Urine Proteomics to Detect Biomarkers for Chronic Allograft Dysfunction

Despite optimal immunosuppressive therapy, more than 50% of kidney transplants fail because of chronic allograft dysfunction. A noninvasive means to diagnose chronic allograft dysfunction may allow earlier interventions that could improve graft half-life. In this proof-of-concept study, we used mass spectrometry to analyze differences in the urinary polypeptide patterns of 32 patients with chronic allograft dysfunction (14 with pure interstitial fibrosis and tubular atrophy and 18 with chronic active antibody-mediated rejection) and 18 control subjects (eight stable recipients and 10 healthy control subjects). Unsupervised hierarchical clustering showed good segregation of samples in groups corresponding mainly to the four biomedical conditions. Moreover, the composition of the proteome of the pure interstitial fibrosis and tubular atrophy group differed from that of the chronic active antibody-mediated rejection group, and an independent validation set confirmed these results. The 14 protein ions that best discriminated between these two groups correctly identified 100% of the patients with pure interstitial fibrosis and tubular atrophy and 100% of the patients with chronic active antibody-mediated rejection. In summary, this study establishes a pattern for two histologic lesions associated with distinct graft outcomes and constitutes a first step to designing a specific, noninvasive diagnostic tool for chronic allograft dysfunction.During the past three decades, the incidence and prevalence of ESRD has increased each year all over the world.¹ Kidney transplantation is the treatment of choice for ESRD because it prolongs survival,² improves quality of life, and is less costly than dialysis³; however, despite these improvements, a substantial proportion of grafts develop progressive dysfunction and fail within a decade, even with the use of appropriate dosages of immunosuppressive drugs to prevent acute rejection.⁴ Chronic allograft dysfunction (CAD) causes more than 50% of graft losses.^5–7 Although patients can return to dialysis after transplant failure, loss of a functioning graft is associated with a three-fold increase in the risk for death,^2,8,9 a substantial decrease in quality of life in survivors, and a four-fold increase in cost.^1,3The decline in function, often associated with hypertension and proteinuria, constitutes a clinical syndrome that has been called chronic allograft nephropathy (CAN). The histopathologic hallmarks of these patients are chronic interstitial fibrosis, tubular atrophy, vascular occlusive changes, and glomerulosclerosis, usually evaluated by the Banff working classification.¹⁰ Major outcomes discussed at the last Banff Conference included the elimination of the nonspecific term CAN and recognition of the entity “chronic active antibody-mediated rejection” (CAAR).¹¹ The rationale for this update was the improper use of “CAN” as a generic term for all causes of chronic renal allograft dysfunction with interstitial fibrosis and tubular atrophy (IF/TA), which hampers accurate diagnosis and appropriate therapy, and increasing recognition of the role of alloantibody in chronic renal allograft deterioration and the corresponding histologic changes, making the identification of an antibody-mediated component of chronic rejection feasible.¹¹Effective strategies to prevent renal function deterioration should focus on the early detection and treatment of patients who develop CAD. In addition to elevated serum creatinine, usually associated with proteinuria and arterial hypertension, more specific and sensitive markers are needed to identify high-risk patients or initial lesions without any changes in serum creatinine or proteinuria.^5,11New analytic tools that allow rapid screening and accurate protein identification in body fluids are now emerging within the field of proteomic science. High-throughput mass spectrometry (MS) methods allow simultaneous detection of a large number of proteins in a large set of biologic tissues or samples. Protein fingerprinting MS methods using modern matrix-assisted laser desorption/ionization-time of-flight MS (MALDI-MS) instrumentation can detect hundreds of peak signals that, as a whole, could be considered a reflex of the body''s physiologic status.¹² To date, MALDI-MS has been successfully used to detect patterns of substantial overexpression of proteins in cancer cells.^13–15 Urine seems to be an ideal source of potential biomarkers, and urine proteomic approaches have been used in numerous attempts to define biomarkers for a variety of nephro-urologic disorders.^16–18 The aim of this study was to evaluate whether chromatography by solid-phase extraction coupled to MS would differentiate urinary polypeptide patterns in patients with pure IF/TA, patients with CAAR, and two control groups: Healthy individuals and stable renal transplant recipients.