Peptides of Matrix Gla Protein Inhibit Nucleation and Growth of Hydroxyapatite and Calcium Oxalate Monohydrate Crystals

Matrix Gla protein (MGP) is a phosphorylated and γ-carboxylated protein that has been shown to prevent the deposition of hydroxyapatite crystals in the walls of blood vessels. MGP is also expressed in kidney and may inhibit the formation of kidney stones, which mainly consist of another crystalline phase, calcium oxalate monohydrate. To determine the mechanism by which MGP prevents soft-tissue calcification, we have synthesized peptides corresponding to the phosphorylated and γ-carboxylated sequences of human MGP in both post-translationally modified and non-modified forms. The effects of these peptides on hydroxyapatite formation and calcium oxalate crystallization were quantified using dynamic light scattering and scanning electron microscopy, respectively. Peptides YGlapS (MGP1-14: YγEpSHEpSMEpSYELNP), YEpS (YEpSHEpSMEpSYELNP), YGlaS (YγESHESMESYELNP) and SK-Gla (MGP43-56: SKPVHγELNRγEACDD) inhibited formation of hydroxyapatite in order of potency YGlapS > YEpS > YGlaS > SK-Gla. The effects of YGlapS, YEpS and YGlaS on hydroxyapatite formation were on both crystal nucleation and growth; the effect of SK-Gla was on nucleation. YGlapS and YEpS significantly inhibited the growth of calcium oxalate monohydrate crystals, while simultaneously promoting the formation of calcium oxalate dihydrate. The effects of these phosphopeptides on calcium oxalate monohydrate formation were on growth of crystals rather than nucleation. We have shown that the use of dynamic light scattering allows inhibitors of hydroxyapatite nucleation and growth to be distinguished. We have also demonstrated for the first time that MGP peptides inhibit the formation of calcium oxalate monohydrate. Based on the latter finding, we propose that MGP function not only to prevent blood-vessel calcification but also to inhibit stone formation in kidney.     

The association of different urinary proteins with calcium oxalate hydromorphs. Evidence for non-specific interactions

It has been proposed that various urinary proteins interact specifically with different calcium oxalate hydromorphs and these interactions have important implications regarding the understanding of the onset and progress of kidney stone disease. Calcium oxalate monohydrate and dihydrate crystals were grown and characterised thoroughly to establish sample purity. These crystals were then incubated in artificial urine samples containing isolated urinary macromolecules. Crystal growth was prevented by saturating the incubation mix with calcium oxalate, and this was confirmed through electron microscopy and calcium measurements of the incubation mix. The surface interactions between the different calcium oxalate hydrates and urinary proteins were investigated by the use of Western blots and immunoassays. The same proteins, notably albumin, Tamm-Horsfall protein, osteopontin and prothrombin fragment 1, associated with both hydrates. There was a trend for more protein to associate with calcium oxalate dihydrate, and greater quantities of different proteins associated with both hydrates when Tamm-Horsfall protein was removed from the incubation mix. There is no evidence from this study to indicate that particular proteins interact with specific calcium oxalate hydrates, which in turn suggests that these protein-mineral interactions are likely to be mediated through non-specific charge interactions.     

Dissolution and growth of calcium oxalate monohydrate I. Effect of magnesium and pH

The rate of dissolution of calcium oxalate monohydrate and of a calcium oxalate renal stone was measured in 0.9% NaCl solution at different levels of magnesium concentration and pH. The growth of calcium oxalate obtained by chemical reaction between Ca²⁺ and oxalate ions at a concentration similar to that existing in normal urine was also investigated as a function of pH and magnesium concentration. It was found that both magnesium and pH exert a fine kinetic control on the precipitation and growth of calcium oxalate monohydrate. Magnesium had no effect on the dissolution. The possible role of magnesium and pH in calcium oxalate urolithiasis has been discussed in the light of previous reports and of the data presented in this study.     

First evidence for the presence of weddellite crystallites in Opuntia ficus indica parenchyma

Calcium oxalate crystallites occur very often in the plants tissues and their role is still poorly known. We report here the experimental protocol leading to the isolation of two forms of calcium oxalate crystallites differing in their hydration level in the parenchymal tissues of Opuntia ficus indica (Miller). Whereas the whewellite crystallites are habitual in all Opuntia species, the weddellite form has never been isolated from these species before, which is probably due to their small size (about 1 microm). We have identified these forms using X-ray diffraction and scanning electron microscopy.     

Quantitative determination of calcium oxalate and oxalate in developing seeds of soybean (Leguminosae)

Developing soybean seeds accumulate very large amounts of both soluble oxalate and insoluble crystalline calcium (Ca) oxalate. Use of two methods of detection for the determination of total, soluble, and insoluble oxalate revealed that at +16 d postfertilization, the seeds were 24% dry mass of oxalate, and three-fourths of this oxalate (18%) was bound Ca oxalate. During later seed development, the dry mass of oxalate decreased. Crystals were isolated from the seeds, and X-ray diffraction and polarizing microscopy identified them as Ca oxalate monohydrate. These crystals were a mixture of kinked and straight prismatics. Even though certain plant tissues are known to contain significant amounts of oxalate and Ca oxalate during certain periods of growth, the accumulation of oxalate during soybean seed development was surprising and raises interesting questions regarding its function.     

Nucleation of calcium oxalate crystals on an imprinted polymer surface from pure aqueous solution and urine

Calcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. In this study, we have used an imprinted 6-methacrylamidohexanoic acid/divinylbenzene co-polymer as a biomimetic surface to nucleate CaOx crystal formation. The polymer was imprinted with either calcium oxalate monohydrate (COM) or dihydrate (COD) template crystals. These were washed out of the polymer, which was then immersed in various test solutions. The test solutions were an aqueous solution of calcium chloride and sodium oxalate, artificial urine and a sample of real urine. Crystals that formed on the polymer surface were characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, atomic absorption spectroscopy and scanning electron microscopy. Results showed that in the aqueous solution the COM-imprinted polymer induced the nucleation of COM. The COD-imprinted polymer induced only trace amounts of COD crystallization, together with larger quantities of COM. In artificial and real urines, COM also specifically precipitated on the COM-imprinted surface. The results show that, at least to some extent, the imprinted polymers direct formation of their morphologically matched crystals. In the case of COD, however, it appears that either rapid hydrate transformation of COD to COM occurs, or the more stable COM polymorph is directly co-precipitated by the polymer. Our results support the hypothesis that heterogeneous nucleation plays a key role in CaOx stone formation and that the imprinted polymer model could provide an additional and superior diagnostic tool for stone researchers to assess stone-risk in urine.Abbreviations COD calcium oxalate dihydrate - COM calcium oxalate monohydrate - COT calcium oxalate trihydrate - dvb divinylbenzene - 6-maaha 6-methylacrylamidohexanoic acid     

Oxalate patinas on ancient monuments: the biological hypothesis

Summary Whewellite and weddellite, calcium oxalate monohydrate and dihydrate respectively, have been found in the form of thin surface layers on limestone and marble monuments and artifacts of various historical periods at different sites. Experimental results indicate that the formation of both minerals must be attributed essentially to the action of oxalic acid secreted by microorganisms (lichens) which live and proliferate on the stone. Oxalic acid attacks the calcium carbonate of the stone surface giving rise to the precipitation of calcium oxalate.     

Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 mumol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 mumol/mg, but stayed constant at about 0.7 mumol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.     

Crystals in woody stems

Scanning electron microscopy has been used to examine the location and form of crystals in the stems of woody perennials. The results are discussed in relation to the effects of physical conditions and "impurities" upon crystal form. Infrared spectroscopy and X-ray diffraction have been used to establish the chemical identity of the crystals in a number of species which were chosen so as to provide a wide variation in form. Those from Populus deltoides and Polyalthia sp. consisted of calcium carbonate; those from ten other species consisted of hydrated calcium oxalate. The infrared spectra of the crystals from Dracaena fragrans, Terminalia balerica, Goniothalamus sp. and Combretum verticillatum were strongly indicative of calcium oxalate monohydrate, but in the other cases the degree of hydration of the oxalate was uncertain.     

Calcium phosphate and calcium oxalate crystal handling is dependent upon CLC-5 expression in mouse collecting duct cells

Defects in an intracellular chloride channel CLC-5 cause Dent's disease, an inherited kidney stone disorder. Using a collecting duct model, mIMCD-3 cells, we show expression of dimeric mCLC-5. Transient transfection of antisense CLC-5 reduces CLC-5 protein expression. Binding of both calcium phosphate (hydroxyapatite) and calcium oxalate monohydrate (COM) crystals overlaid onto mIMCD-3 cultures was affected by altered CLC-5 expression. Calcium phosphate crystal agglomerations (>10 microm) were minimal in control (9%) and sense (13%) CLC-5-transfected cells, compared to 66% of antisense CLC-5-transfected cells (P<0.001). Small calcium phosphate crystals (<10 microm) were found associated with 45% of sense CLC-5-treated cells, of which the majority (11/14 cells) appeared to be internalised within the cell. Calcium oxalate agglomerations (>10 microm) were also largely absent for controls or sense mCLC-5 transfectants (11% and 9% of cells, respectively) with COM crystal agglomerates predominating in antisense CLC-5 transfectants (66%, P<0.0001). We conclude that collecting duct cells with reduced CLC-5 expression lead to a tendency to form calcium crystal agglomeration, which may help explain the nephrocalcinosis and nephrolithiasis seen in Dent's disease.     

Crystal morphology and carbon/carbon composition of solid oxalate in cacti

Morphology, crystal structure, and carbon isotopic composition of calcium oxalate from representative species from the family Cactaceae were determined using scanning electron microscopy, x-ray diffraction, and isotope ratio mass spectrometry. Crystals from one species in the Opuntieae tribe of the Cactaceae were druses with acute points composed of the monohydrate form of calcium oxalate (whewellite). Crystals from three species in the Cereeae tribe were the dihydrate form of calcium oxalate (weddellite) forming druses made up of tetragonal and isodiametric crystallites. The oxalate was relatively enriched in ¹³C isotope (-7.3 to - 8.7 ‰) compared with woody fibers (-13.3 to 14.1 ‰) from the same plants.     

Polysaccharide from coccoliths (CaCO3 biomineral). Influence on crystallization of calcium oxalate monohydrate

A polysaccharide associated with coccoliths of the marine alga Emiliania huxleyi (coccoliths are elaborately shaped calcite biominerals) was isolated and its influence on the crystallization of calcium oxalate monohydrate crystals was studied. Crystallization was monitored in a carefully controlled system by measuring the incorporation of 45Ca tracer from a supersaturated solution into seed crystals of calcium oxalate monohydrate in the absence and in the presence of polysaccharide. The method allowed differentiation between effects on solubility, growth and agglomeration of crystals. At the very low concentrations used in this study, the polysaccharide had no significant effect on the solubility product; it strongly inhibited the growth and strongly stimulated the agglomeration of the crystals. Thus, the two processes of growth and agglomeration, being both crystal-surface-related processes, may react in opposite directions upon surface adhesion of the additive. This finding opens new insights on how a mineralization process may be controlled. The inhibitory effect on growth is shown to proceed through a monolayer type of adsorption of the polysaccharide onto the crystal surface. The portion of the polysaccharide used for the stimulatory effect on agglomeration shows a different type of adsorption, whereby less crystal surface is covered per molecule of polysaccharide. This strongly suggests, that the mechanism whereby agglomeration is stimulated operates through 'viscous binding', with the polysaccharide bridging the gap between two crystal surfaces. In the discussion these findings are related to some possible biological functions of the polysaccharide.     

Biodeterioration of granite monuments by Ochrolechia parella (L.) mass: an FT Raman spectroscopic study

Ochrolechia parella is one of the most abundant lichens colonizing granite monuments in the region of Galicia (N.W. Spain). Its interaction with granite used in the construction of four ancient monuments was studied using FT Raman spectroscopy to evaluate the production of calcium oxalate by this lichen and the relationship of this production with different environmental conditions, particularly humidity. The results obtained showed that Ochrolechia parella is an aggressive colonizer, causing chemical disturbances to the granite through the formation of both calcium oxalate monohydrate and dihydrate. Apothecial development appears to be related to the production of calcium oxalate, and humidity determines the state of hydration of the calcium oxalate in the thallus.     

Characterization of ATP-driven calcium uptake in renal basal-lateral and renal endoplasmic reticulum membrane vesicles

ATP-driven calcium uptake was studied in basal-lateral membranes and in microsomal fractions, isolated from pig kidney cortex. The uptake is strongly enhanced in conditions where calcium inside the vesicles is precipitated by oxalate (5 mM) or phosphate (40 mM). Both anions were equally effective for the stimulation of calcium uptake in the microsomes but oxalate was less effective than phosphate in the basal-lateral membrane fraction. The active calcium pumps in the renal basal-lateral and microsomal fractions are different transport ATPases characterized by phosphorylated intermediates of 135 kDa and 115 kDa respectively. The subcellular distribution of the 135 kDa and 115 kDa phosphointermediates, reflects the distribution of typical marker enzymes for the basal-lateral membrane and for the endoplasmic reticulum. The calmodulin binding to the 135 kDa polypeptide as estimated by 125I-labelled calmodulin overlay, can be used as a specific marker for the basal-lateral plasma membrane calcium pump.     

Biologically induced mineralization in the tree Milicia excelsa (Moraceae): its causes and consequences to the environment

Iroko trees (Milicia excelsa) in Ivory Coast and Cameroon are unusual because of their highly biomineralized tissues, which can virtually transform the trunk into stone. Oxalic acid (C₂O₄H₂) and metal‐oxalate play important roles in their ecosystems. In this study, the various forms of oxalate and carbonate mineralization reactions are investigated by using scanning electron microscopy and X‐ray diffraction. Calcium oxalate monohydrate is associated with stem, bark and root tissues, whereas calcium oxalate dihydrate is found with wood rot fungi in soils, as well as in decaying wood. Laboratory cultures show that many soil bacteria are able to oxidize calcium oxalate rapidly, resulting in an increase in solution pH. In terms of M. excelsa, these transformations lead to the precipitation of calcium carbonate, not only within the wood tissue, but also within the litter and soil. We calculate that c. 500 kg of inorganic carbon is accumulated inside an 80‐year‐old tree, and c. 1000 kg is associated with its surrounding soil. Crucially, the fixation of atmospheric CO₂ during tree photosynthesis, and its ultimate transformation into calcite, potentially represents a long‐term carbon sink, because inorganic carbon has a longer residence time than organic carbon. Considering that calcium oxalate biosynthesis is widespread in the plant and fungal kingdoms, the biomineralization displayed by M. excelsa may be an extremely common phenomena.     

Characterization of crystalline structures in Opuntia ficus-indica

This research studies the crystalline compounds present in nopal (Opuntia ficus-indica) cladodes. The identification of the crystalline structures was performed using X-ray diffraction, scanning electron microscopy, mass spectrometry, and Fourier transform infrared spectroscopy. The crystalline structures identified were calcium carbonate (calcite) [CaCO₃], calcium-magnesium bicarbonate [CaMg(CO₃)₂], magnesium oxide [MgO], calcium oxalate monohydrate [Ca(C₂O₄)•(H₂O)], potassium peroxydiphosphate [K₄P₂O₈] and potassium chloride [KCl]. The SEM images indicate that calcite crystals grow to dipyramidal, octahedral-like, prismatic, and flower-like structures; meanwhile, calcium-magnesium bicarbonate structures show rhombohedral exfoliation and calcium oxalate monohydrate is present in a drusenoid morphology. These calcium carbonate compounds have a great importance for humans because their bioavailability. This is the first report about the identification and structural analysis of calcium carbonate and calcium-magnesium bicarbonate in nopal cladodes, as well as the presence of magnesium oxide, potassium peroxydiphosphate and potassium chloride in these plants. The significance of the study of the inorganic components of these cactus plants is related with the increasing interest in the potential use of Opuntia as a raw material of products for the food, pharmaceutical, and cosmetic industries.     

Scanning electron microscopy of calcium oxalate on mantle hyphae of hybrid larch roots from a farm forestry experimental site

An examination of roots of hybrid larch from a farm forestry site by scanning electron microscopy has revealed crystalline deposits encrusting mantle hyphae of the associated ectomycorrhizal fungus. Electron probe micro-analysis identified calcium in the crystals and X-ray diffraction showed them to be whewellite, the monohydrate form of calcium oxalate. The significance of the finding is discussed.     

SOME ASPECTS OF THE MANAGEMENT OF BILATERAL CALCULUS DISEASE OF THE KIDNEY

In the diagnosis of bilateral calculus disease of the kidneys, it is important to differentiate between cystine, uric acid, calcium oxalate and phosphate renal lithiasis. Methods for distinguishing one from another are described.Dietary therapy is the method of choice for cystine and uric acid lithiasis.In calcium and phosphate urolithiasis, dietary therapy is a very useful adjunct. It must be regulated by careful studies of its effect on urinary calcium precipitability, a new test for which is described based upon the demonstration of the existence of two forms of calcium in the urine.Irrigation therapy for calcium phosphate and phosphate lithiasis is briefly discussed.Surgical therapy for large renal phosphatic calculi is discussed to show how considerations of renal counterbalance and urinary calcium, magnesium and phosphate excretion through damaged kidney substances influence the surgical plan in each case.     

The Isolation and Properties of Oxalate Crystals from Plants

A method of isolation of crystalline inclusions of plant cellsis described. The crystals consist mainly of calcium oxalatein plants grown under normal conditions, but when calcium isreplaced by magnesium, barium, or strontium in the culture solutionthese elements substitute for calcium in the crystals; evenunder normal conditions magnesium occurs in the crystals tothe extent of about 2 per cent. The crystal morphology vanesin the species examined from raphides to complex conglomeratesand X-ray diffraction demonstrates an association of raphideswith calcium oxalate monohydrate whilst other solitary formsand conglomerates are associated with calcium oxalate 2.25H₂O.On this basis the species examined can be divided mto threegroups.