Apolipoprotein A1 and C-terminal fragment of α-1 antichymotrypsin are candidate plasma biomarkers associated with acute renal allograft rejection

Abstract

Background: Current diagnostic methods of renal allograft rejection are neither sensitive nor specific. Needle biopsies are invasive and associated with patient morbidity. Thus, it is desirable to develop noninvasive tests to predict and diagnose rejection.

Methods: Using a case-control approach, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to identify plasma proteins associated with renal allograft rejection. From each rejection patient (n=16), two plasma samples (one near the biopsy date and the other at a time postbiopsy) were compared. Biopsy-confirmed nonrejection patients (n=48) were further analyzed as controls. Antibody-based quantitative enzyme-linked immunosorbent assay was performed to validate candidate biomarker apolipoprotein A1 (Apo A1) in a subset of the original and a second cohort of biopsy-confirmed rejection (n=40) and nonrejection (n=70) patients.

Results: Twenty-two proteins/peptides showed significant differences between rejection and postrejection samples. Peptides 5191 Da and 4467 Da detected rejection with 100% sensitivity and 94% specificity. The 4467 Da peptide was identified as the C-terminal fragment of α-1 antichymotrypsin and a 28 kDa protein was determined as Apo A1. Both protein levels were significantly lower at rejection compared with postrejection. Protein levels of nonrejection patients were similar to the postrejection samples. Apo A1 enzyme-linked immunosorbent assay results showed significantly lower Apo A1 levels (P=0.001 for the original and P=4.14E-11 for the second cohort) at the time of rejection compared with nonrejection which coincides with the SELDI findings.

Conclusions: Together α-1 antichymotrypsin, Apo A1, and the unidentified 5191 Da peptide provide a plasma molecular profile, and this is associated with acute cellular renal allograft rejection.

Figure 1

Candidate plasma proteins which detect renal allograft rejection. (A) Classification and Regression Tree (CART) analysis assessing the biomarker candidates for detecting rejection vs. postrejection samples. (B) Box plots of the adjusted intensities of the 4467 Da and 5191 Da biomarker candidates in rejection (group A, n=17), postrejection (group B, n= 17), and nonrejection (group C, n=48) plasma samples. (C) Box plots of the serum creatinine levels measured at the time of biopsy for rejection patients at the time of rejection and postrejection (n=16) compared with biopsy-proven nonrejection patients (n=19).

Figure 2

Identification of the C-terminal fragment of α-1 antichymotrypsin and apolipoprotein A1 (Apo A1). (A) High-resolution MALDI-TOF mass spectrum of C8 high-performance liquid chromatography (HPLC)-purified fraction containing the 4467 Da biomarker candidate peptide (average mass as measured by SELDI-TOF MS). Monoisotopic mass differences indicate a sequence ladder of SALV (S, serine; A, alanine; L, leucine; and V, valine). (B) Immunoprecipitation of plasma samples with monoclonal antibody against α-1-antichymotrypsin. Numbered peptides with average masses (Da) are presented. (C) Immunoprecipitation of plasma samples with a monoclonal antibody against human Apo A1. (D) Box plots of the SELDI adjusted peak intensities of Apo A1 in rejection (group A, n=17), postrejection (group B, n=17) and nonrejection (group C, n=48) plasma samples.

Figure 3

Validation of apolipoprotein A1 (Apo A1) by enzyme-linked immunosorbent assay (ELISA) for association with acute cellular rejection. (A) Box plots of the ELISA results of plasma samples of a subset of the original cohort. (B) Box plots of the ELISA results of the plasma samples of the second independent cohort. (C) Box plots of Apo A1 levels in recipients that had a sample collected within – 3 to 0 days before the renal biopsy. Twenty-five rejection and 22 nonrejection samples were compared. The mean Apo A1 levels in nonrejection was 0.22 SD±0.06 and 0.13 SD±0.04 in rejection. The levels of Apo A1 were significantly lower at the time of rejection (P value <0.0001) compared with recipients with biopsy-proven nonrejection. (D) Line graph indicating the individual Apo A1 results for 14 patients who had plasma samples from all three time periods (prerejection, rejection, and postrejection). (E) ROC analysis comparing the rejection samples to the nonrejection samples from both the original cohort and the second cohort combined showing a cut point of 0.167 mg/dL and 76% sensitivity and 86% specificity.