G. Einecke

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.

Banff ’09 Meeting Report: Antibody Mediated Graft Deterioration and Implementation of Banff Working Groups

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v‐lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics‐technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

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.

Scoring Total Inflammation Is Superior to the Current Banff Inflammation Score in Predicting Outcome and the Degree of Molecular Disturbance in Renal Allografts

Emerging molecular analysis can be used as an objective and independent assessment of histopathological scoring systems. We compared the existing Banff i‐score to the total inflammation (total i‐) score for assessing the molecular phenotype in 129 renal allograft biopsies for cause. The total i‐score showed stronger correlations with microarray‐based gene sets representing major biological processes during allograft rejection. Receiver operating characteristic curves showed that total‐i was superior (areas under the curves 0.85 vs. 0.73 for Banff i‐score, p = 0.012) at assessing an abnormal cytotoxic T‐cell burden, because it identified molecular disturbances in biopsies with advanced scarring. The total‐i score was also a better predictor of graft survival than the Banff i‐score and essentially all current diagnostic Banff categories. The exception was antibody‐mediated rejection which is able to predict graft loss with greater specificity (96%) but at low sensitivity (38%) due to the fact that it only applies to cases with this diagnosis. The total i‐score is able to achieve moderate sensitivities (60–80%) with losses in specificity (60–80%) across the whole population. Thus, the total i‐score is superior to the current Banff i‐score and most diagnostic Banff categories in predicting outcome and assessing the molecular phenotype of renal allografts.

FOXP3 Expression in Human Kidney Transplant Biopsies Is Associated with Rejection and Time Post Transplant but Not with Favorable Outcomes

Expression of the transcription factor forkhead box P3 (FOXP3) in transplant biopsies is of interest due to its role in a population of regulatory T cells. We analyzed FOXP3 mRNA expression using RT‐PCR in 83 renal transplant biopsies for cause in relationship to histopathology, clinical findings and expression of pathogenesis‐based transcript sets assessed by microarrays. FOXP3 mRNA was higher in rejection (T‐cell and antibody‐mediated) than nonrejection. Surprisingly, some native kidney controls also expressed FOXP3 mRNA. Immunostaining for FOXP3 was consistent with RT‐PCR, showing interstitial FOXP3+ lymphocytes, even in some native kidney controls. FOXP3 expression correlated with interstitial inflammation, tubulitis, interstitial fibrosis, tubular atrophy, C4d positivity, longer time posttransplant, younger donors, class II panel reactive antibody >20% and transcript sets reflecting inflammation and injury, but unlike these features was time dependent. In multivariate analysis, higher FOXP3 mRNA was independently associated with rejection, T‐cell‐associated transcripts, younger donor age and longer time posttransplant. FOXP3 expression did not correlate with favorable graft outcomes, even when the analysis was restricted to biopsies with rejection. Thus FOXP3 mRNA expression is a time‐dependent feature of inflammatory infiltrates in renal tissue. We hypothesize that time‐dependent entry of FOXP3‐positive cells represents a mechanism for stabilizing inflammatory sites.

Changes in the Transcriptome in Allograft Rejection: IFN-γ-Induced Transcripts in Mouse Kidney Allografts

We used Affymetrix Microarrays to define interferon‐γ (IFN‐γ)‐dependent, rejection‐induced transcripts (GRITs) in mouse kidney allografts. The algorithm included inducibility by recombinant IFN‐γ in kidneys of three normal mouse strains, increase in kidney allografts in three strain combinations and less induction in IFN‐γ‐deficient allografts. We identified 40 transcripts, which were highly IFN‐γ inducible (e.g. Cxcl9, ubiquitin D, MHC), and 168 less sensitive to IFN‐γ in normal kidney. In allografts, expression of GRITs was intense and consistent at all time points (day 3 through 42). These transcripts were partially dependent on donor IFN‐γ receptors (IFN‐γrs): receptor‐deficient allografts manifested up to 76% less expression, but some transcripts were highly dependent (ubiquitin D) and others relatively independent (Cxcl9). Kidneys of hosts rejecting allografts showed expression similar to that observed with IFN‐γ injections. Many GRITs showed transient IFN‐γ‐dependent increase in isografts, peaking at day 4–5. GRITs were increased in heart allografts, indicating them as generalized feature of alloresponse. Thus, expression of rejection‐induced transcripts is robust and consistent in allografts, reflecting the IFN‐γ produced by the alloresponse locally and systemically, acting via host and donor IFN‐γr, as well as local IFN‐γ production induced by post‐operative stress.

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.