| Objective: Overtraining cause rhabdomyolysis (RM), which can leadto acute kidney injury (AKI). AKI has been the most important kidneyemergency, therefore the effective treatment is a worldwide difficult problem. How to prevent and treat AKI due to overtraining has become an important research topic in the fields of sports medicine, military medicine and clinical medicine. In the pathogenesis of AKI caused by overtraining, the renal toxicity of myoglobin is an important factor. The absorption of myoglobin relies on the the Megalin glycoprotein expressionin the renal tubular proximal end, but the specific pathogenic molecularmechanisms and the molecular target sites of early intervention treatment rarely reported. In this study, we used the acute renal injury model induced by glycerol which lead to rhabdomyolysis, observed the changesof biochemical indicators, tested the expression of Megalin and a-1,6-fucoseglycosyltransferase (FUT8), which have monitor function in decorating after translation of Megalin, and the influence of Anisodamine (AD)on the expression of these indicators. This research can provide experimental and theoretical basis for further study of the pathogenesis of AKI,early drug intervention target sites and effective prevent.Methods:⑴H ealthy male SD rats(n=42), weighted200-220g, wererandomly divided into three groups: control group (Control, CN, n=6),acute kidney injury model group (AKI, n=18). The Anisodamine intervention group (AD, n=18). AKI group and AD group were divided into three subgroups based on injection time,1h,6h and24h group, each group had six rats.⑵The model of AKI induced by rhabdomyolysis wasdeveloped by injecting glycerol saline to the posterior limbs of rats.⑶Bl ood urea nitrogen(BUN), serum creatinine(Scr), and createnekinase(CK)were determined with the automatic biochemical analyzer, blood musclemyoglobin(Mb) were measured by ELISA, The morphological changes of the kidney tissue were observed with microscope, Immunohistochemistry andimage analysis technology were used to detect the expression ofMegalin and FUT8, the protein expression of FUT8were measured byWestern Blot.⑷Statistical analysis: All of the experimental datas wereanalysed by using SPSS13.0software package for statistical processing,the results were presented as mean±standard deviation (X——±s). Differences were compared by using single factor analysis of variance (one-wayANOVA), Paired t-test was used to compare the differences between the same point time of groups. Correlation analysis using Pearson correlation test, the p<0.05was considered statistically significance.Results:⑴The levels of BUN, Cr, Mb of AKI6h and AKI24h groups were significantly higher than CN group (p<0.05), reached the peak in AKI24h group (p<0.05); the levels of CK were increased in AKI1h group, reached the peak in AKI6h group, and felt in AKI24h; compared with the same period of AKI groups, the level of BUN, Cr, Mb of AD groups were significantly decreased (p<0.05).⑵Light microscopy: The morphological of renal tissues had no changes in CN group, pathological changes were gradually increased in AKI group. These changes including numerous tubular epithelial cells darkly stained and pyknoticnuclei, necrosis, collapse, irregular shedding. Lumen tube can be seen inrenal tubular formed by a large number of the proteins and shedding of epithelial cell debris. The interstitial capillaries congestion, inflammatory cells infiltration, and some tubular blocked. The pathological changesin AD groups reduced compared with the same period of AKI groups.⑶Immunohistochemical results:①The expression of Megalin: Most positive cells were renal tubular epithelial cells, glomerular and interstitial expressed occasionally. The expression of Megalin were weak in CN group, and little in AKI1h group (p<0.05), and significantly increased in AKI6h group (p<0.05), the expression in AKI24h group reached the peak (p<0.01). The expression in AD groups were significantly lower than the same period of AKI groups (p<0.05).②The expression of FUT8:Most positive cells were renal tubular epithelial cells, glomerular and interstitial expressed occasionally. FUT8had weak expression in CN groupWith the prolongation of glycerol action time, the expression of FUT8gradually increased in AKI groups (p<0.05), reached the peak in AKI24h group. All expression in CN groups were lower than AKI groups (p<0.05). The expression in AD groups were significantly lower than thesame period of AKI groups (p<0.05).⑷Western Blot test results: Underthe condition of basically identical expression of internal reference (GAPDH), FUT8band had lighter color in CN group, with the prolongationof glycerol action time, the band of FUT8gradually deepened in AKIgroups (p<0.05), the bands in AD groups was significantly lighter thanthe same period of AKI groups (p<0.05). Statistical significant differences had been found between AD groups and the same period of AKI groups (p<0.05).(5) Correlation analysis results: Respectively, the plasma Mb had significantly positive correlation with the value of renal functionBUN and Scr (r=0.872, p<0.05; r=0.716, p<0.05), and Megalin and FUT8protein (r=0.843, p＜0.05; r=0.802, p＜0.05). The expression levelsof Megalin protein showed a significant positive correlation with FUT8protein (r=0.965, p＜0.05).Conclusions:1The expression of Fut8in renal tissues of rats enhanced when rhabdomyolysis induced by glycerol, furthermore, which cause excessive intake of myoglobin with nephrotoxicity by upregulating the core fucosylated modification of Megalin to cause or aggravate kidneyinjury.2Anisodamine significantly decreased the protein expression of Megalinand FUT8, alleviating AKI induced by overtraining, played an important role in protecti ng the kidney injury. |
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