Impact of a Single Dose of Alpha-1-Antitrypsin in a Rat Model of Bilateral Kidney Ischemia Reperfusion Injury.

INTRODUCTION: Renal ischemia reperfusion injury is a major cause of perioperative acute kidney injury. Alpha-1-antitrypsin (AAT), a protease inhibitor, might improve outcomes by reducing inflammation and apoptosis. We investigated the effects of a single intravenous dose of AAT immediately before ischemia in a rat bilateral renal clamping model. METHODS: Both renal pedicles of male Sprague-Dawley rats were clamped (45 min). Plasma and renal tissue were collected at 3 h, 24 h, and 7 d. Intravenous AAT (60 mg/kg) was administered 5 min before clamping. Controls received saline. Shams underwent surgery without clamping or injection. Kidney function was assessed by plasma creatinine; injury by aspartate aminotransferase, heart-type-fatty-acid-binding-protein, and histopathology. Renal gene expression of tumor necrosis factor α, interleukin (IL)-6, heat shock protein 70, Chemokine (C-X-C motif) ligand 2, cyclo-oxygenase 2, endothelin-1, IL-10, heme oxygenase 1, B-cell lymphoma 2, and bcl-2-like protein 4 were determined by quantitative reverse transcriptase polymerase chain reaction. RESULTS: None of the 3 h and 24 h end points were different between Control and AAT. In Sham, survival was 100% (6/6), 33% in Control (2/6), and 83% (5/6) in AAT (overall log-rank 0.03). At 7 d, plasma creatinine was lower with higher glomerular filtration rate in surviving AAT treated animals compared to Control (P < 0.001, P 0.03, respectively). These also had lower tumor necrosis factor α and IL-6 gene expression (P 0.001, P < 0.001, respectively). CONCLUSIONS: These data suggest that a single intravenous dose of AAT immediately before ischemia might affect proinflammatory gene expression, glomerular filtration rate and animal survival at 1 wk after reperfusion despite an absence of improvement in early renal function and injury. These findings deserve further investigating in sufficiently powered studies including both sexes.

An observational cohort study of histological screening for BK polyomavirus nephropathy following viral replication in plasma.

Systematic screening for BKPyV-DNAemia has been advocated to aid prevention and treatment of polyomavirus associated nephropathy (PyVAN), an important cause of kidney graft failure. The added value of performing a biopsy at time of BKPyV-DNAemia, to distinguish presumptive PyVAN (negative SV40 immunohistochemistry) and proven PyVAN (positive SV40) has not been established. Therefore, we studied an unselected cohort of 950 transplantations, performed between 2008-2017. BKPyV-DNAemia was detected in 250 (26.3%) transplant recipients, and positive SV40 in 91 cases (9.6%). Among 209 patients with a concurrent biopsy at time of first BKPyV-DNAemia, 60 (28.7%) biopsies were SV40 positive. Plasma viral load showed high diagnostic value for concurrent SV40 positivity (ROC-AUC 0.950, 95% confidence interval 0.916-0.978) and the semiquantitatively scored percentage of tubules with evidence of polyomavirus replication (pvl score) (0.979, 0.968-0.988). SV40 positivity was highly unlikely when plasma viral load is below 4 log10 copies/ml (negative predictive value 0.989, 0.979-0.994). In SV40 positive patients, higher plasma BKPyV-DNA load and higher pvl scores were associated with slower viral clearance from the blood (hazard ratio 0.712, 95% confidence interval 0.604-0.839, and 0.327, 0.161-0.668, respectively), whereas the dichotomy positivity/negativity of SV40 immunohistochemistry did not predict viral clearance. Although the pvl score offers some prognostic value for viral clearance on top of plasma viral load, the latter provided good guidance for when a biopsy was unnecessary to exclude PyVAN. Thus, the distinction between presumptive and proven PyVAN, based on SV40 immunohistochemistry, has limited clinical value. Hence, management of BKPyV-DNAemia and immunosuppression reduction should be weighed against the risk of occurrence of rejection, or exacerbation of rejection observed concomitantly.

Data-Driven Chronic Allograft Phenotypes: A Novel and Validated Complement for Histologic Assessment of Kidney Transplant Biopsies.

BACKGROUND: No validated system currently exists to realistically characterize the chronic pathology of kidney transplants that represents the dynamic disease process and spectrum of disease severity. We sought to develop and validate a tool to describe chronicity and severity of renal allograft disease and integrate it with the evaluation of disease activity. METHODS: The training cohort included 3549 kidney transplant biopsies from an observational cohort of 937 recipients. We reweighted the chronic histologic lesions according to their time-dependent association with graft failure, and performed consensus k-means clustering analysis. Total chronicity was calculated as the sum of the weighted chronic lesion scores, scaled to the unit interval. RESULTS: We identified four chronic clusters associated with graft outcome, based on the proportion of ambiguous clustering. The two clusters with the worst survival outcome were determined by interstitial fibrosis and tubular atrophy (IFTA) and by transplant glomerulopathy. The chronic clusters partially overlapped with the existing Banff IFTA classification (adjusted Rand index, 0.35) and were distributed independently of the acute lesions. Total chronicity strongly associated with graft failure (hazard ratio [HR], 8.33; 95% confidence interval [CI], 5.94 to 10.88; P<0.001), independent of the total activity scores (HR, 5.01; 95% CI, 2.83 to 7.00; P<0.001). These results were validated on an external cohort of 4031 biopsies from 2054 kidney transplant recipients. CONCLUSIONS: The evaluation of total chronicity provides information on kidney transplant pathology that complements the estimation of disease activity from acute lesion scores. Use of the data-driven algorithm used in this study, called RejectClass, may provide a holistic and quantitative assessment of kidney transplant injury phenotypes and severity.

Association of Predicted HLA T-Cell Epitope Targets and T-Cell-Mediated Rejection After Kidney Transplantation.

RATIONALE & OBJECTIVE: The relationship between human leukocyte antigen (HLA) molecular mismatches and T-cell-mediated rejection (TCMR) is unknown. We investigated the associations between the different donor HLA-derived T-cell targets and the occurrence of TCMR and borderline histologic changes suggestive of TCMR after kidney transplantation. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: All kidney transplant recipients at a single center between 2004 and 2013 with available biopsy data and a DNA sample for high-resolution HLA donor/recipient typing (N = 893). EXPOSURE: Scores calculated by the HLA matching algorithm PIRCHE-II and HLA eplet mismatches. OUTCOME: TCMR, borderline changes suggestive of TCMR, and allograft failure. ANALYTICAL APPROACH: Multivariable cause-specific hazards models were fit to characterize the association between HLA epitopes targets and study outcomes. RESULTS: We found 277 patients developed TCMR, and 134 developed only borderline changes suggestive of TCMR on at least 1 biopsy. In multivariable analyses, only the PIRCHE-II scores for HLA-DRB1 and HLA-DQB1 were independently associated with the occurrence of TCMR and with allograft failure; this was not the case for HLA class I molecules. If restricted to rejection episodes within the first 3 months after transplantation, only the T-cell epitope targets originating from the donor's HLA-DRB1 and HLA-DQB1, but not class I molecules, were associated with the early acute TCMR. Also, the median PIRCHE-II score for HLA class II was statistically different between the patients with TCMR compared to the patients without TCMR (129 [IQR, 60-240] vs 201 [IQR, 96-298], respectively; P < 0.0001). These differences were not observed for class I PIRCHE-II scores. LIMITATIONS: Observational clinical data and residual confounding. CONCLUSIONS: In the absence of HLA-DSA, HLA class II but not class I mismatches are associated with early episodes of acute TCMR and allograft failure. This suggests that current immunosuppressive therapies are largely able to abort the most deleterious HLA class I-directed alloimmune processes; however, alloresponses against HLA-DRB1 and HLA-DQB1 molecular mismatches remain insufficiently suppressed. PLAIN-LANGUAGE SUMMARY: Genetic differences in the human leukocyte antigen (HLA) complex between kidney transplant donors and recipients play a central role in T-cell-mediated rejection (TCMR), which can lead to failure of the transplanted kidney. Evaluating this genetic disparity (mismatch) in the HLA complex at the molecular (epitope) level could contribute to better prediction of the immune response to the donor organ posttransplantation. We investigated the associations of the different donor HLA-derived T-cell epitope targets and scores obtained from virtual crossmatch algorithms with the occurrence of TCMR, borderline TCMR, and graft failure after kidney transplantation after taking into account the influence of donor-specific anti-HLA antibodies. This study illustrates the greater importance of the molecular mismatches in class II molecules compared to class I HLA molecules.

Missing Self-Induced Microvascular Rejection of Kidney Allografts: A Population-Based Study.

BACKGROUND: Circulating anti-HLA donor-specific antibodies (HLA-DSA) are often absent in kidney transplant recipients with microvascular inflammation (MVI). Missing self, the inability of donor endothelial cells to provide HLA I-mediated signals to inhibitory killer cell Ig-like receptors (KIRs) on recipient natural killer cells, can cause endothelial damage in vitro, and has been associated with HLA-DSA-negative MVI. However, missing self's clinical importance as a nonhumoral trigger of allograft rejection remains unclear. METHODS: In a population-based study of 924 consecutive kidney transplantations between March 2004 and February 2013, we performed high-resolution donor and recipient HLA typing and recipient KIR genotyping. Missing self was defined as the absence of A3/A11, Bw4, C1, or C2 donor genotype, with the presence of the corresponding educated recipient inhibitory KIR gene. RESULTS: We identified missing self in 399 of 924 transplantations. Co-occurrence of missing self types had an additive effect in increasing MVI risk, with a threshold at two concurrent types (hazard ratio [HR], 1.78; 95% confidence interval [95% CI], 1.26 to 2.53), independent of HLA-DSA (HR, 5.65; 95% CI, 4.01 to 7.96). Missing self and lesions of cellular rejection were not associated. No HLA-DSAs were detectable in 146 of 222 recipients with MVI; 28 of the 146 had at least two missing self types. Missing self associated with transplant glomerulopathy after MVI (HR, 2.51; 95% CI, 1.12 to 5.62), although allograft survival was better than with HLA-DSA-associated MVI. CONCLUSION: Missing self specifically and cumulatively increases MVI risk after kidney transplantation, independent of HLA-DSA. Systematic evaluation of missing self improves understanding of HLA-DSA-negative MVI and might be relevant for improved diagnostic classification and patient risk stratification.

Data-driven Derivation and Validation of Novel Phenotypes for Acute Kidney Transplant Rejection using Semi-supervised Clustering.

BACKGROUND: Over the past decades, an international group of experts iteratively developed a consensus classification of kidney transplant rejection phenotypes, known as the Banff classification. Data-driven clustering of kidney transplant histologic data could simplify the complex and discretionary rules of the Banff classification, while improving the association with graft failure. METHODS: The data consisted of a training set of 3510 kidney-transplant biopsies from an observational cohort of 936 recipients. Independent validation of the results was performed on an external set of 3835 biopsies from 1989 patients. On the basis of acute histologic lesion scores and the presence of donor-specific HLA antibodies, stable clustering was achieved on the basis of a consensus of 400 different clustering partitions. Additional information on kidney-transplant failure was introduced with a weighted Euclidean distance. RESULTS: Based on the proportion of ambiguous clustering, six clinically meaningful cluster phenotypes were identified. There was significant overlap with the existing Banff classification (adjusted rand index, 0.48). However, the data-driven approach eliminated intermediate and mixed phenotypes and created acute rejection clusters that are each significantly associated with graft failure. Finally, a novel visualization tool presents disease phenotypes and severity in a continuous manner, as a complement to the discrete clusters. CONCLUSIONS: A semisupervised clustering approach for the identification of clinically meaningful novel phenotypes of kidney transplant rejection has been developed and validated. The approach has the potential to offer a more quantitative evaluation of rejection subtypes and severity, especially in situations in which the current histologic categorization is ambiguous.

Risk factors, histopathological features, and graft outcome of transplant glomerulopathy in the absence of donor-specific HLA antibodies.

Transplant glomerulopathy is established as a hallmark of chronic antibody-mediated rejection in kidney transplant patients with donor-specific HLA antibodies (HLA-DSA). The clinical importance of transplant glomerulopathy in the absence of HLA-DSA is not well established. To help define this, 954 patients (encompassing 3744 biopsies) who underwent kidney transplantation 2004-2013 were studied with retrospective high-resolution HLA genotyping of both donors and recipients. The risk factors, histopathological appearance and prognosis of cases with transplant glomerulopathy in the absence of HLA-DSA were compared to those cases with HLA-DSA, and the impact of the PIRCHE-II score and eplet mismatches on development of transplant glomerulopathy evaluated. In this cohort, 10.3% developed transplant glomerulopathy, on average 3.2 years post-transplant. At the time of glomerulopathy, 23.5% had persistent pre-transplant or de novo HLA-DSA, while 76.5% were HLA-DSA negative. Only HLA-DSA was identified as a risk factor for glomerulopathy development as eplet mismatches and the PIRCHE-II score did not associate. HLA-DSA negative biopsies with glomerulopathy had less interstitial inflammation, less glomerulitis, and less C4d deposition in the peritubular capillaries compared to the HLA-DSA positive biopsies with glomerulopathy. While graft function was comparable between the two groups, HLA-DSA positive glomerulopathy was associated with a significantly higher risk of graft failure compared to HLA-DSA negative glomerulopathy (Hazard Ratio 3.84; 95% confidence interval 1.94-7.59). Landmark analysis three-years post-transplant showed that HLA-DSA negative patients with glomerulopathy still had a significant increased risk of graft failure compared to patients negative for glomerulopathy (2.62; 1.46-4.72). Thus, transplant glomerulopathy often occurs in the absence of HLA-DSA, independent of HLA molecular mismatches, and represents a different phenotype with less concomitant inflammation and better graft survival compared to that developed in the presence of HLA-DSA.

Revisiting the changes in the Banff classification for antibody-mediated rejection after kidney transplantation.

The Banff classification for antibody-mediated rejection (ABMR) has undergone important changes, mainly by inclusion of C4d-negative ABMR in Banff'13 and elimination of suspicious ABMR (sABMR) with the use of C4d as surrogate for HLA-DSA in Banff'17. We aimed to evaluate the numerical and prognostic repercussions of these changes in a single-center cohort study of 949 single kidney transplantations, comprising 3662 biopsies that were classified according to the different versions of the Banff classification. Overall, the number of ABMR and sABMR cases increased from Banff'01 to Banff'13. In Banff'17, 248 of 292 sABMR biopsies were reclassified to No ABMR, and 44 of 292 to ABMR. However, reclassified sABMR biopsies had worse and better outcome than No ABMR and ABMR, which was mainly driven by the presence of microvascular inflammation and absence of HLA-DSA, respectively. Consequently, the discriminative performance for allograft failure was lowest in Banff'17, and highest in Banff'13. Our data suggest that the clinical and histological heterogeneity of ABMR is inadequately represented in a binary classification system. This study provides a framework to evaluate the updates of the Banff classification and assess the impact of proposed changes on the number of cases and risk stratification. Two alternative classifications introducing an intermediate category are explored.

Diagnostic performance of kSORT, a blood-based mRNA assay for noninvasive detection of rejection after kidney transplantation: A retrospective multicenter cohort study.

The Kidney Solid Organ Response Test (kSORT) blood gene expression assay was developed to noninvasively detect acute rejection (AR) after kidney transplantation. Its performance in a setting with natural disease prevalence has not been evaluated. A retrospective, multicenter cohort study was conducted across all single kidney transplant recipients, transplanted between 2011 and 2015, with samples within the first year after transplantation available in existing biobanks. The primary objective was to determine the diagnostic performance of the kSORT assay to detect AR (T cell-mediated and/or antibody-mediated rejection) as compared to a concomitant renal biopsy. AR was reported on the concomitant biopsy in 188 of 1763 (10.7%) blood samples and any rejection (including borderline changes) in 614 of 1763 (34.8%) blood samples. In 320 of 1763 samples (18.2%) the kSORT risk category was indeterminate. The kSORT assay had no diagnostic value for AR (area under the curve [AUC] 0.51, 95% confidence interval [CI] 0.50-0.56; P = .46) overall, or when considering indication biopsies (N = 487) and protocol-specified biopsies (N = 1276) separately (AUC of 0.53, 95% CI 0.50-0.59, P = .44 and 0.55, 95% CI 0.50-0.61, P = .09, respectively). This large retrospective study utilizing samples obtained under real-world clinical conditions, was unable to validate the kSORT assay for detection of AR in the first year after transplantation.

The evolution of histological changes suggestive of antibody-mediated injury, in the presence and absence of donor-specific anti-HLA antibodies.

The interplay between donor-specific anti-HLA antibodies (HLA-DSA), histology of active antibody-mediated rejection (aABMRh ), transplant glomerulopathy (cg), and graft failure in kidney transplantation remains insufficiently understood. We performed a single-center cohort study (n = 1000) including 2761 protocol and 833 indication biopsies. Patients with pretransplant HLA-DSA were more prone to develop aABMRh (OR 22.7, 95% CI, 11.8-43.7, P < 0.001), cg (OR 5.76, 95% CI, 1.67-19.8, P = 0.006), and aABMRh/cg (OR 19.5, 95% CI, 10.6-35.9, P < 0.001). The negative impact of pre-transplant HLA-DSA on graft survival (HR 2.12, 95% CI, 1.41-3.20, P < 0.001) was partially mediated through aABMRh and cg occurrence. When adjusted for time-dependent HLA-DSA (HR 4.03, 95% CI, 2.21-7.15, P = 0.002), graft failure was only affected by aABMRh when cg was evident. In HLA-DSA negative patients, aABMRh was associated with impaired graft outcome only when evolving to cg (HR 1.32, 95% CI, 1.07-1.61, P = 0.008). We conclude that the kinetics of HLA-DSA are important to estimate the rate of graft failure, and that histological follow-up is necessary to discover, often subclinical, ABMR and cg. In the absence of HLA-DSA, patients experience similar histological lesions and the evolution to transplant glomerulopathy associates with impaired graft outcome.

Eplet Mismatch Load and De Novo Occurrence of Donor-Specific Anti-HLA Antibodies, Rejection, and Graft Failure after Kidney Transplantation: An Observational Cohort Study.

BACKGROUND: In kidney transplantation, evaluating mismatches of HLA eplets-small patches of surface-exposed amino acids of the HLA molecule-instead of antigen mismatches might offer a better approach to assessing donor-recipient HLA incompatibility and improve risk assessment and prediction of transplant outcomes. METHODS: To evaluate the effect of number of eplet mismatches (mismatch load) on de novo formation of donor-specific HLA antibodies (DSAs) and transplant outcomes, we conducted a cohort study that included consecutive adult kidney recipients transplanted at a single center from March 2004 to February 2013. We performed retrospective high-resolution genotyping of HLA loci of 926 transplant pairs and used the HLAMatchmaker computer algorithm to count HLA eplet mismatches. RESULTS: De novo DSAs occurred in 43 (4.6%) patients. Multivariable analysis showed a significant independent association between antibody-verified eplet mismatch load and de novo DSA occurrence and graft failure, mainly explained by DQ antibody-verified eplet effects. The association with DQ antibody-verified eplet mismatches was linear, without a safe threshold at which de novo DSA did not occur. Odds for T cell- or antibody-mediated rejection increased by 5% and 12%, respectively, per antibody-verified DQ eplet mismatch. CONCLUSIONS: Eplet mismatches in HLA-DQ confer substantial risk for de novo DSA formation, graft rejection, and graft failure after kidney transplantation. Mismatches in other loci seem to have less effect. The results suggest that antibody-verified HLA-DQ eplet mismatch load could be used to guide personalized post-transplant immunosuppression. Adoption of molecular matching for DQA1 and DQB1 alleles could also help to minimize de novo DSA formation and potentially improve transplant outcomes.

Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies.

BACKGROUND: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype. METHODS: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status. RESULTS: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences. CONCLUSIONS: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.

Clinical importance of extended second field high-resolution HLA genotyping for kidney transplantation.

The need for extended second field high-resolution (2F-HR) HLA genotyping in kidney transplantation is debated. In a cohort of 1000 kidney transplants, we evaluated the impact of different HLA genotyping levels on the assignment of donor-specific anti-HLA antibodies (DSA) and investigated whether inference of 2F-HR genotypes from low-resolution (LR) genotypes could be used to correctly assign DSA. Based on LR genotypes, 224 pretransplant DSAs were present in 140 patients and absent in 860 patients (DSAneg group). With extended 2F-HR HLA genotyping, we confirmed 173 DSA (77.2%) in 108 (77.1%) patients (2F-HRpos LRpos DSA group) and excluded DSA in 32 patients (22.9%) (2F-HRneg LRpos DSA group). Kaplan-Meier curves showed that 10-year graft survival rates were similar between the DSAneg and 2F-HRneg LRpos DSA groups (82.4% vs 93.8%; P = .27) and confirmed that DSA determined using LR typing but not confirmed using 2F-HR typing were indeed misclassified. By inferring 2F-HR genotypes using HaploStats, DSA still could not be correctly assigned in 23.3% of cases. We conclude that extended 2F-HR typing of the donor-recipient pairs is relevant for the correct assessment of DSA. Although inference of 2F-HR genotypes may improve the assessment of DSA in some cases, significant misclassification occurs, and warrants caution in using inferred HLA results for clinical and research purposes.

Development and External Validation of a Multiparametric Magnetic Resonance Imaging and International Society of Urological Pathology Based Add-On Prediction Tool to Identify Prostate Cancer Candidates for Pelvic Lymph Node Dissection.

PURPOSE: We sought to expand current prediction tools for lymph node invasion in patients with prostate cancer using current state-of-the-art available tumor information, including multiparametric magnetic resonance imaging based tumor stage and detailed biopsy information. MATERIALS AND METHODS: We selected patients with prostate cancer for study who had available registered information on ISUP (International Society of Urological Pathology) based biopsy grading and multiparametric magnetic resonance imaging, and who had undergone radical prostatectomy with extended pelvic lymph node dissection. We developed a lymph node invasion prediction tool in 420 patients and externally validated it in 187. A concordance index was estimated to quantify the discriminative performance of the model. RESULTS: In the development cohort a median of 21 lymph nodes were removed per patient and 71 patients (16.9%) were diagnosed with lymph node invasion. Statistically significant predictors of lymph node invasion were the initial prostate specific antigen value, multiparametric magnetic resonance imaging based T stage, maximum tumor length in 1 core in mm and ISUP grade group corresponding to the maximum tumor involvement in 1 core. The predictive accuracy of this lymph node invasion prediction tool was 79.7% after fivefold internal cross validation and 72.5% after external validation. CONCLUSIONS: We report a contemporary, externally validated prediction tool for lymph node invasion in patients with prostate cancer. This prediction tool is a response to the paradigm shift from systematic to targeted biopsies by incorporating additional core specific biopsy information instead of the percent of positive cores. This new tool will also overcome stage migration, which is a potential risk when multiparametric magnetic resonance imaging information is used in digital rectal examination based nomograms.

Replicative senescence and arteriosclerosis after kidney transplantation.

BACKGROUND: Replicative senescence is associated with telomere shortening. In native kidneys, obtained prior to transplantation, we recently described and validated a significant association between shorter intrarenal telomere length and renal arteriosclerosis. After renal transplantation, animal experiments suggested that ischaemia-reperfusion injury, acute rejection episodes and cytomegalovirus disease associate with accelerated renal allograft senescence. The association between post-transplant events and replicative senescence has not yet been evaluated in a human setting. METHODS: In a cohort of 134 kidney allograft recipients, we performed protocol-specified renal allograft biopsies at 3 months, 1 year, 2 years and 5 years after transplantation (n = 579 biopsies). We used quantitative real-time polymerase chain reaction to measure intrarenal relative average telomere length (T/S ratio). The association between donor and recipient demographic factors, post-transplant clinical/histological events, renal allograft histological evolution by 5 years post-transplant and intrarenal telomere length at 5 years after transplantation was studied using multiple regression models. RESULTS: At 5 years after transplantation, shorter intrarenal telomere length was associated with male donor gender, older donor age, donor history of hypertension and donor cardiovascular risk, which confirms the associations observed in native kidneys. Recipient characteristics and post-transplant events like delayed graft function, acute rejection episodes, presence of donor-specific antibodies, cytomegalovirus disease and immunosuppressive regimen did not associate with alterations of intrarenal telomere length at 5 years. Independent of donor age and donor cardiovascular risk, intrarenal arteriosclerosis in protocol biopsies obtained at 5 years after transplantation and progressive arteriosclerosis over time after transplantation associated with shorter telomere length, while this was not the case for other histological lesions. Moreover, telomere attrition augments the association between older donor age and the presence of severe arteriosclerosis. In the group with the oldest donor age and shortest telomere length, there was significantly more severe arteriosclerosis (43%) in protocol biopsies at 5 years after transplantation, compared with other combinations (13-28%) (P = 0.001). Intrarenal arteriosclerosis at 5 years after transplantation did not associate with post-transplant clinical events. CONCLUSIONS: We demonstrate that intrarenal telomere length at 5 years after transplantation, as a marker for replicative senescence, associates with renal arteriosclerosis and reflects kidney donor characteristics, but not post-transplant events.

Antibody-mediated rejection with and without donor-specific anti-human leucocyte antigen antibodies: performance of the peripheral blood 8-gene expression assay.

BACKGROUND: Recently a peripheral blood 8-gene expression assay was developed for non-invasive detection of antibody-mediated rejection (ABMR) after kidney transplantation. Its value has not yet been evaluated in detail in clinical scenarios with different baseline disease probability [human leucocyte antigen donor-specific antibodies (HLA-DSA)-positive versus HLA-DSA-negative cases at the time of stable graft function versus graft dysfunction]. METHODS: Here we investigated the diagnostic accuracy of the 8-gene expression assay for histology of ABMR (ABMRh) with or without HLA-DSA in a cross-sectional cohort study of 387 blood samples with a concomitant graft biopsy. RESULTS: In patients with HLA-DSA (n = 64), the 8-gene expression assay discriminated DSA-positive ABMRh (DSAposABMRh) cases (n = 16) with good diagnostic performance {area under the receiver operating characteristic curve [AUROC] 83.1% [95% confidence interval (CI) 70.8-95.3]}. Also, in HLA-DSA-negative samples (n = 323), a clinically relevant diagnostic performance for DSAnegABMRh cases was found (n = 30) with an AUROC of 75.8% (95% CI 67.4-84.4). The 8-gene assay did not discriminate DSAposABMRh cases from DSAnegABMRh cases. There was a net benefit for clinical decision-making when adding the 8-gene expression assay to a clinical model consisting of estimated glomerular filtration rate, proteinuria, HLA-DSA and age. CONCLUSION: The 8-gene expression assay shows great potential for implementation in the clinical follow-up of high-risk HLA-DSA-positive patients and clinical relevance in HLA-DSA-negative cases.

Whole-body diffusion-weighted magnetic resonance imaging for the detection of bone metastases and their prognostic impact in metastatic renal cell carcinoma patients treated with angiogenesis inhibitors.

Background: Metastatic renal cell carcinoma (mRCC) patients with bone metastases (BM) are at high risk for skeletal related events and have a poorer outcome when treated with vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs). Computed tomography (CT) lacks sensitivity to detect BM in mRCC. We aimed to determine the added value of whole body diffusion-weighted magnetic resonance imaging (WB-DWI/MRI) to CT for the detection of BM in mRCC and to estimate the prognostic impact of the number of BM in mRCC patients treated with VEGFR-TKIs.Material and methods: We conducted a prospective study including consecutive mRCC patients treated with a first-line VEGFR-TKI in the metastatic setting. All patients underwent a pretreatment thoracic-abdominal-pelvic CT and WB-DWI/MRI. CT and WB-DWI/MRI were compared for the detection of BM. The number of detected BM was correlated with response rate (RR), progression-free survival (PFS) and overall survival (OS) after start of the VEGFR-TKI.Results: Ninety-two patients were included. BM were found in 55% of the patients by WB-DWI/MRI and in 43% of the patients by CT (p = .003). Mean number of BM discovered per patient was 6.8 by WB-DWI/MRI versus 1.9 by CT (p = .006). The cutoff of ≤5 versus >5 BM on WB-DWI/MRI had the highest discriminative power for all outcome measures. Patients with >5 BM had a lower RR (10% versus 42%), more frequently early progressive disease (43% versus 13%, p = .003), shorter PFS (4 versus 10 months, p = .006) and shorter OS (10 versus 35 months, p < .0001) compared to patients with ≤5 BM.Conclusion: WB-DWI/MRI detects significantly more BM in mRCC patients than CT, allowing better estimation of the prognostic impact of BM in mRCC patients treated with VEGFR-TKIs. The prognostic impact should now be validated in patients treated with immune checkpoint inhibitors.

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection.

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.

Age-related changes in DNA methylation affect renal histology and post-transplant fibrosis.

During ageing, kidney function decreases due to renal tubular atrophy, interstitial fibrosis, glomerulosclerosis and arteriosclerosis. Recently, changes in DNA methylation were shown to contribute to various ageing processes. However, it is unknown whether such changes also contribute to age-related kidney dysfunction. To assess this, we profiled genome-wide changes in DNA methylation (over 800 000 CpG sites) in 95 renal biopsies obtained prior to kidney transplantation from donors aged 16 to 73 years. Donor age significantly associated with the methylation of 92 778 CpGs (false discovery rate under 0.05), corresponding to 10 285 differentially methylated regions. These regions were most frequently located in genes involved in the Wnt/beta-catenin signaling pathway. Using an independent cohort of 67 biopsies, we autonomously validated these findings. Interestingly, the methylation status of these 92 778 age-related CpGs was associated with glomerulosclerosis (34.4% of CpGs at a false discovery rate under 0.05) and interstitial fibrosis (0.9%) and graft function at one year after transplantation, but not with tubular atrophy and arteriosclerosis. No association was observed with any of these pathologies at the time of transplantation (0% at a false discovery rate under 0.05). Thus, age-associated changes in DNA methylation at the time of transplantation predict future injury of transplanted kidneys. Specifically, our epigenome-wide association study demonstrates that epigenetic renal ageing is implicated in progressive fibrosis in both the glomerulus and the interstitium.

Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation.

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation.