| ObjectiveTo investigate the protective effect and mechanism of gadolinium chloride, which inhibited the activation of restraining Kupffer cell via hepatic ischemia reperfusion (I/R) in rats and released the renal injury.Materials and Methods1. Materials100 male Wistar rats (180-220 g,7-8weeks age) were divided into two groups at random, Gd group:GdCl3 exposure and ischemia/reperfusion; control group:normal saline and ischemia/reperfusion. Gadolinium chloride (bought from Japan); correlated biochemical agents were bought from Boehringer Mannheim Company in Germany; TNF-a kit was bought from American BD Company; MDA kits were purchased from Nanjing Jiancheng Biological Engineering Research Center. MMP-9 antibody (Santa Cruz Company, USA); MMP-2 antibody (Santa Cruz Company, USA). HITACHI-7600A automatic biochemistry analyzer; Finland Multlskan MK-3 automatic multi-function ELIASA; the picture analyses were all finished with MetaMorph/Evolution MP5.0/BX51 micrograph analysis system.2. MethodsIn Gd group, GdCl3 (10mg/kg) was injected via the tail vein at 24,48hours before operation; in control group, the same amount of normal saline was injected. Left portal vein, left hepatic artery and hepatic duct were blocked for 60 min and then reperfused to establish liver I/R model in rats. In each group, samples were collected at 0.5,1,6, 12 and 24hours after reperfusion respectively.10 rats were observed in parallel at each time point. All the rats were treated with 10% Chloral Hydrate (3ml/kg) via the abdominal cavity before the operation.4% Paraformaldehyde-0.1 mol/L phosphate buffer (pH=7.3) was used for immunohistochemisty specimen fixation. Blood was collected from the heart through the diaphragm. The clear supernatant liquid after five-minute centrifugation was preserved in the low temperature refrigerator for the next measuration. The level of TNF-a in the serum was measured by ELIASA. The middle 1/3 part of the left kidney was taken for measuring the concentration of MDA, the lower 1/3 part of the left kidney (about 2×1×0.3cm) was taken for immunohistochemical staining. Immunohistochemical staining was observed under an optical microscope at original magnification×400, and the presence of yellow granules in the cytoplasm was defined positive.5 fields in each section were averaged, and the value was expressed as mean±SD. All data were analyzed by the statistical software SPSS 13.0 with t test.ResultsAfter reperfusion for 1h, the levels of BUN in the exposure group at each time point were significantly lower than those in control group, and there was statistically significant difference between GdCl3 exposure group and control group at the times of 6,12,24 hours (P< 0.05). The levels of Cr in the GdCl3 group were lower than those in control group at all time points after reperfusion, and there was statistically significant difference between Gdcl3 exposure group and control group at the times of 6,12,24 hours (P< 0.05). The levels of TNF-a in the GdCl3 group were lower than those in control group at all time points after reperfusion, and these differences were statistically significant. The tendencies of TNF-a increasing in both groups were basically same. The peaks of TNF-a quantity in both groups were observed at 12 hours, and then the TNF-a quantity began to decrease. The levels of MDA in the GdCl3 group were lower than those in control group at 6,12 and 24 hours after reperfusion (P<0.05). The IOD values of MMP-2 in both groups increased gradually after reperfusion, and then the amplification of the increase became slower. The IOD values of MMP-2 in GdCl3 group were lower than those in control group at all time points after reperfusion, and there was statistically significant difference at 6,12 and 24 hours after reperfusion(P<0.05). The positive cells were mainly the proximal convoluted Tubule epithelial cells in the deep layer of cortex and vascular endothelial cells and interstitial cells near the renal tubule. The IOD values of MMP-9 in both groups increased gradually after reperfusion, but the increasing rate of MMP-9 in the GdCl3 group was slower than it in control group. The IOD values of MMP-9 in the GdCl3 group were lower than those in control group at all time points, and there were statistically significant differences at 6,12 and 24 hours (P<0.05).The parts of positive expression of MMP-9 were similar with those of MMP-2. The positive cells were mainly the proximal convoluted tubule epithelial cells in the deep layer of cortex and vascular endothelial cells and interstitial cells near the renal tubule.ConclusionsDuring the course of hepatic ischemia-reperfusion, Kupffer cells are activated and release a large amount of bioactive substance, which can lead to renal injury. Bioactive substances, such as TNF-a and oxygen radicals, not only cause renal injury directly, but also can upregulate the expression of MMP-2, MMP-9 and aggravate the renal injury. Renal injury can be relieved via inhibiting the activation of Kupffer cells, deceasing the release of TNF-a and oxygen radicals and avoiding the expression upregulation of MMP-2 and MMP-9 in the renal tissue.
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