J. Sellarés

Understanding the Causes of Kidney Transplant Failure: The Dominant Role of Antibody-Mediated Rejection and Nonadherence

We prospectively studied kidney transplants that progressed to failure after a biopsy for clinical indications, aiming to assign a cause to every failure. We followed 315 allograft recipients who underwent indication biopsies at 6 days to 32 years posttransplant. Sixty kidneys progressed to failure in the follow‐up period (median 31.4 months). Failure was rare after T‐cell–mediated rejection and acute kidney injury and common after antibody‐mediated rejection or glomerulonephritis. We developed rules for using biopsy diagnoses, HLA antibody and clinical data to explain each failure. Excluding four with missing information, 56 failures were attributed to four causes: rejection 36 (64%), glomerulonephritis 10 (18%), polyoma virus nephropathy 4 (7%) and intercurrent events 6 (11%). Every rejection loss had evidence of antibody‐mediated rejection by the time of failure. Among rejection losses, 17 of 36 (47%) had been independently identified as nonadherent by attending clinicians. Nonadherence was more frequent in patients who progressed to failure (32%) versus those who survived (3%). Pure T‐cell–mediated rejection, acute kidney injury, drug toxicity and unexplained progressive fibrosis were not causes of loss. This prospective cohort indicates that many actual failures after indication biopsies manifest phenotypic features of antibody‐mediated or mixed rejection and also underscores the major role of nonadherence.

De Novo Donor‐Specific Antibody at the Time of Kidney Transplant Biopsy Associates with Microvascular Pathology and Late Graft Failure

We studied whether de novo donor‐specific antibodies (DSA) in sera from patients undergoing kidney transplant biopsies associate with specific histologic lesions in the biopsy and prognosis. DSA were assessed in 145 patients at the time of biopsy between 7 days to 31 years posttransplant. DSA was detected in 54 patients (37%), of which 32 represented de novo DSA. De novo DSA was more frequent in patients having late biopsies (34%) versus early biopsies (4%), and was usually either against class II alone or class I and II but rarely against class I alone. Microcirculation inflammation (glomerulitis, capillaritis) and damage (glomuerulopathy, capillary basement membrane multilayering), and C4d staining were associated with de novo DSA. However, the degree of scarring, arterial fibrosis and tubulo‐interstitial inflammation did not correlate with the presence of de novo DSA. De novo DSA correlated with reduced graft survival after the biopsy. Thus, de novo DSA at the time of a late biopsy for clinical indication is primarily against class II, and associates with microcirculation changes in the biopsy and subsequent graft failure. We propose careful assessment of de novo DSA, particularly against class II, be performed in all late kidney transplant biopsies.

NK Cell Transcripts and NK Cells in Kidney Biopsies from Patients with Donor-Specific Antibodies: Evidence for NK Cell Involvement in Antibody-Mediated Rejection

To explore the mechanisms of antibody‐mediated rejection (ABMR) in kidney transplants, we studied the transcripts expressed in clinically indicated biopsies from patients with donor‐specific antibody (DSA). Comparison of biopsies from DSA‐positive versus DSA‐negative patients revealed 132 differentially expressed transcripts: all were associated with class II DSA but none with class I DSA. Many transcripts were expressed in DSA‐positive ABMR but were also expressed in T‐cell‐mediated rejection (TCMR), reflecting shared molecular features. Removal of shared transcripts created 23 DSA selective transcripts (DSASTs). Some DSASTs (6/23) showed selective high expression in NK cells, whereas others (8/23) were expressed in endothelium or in endothelium plus other cell types (7/23). Of 145 biopsies ranked by DSAST expression, the 25 with highest DSAST expression primarily consisted of ABMR (22/25, 88%), either C4d‐positive or C4d‐negative. By immunostaining, CD56+ and CD68+ cells in peritubular capillaries, but not CD3+ cells, were increased in ABMR compared to TCMR, compatible with a role for NK cells, as well as macrophages, as effectors in endothelial injury during ABMR. Thus, the strategy of using DSASTs in the biopsy to identify mechanism‐related transcripts in biopsies from patients with clinical phenotypes indicates the selective involvement of NK cells in ABMR.

Molecular Diagnosis of Antibody‐Mediated Rejection in Human Kidney Transplants

Antibody‐mediated rejection is the major cause of kidney transplant failure, but the histology‐based diagnostic system misses most cases due to its requirement for C4d positivity. We hypothesized that gene expression data could be used to test biopsies for the presence of antibody‐mediated rejection. To develop a molecular test, we prospectively assigned diagnoses, including C4d‐negative antibody‐mediated rejection, to 403 indication biopsies from 315 patients, based on histology (microcirculation lesions) and donor‐specific HLA antibody. We then used microarray data to develop classifiers that assigned antibody‐mediated rejection scores to each biopsy. The transcripts distinguishing antibody‐mediated rejection from other conditions were mostly expressed in endothelial cells or NK cells, or were IFNG‐inducible. The scores correlated with the presence of microcirculation lesions and donor‐specific antibody. Of 45 biopsies with scores >0.5, 39 had been diagnosed as antibody‐mediated rejection on the basis of histology and donor‐specific antibody. High scores were also associated with unanimity among pathologists that antibody‐mediated rejection was present. The molecular score also strongly predicted future graft loss in Cox regression analysis. We conclude that microarray assessment of gene expression can assign a probability of ABMR to transplant biopsies without knowledge of HLA antibody status, histology, or C4d staining, and predicts future failure.

Molecular Diagnosis of T Cell-Mediated Rejection in Human Kidney Transplant Biopsies

Histologic diagnosis of T cell‐mediated rejection is flawed by subjective assessments, nonspecific lesions and arbitrary rules. This study developed a molecular test for T cell‐mediated rejection. We used microarray results from 403 kidney transplant biopsies to derive a classifier assigning T cell‐mediated rejection scores to all biopsies, and compared these with histologic assessments. The score correlated with histologic lesions of T cell‐mediated rejection (infiltrate, tubulitis). The accuracy of the classifier for the histology diagnoses was 89%. Very high and low molecular scores corresponded with unanimity among three pathologists on the presence or absence of T cell‐mediated rejection, respectively. The molecular score had low sensitivity (50%) and positive predictive value (62%) for the histology diagnoses. However, histology showed similar disagreement between pathologists—only 45–56% sensitivity of one pathologist with diagnoses of T cell‐mediated rejection by another. Discrepancies between molecular scores and histology were mostly when histology was ambiguous (“borderline”) or unreliable, e.g. in cases with scarring or inflammation induced by tissue injury. Vasculitis (isolated v‐lesion TCMR) was particularly discrepant, with most cases exhibiting low TCMR scores. We propose new rules to integrate molecular tests and histology into a precision diagnostic system that can reduce errors, ambiguity and interpathologist disagreement.

Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM).

In a reference set of 403 kidney transplant biopsies, we recently developed a microarray‐based test that diagnoses antibody‐mediated rejection (ABMR) by assigning an ABMR score. To validate the ABMR score and assess its potential impact on practice, we performed the present prospective INTERCOM study (clinicaltrials.gov NCT01299168) in 300 new biopsies (264 patients) from six centers: Baltimore, Barcelona, Edmonton, Hannover, Manchester and Minneapolis. We assigned ABMR scores using the classifier created in the reference set and compared it to conventional assessment as documented in the pathology reports. INTERCOM documented uncertainty in conventional assessment: In 41% of biopsies where ABMR features were noted, the recorded diagnoses did not mention ABMR. The ABMR score correlated with ABMR histologic lesions and donor‐specific antibodies, but not with T cell–mediated rejection lesions. The agreement between ABMR scores and conventional assessment was identical to that in the reference set (accuracy 85%). The ABMR score was more strongly associated with failure than conventional assessment, and when the ABMR score and conventional assessment disagreed, only the ABMR score was associated with early progression to failure. INTERCOM confirms the need to reduce uncertainty in the diagnosis of ABMR, and demonstrates the potential of the ABMR score to impact practice.

An integrated view of molecular changes, histopathology and outcomes in kidney transplants.

Data‐driven approaches to deteriorating kidney transplants, incorporating histologic, molecular and HLA antibody findings, have created a new understanding of transplant pathology and why transplants fail. Transplant dysfunction is best understood in terms of three elements: diseases, the active injury–repair response and the cumulative burden of injury. Progression to failure is mainly attributable to antibody‐mediated rejection, nonadherence and glomerular disease. Antibody‐mediated rejection usually develops late due to de novo HLA antibodies, particularly anti‐class II, and is often C4d negative. Pure treated T cell‐mediated rejection does not predispose to graft loss because it responds well, even with endothelialitis, but it may indicate nonadherence. The cumulative burden of injury results in atrophy‐fibrosis (nephron loss), arterial fibrous intimal thickening and arteriolar hyalinosis, but these are not progressive without ongoing disease/injury, and do not explain progression. Calcineurin inhibitor toxicity has been overestimated because burden‐of‐injury lesions invite this default diagnosis when diseases such as antibody‐mediated rejection are missed. Disease/injury triggers a stereotyped active injury–repair response, including de‐differentiation, cell cycling and apoptosis. The active injury–repair response is the strongest correlate of organ function and future progression to failure, but should always prompt a search for the initiating injury or disease.

The molecular phenotype of kidney transplants.

Microarray studies of kidney transplant biopsies provide an opportunity to define the molecular phenotype. To facilitate this process, we used experimental systems to annotate transcripts as members of pathogenesis‐based transcript sets (PBTs) representing biological processes in injured or diseased tissue. Applying this annotation to microarray results revealed that changes in single molecules and PBTs reflected a large‐scale coordinate disturbance, stereotyped across various diseases and injuries, without absolute specificity of individual molecules or PBTs for rejection. Nevertheless, expression of molecules and PBTs was quantitatively specific: IFNG effects for rejection; T cell and macrophage transcripts for T cell‐mediated rejection; endothelial and NK transcripts for antibody‐mediated rejection. Various diseases and injuries induced the same injury–repair response, undetectable by histopathology, involving epithelium, stroma and endothelium, with increased expression of developmental, cell cycle and apoptosis genes and decreased expression of differentiated epithelial features. Transcripts reflecting this injury–repair response were the best correlates of functional disturbance and risk of future graft loss. Late biopsies with atrophy‐fibrosis, reflecting their cumulative burden of injury, displayed more transcripts for B cells, plasma cells and mast cells. Thus the molecular phenotype is best described in terms of three elements: specific diseases, including rejection; the injury–repair response and the cumulative burden of injury.

Interpreting NK Cell Transcripts Versus T Cell Transcripts in Renal Transplant Biopsies

NK cell transcripts are increased in biopsies with antibody‐mediated rejection, whereas T cell transcripts are increased in T cell‐mediated rejection. However, NK and T cells share many features, creating potential ambiguity. Therefore to estimate the NK‐ versus T cell transcript burdens separately, we defined nonoverlapping transcripts selective for NK cells (N = 4) or T cells (N = 5). We compared NK‐ versus T cell transcript burdens in microarrays from 403 kidney transplant biopsies (182 early, 221 late). In late biopsies, high NK‐cell transcript expression was associated with antibody‐mediated rejection, correlating with microvascular inflammation and donor specific HLA antibody. However, some early biopsies with T cell‐mediated rejection had high NK‐cell transcript expression, as well as T cell transcripts, without evidence of antibody‐mediated rejection or DSA, correlating with interstitial inflammation and tubulitis. Both NK‐cell and T cell transcripts were moderately increased in many kidneys with inflammation secondary to injury or atrophy scarring. These results support the distinct role of NK cells in late antibody‐mediated rejection, but indicate a role for NK‐transcript expressing cells (NK cells or T cells with NK features) both in T cell‐mediated rejection and in inflammation associated with injury and atrophy scarring.

Disappearance of T Cell-Mediated Rejection Despite Continued Antibody-Mediated Rejection in Late Kidney Transplant Recipients

The prevalent renal transplant population presents an opportunity to observe the adaptive changes in the alloimmune response over time, but such studies have been limited by uncertainties in the conventional biopsy diagnosis of T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). To circumvent these limitations, we used microarrays and conventional methods to investigate rejection in 703 unselected biopsies taken 3 days to 35 years post-transplant from North American and European centers. Using conventional methods, we diagnosed rejection in 205 biopsy specimens (28%): 67 pure TCMR, 110 pure ABMR, and 28 mixed (89 designated borderline). Using microarrays, we diagnosed rejection in 228 biopsy specimens (32%): 76 pure TCMR, 124 pure ABMR, and 28 mixed (no borderline). Molecular assessment confirmed most conventional diagnoses (agreement was 90% for TCMR and 83% for ABMR) but revealed some errors, particularly in mixed rejection, and improved prediction of failure. ABMR was strongly associated with increased graft loss, but TCMR was not. ABMR became common in biopsy specimens obtained >1 year post-transplant and continued to appear in all subsequent intervals. TCMR was common early but progressively disappeared over time. In 108 biopsy specimens obtained 10.2-35 years post-transplant, TCMR defined by molecular and conventional features was never observed. We conclude that the main cause of kidney transplant failure is ABMR, which can present even decades after transplantation. In contrast, TCMR disappears by 10 years post-transplant, implying that a state of partial adaptive tolerance emerges over time in the kidney transplant population.