The Role Of The Key Proteins In Rho/ROCK Signal Pathway In Acute Kidney Injury Induced By Overtraining And The Protective Effects Of Anisodamine And Fasudil In Rats

Objective: Acute kidney injury induced by overtraining in the sports andhigh-intensity military training have high morbidity and mortality in recentyears. Therefore, it has important clinical and military significance toinvestigate the pathogenic mechanisms and protective measures. Studies haveshown that Rho/ROCK signal pathway can induce acute kidney injury bydestructing the cytoskeleton, inducing cell apoptosis, activating inflammatoryreaction and mediating vascular smooth muscle contraction and so on.However, it is not fully elucidated that Rho/ROCK signal pathway take part inthe process or not in the pathogenesis of overtraining-induced acute kidneyinjury(OTIAKI). In this study, an animal model of OTIAKI was developed byexhaustive swim. we research the expression and significance of the keyproteins in Rho/ROCK signal pathway in the kidney tissues in OTIAKI andthe protective effects of anisodamine and fasudil in rats to supply informationfor clinical application.Methods:(1) A total of54male Sprague–Dawley rats weighing180~200g were used, the rats were randomly divided into control group (CN, n=6),exhausted group (ES, n=18), anisodamine group (AD, n=12) and fasudilgroup (FA, n=18). The rats of ES group were divided again into threesubgroups: ESI, ES6h and ES24h according to the recovery time afterexhaustion； The rats in AD group received intraperitoneal injection ofanisodamine at the dose of10mg/kg body weight at20minutes beforeswimming and swim to exhaustive condition, then divided again into twosubgroups：AD6h and AD24h；the rats of FA group received intraperitonealinjection of fasudil at the dose of15mg/kg body weight at20minutesbefore swimming and then swim to exhaustive condition. The rat of FA group were divided again into three subgroups：FAI, FA6h and FA24h. An animalmodel of OTIAKI was developed by exhaustive swim.(2) After anesthesiausing intraperitoneal injection of10%Chloral Hydrate (0.6ml/kg), bloodsamples were collected via the inferior vena cava for determining serumcreatinine (Cr), blood urea nitrogen (BUN). The right kidney was fixed in.4%phosphate-buffered paraformaldehyde at4℃for hematoxylin and eosin toobserve the histopathology changes. The left kidney was store at-80℃forWestern Blotting to detect the expression of ROCK-1and the phosphorylationof MYPT1—a substrate of ROCK-1.(3) Statistical analysis: All statisticalanalysis of the experimental data were used SPSS13.0statistical softwarepackage, the results were expressed as mean±standard deviation (x±s).Differences are compared using single factor analysis of variance (one-wayANOVA), between groups comparison using SNK method, the P<0.05wasconsidered statistically significant.Results:(1) The rats in CN group could have food normally, move freely,fur smooth and shine, sensitive reaction to the outside various stimuli. Theexhausted rats after exercise were tremble, laziness, hard to active and unableto stand and bulbar conjunctiva hemorrhage, unresponsive to external stimuli.AD and FA groups were better than ES group in general changes and themental state after exhaustive swim.(2) The levels of serum BUN and Scrincreased markedly in ESI rats (P<0.05), higher in ES6h rats (P<0.05), andreturned to CN levels in ES24h rats (P>0.05). AD6h, FAI, FA6h groupswere significantly lower than corresponding subgroup of ES group (P<0.05),but still higher than CN group (P<0.05). AD24h and FA24h groups returnedto CN levels (P>0.05).(3) No obvious changes in renal tissue structure weredetected in CN group and some light histopathological changes, such asdilatation of renal tubules, edema in renal tubular epithelial cells and werefound in ESI and ES6h groups. However, in ES24h group rats,which havesignificantly changes, more dilatation in renal tubules, irregular or evendeciduous brush border were observed in renal tubular epithelial cells. Therats in AD and FA groups showed significantly slight in structure changes compared to the corresponding subgroup of ES group.(4) The results ofWestern Blotting: the expression of ROCK-1and P-MYPT1have significantlyincreases in ES group compared to the CN group (P<0.05), and peaked at6hafter exhaustion (P<0.05). Compared with corresponding ES group, theexpression of ROCK-1and P-MYPT1was decreased in AD and FA groupswere significantly decreased (P<0.05).Conclusions:(1)The expression of ROCK-1and P-MYPT1enhanced in the kidneytissues with OTIAKI and pretreatment with Fasudil—a specific inhibitor ofRho-kinase, can prevent the development of the changes, which remind usRho/ROCK signal pathway have an important role in OTIAKI.(2) Administration advance with Anisodamine can decrease theexpression of ROCK-1and P-MYPT1in OTIAKI, which suggestAnisodamine can alleviate kidney injury via inhibit Rho/ROCK signalpathway.(3) Fasudil—a specific inhibitor of Rho-kinase has protective effect onovertraining-induced acute kidney injury, which supply information forclinical application.