Angiotensin II Type 1 Receptor Antibody Research Articles

Antibodies against angiotensin II receptor type 1 and endothelin A receptor are increased in COVID-19 patients.

Increased titers of autoantibodies targeting the G-protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endotelin-1 type A receptor (ETAR) are associated with severe coronavirus disease 2019 (COVID-19) infection. The aim of this study was to determine whether 1) these antibodies are specifically related to COVID-19 disease pathogenesis or increased during any severe respiratory illness, 2) if they are formed during illness, and 3) if they correlate with inflammatory markers or long-term symptoms. Antibodies against AT1R, ETAR, and antinuclear antibodies (ANAs) were measured in n=40 prospectively enrolled COVID-19 patients and n=207 COVID-19 patients included in a biobank. Clinical and laboratory findings were prospectively and retrospectively assessed in both cohorts, and results were combined for analysis. The presence of auto-antibodies against AT1R or ETAR in peripheral blood was compared between hospitalized patients with COVID-19 and controls (n=39). Additionally, AT1R and ETAR titers were compared between patients with an unfavorable disease course, defined as intensive care admission and/or death during hospital admission (n=121), to those with a favorable disease course (n=126). A subset of intubated patients with severe COVID-19 were compared to intubated patients with acute respiratory distress syndrome (ARDS) due to any other cause. Significantly increased AT1R and ETAR antibody titers were found in COVID-19 patients compared to controls, while titers were equal between favorable and unfavorable COVID-19 disease course groups. On ICU, intubated patients with COVID-19 had significantly increased AT1R and ETAR titers compared to patients with ARDS due to any other cause. The titers did not correlate with baseline inflammatory markers during admission or with diffusion capacity, cognitive impairment, or fatigue measured at 3 months follow-up. In patients hospitalized for COVID-19, antibodies against AT1R and ETAR are increased compared to controls and patients with ARDS due to other causes than COVID-19. The baseline antibody titers do not correlate with inflammatory markers or long-term symptoms in this study.

Angiotensin II type 1 receptor antibodies and native kidney function in pediatric liver and intestinal transplant recipients.

Angiotensin II type-1 receptor antibody (AT1R-Ab) has been associated with vascular injury and kidney dysfunction in pediatric kidney transplant recipients. The role of AT1R-Ab in the development of chronic kidney disease in pediatric liver and intestinal transplant recipients has not been explored. Twenty-five pediatric intestinal transplant recipients and 79 pediatric liver transplant recipients had AT1R-Ab levels measured at varying time points in the post-transplant period. Estimated glomerular filtration rate (eGFR) was determined using creatinine based CKiD U25 equation and measured at time of AT1R-Ab measurement, at 1year post-AT1R-Ab measurement, at 5years post-AT1R-Ab measurement, and at the most recent routine clinic visit. The prevalence of hypertension and antihypertensive medication use were also evaluated. Younger age at time of AT1R-Ab measurement was associated with AT1R-Ab positivity in liver transplant recipients. There was no association between AT1R-Ab status and change in eGFR, prevalence of hypertension, or use of antihypertensive medications at the described time points. AT1R-Ab positivity was not associated with a decline in eGFR or hypertension in pediatric liver and intestinal transplant recipients. Further studies are needed using other markers of kidney function, such as cystatin C, to validate this finding. A higher resolution version of the Graphical abstract is available as Supplementary information.

Angiotensin II Type 1 Receptor Antibody-mediated Rejection Following Orthotopic Heart Transplant: A Single-center Experience.

Antibody-mediated rejection (AMR) following orthotopic heart transplant (OHT) causes significant morbidity and mortality. There are limited data on antibodies to the angiotensin II type 1 receptor antibody (AT1R-Ab) causing rejection following OHT. This is a retrospective, single-center study that presents our 2-y experience with a series of 11 patients with evidence of nonspecific graft dysfunction and pathologic levels of AT1R-Ab. The clinical outcomes and treatments were compared to a group of 10 patients, also with evidence of nonspecific graft dysfunction, but who had nonsignificant AT1R-Ab titers. The mean age of the AT1R-Ab cohort was 52% and 73% were bridged to transplant with an left ventricular assist device. The average left ventricular ejection fraction at presentation was 45%, and most were not on an angiotensin receptor blocker (ARB). Endomyocardial biopsies in those with elevated AT1R-Ab levels frequently showed reactive endothelium/endocardium without C4d or intravascular CD68 staining. Ten patients (91%) were started on an ARB. Other therapies included plasmapheresis and IVIg (64%), with 4 patients also receiving rituximab. Most patients had symptom improvement, but minimal change in graft function at an average 6 mo of follow-up. The role of AT1R-Ab-mediated rejection in OHT recipients remains poorly understood. More than half of patients at our center who presented with graft dysfunction in the absence of acute cellular rejection or AMR were found to have elevated AT1R-Ab titers. Empiric AMR treatment in conjunction with ARB therapy may improve patient outcomes. Future studies are needed to better define the optimal treatment modalities for ATR1-Ab-mediated AMR.

Non-HLA AT1R antibodies are highly prevalent after pediatric intestinal transplantation.

The role of angiotensin II type-1 receptor (AT1R) antibodies in intestinal transplantation (ITx) is unclear. The aims were 1) to identify the prevalence of AT1R antibodies in pediatric ITx, compared to pediatric intestinal failure (IF), and 2) to determine whether AT1R antibodies were associated with graft dysfunction. 46 serum samples from 25 ITx patients (3 isolated ITx, 22 liver-inclusive ITx) were collected during routine visits >6months apart and during episodes of graft dysfunction as a result of infectious enteritis or rejection. For comparison, samples were collected from 7 IF control patients. AT1R antibodies were considered positive for levels >17 U/mL. The median (range) AT1R antibody level for ITx patients was 40.0 U/mL (7.2-40.0), compared to 7.0 U/mL (5.7-40.0) for IF patients (p=.02). There was a trend toward higher prevalence of AT1R antibodies in ITx compared with IF patients (68% versus 29%, p=.09). Among ITx patients, the prevalence of AT1R antibodies was not different between periods of active graft dysfunction and normal health (83% versus 67%, p=.31). For 16 patients with >2 samples, AT1R antibodies remained positive in 67% cases, developed in 14% cases, disappeared in 10% cases, and remained negative in 10% cases. The changes in AT1R antibodies did not correlate with de/sensitizing events. This is the first study of AT1R antibodies in pediatric ITx. AT1R antibodies are highly prevalent after ITx and may be triggered by immune activation associated with the transplant. However, their pathogenicity and clinical utility remain in question.

Elevated AT1R Antibody and Morbidity in Patients Bridged to Heart Transplant Using Continuous Flow Left Ventricular Assist Devices

BackgroundWe studied longitudinal levels of angiotensin-II type 1 receptor antibody (AT1R-Ab) and their effects on adverse events (death, treated rejection and cardiac allograft vasculopathy) in patients who were bridged to heart transplant using a continuous flow left ventricular assist device (LVAD). Methods and ResultsSera of 77 patients bridged to heart transplant (from 2009 to 2017) were tested for AT1R-Ab and CRP before and after LVAD. Elevated AT1R-Ab was defined as >10.0 U/mL. The median follow-up after transplant was 3.6 years (interquartile range, 2.2–5.6 years). After LVAD, AT1R-Ab levels increased from baseline and remained elevated until transplant. Freedom from adverse events at 5 years was lower in those with elevated AT1R-Ab levels at time of transplant. In an adjusted, multivariable Cox analysis, an AT1R-Ab level of >10 U/mL was associated with developing the primary end point (adjusted hazard ratio 3.4, 95% confidence interval 1.2–9.2, P = .017). Although C-reactive protein levels were high before and after LVAD placement, C-reactive protein did not correlate with AT1R-Ab. ConclusionsIn LVAD patients bridged to heart transplant, an increased AT1R-Ab level at time of transplant was associated with poor outcomes after heart transplant. Post-LVAD AT1R-Ab elevations were not correlated with serum markers of systemic inflammation. Larger studies are needed to examine the pathologic role of AT1R-Ab in heart transplant.

Targeted Delivery of Recombinant Heat Shock Protein 27 to Cardiomyocytes Promotes Recovery from Myocardial Infarction.

Ischemic heart disease, especially myocardial infarction (MI), is the leading cause of death worldwide. Apoptotic mechanisms are thought to play a significant role in cardiomyocyte death after MI. Increased production of heat shock proteins (Hsps) in cardiomyocytes is a normal response to promote tolerance and to reduce cell damage. Hsp27 is considered to be a therapeutic option for the treatment of ischemic heart disease due to its protective effects on hypoxia-induced apoptosis. Despite its antiapoptotic effects, the lack of strategies to deliver Hsp27 to the heart tissue in vivo limits its clinical applicability. In this study, we utilized an antibody against the angiotensin II type 1 (AT1) receptor, which is expressed immediately after ischemia/reperfusion in the heart of MI rats. To achieve cardiomyocyte-targeted Hsp27 delivery after ischemia/reperfusion, we employed the immunoglobulin-binding dimer ZZ, a modified domain of protein A, in conjunction with the AT1 receptor antibody. Using the AT1 receptor antibody, we achieved systemic delivery of ZZ-TAT-GFP fusion protein into the heart of MI rats. This approach enabled selective delivery of Hsp27 to cardiomyocytes, rescued cells from apoptosis, reduced the area of fibrosis, and improved cardiac function in the rat MI model, thus suggesting its applicability as a cardiomyocyte-targeted protein delivery system to inhibit apoptosis induced by ischemic injury.

Effect of Bortezomib Desensitization Therapy on Levels of AT1R Antibody

Purpose Increased levels of angiotensin II type 1 receptor (AT1R) antibody have been shown to be associated with antibody mediated rejection (AMR), cell mediated rejection (CMR), and early onset of microvasculopathy at one year post heart transplant. MCS patients with the saturated levels of AT1R antibody also trend lower survival. The presence of AT1R antibody pretransplant increases risk of development of de novo donor specific antibodies posttransplant in lung transplantation. Bortezomib, a protease inhibitor, has been used for desensitization or for treatment of AMR. This study aims to determine if Bortezomib treatment can decrease the level of AT1R antibody. Methods 25 heart transplant candidates who were highly sensitized were desensitized with the Bortezomib therapy. Levels of AT1R antibody were evaluated pre-therapy, 1 month and 6 months post-therapy. Results Out of 25 patients who were treated with Bortezomib, 22 patients who were tested for AT1R antibody at all three-time points were evaluated. 8 patients had AT1R levels 17 U). Of these 14 patients, the levels of AT1R antibody decreased below 17 U in 5 patients 1 month post Bortezomib treatment. Of note, 2 patients who had the saturated levels of AT1R antibody (>40 U) remained negative ( Conclusion The Bortezomib therapy can reduce the level of AT1R antibody in some heart transplant patients. Of interest, 2 patients who were still negative for AT1R antibodies 6 month post therapy were implanted with LVAD.

Efficacy of therapeutic plasma exchange on angiotensin II type‐1 receptor antibodies on two kidney transplant recipients

Angiotensin II type-1 receptor antibody (AT1RAb) has been reported to cause antibody mediated rejection (AMR) in kidney transplant recipients possibly by contraction of renal arteries. We here report 2 kidney transplant recipients with elevated AT1RAbs and negative HLA donor specific antibodies (DSA) and anti-major histocompatibility complex class I chain-related gene A (MICA) Abs who received therapeutic plasma exchange (TPE) treatment followed by IVIG. CASE 1: Thirty-eight-year-old patient received second kidney transplant for end stage renal disease (ESRD) with chronic rejection. Three years post-transplant, she developed AMR with AT1RAb level >40 U/mL. She received 5 TPE and AT1RAb decreased by 20%, and biopsy showed improvement of AMR. She received another 3 TPE and AT1RAb decreased by 60%. Her creatinine (Cr) was stabilized at around 1.4 mg/dL. CASE 2: Twenty-four-year-old patient received kidney transplant for ESRD with unclear etiology. Two weeks post-transplant, her Cr rose with AT1RAb level at 18 U/mL and biopsy showed possible AMR. She received 6 TPE treatments and AT1RAb decreased by 55% and biopsy showed improvement of AMR. She received weekly TPE for subsequently rising AT1RAb but TPE was discontinued because of unsuccessful decrease of AT1RAb. Her Cr was stabilized at around 1.7 mL/dL. We reported 2 patients who received TPE treatments to decrease AT1RAbs. A course of TPE treatment successfully decreased AT1RAb. Histological improvement was observed quickly and Cr was also stabilized following the TPE treatment. Further study is necessary to determine the optimal use of TPE in renal transplant recipients with AT1RAbs.

P161 Autoantibodies: Angiotensinogen vs AT1R as a target, Is there a correlation?

Aim Autoantibody directed at the Angiotensin II Type 1 Receptor (AT1R) has been reported as detrimental to solid organ transplants. An ELISA based AT1R antibody test is available, but presents challenges including different methodology, lot-to-lot variability, and cost effectiveness among them. A Luminex bead based assay autoantibody panel is available, currently detecting 33 targets, with more targets in development. One of the targets on the assay is Angiotensinogen (AGT), the precursor to Angiotensin II. This assay overcomes many of the challenges of the AT1R ELISA test. The aim of this study is to compare results of AGT autoantibody detection using the bead panel to the results of the AT1R autoantibody detection using the ELISA based testing. Methods OneLambda AT1R ELISA kits were used to test 30 sera from transplant patients for the presence of AT1R antibody. The trays were read on the VictorX and results were calculated using either the OneLambda 4PL curve calculation software, or the Workout Plus software on the VictorX, (validated as equivalent to the OneLambda software). OneLamda Autoantibody kits were used to test for the presence of antibody to angiotensinogen (AGT). The kit is a Luminex bead-based assay. Results were read on the Luminex 3D and results were analyzed using Fusion Research 6 software. Results The Reference values for the AGT target were determined by the manufacturer in a non-transplant population of 139 people. 75% had MFI of 777 or less, 85% had MFI of 1045 or less, and 95% had MFI of 1739 or less. Download : Download high-res image (578KB) Download : Download full-size image Conclusions These results show that ranges are overlapping and MFI mean values of AGN as a continuous variable are not consistent with the level of AT1R We conclude that there is no suggestion of a correlation between the results of both assays.

Successful Treatment of Anti–angiotensin II Type 1 Receptor Antibody–Associated Rejection in Kidney Transplantation: A Case Report

Angiotensin II type 1 receptor (AT1R) antibody, a non-HLA antibody, has been found to have a detrimental effect on kidney allografts. Similarly to HLA antibodies, recipients who have AT1R antibodies are at risk for allograft rejection and poor long-term graft outcome. Besides mediating allograft rejections via direct effects on endothelial and vascular smooth muscle without complement activation, AT1R antibodies may lead to accelerated hypertension via the renin-angiotensin pathway. There has been no definite level of AT1R antibody that predicts allograft rejection. Because of a low incidence of AT1R antibody–associated rejection, there are few reports on specific treatment. The results of conventional treatment, which aims to remove these pathologic antibodies similarly to the treatment of HLA antibody–associated rejection, have been unsatisfactory. Some studies recommend using angiotensin receptor blocker to attenuate the adverse effects of AT1R antibody on kidney allograft. Herein we present a kidney transplant recipient with AT1R antibody–associated refractory allograft rejection who was successfully treated with the use of steroid, plasmapheresis, intravenous immunoglobulin, and rituximab.

Angiotensin II Type 1 receptor antibodies are associated with inflammatory cytokines and poor clinical outcomes in pediatric kidney transplantation

Angiotensin II type 1 receptor (AT1R) antibody has beenlinked to poor allograft outcomes in adult kidney transplantation. However, its clinical consequences in children are unknown. To study this, we examined the relationship of AT1R antibody with clinical outcomes, biopsy findings, inflammatory cytokines, and HLA donor-specific antibodies (DSA) in a cohort of pediatric renal transplant recipients. Sixty-five patients were longitudinally monitored for AT1R antibody, HLA DSA, IL-8, TNF-α, IL-1β, IFN-γ, IL-17, and IL-6, renal dysfunction, hypertension, rejection, and allograft loss during the firsttwo years post transplantation. AT1R antibody waspositive in 38 of the 65 of children but was not associated with HLA DSA. AT1R antibody was associated with renal allograft loss (odds ratio of 13.1 [95% confidence interval 1.48-1728]), the presence of glomerulitis or arteritis, and significantly higher TNF-α, IL-1β, and IL-8 levels, but not rejection or hypertension. AT1R antibody was associated with significantly greater declines in eGFR in patients both with and without rejection. Furthermore, in patients without rejection, AT1R antibody was a significant risk factor for worsening eGFR over the two-year follow-up period. Thus, AT1R antibody is associated with vascular inflammation in the allograft, progressive decline in eGFR, and allograft loss. AT1R antibody and inflammatory cytokines may identify those at risk for renal vascular inflammation and lead to early biopsy and intervention in pediatric kidney transplantation.

P216 Case of suspected hyperacute rental transplant rejection due to angiotensin II type 1 receptor antibodies

Though the majority of antibody-mediated renal transplant rejections are thought to be due to HLA antibodies, there is increasing evidence that non-HLA antibodies also can mediate rejection. For example, antibodies directed against the Angiotensin II type 1 receptor (AT1R) have been implicated in humoral renal transplant rejection episodes and impaired graft survival. While many cases of acute rejection potentially due to AT1R antibodies have been reported, there is little published evidence relating to AT1R antibodies causing hyperacute rejection. We report the case of a patient with pre-existing AT1R antibodies and no donor-specific HLA antibodies who experienced an episode of hyperacute rejection. The patient is a 47 year old female with a longstanding history of type 1 diabetes and end stage renal disease secondary to diabetic nephropathy. She had received 2 previous renal transplants approximately 21 and 22 years ago. Pre-transplant she was highly sensitized with a T-cell flow PRA of 99% and B-cell flow PRA of 100%, but did not demonstrate any donor- specific HLA antibody. The T- and B-cell flow and T-cell CDC crossmatches performed between the patient and donor were negative. She had a pre-transplant AT1R antibody level of 18.5 Units/ml measured 8 days before transplant. During the surgical procedure the patient experienced a hyperacute rejection episode. Biopsy of the organ demonstrated possible antibody-mediated rejection. The patient was treated with plasmapheresis and IVIG and her renal function stabilized over the following 6 weeks. The patient has also been maintained on Losartan, an AT1R inhibitor. The patient has now continued to demonstrate stable renal function for the previous 2 years. Our case highlights the importance of appreciating the possible peri-transplant risks of AT1R antibody particularly as treatment with the AT1R inhibitor, Losartan, has been shown by others to provide therapeutic benefits.

Angiotensin II type-1 receptor antibody (AT1Rab) associated humoral rejection and the effect of peri operative plasma exchange and candesartan

Angiotensin II type 1 antibodies (AT1Rab) can mediate antibody mediated rejection (AMR). Pre transplant AT1Rab levels, and risk of rejection were assessed in Kidney Transplant Recipients (KTR) transplanted in our centre from 2013 to 2014 (n=145). 14/145 (9.7%) KTR experienced antibody mediated rejection (AMR). The Hazard Ratio for AMR=3.7 [95% CI 2–26] (p=0.009) for KTR with AT1Rab levels >17.5U/ml. 6/11 of KTR with levels >25U/ml experienced AMR.In 2015 (n=80) KTR were transplanted and 6/80 KTR experienced rejection (2 AMR and 4 TCMR with vascular lesions). 7/80 of KTR had AT1Rab 17.5–25U/ml and none experienced rejection and were induced with ATG and candesartan. 7/80 had AT1Rab 25–40U/ml and received pre and post-operative plasma exchange, ATG and candesartan and 1/7 experienced TCMR with a vascular lesion.This perioperative regimen may alter the risk of rejection in patients with high levels of AT1Ab and further studies are needed.

LBP37: The incidence of AT1R antibody in solid organ transplant and correlation with pre-transplant factors

Aim Antibodies to Angiotensin II type 1 receptor (AT1R) have been reported to be a risk factor for graft rejection and loss (Dragun). It is proposed that autoreactive and alloreactive antibodies to AT1R arise through multiple mechanisms, including transfusion, prior transplant and after tapering of immunosuppression (Reinsmoen, 2012). Thus, we hypothesized that AT1R antibodies (AT1R-Ab) would occur at a higher incidence in candidates for retransplantion. If true, this could factor into an effective algorithm screening algorithm. The purpose of this study was to determine the incidence of AT1R-Abs in the solid organ transplant candidates at our institution, comparing first transplant to retransplant candidates. Methods Pre-transplant sera from 101 solid organ transplant candidates, evaluated for transplant between January 2011 and June 2014 were retrospectively tested for AT1R-Ab. The presence of AT1R-Abs was determined using AT1R-Ab EIA test (One Lambda; Division of ThermoFisher, Inc., Canoga Park, CA) according to manufacturer instructions. The manufacturer suggested cut offs were used to categorize Results 17 U/mL as positive. Transplant candidates were sorted into groups based on organ(s) to be transplanted and whether it represented the candidate’s first or subsequent transplant. The percent of candidates with results positive for AT1R-Abs was calculated. Results Overall, 34% of all candidates were positive for AT1R antibodies prior to transplant. Another 14% of patients were classified as ‘At Risk’ with 10–17 U/mL AT1R Ab. The table below shows the breakdown of these results by organ type and transplant number. Conclusion AT1R antibodies were observed in a significant portion of solid organ transplant candidates. However, contrary to our hypothesis, we observed a significantly lower incidence of AT1R antibodies in retransplant candidates (26%), compared to previously untransplanted candidates (43%).

Novel retro-inverso peptide inhibitor reverses angiotensin receptor autoantibody-induced hypertension in the rabbit.

Activating autoantibodies to the angiotensin II type 1 receptor (AT1R) have been implicated in hypertensive disorders. We investigated whether AT1R antibodies produced in immunized rabbits will activate AT1R and contribute to hypertension by a direct contractile effect on the vasculature and whether they can be blocked by a novel decoy peptide. A multiple antigenic peptide containing the AT1R epitope AFHYESQ, which is the receptor-binding epitope of AT1R-activating autoantibodies, was used to immunize 6 rabbits. AT1R antibody activity was analyzed in AT1R-transfected cells, and their contractile effects were assayed using isolated perfused rat cremaster resistance arterioles. A retro-inverso D-amino acid epitope-mimetic peptide was tested for AT1R antibody inhibition in vitro and in vivo. All immunized animals produced high AT1R antibody titers and developed elevated blood pressure. No changes in measured blood chemistry values were observed after immunization. Rabbit anti-AT1R sera induced significant AT1R activation in transfected cells and vasoconstriction in the arteriole assay, both of which were blocked by losartan and the retro-inverso D-amino acid peptide. A single intravenous bolus injection of the retro-inverso d-amino acid peptide (1 mg/kg) into immunized rabbits dropped the mean arterial pressure from 122±11 to 82±6 mm Hg. Rabbit anti-AT1R sera partially suppressed angiotensin II-induced contraction of isolated rat cremaster arterioles, and the pressor response to angiotensin II infusion was attenuated in immunized animals. In conclusion, AT1R-activating autoantibodies and the retro-inverso d-amino acid peptide, respectively, have important etiologic and therapeutic implications in hypertensive subjects who harbor these autoantibodies.

Autoantibodies against β1 Receptor and AT1 Receptor in Type 2 Diabetes Patients with Left Ventricular Dilatation

Objectives: To explore the relationship between the autoantibodies against the β1 and AT1 receptors and left ventricular dilatation in patients with type 2 diabetes (T2DM). Methods: The autoantibodies against the β1 and angiotensin II type 1 (AT1) receptors of T2DM patients with and without hypertension were screened by ELISA. Multiple logistic regression was used to analyze the risk factors for left ventricular dilatation. The reversing effect of left ventricular dilatation was evaluated after receptor blocker treatment. Results: The positive rates of autoantibodies against the β1 and AT1 receptors (43.0 and 44.1%, respectively) in T2DM patients with hypertension were significantly higher than those in normotensive patients (16.0 and 10.4%, respectively; all p < 0.01). Furthermore, among T2DM patients with hypertension, the positive rates (61.4 and 64.9%, respectively) in patients with left ventricular dilatation were remarkably higher than those with normal left ventricular dimensions (34.4 and 36.1%, respectively; all p < 0.01). The presence of β1 receptor antibody and AT1 receptor antibody were risk factors for left ventricular dilatation (p < 0.05). The curative effect of metoprolol tartrate and valsartan in reversing left ventricular hypertrophy in the group positive for autoantibodies was much better than in the negative group. Conclusion: The findings show that autoantibodies against the β1 and AT1 receptors may play a role in predicting left ventricular dilatation in T2DM patients in combination with hypertension. Metoprolol tartrate and valsartan are effective and safe in the treatment of these patients.

OR47: INCIDENCE OF AT1R ANTIBODY IN LIVER TRANSPLANT CANDIDATES WITH FIBROSIS

Aim Liver fibrosis occurs as a result of deposition of collagen in response to an injury. Activation of the renin-angiotensin system increases angiotensin II levels in chronic cirrhosis. Angiotensin II promotes deposition of fibrous tissue and functions through the angiotensin II type 1 receptor (AT1R) which is expressed on hepatic stellate cells (HSC). AT1R antibodies have a similar agonistic effect when bound to the receptor and may be involved in the development of liver fibrosis. The presence of AT1R antibody in liver disease has not been investigated to date. The aim of this study is to determine the prevalence of AT1R antibody in liver transplant patients with fibrosis. Method Stored pre-transplant samples from sixty-three (63) liver transplant candidates and 40 normal healthy controls were tested for presence of AT1R under approved IRB using a sandwich ELISA (One Lambda as part of Thermofisher Scientific). Serum was incubated onto 96 well plates precoated with full length AT1R. Binding of antibody to AT1R was detected by horseradish peroxidase (HRP)-labeled goat anti-human IgG. Standard samples provided in the ELISA kit were tested in parallel in each run to generate a standard curve correlating optical density to AT1R antibody level. Each sample was tested in duplicate and the results expressed as a mean value. Results The average optical density (OD) in the liver group was 0.659 compared to an average OD of 0.354 in the control group (unpaired Student’s t test; p value 17 units/ml) or negative ( Conclusion Preliminary data show that there is a high incidence of AT1R antibody in liver transplant candidates with fibrosis or cirrhosis. This may suggest a link between AT1R antibody and liver fibrosis. The clinical relevance of this antibody will need to be further assessed. Liver fibrosis is usually detected by biopsy; however, tests of a fibrosis-associated serum marker would be less invasive and have a lower risk of complications. Furthermore, these patients may benefit from treatment with an angiotensin receptor blocker.

Angiotensin Type 1 Receptor Antibodies in Transplant Glomerulopathy.

Transplant glomerulopathy (TG) is a common cause of graft failure in kidney transplantation and is associated with donor specific antibodies (DSA). Angiotensin II type 1 receptor antibody (AT1RAb) and endothelin 1 type A antibody (ETARAb), two recently described non-HLA antibodies, are implicated as independent risk factor of acute vascular rejection in the absence of donor specific antibodies. In a single centre retrospective study, we analysed from the ANZDATA registry, all of the TG patients, transplanted between 1980 and 2011, who met with TG Banff criteria. All were tested by a solid phase assay method for AT1RAb and ETARAb and by Luminex single antigen for DSA. Eight percent (141 patients) of the 1729 patients recorded along the period study met the TG biopsy proven criteria. More than 50% of this TG group had a value for AT1RAb and ETARAb >10U/mL. Of the 89 TG patients tested for AT1RAb at transplantation, 38 were positive for AT1RAb. We showed a strong link between the value of AT1AbR and patient survival with a significant risk of death in the high value group (>17U/mL). The presence of DSA did not correlate with AT1RAb and 8 of the 38 patients developed TG were positive for AT1RAb tested at transplantation without DSA or HLA antibodies, suggesting an independent role of AT1RAb in TG. In conclusion, the prevalence of AT1RAb is high in this large cohort of TG and may be involved in the TG development independently of DSA. DISCLOSURE:Coates, P.: Speaker’s Bureau, Alexion, Other, Novartis, Advisory Board. Dragun, D.: Grant/Research Support, OneLambda, Novartis.

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Angiotensin II type 1 receptor (AT1R) is found in lung alveolar epithelium, the stroma surrounding epithelial layer of small airways, fibroblasts, vessel smooth muscle and alveolar macrophages. While the impact of HLA antibody in lung rejection is increasingly recognized, this association has not been studied for non-HLA antibodies such as anti-AT1R antibodies. We studied 112 patients who underwent lung transplantation in our center since the inception of the lung allocation score (LAS) in May 2005. Pre-transplant sera were screened for HLA and AT1R antibodies. 89 patients had clinical data available for analysis. AT1R antibody was tested on an ELISA platform while HLA antibody was defined using solid phase luminometric assays. AT1R levels were examined as continuous values. Test results were interpreted using a dichotomized cut-point of 17 U/ml as recommended by the manufacturer. AT1R antibodies were positive in 31(35%) and negative in 58(65%) patients. Gender was not associated with AT1R sensitization (p = 0.2). Donor HLA specific antibodies were found in 10(11%) patients who were excluded from lung function and survival analysis. AT1R sensitization was not a significant predictor for lung function at 48 hours (p = 0.07) or at 6 month (p = 0.9). Further, presence of AT1R antibodies did not correlate with one year patient survival by Kaplan-Meier method (p = 0.8). AT1R sensitization was not a risk factor for rejection within 1 year (p = 0.5) or >1 year (p = 0.7) post-transplant. Post-transplant acute kidney injury was not associated with presence of AT1R antibodies. Post-operative care was also similar between the two groups (median length of stay in ICU, p = 0.9; median total hospital length of stay, p = 0.6). Our data show that AT1R sensitization does not correlate with early clinical outcomes in lung transplantation.

Recognition of tissue- and subtype-specific modulation of angiotensin II receptors using antibodies against AT1 and AT2 receptors.

Polyclonal antibodies have been prepared against synthetic peptides of human angiotensin II type-1 (AT1) and type-2 (AT2) receptors. Synthetic peptides corresponded to amino acids 15-24 of AT1 receptor and amino acids 241-253 of AT2 receptor. Western blot analysis of membranes from cell homogenates of COS-7 cells transfected with expression plasmids for mouse AT1b receptor, human vascular smooth muscle cells, and rat peripheral tissue demonstrated a major band of MW 44,000 with AT1 receptor antibody. Cell homogenates of COS-7 cells transfected with expression plasmids for human AT2 receptor showed a band of MW 44,000 with AT2 receptor antibody. Tissue homogenate of rat adrenal medulla and human pheochromocytoma presented a major band of MW 52,000 and a minor band of MW 44,000 with AT2 receptor antibody. AT1 receptor expression was in the following order: rat aorta > > lung, kidney, spleen, adrenal cortex > adrenal medulla, heart. Expression of AT2 receptor was in the following order: rat adrenal medulla > cortex, kidney, heart. Three-day treatment with CS866, an AT1 receptor antagonist, and temocapril, an angiotensin-converting enzyme inhibitor, suppressed AT1 receptor expression in the rat adrenal cortex, but not in the heart or adrenal medulla. AT2 expression was not affected by treatment with these drugs. These results suggest that newly developed antibodies for AT1 and AT2 receptors are useful in elucidating the regulation of subtype-specific receptor expression, and that AT1 but not AT2 receptors in adrenal tissue are regulated by angiotensin II.