The Role of Free Radicals in Experimental Nephrolithiasis

• Main Authors: Ho-Shiang Huang, 黃鶴翔
• Other Authors: Chau-Fong Chen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/06275514898223705942

博士 === 國立臺灣大學 === 生理學研究所 === 90 === Free radicals, including superoxide anions, peroxides and hydroxyl radicals, are unstable chemical species and highly reactive. Free radicals damage lipids, proteins and deoxyribonucleic acid with consequent effects ranging from cell dysfunction to neoplasia, and reactive oxygen species have been implicated in the pathogenesis of several diseases. Toxic nature of oxalic acid and its nephrotoxicity was well recognized since the nineteenth century. Its injurious effects were considered to be a result of the physical properties of its calcium salts, the calcium oxalates, which are insoluble at physiological pH. Reviewing the literature, we found that oxalate itself might be injurious to renal epithelial cells and this injury may be exaggerated in the presence of calcium oxalate (CaOx) crystals. Therefore, we designed four experiments to evaluate the possible role of free radical in the rat model of experimental nephrolithiasis. The aim of the first experiment was to evaluate whether free radicals production could be induced in the kidney of chronic hyperoxaluric rats and to examine the correlation between urinary enzymes, including N-acetyl-bate-glucosaminidase (NAG), beta-galactosidase (GAL), and neutral endopeptidase (NEP), and free radical production. We found that urinary oxalate increased significantly and the increased urinary oxalate was associated with elevation of urinary marker enzymes (NAG and GAL) at all time points of observation. However, urinary NEP activity was no change on day 7, though four times elevation of that was found on days 21 and 42 when compared with the control groups. Formazan particles in the renal cortex were scored as 3+ to 4+ in rats treated for 7 days with ethylene glycol (EG). Elevated blood chemiluminescence (CL) levels were also found in this group when compared to the control groups. On day 42, 1+ to 2+ formazan particles were scored in the kidney, but the blood CL level was normal. After vitamin E and potassium citrate treatment, the blood CL levels were decreased when compared with those treated with EG alone. The second study was designed to investigate the possible mechanism of increased free radicals, the role of antioxidant enzymes, and their correlation with renal tubular damage in the kidney after feeding 0.75% EG to male Wistar rats (1、3、5、7、9、21 and 42 days). We found that the CL levels of the renal venous blood samples were elevated on days 1, 3, and 7 (p<0.05), and those of the kidney were elevated only on days 3 and 42 (p<0.05), when compared with controls. The infiltration of CD45-positive cells in the kidney increased on day 7, and a further increase in these positive cells was noted on day 21. Fused ED1-positive cells surrounding the CaOx crystals and adjacent to the NBT positive area were found on day 42. NADPH-dependent oxidase activity was elevated in all experimental groups (p<0.05). The activities of catalase (CAT) and manganese superoxide dismutase (MnSOD) were elevated in the early stage. On day 42, almost all antioxidant enzyme activities were attenuated (p<0.05), except for CAT. The urinary levels of alpha-GST were elevated from day 7 till day 42, whereas levels of mu-GST were elevated from day 3 till day 42, except day 5. The third experiment was designed to evaluate possible changes in renal function in rats with chronic hyperoxaluria and after acute oxalate challenge. We also evaluated the possible association between free radical (FR) production, hyperoxaluria, and CaOx calculi formation. We found that urinary lipid peroxides, and enzymuria had increased since day 7, and increased CaOx crystals in the kidney were noted beginning on day 21, but elevated CL was detectable only on day 7 after 0.75% EG treatment. Decreased urine flow (UV) and urine sodium excretion (UNaV) were noted in the EG 42-day group, though the 24-hour creatinine clearance values were normal in all experimental groups. On the other hand, RBF, GFR, and CMVBF were attenuated with elevated FR when the oxalate concentration was higher than 10 mg/kg/hour in the acute oxalate infusion group. With SOD pretreatment via a renal arterial catheter, the decreased RBF, GFR, and CMVBF could be reversed at 10 mg kg-1hour-1 of oxalate, and be partially reversed at 20. FR was also reduced significantly at 10 and 20 mg kg-1hour-1 of oxalate. The fourth experiment was designed to evaluate the possible correlations between urinary oxalate level, kidney free radical amount, and the formation of renal CaOx calculi in hyperoxaluric rats. We also evaluated the effect of vitamin E supplement on the CaOx nephrolithiasis in this rat model. The rats of group 5 had the same protocol as that of group 4 (EG with low vitamin E diet), except they received supplemental vitamin E for diet shortage. We found the rats received 0.75% EG in drinking water with additional vitamin E supplement (200 mg/kg/each time, twice a week, i.p.) had grade I kidney stone with lowered TBARS and elevated MDA, and tubular enzymuria was also increased significantly. The rats treated with low vitamin E diet with 0.75% EG had marked renal calculi with decreased renal function. The rats treated with low vitamin E diet with 0.75% EG and supplemental vitamin E had the same experimental feature as those treated with EG and standard rat diet. The rats treated with low vitamin E diet and normal distilled water had elevated urine lipid peroxides, normal urinary oxalate levels, low serum vitamin levels, but no renal calculus. From these results we can conclude that free radical production can be happened during the course of nephrolithiasis of rats fed with EG. The possible mechanisms that made the hyperoxaluric kidney under excessive oxidative stress were 1). Exposure to the elevated urinary oxalate levels, 2). Associated with calcium oxalate crystals, 3). Infiltrated leukocyte and macrophage. Decreased urine flow and sodium excretion were the main renal functions injured in chronic hyperoxaluria. With acute oxalate infusion, the major insult to renal function was renal hemodynamics. Pretreated SOD could reverse the attenuated hemodynamics and reduce the elevated FR partly, which implied that FR is responsible for oxalate toxicity. Renal tubules damaged by oxalate may play a major role of CaOx nephrolithiasis. Low serum vitamin E level increased the oxidative stress of the kidney, but no associated tubular enzymuria and renal calculi could be induced. Vitamin E supplement can offer partial protection of the kidney from CaOx nephrolithiasis, and a low vitamin E diet can make the kidney CaOx calculi more prominent in this hyperoxaluric rat model. Some other factors or mechanisms may also participate in inducing nephrolithiasis in this model and these need further investigation.