Rejection In Kidney Transplant Recipients Research Articles

Extracellular vesicles as potential diagnostic markers for kidney allograft rejection.

Kidney transplantation is a highly effective treatment for end-stage kidney disease. However, allograft rejection remains a significant clinical challenge in kidney transplant patients. Although kidney allograft biopsy is the gold-standard diagnostic method, it is an invasive procedure. Since the current monitoring methods, including screening of serum creatinine and urinary protein, are not of sufficient sensitivity, there is a need for effective post-transplant monitoring to detect allograft rejection at an early stage. Extracellular vesicles are vesicles with a lipid bilayer that originate from different cell types in pathological and physiological conditions. The content of extracellular vesicles reflects the status of cells at the time of their production. This review comprehensively summarizes clinical, in vivo, and in vitro reports that highlight the potential of extracellular vesicles as diagnostic biomarkers for kidney allograft rejection. Clarification would facilitate differentiation between rejection and non-rejection and identification of the mechanisms involved in the allograft rejection. Despite increasing evidence, further research is necessary to establish the clinical utility of extracellular vesicles in the diagnosis and monitoring of allograft rejection in kidney transplant recipients. Using extracellular vesicles as non-invasive biomarkers for diagnosis of kidney allograft rejection could have tremendous benefits in improving patient outcomes and reduce the need for invasive procedures.

474 An Investigation of Novel Urinary Cell mRNA Profiles for Noninvasive Diagnosis of Acute Rejection in Kidney Transplant Recipients

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values <0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P<0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.

Pharmacogenomic implications of the differential distribution of CYP3A5 metabolic phenotypes among Latin American populations.

This study shows that the distribution of CYP3A5 alleles (*1, *3, *6 and *7) and genotype-predicted CYP3A5 phenotypes vary significantly across Latin American cohorts (Brazilians and the One Thousand Genomes Admixed American superpopulation), as well as among subcohorts comprising individuals with the highest proportions of Native, European or sub-Saharan African ancestry. Differences in biogeographical ancestry across the study groups are the likely explanation for these results. The differential distribution of CYP3A5 phenotypes has major pharmacogenomic implications, affecting the proportion of individuals carrying high risk CYP3A5 phenotypes for the immunosuppressant tacrolimus and the number of patients that would need to be genotyped to prevent acute rejection in kidney transplant recipients under tacrolimus treatment.

Impact of individual eplets to acute rejection in kidney transplant recipients: machine learning analysis of Korean Organ Transplantation Registry

Impact of individual eplets to acute rejection in kidney transplant recipients: machine learning analysis of Korean Organ Transplantation Registry

Everolimus plus reduced calcineurin inhibitor prevents de novo anti-HLA antibodies and humoral rejection in kidney transplant recipients: 12-month results from the ATHENA study

BackgroundStudies prospectively monitoring de novo donor-specific antibodies (dnDSAs) and their clinical impact are sparse. This substudy of ATHENA was initiated to evaluate the effect of everolimus (EVR) or mycophenolic acid (MPA) in combination with reduced calcineurin inhibitor (CNI, tacrolimus [TAC] or cyclosporine [CsA]) on the formation of human leukocyte antibodies (HLA), including dnDSA, and the impact on clinical outcomes in kidney transplant (KTx) recipients.MethodsAll eligible patients were randomized 1:1:1 to receive either EVR + TAC, EVR + CsA or MPA + TAC, with basiliximab induction plus steroids after transplantation up to Month 12. The incidence of dnDSA by treatment group and the association with clinical events were evaluated descriptively as an exploratory objective in the intent-to-treat (ITT) and per-protocol (PP) populations with at least one antibody assessment.ResultsOverall, none of the patients in the EVR + TAC group had either dnDSA or antibody mediated rejection (PP or ITT population) and only one patient with dnDSA in the TAC + MPA group had antibody mediated rejection.ConclusionThe EVR regimen was comparable to MPA regimen with an extremely low incidence of dnDSA over 1 year of treatment.

318.5: Emerging data from a study of tegoprubart for the prevention of rejection in kidney transplant recipients: Implications for a similar study in islet cell transplantation

318.5: Emerging data from a study of tegoprubart for the prevention of rejection in kidney transplant recipients: Implications for a similar study in islet cell transplantation

P122 High and low resolution-based HLA typing for PIRCHE analysis of molecular mismatch is comparable for prediction of DSA and rejection in kidney transplant recipients

P122 High and low resolution-based HLA typing for PIRCHE analysis of molecular mismatch is comparable for prediction of DSA and rejection in kidney transplant recipients

O9 Impact of induction on acute rejection in kidney transplant recipients with high and low risk class II eplet mismatches

O9 Impact of induction on acute rejection in kidney transplant recipients with high and low risk class II eplet mismatches

198 Novel natural antibodies targeted against cardiolipin are associated with higher risk of rejection in kidney transplant recipients

198 Novel natural antibodies targeted against cardiolipin are associated with higher risk of rejection in kidney transplant recipients

Performance and Advancement of the Kidney Solid Organ Response Test.

The kidney solid organ response test (kSORT) has been investigated for the prediction of acute rejection in kidney transplant recipients with conflicting results. We aimed to investigate if the kSORT assay score is associated with rejection or immune quiescence. The blinded association between rejection and kSORT > 9 were investigated. Optimization of kSORT prediction was evaluated after unblinding to determine the optimal prediction cutoff value of kSORT score. Additionally, the predictive capability of the kSORT gene set was assessed using blinded normalized gene expression data from microarray (Affymetrix) and qPCR assays. Of the 95 blood samples analyzed, 18 patients had blood samples before transplant, 77 patients after transplant and 71 had clinically indicated biopsies of which 15 biopsies showed acute rejection and 16 showed chronic active antibody-mediated rejection. When 31 patients with rejection were compared to the remaining 64 patients, positive predictive value (PPV) was 54.29% and negative predictive value (NPV) was 75% when stratified using a kSORT score > 9, and PPV was 57.89% and NPV was 78.95% when stratified using a kSORT score > 5. Using the kSORT assay for detection of rejection showed an area under the curve value of 0.71. Microarray data improved prediction accuracy with PPV of 53% and NPV of 84% compared to qPCR results (PPV and NPV were 36% and 66%), respectively. The kSORT assay has the potential to be used as a predictive tool for active rejection and/or immune quiescence, but additional studies will be useful in improving and refining the kSORT assay, in particular the prediction algorithm.

Mechanism and treatment for chronic antibody-mediated rejection in kidney transplant recipients.

Chronic antibody-mediated rejection of kidney transplantation is a major cause of late-stage graft loss. Donor-specific antibodies are the main cause of antibody-mediated rejection; in particular, de novo donor-specific antibodies are a risk factor for chronic active antibody-mediated rejection. The level of de novo donor-specific antibodies tends to increase with time throughout long-term graft survival. Donor-specific antibodies induce humoral rejection through complement activation, which results in tissue injury and coagulation. Additionally, complement activation promotes the migration of inflammatory cells through the innate immune response, causing endothelial injury. This inflammatory response may cause persistent glomerulitis and peritubular capillaritis, leading to fixed pathological lesions that impair graft function. No treatment has been established for chronic antibody-mediated rejection, a condition in which antibody-mediated rejection becomes irreversible. Thus, antibody-mediated rejection must be detected and treated while it is still reversible. In this review, we discuss the development of de novo donor-specific antibodies and the mechanisms leading to chronic antibody-mediated rejection and summarize the current treatment options and the latest biomarkers for detecting chronic antibody-mediated rejection at an earlier stage.

Development and validation of urinary exosomal microRNA biomarkers for the diagnosis of acute rejection in kidney transplant recipients.

Acute rejection (AR) continues to be a significant obstacle for short- and long-term graft survival in kidney transplant recipients. Herein, we aimed to examine urinary exosomal microRNAs with the objective of identifying novel biomarkers of AR. Candidate microRNAs were selected using NanoString-based urinary exosomal microRNA profiling, meta-analysis of web-based, public microRNA database, and literature review. The expression levels of these selected microRNAs were measured in the urinary exosomes of 108 recipients of the discovery cohort using quantitative real-time polymerase chain reaction (qPCR). Based on the differential microRNA expressions, AR signatures were generated, and their diagnostic powers were determined by assessing the urinary exosomes of 260 recipients in an independent validation cohort. We identified 29 urinary exosomal microRNAs as candidate biomarkers of AR, of which 7 microRNAs were differentially expressed in recipients with AR, as confirmed by qPCR analysis. A three-microRNA AR signature, composed of hsa-miR-21-5p, hsa-miR-31-5p, and hsa-miR-4532, could discriminate recipients with AR from those maintaining stable graft function (area under the curve [AUC] = 0.85). This signature exhibited a fair discriminative power in the identification of AR in the validation cohort (AUC = 0.77). We have successfully demonstrated that urinary exosomal microRNA signatures may form potential biomarkers for the diagnosis of AR in kidney transplantation recipients.

Extracorporeal Photopheresis Suppresses Transplant Fibrosis by Inducing Decorin Expression in Alveolar Macrophages.

Extracorporeal Photopheresis Suppresses Transplant Fibrosis by Inducing Decorin Expression in Alveolar Macrophages.

Tocilizumab for treatment of chronic active antibody-mediated rejection in kidney transplant recipients.

The optimal treatment for chronic active antibody-mediated rejection (ca-AMR) remains unclear. Tocilizumab (TCZ), a monoclonal antibody against IL-6, has been proposed as a therapeutic option. We reported our experience treating ca-AMR with TCZ either as the first line option or as a rescue therapy. We studied 11 adult kidney transplant recipients with biopsy-proven ca-AMR and preserved kidney function (eGFR 57 ± 18) who were treated with TCZ (8 mg/kg IV monthly). All biopsies were prompted by abnormal surveillance biomarker testing with DSA and/or dd-cfDNA. Clinical monitoring included dd-cfDNA and DSA testing every 3 months during the treatment with TCZ. In this cohort, ca-AMR was diagnosed at a median of 90 months (range 14-224) post-transplant, and 4 of 11 patients had DSA negative ca-AMR. Patients received a minimum of 3 months of TCZ, with 6 patients receiving at least 12 months of TCZ. Dd-cfDNA was elevated in all patients, with a median 2.24% at the start of TCZ treatment. After 6 months of TCZ treatment, 8/11 patients had dd- cfDNA <1%, and 3/11 had values <0.5%. Among those who completed at least 12 months of TCZ, dd-cfDNA decreased by 29% at 6 months (p=.05) and 47% by 12 months (p=.04). DSA also stabilized and, by 12 months, was reduced by 29% (p=.047). Graft function remained stable with no graft loss during treatment. There was a nonsignificant trend towards proteinuria reduction. During the course of treatment with tocilizumab, two patients experienced moderate to severe infections. In our early short-term experience, TCZ appears to reduce graft injury as measured by dd-cfDNA and modulate the immune response as evident by a modest reduction in immunodominant DSA MFI. Allograft function and proteinuria alsostabilized.

Assessment of Cytokines as Potential Biomarkers to Predict Allograft Rejection in Kidney Transplant Recipients

Objectives: Allograft rejection is the most common event that occurs posttransplant and the current diagnostic tools like the measurement of serum creatinine or core kidney biopsy could not serve as a predictive tool and have certain drawbacks. Thus, our target was to find potential, non-invasive, and predictive biomarkers which could identify recipients at the risk of post-transplant allograft rejection. Methods: It is a prospective longitudinal cohort study comprising of 40 live-related kidney transplant recipients whose peripheral blood was collected at three time points viz pre, one and threemonths post-transplant to assess the plasma cytokine levels of FOXP3, IL- 6, IL-17 and TGF-b using Enzyme linked immunosorbent assay (ELISA). The patients were clinically followed for period of two years to determine their posttransplant outcome. Findings: 13 of our recipients had biopsy proven allograft rejection at the end of follow-up period. The pre-transplant concentration of FOXP3 were less in the rejection group whereas the concentration of IL-6, IL- 17 and TGF- b were more in the rejection group (p<0.05). The classification accuracy of the markers FOXP3, TGF- b , IL-17 and IL-6 were assessed and they had an AUC of 1.0, 1.0, 0.96 and 0.84 respectively with high sensitivity, specificity, and statistical significance. Novelty: Pre-transplant levels of plasma cytokines were able to predict recipients at the risk of allograft rejection. Thus, our study confirms the predictive power of our cytokine biomarkers FOXP3, IL- 6, IL-17 and TGF-b and further paves way for personalized immunosuppressive regimen. Keywords: Predictive Biomarkers; Allograft Rejection; FOXP3; IL6; IL17; TGFb

In silico design and evaluation of a novel mRNA vaccine against BK virus: a reverse vaccinology approach.

Human polyomavirus type 1, or BK virus (BKV), is a ubiquitous pathogen belonging to the polyomaviridae family mostly known for causing BKV-associated nephropathy (BKVN) and allograft rejection in kidney transplant recipients (KTRs) following the immunosuppression regimens recommended in these patients. Reduction of the immunosuppression level and anti-viral agents are the usual approaches for BKV clearance, which have not met a desired outcome yet. There are also debating matters such as the effect of this pathogen on emerging various comorbidities and the related malignancies in the human population. In this study, a reverse vaccinology approach was implemented to design a mRNA vaccine against BKV by identifying the most antigenic proteins of this pathogen. Potential immunogenic T and B lymphocyte epitopes were predicted through various immunoinformatic tools. The final epitopes were selected according to antigenicity, toxicity, allergenicity, and cytokine inducibility scores. According to the obtained results, the designed vaccine was antigenic, neutral at the physiological pH, non-toxic, and non-allergenic with a world population coverage of 93.77%. Since the mRNA codon optimization ensures the efficient expression of the vaccine in a host cell, evaluation of different parameters showed our designed mRNA vaccine has a stable structure. Moreover, it had strong interactions with toll-like receptor 4 (TLR4) according to the molecular dynamic simulation studies. The in silico immune simulation analyses revealed an overall increase in the immune responses following repeated exposure to the designed vaccine. Based on our findings, the vaccine candidate is ready to be tested as a promising novel mRNA therapeutic vaccine against BKV.

Tocilizumab for the treatment of chronic antibody mediated rejection in kidney transplant recipients.

Chronic active antibody-mediated rejection (CAAMR) constitutes a dominant form of late allograft failure. Several treatment strategies directed at CAAMR have been attempted but proven ineffective at delaying kidney function decline or reducing donor-specific antibodies (DSA). We describe our single-center experience using tocilizumab in patients with CAAMR. This is a retrospective analysis using electronic medical records. 38 kidney transplant recipients at Columbia University Irving Medical Center who had been prescribed tocilizumab and followed for at least 3 months between August 2013 through December 2019 were included. Tocilizumab use was associated with a decrease in the rate of estimated glomerular filtration rate (eGFR) decline in the 6 months following treatment initiation as compared to the 3 months before tocilizumab was initiated (difference between slopes before and after initiation of treatment = 2.6 mL/min/1.73 m2 (SE=.8, p=.002) per month for up to 6 months following Tocilizumab initiation). Allograft biopsies showed significant improvement in interstitial inflammation scores (score 1(0,1) to 0 (0,1), p = .03) while other histologic scores remained stable. There was no significant change in proteinuria or DSA titers post-treatment with tocilizumab. Treatment of CAAMR with tocilizumab was associated with a decrease in the rate of eGFR decline and a reduction in interstitial inflammation scores in patients with CAAMR.

A comparison of methods of plasmapheresis for the treatment of late antibody mediated rejection in kidney transplant recipients.

We compared the outcomes associated with plasma exchange (PE), double filtration plasmapheresis (DFPP), or immunoadsorption (IA) in the treatment of late antibody mediated rejection (AMR). Sixty-nine kidney transplantation (KTx) recipients with late AMR were retrospectively categorized according to management with PE (n= 30), DFPP (n= 22) or IA (n= 17). Allograft loss was compared across treatment groups by Kaplan-Meier analysis and Cox regression. Study groups were similar regarding age, sex, donor type, kidney function, donor specific antibodies, and post-KTx follow-up time. Five-year graft survival trended higher with IA (70.6%) compared to PE (36.7%) and DFPP (27.3%) (p= 0.06). In multivariate Cox regression, baseline eGFR (HR per ml/min/1.73 m2 [95% CI]; 0.96 [0.94-0.99]), rituximab use (HR [95% CI]; 0.42 [0.21-0.84]), interstitial inflammation (i) (HR [95% CI]; 2.05 [1.13-3.69]), and transplant glomerulopathy (cg) (HR [95% CI]; 1.46 [1.13-1.87]) were associated with graft loss. These results motivate the need for continued assessment of rituximab and plasmapheresis in larger studies.

Deceased-Donor Acute Kidney Injury and Acute Rejection in Kidney Transplant Recipients: A Multicenter Cohort

Donor acute kidney injury (AKI) activates innate immunity, enhances HLA expression in the kidney allograft, and provokes recipient alloimmune responses. We hypothesized that injury and inflammation that manifested in deceased-donor urine biomarkers would be associated with higher rates of biopsy-proven acute rejection (BPAR) and allograft failure after transplantation. Prospective cohort. 862 deceased donors for 1,137 kidney recipients at 13 centers. We measured concentrations of interleukin 18 (IL-18), kidney injury molecule 1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) in deceased donor urine. We also used the Acute Kidney Injury Network (AKIN) criteria to assess donor clinical AKI. The primary outcome was a composite of BPAR and graft failure (not from death). A secondary outcome was the composite of BPAR, graft failure, and/or de novo donor-specific antibody (DSA). Outcomes were ascertained in the first posttransplant year. Multivariable Fine-Gray models with death as a competing risk. Mean recipient age was 54 ± 13 (SD) years, and 82% received antithymocyte globulin. We found no significant associations between donor urinary IL-18, KIM-1, and NGAL and the primary outcome (subdistribution hazard ratio [HR] for highest vs lowest tertile of 0.76 [95% CI, 0.45-1.28], 1.20 [95% CI, 0.69-2.07], and 1.14 [95% CI, 0.71-1.84], respectively). In secondary analyses, we detected no significant associations between clinically defined AKI and the primary outcome or between donor biomarkers and the composite outcome of BPAR, graft failure, and/or de novo DSA. BPAR was ascertained through for-cause biopsies, not surveillance biopsies. In a large cohort of kidney recipients who almost all received induction with thymoglobulin, donor injury biomarkers were associated with neither graft failure and rejection nor a secondary outcome that included de novo DSA. These findings provide some reassurance that centers can successfully manage immunological complications using deceased-donor kidneys with AKI.

P8.054: Out of the Box. Immunosuppressive Therapy With Four Drugs As Rescue Therapy in Kidney Transplant Recipients With Chronic Allograft Rejection

Introduction: Persistent or repeated episodes of acute rejection (AR) may lead to chronic allograft damage. Antibody-mediated rejection (AMR); T-cell mediated rejection (TCMR) and mixed variants (MR) of chronic kidney allograft rejection (CR) are nowadays considered as the main causes of graft loss (GL). This encompasses from endothelial damage up to fibrosis challenging immunosuppressive therapy (IT) management. IT reinforcement with a four-drug scheme (4D) including m-TOR inhibitors, anticalcineurinics, mycophenolate and steroids, could be a good strategy in this scenario, nevertheless there is no consensus about it. We present our experience with 4D in kidney transplant recipients (KT) That presented with CR or recurrent episodes of AR. Objectives: To evaluate kidney graft function (KGF), incidence of rejection and safety of a 4D in KT with CR or recurrent episodes of AR. Methods: KT on treatment with 4D from 2008 to 2021 were included. KGF was evaluated by estimated glomerular filtration rate with CKD-EPI (eGFR) and urinary protein/creatinine ratio (PCR) at 3, 6 and 12 months. Kidney biopsies and panel reactive antibodies (PRA) by Luminex were performed before and after introduction of 4D. BK virus (BKV) incidence, infectious (IE) and neoplastic (NE) events were recorded during the follow up. Results: 16 KT (median age 45 ± 11 years) were treated with 4D during 52.8 (17-106) months. 43.8% of them were women, 50% were deceased KT. The median post transplant time was 108 (60-129) months. Reasons for 4D introduction were CR in 87.4% of the cases (MR 50%; TCMR 31.3%; AMR 6.3%) and in the remaining 12, 6%, recurrent or persistent acute TCMR. Median eGFR was 42 (27-66); 44 (31-62); 48 (33-60) and 48 (32-65) ml/min prior to IT change, at 3, 6 and 12 months respectively. At baseline, mean PCR was 179 (147-664) mg/g without significant changes during follow up. Biopsy proven AR was found in 37% of KT under 4D. No NE were registered. 43% of the patients presented a non major IE, and only one patient was BKV positive during follow up with 4D. There was one GL, one loss of follow up and one death due to sepsis with functioning graft. Conclusions: KT patients with CR or recurrent episodes of AR changed to 4D IT stabilized their renal function and proteinuria. The incidence of new biopsy proven AR was 37%, and no new DSA were found or serious adverse events reported during follow-up. 4D could be a good option to halt the progression of renal damage due to rejection in KT.