Experimental Study And Monitoring Of ESM-1 For The Diagnosis And Treatment Of Rejection After Renal Transplantation

Due to the combination of new and potent immunosuppressive drug, renal transplantation is the major treatment of choice for most patients with end-stage renal failure. However, rejection is now also an inextricability problem. It`s still an ideal goal of immune tolerance between the donor and recipient.The occurrence of acute rejection is correlated not only to the success of transplantation,but also to the incidence of chronic rejection. Even a single episode of acute rejection is a strong predictor of intermediate-and long-term graft failures . Therefore, the goal of current immunosuppressive protocols has been to prevent rather than to treat AR episodes. Thus , the grafts must be carefully monitored in organ transplantation. Up to the moment, an efficient and reliable test for continuous monitoring of the immune activation status of kidney transplant recipients is not available. It`s important to explore a noninvasive , susceptible, specific and safe markers of immune activation.on the prediction of AR of kidney allografts.Nowadays,the diagnosis of acute rejection is based on some factors as listed below: clinical symptoms, result of kidney function test, graft biopsy, the effectiveness after treatment,etc. Although biopsy is now the golden standard of diagnosis.However, it still has some dis- advantages, such as invasive processes, potential damage on the graft. And as a result,some patients refuse any request of graft biopsy.The vascular endothelium is an active barrier between the vascular wall and the blood. Endothelial cells provide the physical interface between blood and surrounding tissue, regulate several important physiological and pathological reactions,such as nutrient and blood component traffic, and participate in many physiologic events such as hemostasis, inflammation, angiogenesis and intercellular communications.The main functions of the endothelium are the control of coagulation, fibrinolysis, vascular tone, and immune responses. Potential causes of endothelial dysfunction are inflammation,oxidative stress, dyslipidemia, hyperglycemia, hypertension. Recently, it was reported that endothelial dysfunction is a feature of patients after renal transplantation. Positioned at the interface between circulating cells and tissues, the endothelial cells play a critical role in the homing and the local accumulation of leukocytes. Initial tethering and rolling, subsequent arrest and adhesion, and transendothelial migration constitute the current view of leukocyte migration.Endothelial cells may appear in the circulation by detaching from activated or damaged vessels. An increase of circulating endothelial cells (CEC) is described in several pathologic conditions that involve vascular injury or instability as myocardial infarction, infectious vasculitis and cancer. The developing view of CECs is that they most likely represent a population of well or perhaps terminally differentiated mature endothelial cells that have desquamated from the intima of the blood vessel. Consequently, it has been suggested that raised CECs reflect a serious damage to the endothelium by a particular disease process, such as cytotoxic preconditioning in transplantation, pulmonary hypertension, septic shock, haemodialysis, inflammatory vasculitis, diabetes, and acute myocardial infarction. Therefor,CECs are a novel marker of vascular damage.In kidney transplantation,the highest numbers of CEC could be shown in patients with acute vascular rejection compared to patients without rejection, with interstitial or borderline rejection. Eliane R had found that CEC present in the blood of kidney allograft recipients are of donor origin,thus reflecting vascular damage in the allograft..Endothelial-cell-specific molecule 1 (ESM-1) is a recently identified endothelial cell molecule.ESM-1 was found to be expressed in many endothelial cells in normal tissues. It appears that it is a general property of the endothelial cells since most of the capillaries, arterioles and venules were similarly stained.ESM-1 is highly restricted to the endothelial cell, and that the ESM-1 is synthetized, distributed,and restricted to the vascular endothelial cells.Therefore, ESM-1 may participate in specialized endothelial functions,and could be a potential novel endothelial cell marker for Endothelial function.The aim of our study is to measure the change of ESM-1express on the CECs in recipient and the graft after renal transplant.And investigate the clinical application of ESM-1 in the diagnosis of acute rejection after renal transplantation.The main research contents: 1. Firstly,the expression of ESM-1 mRNA on the CECs in recipients and ESM-1 protein on the grafts were measured after renal transplantation. Secondly, we discussed whether or not the result could reflect the degree of Endothelial cells damage and the degree of rejection after renal transplantation. Lastly, we investigated the clinical application of ESM-1 and compared with some other marker in the diagnosis of acute rejection after renal transplantation,2. We established a modified rat model of bilateral renal allograft.In this model,the serum creatinine and allograft biopsy were measured.And this model was proved to be succeed for less operation time and cost.3. In the randomized controlled trials of rat kidney transplantation, we studied whether the H0-1 protein induced by hemin pretreatment could contribute to the protection of the kidney after transplantation.After the pretreat with hemin,we measured the expression of ESM-1 mRNA on the CECs in recipients and ESM-1 protein on the grafts. The last, we discussed whether or not the experssion of ESM-1could reflect the degree of Endothelial cells damage and the degree of pathological lesion after renal transplantation.The main results are as follows:1. The result of humen ESM-1mRNA/GAPDH was (3.47±1.05)at preoperative,and it increased significantly ((11.38±2.35)×10-3)during the rejective time.The result of humen ESM-1 protein/β-actin on normal kidney is (0.18±0.04),and it increased significantly (0.32±0.06) on kidney with acute rejection(Western-blot).2. The expression of humen ESM-1mRNA in the transplantation group with stable renal function increased and changed moderately after reducing,but there were no significant differences as compared with preoperative.3. In the group with renal insufficiency due to some other reason such as ATN,there were no significant differences of humen ESM-1mRNA as compared with preoperative.4. The rat model of bilateral renal allograft was established with the classic technique of microsurgery .It can be fulfilled with only one person,and with better achievement ratio. Therefor,it has less operation time and cost as compared with the model of unilateral renal allograft.5. After the pretreatment with hemin, induction of HO-1 protein and bioactivity levels were observed ,the up-regulation was stronger (bioactivity: p < 0.05, protein: p < 0.05, respectively).The apoptosis of rat renal allograft was reduced (19.52±2.73%).The degree of pathological lesion were more light in group B(pretreat with Hemin) than in group A(acute rejection). In contrast , the degree of pathological lesion were more severe in group B(pretreat with Hemin) than in group C(treat with CsA).6. The expression of esm-1mRNA and ESM-1 protein increased moderately afteroperation, and increased significantly when acute rejection occurred. In contrast , The expression of esm-1mRNA and ESM-1 protein decreased after pretreat with Hemin.The main conclusions:1. ESM-1 is highly restricted to the endothelial cell, and that the ESM-1 is synthetized, distributed,and restricted to the vascular endothelial cells.Therefore, ESM-1 may participate in specialized endothelial functions,and could be a potential novel endothelial cell marker for Endothelial function. The expression of ESM-1mRNA and ESM-1 protein could reflect the degree of Endothelial cells damage and the degree of rejection after renal transplantation.2. The expression of ESM-1mRNA and ESM-1 protein could be an useful marker with highly sensitivity and specificity for the diagnosis of rejection after renal transplantation.3. The H0-1 protein induced by hemin pretreatment could contribute to the protection of the kidney after transplantation.4. There are many differences between the different recipients when compared for the expression of ESM-1mRNA and ESM-1 protein.Therefore,it should be careful and individuals for the diagnosis and treatment of rejection after renal transplantation.