Tara K. Sigdel

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. The gene expression profiles of living-donor kidneys and pristine deceased-donor kidneys (normal histology, young age) were significantly different before reperfusion at implantation. Deceased-donor kidneys exhibited a significant increase in renal expression of complement genes; posttransplantation biopsies from well-functioning, nonrejecting kidneys, regardless of donor source, also demonstrated a significant increase in complement expression. Peritransplantation phenomena, such as donor death and possibly cold ischemia time, contributed to differences in complement pathway gene expression. In addition, complement gene expression at the time of implantation was associated with both early and late graft function. These data suggest that complement-modulating therapy may improve graft outcomes in renal transplantation.

Integrative Urinary Peptidomics in Renal Transplantation Identifies Biomarkers for Acute Rejection

Noninvasive methods to diagnose rejection of renal allografts are unavailable. Mass spectrometry followed by multiple-reaction monitoring provides a unique approach to identify disease-specific urine peptide biomarkers. Here, we performed urine peptidomic analysis of 70 unique samples from 50 renal transplant patients and 20 controls (n = 20), identifying a specific panel of 40 peptides for acute rejection (AR). Peptide sequencing revealed suggestive mechanisms of graft injury with roles for proteolytic degradation of uromodulin (UMOD) and several collagens, including COL1A2 and COL3A1. The 40-peptide panel discriminated AR in training (n = 46) and test (n = 24) sets (area under ROC curve >0.96). Integrative analysis of transcriptional signals from paired renal transplant biopsies, matched with the urine samples, revealed coordinated transcriptional changes for the corresponding genes in addition to dysregulation of extracellular matrix proteins in AR (MMP-7, SERPING1, and TIMP1). Quantitative PCR on an independent set of 34 transplant biopsies with and without AR validated coordinated changes in expression for the corresponding genes in rejection tissue. A six-gene biomarker panel (COL1A2, COL3A1, UMOD, MMP-7, SERPING1, TIMP1) classified AR with high specificity and sensitivity (area under ROC curve = 0.98). These data suggest that changes in collagen remodeling characterize AR and that detection of the corresponding proteolytic degradation products in urine provides a noninvasive diagnostic approach.

A Randomized, Prospective Trial of Rituximab for Acute Rejection in Pediatric Renal Transplantation

We report 1‐year outcomes of a randomized study of Rituximab versus standard‐of‐care immunosuppression (Thymoglobulin and/or pulse steroids) for treatment of biopsy confirmed, acute transplant rejection with B‐cell infiltrates, in 20 consecutive recipients (2–23 years). Graft biopsies, with Banff and CADI scores, CD20 and C4d stains, were performed at rejection and 1 and 6 months later. Peripheral blood CMV, EBV and BK viral loads, graft function, DSA, immunoglobulins, serum humanized antichimeric antibody (HACA) and Rituximab, and lymphocyte counts were monitored until 1 year posttreatment. Rituximab infusions were given with a high index of safety without HACA development and increased infections complications. Rituximab therapy resulted in complete tissue B‐cell depletion and rapid peripheral B‐cell depletion. Peripheral CD19 cells recovered at a mean time of ∼12 months. There were some benefits for the recovery of graft function (p = 0.026) and improvement of biopsy rejection scores at both the 1‐ (p = 0.0003) and 6‐month (p < 0.0001) follow‐up biopsies. Reappearance of C4d deposition was not seen on follow‐up biopsies after Rituximab therapy, but was seen in 30% of control patients. There was no change in DSA in either group, independent of rejection resolution. This study reports safety and suggests further investigation of Rituximab as an adjunctive treatment for B‐cell‐mediated graft rejection.

Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

Purpose: Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non‐invasive diagnostic biomarkers for AR is an unmet need.

Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures

We have conducted an integrative genomics analysis of serological responses to non-HLA targets after renal transplantation, with the aim of identifying the tissue specificity and types of immunogenic non-HLA antigenic targets after transplantation. Posttransplant antibody responses were measured by paired comparative analysis of pretransplant and posttransplant serum samples from 18 pediatric renal transplant recipients, measured against 5,056 unique protein targets on the ProtoArray platform. The specificity of antibody responses were measured against gene expression levels specific to the kidney, and 2 other randomly selected organs (heart and pancreas), by integrated genomics, employing the mapping of transcription and ProtoArray platform measures, using AILUN. The likelihood of posttransplant non-HLA targets being recognized preferentially in any of 7 microdissected kidney compartments was also examined. In addition to HLA targets, non-HLA immune responses, including anti-MICA antibodies, were detected against kidney compartment-specific antigens, with highest posttransplant recognition for renal pelvis and cortex specific antigens. The compartment specificity of selected antibodies was confirmed by IHC. In conclusion, this study provides an immunogenic and anatomic roadmap of the most likely non-HLA antigens that can generate serological responses after renal transplantation. Correlation of the most significant non-HLA antibody responses with transplant health and dysfunction are currently underway.

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

Pretransplant antibodies, particularly anti-CD40, correlate with risk of recurrent focal segmental glomerulosclerosis after kidney transplant. Focusing on Kidney Damage Focal segmental glomerulosclerosis (FSGS) is a disease that damages podocytes, specialized cells that help the kidneys filter blood and produce urine. Over time, this condition leads to progressive deterioration of kidney function, and patients eventually require dialysis or kidney transplant. Unfortunately, the disease often recurs in the transplanted kidneys, causing them to fail as well. Now, Delville et al. present a panel of antibodies (Abs) that can be detected in the blood of patients whose FSGS is more likely to recur after transplant. The authors also highlight one specific Ab that appears to play the largest role in causing podocyte damage and demonstrate how its presence contributes to disease recurrence. 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. A panel of seven Abs (CD40, PTPRO, CGB5, FAS, P2RY11, SNRPB2, and APOL2) could predict posttransplant FSGS recurrence with 92% accuracy. Pretransplant elevation of anti-CD40 Ab alone had the best correlation (78% accuracy) with rFSGS risk after transplantation. Epitope mapping of CD40 with customized peptide arrays and rFSGS sera demonstrated altered immunogenicity of the extracellular CD40 domain in rFSGS. Immunohistochemistry of CD40 demonstrated a differential expression in FSGS compared to non-FSGS controls. Anti-CD40 Abs purified from rFSGS patients were particularly pathogenic in human podocyte cultures. Injection of anti-CD40/rFSGS Ab enhanced suPAR (soluble urokinase receptor)–mediated proteinuria in wild-type mice, yet no sensitizing effect was noted in mice deficient in CD40 or in wild-type mice that received blocking Ab to CD40. In conclusion, a panel of seven Abs can help identify primary FSGS patients at high risk of recurrence before transplantation. Intrarenal CD40 (and possibly other specific glomerular antigens) is an important contributor to FSGS disease pathogenesis. Human trials of anti-CD40 therapies are warranted to evaluate their efficacy for preventing rFSGS and improving graft survival.

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

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. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers.

Complete Steroid Avoidance Is Effective and Safe in Children With Renal Transplants: A Multicenter Randomized Trial With Three‐Year Follow‐Up

To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid‐free (SF) or steroid‐based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow‐up was 3 years posttransplant. Standardized height Z‐score change after 3 years follow‐up was –0.99 ± 2.20 in SF versus –0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z‐score at 3 years –0.43 ± 1.15 vs. –1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy‐proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow‐up. Over the 3 year follow‐up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants.

Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes.

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.