Oxalate Binding Protein Research Articles

StoneMod 2.0: Database and prediction of kidney stone modulatory proteins

Stone modulators are various kinds of molecules that play crucial roles in promoting/inhibiting kidney stone formation. Several recent studies have extensively characterized the stone modulatory proteins with the ultimate goal of preventing kidney stone formation. Herein, we introduce the StoneMod 2.0 database (https://www.stonemod.org), which has been dramatically improved from the previous version by expanding the number of the modulatory proteins in the list (from 32 in the initial version to 17,130 in this updated version). The stone modulatory proteins were recruited from solid experimental evidence (via PubMed) and/or predicted evidence (via UniProtKB, QuickGO, ProRule, STITCH and OxaBIND to retrieve calcium-binding and oxalate-binding proteins). Additionally, StoneMod 2.0 has implemented a scoring system that can be used to determine the likelihood and to classify the potential stone modulatory proteins as either “solid” (modulator score ≥ 50) or “weak” (modulator score < 50) modulators. Furthermore, the updated version has been designed with more user-friendly interfaces and advanced visualization tools. In addition to the monthly scheduled update, the users can directly submit their experimental evidence online anytime. Therefore, StoneMod 2.0 is a powerful database with prediction scores that will be very useful for many future studies on the stone modulatory proteins.

OxaBIND: A tool for identifying oxalate-binding domain(s)/motif(s) in protein(s)

High oxalate level in blood and urine may cause oxalate-related disorders, particularly kidney stone disease. To unravel disease mechanisms, investigations of oxalate level and its binding proteins are required. However, the information on oxalate-binding proteins is limited due to a lack of appropriate tool for their investigations. Therefore, we have developed a freely accessible web-based tool, namely OxaBIND (https://www.stonemod.org/oxabind.php), to identify oxalate-binding site(s) in any proteins of interest. The prediction model was generated by recruiting all of the known oxalate-binding proteins with solid experimental evidence (from PubMed and RCSB Protein Data Bank). The potential oxalate-binding domains/motifs were predicted from these oxalate-binding proteins using PRATT tool and used to discriminate these known oxalate-binding proteins from the known non-oxalate-binding proteins. The best one, which provided highest fitness score, sensitivity and specificity, was then implemented to create the OxaBIND tool. After inputting protein identifier or sequence (which can be single or multiple), details of all the identified oxalate-binding site(s), if any, are presented in both textual and graphical formats. OxaBIND also provides theoretical three-dimensional (3D) structure of the protein with oxalate-binding site(s) being highlighted. This tool will be beneficial for future research on the oxalate-binding proteins, which play important roles in the oxalate-related disorders.

Differential bound proteins and adhesive capabilities of calcium oxalate monohydrate crystals with various sizes

Adhesion of calcium oxalate (CaOx) crystals onto renal tubular epithelial cells is one of the critical steps in kidney stone formation. However, effects of crystal size on the crystal adhesive capability remained unclear. This study compared the adhesive capabilities of CaOx monohydrate (COM) crystals with various sizes (<10 μm, 20–30 μm, 50–60 μm, and > 80 μm). Crystal-cell adhesion assay showed size-dependent increase of COM crystal adhesion onto epithelial cell surface using the larger crystals. Identification of apical membrane proteins that could bind to COM crystals by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) demonstrated size-specific sets of the COM crystal-binding proteins. Among these, numbers of known oxalate-binding proteins and COM crystal receptors were greatest in the set of the largest size (>80 μm). Atomic force microscopy (AFM) revealed that adhesive forces between carboxylic-immobilized AFM tip and COM crystal surface and between COM-mounted AFM tip and renal epithelial cell surface were size-dependent (greater for the larger crystals). In summary, the adhesive capability of COM crystals is size-dependent – the larger the greater adhesive capability. These data may help better understanding of the pathogenic mechanisms of kidney stone formation at an initial stage when renal tubular cells are exposed to various sizes of COM crystals.

In vitro and in silico study of antioxidant effect of Bergenia ciliata and Terminalia chebula against sodium oxalate induced oxidative stress

Previous experimental evidence suggested that Bergenia ciliata (Saxifragaceae) and Terminalia chebula Retzius(Combretaceae) are effective against nephrolithiasis and have potential antioxidant activity. In this regard, hydro-methanolic extracts of B. ciliata rhizomes and T. chebula fruits were investigated for antioxidant potential against sodium oxalate induced oxidative imbalance in the kidney of female Wistar rats. We also performed molecular docking studies of all the reported phenolic compounds of both the plants to evaluate its interaction with the active site of oxalate binding protein (OBP). In summary, our findings showed that sodium oxalate caused significant increase in lipid peroxidation with concurrent decrease in activities of superoxide dismutase and catalase as well as in total reduced glutathione content in a concentration-dependent manner. The hydro-alcoholic extracts, however, when co-administered with sodium oxalate resulted in significant protection with maximum percent protection was achieved by B. ciliata. Thereafter, daucosterol showed best binding efficiency with OBP. However, further work on the purification of isolated bioactive components and pharmacological testing can reveal the therapeutic potential of the plant.

Isolation and characterizations of oxalate-binding proteins in the kidney

Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed “L-x(3,5)-R-x(2)-[AGILPV]” as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.

Development of an oxalate-affinity chromatographic column to isolate oxalate-binding proteins

Calcium oxalate (CaOx) is the major crystalline component in kidney stones. Many previous studies on stone modulation have focused mainly to calcium-binding proteins, whereas oxalate-binding proteins remain under-investigated due to a lack of a simple method to purify oxalate-binding proteins. Therefore, we have developed an affinity chromatographic column to isolate or purify oxalate-binding proteins. The oxalate-affinity column developed contained EAH-Sepharose 4B beads conjugated with oxalic acid by carbodiimide reaction using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) as an activator. The coupling efficacy of coupled beads (oxalic acid-EAH Sepharose 4B) was determined by a competitive ninhydrin assay to quantify the remaining amino group on EAH-Sepharose 4B. In addition, the oxalate-affinity column showed high specific binding to a known oxalate-binding protein (p62) but not to an irrelevant protein (carbonic anhydrase). In conclusion, we have established the oxalate-affinity chromatographic column, which showed the high efficiency to isolate oxalate-binding proteins. This novel chromatographic column will be very useful for future stone research, particularly in the area of stone modulation by oxalate-binding proteins.

Characterization of histone (H1B) oxalate binding protein in experimental urolithiasis and bioinformatics approach to study its oxalate interaction

The rat kidney H1 oxalate binding protein was isolated and purified. Oxalate binds exclusively with H1B fraction of H1 histone. Oxalate binding activity is inhibited by lysine group modifiers such as 4′,4′-diisothiostilbene-2,2-disulfonic acid (DIDS) and pyridoxal phosphate and reduced in presence of ATP and ADP. RNA has no effect on oxalate binding activity of H1B whereas DNA inhibits oxalate binding activity. Equilibrium dialysis method showed that H1B oxalate binding protein has two binding sites for oxalate, one with high affinity, other with low affinity. Histone H1B was modeled in silico using Modeller8v1 software tool since experimental structure is not available. In silico interaction studies predict that histone H1B-oxalate interaction take place through lysine121, lysine139, and leucine68. H1B oxalate binding protein is found to be a promoter of calcium oxalate crystal (CaOx) growth. A 10% increase in the promoting activity is observed in hyperoxaluric rat kidney H1B. Interaction of H1B oxalate binding protein with CaOx crystals favors the formation of intertwined calcium oxalate dehydrate (COD) crystals as studied by light microscopy. Intertwined COD crystals and aggregates of COD crystals were more pronounced in the presence of hyperoxalauric H1B.

Epitaxial deposition of calcium oxalate on uric acid rich stone matrix is induced by a 29 kDa protein

Background Association of macromolecules particularly the role of proteins in urolithiasis has been studied for last few centuries, but still a complete profile of stone matrix proteins that mediate co-precipitation of uric acid and calcium oxalate has not been characterized. We isolated and characterize proteins from uric acid rich stone matrix, which have oxalate binding activity. Methods Matrix proteins were isolated from uric acid rich stone matrix using EDTA as a demineralizing agent. The radiolabelled solubilized proteins were fractionated with increasing ionic concentration by DEAE cellulose column chromatography to identify the oxalate binding protein. It was purified using Sephadex G-200 column chromatography. Amino acid composition was determined and monoclonal antibody was produced against the oxalate binding uric acid rich stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers urine, their oxalate binding activity assayed and cross reactivity with the produced monoclonal antibody were checked using ELISA and Western blotting. Results Matrix on DEAE column chromatography elution yielded 3 protein peaks and they were named as fraction I, II and III among which fraction I had higher oxalate binding activity which was further purified with Sephadex G-200 column which yielded 2 protein peaks designated as Ia and Ib. Fraction Ib with molecular weight 29 kDa exhibited the maximum oxalate binding activity. Forty percent of this 29 kDa protein is comprised of basic amino acids. Monoclonal antibody (IgG 1) was produced against the 29 kDa stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers, 4 protein peaks were obtained named as fraction I to IV. Among them, fraction IV having molecular weight of ∼29 kDa cross reacted up to 85.6% with 29 kDa stone matrix protein. Moreover, urinary 29 kDa protein exhibited oxalate binding activity of 94.16 ± 6.08 pmol/mg protein at pH 5.5. Conclusion The 29 kDa protein isolated from uric acid rich stone matrix and urine are one and the same, thereby insinuating that 29 kDa protein might play a major role in epitaxial deposition of calcium oxalate over uric acid core, consequently favoring the lithogenic events like uric acid and calcium oxalate nucleation, aggregation and retention.

Nuclear pore complex oxalate binding protein p62: its expression on oxalate exposure to VERO cells.

Oxalate rich stones are the most common among the various stones. Oxalate binding protein plays a vital role in the transport of oxalate. Nuclear pore complex (NPC) contains a protein of molecular weight 62 kDa and it has maximum oxalate binding activity. The physiological significance of the presence of oxalate binding protein in the NPC is not well understood. In order to study its function, the expression of this protein during oxalate stress condition and the morphological changes on oxalate exposure to synchronized VERO cells have been determined. VERO cells were synchronized at different stages of cell cycle using cell cycle blockers and expression of the NPC p62 was assessed using enzyme linked immunosorbent assay (ELISA) technique with p62 antibody (MAb 414). Expression of NPC p62 was more pronounced in 1.0 mM oxalate concentration in mitotic phase than in S phase, suggesting cell cycle dependency. During oxalate exposure there is cell aggregation and complete degeneration of cell morphology occurs, which in turn lead to the expression of certain genes, including the NPC oxalate binding protein p62. Thus, oxalate induces degeneration of cells (may be due to the lipid peroxidation) and leads to the expression of NPC oxalate binding protein and the expression is of cell cycle dependent manner.

Expression of Nuclear Pore Complex Oxalate Binding Protein p62 in Experimental Hyperoxaluria

Proteins are thought to play a major role in stone formation. Oxalate binding protein plays a vital role in the transport of oxalate. This study was aimed at determining whether hyperoxaluria induces the expression of nuclear pore complex oxalate binding protein p62 which has the transport function. Hyperoxaluria was induced in male Wistar rats by feeding 0.75% ethylene glycol in water. The oxalate binding activity of the nuclear pore complex protein increased markedly during experimental hyperoxaluria, whereas nuclear lamina had no binding at all. There was an alteration in the elution profile of the nuclear pore complex oxalate binding protein during the hyperoxaluric condition. The protein was purified and had a molecular weight of 62 kDa (data not shown). The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and it showed homogeneity. The expression of this protein (p62) during the hyperoxaluric condition was determined by ELISA and a 3-fold increase was observed when compared to control rats. The increased expression is further confirmed by Western blotting and immunohistochemistry. The increase in p62 protein expression may be either due to increased expression of certain genes or degradation of the cell membrane by oxalate-induced cell injury. Thus, the present study suggests that the increased expression of this protein (p62) may be due to the oxalate induction.

Nuclear pore complex oxalate binding protein p62: expression in different kidney disorders

Background: Urolithiasis is a multifactorial process that starts with the formation of microcrystals in the urine and terminates as mature renal calculi. The oxalate binding protein plays a vital role in the transport of oxalate. The physiological significance of the presence of oxalate binding protein in the nuclear pore complex is not well understood. Methods: The nuclear envelope was extracted from human cadaver kidneys. 14C oxalate was labeled, nuclear pore complex proteins were extracted and loaded onto Sephadex G-200, and further purified in DEAE-Sephadex A-50 column. The radioactive protein peak was pooled, concentrated and checked for purity in SDS-PAGE. The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and was homogeneous. Urine samples of healthy individuals with no history of kidney disease served as control. Blood and urine samples were collected from kidney and autoimmune disorder patients and checked for the expression of p62 protein by ELISA. Results: Extracted and purified nuclear pore complex oxalate binding protein had a molecular weight of 62 kDa. A threefold increase in oxalate excretion was observed in hyperoxaluric patients compared to control subjects. The protein expression was found to be higher in hyperoxaluric patients vs. controls, chronic renal failure (CRF) and acute renal failure (ARF), whereas decreased expression was observed in nephrotic syndrome (NS) patients. p62 autoantibodies was observed in hyperoxaluria (HO), systemic lupus erythematosus (SLE) and primary biliary cirrhosis (PBC), whereas it was absent in controls. Conclusion: Increased expression of p62 may be due to membrane damage induced by oxalate stress, and may be used as a diagnostic marker. This study also confirms the presence of p62 autoantibodies in HO patients.

Oxalate binding proteins in calcium oxalate nephrolithiasis.

The existence of several oxalate specific binding proteins have been demonstrated in human and rat kidney. These occur in both cortical and medullary cells and are distributed mostly in the subcellular organelles. About 1/3 of the total cellular oxalate binding was localised in the inner mitochondrial membrane while the rest was in the nucleus. The purified mitochondrial oxalate binding protein (62 kDa) was composed, with a higher molar proportion, of basic amino acids, and could accumulate oxalate on incorporation into liposomes. In the nucleus, histone H(1B) (27.5 kDa), nuclear membrane protein (68 kDa) and nuclear pore complex protein (205 kDa) were present with oxalate binding activities. In addition, a 45 kDa calcium oxalate binding protein was identified in most of the subcellular organelles. Both mitochondrial and nuclear oxalate binding proteins and calcium oxalate binding protein have shown the kinetic properties of specificity, saturability, pH and temperature dependency, energy of activation and inhibition by substrate analogues. All oxalate binding proteins were sensitive to the transport inhibitor 4'-4' diisothiocyano stilbene-2-2 disulphonic acid (DIDS), which is known to interact with the lysine moiety of the proteins. Calcium oxalate monohydrate (COM) crystals adsorbed oxalate binding proteins from human and rat kidney, and oxalate binding proteins isolated from human kidney stone matrix also exhibited the above kinetic properties. In experimental hyperoxaluria, all of the renal oxalate binding proteins showed enhanced oxalate binding activity with increased protein concentration. This enhanced oxalate binding activity was also attributed to increased lipid peroxidation, which correlated positively, and to decreased thiol status, which correlated negatively. A positive correlation was observed between the lipid peroxidation and both the oxalate binding activity of the in vitro peroxidised subcellular organelles and the purified protein. Similarly, in an in vivo hyperoxaluric condition, a negative correlation was observed between thiol content and both the oxalate binding activity of the peroxidised subcellular organelles and the purified protein. In the calcium oxalate crystal growth system, the oxalate binding proteins behaved either as promoters or inhibitors of the nucleation and aggregation of crystals. Following the peroxidation of the proteins, the degree of effect of the promoter protein was further stimulated while the degree of inhibition caused by the inhibitor protein further declined. Similar observations were duplicated with the proteins derived from hyperoxaluric rat kidney or kidney homogenate subjected to in vitro lipid peroxidation. The oxalate binding proteins were thought to modulate the crystallisation process in an hyperoxaluric condition similar to calcium specific binding protein modulators.

Characterisation of nuclear pore complex oxalate binding protein from human kidney.

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4'-dithiocyanostilbene-2,2'-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton-high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl-Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 microM oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.

An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix.

To fractionate renal-stone matrix proteins, identify the presence of oxalate-binding protein and assess its effect in a calcium oxalate (CaOx) crystal growth system. Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent, using EDTA as a demineralizing agent. The solubilized proteins were subjected to cellulose-column chromatography by eluting with increasing sodium chloride concentrations in Tris-HCl buffer. Three protein fraction peaks were eluted, i.e. fraction I in buffer, fraction II in 0.05 mol/L NaCl in buffer and fraction III in 0.3 mol/L NaCl in buffer. The protein fractions were tested for their effects on CaOx crystal growth. All three fractions had maximum CaOx binding activity at pH 7.4 but fraction II also had activity at pH 4.5. Fraction I promoted in vitro CaOx crystal growth, while fractions II and III were inhibitory. When fraction I was further separated on a Sephadex G-200 column, two protein fractions (Ia and Ib) were obtained. Fraction Ia protein had high and fraction Ib low CaOx-binding activity. Fraction Ia had a molecular weight of 48 kDa on gel electrophoresis and Western blotting. The 48 kDa protein did not cross-react with crystal matrix protein antibody, band-3 protein antibody, or albumin. The protein promoted CaOx crystal growth, with an optimum temperature of 37 degrees C and pH 6.5. The inhibitory effect of citrate on crystal growth was significantly lower in the presence of the 48 kDa protein. The protein promoted nucleation and aggregation of CaOx crystals in the in vitro crystallization system at pH 6.5, whereas fraction Ib (29 kDa) inhibited both nucleation and aggregation. Using the 48 kDa antibody, the yield of the protein from the stone matrix was 32% by EDTA extraction and only 3% with other methods. The protein was also detected in the nucleus and mitochondria, and in other matrix fractions of calcium phosphate and uric acid stones. The 48 kDa protein isolated from stone matrix is a potent promoter of CaOx crystal growth with high oxalate-binding activity; it is enriched in the nucleus and mitochondria.

Effect of cyclosporin A treatment on renal calcium oxalate binding in experimental hyperoxaluria.

This study was aimed to investigate the effect ofCyclosporin A administration on renal calcium oxalate binding under hyperoxaluric condition. Cyclosporin A administration or ammonium oxalate treatment increased calcium oxalate binding, which was further increased in kidney treated with cyclosporin A and ammonium oxalate together. The increase of calcium oxalate binding was associated with lipid peroxidation as well as with a concomitant decrease in total thiol in both rat and human kdiney homogenate. Cyclosporin A administration to hyperoxaluric rats resulted with increased calcium oxalate binding protein. However there was no change with specific activity of the protein. In conclusion, Cyclosporin A administration either to normal or hyperoxaluric rats is resulted with increased concentration of calcium oxalate binding protein as well as enhanced activity due to membrane lipid peroxidation.

Studies on Calcium Oxalate Binding Proteins: Effect of Lipid Peroxidation

Objective: Urolithiasis and free radicals have long been associated. In this study, we have isolated calcium oxalate monohydrate (COM) binding proteins from rat kidney before and after lipid peroxidation (LPO) and studied its properties on calcium oxalate crystal growth. Materials and Methods: LPO was carried out using t-butyl hydroperoxide, cumene hydroperoxide and an ascorbate system. The COM binding proteins from control and peroxidised tissues were isolated using a modified procedure. Protein was extracted using 25 mM EDTA, and the extract was loaded onto a DEAE cellulose column and eluted with Tris-HCl buffer (pH 6.5), 0.05 M NaCl in the above buffer and 0.3 M NaCl in the same buffer. Three major protein fractions were obtained, and they were designated as fractions I, II and III according to their order of elution. The proteins were subjected to calcium oxalate crystal nucleation and aggregation. Results: A positive correlation was observed between LPO and COM adsorption, while a negative correlation was observed between reduced glutathione and COM adsorption. Peroxidised protein did not show any alteration in the elution profile on the DEAE cellulose column. The –SH content of the peroxidised fractions were lower than that of the control fractions, but their oxalate binding activities were increased. Peroxidised fraction I promoted crystal growth to a greater extent than the control fraction I. Peroxidised fractions II and III were less inhibitory in nature compared to their control fractions. Light-microscopic examination of the crystals formed in the presence of the peroxidised fractions showed the formation of large aggregates of COM. Conclusion: Peroxidation of the renal proteins favoured their adsorption to COM crystals. –SH depletion increased the oxalate binding activity and also their affinity to the COM crystals. The peroxidised fraction I was found to favour the formation of large aggregates, suggesting that peroxidation may be one of the mechanisms altering the crystal inhibitory activity of the proteins in hyperoxaluria.

Effect of experimental hyperoxaluria on renal calcium oxalate monohydrate binding proteins in the rat.

To determine the functional role of calcium oxalate binding proteins in the nucleation, aggregation and retention of calcium oxalate crystals under physiological and hyperoxaluric conditions. Materials and methods Hyperoxaluria was induced in rats using 0.75% of ethylene glycol in drinking water. Calcium oxalate binding proteins were isolated and fractionated by cellulose column chromatography. Three major protein peak fractions were obtained (73 kDa in Tris-HCl buffer, 20 kDa in 0.05 mol/L NaCl buffer and 23 kDa in 0.3 mol/L buffer). Oxalate binding and the inhibition of crystal nucleation and aggregation by these fractions were determined. The adsorption of calcium oxalate monohydrate (COM) was ubiquitous in rat tissues and subcellular organelles, but the percentage adsorption varied; maximum absorption occurred in kidneys and pancreas, with microsomes showing maximal adsorption in the kidney. Hyperoxaluric rat tissues showed a greater percentage adsorption. Microsomes were enriched with the 20 kDa protein, while nuclei contained the 23 kDa protein in higher concentrations. COM-binding proteins derived from hyperoxaluric rat kidney had a greater content of 74 kDa and 23 kDa proteins with increased oxalate-binding activities. In the crystal-growth studies, the 74 kDa protein was a promoter, while the other protein fractions inhibited crystallization. In hyperoxaluria, the crystal-growth promoting activity of the 74 kDa protein was further increased, while the inhibition by the 20 and 23 kDa proteins was decreased. The 74 kDa protein derived from control rats formed single COM crystals in a crystal growth system, while the hyperoxaluric rat fraction induced the aggregation of COM crystals. COM-binding proteins (the 74 and 23 kDa fractions) were expressed more in hyperoxaluric rats. In hyperoxaluria the 74 kDa protein tended to promote crystal nucleation and aggregation, and the 20 and 23 kDa proteins were less inhibitory, which increases the risk of stone formation.

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.

Oxalate-Induced and Cell-Cycle-Dependent Expression of Nuclear Pore Complex Oxalate Binding Protein gp210

The effect of oxalate, a constituent of renal stone, on the expression of nuclear pore complex oxalate binding protein (gp210) in Vero monkey kidney cells was examined. The expression of this protein was found to increase more in mitotic phase than in S phase, suggesting cell cycle dependency. Exposure of cells to oxalate-containing growth medium resulted in a relative increase in nuclear pore complex oxalate binding protein in each stage of cell cycle. The concentration of this protein was found to increase six times in the telophase stage of the cells exposed to high concentrations of oxalate in the growth medium, though slight reduction in cell density was observed. Structural analogues of oxalate did not show any stimulatory effect on expression of this oxalate binding protein. Hence, the expression of the nuclear pore complex oxalate binding protein gp210 was specific to oxalate and is cell cycle dependent.

Renal calcium oxalate binding protein: Studies on its properties

To understand the mechanism of calcium oxalate (CaOx) crystal retention within the kidneys, calcium oxalate binding protein was isolated and characterized. The specific activity of calcium oxalate binding protein in the homogenate was 4.54 nmol/mg protein. The renal medulla showed higher CaOx binding activity than that of papilla or cortex, and among the cellular fractions the nucleus exhibited highest specific activity. Several tissues showed CaOx binding activity suggesting its ubiquitous nature. After being subjected to acetone precipitation, ethanol precipitation and HPLC chromatography, the renal protein revealed a 57-fold purity with a specific activity of 260 nmol/mg protein and a molecular weight of 45 kDa. The CaOx binding protein had the kinetic properties of concentration and time dependency, optimum temperature and substrate saturability. Scatchard plot analysis showed a single affinity site with a kDa of 41 nM and Bmax of 6.7 nmol/mg protein. The binding activity was inhibited by the anion transport inhibitor DIDS and substrate analogs like succinate and oxamide, while EGTA or ruthenium red had no effect on binding, suggesting that the protein binding was oxalate site specific. The molecular weight of the CaOx binding protein of different tissues was similar to that of renal cells. In conclusion, the presence of CaOx binding protein is demonstrated in rat and human kidneys, as well as other rat tissues.