The Molecular Mechanisms Of Calcium Dobesilate In Protecting Renal Ischemia Reperfusion Injury Through Regulating ER Stress GRP78/CHOP Pathway

Background:Ischemia reperfusion injury（IRI）is a common phenomenon during renal surgery and kidney transplantation.Renal IRI not only causes acute kidney injury,but also is an important cause of delayed recovery of transplanted kidney function,acute rejection and chronic renal disease,which has become a bottleneck restricting long-term survival of transplanted kidney.In recent years,with the extensive development of donation after citizen death（DCD）,the number of kidney transplantation cases has become the second largest in the world,while kidney IRI,as a key factor hindering the long-term survival of kidney transplantation recipients,is still a worldwide issue.A large number of studies have shown that renal IRI is a multi-factor and multi-pathway complex pathological process,involving various molecular and cellular mechanisms causing different consequences,such as oxygen free radical injury,inflammatory reaction,cell apoptosis,endoplasmic reticulum stress,mitochondrial injury,intracellular Ca²⁺overload and microvascular dysfunction.How to prevent and alleviate renal IRI is an important topic in renal protection research.Ischemic preconditioning is an effective strategy to alleviate renal IRI,but its clinical application is limited because of its invasive intervention at the cost of multiple renal ischemia.Active search for new drugs to simulate ischemic preconditioning is a current research focus,but the intervention measures for a single agent or mechanism are not ideal.Calcium dobesilate is a new type of microvascular protective agent.Its main functions are to reduce the permeability of capillaries,inhibit platelet aggregation,reduce blood viscosity and improve microcirculation disorders.Besides,it is also known as a strong deoxidizer.Studies have confirmed that Calcium Dobesilate has a similar effect on renal IRI after improving myocardial IRI,liver IRI and skeletal muscle IRI in lung injury.So far,no literatures related to the prevention and treatment of renal IRI by Calcium Dobesilate.We speculate that Calcium Dobesilate may have a protective effect on renal IRI.Through in vivo and in vitro experiments,this study explored the effect and mechanism of Calcium Dobesilate on renal IRI from different perspectives.Objective:To study the Calcium Dobesilate in the renal protective effect of IRI and clarify Calcium Dobesilate may through anti-oxidation stress,regulation of endoplasmic reticulum stress way relieve renal IRI mechanisms such as apoptosis related factors,explore the Calcium Dobesilate as a cheap,efficient and low toxicity of the possibility of potential renal IRI antagonists,for clinical prevention and treatment of renal IRI to provide sufficient theoretical foundation and basis.Methods:Part one:Establishing and evaluating the model of renal ischemia reperfusion injury in SD ratsRight nephrectomy and left renal pedicle clamp were used to establish the classic renal IRI model in rats.By observing the effects of different renal ischemia time（0min,5min,15min,30min,45min,60min）reperfusion for 24h on renal function,pathology and apoptosis level,the degree of renal injury was evaluated,and the rational model of acute IRI in rats was established.Part two:The protective effects and mechanisms of Calcium Dobesilate preconditioning against renal ischemia reperfusion injury in ratsCalcium Dobesilate was used to pretreat the IRI model of rat kidney,and blood samples were collected to determine renal function（BUN,Scr,Cystatin C）before the rats were sacrificed.The morphological changes of renal tissue were observed by light microscope and the ultrastructural changes of renal tubular epithelium were observed by transmission electron microscope.The concentration of malondialdehyde（MDA）、superoxide dismutase（SOD）、glutathione peroxidase（GSH-Px）and catalase（CAT）activity in renal tissue were determined by corresponding kits.The expression levels of TNF-α,IL-6 and IL-8 in serum were quantitatively detected by ELISA.TUNEL assay was used to detect the level of apoptosis in renal tissue.The expression and distribution of GRP78,CHOP,Cleaved-Caspase-3 and Cleaved-Caspase-12 were determined by immunohistochemistry.Part three:The molecular mechanisms of Calcium Dobesilate in alleviating HK-2 cells ischemia reperfusion injury through regulating ER stress GRP78/CHOP pathwayThe IRI model of HK-2 cells was established by the induction of anti-mycin A and A23187,and the pretreatment was conducted with Calcium Dobesilate,the cell mortality was calculated by MTT method,and the effect of Calcium Dobesilate on cell growth was evaluated by the observation of cell morphology,so as to determine whether Calcium Dobesilate had a protective effect on the damage of HK-2 cells.The apoptosis rate was detected by flow cytometry.The concentration of malondialdehyde（MDA）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）and catalase（CAT）activity in the supernatant of cell culture were determined by corresponding kits.In ord to evaluate the intervention and influence of Calcium Dobesilate on oxidative stress of HK-2cells.The expression levels of TNF-α,IL-6 and IL-8 in the culture supernatant were quantitatively detected by ELISA,revealing the possible inhibitory effect of Calcium Dobesilate on inflammatory response.The expressions of GRP78,CHOP,Cleaved-Caspase-3 and Cleaved-Caspase-12 proteins in the apoptosis signaling pathway were detected by Western blot.The effects of Calcium Dobesilate on the stress induced apoptosis of endoplasmic reticulum were evaluated.Results:Part one:Establishing and evaluating the model of renal ischemia reperfusion injury in SD ratsRight nephrectomy and left renal pedicle clamp were used to establish the model of renal IRI injury in rats.Different renal ischemia time had different effects on renal function,pathology and apoptosis.The longer the ischemia time expose,the more serious the injury.Renal ischemia for 45min and reperfusion for 24h is a classic model of acute IRI in rats,laying a foundation for the establishment of rat renal IRI model and animal experiments in this study.Part two:The protective effects and mechanisms of Calcium Dobesilate preconditioning against renal ischemia reperfusion injury in ratsThe results of renal function showed that compared with the IRI group,BUN,Scr,Cystain C in the Calcium Dobesilate intervention group was much lower（P<0.05）,with a significant dose-effect relationship,indicating that Calcium Dobesilate had a protective effect on renal function.Pathological manifestations of renal tissue:under light microscope and transmission electron microscope,compared with the IRI group,the damage of renal tubular epithelial cells in the Calcium Dobesilate group was significantly reduced,and the pathological score was significantly reduced（P<0.05）,indicating that Calcium Dobesilate could improve the renal histopathological injury caused by renal IRI.Compared with the I/R group,the MDA level in the Calcium Dobesilate intervention group was significantly lower than that in the I/R group（P<0.05）.However,the SOD,GSH-Px and CAT levels were significantly higher than I/R group（P<0.05）,suggesting that Calcium Dobesilate alleviates renal IRI through antioxidant stress.The same phenomenon was observed on effects of inflammatory responses:the TNF-a,IL-6 and IL-8 level of Calcium Dobesilate intervention group was significantly lower than that in the I/R group.Endoplasmic reticulum stress induced apoptosis:compared with IRI group,Calcium Dobesilate intervention group kidney endoplasmic reticulum stress marker protein GRP78,CHOP,Cleaved-Caspase-3,Cleaved-Caspase-12 protein expression and distribution of decreased significantly（P<0.05）,indicating that Calcium Dobesilate can decrease the endoplasmic reticulum stress marker protein expression levels,inhibition of endoplasmic reticulum stress response.Part three:The molecular mechanisms of Calcium Dobesilate in alleviating HK-2 cells ischemia reperfusion injury through regulating ER stress GRP78/CHOP pathwayEffect of Calcium Dobesilate on the IRI model activity of HK-2 cells:there was no significant difference in the activity of HK-2 cells in the first 3 days after treatment（P>0.05）.On the fourth day,compared with the IRI group,the activity of the Calcium Dobesilate intervention group was significantly increased（P<0.05）and showed a significant dose-dependent relationship.The level of apoptosis rate:compared with the normal control group,the level of apoptosis in IRI group was increased.Calcium Dobesilate can reduce the apoptosis of HK-2 cells in a dose-dependent manner.The apoptosis rate in the Calcium Dobesilate group was lower than HK-2 IRI group（P<0.05）.Compared with the I/R group,the MDA level in the Calcium Dobesilate intervention group were significantly lower than that in the I/R group（P<0.05）,the SOD,GSH-Px and CAT levels were significantly higher than I/R group（P<0.05）,suggesting that Calcium Dobesilate alleviates HK-2 cells IRI through antioxidant stress.The effects of inflammatory responses:the level of TNF-a,IL-6 and IL-8 with Calcium Dobesilate intervention groups were significantly lower than that in the I/R group（P<0.05）.Apoptosis induced by the ER stress:the expressions of GRP78,CHOP,Cleaved-Caspase-3 and Cleaved-Caspase-12 proteins were detected by Western blot.The results verify that the expressions of GRP78,CHOP,Cleaved-Caspase-3 and Cleaved-Caspase-12 proteins in the IRI group and the Calcium Dobesilate treatment groups were significantly higher,but the expressions of GRP78,CHOP,Cleaved-Caspase-3 and Cleaved-Caspase-12 proteins in the Calcium Dobesilate treatment group were all lower than those in the IRI group,with significant differences（P<0.01）.Conclusion:1.The longer the time of renal ischemia,the more serious the degree of renal injury.Renal ischemia for 45min and reperfusion for 24h is an ideal model for acute kidney I/R injury in rats.2.Calcium Dobesilate has protective effects on the renal IRI of rats and the IRI of HK-2 cells.3.Calcium Dobesilate has significant anti-oxidative stress effect,inhibiting the inflammatory reaction and anti-apoptosis effect in the process of renal IRI.4.Calcium Dobesilate plays a protective role in renal ischemia reperfusion injury by regulating the ER stress GRP78/CHOP signaling pathway.