The effect of glycosaminoglycans on calcium oxalate crystal formation

The effect of glycosaminoglycans on calcium oxalate crystal formation in the supersaturated solution was studied by examining the size and shape of calcium oxalate crystals generated under an optical microscope. It was found that heparan sulfate and heparin were more effective growth inhibitors than chondroitin sulfate and hyaluronic acid at concentrations within their respective urinary range. With increasing calcium and/or glycosaminoglycans concentration in the solution, the degree of growth inhibition caused by glycosaminoglycans was enhanced. Calcium oxalate crystal shapes generated with various glycosaminoglycans varied with glycosaminoglycan species. One of the causes of those differences in the shape and degree of growth inhibition might be the structural differences between them, that is, the number of sulfate residue and O- or N-form they contain. Calcium oxalate crystal shapes in the presence of heparin or heparan sulfate at higher concentrations were similar to those of calcium oxalate monohydrate crystals in the urinary sediments of hyperoxaluric patients. These facts might suggest the possibility that heparin and/or heparan sulfate were present in the crystal forming region.     

Structural characteristics of osteopontin for calcium oxalate crystal]

BACKGROUND: I investigated which structural segment of osteopontin (OPN), a matrix component of urinary stones, is significantly related to the formation of urinary stones. METHODS: I prepared several kinds of OPNs under various conditions and compared the effects of these OPNs on calcium oxalate (CaOx) crystal using RI counts obtained by the seed crystal method and diluted urine method. Furthermore, I performed scanning electron microscopic (SEM) observation of CaOx crystals used in these experiments and evaluated the effects of OPN based on morphological changes in CaOx crystals. The following OPNs were used in this study: human recombinant OPN (rOPN), human native OPN (nOPN) purified from human milk, denatured OPN (dOPN) obtained by adding organic solvent during the course of nOPN purification, and asiaro OPN (aOPN) obtained by removing sialic acid after enzymatic digestion of nOPN. RESULTS: When the effects of OPNs (15 micrograms/ml) were evaluated by the seed crystal method, the following inhibitory activities were observed: nOPN (82%), aOPN (56%), dOPN (49%) and rOPN (15%). When the effects of OPNs (150 micrograms/ml) were evaluated by the undiluted urine method, the following inhibitory activities were observed: nOPN (38%), aOPN (27%), dOPN (21%) and rOPN (0%). Furthermore, using nOPN, I performed SEM observation of CaOx crystals and found that nOPN mainly inhibited CaOx crystal aggregation. CONCLUSION: Since the inhibitory activity of nOPN was observed not only in the seed crystal method, but also in the undiluted urine method, it was suggested that nOPN may play an important role in the living body during the course of urinary stone formation. Moreover, the inhibitory activity of OPN was not due to its primary structure, but it was closely related to its higher-order structure and side chains including sialic acid. Furthermore, it was clarified that the inhibitory activity of OPN mainly resulted from inhibition of CaOx crystal aggregation rather than growth inhibition in these crystals.     

Studies on the role of calcium phosphate in the process of calcium oxalate crystal formation

Crystals of calcium phosphate (CaP) added to solutions with a composition corresponding to that at different levels of the collecting duct (CD) and with different pH were rapidly dissolved at pH 5.0, 5.25 and 5.5. Only minor or no dissolution was observed at higher pH levels. Despite this effect, CaP crystals induced nucleation or heterogeneous crystallization of CaOx up to a pH of 6.1, whereas CaP was the type of crystalline material that precipitated at higher pH. Accordingly, small crystal volumes were recorded at pH 5.5 and great volumes at pH 6.7 4 h after the addition of CaP crystals to the solutions. Dialyzed urine appeared to counteract the dissolution of CaP and to reduce the rate of secondary crystallization. The CaP induced crystallization of CaOx was confirmed by a reduction of ¹⁴C-labeled oxalate in solution. The AP_CaOx required for a nucleation or heterogeneous crystallization of CaOx in the presence of CaP was around 1.5 × 10⁻⁸ (mol/l)². For CaP crystal formation on CaP, an AP_CaP (^aCa²⁺ × ^aPO₄ ³⁻) of approximately 50 × 10⁻¹⁴ (mol/l)² appeared to be necessary. The CaOx crystals formed were microscopically found in association with the CaP crystalline material and were most frequently of CaOx dihydrate type. Step-wise crystallization experiments comprising supersaturation with CaP (Step A), supersaturation with CaOx (Step B) and subsequently acidification (Step C) showed that CaOx crystal formation occurred when CaP crystals were dissolved and thereby served as a source of calcium. The ensuing formation of CaOx crystals is most likely the result from high local levels of supersaturation with CaOx caused by the increased concentration of calcium. These experimental studies give support to the hypothesis that crystallization of CaOx at lower nephron levels or in caliceal urine might be induced by dissolution of CaP formed at nephron levels above the CD, and that a low pH is prerequisite for the precipitation of CaOx. The observations accordingly provide additional evidence for the important role of calcium phosphate in the crystallization of calcium oxalate, that might occur both at the surface of Randall’s plaques and intratubularly at the papillary tip. Parts of these studies were presented at the Scanning Microscopy Meeting 1996, at the International Symposium on Urolithiasis, Dallas 1996 and at the Eurolithiasis meeting in Istanbul 1998.     

The role of glycoproteins in calcium oxalate crystal development

OBJECTIVE: To assess the effects of a glycoprotein (mucine) on calcium oxalate crystal development in different conditions and situations, to clarify some of its possible effects. MATERIALS AND METHODS: Crystallization was assessed using a batch system in presence of mucine suspensions, by kinetic-turbidimetric measurements, and using a flow system in the presence of retained agglomerates of mucine, evaluating the precipitated calcium oxalate. RESULTS: In batch conditions low mucine concentrations (<15 mg/L) inhibited calcium oxalate nucleation and higher concentrations (<250 mg/L) inhibited calcium phosphate nucleation, whereas at high concentrations there was also promotion. The presence of an aggregate of mucine in the flow system provoked calcium oxalate monohydrate crystallization at 0.691 microg/h per mg of mucine. In flow conditions pyrophosphate at 11.5 micromol/L caused a decrease of 84% in the calcium oxalate crystallized on mucine, 1.32 mmol/L of citrate a decrease of a 83%, 20 mg/L of pentosan polysulphate a decrease by 80%, and 7.58 micromol/L phytate totally prevented the crystallization of calcium oxalate on mucine. CONCLUSION: All substances inhibiting calcium oxalate crystallization with the capacity to interact with calcium ions also have crystallization promoting properties when they are at sufficiently high concentrations, because of their capacity to form agglomerates or the insolubility of their calcium salts.     

Influence of urinary sialic acid on calcium oxalate crystal formation

Using seed crystal method, whole-urine method, and scanning electron microscopy, the inhibitory effects of sialic acid and osteopontin (OPN) on aggregation/growth of CaOx crystals were investigated. Using the seed crystal method, sialic acid showed an inhibitory effect on CaOx crystal aggregation/growth in a concentration-dependent manner, but almost no effect was observed using the whole-urine method. OPN showed an inhibitory effect on aggregation/growth in both experimental systems. The inhibitory effect of asialo-OPN on aggregation/growth was approximately 20% lower than that of OPN in the experiment using the seed crystal method and approximately 15% lower in the experiment using the whole-urine method. Scanning electron microscopy showed that OPN and sialic acid inhibit the aggregation of CaOx crystals. The above findings show that sialic acid accounts for about 15-20% of the involvement of OPN in CaOx crystallization.     

Interaction between osteopontin on madin darby canine kidney cell membrane and calcium oxalate crystal.

We recently reported that the addition of the protein osteopontin (OPN) resulted in an increase in the deposition of calcium oxalate (CaOx) crystals on the surface of Madin Darby canine kidney (MDCK) cells. To determine the degree to which this increased deposition is caused by OPN, we investigated the extent to which the CaOx crystal deposition produced by the expression of OPN at the cell surface was suppressed by 4 different methods prior to the determination of the level of CaOx crystal binding. MDCK cells (2 x 10(6) cells/well) were cultured to a confluent state, and the binding of OPN to the cellular surface was then inhibited by adding one of the following 4 substances: human OPN polyclonal antibody, thrombin, cyclic Arg-Gly-Asp (RGD) peptides and tunicamycin. The cells were cultured for 24 h. We then used a fluorescent antibody technique with an OPN polyclonal antibody to determined whether the expression of OPN at the cell surface was inhibited, and we measured the degree of CaOx crystal deposition using the isotope (45)Ca. The degree of CaOx crystal deposition was inhibited by 80% or more in the antibody-treated group, by 50-80% in the thrombin-treated group, by 60-80% in the cyclic RGD-treated group, and by 50-60% in the tunicamycin-treated group. These results suggest that OPN in the extracellular matrix is the main cause of CaOx crystal deposition on the surface of MDCK cells.     

正常人和草酸钙结石病人尿草酸钙晶体基质

以改良Ｍｏｒｓｅ和Ｒｅｓｎｉｃｋ法提取１０例上尿路草酸钙结石病人和１１例正常人的尿草酸钙晶体基璺，用双向聚丙烯酰胺凝胶电泳对晶体基质及结晶前后大分子物的蛋白质组成进行了比较分析。     

Effect of angiotensin II receptor blockage on osteopontin expression and calcium oxalate crystal deposition in rat kidneys

Hyperoxaluria leads to calcium oxalate (CaOx) crystallization and development of tubulointerstitial lesions in the kidneys. Treatment of hyperoxaluric rats with angiotensin II (Ang II) type I receptor blocker (ARB) reduces lesion formation. Because Ang II mediates osteopontin (OPN) synthesis, which is involved in both macrophage recruitment and CaOx crystallization, it was hypothesized that ARB acts via OPN. Hyperoxaluria was induced in 10-wk-old male Sprague-Dawley rats, and they were treated with ARB candesartan. At the end of 4 wk, kidneys were examined for crystal deposits, ED-1-positive cells, and expression of OPN mRNA. PCR was used to quantify OPN, renin, and angiotensin-converting enzyme (ACE) mRNA in kidneys. RIA was used to determine renal, plasma, and urinary OPN; plasma renin; Ang II and ACE; and renal Ang II. For evaluating oxidative stress, malondialdehyde was measured. Urinary calcium, oxalate, creatinine, and albumin were also determined. Despite similar urinary calcium and oxalate levels, kidneys of hyperoxaluric rats on candesartan had fewer CaOx crystals, fewer ED-1-positive cells, reduced OPN expression, and reduced malondialdehyde than hyperoxaluric rats. Urinary albumin excretion and serum creatinine levels improved significantly on candesartan treatment. mRNA for OPN, renin, and ACE were significantly elevated in hyperoxaluric rats. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in candesartan-treated hyperoxaluric rats. These results show for the first time that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via renal renin-angiotensin system.     

Inhibitory effect of glutamic acid and aspartic acid on calcium oxalate crystal formation

The effects of Glu and Asp on calcium stone formation was evaluated in three experiments. Studies using mixed suspension, mixed product removal crystallization and scanning electron microscopy showed that Glu and Asp inhibited the nucleation rate, growth rate and suspension density (crystal mass produced) in proportion to the concentration. The main amino acids in calcium oxalate stones and calcium phosphate stones were Glu and Asp. However, the main amino acids in uric acid stones were glycine and urea, and there were no specific amino acids in struvite stones. The activity of urinary GOT and GPT, which convert Asp and alanine, respectively, to Glu in normal subjects was significantly greater than in calcium stone formation.     

Ultrastructural events in early calcium oxalate crystal formation in rats.

A model system is described for the induction of renal calcium oxalate crystals with intraperitineal injections of sodium oxalate in rats. Early crystals are formed predominantly in cortical areas. Massive amounts of calcium are associated with this process, as demonstrated by potassium pyroantimonate staining. Actual crystal formation appears to be an involved process associated with calcium, oxalate, and cellular membranes. Although overt stone formation was not observed, we feel that the intimate involvement of membranes during crystal formation may be similar to that found in renal stones.     

The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.

Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 micrograms/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x 10(-8) M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 micrograms/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.     

Raising urinary citrate lowers calcium oxalate and calcium phosphate crystal formation in whole urine

Crystal formation in whole urine was studied by the technique of rapid evaporation to 1,250 mosmol/l with and without raising citrate concentration by 40-50%. The added citrate reduced calcium oxalate crystal formation at pH 5.3 by about 25% and reduced calcium phosphate crystal formation at pH 6.8 by some 42%. These results support the view that citrate is important in maintaining calcium in solution in whole urine, and that raising the urinary citrate could be effective treatment for calcium oxalate/phosphate urolithiasis.     

The effects of citrate and urine on calcium oxalate crystal aggregation

Summary The rate of crystal sedimentation in a suspension of calcium oxalate monohydrate (COM) crystals was determined spectrophotometrically in the presence and absence of dialysed urine and citrate. A reduced rate of crystal sedimentation after stirring was recorded in suspensions containing citrate in concentrations between 0.33 and 1.67 mmol/l. The sedimentation rate was reduced in the presence of a 0.3–3.3% concentration of dialysed urine, with increased inhibition of crystal sedimentation when the concentration of urine was increased. A comparison of the inhibition of COM crystal sedimentation in whole urine and in dialysed urine from normal subjects and stone-formers disclosed significantly higher values (P<0.05) in the dialysed urine. The results support previous observations that physiological concentrations of citrate might efficiently inhibit the aggregation of COM crystals. Furthermore even low concentrations of both whole urine and dialysed urine are apparently very efficient inhibitors of COM crystal aggregation.     

The influence of urinary macromolecules on calcium oxalate monohydrate crystal growth

The Constant Composition (CC) kinetics method has been used for studying the mineralization of calcium oxalate monohydrate (COM) at sustained supersaturations in the presence of pre-bladder urine and macromolecules isolated from normal urine and kidney and bladder stones. The method is especially sensitive for investigating the inhibitory activities of these urinary macromolecular components (UMMC) and matrix macromolecular components (MMMC) with a coefficient of variation in growth rate of approximately 2%. Significant COM mineral inhibition was observed in a wide molecular weight region of urine components. Urine removed directly from the kidney showed appreciable inhibitory activity towards COM crystallization. Normal urinary proteins and the dissolved precipitate resulting from urine centrifugation were fractionated by gel filtration. The resulting solutions were mostly COM mineralization inhibitors. Electrodialysis was utilized to isolate the MMMC (greater than 7000 d) of renal and bladder calculi. While these solutions inhibited COM crystallization, they were also found to be calcium binders as measured by the calcium electrode.     

The role of magnesium in calcium oxalate urolithiasis

The aim of this study was to evaluate the effect of magnesium on calcium oxalate crystal formation, both in physiological conditions and at slightly higher oxalate concentrations, using a mixed suspension mixed product removal crystallizer and scanning electron microscopy. True supersaturation ratios were calculated by allowing for complexation in solution. Magnesium inhibited the nucleation rate at all oxalate concentrations. It also inhibited the growth rate at oxalate concentrations of less than approximately 2.0 mmol/l but promoted the growth rate at higher concentrations. This suggests that, provided the oxalate concentration is sufficiently high, increase of magnesium concentration can increase the crystal growth rate. At physiological concentrations of oxalate, however, magnesium decreases both nucleation and growth rates. The SEM photographs showed that the predominant crystal was calcium oxalate trihydrate at low magnesium concentrations, with calcium oxalate dihydrate being observed in larger quantities at high magnesium concentrations.     

Pyrophosphate does not influence calcium oxalate or calcium phosphate crystal formation in concentrated whole human urine

1) Low pyrophosphate urine was generated by passage through a nylon coil bearing immobilised inorganic pyrophosphatase. High pyrophosphate urine was made by addition of inorganic pyrophosphate. 2) Urine samples of low, normal, and high pyrophosphate content were rapidly evaporated at 37 degrees C to 1,050 or 1,250 mosmol/L and the crystals formed studied by microscope, isotope and chemical methods. 3) Urinary pyrophosphate level had no significant effect upon calcium oxalate crystals formed in whole urine at pH 5.3 or 6.1, or calcium phosphate crystals formed at pH 6.8.     

The effect of magnesium on calcium oxalate crystal shapes generated with glycosaminoglycan

Our previous report has shown that the shapes of calcium oxalate crystals formed in a supersaturated solution with glycosaminoglycan varied with glycosaminoglycan species. The present study was conducted to demonstrate the effect of magnesium on the shapes of calcium oxalate crystals with glycosaminoglycan by optical and scanning electron microscopy. In the presence of magnesium, the phenomenon of the plate- or sheet-like crystals parallel growing on the surface of other crystals was observed with each glycosaminoglycan and was enhanced with hyaluronic acid, chondroitin sulfate, heparan sulfate or heparin, more marked in this order. On the other hand, with heparan sulfate or heparin the stratification of the plate-like crystals was observed, whereas with hyaluronic acid or chondroitin sulfate it was not observed. Moreover, the stratification of the crystals with heparin was stimulated more markedly with increasing magnesium concentrations, and the lamella-shaped crystals at low heparin concentrations in the presence of magnesium was similar to the crystals at high heparin concentrations. These results suggest that magnesium stimulates the effect of glycosaminoglycan on calcium oxalate crystal shapes.     

Urinary crystal surface binding substances on calcium oxalate crystals

Summary In order to study the effect of urinary crystal surface binding substances (CSBS), we extracted the naturally existing CSBS from urine from healthy individuals by conducting homogeneous crystallization of calcium oxalate. CSBS proved not to be promoters but rather strong inhibitors of calcium oxalate crystal growth and aggregation. It is suggested that CSBS exhibited their inhibitory effect by masking the growing sites and aggregating sites on the crystal surface. As for the characteristics of CSBS, we found around 10 peaks of molecular weight, and all of them contained both peptides and saccharides. The findings suggest that CSBS are composed of various kinds of glycoproteins and proteoglycans.