| Objective: Oxygen free radicals (OFR) are produced through an oxidizing agent or during corpuscular aerobic oxidation. Superoxide dismutase and catalase clear free oxygen radicals quickly in vivo and lessen the impact of accumulating OFR in an organism's cells. Ischemia and anoxia directly decrease nourishment material and ATP within the cells, which result in increased OFR. This surge in OFR under these conditions exceeds the cells capacity to clear these harmful molecules. Unscavenged OFR can cause immediate damage and induce apoptosis. Previous study showed that oxygen free radicals play an important role in the occurrence and development of kidney disease. Reducing the synthesis or release of OFR within the cell could prevent or correct damage to the kidney. The present study observed the role of lipoic acid (LA) and vitamin E (VE) on renal ischemia-reperfusion injury (RIRI) and their underlying mechanisms through use of physiological, biochemical, histological and flow cytometer methods.The results were as follows: 1 The effect of lipoic acid on expression of HSP70 and NO in renal ischemia-reperfusion injury of aged ratsMethods: The model of RIRI was induced by bilateral clamping the renal artery and vein for 45 min followed by reperfusion and observing the effect of LA on RIRI in aged rats. Rats were divided three groups: Sham; IR and IR+LA groups. For the LA treatment group, four weeks prior to the RIRI surgery, rats were started on oral LA treatment (250 mg/kg/d). After RIRI surgery, each layer opened was closed with suture, including the peritoneum, abdominal wall muscle and skin. IR group rats were fed the same doses solution in the same way. The rats in the sham group were treated identically except for the clamping. After 24 hours of reperfusion, the blood and urine samples were taken for detection of serum creatinine (Scr), blood urea nitrogen (BUN), urea N-Acety-P-beta-Glucosaminidase (UNAG), superoxide dismutase (SOD), nitric oxide (NO), and malondialdehyde (MDA). After blood was taken, the left kidney was excised for histological examination. Immunohistological and flow cytometry were used to determine the hot shock protain 70 and apoptosis rate of renal cortex cells.Results: Renal function was impaired in aged rats. Levels of BUN, Scr, MDA, NO and UNAG were significantly increased in RIRI rats compared to sham animals (80.55±22.13 vs 20.06±3.43, 2.68±0.70 vs 1.08±0.21; 3.61±0.74 vs 2.41±0.41, 4.67±1.06 vs 1.87±0.41, 48.31±6.82 vs 15.43±5.97, P<0.01). IR group rats showed decreased SOD compared to sham rats (471.51±15.60 vs 639.09±27.45, P<0.01). The IR group rats'renal tubule epithelial cells showed signs of damage, especially in the proximal convoluted tubule, in which the lumina was enlarged; also there were some cast and caduceus cells in many of the renal tubules. Chromatin was localized in the cell nucleus periphery. Flow cytometric analysis revealed a conspicuous apoptotic peak in the IR group, as well as showing an increased rate over sham group (0.39±0.02 vs 0.16±0.03, P<0.01). In IR group, HSP70 was highly expressed. A large number of brown particles were detected in the IR group in the renal tubular epithelial cells. These particles had a diffuse distribution, but staining was absent in the glomeruli. Pre-treatment with LA normalized many of the abnormal values found than in the IR alone group. BUN, Scr, MDA, UNAG were significantly decreased in IR+LA group (49.46±4.85 vs 80.55±22.13, 1.27±0.10 vs 2.68±0.70; 2.69±0.38 vs 3.61±0.74, 21.09±4.29 vs 48.31±6.82, P < 0.01), SOD was significantly increased (619.61±13.84 vs 471.51±15.60, P<0.01), and serum NO was increased (8.00±0.72 vs 4.67±1.06, P < 0.01). From the histological assessment, renal damage was less severe in IR+LA than IR alone. The nucleus appeared normal, and the few cast cells present were localized to the tubule cells only. The rate of apoptosis was lower in the LA treated group (0.23±0.05 vs 0.39±0.02, P<0.01). The IR+LA group also showed a high expression of HSP70. Experimental results suggest that HSP70 plays an important role in the pathogenesis of RIRI in rats. LA might have a protective effect on RIRI rat's kidney through decreased OFR and increased expression of HSP70.2 The effect of vitamin E on renal ischemia reperfusion in ratsMethods: The animal model of RIRI and experimental methods were made as describe in part 1. Four weeks prior to RIRI, rats were started on oral VE (500mg/kg/d) to comprise the in IR+VE.Results: Renal function was significantly damaged in aged rats. Compared with those in sham group, contents of BUN, Scr, MDA NO and UNAG were significantly increased in RIRI rats (80.55±22.13 vs 20.06±3.43, 2.68±0.70 vs 1.08±0.21; 3.61±0.74 vs 2.41±0.41, 4.67±1.06 vs 1.87±0.41, 48.31±6.82 vs 15.43±5.97, P<0.01). Contents of SOD were significantly decreased in RIRI rats (471.51±15.60 vs 639.09±27.45, P<0.01) than in sham group rats. In IR group, renal tubule epithelial cells obviously damaged especially proximal convoluted tubules. It was also showed that lumina of renal tubule extended in IR rats. There were some cast in renal tubules. Chromatin went together to the periphery of cell nucleus. It was found that some vacuolus and necrosis in renal cortex tobular cells. Renal cortex cells appeared conspicuous apoptotic peak in IR group. Compared with sham group, apoptosis rate significant increased (0.39±0.02 vs 0.16±0.03, P<0.01). HSP70 expressed obviously increased in IR group. Compared with IR group, contents of BUN, Scr, MDA, UNAG were significantly decreased in IR+VE group (37.97±9.15 vs 80.55±22.13, 1.34±0.10 vs 2.68±0.70; 2.75±0.29 vs 3.61±0.74, 23.92±2.73 vs 48.31±6.82, P<0.01). Contents of SOD were significantly increased in IR+VE group (570.92±19.76 vs 471.51±15.60, P<0.01). Contents of NO in serum were significantly increased in IR+VE group (7.07±0.94 vs 4.67±1.06, P<0.01). The degree of renal tubule injury in IR+VE group were obviously less and lighter than IR group. Nucleus fundamentally was normal. A small quantity of Cast was merely seen in renal tubules. The apoptosis rate of renal cortex cell was obviously attenuated than IR group (0.25±0.05 vs 0.39±0.02, P < 0.01). The expression of HSP70 was upragulated in IR+VE group than in IR group rats.The above results suggested that vitamin E possiblely had beneficial effects through decrease OFR and the apoptosis rate of renal cortex cell.Conclusions:1. BUN, Scr, UNAG, MDA, and apoptosis are greatly increased in the renal cortex of IR exposed rats compared to shams, revealing severe impairment of renal function. HSP70 immunoreactivity was increased in the kidneys of IR rats, suggesting a protective role for HSP70 in response to the stress of RIRI.2. LA and VE normalized renal function in the face of RIRI, and renal immunoreactivity of HSP70 was increased. This indicates HSP70 is elevated by a different mechanism than RIRI alone.3. LA and VE could produce their protective effect by decreasing intracellular OFR damage and/or upregulation of HSP70, causing renal protection and systemic vasodilation, respectively.
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