Quantitative analysis of glycosaminoglycans in urothelium and bladder wall of calf

doi:10.1016/0090-4295(83)90321-7

Urology

Volume 22, Issue 6, December 1983, Pages 655-657

https://doi.org/10.1016/0090-4295(83)90321-7 Get rights and content

Abstract

Glycosaminoglycans (GAG) were described by histochemical staining procedures in the normal urothelium of the urinary bladder; they are supposed to be involved in the antibacterial defense mechanism. Our quantitative analysis, however, demonstrated only heparan sulfate in the normal calf urothelium (< 600 nmol/Gm d.wt.); only trace amounts of other GAG were to be analyzed. High concentrations of GAG were found in the submucosa and muscle layers as to be expected in mesoderm originating tissues. According to these results there were no GAG with the exception of heparan sulfate at the surface of the normal urothelium; therefore, glycoproteins detected in μmol/Gm d. wt. ranges are more likely to be involved in the antibacterial defense mechanism.

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Cited by (16)

Use of glycosaminoglycans in the treatment of interstitial cystitis: A strategy to improve efficacy
2001, International Congress Series
Interstitial cystitis (IC) is a chronic and incurable disease with symptoms including urinary urgency, frequency, nocturia and bladder pain. A recent survey estimated between 130,000 and 170,000 cases of the disease in the United States. The most popular etiological theory on interstitial cystitis is that the glycocalyx of the bladder epithelium is deficient. Accordingly, some of the current treatments are aimed at replacing the missing glycoconjugates by the intravesical administration of heparin, hyaluronic acid, or pentosan polysulfate (PPS) (Elmiron). It is presumed that these anionic polysaccharides act by replacing the missing coat of endogenous glycosaminoglycans (GAGs) on the epithelium. However, biotinylated pentosan polysulfate and heparin showed either no or weak binding to paraffin sections of rabbit and human bladder. The binding of radiolabeled heparin and pentosan sulfate to fresh rabbit bladders was also negligible. In contrast, mucins showed strong staining of the epithelium, indicative of lectin-mediated interactions. In other studies, the binding of biotinylated neoglycoconjugates demonstrated the presence of galactose- and N-acetylglucosamine-binding lectins in bladder epithelium. The presence of galectin-3 and galectin-4 in bladder mucosa was confirmed by molecular biological techniques. Based on these findings, heparin and pentosan polysulfate were modified with galectin-specific saccharide ligands in order to improve their binding to the endogenous lectins in the bladder.
Urinary chondroitin sulfates, heparan sulfate and total sulfated glycosaminoglycans in interstitial cystitis
1997, Journal of Urology
We compared urinary glycosaminoglycan levels in patients with interstitial cystitis and healthy controls.
Total sulfated glycosaminoglycans assayed by dimethylmethylene blue binding and individual glycosaminoglycans analyzed by cellulose acetate electrophoresis were compared in patients with interstitial cystitis and healthy controls. Also, multiple urine samples were obtained from healthy female controls for 2 months to assess the relationship of urinary glycosaminoglycan and creatinine concentrations, and to determine whether glycosaminoglycan excretion changes during the menstrual cycle.
Total sulfated glycosaminoglycan and creatinine concentrations correlated well in random voided samples. Menstrual cycle day did not affect total sulfated glycosaminoglycan levels. Cellulose acetate electrophoresis revealed 3 bands corresponding to chondroitin sulfates, heparan sulfate and acidic glycoprotein. Patients with interstitial cystitis had decreased urinary concentrations of each of these individual components and total sulfated glycosaminoglycans. However, glycosaminoglycan-to-creatinine ratios were similar in interstitial cystitis and control urine.
Using these assays total and individual urinary glycosaminoglycan levels normalized to creatinine were not altered in interstitial cystitis.
Characterization and immunohistochemical localization of the glycoconjugates of the rabbit bladder mucosa
1996, Archives of Biochemistry and Biophysics
An impairment of the mucosal glycoconjugates could be an important factor in the development of bladder disorders such as interstitial cystitis. However, very little definitive biochemical information is available on the glycoconjugate components of the mammalian bladder mucosa. In this study, the mucosa from metabolically radiolabeled rabbit bladder was separated, delipidated, and digested with protease, and the released glycosaminoglycans and glycopeptides were fractionated. About 80 and 36% of the nondialyzable tritium and³⁵S activities, respectively, was associated with the sialoglycopeptide fractions. The balance of the total tritium activity in the protease digest was in glycosaminoglycans identified as hyaluronan, chondroitin sulfates, and heparan sulfate. Immunohistochemical examination using anti-heparan sulfate antibodies, including one against mouse syndecan-1, indicated the presence of heparan sulfate proteoglycan in the epithelium. In contrast, there was no significant staining of the bladder epithelium with anti-chondroitin-4- and 6-sulfate antibodies or hyaluronan-binding protein. The lamina propria and muscle layers showed strong staining with anti-chondroitin-4-sulfate antibody and hyaluronan-binding protein and weak staining with anti-chondroitin-6-sulfate antibody. The insignificant levels of glycosaminoglycans in the glycocalyx of bladder mucosa epithelium suggest that glycosaminoglycans may be less important than other glycoconjugates in maintaining normal epithelial function and in bladder disorders such as interstitial cystitis.
Identification of a mucin layerin the urinary bladder
1994, Urology
The specific goal of this study was to establish a simple histochemicaltechnique by which the glycosaminoglycan (GAG) lining in the rabbit bladder can be routinely identified.
Rabbit bladder tissues were fixed in zinc formal, 10% neutral buffered formalin (NBF), 20% NBF, 40% NBF, 2% calcium acetate in 10% NBF, 2% sodium acetate in 10% NBF, 1 % cetylpyridinium chloride in 10% NBF, 80% alcohol, histochoice, and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. The following histochemical staining was used: mucicarmine/metanil yellow, colloidal iron, deamination followed by colloidal iron, periodic acid-Schiff, saponification followed by colloidal iron, Alcian blue (AB) at variable pH values, combined aldehyde-fuchsin/AB, performic acid/AB, AB/Alcian yellow, high temperature (60°C) methylation/saponification/AB, and AB/nuclear fast red at pH 5.8 with critical electrolyte concentrations with or without deamination.
AB with sodium acetate buffer at pH 5.8 containing 0.6 M or 0.8 M magnesium chloride (MgCl₂) showed a well-defined thin GAG lining on the surface of the urothelium, whereas histochemical staining used by previous investigators showed only patchy distribution. There was no observable difference due to the gender, fixative, or region of the bladder from which the tissue was obtained.
A very thin lining of GAG exists in the rabbit bladder which can be localized by AB in sodium acetate buffer at pH 5.8 containing 0.6 M or 0.8 M MgC1₂ but not by conventional histochemical techniques. This method now can be applied to answer many questions regarding urothelial function.
Adherence of urease-induced crystals to rat bladder epithelium following acute infection with different uropathogenic microorganisms
1988, Journal of Urology
Apart from urine supersaturation with respect to struvite and calcium phosphate, crystal retention is considered to be necessary for the formation of infection stones. This study was performed to investigate the role of the mucous coat in rat bladders in the adhesion of sterile urease-induced crystals and to determine to what extent the adhesion was influenced by infection. E1imination of the mucous coat with 0.1 M HCl increased the adherence of crystals six times compared to that in bladders with an intact mucous coat. Infection with Proteus mirabilis, Escherichia coli, enterococci and Ureaplasma urealyticum increased the adherence six, five, four and two times, respectively. Injury to the mucous coat may thus be one mechanism by which microorganisms can contribute to the formation of infection stones in the urinary tract.
Functional and structural characteristics of the glycosaminoglycans of the bladder luminal surface
1987, Journal of Urology
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. J Urol, 138: 433-437, 1987

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^☆: Supported by a grant of the Minister fur Wissenchaft and Forschung des Landes Nordrhein-Westfalen.

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Quantitative analysis of glycosaminoglycans in urothelium and bladder wall of calf☆

Abstract

Urology

J Urol

Glycosaminoglycans

Eine bemerkenwerte Anomalie der menschlichen Harnblase

Arch Pathol Anat

Some histochemical observations in transitional epithelium of man

J Histochem Cytochem

Histochemistry of mucus and other polysaccharides in tumor I. Carcinoma of the bladder

Lab Invest

Histochemische Untersuchungen über Sekretionserscheinungen in Papillomen and Karzinomen der Harnblase

Beitr Pathol Anat