A urinary Common Rejection Module (uCRM) score for non-invasive kidney transplant monitoring.

A Common Rejection Module (CRM) consisting of 11 genes expressed in allograft biopsies was previously reported to serve as a biomarker for acute rejection (AR), correlate with the extent of graft injury, and predict future allograft damage. We investigated the use of this gene panel on the urine cell pellet of kidney transplant patients. Urinary cell sediments collected from patients with biopsy-confirmed acute rejection, borderline AR (bAR), BK virus nephropathy (BKVN), and stable kidney grafts with normal protocol biopsies (STA) were analyzed for expression of these 11 genes using quantitative polymerase chain reaction (qPCR).

Targeted Transcriptional Profiling of Kidney Transplant Biopsies.

Introduction: Studies are needed to assess the quality of transcriptome analysis in paired human tissue samples preserved by different methods and different gene amplification platforms to enable data comparisons across experimenters.

Methods: RNA was extracted from kidney biopsies, either submerged in RNA-stabilizing solution (RSS) or stored in formalin-fixed, paraffin-embedded (FFPE) blocks. RNA quality and integrity were compared.

Expression of Mitochondrial-Encoded Genes in Blood Differentiate Acute Renal Allograft Rejection.

Despite potent immunosuppression, clinical and biopsy confirmed acute renal allograft rejection (AR) still occurs in 10-15% of recipients, ~30% of patients demonstrate subclinical rejection on biopsy, and ~50% of them can show molecular inflammation, all which increase the risk of chronic dysfunction and worsened allograft outcomes. Mitochondria represent intracellular endogenous triggers of inflammation, which can regulate immune cell differentiation, and expansion and cause antigen-independent graft injury, potentially enhancing the development of acute rejection. In the present study, we investigated the role of mitochondrial DNA encoded gene expression in biopsy matched peripheral blood (PB) samples from kidney transplant recipients.

Molecular and Functional Noninvasive Immune Monitoring in the ESCAPE Study for Prediction of Subclinical Renal Allograft Rejection.

Background: Subclinical acute rejection (sc-AR) is a main cause for functional decline and kidney graft loss and may only be assessed through surveillance biopsies.

Methods: The predictive capacity of 2 novel noninvasive blood biomarkers, the transcriptional kidney Solid Organ Response Test (kSORT), and the IFN-γ enzyme-linked immunosorbent spot assay (ELISPOT) assay were assessed in the Evaluation of Sub-Clinical Acute rejection PrEdiction (ESCAPE) Study in 75 consecutive kidney transplants who received 6-month protocol biopsies. Both assays were run individually and in combination to optimize the use of these techniques to predict sc-AR risk.

Intragraft Antiviral-Specific Gene Expression as a Distinctive Transcriptional Signature for Studies in Polyomavirus-Associated Nephropathy.

Background: Polyomavirus nephropathy (PVAN) is a common cause of kidney allograft dysfunction and loss. To identify PVAN-specific gene expression and underlying molecular mechanisms, we analyzed kidney biopsies with and without PVAN.

Methods: The study included 168 posttransplant renal allograft biopsies (T cell-mediated rejection [TCMR] = 26, PVAN = 10, normal functioning graft = 73, and interstitial fibrosis/tubular atrophy = 59) from 168 unique kidney allograft recipients.

A Computational Gene Expression Score for Predicting Immune Injury in Renal Allografts.

Background: Whole genome microarray meta-analyses of 1030 kidney, heart, lung and liver allograft biopsies identified a common immune response module (CRM) of 11 genes that define acute rejection (AR) across different engrafted tissues. We evaluated if the CRM genes can provide a molecular microscope to quantify graft injury in acute rejection (AR) and predict risk of progressive interstitial fibrosis and tubular atrophy (IFTA) in histologically normal kidney biopsies.

Methods: Computational modeling was done on tissue qPCR based gene expression measurements for the 11 CRM genes in 146 independent renal allografts from 122 unique patients with AR (n = 54) and no-AR (n = 92).

Transcriptional Perturbations in Graft Rejection.

Background: Understanding the regulatory interplay of relevant microRNAs (miRNAs) and messenger RNAs (mRNAs) in the rejecting allograft will result in a better understanding of the molecular pathophysiology of alloimmune injury.

Methods: One hundred sixty-seven allograft biopsies, with (n = 47) and without (n = 120) central histology for Banff scored acute rejection (AR), were transcriptionally profiled for mRNA and miRNA by whole genome microarrays and multiplexed microfluidic quantitative polymerase chain reaction, respectively. A customized database was curated (GO-Elite) and used to identify AR-specific dysregulated mRNAs and the role of perturbations of their relevant miRNAs targets during AR.

A Three-Gene Assay for Monitoring Immune Quiescence in Kidney Transplantation.

Organ transplant recipients face life-long immunosuppression and consequently are at high risk of comorbidities. Occasionally, kidney transplant recipients develop a state of targeted immune quiescence (operational tolerance) against an HLA-mismatched graft, allowing them to withdraw all immunosuppression and retain stable graft function while resuming immune responses to third-party antigens. Methods to better understand and monitor this state of alloimmune quiescence by transcriptional profiling may reveal a gene signature that identifies patients for whom immunosuppression could be titrated to reduce patient and graft morbidities.

Endothelial cell antibodies associated with novel targets and increased rejection.

The initial contact point between a recipient's immune system and a transplanted graft is the vascular endothelium. Clinical studies suggest a pathogenic role for non-HLA antiendothelial cell antibodies (AECAs) in allograft rejection; however, evidence linking AECAs of known specificity to in vivo vascular injury is lacking. Here, we used high-density protein arrays to identify target antigens for AECAs isolated from the sera of recipients of kidney transplants experiencing antibody-mediated rejection in the absence of donor-specific HLA antibodies.

A circulating antibody panel for pretransplant prediction of FSGS recurrence after kidney transplantation.

Recurrence of focal segmental glomerulosclerosis (rFSGS) after kidney transplantation is a cause of accelerated graft loss. To evaluate pathogenic antibodies (Abs) in rFSGS, we processed 141 serum samples from 64 patients with and without primary rFSGS and 34 non-FSGS control patients transplanted at four hospitals. We screened about 9000 antigens in pretransplant sera and selected 10 Abs targeting glomerular antigens for enzyme-linked immunosorbent assay (ELISA) validation.

Optimization for peptide sample preparation for urine peptidomics.

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput 'peptidomics' methods , an attractive approach for investigating the pathogenesis of renal disease.

Identification of common blood gene signatures for the diagnosis of renal and cardiac acute allograft rejection.

To test, whether 10 genes, diagnostic of renal allograft rejection in blood, are able to diagnose and predict cardiac allograft rejection, we analyzed 250 blood samples from heart transplant recipients with and without acute rejection (AR) and with cytomegalovirus (CMV) infection by QPCR. A QPCR-based logistic regression model was built on 5 of these 10 genes (AR threshold composite score >37%  = AR) and tested for AR prediction in an independent set of 109 samples, where it correctly diagnosed AR with 89% accuracy, with no misclassifications for AR ISHLT grade 1b. CMV infection did not confound the AR score.

The identification of novel potential injury mechanisms and candidate biomarkers in renal allograft rejection by quantitative proteomics.

Early transplant dysfunction and failure because of immunological and nonimmunological factors still presents a significant clinical problem for transplant recipients. A critical unmet need is the noninvasive detection and prediction of immune injury such that acute injury can be reversed by proactive immunosuppression titration. In this study, we used iTRAQ -based proteomic discovery and targeted ELISA validation to discover and validate candidate urine protein biomarkers from 262 renal allograft recipients with biopsy-confirmed allograft injury.

A rapid noninvasive assay for the detection of renal transplant injury.

Background: The copy number of donor-derived cell-free DNA (dd-cfDNA) in blood correlates with acute rejection (AR) in heart transplantation. We analyzed urinary dd-cfDNA as a surrogate marker of kidney transplant injury.

Methods: Sixty-three biopsy-matched urine samples (41 stable and 22 allograft injury) were analyzed from female recipients of male donors for chromosome Y (donor)-specific dd-cfDNA.

The clinical impact of humoral immunity in pediatric renal transplantation.

The development of anti-donor humoral responses after transplantation associates with higher risks for acute rejection and 1-year graft survival in adults, but the influence of humoral immunity on transplant outcomes in children is not well understood. Here, we studied the evolution of humoral immunity in low-risk pediatric patients during the first 2 years after renal transplantation. Using data from 130 pediatric renal transplant patients randomized to steroid-free (SF) or steroid-based (SB) immunosuppression in the NIH-SNSO1 trial, we correlated the presence of serum anti-HLA antibodies to donor HLA antigens (donor-specific antibodies) and serum MHC class 1-related chain A (MICA) antibody with both clinical outcomes and histology identified on protocol biopsies at 0, 6, 12, and 24 months.

Significance and suppression of redundant IL17 responses in acute allograft rejection by bioinformatics based drug repositioning of fenofibrate.

Despite advanced immunosuppression, redundancy in the molecular diversity of acute rejection (AR) often results in incomplete resolution of the injury response. We present a bioinformatics based approach for identification of these redundant molecular pathways in AR and a drug repositioning approach to suppress these using FDA approved drugs currently available for non-transplant indications. Two independent microarray data-sets from human renal allograft biopsies (n = 101) from patients on majorly Th1/IFN-y immune response targeted immunosuppression, with and without AR, were profiled.

Non-HLA antibodies to immunogenic epitopes predict the evolution of chronic renal allograft injury.

Chronic allograft injury (CAI) results from a humoral response to mismatches in immunogenic epitopes between the donor and recipient. Although alloantibodies against HLA antigens contribute to the pathogenesis of CAI, alloantibodies against non-HLA antigens likely contribute as well. Here, we used high-density protein arrays to identify non-HLA antibodies in CAI and subsequently validated a subset in a cohort of 172 serum samples collected serially post-transplantation.

Phenotypic evaluation of B-cell subsets after rituximab for treatment of acute renal allograft rejection in pediatric recipients.

Background: Recently published pediatric trial of Rituximab for the treatment of CD20+ acute renal allograft rejection (AR) demonstrated transient depletion of circulating/intragraft B cells (Zarkhin et al., Am J Transplant 2008; 8: 2607). In this study, we have evaluated phenotypic definition of circulating B-cell subsets before and after standard of care and B-cell depletional AR therapies.

Differentially expressed RNA from public microarray data identifies serum protein biomarkers for cross-organ transplant rejection and other conditions.

Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases.

Insights into novel cellular injury mechanisms by gene expression profiling in nephropathic cystinosis.

Nephropathic cystinosis is a rare, inherited metabolic disease caused by functional defects of cystinosin associated with mutations in the CTNS gene. The mechanisms underlying the phenotypic alterations associated with this disease are not well known. In this study, gene expression profiles in peripheral blood of nephropathic cystinosis patients (N = 7) were compared with controls (N = 7) using microarray technology.

Expression of complement components differs between kidney allografts from living and deceased donors.

A disparity remains between graft survival of renal allografts from deceased donors and from living donors. A better understanding of the molecular mechanisms that underlie this disparity may allow the development of targeted therapies to enhance graft survival. Here, we used microarrays to examine whole genome expression profiles using tissue from 53 human renal allograft protocol biopsies obtained both at implantation and after transplantation.

Characterization of intra-graft B cells during renal allograft rejection.

Intra-graft CD20(+) B-cell clusters are found during acute rejection of renal allografts and correlate with graft recovery following rejection injury. Here using archived kidney tissue we conducted immunohistochemical studies to measure specific subsets of pathogenic B cells during graft rejection. Cluster-forming CD20(+) B cells in the rejected graft are likely derived from the recipient and are composed of mature B cells.

Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance.

Long-term allograft survival generally requires lifelong immunosuppression (IS). Rarely, recipients display spontaneous "operational tolerance" with stable graft function in the absence of IS. The lack of biological markers of this phenomenon precludes identification of potentially tolerant patients in which IS could be tapered and hinders the development of new tolerance-inducing strategies.

Transcriptional analysis of the molecular basis of human kidney aging using cDNA microarray profiling.

Background: The molecular basis of renal aging is not completely understood.

Methods: We used global gene expression monitoring by cDNA microarrays to identify age associated genes in human kidney samples. Our samples included young (8 weeks-8 years, N= 4), adult (31-46 years, N= 7), and old kidneys (71-88 years, N= 9).

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival.

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells.