Effect of chronic nitrofurantoin on the rabbit urinary bladder

Interstitial cystitis is a pathological condition whose symptoms mimic urinary tract infection and include urgency, frequency, and moderate to severe pain. Many more women than men are affected, with antibiotic therapy being the usual first treatment approach based on symptomology. Some clinicians believe that chronic antibiotic therapy may play an etiological role in interstitial cystitis; however neither clinical nor experimental data support their opinion. The implied pathogenesis of antibiotic injury is an alteration of the bladder mucosa and its protective mucin coating to allow urine-mediated damage to the bladder wall. The purpose of this study is to evaluate rabbit urinary bladder function and morphology during chronic nitrofurantoin administration. The results demonstrate that up to twelve months of chronic nitrofurantoin administration produce no changes in 1) bacterial adherence to the rabbit bladder mucosa, 2) specific antibacterial adherence activity of the bladder mucin, and 3) ultrastructure of the mucosa, submucosa, and muscularis.     

Glycosaminoglycans Excretion in Interstitial Cystitis

Glycosaminoglycans (GAGs) have been identified histochemically and biochemically in urothelium. They have been suggested to have an anti-adherence effect at the bladder surface important in the urothelial defence against bacterial and carcinogenic insult. The aim of this study was to investigate urinary GAGs excretion in patients with interstitial cystitis.Urinary GAG excretion was determined in patients with interstitial cystitis (n:34; 16 males, 18 females) and healthy subjects (n:34; 16 males, 18 females). Urinary GAG determinations were made by the dimethymethylene blue method. Student's t-test was used for statistical analysis of the results.Urinary GAG excretion was found to be elevated significantly in patients with interstitial cystitis (14.45±2.02 mg/g Cr) as compared to healthy subjects (10.11±2.3 mg/g Cr) (p<0.01). There was no significant difference in urinary GAG excretion between males and females in either the healthy subjects or in the patients.Determination of urinary GAG excretion may be an important non-invasive test in the investigation of patients with interstitial cystitis.     

A model for the function of glycosaminoglycans in the urinary tract

Summary Investigations into the antibacterial defense mechanisms of the urinary tract revealed an important function for cell surface glycosaminoglycans (GAG), that of a generalized antiadherent activity. This activity was found to prevent bacterial, protein, and ionic adherence to the cell membrane. A model was developed to explain mechanically the activity of sulfated polysaccharides at the bladder surface. The model predicted injurious effects of quaternary amines and also that the mucus would be the so-called blood-urine barrier. It also led to the hypothesis that exogenous polysaccharides may be important in treating bladder disease such as infection and interstitial cystitis. For the first clinical test of these concepts, a polysaccharide was employed in several double-blind studies and was shown to ameliorate significantly the symptoms of interstitial cystitis. These discoveries suggest new methods to manipulate the microenvironment between the transitional cell surface and the urine, leading to novel therapies in regulating disease of the genitourinary tract. They also stress the importance of understanding the mechanisms by which GAGs exert their effect in the urinary tract and how they are produced, maintained, and even inactivated (e.g., by urinary substances such as protamine).Supported by the Medical Research Service of the Veterans Administration, Urological Research and Education Foundation, and by National Institutes of Health grant R01DK39239.     

The use of dimethyl sulfoxide in the treatment of intractable urinary frequency

Intravesical instillation of dimethyl sulfoxide (DMSO) was used in the treatment of patients with intractable urinary frequency due to chronic prostatitis, chronic cystitis, tuberculous contracted bladder and interstitial cystitis. Before the application of this therapy, all 4 patients were examined carefully to rule out cases of acute infectious diseases of the urinary tract, active urinary tuberculosis, neurogenic bladder and carcinoma in situ of the bladder. Three of the four patients achieved an excellent response both subjectively and objectively. In the United States, intravesical instillation of DMSO had already been established as the specific method in the treatment of interstitial cystitis and no side effects have been reported so far. Therefore, we recommend the use of intravesical instillation of DMSO more commonly in various forms of intractable urinary frequency.     

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.).     

Trypan blue as an indicator of urothelial integrity

Defects in the urinary bladder urothelium may be an etiological factor in conditions such as recurrent urinary tract infection and interstitial cystitis. Initial studies have demonstrated that the colloidal dye trypan blue can be used as a marker for defects in the integrity of the mucosal epithelium. Male white New Zealand rabbits were separated into 4 groups: Control, in vivo overdistension (via saline infusion), intravesical acetone (20 ml 50% for 1 minute), and intravesical 50% DMSO (20 ml “RIMSO-50” for 30 minutes). Both intravesical instillations were followed by 3–20 ml saline washes. Immediately following acetone or DMSO, 20 ml 1% trypan blue in saline was instilled and left in the bladder for up to 1 hour. After overdistension, 1% trypan blue in saline was infused into the bladder at the same rate until the volume reached that previously defined as the overdistension volume. Control bladders were rinsed with 20 ml saline, twice, before being filled with 20 ml of the trypan blue solution. There was no evidence of trypan blue's staining of tissues in any of the control bladders. After DMSO treatment, there was usually only slight staining of the bladder surface urothelium and subjacent connective tissues. After acetone treatment, microscopic examination of bladder wall showed that nuclei were stained in all tissues of the wall (e.g., urothelium, connective tissue and smooth muscle). After acute frank overdistension, the bladder wall was deep blue throughout, very similar to the acetone-treated bladder. Microscopic analysis demonstrated that the smooth muscle elements were the most heavily stained. In summary 1) trypan blue does not penetrate the normal bladder mucosa, nor is any dye absorbed into the blood stream. 2) Frank overdistension of the bladder defeats the physical integrity of the urothelium as evidenced by penetration of the dye into the submucosal tissue, the peritoneal cavity, and bloodstream. 3) Where DMSO penetrates the urothelium itself, it appears to leave no permanent route by which other molecules can traverse the urothelium after it is removed. 4) Only after the integrity of the urothelial surface is disturbed are the cells of the urothelium susceptible to staining.     

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.     

Intravesical chemotherapy: Combination with dimethyl sulfoxide does not enhance cytotoxicity in vitro

Summary There is evidence that dimethyl sulfoxide (DMSO) can increase the anticancer activity of chemotherapeutic drugs. As DMSO is instilled into the bladder for interstitial cystitis, it could be readily adopted in clinical practice if it was found to enhance the effectiveness of the drugs used for intravesical chemotherapy. The purpose of this study was to investigate, using a human bladder cancer cell line, the hypothesis that DMSO enhances the activity of these agents. However, the addition of 4% DMSO to the four drugs most frequently used for intravesical chemotherapy (adriamycin, epodyl, mitomicin-c, thiotepa) did not increase tumour cell kill in vitro.     

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.     

Scanning electron microscopic findings in interstitial cystitis

Scanning electron microscopy (SEM) was performed on transurethral resection biopsies from 13 patients with classic interstitial cystitis. Biopsies from 9 patients with stress incontinence served as controls. The SEM appearance of the bladder surface in interstitial cystitis varied considerably, exhibiting small round and large polygonal cells. The proportion of cells displaying round uniform and pleomorphic microvilli was high and sometimes dominated the area examined. SEM characteristics earlier assigned to bladder tumours were detected in patients with interstitial cystitis and, at a lower frequency, also in control patients. The mucin layer covering the urothelial cells seemed reduced in interstitial cystitis compared with control specimens. Surface characteristics specific for interstitial cystitis were, however, not detected by SEM.     

Urinary neutrophil chemotactic factors in interstitial cystitis patients and a rabbit model of bladder inflammation.

The present study investigates a possible source of inflammatory mediators involved in the pathogenesis of bladder inflammation characteristics of interstitial cystitis disease. Our tested hypothesis is that in response to injury, tissues of the urinary bladder participate in the initiation of bladder inflammation by releasing inflammatory mediators such as neutrophil chemotactic factors. Bladders of anesthetized rabbits (n = 7) were instilled with an acidic solution (pH 4.5) for 15 minutes, then washed with saline and instilled with sterile phosphate buffered saline (PBS) (pH 7.2) for an additional 45 minutes prior to sacrificing the rabbits. Control rabbits (n = 7) were instilled with sterile PBS (pH 7.2) for 15 minutes, then 45 minutes. The levels of neutrophil chemotactic factors were measured using modified Boyden chambers and rabbit peritoneal neutrophils as indicator cells. Results indicated the release of high levels of neutrophil chemotactic factors (via a checkerboard analysis) from acid-treated bladders after 15 minutes (70 +/- 4% of standard) and 45 minutes (80 +/- 7%). Electron microscopy analysis of these acid-treated bladders revealed the infiltration of a large number of neutrophils, which correlates with the recovery of neutrophil chemotactic factors. Control rabbits, on the other hand, showed low levels of chemotactic activity (less than 10 percent) and exhibited normal bladder morphology with absence of neutrophils. The glycosaminoglycan (GAG) layer was intact in both acid-treated and control bladders. High levels of neutrophil chemotactic factors were also detected in urine samples from eleven patients with interstitial cystitis (113 +/- 25%) (not due to interleukin-1 or leukotriene B4) which were not detected in urine samples from healthy volunteers (n = 9) or from thirteen control patients with bladder diseases other than interstitial cystitis. These preliminary studies indicate the capability of injured bladder tissues to release neutrophil chemotactic factors which contribute to the initiation of bladder inflammation. The presence of neutrophil chemotactic factors in urine samples of interstitial cystitis patients suggests a possible role of these mediators in the pathogenesis of the disease.     

Dimethyl sulfoxide: does it change the functional properties of the bladder wall?

PURPOSE: Dimethyl sulfoxide (DMSO) is used in a 50% solution to treat interstitial cystitis. Symptomatic relief occurs in about two-thirds of cases. The mechanism of action and effects of DMSO on bladder tissue function are poorly understood. Therefore, the effect of DMSO on bladder muscle compliance and contractility was evaluated. MATERIALS AND METHODS: Contractility and compliance were evaluated in rat bladder strips exposed to various concentrations of DMSO for 7 minutes, followed by 7 to 60-minute washout periods. The effect of DMSO at concentrations of 25%, 30%, 35%, 40% and 50% on electrical field stimulation induced contractions was assessed. Acetylcholine and high KCl (Sigma Chemical Co.) induced contractions were measured after exposure to 30% DMSO. Compliance was evaluated after exposure to 30% and 50% DMSO. RESULTS: Exposure to 40% DMSO completely abolished electrical field stimulation contractions, while 30% DMSO decreased the electrical field stimulation contraction to 40% +/- 6% of the initial force but there was almost complete recovery within 30 minutes. Contractile force was unaltered by 25% DMSO. Acetylcholine and KCl stimulation after exposure to 30% DMSO produced contractile forces of 78% +/- 6% and 39% +/- 6% of pre-DMSO control contractions, respectively. Compliance decreased by 2.4 and 4.6-fold following 30% and 50% DMSO exposure, respectively. CONCLUSIONS: DMSO completely and irreversibly abolishes contractions at a 40% concentration. Compliance is altered at even lower concentrations (30%). These findings bring into question the current practice of treating patients who have IC with 50% DMSO. Lower concentrations (25%) of DMSO may serve as a safe, effective analgesic and anti-inflammatory treatment for IC and other bladder pathologies.     

Glycosaminoglycans on the surface of the human urothelium: A preliminary report

Glycosaminoglycans (GAGs) are thought to be present in the mucous surface coat of the human urinary bladder and to play a role in the environmental adaptation of the bladder urothelium. A defective mucous surface coat might be involved in the pathogenesis of painful bladder disease (interstitial cystitis) of unknown etiology. By a new cystoscopic scraping method for obtaining the mucous surface coat of the bladder, we investigated four painful bladder patients and three controls (prostatic hypertrophy). Electrophoretic analysis of the surface coat revealed differences in the biochemical GAG-composition of the surface coat between patients and controls. This preliminary study suggests the existence of GAGs in the mucous surface coat of the bladder and suggests that qualitative differences in the GAG composition might play a role in painful bladder disease.     

Effects of dimethyl sulphoxide and heparin on stretch-activated ATP release by bladder urothelial cells from patients with interstitial cystitis

OBJECTIVE: To determine whether dimethyl sulphoxide (DMSO) and heparin reduce the greater stretch-activated ATP release in interstitial cystitis (IC), as ATP serves as a nocio-neurotransmitter in the bladder, and thus explain their beneficial effects in patients with IC (a disease characterized by hypersensory bladder symptoms). MATERIALS AND METHODS: Bladder epithelia in IC release more ATP in response to stretch than do control samples. Both DMSO and heparin are used intravesically to treat IC; such agents can modulate urothelial function because they directly contact bladder urothelium. Biopsies taken from patients with IC and from control subjects were grown in primary cultures using established cell-culture techniques. Cultured urothelial cells were stretched with the Flexcell device (Flexcell International Corp., McKeesport, PA, USA) and supernatant ATP was measured, using a luciferin-luciferase assay. DMSO (0.1%, 0.5% and 1%) or heparin (50, 200, 800 and 1600 U) was added to the cells at the start of the stretch experiments and the ATP released into the supernatant measured. Cell viability was also determined using Trypan Blue staining. RESULTS: IC cells released significantly more ATP in response to stretch than did control cells. This increased release of ATP by stretched IC cells was significantly blocked by adding DMSO or heparin at all concentrations used. Heparin appeared to have a greater dose-dependent effect on ATP release than did DMSO. CONCLUSIONS: These findings are consistent with the hypothesis that the urothelium provides sensory input via ATP release and that this process is increased in IC. Furthermore, stretch-activated ATP release was blocked by adding DMSO and heparin, both intravesical agents commonly used to treat the symptoms of IC. This study supports the notion that purinergic-targeted therapy is warranted in treating IC. Further studies are needed to determine the mechanisms of increased ATP release by IC urothelial cells.     

Eosinophilic cystitis after bladder instillation with dimethyl sulfoxide

Eosinophilic cystitis is a rare and poorly understood disorder. We report the first case of an acute flare of eosinophilic cystitis in a 51-year-old woman after bladder instillation with dimethyl sulfoxide (DMSO) for presumed interstitial cystitis. The patient presented with severe bladder pain, fever, and eosinophilia several hours after instillation. These symptoms were unresponsive to conventional analgesic and antibiotic treatments. Cystoscopy revealed erythema and exudate at the bladder walls, along with edema of both ureteral orifices. Bladder biopsies demonstrated massive eosinophilic infiltration of the bladder, confirming the diagnosis of eosinophilic cystitis. Urologists should bear in mind this clinical entity, particularly when DMSO is administered to patients with multiple drug allergies.     

Cellular immunity in interstitial cystitis.

It has been suggested that interstitial cystitis is an autoimmune disease. The evidence for this hypothesis, based on studies of humoral immune factors, has been contradictory. We assessed the immune response in interstitial cystitis by evaluating lymphocyte populations in the peripheral blood and bladder tissue of interstitial cystitis patients. The lymphocyte phenotypes in peripheral blood were entirely normal, including the CD4 (cluster designation nomenclature) and CD8 subsets, and the CD4:CD8 ratio. Bladder lamina propria showed a predominance of CD4 over CD8 lymphocytes in interstitial and other forms of cystitis. Bladder epithelium showed a similar pattern in bacterial or mechanical cystitis but specimens from patients with interstitial cystitis had a predominance of CD8 cells. The findings of normal lymphocyte populations in the peripheral blood are not supportive of an autoimmune mechanism in the disease. The findings in bladder tissue show that the urothelium is not involved in the inflammatory reaction, as is the lamina propria, and they would suggest, therefore, that the initiating factor does not originate from the bladder lumen. The CD8 predominance in the urothelium along with a CD4 predominance in the lamina propria may form a characteristic pattern for the diagnosis of interstitial cystitis and merits further study.     

Tamm-Horsfall protein as a marker in interstitial cystitis.

It has been suggested that immunohistochemical staining for Tamm-Horsfall protein in bladder epithelium may be a marker for interstitial cystitis. Of bladder biopsies from 14 interstitial cystitis patients only 3 demonstrated positive staining for Tamm-Horsfall protein within the mucosa, whereas 2 of 11 control biopsies showed positive Tamm-Horsfall protein results. In addition, staining in 4 ureteral specimens from interstitial cystitis patients and 4 controls was negative in all cases. An effort to detect this protein in enterocystoplasty specimens also showed a negative pattern. Our study does not confirm the Tamm-Horsfall protein as permeating either the bladder epithelium in interstitial cystitis or bowel mucosa in enterocystoplasty. This finding does not necessarily mean that these surfaces are not permeable to substances of smaller molecular size, nor does it define whether this is of importance in the etiology of interstitial cystitis. However, it does suggest that this technique does not allow detection of a marker for the disease.     

Functional and structural characteristics of the glycosaminoglycans of the bladder luminal surface

The glycosaminoglycan layer of bladder has been proposed to play a crucial role in protecting the bladder from harmful substances in urine. Rats were partially cystectomized to determine whether bladder glycosaminoglycans are routinely eluted from the bladder surface in detectable quantities. Cystectomy produced no detectable qualitative or quantitative changes in excreted GAG thereby showing that most urinary glycosaminoglycan originates in the kidney and not from the bladder. Damaging the glycosaminoglycan layer by a dilute acid wash, however, leads to a consistent decrease in the output of urinary GAG which recovers to normal at the same rate as the layer regenerates. This suggests that the newly exposed sites tightly bind urinary GAG. We suggest that such binding may be a component of the normal physiological defense mechanism of the bladder. The bladder glycosaminoglycan layer was isolated, dilute acid being used to elute ionically-bound material and brief trypsinization to elute intercalated proteoglycans from the luminal surface. The GAG from the luminal surface, which was present at a density of one chain per 50 nm.2 of bladder surface, was quite different in composition from that isolated from the whole bladder.     

Heparin prevention of tumor cell adherence and implantation on injured urothelial surfaces

Increasing laboratory and clinical evidence supports the concept of tumor cell implantation as a cause of intravesical recurrence in transitional cell carcinoma of the bladder. The integrity of the surface mucopolysaccharide layer of the bladder has been shown to be crucial in preventing bacterial or crystalline adherence. The purpose of this study was to evaluate the effects of bladder surface mucin integrity on tumor cell adherence and implantation. The adherence of tumor cells to in situ bladders which were either normal, injured, or injured and subsequently heparin-coated was studied using a radiolabeled tumor cell adherence assay. The mechanism of action of heparin in reducing tumor cell adherence and cytotoxic effects of heparin on tumor cells were evaluated. Finally, the ability of topical heparin to reduce tumor implantation rates on injured urothelial surfaces was studied. Urothelial injury by either dilute acid or fulguration resulted in markedly increased tumor cell adherence compared to normal. Topical heparin was capable of reducing cell adherence in injured bladders to control levels. Bladder coating with heparin rather than tumor cell coating was found to be the critical determinant in preventing tumor cell adherence. Heparin had no cytotoxic effects in any of the assays used. The implantation rate in heparin coated bladder was 19% compared to 50% in non-coated bladders. This study suggests that the bladder surface mucopolysaccharide layer plays a critical role in preventing tumor cell adherence and subsequent implantation. Restoration of this layer via exogenous topical heparin is capable of restoring the anti-adherence integrity to the bladder surface.     

Effects of l-arginine treatment on symptoms and bladder nitric oxide levels in patients with interstitial cystitis

Objectives. Nitric oxide (NO) is involved in host defense reactions, and NO production is elevated in various inflammatory disorders. We have found very high levels of luminal NO in the urinary bladder of patients with interstitial cystitis. Oral treatment with low doses of -arginine, the substrate for NO production, has been reported to alleviate symptoms in patients with interstitial cystitis. The aim of our investigation was to evaluate the effect of higher doses of -arginine in patients with interstitial cystitis and to study the effects of -arginine on NO production in the bladder.Methods. Nine women (age 69 ± 3 years) with interstitial cystitis were treated daily with 3 or 10 g of -arginine for 5 weeks. Symptoms were evaluated with an interstitial cystitis symptom score index, and NO production was measured. Patients with stress incontinence (n = 18) were used as control subjects for measurement of NO levels.Results. NO concentration in the urinary bladder was markedly elevated in the patients with interstitial cystitis (239 ± 60 ppb) compared with the control patients (15 ± 2 ppb). NO levels did not change in the patients with interstitial cystitis after oral treatment with -arginine (189 ± 72 ppb). There was no significant change in the symptom scores at either dose after 5 weeks of -arginine treatment.Conclusions. -arginine treatment in the doses used in this study did not change NO production in the urinary bladder in patients with interstitial cystitis. Furthermore, the patients in our study did not notice any relief of their symptoms.