The Identification of Novel Potential Injury Mechanisms and Candidate Biomarkers in Renal Allograft Rejection by Quantitative Proteomics

Abstract

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. Urine samples were randomly split into a training set of 108 patients and an independent validation set of 154 patients, which comprised the clinical biopsy-confirmed phenotypes of acute rejection (AR) (n ‫؍‬ 74), stable graft (STA) (n ‫؍‬ 74), chronic allograft injury (CAI) (n ‫؍‬ 58), BK virus nephritis (BKVN) (n ‫؍‬ 38), nephrotic syndrome (NS) (n ‫؍‬ 8), and healthy, normal control (HC) (n ‫؍‬ 10). A total of 389 proteins were measured that displayed differential abundances across urine specimens of the injury types (p < 0.05) with a significant finding that SUMO2 (small ubiquitin-related modifier 2) was identified as a "hub" protein for graft injury irrespective of causation. Sixtynine urine proteins had differences in abundance (p < 0.01) in AR compared with stable graft, of which 12 proteins were up-regulated in AR with a mean fold increase of 2.8. Nine urine proteins were highly specific for AR because of their significant differences (p < 0.01; fold increase >1.5) from all other transplant categories (HLA class II protein HLA-DRB1, KRT14, HIST1H4B, FGG, ACTB, FGB, FGA, KRT7, DPP4). Increased levels of three of these proteins, fibrinogen beta (FGB; p ‫؍‬ 0.04), fibrinogen gamma (FGG; p ‫؍‬ 0.03), and HLA DRB1 (p ‫؍‬ 0.003) were validated by ELISA in AR using an independent sample set. The fibrinogen proteins further segregated AR from BK virus nephritis (FGB p ‫؍‬ 0.03, FGG p ‫؍‬ 0.02), a finding that supports the utility of monitoring these urinary proteins for the specific and sensitive noninvasive diagnosis of acute renal allograft rejection. Molecular & Cellular Proteomics

Brain Insulin Lowers Circulating BCAA Levels by Inducing Hepatic BCAA Catabolism

“…Since antibodies to these proteins are not commercially available, we performed a global quantitative proteomic profiling by two-dimensional LC-MS/MS coupled with iTRAQ (isobaric tag for relative and absolute quantitation) labeling (Sigdel et al, 2014) to investigate whether MBH insulin induced downstream BCAA-catabolizing enzymes. This proteomic analysis led to the identification and quantification of 2418 proteins in these liver extracts, of which ~250 were significantly altered by MBH insulin infusion.…”

Antibody-Mediated Rejection in Pediatric Kidney Transplantation: Pathophysiology, Diagnosis, and Management

“…A recent study by our group identified novel antigenic targets [endoglin, epidermal growth factor (EGF)-like repeats and discoidin I-like domains 3, intercellular adhesion molecule 4, and FMS-like tyrosine kinase-3 ligand], which were relevant to the development of acute ABMR as evidenced by a positive endothelial positive cross-match [37]. In addition, upregulation of nHLA proteins in acute rejection in transplant [38] have been described; it would be important to know whether these proteins serve as alloantigens.…”

“…The ability to diagnose rejection at a molecular level from a blood or urine specimen is beneficial because it allows for non-invasive diagnosis and risk stratification of rejection without the traumatic biopsy of an organ that is already undergoing the trauma of rejection. Recently, studies have described patterns of gene expression [53,54] and microarray analysis [55][56][57], urine proteomics [38], and a constitution of both molecular and clinical markers specific to acute rejection [58,59] in solid organ transplants, including kidney transplants. Some of these studies [52,59] have led to the development of the kidney Solid Organ Response Test [60], which can accurately diagnose acute kidney rejection with a peripheral blood sample.…”

The Use of Genomics and Pathway Analysis in Our Understanding and Prediction of Clinical Renal Transplant Injury

“…Other studies have applied a variety of proteomic techniques to identify protein expression in an unbiased high-throughput manner in allograft rejection, before utilizing targeted ELISA for validation of biomarker panels. 93,97,102 However as with the majority of promising proteomic studies to date, multicenter prospective trials are required to validate these candidate markers before meaningful conclusions can be made.…”