Restoring barrier function to acid damaged bladder by intravesical chondroitin sulfate

Abstract

Purpose: Chondroitin sulfate (Stellar Pharmaceuticals, London, Ontario, Canada), which is less expensive and more inert than heparinoids, hyaluronan or pentosan polysulfate, has been introduced to restore the barrier function lost due to epithelial dysfunction in interstitial cystitis cases. To our knowledge chondroitin sulfate binding to damaged bladder as a function of the urinary pH range, its efficacy in restoring the bladder permeability barrier and the capacity of the damaged bladder to bind chondroitin sulfate have not been determined previously.

Materials and methods: Chondroitin sulfate binding to bladder urothelium was investigated quantitatively using chondroitin sulfate highly labeled with Texas Red(R) and quantitative fluorescence microscopy in a mouse model of urothelial acid damage. The efficacy of restoring barrier function was determined using the passage of intravesically instilled (86)Rb, a potassium ion mimetic, through the urothelium into the bloodstream in a rat model of bladder damage. The binding capacity of acid damaged bladder was determined by fluorometry.

Results: Chondroitin sulfate bound tightly and exclusively to the mouse bladder surface damaged by acid but showed only minimal binding to undamaged bladder. There was no systematic variation in pH. The model showed some variability in the degree of damage induced. In rats chondroitin sulfate instillation restored permeability to (86)Rb to control levels. Binding was saturable at a mean +/- SEM 0.67 +/- 0.13 mg/cm(2) of the bladder surface.

Conclusions: Chondroitin sulfate binds preferentially to damaged urothelium and restores the impermeability barrier. This suggests that the glycosaminoglycan layer is a major contributor to bladder urothelial impermeability. As determined by binding capacity, the dose applied in humans in Canada (400 mg per instillation) is sufficient to achieve maximum efficacy.

Figure 1

Alcian Blue stained sections of (A) control (20 × magnification) and (B) rat bladder damaged with 10 mM HCl for 10 min. (40 × magnification). Orange arrows are used to denote areas of minimal damage. Yellow arrows point to areas where umbrella cells are denuded.

Figure 1

Alcian Blue stained sections of (A) control (20 × magnification) and (B) rat bladder damaged with 10 mM HCl for 10 min. (40 × magnification). Orange arrows are used to denote areas of minimal damage. Yellow arrows point to areas where umbrella cells are denuded.

Figure 2

(A) Mouse Bladder, Control, No acid damage, treated with TR-Chondroitin sulfate at pH 6.5 (B) Mouse Bladder, Acid damaged, treated with TR-Chondroitin sulfate at pH 6.5

Figure 2

(A) Mouse Bladder, Control, No acid damage, treated with TR-Chondroitin sulfate at pH 6.5 (B) Mouse Bladder, Acid damaged, treated with TR-Chondroitin sulfate at pH 6.5

Figure 3

Dot plots of normalized integrated red intensities (IRI) of mouse bladder labeled with Texas Red-labeled chondroitin sulfate as a function of pH and treatment. Each symbol represents one measurement of an area of interest. Horizontal bars are mean values.

Figure 4

Dot plot of the concentration of ⁸⁶Rb measured in rat serum as a function of treatment. Horizontal bars represent mean values. A one-way ANOVA showed the differences between the means within the data set were significant at p=0.0056. Bonferroni's Multiple Comparison Test showed the differences between means of negative control and acid-damaged bladders (126.4, t = 4.3) and ChS-treated and acid damaged bladders (117.5, t = 4.0) were both statistically significant (p<0.05, two tailed); the difference between chondroitin sulfate-treated and acid-damaged bladders (8.9, t = 0.28) was not statistically significant. In pair-wise comparisons the two-tailed p-values of t-tests using Welch's correction for unequal variances were p = 0.0203, 0.0196 and 0.5991, respectively.

Figure 5

Binding capacity of rat bladder for Texas Red-labeled chondroitin sulfate following surface damage with 10 mM HCl. The arrows represent the equivalent doses in human bladder (left to right) of 80, 200, 400 and 800 mg, respectively, and were calculated assuming a 350 ml bladder for IC patients. The 400 mg dose currently is in use in Canada.