The Research Of Human Solubie Death Receptor-Fc Protection For Renal Ischemia Reperfusion Injury In Mice

Background: Tumor necrosis factor related apoptosis including ligand is a member of TNF superfamily, In cell and organization, if TRAIL and Death receptor combine, it would cause inflammation and the death of thymus derived. It usually has five important death receptors, among them, DR4 and DR5 contain protein area which is known as death domain. These death domain is quite important for DR4 and DR5. They are important material to deliver death signal. Under the stimulation of TRAIL, DR4 and DR5 combine with them. And then FADD recruits procaspase8 and procaspase10, finally forming Death induced signal complex, DISC. Then, under the processing and catalyst of procaspase8, caspase8 is activated and released from DISC and further activate caspase3, caspase6 and caspase7. Moreover, the activated caspase-3 would further induces death of cell. In addition, The TRAIL apoptosis research of signal path in lung cancer, kidney cancer and liver cancer has already been proved and especially in acute kidney injury, it has become research hotpot.Acute kidney injury, AKI is rapid renal disfuction caused by multiple etiology and pathological syndromes characterized by high mortality. Ischemia reperfusion, IR injury is one of the main cause of AKI. Ischemia reperfusion injury is very complicated in renal pathology and it would happen in many medical condition and has high morbidity. This injury process is usually accompanied by many kind of injury of mixed renal cells including degeneration, apoptosis and necrosis of cell and lymphocyte infiltration and so on. Although renal transplantation is frontier discipline which develops rapidly in recent years to treat renal failure and the most effective treatment way of renal failure, Ischemia reperfusion injury is inevitable in renal transplantation. Researches shows that the study and intervention of IR damage mechanism is helpful to weaken IR injury. Therefore, we research the effect on IR injury after sDR5-Fc block TRAIL signal by building renal IR injury model, testify if sDR5-Fc can protect kidney by testing all kinds of index of kidney function and form of organization to provide good protective medicine for clinical renal transplantation operation.Purpose: Establish animal model of kidney IR injury, testing the serum index of renal function, observing the change of kidney slice morphology and the apoptosis of kidney cell, researching the distribution of every related protein in signal path and the situation changes of gene.Method: Establish kidney IR injury model by clipping left and right renal pedicle of healthy 6-8 weeks adult male C57BL/6 mice. Mice model are divided as: A Normal group, which are raised normally without operation; B. Shame group, which just open back muscles and denude but do not have ischemia; C. IR group, which open their back muscles skin and clip renal pedicle for 22 minutes to make the kidneys have ischemia; D. IR+sDR5-Fc group, open back muscles skin and close renal muscles for 22 minutes to make the kidneys have ischemia. after 2h and 6h, infuse sDR5-Fc protein to abdomen. After 24 h, take the blood of the mouse from their hearts, separate their blood serum, show the kidney function of the mouse by testing the concentration of BUN and Cr in every serum to testify kidney injury situation of every group, meanwhile collect the kidney tissue and keep them in 4% paraformaldehyde and-80?. Observe the apoptosis of kidney and the situation changes of the renal histological with TUNEL method and HE dye separately, make sure the expression situation of Cleaved Capase8 in tissue with Western blot, test the distribution and expression situation of DR5, TRAIL, Caspase8 and Caspase3 with immunohistochemistry, test the expression situation of gene of DR5 and TRAIL protein with RT-PCR method.Results: Successfully established renal IR injury model in mice: model one and model two.Through testing serum BUN concentration found that no significant difference in Sham group and the Normal group; PBS group than in the Normal group and Sham group increased; BUN concentration decreased compared with PBS group After infuse sDR5-Fc protein to abdomen. Through testingserum Cr concentration, the concentration of each group findings are consistent with the results of BUN concentration, consistent with a model and two model results. the difference was significant.By HE staining, Normal group and Sham group normal morphology, renal tubular epithelial cell injury rarely; PBS group tubular structural damaged, renal tubular epithelial cell degeneration and necrosis and cell nuclear shedding, Cells disappeared off from the basement membrane, but the basement membrane contour surviving; The above is not obvious in sDR5-Fc group, The results damage area decreased and significantly less than the PBS group, consistent with a model and two model results.In TUNEL apoptosis assay, Normal group and Sham group can see little green fluorescent dots, but a larger number of green fluorescent dots in PBS group, the difference was significant, sDR5-Fc groups can see the green fluorescent dots also than PBS group decreased, the difference was significant, consistent with a model and two model results.Immunohistochemistry was used to detect distribution and expression of DR5, TRAIL, Cleaved Caspase8 and Cleaved Caspase3 protein, consistent with four protein results, Which expression occasionally in Normal group and Sham group, But PBS groups more positive distribution and sDR5-Fc group lower distribution than PBS group, consistent with a model and two model results.WB showed that activated Cleaved Caspase8 of TRAIL signaling pathway downstream molecules was expressed in each group, PBS group more expression than Normal group and Sham group, sDR5-Fc group were lower expression than the PBS group. consistent with a model and two model resultsRT-PCR validation mRNA levels of DR5 and TRAIL gene, confirmed that the DR5 gene in PBS group increased than Normal group and Sham group of in model one, sDR5-Fc group was lower than PBS group, while TRAIL gene, PBS group increased expression than Normal and Sham group, but sDR5-Fc group no significant decrease expression than PBS group. And DR5 gene and consistent in model one and model two, TRAIL gene exhibited PBS group were higher expression than that Normal and Sham group, sDR5-Fc group was significantly lower expression than the PBS group.Conclusion: The mouse model of renal IRI, sDR5-Fc protein has a good protective effect on acute kidney injury.