heparitin-sulfate and Urinary-Calculi

Topics: Animals; Glycosaminoglycans; Heparitin Sulfate; Humans; Kidney; Parathyroid Hormone; Pentosan Sulfuric Polyester; Urinary Calculi

Proteus mirabilis cause urinary tract infections which are recurrent and can lead to formation of urinary calculi. Both bacterial and the host factors are involved in the development of urolithiasis. To determine the impact of glycosaminoglycans (GAGs) in the formation of P. mirabilis-induced urinary stones, we investigated the in vitro crystallization, aggregation and adhesion of crystals in the presence of GAGs naturally appearing in urine. Crystallization experiments were performed in synthetic urine infected with P. mirabilis in the presence of: hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate A, B and C (ChSA, ChSB, ChSC). The intensity of crystallization and aggregation were established by counting particles and phase-contrast microscopy. To analyze the adhesion of crystals, we used normal urothelium and (45)Ca isotope-labeled crystals. In the presence of ChSC, both the size of the crystals formed and their number were higher compared with the control. GAGs increased crystals adhesion to the cells, but only for ChSA this effect was significant. Chondroitin sulfates, which accelerate the first stages of infection-induced stones formation, may play an important role in the pathogenesis of infectious urolithiasis.

Topics: Adhesiveness; Apatites; Cell Line; Chondroitin Sulfates; Crystallization; Dermatan Sulfate; Glycosaminoglycans; Heparitin Sulfate; Host-Pathogen Interactions; Humans; Hyaluronic Acid; Magnesium Compounds; Microscopy, Phase-Contrast; Models, Biological; Phosphates; Proteus Infections; Proteus mirabilis; Struvite; Urinary Calculi; Urinary Tract Infections; Urothelium; Virulence

Urinary polyanions recovered from the urine samples of kidney stone-formers and normal controls were subjected to preparative agarose gel electrophoresis, which yielded fractions 1-5 in a decreasing order of mobility. In both groups, chondroitin sulfates were identified in the fast-moving fractions and heparan sulfates in the slow-moving fractions. Furthermore, two types of heparan sulfates were identified based on their electrophoretic mobility: slow-moving and fast-moving. The fractionated urinary polyanions were then tested in an in vitro calcium oxalate crystallization assay and compared at the same uronic acid concentration, whereby, the chondroitin sulfates of stone-formers and heparan sulfates of normals enhanced crystal nucleation. Fraction 5 of the normals, containing glycoproteins (14-97 kDa) and associated glycosaminoglycans, were found to effectively inhibit crystallization. Papainization of this fraction in stone-formers revealed crystal-suppressive effects of glycoproteins, which was not seen in similar fractions of normals. It was concluded that glycoproteins could modulate the crystal-enhancing glycosaminoglycans such as chondroitin sulfates of stone-formers but not in normals. The differing crystallization activities of electrophoretic fraction 1 of normals and stone-formers revealed the presence of another class of glycosaminoglycan-hyaluronan. Hence, in the natural milieu, different macromolecules combine to have an overall outcome in the crystallization of calcium oxalate.

Topics: Adult; Calcium Oxalate; Chondroitin Sulfates; Crystallization; Electrophoresis, Agar Gel; Electrophoresis, Cellulose Acetate; Glycoproteins; Heparitin Sulfate; Humans; Kidney Calculi; Middle Aged; Urinary Calculi; Urolithiasis

The aim of this study was to examine the effect of sodium pentosan polysulfate (SPP) in an undiluted urine system and to study its relative affinity to calcium oxalate (CaOx) crystals in the presence or absence of heparan sulfate (HS). CaOx crystals were induced with an overload of oxalate above the metastable limit in spun and filtered urine (SF) and ultrafiltered urine (UF). Then, the crystals were dissolved with EDTA (ethylenediaminetetraacetic acid), electrodialysed and lyophilized. The polyanions, HS or SPP were added to the UF prior to the addition of oxalate. Polyanions in crystal matrices were examined by cellulose acetate electrophoresis. Crystal volume and size were suppressed according to the increase of the concentration of SPP when compared with those of the UF. Scanning electron microscopy (SEM) showed marked aggregation of the crystals in the UF and no aggregation in the presence of SPP. HS was the only polyanion found in CaOx crystals formed after overload of oxalate in SF. Crystals formed in UF did not contain any polyanions. When SPP was added to UF, SPP appeared in the crystal matrix in accordance with its concentration. Once HS in physiological concentration was added to the UF containing SPP, HS and SPP obtained from crystals were strongly stained with Alcian blue in electrophoretic study, where SPP is stained stronger than HS. These results suggest that SPP strongly binds to CaOx crystals as well as HS and that HS and SPP competitively bind to the crystal, then, as a result, they are incorporated into the crystals. The fact that SPP suppressed the aggregation of CaOx crystals in undiluted urine showed the possibility that SPP might be one of the useful drugs for preventing CaOx urolithiasis.

Topics: Adult; Calcium Oxalate; Crystallization; Heparitin Sulfate; Humans; Male; Microscopy, Electron, Scanning; Pentosan Sulfuric Polyester; Urinary Calculi

1. Urinary glycosaminoglycans were recovered from the papain digest of polyanions precipitated sequentially by cetylpyridinium chloride and sodium acetate-saturated ethanol. Those from the early morning urine of 48 stone formers and 43 normal control subjects measured 11 and 16 micrograms of uronic acid/ml of urine, respectively. 2. Preparative agarose gel electrophoresis of the recovered glycosaminoglycans in barium acetate buffer (pH 5.8) yielded fractions containing purely chondroitin sulphate, co-polymeric chondroitin/dermatan sulphates and heparan sulphate. Identification was based on the susceptibility of the fractions to chondroitinase or nitrous acid treatment. Similar compositions of glycosaminoglycan classes were observed in samples from stone formers and normal control subjects. 3. The fractionated glycosaminoglycans were dissolved in urine ultrafiltrate to assay for nucleation-promoting and growth-inhibiting activities towards crystallization of urinary calcium oxalate. When compared at the same uronic acid concentration, both the urinary chondroitin sulphate isomers and heparan sulphates of stone formers demonstrated the capacity to enhance crystal nucleation from calcium oxalate endogenous in urine ultrafiltrates, whereas only urinary heparan sulphates of normal control subjects demonstrated this capacity. 4. Tissue-derived reference chondroitin sulphate, dermatan sulphate and heparin, when similarly tested, showed negligible crystal nucleation-promoting activity. The tissue-derived heparan sulphate was similar to the urinary heparan sulphates in showing marked crystal nucleation-promoting activity. 5. Crystal-growth inhibitory activity was evident in all urinary glycosaminoglycan fractions studied. In particular, urinary heparan sulphate of normal control subjects showed higher activity than that of stone formers or the chondroitin sulphate isomers of both stone formers and normal control subjects (P < 0.005).

Topics: Adult; Calcium Oxalate; Chondroitin Sulfates; Crystallization; Electrophoresis, Agar Gel; Electrophoresis, Cellulose Acetate; Heparitin Sulfate; Humans; Middle Aged; Urinary Calculi

The nature of the soluble stone matrix and its possible role in urinary stone formation was studied. For this purpose we performed two-dimensional cellulose acetate membrane electrophoresis of the glycosaminoglycans (GAGs) which were contained in the soluble stone matrix, substances adsorbed onto calcium oxalate crystals in vitro (crystal surface binding substances, CSBS) and urinary macromolecules (UMMs). The main GAG in the soluble stone matrix and CSBS was found to be heparan sulfate, whereas the UMMs contained various GAGs usually seen in urine. An inhibition assay showed the soluble stone matrix to have the strongest inhibitory activity among these macromolecular substances when inhibitory activity was expressed in terms of uronic acid concentration. It is suggested that the main GAG in the soluble stone matrix consists of heparan sulfate, which has a strong inhibitory activity on calcium oxalate crystal growth and aggregation and constitutes part of the CSBS.

Topics: Adult; Calcium Oxalate; Crystallization; Electrophoresis, Cellulose Acetate; Heparitin Sulfate; Humans; Macromolecular Substances; Male; Solubility; Urinary Calculi; Urine

The role of urinary glycosaminoglycans (GAGs) in lithogenesis is a topic of current interest in urologic research. One GAG, chondroitin sulfate, has previously been shown to inhibit calcium oxalate crystal formation. It has long been known that the chemical components of GAGs are present in the matrix of urinary concretions, but it has not been determined whether these components exist in free form or as constituents of GAG. This study was undertaken to determine whether GAGs are present in urinary stone matrices and, if so, to characterize them. Matrices of nine single urinary stones of various compositions and of three stone pools (calcium oxalate, magnesium ammonium phosphate) were isolated by exhaustive dialysis. The techniques of cellulose acetate electrophoresis, Alcian blue staining and enzymatic degradation were used to identify various GAGs. Material that stained Alcain blue was present in eleven of twelve samples. GAG was detected as this material in ten samples. The GAGs identified are heparan sulfate, hyaluronic acid and possibly keratan sulfate. The most prominent urinary GAG, chondroitin sulfate, was notably absent from urinary stone matrix. GAG seems to be incorporated into matrix on a selective basis. This finding may be due to differences in the affinities of different GAG species for the crystals which comprise the calculi. It has been proposed that the inhibitory activity of GAGs lies in their ability to bind to (and therefore block) the growth sites of crystals. It is apparent from this study that certain GAG species are incorporated into the structure of the stone and they may be intimately related to stone development and growth.

Topics: Calcium Oxalate; Calcium Phosphates; Electrophoresis, Cellulose Acetate; Glycosaminoglycans; Heparitin Sulfate; Hexosamines; Humans; Magnesium; Magnesium Compounds; N-Acetylneuraminic Acid; Phosphates; Sialic Acids; Struvite; Uric Acid; Urinary Calculi; Uronic Acids

At first, urinary stones were classified according to their inorganic components (apatite, struvite, calcium oxalate monohydrate, calcium oxalate dihydrate and uric acid). Then, matrix glycosaminoglycan was extracted from the stones in each group and was analyzed by 2-dimensional electrophoresis. There were differences in the glycosaminoglycan content of matrices among different groups of urinary stones. The principal matrix glycosaminoglycan content consisted of hyaluronic acid in apatite and struvite stones, heparan sulfate in calcium oxalate monohydrate and uric acid stones, and hyaluronic acid and heparan sulfate in calcium oxalate dihydrate stones. We conclude that hyaluronic acid and/or heparan sulfate has an important role in urinary stone formation.

Topics: Electrophoresis; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Urinary Calculi

This study presents a method for the isolation and identification of urinary glycosaminoglycans. Aliquots of 24-hour urine specimens are ultrafiltered through a Biogel filtration column to obtain the macromolecular fractions. These fractions are then subjected to electrophoretic, enzymatic and chemical analyses to obtain qualitative and quantitative information about the glycosaminoglycans contained. The methodology presented is intended to be used in subsequent studies where data can be analyzed and determinations can be made concerning the character of glycosaminoglycans in the urine of stone formers and non-stone formers.

Topics: Chondroitin Sulfates; Densitometry; Electrophoresis; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Macromolecular Substances; Methods; Urinary Calculi; Uronic Acids

Topics: Adult; Chondroitin Sulfates; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Urinary Calculi

Mucopolysaccharides were extracted from both normal and stone-forming urines, and those from the stone-forming samples showed a higher degree of sulfation than those from normal urines, as determined by sulfate analysis and electrophoretic measurement. The sulfated mucopolysaccharides from stone-forming urines formed insoluble calcium salts, whereas those from normal urines generally remained soluble in the presence of calcium ion. Rachitic rat cartilage was found to have more highly sulfated mucopolysaccharides than normal rat cartilage. Highly sulfated mucopolysaccharides appear to be a significant factor in calcium stone formations.

Topics: Animals; Cattle; Chondroitin Sulfates; Electrophoresis; Glycosaminoglycans; Heparitin Sulfate; Rats; Sulfates; Urinary Calculi