Study On The Action Mechanism Of AT1 Receptor Autoantibodies In Mediating Vascular Rejection After Renal Transplantation

Renal transplantation has been an effective and radical treatment for end-stage renal diseases. With the development of technology in tissue matching and the wide application of immunodepressants, the incidence of acute rejection after renal transplantation has decreased and the short-term survival after transplantation has dramatically increased. However, acute rejection is still one of the major complications after renal transplantation, and is also the main risk factor leading chronic rejection and graft dysfunction. There are two main mechanisms of rejection after allograft renal transplantation, one is antibodies-mediated humoral immunity, the other is T cell-mediated cytoimmunity. The basic difference of these two mechanisms is that, for the former the graft injury is caused by antibodies, while for the latter the graft injury is caused by direct cytotoxic actions induced by T cell or NK cell or tardive hypersensitivity reaction. If there are preexisting or newly developed antibodies in the blood of recipient specifically against the donor, the acute rejection after renal transplantation is called acute humoral rejection(AHR) or vascular rejection. Humoral rejection induced graft dysfunction is still a problem in clinical practice. The standardized criteria for the pathological diagnosis of AHR was discussed on the Sixth Banff Conference on Allograft Pathology in 2001. Diagnostic criteria for acute antibody-mediated rejection includes: a) morphologic evidence of acute tissue injury; b)Immunopathologic evidence of antibody action; c)Serologic evidence of circulating antibodies against donor HLA or other anti-donor endothelial antigen. In the past decade, four forms of antibody-mediated graft injury have been defined as hyperacute rejection, acute humoral rejection, chronic humoral rejection and accommodation. Most of the antibodies that mediate humoral rejection are against HLA and can be detected, and the rejection mediated by them can be avoided. While, although the low incidence, there is still no specific treatment for the rejection mediated by other unknown antibodies. For this reason, it is important to identify and elucidate the mechanisms by which non anti-HLA antibodies contribute to humoral rejection to provide effective treatment for humoral rejection. Non anti-HLA antibodies include anti-MICA antibody, anti-MICB antibody, anti-endothelial cell antibody and autoantibodies against the angiotensinⅡtype 1 receptor(AT1-AA), which was discovered in 1999 in patients of preeclampsia. Until 2005 Dragun et al reported the presence of AT1-AA in recipients of renal allografts who had severe vascular rejection and concluded that AT1-AA played an important role in mediating humoral rejection. In order to find out whether AT1-AA can induce vascular rejection, the serum containing human AT1-AA was injected into rat recipients(Fischer344-Lewis renal transplantation model). One week after the surgery, endarteritis and intravascular infiltrates were observed in the graft. For the fact that human serum is heteroantigen for rat, it is still not clear whether it is AT1-AA which specifically causes this injury. A better strategy is to use rat-derived AT-AA to see whether this injury will happen again. We presume that the possible role of AT1-AA in mediating humoral rejection is to bind to AT1R on the target cell surface and then initiate intracellular signal transduction, to promote cell proliferation or apoptosis, which is different from the role of anti-HLA antibodies which activate complement after binding to cell surface antigen. In order to verify our presumption, functional exchange of endothelial cell(EC) stimulated by AT1-AA will be observed and intercellular adhesion molecule-1(ICAM-1), a marker of acute rejection and endothelial cell activation, will be detected.In the theory of humoral rejection, EC is the primary target of antibodies. EC is a type of cell equipped with unique biological functions, serving as a barrier between blood and tissue, a place where cells from the donor and the receptor first meet, where the battle between them first occurs. The rejection mediated by antibody post renal transplantation is characterized by inflammation in EC, and subsequent cell infiltration and broken EC, followed by the activation of EC and apoptosis. There are wide exchanges of molecules between EC and blood, or tissue fluid. EC has many biological functions, such as selective penetration, anti-thrombosis, regulation of tension of blood vessels, involved in formation of capillary, producing cytokines, adhesions and anti-tumor, etc. ICAM-1 is a member of immunoglobulin superfamily, and has been reported to be over-expressed by tubular epithelium and vascular EC during acute rejection. The increase expression of ICAM-1 is also positively correlated with the severeness of the rejection. This suggests that ICAM-1 is an important factor for acute rejection initiating and immunologic response. It is still unknown whether AT1-AA can promote EC proliferation and lead to increased ICAM-1 synthesis and secretion, accordingly trigger or aggravate humoral rejection. In this study, cultrued rat artery EC is stimulated by AT1-AA and whether ICAM-1 synthesis and secretion is increased will be detected. It has been reported that AngII exerts its biological functions through binding to AT1R and activating signal transduction pathways such as MAPK and JAK/STAT, playing a significant role in EC injury, vascular smooth muscle cell proliferation and atherosclerosis forming. We suspect that AT1-AA probably though binding to sell surface ligand(AT1R) to transducts intracellular signal. In this study the role of MAPKs signal transduction pathway in AT1-AA mediated EC functional changes is also evaluated.It is necessary to obtain adequate AT1-AA and to identify and detect it. In this study, rats are immumized with rat-derived AT1R polypeptide and whether AT1-AA can be induced is observed. Then AT1-AA is injected into rat recipients of renal allograft and whether there is a rejection is observed. The signal transduction pathways AT1-AA involved in kidney vascular EC are determined to help to find possible blocking methods. This study aims to demonstrate that homologous AT1-AA can definitely mediate early humoral rejection with graft EC undergoing ischemia-reperfusion injury as the main target cell and to find out the main signal pathways AT1-AA activated following binding to its receptor. The study will provide important evidence for elucidating the mechanism underpinning AT1-AA mediated humoral rejection and also offer theoretical support for the treatment of this type of rejection in clinical practice.Main contents:1. Rat-derived AT1R peptides were coupled with keyhole limpet hemocyanin(KLH) as the antigen to immunize rats. AT1-AA was examined, isolated, purified and identified from immunized rats serum. The toxicity of AT1-AA obtained was also evaluated by examining the tissue damage in immunized rats.2. Rat-derived AT1-AA was injected into rat recipients of renal allograft and whether acute vascular rejection occured were observed. Vascular EC injury was espcially monitored. Whether oarl administration of Losartan was protective was also observed.3. The effect of AT1-AA on cultured EC was observed in vitro studies and ICAM-1 expression change in EC stimulated by AT1-AA was detected. The signal pathways and molecular mechanism of ICAM-1 expression in EC stimulated by AT1-AA were studied. Whether Losartan had a blocking effect on signal pathways and ICAM-1 synthesis and whether it could contribute to prevention and treatment of AT1-AA mediated rejection were evaluated.Main methods and results:1.Rat-derived AT1R peptide coupled with KLH was used as immunogen to immunize rats. After the second enhanced immunization, suspicious positive result for AT1-AA was obtained from rat serum. After the third enhanced immunization, the positive rate for AT1-AA from serum was 90%; after the fourth, 100%. The concentration of AT1-AA peaked at the 13th week. AT1-AA was purified by protein G-sephrose 4B affinity chromatograph and subsequently identified by biological function. No significant changes were observed in heart, kidney and liver in rats as assessed by light microscopy and electon microscopy.2. Rat renal transplantation model was successfully established. Since little difference in MHC existed between individual SD rats, no obvious pathological changes were observed after the surgery. Kidney biopsy on the 7th day post-transplantation indicated a level lower than I A according to Banff'97. The injury in renal tubules, glomeruli, and microvessel was negligible, which was served as the negative control for acute rejection.3. Injection of AT1-AA into rat recipients of renal allograft induced acute vascular rejecion. Under light microscope, it was featured by infiltration of mononuclear cells and lymphocytes into microvessels, endarteritis occured, neutrophils and mononuclear cells in glomeruli, but not capillary thrombosis and not fibrinoid necrosis. Under electron microscope, it was featured by EC swelling, breaking into pieces and desquamating from vessel wall, enlargement of interspace among Ecs, uncompleted conjunction, edema around the vessels and external migration from vessel of inflammatory cells. Losantan exerted an obvious protective effect on AT1-AA induced pathology with improved renal function.4. Rat aorta EC was primary cultured using tissue transplant method, purified and then secondary cultured. The high quality of EC was ascertained by immunofluorescent staining of vWF.5. EC was stimulated by AT1-AA of different concentration(1:200,1:100,1:50) and expression of ICAM-1 was detected at different time after stimulation. The result revealed that expression of ICAM-1 was gradually upregulated as time passed within the first 24 hours, peaking at hour 24 and began to wane from hour 36. It was also found that this effect was AT1-AA dose-dependent: the higher the concentration of AT1-AA, the higher increase of expression of ICAM-1, with the concentration of 1:50 being the most effective one.6. AT1-AA was used to stimulate cultured EC. ERK1/2, p38MAPK and JNK/SAPK were examined respectively at different time point after stimulation. Results revealed that AT1-AA could activate ERK1/2 and p38MAPK with the most powerful activation at 20 minutes after stimulation. Thereafter the level of ERK1/2 and p38MAPK was attenuated. However AT1-AA had no obvious stimulating effect on JNK/SAPK.7. Different concentrations of PD98059(30μM and 60μM)and losantan (10-6mol/L and 10-5mol/L)could dramatically suppress the activity of ERK1/2, with 60μM PD98059 and 10-5mol/L losantan being the most potent. Different concentrations of SB203580(20μM and 40μM)and losantan (10-6mol/L and 10-5mol/L)could also suppress the activity of p38MAPK, with 40μM SB203580 and 10-5mol/L losantan being the most effective.8. EC was pretreated with SB203580, PD98059 and losantan. Then AT1-AA was added to stimulate the increasing expression of ICAM-1. EC was collected to examine the level of ICAM-1 24 hours after the addition of AT1-AA. Results revealed that the combination of losantan and SB203580 had the most powerful suppression on the increase of ICAM-1, which was 97%. While either PD98059 or losantan alone could only achieve 55% and 40%.Conclusion:1. Rat-derived AT1R peptide has the ability of inducing AT1R auto-antibodies, which has no apparent damaging effect on host tissues. The study confirms the exist of rat-derived AT1R and successful isolation and purification of AT1R auto-antibodies, which provides the experimental basis for further study on effect of AT1R auto-antibodies on transplantation.2. Injection of rat-derived AT1-AA into rat recipients of renal allograft can induce acute vascular rejection and obvious damages in vascular EC of the graft under light microscope and electron microscope. 3. AT1-AA can induce partly refractory vascular rejection. The possible mechanism of rejection might be AT1-AA binding to AT1R on the surface of EC and subsequent activation of intra-cellular signal transduction, which leads to EC dysfunction with increasing synthesis and secretion of ICAM-1 being one of the features. Increased ICAM-1 induces leukocytes infiltration and disorder of EC structure and function. The vicious circle finally leads to graft function failure. Losartan partly prevents vascular rejection and is effective on protection of the graft. It suggests that AT1-AA should be detected before and after transplantation, especially for patients who used to have pregnancy hypertension and malignant hypertension. ARBs is a good choice for early treatment of rejection induced by AT1-AA.4. Synthesis and secretion of ICAM-1 is increased in cultured rat aorta EC stimulated by AT1-AA. AT1-AA can activate ERK1/2 and p38MAPK, while ERK1/2 antagonist PD98059, p38MAPK antagonist SB203580 and Losartan can inhibit the increasing of ICAM-1. This study indicates the possible use in prevention and treatment of AT1-AA induced rejection of ARBs, ERK/p38MAPK antagonists and blocking the binding site of ICAM-1 and LFA-1, which lie in different stages of receptor binding stage, signal transduction stage and effectors stage. This will provide important theoretical references for further treatment by AT1-AA.