Inhibition Of Calcium Oxalate Crystal Growth Research Articles

Resveratrol inhibits calcium oxalate crystal growth, reduces adhesion to renal cells and induces crystal internalization into the cells, but promotes crystal aggregation

Resveratrol is a natural phenolic compound that belongs to stilbenoid group found in diverse plants. Health benefits and therapeutic potentials of resveratrol have been widely recognized in various diseases. In kidney stone disease, it can alleviate oxalate-induced hyperproduction of free radicals in renal epithelial cells. Nevertheless, its direct effects on calcium oxalate (CaOx) crystal, which is the major stone component, remained unclear. This study therefore addressed the direct effects of resveratrol (at 1, 10 or 100 μM) on each step of CaOx kidney stone formation. The results revealed that resveratrol had no significant effects on CaOx crystallization. However, resveratrol significantly decreased CaOx crystal growth and adhesion to renal epithelial cells at all concentrations, and induced crystal internalization into the cells (a process related to crystal degradation by endolysosomes) in a concentration-dependent manner. On the other hand, resveratrol promoted crystal aggregation. These data indicate that resveratrol serves as a dual modulator on CaOx stone formation. While it inhibits CaOx stone development by reducing crystal growth and adhesion to renal cells and by inducing crystal internalization into the cells, resveratrol promotes crystal aggregation, which is one of the mechanisms leading to kidney stone formation.

Nucleation and growth inhibition of biological minerals by cementum attachment protein-derived peptide (CAP-pi).

Biomineralization is a highly regulated process where proteins/peptides-crystal interactions contribute to the shaping, phasing and aggregation of minerals. We have identified and synthesized a cementum attachment protein-derived peptide (CAP-pi), which corresponds to amino acids 40-53 of the N-terminal CAP domain (MASSDEDGTNGGAS) and its phosphorylated variant (MASpSpDEDGTNGGASp) (CAP-pip). The peptide is composed of polar and negatively charged amino acids, which are disordered, according to in silico analysis. Our results show that CAP-pi inhibits hydroxyapatite (HA) formation and growth. However, it possesses low capacity to inhibit calcium oxalate crystal growth. CAP-pip showed a stronger inhibitory effect on the formation and growth of HA. As well as a high capacity to inhibit calcium oxalate monohydrate growth, mainly due to adsorption on specific growth faces. Small peptides have many advantages over the full-size protein, including low-cost production and modulation characteristics that allow for structural changes. Our findings suggest that CAP-pip-derived peptide could possess therapeutic potential to prevent or treat pathological calcifications such as renal stones and vascular calcification.

Aqueous extract of Costus arabicus inhibits calcium oxalate crystal growth and adhesion to renal epithelial cells.

Costus arabicus L. (C. arabicus) is a plant used in Brazilian folk medicine to treat urolithiasis; however, its mechanism of action is unclear. The interaction between calcium oxalate (CaOx) crystals and the renal epithelium is important in calculogenesis, and compounds that modulate this process represent candidate therapeutic agents for stone prevention. Therefore, we assessed the inhibitory activity of C. arabicus on CaOx crystallization and the interaction of CaOx crystals with the renal epithelium. A seeded CaOx monohydrate (COM) crystallization system was used to study the effect of C. arabicus on crystal growth. Madin Darby canine kidney (MDCK) cells were used to study [(14)C] COM crystal adhesion in the presence and absence of an aqueous extract of C. arabicus. Cytotoxicity was assessed using a tetrazolium (MTS) cell proliferation assay. Aqueous extracts of C. arabicus decreased crystal growth in a concentration-dependent fashion. Precoating crystals with C. arabicus extract prevented their adhesion to MDCK cells, while pretreating cells did not show any effect. The extract was non-cytotoxic in concentrations of at least 1 mg/ml, which is likely above concentrations achievable in the urine following oral ingestion and excretion. No inhibitory activity was found in hexane, methyl chloride, n-butanol and ethyl acetate fractions of an ethanol extract of the herb. An aqueous extract of C. arabicus may disrupt calculogenesis by interacting with CaOx crystal surfaces. Activity was present in the aqueous extract; therefore, this agent may be bioavailable when administered orally. Fractionation results suggest that the active agent might be a polar polysaccharide. Further identification and characterization along these lines may be warranted.

Phosphate inhibits calcium oxalate crystal growth and crystallization through reducing free calcium ions: a morphological analysis and calcium consumption assay

Phosphate inhibits calcium oxalate crystal growth and crystallization through reducing free calcium ions: a morphological analysis and calcium consumption assay

Mode of interaction of calcium oxalate crystal with human phosphate cytidylyltransferase 1: a novel inhibitor purified from human renal stone matrix

Nephrolithiasis is a common clinical disorder, and calcium oxalate (CaOx) is the principal crystalline component in approximately 75% of all renal stones. It is widely believed that proteins act as inhibitors of crystal growth and aggregation. Acidic amino acids present in these proteins play a significant role in the inhibition process. In this study, interaction of cal-cium oxalate with human phosphate cytidylyltrans-ferase 1(CCT), a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix has been elucidated in silico and involvement of acidic amino acids in the same. As only sequence of CCT is available, henceforth its 3-D structure was modeled via Homology modeling using Prime module of Schrodinger package. Molecular dynamic simulation of modeled protein with solvation was done by mac-romodel (Schrodinger). The quality of modeled pro-tein was validated by JCSG protein structure valida-tion (PROCHECK & ERRAT) server. To analyze the interaction of modeled protein CCT with calcium oxalate along with role played by acidic amino acids, ‘Docking simulation’ was done using MOE–Dock. Interaction between calcium oxalate and CCT was also studied by substituting acidic amino acid in the active sites of the protein with neutral and positively charged amino acids. The in silico analysis showed the bond formation between the acidic amino acids and calcium atom, which was further substantiated when substitution of these acidic amino acids with alanine, glycine, lysine, arginine and histidine com-pletely diminished the interaction with calcium ox-alate.

Mass spectrometric identification of human phosphate cytidylyltransferase 1 as a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix

Background A relatively small number of well-characterized inhibitors of kidney stone formation have been identified from the previous research involved in its formation. In this study conventional biochemical methods have been combined with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in human renal stone matrix. Methods Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW > 10 kDa were subjected to anion exchange and molecular-sieve chromatography. Protein fractions were tested for their effects on CaOx crystal growth. Most potent fraction P2′ was excised, in-gel tryptic digested and identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Results An anionic protein (MW ~ 42 kDa) with potent inhibitory activity against CaOx crystal growth was purified. Its homogeneity was confirmed by RP-HPLC. It was identified by MALDI-TOF-MS followed by database search on MASCOT server as human phosphate cytidylyltransferase 1, beta. Molecular weight of this novel CaOx crystal growth inhibitor from human renal stone matrix is also the same as that of human phosphate cytidylyltransferase 1, choline, beta. Conclusions Human phosphate cytidylyltransferase 1, choline, beta is a novel CaOx crystal growth inhibitor. It is involved in the biosynthesis of phosphatidylcholine which happens to be an important constituent of human renal stones and is also reported to have an antilithiatic effect.

Models for protein binding to calcium oxalate surfaces

It is widely believed that proteins rich in Asp, Glu or Gla (gamma carboxyglutamic acid) interact strongly with calcium oxalate surfaces and inhibit calcium oxalate crystal growth. An alternative hypothesis would be that the interaction of Asp, Glu and Gla residues with surfaces could facilitate nucleation and crystal aggregation. Prothrombin fragment 1 and bikunin have been studied extensively as inhibitors, beta-microglobulin, transferrin and antitrypsin have been found in stone matrix and tubulin has been observed in the attachment of crystals to cell surfaces. The aim of this study is to examine how well carboxylate groups in proteins found either in stone matrix, or proposed as inhibitors, could fit with the calcium ion sub-lattice of both calcium oxalate monohydrate and dihydrate surfaces. The carboxylate groups in the acidic Asp, Glu and Gla residues were marked in the Protein Data Bank structures and matched to calcium oxalate surfaces using the Cerius 3D molecular modeling program. A contact was defined if a carboxylate oxygen atom approached a surface calcium atom in such a way that the separation was less than 6 Angstrom but greater than 2.4 Angstrom, the sum of the ionic radii. If the proteins maintain their 3D structure, the number of contacts was no more than 3 or 4 for all the proteins studied, irrespective of the calcium oxalate surface.

Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor

Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization-time-of-flight MS and electrospray ionization-quadrupole-time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti-C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis.

EFFECTS OF URINARY PROTHROMBIN FRAGMENT 1 IN THE FORMATION OF CALCIUM OXALATE CALCULUS

We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis. Fresh urine and renal parenchyma from patients with calcium oxalate calculus and normal controls were collected. Urinary prothrombin fragment 1 was isolated and purified from urine. It was identified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and analysis of its first 13 N-amino acids. The inhibitory activity of urinary prothrombin fragment 1 on calcium oxalate crystal growth was tested by the seeded crystallization technique. Meanwhile, the gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was analyzed by a previously described method and genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 from renal parenchyma was detected by polymerase chain reaction-single strand conformational polymorphism sequencing. The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was significantly decreased from normal (24.4 to 1.7 mol/1,000 amino acids) in patients with calcium oxalate calculus. The mean growth index +/- SD of urinary prothrombin fragment 1 to calcium oxalate crystals was 42.3 +/- 4.2 compared with the normal index of 19.2 +/- 2.8 (p <0.01). The polymerase chain reaction-single strand conformational polymorphism sequencing technique revealed no genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 in patients with calcium oxalate calculus. The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 as well as its ability to inhibit calcium oxalate crystal growth was significantly decreased in patients with calcium oxalate calculus. This was not caused by genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1. It is important to elucidate the mechanisms of calcium oxalate stones in view of urinary prothrombin fragment 1.

Identification of bikunin isolated from human urine inhibits calcium oxalate crystal growth and its localization in the kidneys.

We previously reported a high molecular weight substance purified from human urine that strongly inhibited calcium oxalate (CaOx) crystal growth in vitro. In the study present herein, we identified and investigated a protein purified from human urine that strongly inhibits CaOx crystal growth using a column chromatography series. The protein was identified by amino acid sequencing and was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and immunohistochemical staining. The molecular weight of this protein was approximately 35 KDa, and it also had another band around 20 KDa. We determined that the amino acid sequence of the protein was homologous with that of bikunin, the light chain of the inter-alpha-trypsin inhibitor, which is known as a strong CaOx crystallization inhibitor in vitro. On western blotting analysis, the molecular weight was also found to be around 35K Da, the same as that of bikunin. Immunohistochemical staining revealed that it was mainly located in the epithelial cells of the proximal tubules and the thin descending segment near the loop of Henle, but not in the glomeruli, distal tubules or the collecting ducts. In the present study, the protein extracted from human urine was identical to bikunin, which may be expressed mainly in the proximal tubules and the thin descending segment near the loop of Henle, and which prevents CaOx crystallization in vitro.

Influence of Gender and Age on Calcium Oxalate Crystal Growth Inhibition by Urine from Relatives of Stone Forming Patients

We define the relationships between urine inhibition of calcium oxalate crystal growth and age, gender, urine chemistries and stone formation among relatives of calcium stone forming patients. We collected 24-hour urine samples from 366 first degree relatives of calcium stone formers. Calcium oxalate crystal growth inhibition was studied using a constant amount of dialyzed urine protein in a seeded crystallization system. Standard stone risk measurements were also performed on the urine, including supersaturation for calcium oxalate, calcium phosphate and uric acid. By multivariate analysis crystal growth inhibition is strongly inversely related to the amount of protein excreted per day, and the age of the subject. When corrected for protein excretion and age, urine proteins from nonstone forming male subjects inhibited crystal growth more strongly than those from corresponding female subjects. Among stone formers the sex difference was not present. Inhibition of calcium oxalate crystal growth is influenced by a complex combination of gender, age, stone formation and assay conditions. The effect of daily protein excretion is most likely a consequence of using a fixed amount of urine protein per assay. The influence of age is significant and unexplained, with the urine of young people (less than 20 years) demonstrating a vigorous ability to inhibit crystallization. In addition, the urine of nonstone forming male relatives appears to have a greater ability to inhibit crystallization than that of nonstone forming female relatives. Further use of this assay in clinical investigations must take age and gender into proper account.

Influence of Gender and Age on Calcium Oxalate Crystal Growth Inhibition by Urine from Relatives of Stone Forming Patients

Influence of Gender and Age on Calcium Oxalate Crystal Growth Inhibition by Urine from Relatives of Stone Forming Patients

A novel basic protein from human kidney which inhibits calcium oxalate crystal growth.

To isolate calcium oxalate-binding proteins from human kidney and characterize the functional properties. Calcium oxalate crystals were prepared and allowed to interact at two different pH values with Triton-extracted human kidney homogenate. The proteins in the homogenate were isolated and fractionated on a cellulose column, and purified by high-performance liquid chromatography. The protein with the greatest oxalate binding activity at pH 4.5 was analysed for its amino-acid composition and characterized by Scatchard plot analysis, crystal growth, nucleation and aggregation studies. Three major protein fractions were eluted when calcium oxalate monohydrate was adsorbed at both pH values (designated as fractions I-III, according to their order of elution). The yield of fraction I and III was increased when adsorbed at an acidic pH. However, only fraction III had maximum oxalate binding activity at pH 4.5. When purified, this protein had maximum oxalate binding activity of approximately 270 pmol/mg protein and a molecular weight of approximately 23 kDa. Amino acid analysis showed that 18% of the total molar proportion was of basic amino acids, e.g. lysine and arginine, while acidic amino acids accounted for only 11%. Both alanine and glycine constituted approximately 41% of the total molar proportion. Modifications to the lysine group abolished oxalate-binding activity of the protein. The protein inhibited crystal growth by 82% at 0.8 micromol/L, while it inhibited the nucleation and aggregation of the crystals by 6% and 28%, respectively, at 49 nmol/L. The inhibition of both nucleation and aggregation was higher at pH 5.7 than at pH 7.4. Significantly, the protein induced the formation of intertwined calcium oxalate dihydrate crystals in a medium known to induce the formation of individual dihydrate crystals. The protein described here is the first reported basic inhibitor of calcium oxalate crystal growth with oxalate-binding activity at pH 4.5 that modulates calcium oxalate crystallization. It is suggested that this protein may play a physiologically significant role in inhibiting stone formation in acidic urine.

Mannan-binding lectin (MBL)-associated plasma protein present in human urine inhibits calcium oxalate crystal growth

Mannan-binding lectin (MBL)-associated plasma protein (MAp19) is an alternatively spliced form of MBL-associated serine protease-2, a component of a complement activation cascade. We observed that MAp19 is excreted in human urine. Interestingly, the amount of MAp19 was higher in urine of renal cell carcinoma patients than healthy people. Pretreatment of urine dialysate with 50 mM EDTA increased the recovery of MAp19, suggesting that MAp19 is a calcium-binding protein. The recombinant MAp19 showed a strong inhibition of calcium oxalate crystal growth in vitro in a concentration-dependent manner. Thus, we conclude that MAp19 plays a role in the inhibition of calcium oxalate renal stone formation.

Possible causes for the low prevalence of pediatric urolithiasis

Objectives. To determine why the incidence of pediatric urolithiasis is less than that of adult urolithiasis, we investigated the difference in inhibition of calcium oxalate (CaOX) crystallization between pediatric and adult urinary macromolecules (UMMs). Methods. Urinary parameters in relation to urolithiasis, the inhibition of CaOX crystallization of original urine and urine from which UMMs (greater than 3 kDa) had been removed, and the inhibition of CaOX crystal growth and aggregation of UMMs alone were measured. These inhibitory activities were compared between children and adults. Results. In the original urine, the inhibition of CaOX crystallization was significantly stronger for children than for adults, but was the same in urine from which the UMMs had been removed. The inhibition of CaOX crystal growth by UMMs alone showed no significant differences between children and adults; their inhibition of CaOX crystal aggregation was significantly stronger for children than for adults. Much more glycosaminoglycan (GAG) was included in pediatric UMMs than in adult UMMs, although there was no difference in UMM concentration between urine from children and urine from adults. Conclusions. The lower incidence of CaOX lithiasis in children may be attributed, among other factors, to the stronger inhibition of CaOX crystal aggregation by pediatric UMMs, which in turn might be affected by the higher concentration of GAGs in children’s urine.

Inhibition of Calcium Oxalate Urolithiasis in a Rat Model of Lithogenesis Using Bisphosphonates

Bisphosphonates bind renal calculi and inhibit calcium oxalate crystal growth in vitro. We evaluated their ability to inhibit calcium oxalate urolithiasis in a lithogenic rat model. Male Sprague-Dawley rats (four groups, eight rats each) were fed 1.0% ethylene glycol in their drinking water for 6 weeks. All rats had implantation of a 50- to 60-mg zinc pellet in their urinary bladder at the beginning of the 6-week period. The control group received no treatment. The other three groups received six weekly intraperitoneal injections of one of three bisphosphonates: pamidronate (APD), clodronate (CLO), or methylene diphosphonate (MDP). At the end of 6 weeks, the zinc pellet was retrieved and weighed; the kidneys were sectioned and stained to evaluate inflammation, tubular dilation, and crystal deposition; and blood and urine samples were analyzed for calcium and creatinine. There were no detectable biochemical differences between the control and the treatment groups. Zinc pellets removed from control animals had a significantly greater increase in weight secondary to crystal deposition than those from the treatment groups (mean 28.4% for control v 18.9%, 15.3%, and 18.6%, respectively, for animals given APD, CLO, and MDP). The control animals also had significantly higher scores for inflammation, tubular dilation, and crystal deposition than animals treated with MDP and CLO. Older and newer-generation bisphosphonates have an inhibitory effect on calcium oxalate urolithiasis that is demonstrable at relatively infrequent dosing intervals in vivo. More frequent dosing or higher doses may allow greater inhibition of stone formation.

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Inhibition of calcium oxalate urolithiasis in a rat model of lithogenesis using bisphosphonates.

Bisphosphonates bind renal calculi and inhibit calcium oxalate crystal growth in vitro. We evaluated their ability to inhibit calcium oxalate urolithiasis in a lithogenic rat model. Male Sprague-Dawley rats (four groups, eight rats each) were fed 1.0% ethylene glycol in their drinking water for 6 weeks. All rats had implantation of a 50- to 60-mg zinc pellet in their urinary bladder at the beginning of the 6-week period. The control group received no treatment. The other three groups received six weekly intraperitoneal injections of one of three bisphosphonates: pamidronate (APD), clodronate (CLO), or methylene diphosphonate (MDP). At the end of 6 weeks, the zinc pellet was retrieved and weighed; the kidneys were sectioned and stained to evaluate inflammation, tubular dilation, and crystal deposition; and blood and urine samples were analyzed for calcium and creatinine. There were no detectable biochemical differences between the control and the treatment groups. Zinc pellets removed from control animals had a significantly greater increase in weight secondary to crystal deposition than those from the treatment groups (mean 28.4% for control v 18.9%, 15.3%, and 18.6%, respectively, for animals given APD, CLO, and MDP). The control animals also had significantly higher scores for inflammation, tubular dilation, and crystal deposition than animals treated with MDP and CLO. Older and newer-generation bisphosphonates have an inhibitory effect on calcium oxalate urolithiasis that is demonstrable at relatively infrequent dosing intervals in vivo. More frequent dosing or higher doses may allow greater inhibition of stone formation.

Urinary calcium oxalate crystal growth inhibitors.

Calcium stones occur because renal tubular fluid and urine are supersaturated with respect to calcium oxalate and phosphate. The process of stone formation includes crystal nucleation, growth, aggregation, and attachment to renal epithelia. Urine contains macromolecules that modify these processes and may protect against stone formation. Attention has focused especially on inhibitors of crystal growth, and several have been isolated from urine, including nephrocalcin, an acidic phosphorylated glycoprotein that contains several residues of gamma-carboxyglutamic acid per molecule; osteopontin (uropontin), a phosphorylated glycoprotein also found in bone matrix; uronic acid-rich protein, which contains a covalently bound glycosaminoglycan residue; and several others. Abnormalities in structure and/or function have been detected in some of these proteins in stone formers' urine. However, the overall ability of urinary macromolecules to inhibit calcium oxalate crystal growth is often normal in stone formers. Recently, attention has been focused on the ability of these molecules to inhibit other stages in stone formation. Nephrocalcin can inhibit crystal nucleation, for example, and both nephrocalcin and Tamm-Horsfall protein inhibit crystal aggregation. Nephrocalcin and Tamm-Horsfall protein from stone formers are less active in preventing aggregation, and under some conditions, Tamm-Horsfall protein may promote the formation of crystal aggregates, especially in the presence of high concentrations of calcium. The structural abnormalities responsible for impaired inhibitory activity are not completely understood.

Isolation and purification of a high molecular weight substance from human urine which inhibits calcium oxalate crystal growth.

A high molecular weight inhibitor of calcium oxalate crystal growth in human urine was investigated. Three inhibitors were isolated by DEAE-Sephacel ion-exchange chromatography and, of these, the substance we named "Peak 3 protein" seemed to be the main inhibitor in human urine. Peak 3 protein and several was purified by fast protein liquid chromatography and polyacrylamide gel electrophoresis. This substance, with a molecular weight of 30 kDa, did not contain uronic acid and its inhibitory activity decreased after digestion with proteinase. The difference between Peak 3 protein and several inhibitors previously reported was investigated but no clear difference could be found. The fact that it was the protein structure which was responsible for the inhibitory activity and the fact that Peak 3 protein probably possessed many side-chains which did not contribute to the inhibitory activity influenced the outcome of the investigation.