The Presence of Anti-Angiotensin II Type-1 Receptor Antibodies Adversely Affect Kidney Graft Outcomes

The aim of this study was to determine whether anti-angiotensin type 1 receptor antibodies (AT1R-Abs) are related to acute rejection (AR) and kidney graft failure in renal transplantation. We searched electronic databases including MEDLINE, EMBASE, and the ISI Web of Science databases for all studies on the association between anti-angiotensin type 1 receptor antibodies and kidney allograft outcomes updated to November 2016. Reference lists from included articles were also reviewed. The pooled relative risks (RRs) with 95% confidence intervals (CIs) were extracted or calculated using a random-effects model. The potential sources of heterogeneity and publication bias were estimated. Nine studies enrolling 1771 subjects were retrieved in the meta-analysis. AT1R-Abs showed significant associations with increased risk of AR (RR = 1.66; 95% CI, 1.23–2.09). In addition, a significant relationship was found between AT1R-Abs and kidney graft failure compared with AR (RR = 3.02; 95% CI, 1.77–4.26). The results were essentially consistent among subgroups stratified by participant characteristics. These results demonstrated that the AT1R-Abs were associated with an elevated risk of kidney allograft outcomes, especially with kidney graft failure. Large-scale studies are still required to further verify these findings.


Introduction
Kidney transplantation following end-stage renal disease has proved to be the optimal treatment providing notable improvement in patient "quality of life" [1]. Acute vascular rejection after kidney transplantation is always the most important challenge for sustaining continued long-term function of the allograft [2]. Advances in human leukocyte antigen (HLA) tissue typing and HLA-antibody detection have remarkably improved antibody-mediated rejection (AMR) prediction and recognition [3]. However, acute vascular rejection that is refractory to therapy still occurs in HLA-identical sibling transplants. A variety of non-HLA antibodies have been identified in serum obtained before transplantation from patients in whom refractory rejection developed after they received kidney transplants from HLA-identical siblings [4]. Elucidation of the association between non-HLA antigens and vascular rejection might provide new insight into potential mechanisms, and facilitate the development of specific therapies.
As special non-HLA antibodies, anti-angiotensin type 1 receptor antibodies (AT1R-Abs) are proposed to set up an alternative mechanism for renal graft injury and acute rejection [5]. Anti-angiotensin II type 1 receptor (AT1R) belongs to the type A family of G protein-coupled receptor (GPCR) and is responsible for most angiotensin II-mediated physiological activities, including blood pressure regulation and fluid and electrolyte balance [6]. AT1R-Abs were characterized as immunoglobulin G1 (IgG1) and IgG3 subclass antibodies, which recognize conformational antigens contained in the second extracellular loop of the AT1R. The binding of antibodies to AT1R appears to be capable of inducing excessive activation of signal transduction in vessel endothelial and smooth muscle cells, which was associated with vascular inflammatory damage [7]. Previous studies have shown that AT1R-Abs were directly involved in the vascular disease pathology of hypertension, preeclampsia, and systemic sclerosis [8][9][10], which may share a similar inflammatory mechanism with acute rejection (AR) in transplant recipients with AT1R-Abs. Moreover, AR after kidney allograft transplantation frequently progresses to persistent chronic rejection and dysfunction of kidney allograft, and ultimately results in kidney graft failure [11], indicating that AT1R-Abs may also influence the long-term outcomes of kidney allograft transplantation. Currently, several studies have shown that AT1R-Abs might be associated with an increased risk of acute rejection and kidney graft failure [12][13][14]; however, the results are inconsistent and inconclusive [15,16].
Therefore, we conducted a meta-analysis of the evidence to evaluate the relationship between AT1R-Abs and the risk of AR in renal transplantation. Furthermore, we also examined the impact of AT1R-Abs on long-term kidney graft outcomes.

Search Strategy
We searched electronic databases including MEDLINE, EMBASE, and the ISI Web of Science databases for all studies on the association between AT1R-Abs and kidney allograft outcomes updated in November 2016, using the search terms: ("angiotensin II type-1 receptor antibody" or "AT1R-Ab" or "AT1R antibody" or "anti-AT1R antibodies" or "AT1Rab") and ("renal" or "kidney") and ("transplantation" or "transplant"). No language restriction was imposed on searching and study inclusion. We also checked the reference lists of retrieved articles and relevant reviews to find other potential articles. We attempted to contact the authors and experts of ongoing research when more detailed information was necessary.

Study Selection
To identify all eligible studies, we used a two-step selection strategy. In the first step, we performed an initial review of all identified abstracts and titles to exclude any clearly unrelated articles. Then, the full texts of the remaining studies were further examined for their suitability for the present meta-analysis. The selected references of the full-text articles were checked using the same criteria. Studies included in the present meta-analysis have to meet the following criteria: (1) assessed the association between AT1R-Abs and kidney allograft outcomes (AR or kidney graft failure) among adult renal transplants; (2) used cohort, case-control, or cross-sectional design; (3) provided adjusted relative risk (RR) or odds ratios (OR) with the corresponding 95% confidence interval (CI) or sufficient data to estimate them. When multiple publications reported on the same or overlapping data, we only selected the most updated data. Two reviewers independently screened and assessed publications for potential inclusion in the analysis according to the same criteria. Discrepancies were resolved by a third author (Le Ma).

Data Extraction and Quality Assessment
The following information was extracted from each article: first author; year of publication; study design; research center; sex distribution; average age; first transplant rate; patients with living donors; detection of AT1R-Abs; follow-up period; diagnosis of AR; classification of AR; induction and maintenance regimens; controlled variable. The RRs (or ORs) with 95% CI in the studies were also extracted. If a study provided several risk estimates, we extracted the estimate that reflected the greatest degree of adjustment. The methodological quality of the eligible studies was assessed using Newcastle-Ottawa Quality Scale (NOS) [17]. The quality of each study was assessed and awarded stars for indicators of quality, including three aspects: subject selection (0-4 scores), comparability (0-2 scores) and exposure (0-3 scores). Total scores ranged from 0 (worst) to 9 (best). Studies with a score of 5-9 were considered to be of high quality and studies with a score of 0-4 were considered to be of low quality. Two authors independently extracted data from each study included in the present meta-analysis using standardized data extraction forms. Discrepancies between two authors were resolved by discussion with a third investigator (Le Ma).

Statistical Analysis
RRs with corresponding 95% CI were used to assess the strength of the association between AT1R antibodies and kidney allograft outcomes. Because the absolute risk of AR or kidney graft failure in renal transplantation was low, ORs and hazard ratios (HRs) could also be considered an approximation of relative risk. Summary RRs were calculated using a random effects model. We evaluated heterogeneity between studies with the I 2 statistic (I 2 > 50% indicated evidence of heterogeneity) [18]. We explored potential sources of heterogeneity with stratified analyses. Subgroup analysis was conducted by mean age, study design, living donors rate, first transplant rate, adjustment, country of origin. We also performed sensitivity analyses by removing each study one at a time to confirm the stability of the results. Potential publication bias was assessed using Begger funnel plots and the Egger linear regression test (p < 0.05 was considered statistically significant) [19,20]. All statistical analyses were performed using the software Stata version 11.0 (StataCorp, College Station, TX, USA).

Characteristics of the Studies
The characteristics of the included studies are presented in Table 1. Of the nine studies, five were conducted in America, two in Europe, one in Asia and one in Australia. Six studies were cohort studies, and three were case-control studies. The number of subjects ranged from 70 to 599. In six studies, more than 90% of subjects were receiving a first kidney transplant. The average age of subjects ranged from 27.7 years to 51.3 years. AR was biopsy-proven in all studies, except one study which reported that a 25% increase in serum creatinine was diagnosed as acute rejection. Seven studies employed an induction regimen strategy including anti-thymocyte globulin (ATG) and anti-human interleukin-2 receptor (anti-IL2R) antibody, whereas two studies did not report the induction regimen used. Five studies included reported a triple immunosuppressive therapy with tacrolimus/cyclosporine A (TAC/CsA), mycophenolate mofetil (MMF), and steroids. Two studies reported that TAC/MMF were used; two studies did not report the immunosuppressive therapy employed. All studies included were classified as high quality.

The Presence of AT1R-Abs and AR Risk
Nine studies with a total of 1771 participants reported the relationship between AT1R-Abs and AR. Five included studies show an association between AT1R-Abs and a significantly increased risk of AR, whereas other studies show no relationship between them. Across the nine studies included, patients with AT1R-Abs were associated with a higher RR of developing AR compared with patients without AT1R-Abs (pooled RR, 1.66; 95% CI, 1.23-2.09), using the random effects model (see Figure 2). No evidence of heterogeneity was detected across these studies (I 2 = 20.7%; p = 0.26). Stratified analysis found that none of the participant characteristics substantially altered the shape of the association (see Table 2). Sensitivity analyses indicated that the pooled RRs were not influenced excessively by any single study. The funnel plot for the studies evaluating AT1R-Abs and its association with AR risk did not show asymmetry (see Figure 3). The Egger test (p = 0.47) and Begg test (p = 0.15) revealed no evidence of publication bias.

The Presence of AT1R-Abs and Risk of Kidney Graft Failure
The association between AT1R-Abs and kidney graft failure was investigated in four studies comprising a total of 1208 participants [14,16,21,22]. Studies showed no existence of significant heterogeneity (I 2 = 0.00%; p = 0.78), and the random-effects pooled incidence of kidney graft failure was significantly higher among patients with AT1R antibodies than those without AT1R antibodies (pooled RR, 3.02; 95% CI, 1.77-4.26; see Figure 4). We analyzed the effect of the difference in the endpoint of kidney graft failure between studies on the results in subgroup analysis. Inconsistencies in the endpoint did not alter the shape of the association (p = 0.55). Moreover, we conducted stratified analyses to evaluate whether the association of the presence of AT1R-Abs differs significantly between AR and kidney graft failure. The association of AT1R antibodies seemed to be slightly stronger with kidney graft failure than AR (p = 0.08), although statistical significance was not reached.

Discussion
In the present study, we evaluated the effects of AT1R-Abs on renal allograft outcome based on data from included studies. The results showed that AT1R-Abs were associated with an increased risk of AR and kidney graft failure. In addition, a significant correlation was found between a decrease in graft survival and the presence of AT1R-Abs, indicating that pretransplant detection of AT1R-Abs may be useful for identifying immunologic risks and kidney allograft outcome. Some stratified analyses across participant characteristics were conducted, with essentially no change in the results of the present study.
AT1R, a seven transmembrane-spanning G-protein-coupled receptor, is distributed in many cell types, including vascular endothelial cells and smooth muscle cells, and mediates physiological activities of angiotensin II under normal circumstances [25]; however, excessive activation of AT1R can trigger transcription factor expression in endothelial and vascular smooth muscle cells, which further results in the secretion of proinflammatory messengers. These inflammatory factors contribute to the pathological progression of vascular and renal diseases [26]. AT1R, as a vascular endothelium antigen, can be invariably recognized and combined by AT1R-Abs, which are produced by the host immune system after renal allograft transplant. As allosteric activators, AT1R-Abs could motivate AT1R in a similar manner as angiotensin II and further result in renal allograft injury [27,28]. The study conducted by Dragun et al. showed that the passive transfer of AT1R-Abs isolated from affected renal transplant patients into low-responder allogeneic rat transplant induced similar vascular rejection phenotypes as observed in their transplant biopsies [29]. In addition, the removal of AT1R-Abs by plasmapheresis in combination with AT1R blockade by Losartan has been proven to improve renal function and graft survival [30]. The results of our meta-analysis suggested that AT1R-Abs were significantly associated with increased risk of AR. The mechanism for such an observed relationship might relate to inflammation and coagulation induced by AT1R-Abs [31,32]. AT1R-Abs can regulate phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and further activate various nuclear transcription factors, including nuclear factor-kappa B (NF-κB) and the activator protein-1 (AP-1) in endothelial and vascular smooth muscle cells [33]. As important nuclear transcription factors, NF-κB can regulate several genes, including cytokines, adhesion molecules, and angiotensinogen involved in the pathogenesis of vascular inflammatory and acute rejection [34]. Furthermore, expression of tissue factor as regulated by NF-κB and AP-1 may increase procoagulatory activity of the injured vessels. Adhesion and chemotaxis of lymphocytes in the vascular bed of the allograft in response to vascular clotting may trigger acute rejection [35].
The present study showed that AT1R-Abs were also correlated with a significantly increased risk of kidney graft failure. The reasons for this might be that chronic rejection arises when low titer AT1R-Abs deposit on capillary endothelial surfaces and could activate inflammation and coagulation, which correlated with chronic allograft injury and transplant failure [11]. Moreover, this effect could be partly explained by the higher incidence of AR in patients with AT1R-Abs. Previous studies had suggested that AR was frequently accompanied by vascular or antibody-mediated rejection, which could initiate persistent chronic rejection followed by progressive functional decline to end-stage failure [36,37].
Our findings have important clinical health significance. Transplant rejection is closely associated with early and late graft loss. The prevalence of rejection episodes related to AT1R-Abs is thought to be 3.6% [29]. The present meta-analysis showed that the AT1R-Abs were significantly correlated with an elevated risk of AR and kidney graft failure, indicating that AT1R-Abs may be a potential biomarker for identification of patients at risk of AR and vascular injury that would be ignored by normal criteria. The assessment of pretransplantation AT1R-Abs status in patients could more effectively stratify the immunologic risk and optimize treatment strategy.
Several limitations of this study need to be considered. First, the present study was based on observational studies, which might be inherently biased by various factors. Second, although several confounding factors had been adjusted for in the included studies, the possibility of other uncontrolled or potential confounding factors could not be completely excluded in the present meta-analysis. Previous studies have shown that genetic or microenvironmental factors might also influence the severity and velocity of AT1R-Abs-associated pathologies, but we could not evaluate the effect of these factors due to insufficient data in the studies included [38]. Fourth, the relatively small sample sizes might influence the statistical power to assess the relationship. However, with negligible heterogeneity across studies, the results from the present meta-analysis were reliable. Fifth, although subgroup analysis showed that the difference in the endpoint of kidney graft failure between studies did not alter the shape of the association, the effect of the endpoint on the results needed further validation because of the small number of articles. Finally, although no publication bias was examined in the present study, it was still difficult to completely rule out such bias because there was not a sufficient number of studies to detect it adequately.

Conclusions
In conclusion, the present meta-analysis demonstrated that the AT1R-Abs were significantly associated with elevated risk of AR and kidney graft failure. Meanwhile, considering that only a few studies have examined this relationship, further well-designed, larger studies with prospective cohort design are required to validate the correlations between AT1R-Abs and renal graft outcomes.