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      Structural and Functional Modification of THP on Nitration: Comparison with Stone Formers THP

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          Objective: The crucial steps involved in the lithogenic process are governed by the macromolecular components of urine, of which proteins play a major role. Structurally abnormal proteins have been reported to be present in the urine of stone formers. Free radical injury has come a long way in explaining some of the pathophysiological events of renal lithiasis. Thus, our present work was designed to study the impact of the potent oxidant peroxynitrite on the biochemical components of the urinary Tamm-Horsfall glycoprotein (THP). Materials and Methods: Nitration on THP was carried out using peroxynitrite (ONOO<sup>–</sup>). After nitration, biochemical components like thiols, S-nitrosothiol, hexose, hexosamine and sialic acid were determined and these factors were compared with those of stone formers and normal THP. Crystallization behavior of control, nitrated NS-THP and stone formers THP was studied. Results: There was a significant decrease in thiol, hexose, hexosamine and sialic acid contents in stone formers and nitrated NS-THP, when compared to that of the control THP. In contrast to this, S-nitrosothiol content was significantly increased in stone formers and nitrated NS-THP (p < 0.001) when compared with the control THP. NO<sub>x</sub> metabolites were significantly elevated in stone formers THP when compared with that of control THP. When subjected to CaOx crystallization, stone formers THP and nitrated NS-THP promoted both CaOx nucleation and aggregation, while normal THP was found to be an inhibitor of the above processes. Conclusion: From our results we conclude that nitration of THP could represent one of the prime events in modifying kinetic behavior of THP, thus converting THP into a heterogeneous nucleator of renal calculi formation.

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          Most cited references 26

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          Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy.

          Medullary cystic kidney disease 2 (MCKD2) and familial juvenile hyperuricaemic nephropathy (FJHN) are both autosomal dominant renal diseases characterised by juvenile onset of hyperuricaemia, gout, and progressive renal failure. Clinical features of both conditions vary in presence and severity. Often definitive diagnosis is possible only after significant pathology has occurred. Genetic linkage studies have localised genes for both conditions to overlapping regions of chromosome 16p11-p13. These clinical and genetic findings suggest that these conditions may be allelic. To identify the gene and associated mutation(s) responsible for FJHN and MCKD2. Two large, multigenerational families segregating FJHN were studied by genetic linkage and haplotype analyses to sublocalise the chromosome 16p FJHN gene locus. To permit refinement of the candidate interval and localisation of candidate genes, an integrated physical and genetic map of the candidate region was developed. DNA sequencing of candidate genes was performed to detect mutations in subjects affected with FJHN (three unrelated families) and MCKD2 (one family). We identified four novel uromodulin (UMOD) gene mutations that segregate with the disease phenotype in three families with FJHN and in one family with MCKD2. These data provide the first direct evidence that MCKD2 and FJHN arise from mutation of the UMOD gene and are allelic disorders. UMOD is a GPI anchored glycoprotein and the most abundant protein in normal urine. We postulate that mutation of UMOD disrupts the tertiary structure of UMOD and is responsible for the clinical changes of interstitial renal disease, polyuria, and hyperuricaemia found in MCKD2 and FJHN.
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            Plasma protein thiol oxidation and carbonyl formation in chronic renal failure.

            Myeloperoxidase-catalyzed oxidative pathways have recently been identified as an important cause of oxidant stress in uremia and hemodialysis (HD), and can lead to plasma protein oxidation. We have examined patterns of plasma protein oxidation in vitro in response to hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). We measured thiol oxidation, amine oxidation, and carbonyl concentrations in patients on chronic maintenance HD compared with patients with chronic renal failure (CRF) and normal volunteers. We have also examined the effect of the dialysis procedure on plasma protein oxidation using biocompatible and bioincompatible membranes. Plasma proteins were assayed for the level of free thiol groups using spectrophotometry, protein-associated carbonyl groups by enzyme-linked immunosorbent assay, and oxidation of free amine groups using a fluorescent spectrophotometer. In vitro experiments demonstrate HOCl oxidation of thiol groups and increased carbonyl formation. In vivo, there are significant differences in plasma-free thiol groups between normal volunteers (279 +/- 12 micromol/L), CRF patients (202 +/- 20 micromol/L, P = 0.005) and HD patients (178 +/- 18 micromol/L, P = 0.0001). There are also significant differences in plasma protein carbonyl groups between normal volunteers (0.76 +/- 0.51 micromol/L), CRF patients (13.73 +/- 4.45 micromol/L, P = 0.015), and HD patients (16.95 +/- 2.62 micromol/L, P = 0.0001). There are no significant differences in amine group oxidation. HD with both biocompatible and bioincompatible membranes restored plasma protein thiol groups to normal levels, while minimally affecting plasma protein carbonyl expression. First, both CRF and HD patients have increased plasma protein oxidation manifested by oxidation of thiol groups and formation of carbonyl groups. Second, HD with biocompatible and bioincompatible membranes restored plasma protein thiol groups to normal levels. Third, these experiments suggest that there is a dialyzable low molecular weight toxin found in uremia that is responsible for plasma protein oxidation.
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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.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                January 2005
                27 December 2004
                : 99
                : 1
                : p28-p34
                Department of Medical Biochemistry, University of Madras, Dr. ALM PGIBMS, Taramani, Chennai, India
                81800 Nephron Physiol 2005;99:p28–p34
                © 2005 S. Karger AG, Basel

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                Figures: 3, Tables: 2, References: 47, Pages: 1
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