The Study About The Role And Mechanism Of Autophagy In Kidney EMT Induced By Calcium Oxalate Crystal

Urinary calculi have become a global health care problem,the incidence of which is increasing year by year,and the recurrence rate of calculi after surgical treatment is high.Obstructive nephropathy caused by calculi has become one of the main causes of chronic kidney disease.Calcium oxalate is the main component of urinary calculi,calcium oxalate calculi accounted for more than 80% of urinary calculi.However,the mechanism of renal calcium oxalate stone formation is still not very clear,and the widely recognized theories include: renal papillary calcium spot theory,urinary supersaturated crystal theory,suppressive factor deficiency theory,immunosuppression theory,multi-factor theory,etc.These mechanism studies suggest that renal tubular damage caused by multiple factors may further promote nucleation and deposition of crystals.Therefore,renal tubular injury may be a new direction in the study of kidney stone formation.The previous research results of our stone team suggested that as early as the calcium oxalate crystal formation stage,oxidative stress,apoptosis and autophagy involved in,and the occurrence of renal tubular epithelial cell interstitial differentiation(EMT)was found.These factors could influence each other to further lead to renal interstitial fibrosis and aggravate the development of renal disease.Based on these,our topic was proposed to the further research on calcium oxalate crystal formation,to explain cell autophagy how to participate in the process of renal tubular epithelial cell damage.We hope to inhibit the occurrence of EMT crystallization of renal injury,delay the development of chronic obstructive nephropathy through regulating autophagy activity,which can provide a new thought and theoretical basis for clinical treatment.Part ?: Expression of autophagy in a calcium oxalate crystal renal injury modelObjective: By detecting the expression of autophagy-related proteins in the animal model and cell model of calcium oxalate crystal kidney injury,to make it clear whether autophagy involved in the formation of calcium oxalate crystal and further leads to renal tubular injury.Methods:1.We constructed crystal renal injury animal models in mice established by successive intraperitoneal injections of glyoxylate(100mg/kg,p H=7.4)for one week whereas the control mice received an equal volume of normal saline.After one week,the animals were euthanized and immediately underwent heart perfusion.The right kidney was fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining,Von Kossa staining,immunofluorescence and immunohistochemistry.The left kidney was stored at-80°C for western blot.HE and Von Kossa staining was used to observe the changing of organization structure and calcium salt deposition,and immunofluorescence and immunohistochemical was used to detect the autophagy key protin LC3 B expression in renal tissue.2.We use sodium oxalate to stimulate renal tubular epithelial cells(HK2)to construct calcium oxalate crystal cell model,through the observation of the calcium oxalate crystallization time,setup time gradient for 6 h,12 h,24 h,48 h,72 h,oxalate sodium concentration gradient of 0 m M,0.5 m M and 1 m M,2 m M.At first we used CCK8 kits to detect HK2 cells activity,secondly used western-bolt to observe the expression of autophagy key protein--LC3 B,Beclin 1,ATG5 in different experimental conditions.Results:1.HE staining showed that,compared with the normal control group,the proximal renal tubular lumen of the mice in the a glyoxylate model group was swollen and the epithelial cells of the renal tubular were shed.Von Kossa staining showed a large amount of calcium salt deposition at the dermal medullary junction of the mouse kidney in the model group.2.Immunohistochemical and immunofluorescence test showed that the expression level of LC3 was generally higher in the kidney tissues of mice in the model group.3.In the HK2 cell model constructed by sodium oxalate,the cell activity decreased gradually with the increase of sodium oxalate concentration and/or the prolongation of the stimulation time,and significantly decreased at 48h;The expression of autophagy-related proteins increased with the extension of time,reached the highest expression level at 24 h,and then gradually decreased.Conclusion: Both in vivo and in vitro experiments we confirmed that autophagy has been over-activated as early as the calcium oxalate crystal formation stage,which is involved in and further affects the occurrence and development of kidney stones.Part ?: The expression of EMT in calcium oxalate crystal kidney and the interaction effect about autophagy and EMTObjective:To verify that at the early stage of kidney stone formation--calcium oxalate crystallization stage,EMT has already appeared in the renal tubular epithelial cell interstitial differentiation,and autophagy has involved in the process of renal tubular epithelial EMT.By regulating autophagy activity,the injury of renal tubular epithelial cells can be improved and the development of EMT in calcium oxalate crystallization stage can be delayed.Methods : 1.Immunohistochemistry and Western-blot were used to detect the expressions of EMT-related proteins and autophagy-related proteins in the kidney tissues of mice in the control group and the model group.2.The groups were grouped according to the time gradient of HK2 cell lines stimulated by sodium oxalate,and the expression of EMT-related proteins was detected by Western-blot;Then the cells were further divided into four groups: control group,sodium oxalate group,model +3MA group,and model + rapamycin group.The expression of autophagy-related proteins LC3 B and EMT-related proteins were detected by Wester-blot.Results : 1.Animal experimental results showed that compared with the blank control group,E-cadherin expression was significantly down-regulated,while Vimentin and LC3 B expression was significantly up-regulated.2.The results of cell experiments in vitro showed that compared with the control group,the expression of E-cadherin was down-regulated and the expression of ?-SMA and Vimentin was up-regulated with the increase of the stimulation time of sodium oxalate,and the trend was most significant at 24 h.After the addition of autophagy inhibitor 3MA,the expression levels of autophagyrelated proteins were significantly decreased,and the expressions of mesothelial cells markers ?-SMA and Vimentin were also significantly down-regulated,while the expression of epithelial marker E-cadherin was significantly retracement.Conclusion: Through experiments in vivo and in vitro studies,we have found that calcium oxalate stimulation can activate renal tubular epithelial mesenchymal transition(EMT)process,and found that EMT key protein expression levels consistent with autophagy key protein expression,so that to induce autophagy excessive activation of calcium oxalate so as to promote the crystallization of renal tubular epithelial cells of kidney injury EMT development.Part ?: The study of the mechanism about autophagy and EMT in calcium oxalate-induced renal tubular epithelial injuryObjective: To explore the potential mechanism of autophagy activity changing in regulating EMT in calcium oxalate crystal kidney injuryMethods: We detected the protein levels of P-MTOR and P-Smad3 in the kidney tissues of mice in the two groups,as well as the protein levels of p-m TOR and p-smad3 in the four groups of cells(control group,sodium oxalate crystal group,3MA+ sodium oxalate crystal group,and rapamycin + sodium oxalate crystal group).Results:Both cell and animal experiments suggested that p-m TOR was significantly down-regulated in the model group,while p-smad3 was up-regulated.After the autophagy inhibitor 3MA was given in the cell model,p-m TOR protein level was up-regulated,which led to the down-regulation of p-smad3 protein expression.Treatment with rapamycin in the model group further inhibited the activity of p-m TOR,leading to further up-regulation of p-smad3 expression.Conclusion:Calcium oxalate crystals stimulate renal tubules,inhibit m TOR level induced autophagy,and promote the occurrence of renal tubules EMT by regulating smad molecular pathways.