Molecular Mechanism Of Overtraining-induced Acute Kidney Injury And The Protective Effects Of Natural Effective Compounds On OTIAKI In Rats

Objective : Overtraining is the pathological states characterized by multiorgan dysfunction induced by overload of exercise and beyond the body ability. In recent years, the studies on overtraining-induced organ injury is paid great attention. However, the studies on overtraining-induced acute kidney injury(OTIAKI) have a little progression. The pathological pattern and cellular and molecular pathogenesis responsible for OTIAKI is unclear. Up to the present, there is few effective drug therapy in clinical practice. Limited evidence indicates that renal cell apoptosis is involved in the development of exercise-induced proteinuria, but the significance and molecular mechanism of renal cell apoptosis in OTIAKI is unknown. Therefore, it is very important to clarify the pathogenesis and to find the methods to prevent and treat OTIAKI. It has been reported that Anisodamine relieves angiospasm, improves microcirculation and has the protective effects on ischemic acute renal failure, Inula Britannica regulates the cell apoptosis by anti-inflammation, Ixeris Sonchifolia Hance has the effect of anti-oxidative stress. However, the protective effect of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAKI and its molecular mechanism were not yet reported. In the present study, we investigated the pathological feature and pathogenesis of OTIAKI, and the role of renal cell apoptosis in development of OTIAKI, and inducing factor, gene regulating and cell signal transduction pathway mediating renal cell apoptosis in process of OTIAKI, by detecting the changes of expression of the key signaling molecules and cytokine in the renal tissue of OTIAKI rats. In addition, the protective effect and molecular mechanism of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on the development of OTIAKI were studied in the OTIAKI rats. Methods: Eighty male Wistar rats were randomly divided into five groups: control group(CN, n=8), exhaustive swimming(ES, n=24), Anisodamine(AD, n=16), Inula Britannica(IB, n=16) and Ixeris Sonchifolia( IS, n=16). The rats of CN were quiet and no swimming. The rats of ES swimed to exhaustive state and sacrificed at immediately(ESI), 6 hour(ES 6 h) and 24 hour(ES 24 h) after exhaustive swimming. The rats of AD received intraperitoneal injection of Anisodamine at the dose of 10 mg/kg body weight at 20 minutes before swimming and then swimed to exhaustive condition. The rats of IB took orally Inula Britannica at the dose of 25 ml/kg body weight at 24 h before swimming and then swimed to exhaustive condition. The rats of IS received intraperitoneal injection of Ixeris Sonchifolia Hance at the dose of 20 g/kg body weight at 20 minutes before swimming and then swimed to exhaustive condition. The rats of AD, IB and IS group were sacrificed at 6 hour ( AD 6 h, IB 6 h, IS 6 h) and 24 hour (AD 24 h, IB 24 h, IS 24 h) after exhaustive swimming. The animal model of overtraining-induced acute kidney injury was developed by exhaustive swimming. The creatine, urea, CK and urineγ-GT levels in serum were measured by biochemistry analyzer. The renal tissue morphological changes were observed by Olympus microscopy and transmission electron microscopy. The renal cell apoptosis was measured by the method of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling(TUNEL) and flow cytometry. The changes of NF-КB, FasL, caspase-8, caspase-9 in renal tissue were measured by the way of flow cytometry and immunohistochemistry. The expression of TNF-α, Bax, Bcl-2, Bid and E-Cadherin in renal tissue were observed by immunohistochemistry. The changes of SOD and MDA in serum and renal tissue were measured. All data were expressed asˉx±s. F and T test was separately used to analyse the difference between the two groups and within groups, which was finished with SPSS 12.0 software.Results:1. The pathological pattern of overtraining-induced acute kidney injury and the role of proinflammatory cytokine and NF-КB To deterimine the pathological pattern and the pathogenesis of OTIAKI, the renal morphorological, functional, metablic changes and SOD and MDA levels in serum and renal tissue and the expression of TNF-αand NF-КB in renal tissue were investigated in this section. The results were as follows:The level of serum Ur, Cr and CK increased markedly in ESI rats( p<0.05), higher in ES 6 h rats( p<0.05), and decreased to CN levels in ES 24 h rats. The level of urineγ-GT increased progressively from 6 h to 24 h after exhaustive swimming( p<0.05). HE staining showed that the morphological slight changes were observed in ESI and ES 6 h rats, the brush borders of tubular epithelial cells were in poor order and partly fallen off, there were a number of tubular epithelial cells whose nuclei were stained heavily and condensed in the medulla and the area between the cortical and medulla in ES24 h rats. Transmission electron microscopy showed that glomerular filtration membrane foot processes blended, the microvilli of the proximal convoluted tubular were not in order, the epithelial cytoplasm was swollen, the protruding and membrane of mitochondrion were entirely and partly blended and disappeared, the chromatin of nuclei condensed and the higher electronic dense chromatins were gathered beneath the nuclear membrane, which indicated the early stage of epithelial cell apoptosis was developed, the changes of renal tissue ultrastructure were progressively serious, and typical changes of apoptosis of renal tubular epithelial cells were observed in ES 6 h and ES 24 h rats. It was revealed by TUNEL that the number of renal tissue apoptotic cells was increased in ESI, ES 6 h and ES 24 h rats. Compared with CN, the renal tissue cell apoptotic ratio also significantly increased in ESI, ES 6 h and ES 24 h rats( p<0.05) by the way of flow cytometry. The urineγ-GT had significantly positive correlation with renal cell apoptosis ratio(r=0.98, p<0.01). The SOD decreased and MDA increased significantly in serum and renal tissue of ESI and ES 6 h rats (p<0.05). The serum SOD was increased to CN levels and MDA decreased to CN levels in ES 24 h rats. The renal tissue SOD was increased but was lower than CN, and renal tissue MDA decreased but was higher than CN levels. Immunohistochemistry staining showed that positive stainings for TNF-αand NF-КB were observed. The positive expressions of TNF-αand NF-КB were increased progressively at 0 h, 6 h and 24 h after exhaustive swimming( p<0.05), It was revealved that the values of NF-КB were significantly increased in ESI, ES 6 h and ES 24 h rats by flow cytometry( p<0.05). These results suggest that the overtraining could induced acute kidney injury involving the renal dysfunction, renal tissue morphological slight changes and obvious renal cell apoptosis by increasing oxidative stress and inflammatory signal pathway.2. The protective effects of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAKIAnisodamine, Inula Britannica and Ixeris Sonchifolia Hance are common natural effective compounds which have relieve angiospasm, anti-inflammation and anti-oxidative stress. They have counter tissue ischemia, anoxia and apoptosis caused by various cause. To find the drug therapy, their protective effects of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAKI were studied in this part.The swimming times were longer in AD, IB and IS rats than ES rats( p<0.05). The level of serum Ur, Cr and CK and urineγ-GT at 6 h of AD, IB and IS rats and urineγ-GT at 24 h of AD, IB and IS rats was decreased remarkably compared with ES rats. The renal tissue structural and ultrastructural changes were slight at 6 h and 24 h of AD, IB and IS rats. TUNEL and flow Cytometry showed that renal cell apoptotic ratio were lower than ES rats but higher than CN rats at 6 h and 24 h of AD, IB and IS rats, especially in AD rats. Compared with CN, the level of SOD in serum increased markedly in AD 6 h, IB 6 h and IS 6 h rats, the level of MDA in serum decreased significantly in AD 6 h, IB 6 h and IS 6 h rats( p<0.05), the level of SOD in serum increased to CN levels and the level of MDA in serum decreased to CN levels at 24 h of AD, IB and IS rats. The level of SOD in renal tissue increased markedly at 6 h of AD, IB and IS rats( p<0.05), especially in AD rats.The level of MDA in renal tissue decreased remarkably at 6 h of AD, IB and IS rats( p<0.05), especially in AD and IS rats. The level of SOD and MDA in renal tissue returned to CN levels at 24 h of AD, IB and IS rats. Immunohistochemistry staining showed that the expression of renal tissue TNF-αdecreased markedly but was higher than CN rats at 6 h and 24 h of AD, IB and IS rats, the expression of renal tissue NF-КB decreased markedly but was higher than CN rats at 6 h of AD, IB and IS rats, and also decreased significangtly but was higher than CN rats at 24 h of AD, IB and IS rats, especially in AD rats. Flow cytometry showed that the values of NF-КB decreased markedly but were higher than CN rats at 6 h of AD, IB and IS rats, and also decreased significangtly but were higher than CN rats at 24 h of AD, IB and IS rats, especially in AD and IB rats. These data indicates that Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance could improve renal function, extenuated renal morphological changes and apoptosis, increased the exercise ability by anti-inflammation and anti- oxidative stress.3. Gene regulation of renal cell apoptosis and effects of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance on overtraining ratsTo understand the pathogenesis of renal cell apoptosis induced by overtraining, in this part, the expression of apoptosis-related genes and influences of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance were studied. The results were as follows:Compared with CN, the expression of Bax in renal tissue was stronger(p<0.05), the expression of Bcl-2 in renal tissue was weaker( p<0.05), the Bax/Bcl-2 ratio increased remarkably in ESI rats, and these changes were more prominent in ES 6 h rats and reached the higher level in ES 24 h rats. The Bax/Bcl-2 ratio had significantly positive correlation with renal cell apoptosis ratio (r=0.674, p<0.01). Compared with ES, the expression of Bax in renal tissue decreased remarkably at 6 h of AD, IB and IS rats( p<0.05), especially in AD and IB rats, the expression of Bcl-2 in renal tissue increased significantly( p<0.05), the Bax/Bcl-2 ratio decreased remarkably at 6 h of AD, IB and IS rats( p<0.05), and these changes were more obvious in AD and IB groups. The expression of Bax in renal tissue decreased remarkably(p<0.05), the expression of Bcl-2 increased significantly( p<0.05), especially in AD rats, the Bax/Bcl-2 ratio decreased remarkably at 24 h of AD, IB and IS rats( p<0.05). These results suggest that the imbalance of Bax and Bcl-2 is one of the important pathogenesis in development of overtraining-induced renal cell apoptosis, Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance have the obvious anti-apoptosis effect by regulating the expression of Bax, Bcl-2 and the Bax/Bcl-2 ratio.4. The changes of caspase-related cell apoptosis signal transduction pathway in OTIAKI ratsTo study apoptosis signal transduction pathway and find the molecular target of drug therapy, the expressions of a group of caspase-related cell apoptosis signal molecular were observed in this part. The results showed that the positive stainings for renal tissue FasL, caspase-8, caspase-9 and Bid were observed, the positive expressions of renal tissue FasL, caspase-8, caspase-9 and Bid were increased progressively at 0 h, 6 h and 24 h after exhaustive swimming by Immunohistochemistry staining compared with CN ( p<0.05). It was revealved that the values of renal tissue FasL, caspase-8 and caspase-9 were significantly increased in ESI, ES 6 h and ES 24 h rats by flow cytometry( p<0.05). These results suggest that caspase-related apoptosis signal transduction pathway plays an important role in the development of overtraining-induced renal cell apoptosis.5. The molecular mechanism of protective effect of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance on OTIAKI in ratsTo understand the molecular mechanism of protective effect of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance on OTIAKI in rats, the influences of above three drugs on the expression of apoptosis-related key signal molecular were observed.Immunohistochemistry staining and flow cytometry showed that the positive expressions of renal tissue FasL, caspase-8, caspase-9 and Bid decreased markedly but were higher than CN at 6 h of AD, IB, IS rats and in AD 24 h, IB 24 h and IS 24 h rats compared with ES rats, and those changes were more prominent in AD 24 h, IB 24 h rats. These results suggest that above three drugs can decrease the apoptosis signal transduction in caspase-related signal pathway.Immunohistochemistry staining showed that the positive expressions of E-cadherin in renal tissue decreased progressively in ESI, ES 6 h and ES 24 h rats compared with CN ( p<0.05). Compared with ES, immunohistochemistry staining showed that the positive expression of E-cadherin in renal tissue increased markedly to CN level at 6 h and 24 h of AD, IB, IS rats(p<0.05). These results indicate that overtraining-medicated the loss of E-cadherin in renal tissue can induce acute kidney injury, three drugs can inhibit above changes and extenuate OTIAKI.Conclusions:1 The overtraining could induce acute kidney injury including the renal dysfunction, renal tissue morphological changes by inducing renal cell apoptosis.2 The overtraining could induce renal cell apoptosis by increasing oxidative stress, impairing the balance of Bax and Bcl-2, up-regulating the expression of apoptosis-related key signal molecular in caspase-related signal pathway, inflammatory signal pathway and E-Cadherin signal pathway.3 Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance could decrease renal cell apoptosis and have obvious protective effects on OTIAKI by anti- oxidative stress, anti- inflammation, balancing the ratio of Bax and Bcl-2, down-regulating the expression of apoptosis-related key signal molecular in caspase-related signal pathway, inhibiting the loss of E-Cadherin in renal tissue. The protective effects of Anisodamine and Inula Britannica on kidney could be better than Ixeris Sonchifolia Hance due to the difference of their molecular mechanism in OTIAKI rats.