Induction of Renal Nuclear Oxalate Binding Activity in Experimental Hyperoxaluric Rats

Каннабиран, Кришнан; Selvam, Ramasamy

doi:10.1159/000189535

Cited by 4 publications

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References 14 publications

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“…Earlier studies have shown the presence of oxalate-binding proteins in rat and human renal inner mitochondrial membranes [22]and histone H 1 B [23]and their excessive activities in hyperoxaluria. Further, these proteins have been shown to have an enhanced oxalate-binding activity in hyperoxaluric state or when subjected to lipid peroxidation [24, 25]. Based upon the above findings, we have studied the role of the 23-kD COM crystal adsorbed protein to resolve some aspects in the etiology of calcium oxalate stone pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

Modulatory Effect of the 23-kD Calcium Oxalate Monohydrate Binding Protein on Calcium Oxalate Stone Formation during Oxalate Stress

Devarajan

Kalaiselvi²,

Varalakshmi³

2004

Nephron Physiol

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Aims: To isolate, characterize, and quantify the 23-kD calcium oxalate monohydrate (COM) binding protein in the urine of controls and calcium oxalate stone formers and to study its role in kidney stone formation. Methods: Calcium oxalate crystals were prepared and allowed to interact with human control kidney homogenate as well as urine of controls and calcium oxalate stone formers. EDTA extract was used for the separation of the 23-kD COM-binding protein (partially purified). This partially purified 23-kD COM-binding protein was further separated by DEAE-cellulose column chromatography. SDS-PAGE confirmed the molecular weight. An antibody was raised against the renal 23-kD COM-binding protein in rabbits. The 23-kD COM-binding protein was quantified in the urine from controls and stone formers by ELISA. Thiol group quantification, oxalate-binding assay, and calcium oxalate crystal nucleation and aggregation were performed. Morphological changes of the calcium oxalate crystals induced by the urinary 23-kDa protein were determined using scanning electron microscopy. The expression of this protein using different concentrations of oxalate was also determined in an in vitro model. Results: The urinary excretion of the 23-kD COM-binding protein varies between 0.5 and 1.5 mg/24 h in controls, while in stone former its excretion was found to range from 5 to 7 mg/24 h. The protein isolated from urine was found to inhibit crystal nucleation and aggregation in controls, while the protein isolated from stone formers exhibited less inhibitory activity with reduced thiol groups. The 23-kD COM-binding protein derived from control urine formed COM crystals and intertwined calcium oxalate dihydrate crystals in a crystal growth system, while protein isolated from stone formers’ urine induced aggregation of COM crystals. This protein expression was found to be increased with increasing concentration of oxalate in renal epithelial cells of the African green monkey kidney (VERO) cell line. Conclusions: Increased expression and excretion of the 23-kD protein was observed in oxalate stress conditions, and in stone formers this protein exhibited a promoting activity. The increased excretion of this protein with promoting activity favors the lithogenic process in stone formers.

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Section: Discussionmentioning

confidence: 99%

Modulatory Effect of the 23-kD Calcium Oxalate Monohydrate Binding Protein on Calcium Oxalate Stone Formation during Oxalate Stress

Devarajan

Kalaiselvi²,

Varalakshmi³

2004

Nephron Physiol

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Oxalate binding proteins in calcium oxalate nephrolithiasis

Selvam

Kalaiselvi

2003

Urological Research

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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.

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Studies on Calcium Oxalate Binding Proteins: Effect of Lipid Peroxidation

Selvam

Kalaiselvi²

2001

Nephron

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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.

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Induction of Renal Nuclear Oxalate Binding Activity in Experimental Hyperoxaluric Rats

Cited by 4 publications

References 14 publications

Modulatory Effect of the 23-kD Calcium Oxalate Monohydrate Binding Protein on Calcium Oxalate Stone Formation during Oxalate Stress

Modulatory Effect of the 23-kD Calcium Oxalate Monohydrate Binding Protein on Calcium Oxalate Stone Formation during Oxalate Stress

Oxalate binding proteins in calcium oxalate nephrolithiasis

Studies on Calcium Oxalate Binding Proteins: Effect of Lipid Peroxidation

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