Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine (original) (raw)
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Urological Research, 1993
A calcium oxalate crystal growth inhibitor was isolated from human urine using DEAE-Sephacel gel followed by Sephacryl S-300 chromatography and FPLC column. The isolated inhibitor was a uronic-acid-rich protein (UAP). It was found to be a glycoprotein with a molecular weight of 35000Da as determined by SDSpolyacrylamide gel electrophoresis. Inhibitory activity was demonstrated using a calcium oxalate crystallization system. In addition UAP, nephrocalcin (NC) or nephrocalcin-like (NC-like) activity was an effective inhibitor in this system. However, the inhibitory activity of UAP appeared to be higher than that of NC or NC-like activity. This finding suggests that NC or NC-like activity is not only urinary protein with strong inhibitory activity. UAP and probably other proteins also play a role in the control of urinary crystal growth.
Bju International, 2007
Objectives To isolate calcium oxalate-binding proteins from human kidney and characterize the functional properties.Materials and methods 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.Results 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 ≈ 270 pmol/mg protein and a molecular weight of ≈ 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 ≈ 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 µmol/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.Conclusion 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.
ROLE OF URINARY INHIBITORS AND PROMOTERS IN CALCIUM OXALATE CRYSTALLISATION
Urine of most people is supersaturated with stone forming constituents, including calcium,oxalate, phosphate and uric acid. Crystals or foreign bodies can act as nidi, upon which ions from the supersaturated urine form microscopic crystalline structures. The majority of urinary calculi contain calcium. Calcium stones occur when urine becomes supersaturated with calcium oxalate and phosphate. They form crystals that bind into hardened mineral deposits known as renal stones. The process of stone formation includes crystal nucleation, growth, aggregation and retention. Various substances in our urine modify these stone forming processes, thereby influencing a person’s ability to promote or inhibit stone formation. Promoters of stone formation facilitate stone formation while inhibitors prevent it. Low urine volume, low urine pH, calcium, sodium, oxalate, and urate are known to promote stone formation. Many inorganic (eg. Citrate, magnesium, pyrophosphate etc.) and organic (eg. Tamm-Horsfall protein, glycosaminoglycans, uropontin, nephrocalcin, renal lithostathine etc.) Substances, high urine volume are known to inhibit stone formation. This paper presents role of urinary inhibitors and promoters in calcium oxalate crystallization.
Journal of Cellular Biochemistry, 2003
Standardized calcium oxalate monohydrate (COM) crystal growth assay system was employed to study the ability of various test samples to influence growth rates of COM crystals. The inhibitory activity (IA) of various samples was expressed in terms of inhibitory units. Urine samples obtained from normal persons and kidney stone patients were found to have IA of 3.18 ± 0.62 and 1.02 ± 0.08, respectively. A potent inhibitor having molecular weight between 14.2 and 16.2 kDa was found to be primarily responsible for the differences observed in the urinary IAs between normal persons and kidney stone patients. The potent inhibitor was found to be tightly associated with a chromophore resembling Urobilirubin. An ELISA based assay system, using monoclonal antibodies against the above most potent inhibitor confirmed the difference observed in the urinary IA between the normal persons and kidney stone patients. This assay system has the potential to be routinely used to screen human beings for potential stone formers. © 2003 Wiley-Liss, Inc.
Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro
Clinica Chimica Acta, 1973
A sensitive method has been developed for measuring the effect of inhibitors of crystallisation on the growth and aggregation of calcium oxalate crystals i?z vitro. Of the known inhibitors tested the highly charged anions pyrophosphate and ethane-I-hydroxy-r,r-diphosphonate (EHDP) were effective down to concentrations of 0.1-1 pmole/l and the polyanions heparin and chondroitin sulphate A down to concentrations of I nmole/l. Within the normal range of urea concentrations found in urine the neutral molecule urea caused a small but significant degree of inhibition. At higher concentrations the degree of inhibition increased sharply. In the concentrations tested the cations magnesium and methylene blue had no effect on the growth and aggregation of crystals. These findings suggest that highly charged anions and polyanions may have some clinical value as a possible treatment for recurrent calcium oxalate stone-formation in man.
Journal of Clinical Investigation, 1985
One reason that some people are prone to calcium oxalate nephrolithiasis is that they produce urine that is subnormal in its ability to inhibit the growth of calcium oxalate crystals. We have identified in human urine a glycoprotein (GCI) that inhibits calcium oxalate crystal growth strongly, and at low concentrations (10-' M); in this study, we have isolated GCI molecules from the urine of normal people and patients with calcium oxalate stones. GCI from stone formers is abnormal in three ways: (1) it contains no detectable y-carboxyglutamic acid (Gla), whereas normal GCI contains 2-3 residues of Gla per mole; (2) about half of the GCI in urine of patients inhibits crystal growth 4-20 times less than normal GCI as judged by its performance in a kinetic growth assay, in vitro; (3) at the air-water interface, patient GCI has a film collapse pressure approximately half of normal. GCI molcules from the urine of patients with calcium oxalate nephrolithiasis are intrinsically abnormal, and these abnormalities could play a role in the genesis of stones.
Immunochemistry of urinary calcium oxalate crystal growth inhibitor (CGI)
Kidney International, 1986
Immunochemistry of urinary calcium oxalate crystal growth inhibitor (CGI). Calcium oxalate crystal growth inhibitor (CGI) was isolated from human urine in monomeric form (14,000 daltons). Antibody was elicited and purified to monospecificity by affinity chromatography. Tamm-Horsfall protein was isolated from human urine and an antibody to Tamm-Horsfall protein compared to anti-CGI. The anti-COl reacted with its antigen on immunodiffusion, by ELISA and by Western Blotting of polyacrylamide gel electrophoresis-separated antigen. Immunofluorescent localization of CGI was found in distal renal tubules. This was precisely the localization of Tamm-Horsfall protein. Isolated Tamm-Horsfall protein was found to bind CGI which could only be partially removed with EDTA. While anti-CGI is suitable to assay CUl in human urine by ELISA techniques, it will also detect CGI that is complexed to THP. While the CGI found in human urine possesses 90% of the urinary macromolecular crystal growth inhibitor activity, THP is without effect on crystal growth, in spite of bound CGI. The balance between free CGI and that bound to Tamm-Horsfall protein may be important in the overall balance of urinary macromolecules that affect calcium oxalate nephrolithiasis.
The calcium oxalate crystal growth inhibition by Tamm-Horsfall protein
International Journal of Integrative Biology
A modified Schneider's gel slide method was developed to evaluate the inhibition of urinary Tamm-Horsfall protein (THP) on the growth of calcium oxalate crystal. The growth of these crystals is an important phenomenon for formation of kidney stones, in vitro gel slide technique followed by monitoring using image analyser was used to study the growth of the crystals. Size related parameters i.e. area, equivalent diameter, convex area and shape related parameters i.e. roundness, aspect ratio and full ratio were measured. These six parameters were processed with multivariate analysis of principal component analysis (PCA) as well as selforganizing map (SOM). The results showed an inhibitory effect on crystal growth by the four concentrations of THP used and positive control of sodium citrate. Both the parametric ANOVA and non-parametric Kruskal-Wallis test together with the inhibition index confirmed the inhibitory effect of the THP. The inhibition index for the four protein concentrations of 2.5, 5.0, 10.0 and 20.0 μg/ml were 0.12, 0.15, 0.25 and 0.25, respectively. ANOVA was more sensitive when compared to Kruskal-Wallis since the latter did not show a significant difference between blank and positive control (inhibition index = 0.204) whereas ANOVA indicated that both were significantly different.
Inhibition of Calcium Oxalate Monohydrate Crystal Growth in High and Low Ionic Strength Solutions
Crystal Research and Technology, 1998
A large number of people in this world are suffering from urinary stone problem. Calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) containing stones (calculi) are commonly found. In the present study, COM crystals were grown by artificial urine is prepared by mixing and stirring two equal volumes of solutions A and B. A screening of plant extracts from wild species antilithic agents using a urolithic could lead to the urinary risk factors of urolithiasis. Ten extracts of different parts of plant species from West and South of Algeria, belonging to different botanical families, were harvested in 2006 and 2007. From each sample aqueous extract is obtained using decoction. Activity was tested in bioassays using different parts of the plants according to the traditional medicine way. Crystal of calcium oxalate growth inhibition was significantly induced when extracts were incorporated at 100%. The most active plants were: Ammodaucus leucotrichus, Ajuga iva, Erica multiflora and Stipa tenacissima . However, only with A. leucotrichus inhibition was 97.94% and 97.68% at (75%) and (100%) respectively. The extracts of Globularia alypum, Atriplex halimus, Tetraclinis articulata, Chamaerops humilis and Erica arborea give percentages of inhibition which varies with the concentration of plant and the time to crystallisation.
Membranes and Their Constituents as Promoters of Calcium Oxalate Crystal Formation in Human Urine
Calcified Tissue International, 2000
We have proposed that membranes of cellular degradation products are a suitable substrate for the nucleation of calcium oxalate (CaOx) crystals in human urine. Human urine is generally metastable with respect to CaOx. To demonstrate that cellular membranes present in the urine promote nucleation of CaOx we removed these substrates by filtration or centrifugation and induced crystallization by adding sodium oxalate, before and after filtration or centrifugation. In a separate experiment, membrane vesicles isolated from rat renal tubular brush border were added into the filtered or centrifuged urine before crystal induction. Crystals were counted using a particle counter. Urine, the pellet, and retentate were analyzed for the presence of membranes, lipids, and proteins. Lipids were further separated into different classes, identified, and quantified. Both filtration and centrifugation removed lipids, proteins, and membrane vesicles, causing a reduction in lipid and protein contents of the urine. More crystals formed in whole than in filtered or centrifuged urine. The number of crystals significantly increased when filtered urine was supplemented with various urinary components such as the retentate and phospholipids, which are removed during filtration. We also determined the urinary metastable limit with respect to CaOx. Filtration and centrifugation were associated with increased metastable limit which was reduced by the addition of membrane vesicles. These results support our hypothesis that urine normally contains promoters of CaOx crystal formation and that membranes and their constituents are the most likely substrate for crystal nucleation in the urine.