Agonistic antibody-triggered stimulation of Angiotensin II type 1 receptor and renal allograft vascular pathology

Despite the substantial improvements in post-transplantation management and novel immuno-suppressive modalities that have resulted in improved overall survival, rejection with vascular involvement remains a challenging problem in organ allotransplantation. Acute vascular rejections have an aggressive clinical course and frequently lead to a loss of allograft [ 1 ]. The histopathology of vascular rejection applies to a wide variety of vascular lesions in the allograft, ranging from thrombosis, fibrinoid vascular necrosis and endarteritis to myointimal proliferation with fibrosis [ 2 ]. A definitive causal relationship that would explain whether overwhelming anti-donor T-cell response, or alloantibodies, or both are responsible for the development of vascular allograft lesions is still missing. Resistance to therapy with anti-lymphocyte antibody preparations or steroids [ 3 ], implicates the contribution of anti-donor humoral reactivity against human leukocyte antigens (HLA) and vascular rejection. Donor-specific anti-HLA alloantibodies initiate rejection through complement-mediated and antibody-dependent, cell-mediated, cytotoxicity [ 4 ]. The accumulation of the complement degradation product, C4d, is generally regarded as a marker for an antibody-mediated alloresponse and is associated with poor graft survival [ 5 ]. Nevertheless, 40–50% of rejections with severe vascular changes and poor prognosis such as fibrinoid necrosis are C4d negative, implicating involvement of non-complement fixing antibodies [ 6 ]. Obviously, vascular rejection forms a heterogeneous group of clinical, aetiological, and histopathological entities. One way to improve the outcome of rejections with vascular pathology is to detail the subtle diagnostic and mechanistic differences. This approach would subcategorize patients who might respond to a particular therapy. Recipients of HLA identical kidneys can develop features of refractory rejection with vascular pathology, implicating putative pathogenic antibodies that are not directed against the HLA system [ 7 ]. Unknown immune targets and consecutive lack of detection methods make non-HLA antibody-mediated rejection particularly difficult to diagnose and treat. The clinical course of non-HLA antibody-mediated rejection is not well defined. In renal transplant rejection, the presence of antibodies to non-HLA antigens has been associated with antibodies against endothelial cells, tubular epithelial cells, podocytes, mesangial cells and monocytes [ 8 ]. Many of these antibodies appear to be auto-antibodies. Most attention has been focused on anti-endothelial cell antibodies. However, antigen heterogeneity of endothelial cells derived from different vascular beds limits the development of standardized tests [ 9 ]. Moreover, initially detected target antigens of anti-endothelial cell antibodies were of unclear functional relevance. For this reason, it is important to identify non-HLA antibodies, determine their antigen specificity, and clarify the pathogenic mechanisms by which they contribute to rejection with vascular lesions or other forms of allograft injury. We recently reported the presence of agonistic antibodies against the Angiotensin II type 1 receptor (AT 1 R-AA) in 16 recipients of renal allografts, who had severe vascular rejection and malignant hypertension, but who did not have anti-HLA antibodies [ 10 ]. AT 1 R-AA have also been associated with pre-eclampsia [ 11 ] and malignant hypertension [ 12 ]. Pregnancies complicated by pre-eclampsia and graft rejection bear some immunologic similarities [ 13 ]. The decision to seek out and isolate AT 1 R-AA were sparked by the observation that the first patient in our study developed vascular rejection accompanied by malignant hypertension and seizures in a ‘zero-mismatch’ kidney, during the first post-transplant week. The observed clinical picture was so reminiscent of eclamptic crisis in pregnancy, a condition that she had developed two decades before transplantation, that we began to prospectively study patients with similar clinical features. The clinical presentation of another 15 transplant recipients with AT 1 R-AA was at first similar to that of anti-HLA antibodies, albeit with more severe histology [ 10 ]. All their transplants exhibited endarteritis, transmural arteritis and/or fibrinoid vascular necrosis (Banff IIb or Banff III rejection). Apart from arterial changes, we also noticed tubulitis and interstitial infiltrates, characteristic for acute cellular rejection. In contrast to anti-HLA antibody positive patients, AT 1 R-AA positivity was invariably associated with severe hypertension in all and seizures in some of the patients. Most patients (13/16) did not have hypertension before vascular rejection occurred, implying that the post-transplantation hypertension was secondary to rejection. AT 1 R-AA-related vascular rejections occurred during the first week after transplantation, equally frequent in female and male renal transplant recipients. Causes of end-stage renal disease were not primarily attributed to hypertensive nephrosclerosis, but instead to a variety of tubulo-interstitial or glomerular diseases. The incidence of severe rejection among patients positive for AT 1 R-AA in our study was similar to that of rejection due to donor-specific anti-HLA antibodies Seven among a total of 16 AT 1 R-AA patients were treated with combination therapy consisting of plasmapheresis, intravenous immune globulin infusions, and the AT 1 R-blocker, losartan. The treatment significantly prolonged allograft survival, compared with AT 1 R-AA-positive patients who received a standard treatment option [ 10 ]. Admittedly, our study was not a prospective randomized and controlled trial on this issue and all the treated patients were from one center. However, we believe our findings, even with their limitations, are highly suggestive. Some transplant nephrologists are perhaps the only remaining clinicians skeptical about the use of anti-renin-angiotensin system (RAS) drugs in the early post-transplant period. Given the reported beneficial effects of blockade of RAS on early outcomes of renal transplants, this view seems to be outdated [ 14 ]. Moreover, AT 1 R-antagonists may exert a clinically relevant immunomodulatory role by blocking IFN-γ production by T-cells [ 15 ]. All 16 patients from our initial study had pre-formed AT 1 R-AA and some of them were treated, either with ACE-inhibitors or AT 1 R-antagonists, while on dialysis. We speculate that the absence or discontinuation of RAS blockade in combination with permissive factors surrounding renal transplantation may have predisposed them to develop AT 1 R-AA-related vascular rejection. Thus, continous pharmacological blockade of AT1R in patients on a waiting list for kidney transplantation detected as AT 1 R-AA- positive, may be a useful approach to prevent or attenuate vascular rejection. Detection of AT 1 R-AA activity initially relied on the bioassay that measures the chronotropic responses to AT 1 R-IgG-mediated stimulation of cultured cardiomyocytes coupled with receptor-specific antagonists. High costs and a time-consuming test setting precluded screening of larger patient cohorts by bioassay at the time when the initial study was performed. We have now established and validated a cell-based ELISA in collaboration with biotech partners, for the detection of AT 1 R-AA in serum [ 16 ]. Pretransplantation screening for AT 1 R-AA detects a subset of end-stage renal disease (ESRD) patients who are similar but not identical to patients with anti-HLA-panel reacitivity. Whether or not all AT 1 R-AA-positive patients who are continuously treated with AT 1 R-blockade will develop milder or no vascular rejection is the subject of current studies. In contrast to HLA-antibodies, AT 1 R-AA seem to operate through complement-independent mechanisms, as C4d was detected in biopsy specimens from only 5 of our 16 patients. According to our working concept, AT 1 R-AA bind to second extracellular loop of AT 1 R and acts as an allosteric receptor agonist. AT 1 R-AA-AT 1 R interaction initiates signal transduction cascades, by inducing Erk 1/2 phosphorylation in endothelial and vascular smooth muscle cells ( Figure 1 ). Consequent increasing DNA-binding activity of transcription factors activated activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) is responsible for increased expression of their target genes involved in inflammatory responses and coagulation. Increased synthesis of chemokines MCP-1 and RANTES may probably explain intravascular inflammatory cell infiltration, while augmented activity and expression of Tissue Factor may account for thrombotic angiopathy. Renal transplant biopsy specimens obtained during an AT 1 R-AA-mediated rejection episode revealed intense diffuse Tissue Factor staining of epithelial, endothelial and mesangial cells in the absence of complement activation. As human mononuclear cells express AT 1 R [ 17 ], an effect of AT 1 R-AA on T-cells or monocytes appears to be likely, but needs to be evaluated. Model for AT 1 R-AA related signaling in vascular cells. AT 1 R-AA binds to second extracellular loop of the AT 1 R and acts as an allosteric receptor agonist. Target receptor-antibody interaction induces cell signalling via Erk1/2 and increased expression of pro-inflammatory (MCP-1, RANTES) and pro-coagulatory genes (Tissue Factor, TF) via activation of transcription factors AP-1 and NF-κB. Neither do we know why AT 1 R-AA induce severe vascular pathology in renal allografts and not in other AT 1 R expressing tissue. Target cells may ‘fine-tune’ levels of the receptors, change dominant splice variants to control signalling pathway activation, and change the stoichiometry of cytosolic reactants [ 18 ] Another important question is why AT 1 R-AA develop in patients with pre-eclampsia and ESRD and what is the role of antigen mimicry (cross-reactivity with bacterial or viral antigens) or genetic predispositions? Our current working hypothesis is that factors surrounding the organ transplantation process may contribute to the overall reactivity of the target cells by enhancing AT 1 R-AA binding to AT 1 R. An allogeneic background, brain death associated ‘cytokine storm’, reperfusion injury to the transplant and/or use of calcineurin inhibitors or steroids are probably permissive factors responsible for an increased AT 1 R density on target cells. For example in heart transplantation, systemic upregulation of AT 1 R can be found in donors with spontaneous intracerebral haemorrhage which was associated with subsequent development of cardiac vasculopathy [ 19 ]. However, their relative individual contribution needs to be further elucidated, in order to better understand and prevent AT 1 R-AA-related clinical syndrome. As happens in studies with unexpected findings, our work has raised more questions than it has answered. For the moment, we provide a new view on the complexity of vascular rejection process, where auto-immune receptor activation induces severe vascular pathology in the situation of allogeneic transplantation. Whether or not an AT 1 R-AA-mediated process represents a ‘true-rejection’ or an auto-immune phenomenon triggered in the permissive allogeneic and post-ischaemic inflammatory environment is the subject of our current investigations. The role of AT 1 R-AA during native renal disease and putative effects on cardiovascular comorbidity remains to be determined. A new cell-based AT 1 R-AA ELISA should help in the pre-transplant individual risk assessment of renal-allograft recipients and offer patients with AT 1 R-AA preemptive specific treatment. Conflict of interest statement . None declared.