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 followup 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 antibodymediated 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.

We studied the phenotype of late kidney graft failure in a prospective study of unselected kidney transplant biopsies taken for clinical indications. We analyzed histopathology, HLA antibodies and death-censored graft survival in 234 consecutive biopsies from 173 patients, taken 6 days to 31 years posttransplant. Patients with late biopsies (>1 year) frequently displayed donor-specific HLA antibody (particularly class II) and microcirculation changes, including glomerulitis, glomerulopathy, capillaritis, capillary multilayering and C4d staining. Grafts biopsied early rarely failed (1/68), whereas grafts biopsied late often progressed to failure (27/105) within 3 years. T-cell-mediated rejection and its lesions were not associated with an increased risk of failure after biopsy. In multivariable analysis, graft failure correlated with microcirculation inflammation and scarring, but C4d staining was not significant. When microcirculation changes and HLA antibody were used to define antibody-mediated rejection, 17/27 (63%) of late kidney failures after biopsy were attributable to antibody-mediated rejection, but many were C4d negative and missed by current diagnostic criteria. Glomerulonephritis accounted for 6/27 late losses, whereas T-cell-mediated rejection, drug toxicity and unexplained scarring were uncommon. The major cause of late kidney transplant failure is antibody-mediated microcirculation injury, but detection of this phenotype requires new diagnostic criteria.

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 donorspecific 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.

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.

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.

Microarrays offerpotential for objective diagnosis and insights into pathogenesis of allograft rejection. We used mouse transplants to annotate pathogenesisbased transcript sets (PBTs) that reflect major biologic events in allograft rejection-cytotoxic T-cell infiltration, interferon-c effects and parenchymal deterioration. We examined the relationship between PBT expression, histopathologic lesions and clinical diagnoses in 143 consecutive human kidney transplant biopsies for cause. PBTs correlated strongly with one another, indicating that transcriptome disturbances in renal transplants have a stereotyped internal structure. This disturbance was continuous, not dichotomous, across rejection and nonrejection. PBTs correlated with histopathologic lesions and were the highest in biopsies with clinically apparent rejection episodes. Surprisingly, antibody-mediated rejection had changes similar to T-cell mediated rejection. Biopsies lacking PBT disturbances did not have rejection. PBTs suggested that some current Banff histopathology criteria are unreliable, particularly at the cut-off between borderline and rejection. Results were validated in 51 additional biopsies. Thus many transcriptome changes previously described in rejection are features of a large-scale disturbance characteristic of rejection but occurring at lower levels in many forms of injury. PBTs represent a quantitative measure of the inflammatory disturbances in organ transplants, and a new window on the mechanisms of these changes.

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.

Late antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. Uncontrolled studies have suggested efficacy of the proteasome inhibitor bortezomib, but no systematic trial has been undertaken to support its use in ABMR. In this randomized, placebo-controlled trial (the Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection [BORTEJECT] Trial), we investigated whether two cycles of bortezomib (each cycle: 1.3 mg/m intravenously on days 1, 4, 8, and 11) prevent GFR decline by halting the progression of late donor-specific antibody (DSA)-positive ABMR. Forty-four DSA-positive kidney transplant recipients with characteristic ABMR morphology (median time after transplant, 5.0 years; pretransplant DSA documented in 19 recipients), who were identified on cross-sectional screening of 741 patients, were randomly assigned to receive bortezomib (=21) or placebo (=23). The 0.5-ml/min per 1.73 m per year (95% confidence interval, -4.8 to 5.8) difference detected between bortezomib and placebo in eGFR slope (primary end point) was not significant (=0.86). We detected no significant differences between bortezomib- and placebo-treated groups in median measured GFR at 24 months (33 versus 42 ml/min per 1.73 m; =0.31), 2-year graft survival (81% versus 96%;=0.12), urinary protein concentration, DSA levels, or morphologic or molecular rejection phenotypes in 24-month follow-up biopsy specimens. Bortezomib, however, associated with gastrointestinal and hematologic toxicity. In conclusion, our trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA, despite significant toxicity. Our results reinforce the need for systematic trials to dissect the efficiency and safety of new treatments for late ABMR.