Objectives: Anti-glutathione S transferase T1 (GSTT1) antibodies, a type of non-HLA antibody, have been associated with chronic hepatic graft rejection. Despite the presence of this enzyme in the kidney, there are not enough studies on the development of anti-GSTT1 antibodies and their impact on renal grafts. Our objective was to evaluate the presence of anti-GSTT1 antibodies after renal transplant and their impact on graft outcomes.
Materials and Methods: We conducted an ambispective cohort study. We performed real-time polymerase chain reaction to screen for GSTT1 alleles in 293 recipients and their donors. In null GSTT1 (GSTT1*0) genotype recipients of GSTT1-positive donors, the presence of anti-GSTT1 antibodies was evaluated using indirect immunofluorescence and Luminex assays, and their effects on graft function were evaluated. The median follow-up period was 54.3 months.
Results: Of the 293 patients studied, 42 recipients (14.4%) with GSTT1-positive donors did not have the GSTT1 allele (GSTT1-positive donor/GSTT1*0 recipient). Using Luminex assay, we detected antibodies in 16 patients (38.1%), 12 of which were already present at the time of transplant. Of these cases, 37.5% with antibodies had undergone a previous renal transplant. Using indirect immunofluorescence, we found that only 12 patients tested positive, 4 at the time of transplant. Antibody presence did not effect graft glomerular filtration rates or graft loss at 1 year, at 2 years, or end of follow-up.
Conclusions: The presence of anti-GSTT1 antibodies is frequent in renal transplant GSTT1*0 recipients of GSTT1-positive donors but has no effects on graft outcome.
Key words : Anti-GSTT1 antibody, GSTT1 genotype, Kidney transplant outcome, Non-HLA antibodies
Renal transplantation is the most effective treatment option for chronic end-stage kidney disease. In recent decades, improvements in immunosuppression regimens with more powerful agents have signi-ficantly increased immediate graft survival rates.1,2 Developments in the prevention and treatment of immunosuppression-related infections have also led to a decline in the incidence and severity of posttransplant infections, increasing the patient’s ability to tolerate more efficient immunosuppression regimens.3
Nevertheless, despite advances in short-term graft survival, long-term survival rates have remained unchanged, and chronic deteriorating graft function still stands as one of the primary causes of graft loss.2,4 A major cause of chronic graft dysfunction is antibody-mediated rejection (AMR) of donor-specific antigens. Specifically, AMR has been commonly associated with donor-specific HLA antibodies,5-8 although in recent years there has been an increased interest in the role of non-HLA antibodies in AMR.9 Non-HLA antigens have been identified as potential alloantigens in transplantation and are a risk factor for chronic allograft nephropathy.10-12
Glutathione S-transferase theta 1 (GSTT1) is one type of non-HLA antigen. This enzyme belongs to a superfamily of proteins responsible for catalyzing the conjugation of reduced glutathione to different electrophilic and hydrophobic compounds, which play roles in cellular 5 detoxification and oxidative stress. The GSTT1 enzyme is mostly expressed in the liver and kidneys, with low levels also found in the prostate and small bowel. In the blood, it is only expressed in red blood cells.13,14 The GSTT1 enzyme is coded by a polymorphic gene with 2 alleles: GSTT1*A (positive) and GSTT1*0 (null). The GSTT1*0 genotype, associated with the total absence of the enzyme, is present in 20% of White populations and 11% to 58% of other ethnic and racial groups.15-17 Although this enzyme has been proven to have a protective role against free radicals, acting as a significant shield against oxidative stress, there is no evidence to suggest that its absence is linked to chronic renal graft dysfunction.18 However, GSTT1*0 transplant recipients of GSTT1-positive donors may develop an immune response leading to anti-GSTT1 antibody production, which could in turn cause graft rejection.19
Most studies on the influence of anti-GSTT1 antibodies have been conducted on liver transplant recipients. These antibodies have been associated with de novo immune hepatitis after liver transplant in GSTT1*0 recipients who were matched with a GSTT1-positive donor.20-23 Research on the influence of anti-GSTT1 antibodies in renal graft evolution is insufficient. Although it is known that anti-GSTT1 antibodies can develop after kidney transplantation in GSTT1-positive donor/GSTT1*0 recipient matches, their impact on graft outcome is contradictory and inconclusive.24-27 The high prevalence of GSTT1 positivity in our population increases the likelihood of a GSTT1 mismatch between donor and recipient, which could lead to decreased graft function. The purpose of our study was to analyze the presence of anti-GSTT1 antibodies after renal transplant and their impact on graft outcomes.
Materials and Methods
We performed an ambispective cohort study of patients who received a kidney transplant between 2009 and 2019. The study considered all patients who underwent transplant during the study period and had a minimum follow-up period of 3 months, with this time spanning up to December 2021.6 Renal transplant was performed to ensure ABO blood type compatibility between organ donors and recipients and with no donor-specific anti-HLA antibodies. All donors and recipients were White. The study was approved by the Ethics and Research Committee of the Hospital Universitario de Badajoz.
Genotyping of the GSTT1 allele and detection of anti-GSTT1 antibodies
All recipients were screened for the GSTT1 allele. For GSTT1*0 recipients only, GSTT1 allele genotyping of their donors was performed to determine whether the match was GSTT1-postive donor to GSTT1*0 recipient. In this group, we determined whether anti-GSTT1 antibodies were present in the recipient.
We performed GSTT1 allele detection by real-time polymerase chain reaction (GVS-GSTT1-48; Blackhills Diagnostic Resources) after DNA extraction (MagCore Genomic ADN whole blood kit, Cartridge Code 102, RBC Bioscience) of peripheral blood samples collected in tubes with EDTA anticoagulant. Donor DNA extraction was performed on peripheral blood samples or on isolated lymphatic or splenic cellular specimens. In the GSTT1-positive donor/GSTT1*0 recipient group, we used indirect immunof-luorescence (IIF) and Luminex assays in pretransplant and posttransplant serum samples to establish the presence of anti-GSTT1 antibodies. We performed IIF on triple rodent tissue (liver, kidney, and stomach) (Biocientífica, Menarini Diagnostics) using the automated Zenit UP system (A. Menarini Diagnostics) and Zenit HUB 2.1.3 Rev2n software (A. Menarini Diagnostics). The prepared slides were observed under a LED fluorescence microscope (×100 to ×400 magnification). Specific markers in perivenous hepatocytes (primarily in centrolobular areas) in the liver (Figure 1) and P2 proximal tubules in the kidney (Figure 2) indicated a positive result. For the Luminex assay, we used the LIFECODES non-HLA antibody (Immucor) detection kit.
Clinical and analytical variables
We adjusted for potential confounding variables in our analyses of the effects of anti-GSTT1 antibodies on graft function. We collected the following variables: (1) donor and recipient age; (2) donor biopsy score (a renal biopsy was performed on all donors with expanded criteria [deceased donors aged ≥60 y or aged 50-59 with stroke as the cause of death, history of arterial hypertension, or creatinine >1.5 mg/dl at the time of donation] and on donors after cardiac death); and (3) number of HLA mismatches between donors and recipients in HLA-A, HLA-B, and HLA-DR (0-6). We also collected the type of immunosuppression, which included (1) corticoids, mycophenolate, and delayed dose tacrolimus for donors with brain death without expanded criteria; (2) corticoids, basiliximab, mycophenolate, and delayed dose tacrolimus for donors with expanded criteria and recipients with low immunologic risk; (3) corticoids, basiliximab, mycophenolate, and tacrolimus for donors with expanded criteria and recipients with moderate immunologic risk; and (4) thymoglobulin, corticoids, mycophenolate, and tacrolimus for donors after cardiac death and recipients with high immune compromise. We also collected recipient sex; recipient’s history of diabetes mellitus, ischemic cardiopathy, arterial hypertension, or peripheral vascular disease (intermittent claudication or ischemic stroke); type of dialysis pretransplant (hemodialysis or peritoneal dialysis); recipient’s renal disease etiology; potential presence of delayed graft function (defined as the need for hemodialysis during the immediate posttransplant period); BK virus infection posttransplant; acute rejection; and development of donor-specific anti-HLA antibodies.
The renal graft function was calculated using the CKD-EPI formula, in which plasma creatinine levels were considered at the following time points: 1 year, 2 years, and end of follow-up. Graft loss was defined as the return to chronic dialysis but excluded functioning grafts at the time of recipient’s death.
Using the Shapiro-Wilk test, we expressed normally distributed quantitative variables as means and standard deviation (SD) and those that failed the normality test as median and interquartile range (IQR; 25th to 75th percentile). We compared the 2 groups using t tests or Mann-Whitney U test, depending on the case.
We expressed categorical variables as frequency (number of observations) and proportions (percent of the whole or within the category). We compared groups using the Pearson chi-square or the Fisher exact test for 2 × 2 tables, according to the number of expected cases per category, and the likelihood ratio for tables with more than 2 categories.
The impact of anti-GSTT1 antibodies on graft function, as assessed by estimated glomerular filtration rate (eGFR) or graft loss at the end of the follow-up period, was analyzed by multivariable linear or adjusted logistic regression, respectively.
Statistical significance was established at P < .05. We used SPSS 26.0 for Windows for data analyses.
Initially, 301 renal transplant patients were considered for this study, 8 of whom were excluded from analysis because of graft loss during follow-up or lack of donor sample availability. The 293 patients included had a median follow-up period of 54.3 months after transplant (Table 1). The mean ± SD age was 54.8 ± 11.8 years, with a greater number of male patients (n = 186, 63.5%) and patients on hemodialysis (n = 238, 81.2%).
The most frequent donation type was donation after brain death (n = 232, 79.2%). More than half of all patients received immunosuppression based on induc-tion with basiliximab and delayed dose tacrolimus, combined with steroids and mycophenolate.
The median eGFR at 1 year, 2 years, and end of follow-up was 51.1 mL/min, 51.1 mL/min, and 47.4 mL/min, respectively. During the follow-up period, 23 patients (7.9%) experienced graft loss and returned to dialysis.
GSTT1 allele genotyping
Of the 293 transplant recipients, 56 (19%) had the GSTT1*0 allele. GSTT1 genotyping of their matched donors was performed in these cases, with 42 donors (75%) having the GSTT1*A allele (GSTT1-positive donor/GSTT1*0 recipient match) and 14 donors (25%) having the GSTT1*0 allele (GSTT1*0 donor/GSTT1*0 recipient match).
Indirect immunofluorescence anti-GSTT1 antibody detection
In the GSTT1-positive donor/GSTT1*0 recipient group, 12 patients (28.6%) screened positive for anti-GSTT1 antibodies. Antibody titer ranged from 1/40 to 1/320, with a median of 1/160. In 4 of these patients (33.3%), antibodies were present at the time of transplant; the median time for detection during follow-up in the remaining patients was 20.8 months (IQR, 4.9-71.2 mo).
Luminex assay anti-GSTT1 antibody detection
Luminex assay results showed that 16 of 42 patients (38.1%) in the GSTT1-positive donor/GSTT1*0 recipient group tested positive for anti-GSTT1 antibodies, with a mean ± SD median fluorescence intensity of 11 700 ± 6360). Among them, 12 patients (75%) were positive at the time of transplant, with median time for detection in the remaining cases of 34.5 months (IQR, 22.0-68.3 mo) posttransplant. A positive anti-GSTT1 antibody IIF screen was corroborated by the Luminex assay in all cases; 37.5% of patients with a positive Luminex assay screen had a history of previous transplant.
The presence of anti-GSTT1 antibodies was also analyzed by both methods in the GSTT1*0/GSTT1*0 group (n = 14), with 1 of the patients testing positive by both techniques. The recipient was a female patients with no previous transplants. She was excluded from the study given that her anti-GSTT1 antibodies were not donor specific (GSTT1*0 donor) and therefore not secondary to the graft.
Effect of anti-GSTT1 antibodies on graft outcome
Table 1 shows the characteristics of patients according to positive or negative anti-GSTT1 antibody screenings as determined by the Luminex assay, which in our study was considered the reference technique due to its higher sensitivity.
Patients with antibodies were younger (mean age of 48.6 vs 55.2 y; P = .030) and had longer dialysis duration before transplant (median of 66.2 vs 40.9 mo; P = .026); there was also a greater percentage with ischemic cardiopathies (31.3% vs 7.3%; P = .007). No statistically significant differences were observed in terms of sex or type of immunosuppression used.
The eGFR at 1 year, 2 years, and end of follow-up was similar between the 2 groups, with no statistically significant differences found, respectively showing 51.2 versus 50.7 mL/min (P = .997), 51.1 versus 49.9 mL/min (P = .842), and 48.2 versus 43.4 mL/min (P = .635). Although the rate of graft loss was higher in the group with positive antibodies, this difference was not statistically significant (18.8% vs 7.3%; P = .122).
The results of the multivariable linear regression analysis did not identify any effects from the presence of anti-GSTT1 antibodies on eGFR at 1 year, 2 years, or end of follow-up, respectively showing β coefficient = 1.4 (95% CI, -8.8 to 11.7; P = .782). β coefficient = 0.9 (95% CI -10.0 to 11.9; P = .866); and β coefficient = -2.6 (95% CI, -13.4 to 8.2; P = .636).
With multivariable logistic regression analysis, we observed no significant relation between a positive test for anti-GSTT1 antibodies and graft loss (odds ratio = 3.5; 95% CI, 0.8-16.0; P = .099).
Genetic differences between donors and recipients are one of the main obstacles affecting graft success and outcome. Mismatches in diverse polymorphic antigen systems may lead to an immune response or graft rejection as antibodies develop to target these polymorphic antigens.
With literature documenting histologic cases of AMR in renal transplant biopsies without circulating anti-HLA antibodies, recent research has turned to other types of antibodies as the potential culprits of these findings.9
Data from renal transplant recipients who met the histologic criteria for AMR in their renal biopsy and who had no circulating anti-HLA antibodies showed positive findings for anti-GSTT1 antibodies in serum in 16% of cases at the time of biopsy.19 This finding could be responsible for a higher rate of renal graft loss, as is the case with liver transplant recipients28; however, further studies are necessary to confirm this relation.
Evaluations of the effect of anti-GSTT1 antibodies have found no influence on graft function. Nevertheless, these evaluations had small cohorts and used IIF as the detection method, which is less sensitive than the Luminex assay, both of which factors could have affected the results.24,27 Other evaluations have shown a relation between the development of anti-GSTT1 antibodies and AMR, although cohort sizes were small and used IIF, thus warranting a cautious interpretation of their conclusions.25,26 Our study looked into a larger sample of patients and used Luminex assay for antibody detection. We did not observe a connection between the development of anti-GSTT1 antibodies and the renal graft outcome. At the end of follow-up, we observed no significant differences in the eGFR between antibody-positive and -antibody negative patients; although the multivariate analysis showed that the presence of antibodies did increase the risk of graft loss, the difference was not statistically significant.
It is worth noting that all the studies we referenced only described the development of anti-GSTT1 antibodies during follow-up posttransplant. Nevertheless, our study found anti-GSTT1 antibodies at the time of transplant in 75% of patients who would go on to develop an immune response against GSTT1. Some of the patients with pretransplant anti-GSTT1 antibodies had a history of prior transplant, which could have substantiated the existence of antibodies if they were originally GSTT1*0 recipients matched with GSTT1-positive donors at first transplant. Alternatively, the presence of GSTT1 antibodies at the time of transplant could be explained by the blood transfusions, as previously reported.29 The fact that patients with antibodies in our study had been on dialysis longer than their negative counterparts could be consistent with this explanation. Some have suggested that GSTT1*0 women who were pregnant with GSTT1-positive fetuses could ultimately go on to develop antibodies,29 but our study found no significant differences between the sexes. Nonetheless, this could explain the presence of anti-GSTT1 antibodies in our female patient from the GSTT1*0 donor/GSTT1*0 recipient group, who had no history of previous renal transplant.
The studies referenced found no significant differences in terms of chronic graft dysfunction between GSTT1-positive and GSTT1*0 recipients.18 This enzyme has a role in the elimination of free radicals and provides protection against oxidative stress; therefore, it could play a crucial role in preventing endothelial dysfunction. In our cohort, the rate of ischemic cardiopathies was higher among patients with anti-GSTT1 antibodies. We believe that, rather than being explained by the presence of these antibodies, the finding is likely consistent with the GSTT1*0 condition. The absence of this enzyme would, in turn, deprive the patient of its important protective role against oxidation and endothelial dysfunction, thus increasing the risk for ischemic cardiopathy.30-32
Another suggestion is that antibody development against this enzyme could lead to a dysfunction in the enzyme itself and eventually to calcineurin inhibitor nephrotoxicity.26 In liver transplant recipients, antibody production is reported to increase when cyclosporine versus tacrolimus is used as immunosuppression.33 Our study found no statis-tically significant differences in terms of the type of immunosuppression received by the 2 groups; all patients, however, were given both tacrolimus and a calcineurin inhibitor. Nevertheless, the percentage of anti-GSTT1-positive patients was higher in our study than in a previous study that used cyclosporine,24 which could mean that antibody production is unrelated to the calcineurin inhibitor used, but clinical trials would be required to confirm this.
Our study used 2 anti-GSTT1 antibody detection methods, IIF and Luminex assay, whereas most previous studies only used the former. Given the higher sensitivity of Luminex, we were able to detect antibodies in a larger proportion of patients.
The main limitation of our study was the small number of patients with antibodies. Albeit the rate of GSTT1-positive donor/GSTT1*0 matches was similar to that described in literature for our population; in addition, despite the number of patients who went on to develop antibodies being higher than in other studies, the total sample of patients with anti-GSTT1 antibodies was small. Therefore, conclusions should be interpreted with caution. Multicentric and multiracial studies are needed to confirm our findings.
Anti-GSTT1 antibody detection is a frequent discovery among renal transplant patients with the GSTT1*0 (null) allele whose organ came from a donor with a GSTT1*A (positive) allele. The use of Luminex assay for detection is preferrable to IIF given its higher sensitivity. A large number of renal transplant recipients test positive for anti-GSTT1 antibodies at the time of transplant. Development of these antibodies does not affect graft outcome, although further studies are required to confirm these findings.
Volume : 20
Issue : 10
Pages : 901 - 907
DOI : 10.6002/ect.2022.0189
From the 1Department of Immunology, Hospital Universitario de Badajoz, and the 2Department of Nephrology, Hospital Universitario de Badajoz, Badajoz, Spain
Acknowledgements: This study was funded by the Junta de Extremadura and co-funded by the European Union (European Regional Development Fund; “A way of making Europe”) through the IB18077 “Incidencia de aparición de anticuerpos anti-GSTT1 y su repercusión clínica en la evolución del trasplante renal” (Incidence of antiGSTT1 antibody development and its clinical repercussion on renal transplant outcome) project. The authors have no declarations of potential conflicts of interest.
Corresponding author: Sergio Barroso Hernández, Department of Nephrology, Hospital Universitario de Badajoz, 06080 Badajoz, Spain
Figure 1. Indirect Immunofluorescence, Showing Centrolobular Hepatocytes
Figure 2. Indirect Immunofluorescence, Showing Proximal Renal Tubules
Table 1. Characteristics of Total Study Sample, Negative Antibody Patients, and Positive Antibody Patients