The Role Of Exogenous Carbon Monoxide On Epithelial-mesenchymal Transition Of Transplant Kidney Tubules And Its Mechanism

Background:Kidney transplantation is an effective and final treatment of patients with renal failure. Now, The1-year graft survival rate is more than90percent, but its long-term survival has no significant improvement. In nearly half of transplant patients’kindney will be in fibrosis and failure within10years, which is mainly attributed to chronic kidney transplant rejection. Although the specific molecular and cellular mechanisms are unclear, but the non-immunological factors and immune factors plays an important role are certainly.The main pathological features of chronic renal rejection are the excessive accumulation of parenchymal cells and the progressive reduction of extracellular matrix (ECM). ECM is mainly generated from the fibroblasts. The over-expression fibroblasts derived from the phenotypic transition of tubular epithelial cells. In certain conditions, the renal tubular epithelial cells change it’s morphological and functional, and transformate into the activity of mesenchymal cells, named the epithelial-mesenchymal transition (epithelial-mesenchymal transition EMT). EMT molecular manifestations of decreased expression of epithelial cell marker E-cadherin (E-cadherin) and continuous myofibroblast cell marker a-SMA.The TGF-β1is a key cytokine in the regulation of renal tubular EMT. Its role is inhibit the expression of epithelial cell marker E-cadherin, raising the expression of myofibroblast marker α-SMA, and increase the ECM accumulation. The physiological expression of TGF-β1is a potent immunosuppressive and anti-inflammatory factor, it can inhibit acute rejection and induce the immune tolerance significantly. So, it is not wise to block its expression. Studies have indicated that the overexpressed TGF-β1is considerablly part from the T cells and monocytes-macrophage cells. The EMT progression should be limited by inhibiting the infiltration of immune cells.Heme oxygenase-1(HO-1) was found to have the action of suppress of immune cell infiltration. It can catalysis heme to generate biliverdin, carbon monoxide (CO) and free iron in body. CO can completely or partly replace the protective effect of HO-1in almost all of the injury and disease models. On this basis, we speculate that the CO should have the exact suppression of immune cells action in the context of renal transplantation. Further speculated that the CO have the significantly action of inhibit or reverse the renal tubular EMT.Objective:1. Exploration and optimization of the operative techniques to establish a stable DA-WF rats transplanted kidney animal model.2. Identify the graft interstitial fibrosis appears by relevant stained test on the basis of established renal transplantation model, and confirm the presence of epithelial-mesenchymal transition (EMT) in transplanted kidney.3. Interference the DA-WF model with carbon monoxide released molecule (CORM-2), and gain the tissue.4. Study CO inhibit or reverse the transplanted kidney’s EMT, significantly delay the process of renal allograft fibrosis is in the mechanism of suppress the immune cell infiltration by testing this tissues.MethodThis topic is divided into four parts:1. Exploration and optimization of the operative techniques of kidney transplantation surgery with SD rats. Chloral hydrate anesthetize the rats, the donor kidney perfusion with abdominal aortic intubation in situ. The donor kidney are transplant to the receptor in the same name renal vascular and ureteral. The receptor’s another kidney is deal with the manner of blood vessels in vitro delayed ligation.2. To establishe the model of kidney transplantation with Dark Agouti (DA)(RTlavl) rats for the donor, Wistar-Furth (WF)(RTlu) rats for the receptor, and marked as the experimental group. The WF-WF rats kidney transplantation marked as the control group. The WF receptors receive10mg/kg/d of cyclosporine subcutaneous injection preoperative3d and postoperative2w. Measure the body weight of rats, blood serum creatinine and blood urea nitrogen at postoperative2w,4w,8w,12w, to dynamic monitor the general situation of rats and kidney function. Take the12w experimental group and control group transplanted kidney tissue to perform H&E staine, Masson staine, E-cadherin immunohistochemistry, α-SMA immunohistochemistry. Analysis with HPIAS-1000analyzer, and SPSS15.0statistical software processing data. 3. Establishe the DA-WF kidney transplant model, random numbers and divide into two groups, one group of CORM-2intervention, another of iCORM-2intervention. The WF-WF are blank reference. Select a fixed number of rats in each group to weigh and sacrifice at postoperative2w,4w,8w,12w. Remain the sacrificed receptors’s kidney and cavity blood. The kidney allograft tissues are equipped for further experiments, the cavity blood is to detect blood serum creatinine and urea nitrogen. Analysis the data with SPSS15.0statistical software.4. Take the kidney allograft tissues intervention CORM-2, iCORM-2and blank reference at postoperative2w,4w.8w,12w, to perform H&E staine, Masson staine, E-cadherin immunohistochemistry, a-SMA immunohistochemistry, CD3immunohistochemistry, CD163immunohistochemistry. Analysis with HPIAS-1000analyzer, and SPSS15.0statistical software processing data.Conclusion1. Through the early exploration and practice, grasp the technic of establish the rat kidney transplant model, to guarantee the survival rate of the model, and to provide technical support and relevant experience for the subject of experiments2. To establish and compare the two groups of kidney transplantation of DA-WF, WF-WF postoperative’s general condition, graft function, and the12w transplanted kidney tissue HE staine, Masson staine, E-cadherin immunohistochemical, α-SMA immunohistochemical to confirm that of DA-WF rat renal transplantation model induce chronic rejection, a renal interstitial fibrosis, and confirm the presence of the EMT in the kidney transplant model.3. By the results of detection of the three groups’(CORM-2intervention, iCORM-2intervention, blank reference) renal function, we can confirm that the carbon monoxide release molecule (CORM-2) can inhibit the process EMT of kidney transplant, prediction that the CO released by CORM-2in vivo should have some sort of transplant renal protective effects.4. By the analysis of CORM-2intervention, iCORM-2intervention and blank reference tissue’s H&E staine, Masson staine, E-cadherin immunohistochemistry, α-SMA immunohistochemistry, CD3immunohistochemistry, CD163immunohistochemistry. We can confirm that the CO released by CORM-2should significantly inhibit or reverse the transplant renal tubular EMT. and the possible mechanism of this effect should the exact suppression of T lymphocytes, macrophages in the context of renal transplantation.