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

      , ,

      Nephron Physiology

      S. Karger AG

      Oxalate stone formation, Oxalate stress, 23-kD protein

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          Abstract

          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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          Most cited references 16

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          Calcium oxalate stone disease: role of lipid peroxidation and antioxidants.

           R. Selvam (2002)
          Membrane injury facilitated the fixation of calcium oxalate crystals and subsequent growth into kidney stones. Oxalate-induced membrane injury was mediated by lipid peroxidation reaction through the generation of oxygen free radicals. In urolithic rat kidney or oxalate exposed cultured cells, both superoxide anion and hydroxyl radicals were generated in excess, causing cellular injury. In hyperoxaluric rat kidney, both superoxide and H2O2-generating enzymes such as glycolic acid oxidase (GAO) and xanthine oxidase (XO) were increased, and hydroxyl radical and transition metal ions, iron, and copper were accumulated. The lipid peroxidation products, thiobarbituric acid-reactive substances (TBARS), hydroperoxides, and diene conjugates were excessively released in tissues of urolithic rats and in plasma of rats as well as stone patients. The accumulation of these products was concomitant with the decrease in the antioxidant enzymes, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glucose-6 phosphate dehydrogenase (G6PD) as well as radical scavengers, vitamin E, ascorbic acid, reduced glutathione (GSH), and protein thiol. All the above parameters were decreased in urolithic condition, irrespective of the agents used for the induction of urolithiasis. Oxalate binding activity and calcium oxalate crystal deposition were markedly pronounced, along with decreased adenosine triphosphatase (ATPase) activity. Lipid peroxidation positively correlated with cellular oxalate, oxalate binding, gamma-glutamyl carboxylase, and calcium level and negatively correlated with GSH, vitamin E. ascorbic acid, and total protein thiol. Antioxidant therapy to urolithic rats with vitamin E, glutathione monoester, methionine, lipoic acid, or fish oil normalised the cellular antioxidant system, enzymes and scavengers, and interrupted membrane lipid and protein peroxidation reaction, ATPase inactivation, and its associated calcium accumulation. Antioxidant therapy prevented calcium oxalate precipitation in the rat kidney and reduced oxalate excretion in stone patients. Similarly, calcium oxalate crystal deposition in vitro to urothelium was prevented by free radical scavengers such as phytic acid and mannitol by protecting the membrane from free radical-mediated damage. All these observations were suggestive of the active involvement of free radical-mediated lipid peroxidation-induced membrane damage in the pathogenesis of calcium oxalate crystal deposition and retention.
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            Oxalate toxicity in LLC-PK1 cells: role of free radicals.

            Oxalate, the most common constituent of kidney stones, is an end product of metabolism that is excreted by the kidney. During excretion, oxalate is transported by a variety of transport systems and accumulates in renal tubular cells. This process has been considered benign; however, recent studies on LLC-PK1 cells suggested that high concentrations of oxalate are toxic, inducing morphological alterations, increases in membrane permeability to vital dyes and loss of cells from the monolayer cultures. The present studies examined the basis for oxalate toxicity, focusing on the possibility that oxalate exposure might increase the production/availability of free radicals in LLC-PK1 cells. Free radical production was monitored in two ways, by monitoring the reduction of nitroblue tetrazolium to a blue reaction product and by following the conversion of dihydrorhodamine 123 (DHR) to its fluorescent derivative, rhodamine 123. Such studies demonstrated that oxalate induces a concentration-dependent increase in dye conversion by a process that is sensitive to free radical scavengers. Specifically, addition of catalase or superoxide dismutase blocked the oxalate-induced changes in dye fluorescence/absorbance. Addition of these free radical scavengers also prevented the oxalate-induced loss of membrane integrity in LLC-PK1 cells. Thus it seems likely that free radicals are responsible for oxalate toxicity. The levels of oxalate that induced toxicity in LLC-PK1 cells (350 microM) was only slightly higher than would be expected to occur in the renal cortex. These considerations suggest that hyperoxaluria may contribute to the progression of renal injury in several forms of renal disease.
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              Aging and oxidation of reactive protein sulfhydryls.

              Protein sulfhydryls are potential sites of reversible oxidative modification by S-glutathiolation, and S-nitrosylation, but they are also susceptible to irreversible damage by oxidative conditions. In the absence of adequate antioxidant protection, these reactive sites may become useless because of this irreversible damage. It has recently become possible to directly access the nature and amount of irreversibly oxidized protein sulfhydryls by both gel-based methods and direct amino acid analysis. Results are in keeping with the concept that irreversible oxidation of protein sulfhydryls is more extensive in aged tissue samples. It is proposed that an adequate pool of glutathione is essential to prevent this increase in sulfhdryl oxidation. The increased amount of protein sulfhydryl damage may be critically important to the function of signal-transduction and transcription events that utilize proteins containing these reactive sites.
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                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                1660-2137
                2004
                May 2004
                24 May 2004
                : 97
                : 1
                : p23-p30
                Affiliations
                Department of Medical Biochemistry, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Chennai, India
                Article
                77599 Nephron Physiol 2004;97:p23–p30
                10.1159/000077599
                15153748
                © 2004 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 7, Tables: 4, References: 32, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/77599
                Categories
                Original Paper

                Cardiovascular Medicine, Nephrology

                Oxalate stress, 23-kD protein, Oxalate stone formation

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