal controls. We also examined different HA species present in the urine of interstitial cystitis patients. MATERIALS AND METHODS: The total GAG and sulfated GAG levels in urine specimens of normal individuals (n = 20) and interstitial cystitis patients (n = 25) were determined by utilizing the carbazole reaction assay and the Farndale method, respectively, and were expressed as microg./mg. creatinine. Urinary HA levels were measured by applying the HA test and were expressed as ng./mg. creatinine. Gel filtration column chromatography was used to examine the profile of urinary GAGs and HA species. RESULTS: Total urinary GAGs were 2.5 to 4-fold elevated in interstitial cystitis patients with moderate to severe symptoms (Group 2; 76.2 +/- 24.8) when compared with those in normal individuals (19.9 +/- 2.5) and patients with mild symptoms (Group 1; 30.4 +/- 5.1) (p <0.001). Three urinary GAG peaks were detected in both normal and interstitial patients. However, each GAG peak from interstitial cystitis patient urine was 3 to 5-fold higher than that from normal patient urine. The sulfated GAG levels, however, remained unchanged among normal individuals (1.4 +/- 0.22), Group 1 (2.2 +/- 0.96) and Group 2 (1.6 +/- 0.38) patients (p >0.05). Consequently, the ratio of total GAGs to sulfated GAGs was elevated 3 to 3.5-fold in Group 2 patients (49.9 +/- 13.9) in comparison to that in normal individuals (16.7 +/- 2.5) and group 1 patients (14.4 +/- 4.6) (p <0.001). Urinary HA levels were marginally elevated in Group 2 patients (821. 4 +/- 247.9) when compared with those in the normal group (337.3 +/- 106.1) and Group 1 patients (540.9 +/- 166.5). In addition, a distinct high molecular mass HA species was present only in Group 2 patients. CONCLUSIONS: The increased ratio of total GAGs to sulfated GAGs and marginally elevated HA levels in urine indicate that the GAG layer is altered in interstitial cystitis patients. However, these results are in contrast to the accepted concept that a reduction in urothelial GAGs causes interstitial cystitis. The high molecular mass HA species detected in patients with severe symptoms may play a role in the pathophysiology of this disease.     

The role of the glycosaminoglycan layer in bladder defense mechanisms and interstitial cystitis

The bladder surface glycosaminoglycan layer is an important defense mechanism for the transitional epithelium. It allows adaptation to the constant exposure to urine, and controls the permeability of urinary substances to the transitional cell. This layer seems to be impaired in some patients with interstitial cystitis.     

Bladder cancer cell implantation in reconstructed bladder in vitro: a model of tumour recurrence

OBJECTIVE: To evaluate the involvement of umbrella cells in tumour adhesion and growth, by examining whether human urinary bladder carcinoma cells (HUBCC) can grow on reconstructed urinary bladder mucosa in vitro, as the implantation of tumour cells after resection is thought to be a cause of bladder tumour recurrence. MATERIALS AND METHODS: Normal transitional epithelial cells isolated from porcine bladder were cultured on reconstructed lamina propria using fibroblasts in type I collagen gel. The urothelium thus reconstructed was artificially injured either by a scalpel or by dilute acid, after which transitional epithelial cells began to grow in a stratified fashion within a few days of culture. A HUBCC line (HT-1197) was seeded onto this impaired mucosa to determine whether the cells could become implanted. Cultured cells on the reconstructed mucosa were evaluated by histological observation of vertical paraffin sections. RESULTS: The inoculated transitional epithelial cells grew in a stratified fashion and closely resembled urothelium in vivo. The superficial cells that were in contact with the medium solution differentiated into umbrella cells. HUBCC were unable to adhere to reconstructed mucosa which had not been injured, but these cells could adhere to and become implanted on the reconstructed mucosa after it had been injured either by a scalpel or by dilute acid. After acid injury, only the surface-covering cells were removed sporadically, while the lower epithelial cell layer remained intact. The bladder cancer cells adhered to and proliferated within these stripped regions. CONCLUSION: These results suggest that the urothelium, especially umbrella cells, seems to be important in preventing the adhesion and growth of urinary bladder tumour cells.     

A system for the enhancement of adenovirus mediated gene transfer to uro-epithelium

PURPOSE: Recombinant adenovirus has been used widely as an in vivo gene transfer vector, although its transfection efficiency in bladder tissue is limited. Several studies have indicated that the bladder surface glycosaminoglycan (GAG) layer functions as a nonspecific anti-adherence factor and possibly as a first line anti-infection defense mechanism. We determined whether recombinant adenovirus mediated gene transfer could be enhanced in intact bladders by HCl pretreatment and by alterations in the GAG layer. MATERIALS AND METHODS: In vitro viral transfection efficiencies with and without the GAG analog pentosan polysulfate (Sigma Chemical Co., St. Louis, Missouri) were determined in bladder muscle and urothelial cells. Immunocytochemical studies and Western blot analysis were performed to determine whether urothelial cells possessed the Coxsackievirus and adenovirus receptor. Rat bladders were intravesically pretreated with HCl at various concentrations and for various periods. After 60 mM. HCl pretreatment for 10 minutes 2 x 109 pfu of recombinant adenovirus carrying the Escherichia coli LacZ gene were intravesically instilled into the bladders. RESULTS: Adenoviral infection of urothelial cells was significantly reduced in the presence of pentosan polysulfate in vitro. Coxsackievirus and adenovirus receptor expression was detected in urothelial cells in vivo and in vitro. Bladders pretreated with HCl resulted in an alteration of the bladder GAG layers. After intravesical gene instillation reporter gene analyses using X-5-bromo-4-chloro-3-inodolyl beta-D-galactopyranoside (Sigma Chemical Co.) showed approximately 80% urothelial cell transfection efficiency in bladders pretreated with HCl. However, less than 10% of the urothelial cells expressed the transfected gene in control HCl untreated bladders. CONCLUSIONS: Primary urothelial cells and bladder carcinoma cells can be efficiently transfected using an adenoviral vector with similar infectivity. In vitro viral infection shows that the efficiency of adenoviral transfection is significantly reduced in the presence of pentosan polysulfate, a GAG analog. Adenoviral mediated gene transfer to bladder urothelium is enhanced by HCl pretreatment.     

Pentosanpolysulphate for interstitial cystitis

Interstitial Cystitis (IC) is a chronic disease of unknown etiology which primarily affects women aged 40–60 years. Many plausible theories for the development of IC have been postulated, and one current theory is that these patients have a quantitative and qualitative defect in the glycosaminoglycan (GAG) layer of the urothelium. Such a defect may allow toxic substances in the urine to gain access to the lamina propria and initiate a chronic inflammatory process. Pentosanpolysulphate (PPS) is a sulphated proteoglycan similar in structure to heparin sulphate, which is quantitatively the major GAG on cell surfaces. Exogenously administered PPS has been shown in several studies to decrease bladder pain and urinary frequency and to increase the voided volume. Further studies are required to evaluate the role of PPS in the management of IC patients, with particular emphasis on dosage, route of administration and combination with other compounds.