Detection of endothelial nitric oxide synthase and NADPH-diaphorase in experimentally induced hyperoxaluric animals.

Nitrosative stress plays a role in calcium oxalate stone formation, as nitrosated proteins have been identified in stone formers. Nitric oxide (NO(*)), the common precursor for reactive nitrogen species, is synthesized in the juxtaglomerular apparatus of the kidneys. The present study is aimed to determine the role of nitric oxide synthase (NOS) in an experimental hyperoxaluric condition by histological and biochemical techniques. Hyperoxaluria was induced by 0.75% ethylene glycol in drinking water. L-arginine (L-arg) was supplemented at a dose of 1.25 g/kg body weight orally for 28 days. Nitric oxide metabolites (NOx), protein content in the urine and lipid peroxidation in the kidney were determined at the end of the experimental period. Histopathological examination of the rat kidneys was then carried out. NADPH-diaphorase and eNOS expression studies were carried out in control and hyperoxaluric rat kidneys using histochemical and immunohistochemical techniques. Significant amounts of NOx were present in the urine of hyperoxaluric animals when compared to control rats. Histopathological examinations revealed membrane injury, tubular dilatation and edema in the hyperoxaluric rats, whereas co-supplementation of L-arg to the hyperoxaluric rats significantly reduced these changes. The results of histochemical analysis for NADPH-diaphorase staining demonstrate the role of NOS in hyperoxaluric rats. Hyperoxaluric rats showed intense staining for NADPH-diaphorase when compared to control and L-arg co-supplemented hyperoxaluric rats. Immunohistochemical demonstration confirmed that eNOS expression was markedly increased in L-arg supplemented rats, when compared to EG treated rat kidney sections. Thus, from the present study, we conclude that supplementation of L-arg to the hyperoxaluric animals minimizes the cellular injury mediated by ethylene glycol, prevents oxidative/nitrosative damage to the membranes and reduces the incidence of calcium oxalate stone formation.

Beneficial effect of vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein in hypertensive and hyperoxaluric patients.

BACKGROUND: This study aimed to assess the therapeutic efficacy of oral vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein (THP) in hypertensive and hyperoxaluric patients. METHODS: Newly detected hypertensives (n = 200) and stone formers (n = 200) were each subdivided into two groups. One group (n = 100) was administered the antioxidant vitamin E at 400 mg/day given as an oral supplement along with standard therapeutic drugs for hypertension and hyperoxaluria and the patients were followed for a period of 9 months. The other group (n = 100) did not receive vitamin E (placebo controls). Age and sex-matched controls (n = 100) were monitored simultaneously. THP was isolated from 24 h urine samples before and at the end of every third month during a period of 9 months from the vitamin E-treated hypertensive and hyperoxaluric groups. THP samples were also collected from control subjects, and at the end of the ninth month from placebo controls. The isolated protein was assessed for purity by SDS-PAGE. The purity-checked proteins were subjected to spectrophotometric crystallization assay, calcium oxalate (CaOx) crystal interaction studies, and biochemical analysis of sialic acid, thiol and carbonyl content. Plasma superoxide, hydroxyl radical, hydrogen peroxide and vitamin E levels as well as superoxide dismutase and catalase activities were also monitored. RESULTS: The THP from the hypertensive and hyperoxaluric subjects exhibited a significant promoting effect on the nucleation and aggregation phases and caused a concomitant increase in CaOx crystal interaction. The altered kinetic properties of THP in these subjects were strongly associated with increased carbonyl content and with decreased thiol and sialic acid contents. Oral administration of vitamin E to these patients caused near normalization of these biochemical alterations and satisfactorily restored the kinetic properties of THP to near normal activity. At the end of 9 months, THP isolated from placebo controls (hypertensive and hyperoxaluric) showed highly aggregated calcium oxalate monohydrate crystals as observed by light microscopy. In contrast, vitamin E-supplemented patients showed CaOx dihydrate crystals that were similar to control THP. There was an imbalance in the oxidant and antioxidant levels. For the oxidants, superoxide, hydrogen peroxide and hydroxyl radical levels were increased, and for the antioxidants, there was loss of antioxidant enzyme activities and a decline in plasma vitamin E level in both hypertensive and hyperoxaluric patients. Supplementary antioxidant (vitamin E) corrected this imbalance to near normal conditions. CONCLUSION: We hypothesize that the loss of THP inhibitory activity in the hypertensive and hyperoxaluric patients in a crystallizing medium is mediated primarily by oxidative damage to this protein. The possible occurrence of renal stones in essential hypertensive subjects, and the risk of recurrence in hyperoxaluric subjects, may be explained by oxidative damage to renal tissues that remained unchecked by standard drug therapies. The normalization of the kinetic properties of THP following vitamin E supplementation is in support of our hypothesis.