Gian S. Jhangri

Endothelial Gene Expression in Kidney Transplants with Alloantibody Indicates Antibody‐Mediated Damage Despite Lack of C4d Staining

Anti‐HLA alloantibody is a risk factor for graft loss, but does not indicate which kidneys are experiencing antibody‐mediated rejection (ABMR). C4d staining in biopsies is specific for ABMR but insensitive. We hypothesized that altered expression of endothelial genes due to alloantibody acting on the microcirculation would be sensitive indicator of ABMR. We identified 119 endothelial‐associated transcripts (ENDATs) from literature, and studied their expression by microarrays in 173 renal allograft biopsies for cause. Mean ENDAT expression was increased in all rejection but was higher in ABMR than in T‐cell‐mediated rejection and correlated with histopathologic lesions of ABMR, and alloantibody. Many individual ENDATs were increased in ABMR and predicted graft loss. Kidneys with high ENDATs and antibody showed increased lesions of ABMR and worse prognosis in comparison to controls. Only 40% of kidneys with high ENDAT expression and chronic ABMR or graft loss were diagnosed by C4d positivity. High ENDAT expression with antibody predicts graft loss with higher sensitivity (77% vs. 31%) and slightly lower specificity (71% vs. 94%) than C4d. The results were validated in independent set of 82 kidneys. High renal endothelial transcript expression in patients with alloantibody is indicator of active antibody‐mediated allograft damage and poor graft outcome.

Improving gene set analysis of microarray data by SAM-GS

BackgroundGene-set analysis evaluates the expression of biological pathways, or a priori defined gene sets, rather than that of individual genes, in association with a binary phenotype, and is of great biologic interest in many DNA microarray studies. Gene Set Enrichment Analysis (GSEA) has been applied widely as a tool for gene-set analyses. We describe here some critical problems with GSEA and propose an alternative method by extending the individual-gene analysis method, Significance Analysis of Microarray (SAM), to gene-set analyses (SAM-GS).ResultsUsing a mouse microarray dataset with simulated gene sets, we illustrate that GSEA gives statistical significance to gene sets that have no gene associated with the phenotype (null gene sets), and has very low power to detect gene sets in which half the genes are moderately or strongly associated with the phenotype (truly-associated gene sets). SAM-GS, on the other hand, performs very well. The two methods are also compared in the analyses of three real microarray datasets and relevant pathways, the diverging results of which clearly show advantages of SAM-GS over GSEA, both statistically and biologically. In a microarray study for identifying biological pathways whose gene expressions are associated with p53 mutation in cancer cell lines, we found biologically relevant performance differences between the two methods. Specifically, there are 31 additional pathways identified as significant by SAM-GS over GSEA, that are associated with the presence vs. absence of p53. Of the 31 gene sets, 11 actually involve p53 directly as a member. A further 6 gene sets directly involve the extrinsic and intrinsic apoptosis pathways, 3 involve the cell-cycle machinery, and 3 involve cytokines and/or JAK/STAT signaling. Each of these 12 gene sets, then, is in a direct, well-established relationship with aspects of p53 signaling. Of the remaining 8 gene sets, 6 have plausible, if less well established, links with p53.ConclusionWe conclude that GSEA has important limitations as a gene-set analysis approach for microarray experiments for identifying biological pathways associated with a binary phenotype. As an alternative statistically-sound method, we propose SAM-GS. A free Excel Add-In for performing SAM-GS is available for public use.

Microarray Analysis of Rejection in Human Kidney Transplants Using Pathogenesis-Based Transcript Sets

Microarrays offer potential for objective diagnosis and insights into pathogenesis of allograft rejection. We used mouse transplants to annotate pathogenesis‐based transcript sets (PBTs) that reflect major biologic events in allograft rejection—cytotoxic T‐cell infiltration, interferon‐γ 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.

Herpes Zoster Infection Following Solid Organ Transplantation: Incidence, Risk Factors and Outcomes in the Current Immunosuppressive Era

Herpes zoster (HZ) infection is a frequent and serious complication of organ transplantation that has not been examined in the current era of immunosuppression.

A New Diagnostic Algorithm for Antibody‐Mediated Microcirculation Inflammation in Kidney Transplants

We studied the significance of microcirculation inflammation in kidney transplants, including 329 indication biopsies from 251 renal allograft recipients, who were mostly nonpresensitized (crossmatch negative). Glomerulitis (g) and peritubular capillaritis (ptc) were often associated with antibody‐mediated rejection (65% and 75%, respectively), but were also found in other diseases in the absence of donor‐specific antibody (DSA): T‐cell‐mediated rejection (ptc, g), glomerulonephritis (g) and acute tubular necrosis (ptc). To develop rules for reducing the nonspecificity of microcirculation inflammation and defining the best grading thresholds associated with DSA, we built and validated a decision tree to predict DSA. The decision tree revealed that g + ptc sum (addition of g‐score plus ptc‐score) was the best predictor of DSA, followed by time posttransplant, then C4d, which had a small role. Late biopsies with g + ptc > 0 showed higher frequency of DSA compared to early biopsies with g + ptc > 0 (79% vs. 27%). Microcirculation inflammation in early biopsies was often false positive (antibody‐independent). The decision tree predicted DSA with higher sensitivity and accuracy than C4d staining. Microcirculation inflammation sum score predicted graft failure independently of time, C4d and transplant glomerulopathy. Thus any degree of microcirculation inflammation in late kidney transplant biopsies strongly indicates presence of DSA and predicts progression to graft failure.

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.

The Stability of the Glomerular Filtration Rate after Renal Transplantation Is Improving

The 6-mo function and the stability of function posttransplantation in 429 cadaver renal transplants was investigated from 1990 to 2000. The 6-mo creatinine clearance (CrCl) and the rate of change of CrCl beyond 6 mo posttransplantation were calculated. The mean 6-mo CrCl was 64.6 +/- 1.1 ml/min and was stable between 1990 and 2000. The net slope of CrCl was -1.4 +/- 0.5 ml/min per yr. The slope has improved in recent years, such that the mean slopes in the period after 1997 are actually positive (+3.5 ml/min per yr). The slope of CrCl beyond 6 mo was not related to the actual value of the 6 mo CrCl, i.e., there was no accelerated loss of function at low CrCl levels. The 6-mo CrCl was independently determined by donor factors (age, gender), recipient factors (age, gender), and immune factors (rejection episodes, regraft status). The slope of the CrCl correlated independently with the transplant year, recipient gender, rejection episodes, diastolic BP, and the choice of immunosuppressive drugs. Cytomegalovirus infection and mismatch status and lipid levels and treatment were not independently associated with slope or 6-mo CrCl. Thus, the most striking change in the course of renal transplants over the past decade is the new stability of function, correlating with reduced rejection and probably due at least in part to the new immunosuppressive agents. Despite continued calcineurin inhibitor use, late improvement in function now occurs in many cadaver kidney transplants, suggesting a previously unappreciated capacity for functional adaptation.

The Transcriptome of the Implant Biopsy Identifies Donor Kidneys at Increased Risk of Delayed Graft Function

Improved assessment of donor organ quality at time of transplantation would help in management of potentially usable organs. The transcriptome might correlate with risk of delayed graft function (DGF) better than conventional risk factors. Microarray results of 87 consecutive implantation biopsies taken postreperfusion in 42 deceased (DD) and 45 living (LD) donor kidneys were compared to clinical and histopathology‐based scores. Unsupervised analysis separated the 87 kidneys into three groups: LD, DD1 and DD2. Kidneys in DD2 had a greater incidence of DGF (38.1 vs. 9.5%, p < 0.05) than those in DD1. Clinical and histopathological risk scores did not discriminate DD1 from DD2. A total of 1051 transcripts were differentially expressed between DD1 and DD2, but no transcripts separated DGF from immediate graft function (adjusted p < 0.01). Principal components analysis revealed a continuum from LD to DD1 to DD2, i.e. from best to poorest functioning kidneys. Within DD kidneys, the odds ratio for DGF was significantly increased with a transcriptome‐based score and recipient age (p < 0.03) but not with clinical or histopathologic scores. The transcriptome reflects kidney quality and susceptibility to DGF better than available clinical and histopathological scoring systems.

Peritubular Capillaritis in Renal Allografts: Prevalence, Scoring System, Reproducibility and Clinicopathological Correlates

While glomerulitis is graded according to the Banff classification, no criteria for scoring peritubular capillaritis (PTC) have been established. We retrospectively applied PTC‐scoring criteria to 688 renal allograft (46 preimplantation, 461 protocol, 181 indication) biopsies.