| Objective:Over-training can induce acute kidney injury, sever cases arelife-threatening. So, it is very important to clarify the pathogenesis and to findmeasures to prevent and treat over-training induced acute kidney injury(OTIAKI) in clinic. Our previous studies have shown that inflammatoryreaction took part in the occurrence and development of OTIAKI. It has beenreported that anisodamine could alleviate kidney injury induced byover-training. However, the exact mechanism hasn’t been reported. Studieshave shown that nucleotide-binding oligomerization-2(NOD2) andinterleukin-6(IL-6) play important role in many inflammatory diseasesprocesses and participate in the inflammatory reaction of renal ischemiareperfusion injury, but the role of NOD2and IL-6in the OTIAKI is unclear uptill now. In present study, we established the animal model of OTIAKI byexhaustive swimming, detected the changes of expression of NOD2and IL-6,clarified the mechanism of them in OTIAKI, learned the effects of anisodaminon the expression of NOD2and IL-6in OTIAKI, and provided new ideas forpreventing and treating OTIAKI in clinic.Methods:Thirty-six male SD rats, weighing180-200g, were randomly divided intothree groups: control group (CN, n=6), exhaustive swimming group (ES, n=18)and anisodamine group (AD, n=12). According to the different recovery timeafter exhaustion, The rats of ES groups were further divided into ESI, ES6hand ES24h, AD groups into AD6h and AD24h. Rats in CN group didn’tparticipate in exhaustion exercise. Rats in AD groups were injectedanisodamine at a dose of10mg/kg20minutes before swimming, rats in ESgroups were injected sterile water at a dose of2ml/kg in the same way.(2) The animal model of overtraining-induced acute kidney injury was developedby exhaustive swimming. Experiments are conducted in a quiet environment.To reduce experimental error, rats in ES and AD groups took adaptiveswimming thirty minutes the day before experiment. In order to avoid ratsdrawing because of carelessness, one rat in every group was chosen in eachexperiment.(3) Rats were sacrificed after anesthesia induced byintraperitoneal injection of10%Chloral Hydrate (3ml/kg). Blood sampleswere collected via the inferior vena cava for determining serum creatinine (Cr)and blood urea nitrogen (BUN). The right kidney was fixed in4℃4%triformol, the left kidney was placed in an EP tube and stored at-80℃fordetecting the expression of NOD2and IL-6. Renal pathological changes wereobserved with light microscope in HE staining. Cr and BUN were measuredby AU2700automatic biochemical analyzer. Expression of NOD2wasdetected by immunohistochemistry and western blotting.IL-6was detected byELISA.(4) Statistical analysis: All experimental were analyzed by SPSS13.0statistical software package, the results were expressed as mean±standarddeviation (X±S). One-way ANOVA and SNK tests were separately used toanalyze the differences within groups and between groups. P<0.05indicatesstatistically significant.Results：(1) General condition: rats in CN group could diet, responsive andactive freely. Rats in ES groups were unresponsive, mental fatigue, shortnessof breath, unstable, loss of coordination, conjunctiva congestion, decreased indiet significantly. Compared with the same period ES groups, rats in ADgroups were better.(2) Kidney pathological changes: there was no obviouspathological changes in CN group; in ESI group, renal tubular epithelialcells were mild edema; Small set of glomerular balloon expansion, proximaltube cavity mild expansion, tubular epithelial cell edema, free vesiculardegeneration, local hyperemia, part of the brush border were not neat andpartial loss, inflammatory cells infiltration were observed in ES6h group; InES24h group congestion and edema were observed in glomeruli significantly, the proximal tubular epithelial cells degeneration, protein casts can be seenoccasionally. Pathological changes in AD groups were lighter than that of thesame period of ES groups.(3)Compared with CN group, the level of serum Crand BUN in ESI and ES6h were significantly increased, and peaked at6h(P<0.05); The value of BUN and Cr in AD6h decreased than ES6h, but stillhigher than CN group(P<0.05)；(4) ELISA results: The level of IL-6in ESIand ES6h group were obviously increased than CN, and up to peak at6h(P<0.05), decreased at24h; Compared with ES6h group, the expression ofIL-6in AD group were decreased(P<0.05)；(5) The results of the immunologyshowed that NOD2mainly expressed in the renal tubular epithelial cellcytoplasm. Image analysis and western blotting results: Compared with CNgroup, the expression of NOD2in ES groups increased obviously, and reachedthe high level in ES6h group(P<0.05). Compared with the same period of ESsubgroups, expression of NOD2in AD groups was markedlydecreased(P<0.05).Conclusions:1The expression of NOD2and IL-6are up-regulated in the model ofOTIAKI, this may indicate that inflammatory reaction plays an important rolein OTIAKI;2Anisodamine can protect kidney from injury in OTIAKI, one of itsimportant molecular mechanisms is that anisodamine can partly down-regulatethe expression of NOD2and IL-6in kidney tissue. |
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