Protein Microarrays Discover Angiotensinogen and PRKRIP1 as Novel Targets for Autoantibodies in Chronic Renal Disease

Abstract

Biomarkers for early detection of chronic kidney disease are needed, as millions of patients suffer from chronic diseases predisposing them to kidney failure. Protein microarrays may also hold utility in the discovery of autoantibodies in other conditions not commonly considered auto-immune diseases. We hypothesized that proteins are released as a consequence of damage at a cellular level during end-organ damage from renal injury, not otherwise recognized as self-antigens, and an adaptive humoral immune response to these proteins might be detected in the blood, as a noninvasive tracker of this injury. The resultant antibodies (Ab) detected in the blood would serve as effective biomarkers for occult renal injury, enabling earlier clinical detection of chronic kidney disease than currently possible, because of the redundancy of the serum creatinine as a biomarker for early kidney injury. To screen for novel autoantibodies in chronic kidney disease, 24 protein microarrays were used to compare serum Ab from patients with chronic kidney disease against matched controls. From a panel of 38 antigens with increased Ab binding, four were validated in 71 individuals, with (n ‫؍‬ 50) and without (n ‫؍‬ 21) renal insufficiency. Significant elevations in the titer of novel auto-Ab were noted against angiotensinogen and PRKRIP1 in renal insufficiency. Current validation is underway to evaluate if these auto-Ab can provide means to follow the evolution of chronic kidney disease in patients with early stages of renal insufficiency, and if these rising titers of these auto-Ab correlate with the rate

Non-HLA Antibodies to Immunogenic Epitopes Predict the Evolution of Chronic Renal Allograft Injury

“…As shown by our group in a previous publication, 15 chronic renal injury and end stage renal failure result in uncovering of kidney-specific epitopes, mostly in the renal cortex and the renal pelvis, and the immunologic recognition of these newly discovered non-HLA antigens with humoral responses in the form of non-HLA antibodies, specific to chronic renal injury. To evaluate if this repertoire of preformed non-HLA antibodies specific to chronic renal failure could predict the subsequent development of CAI after kidney transplantation, the level of these non-HLA antibodies was evaluated at the baseline (day 0 sera) of each patient.…”

“…We followed a previously published protocol developed by our laboratory. 11,15 Briefly, the 96-well microwell ELISA plates were coated with corresponding protein in 50 ml of coating buffer (15 mM Na 2 CO 3 , 30 mM NaHCO 3 , 0.02% NaN 3 , pH 9.6) and incubated overnight at 4°C. Standard curves were generated using anti-GST tag (mouse monoclonal IgG) (Millipore, Temecula, CA) and AP-conjugated AffiniPure goat antimouse IgG (Jackson ImmunoResearch, West Grove, PA).…”

“…11,15 In brief, purified proteins, CSNK2A2 (cat# PV3624), PDGFRA (cat# PV3811), ITAC (cat# PHC1694), and MIG (cat# OHC1604) were acquired from Invitrogen (Carlsbad, CA). A titration with various coated amounts starting at 30.00, 15.00, 7.5, 3.75, 1.87, 0.94, 0.47, and 0 ng, respectively, was performed to determine the optimal amount to be coated onto the immunosorbent 96-well plate.…”

“…The subsequent washing and antibody incubation followed the method previously published. 11,15 The color was developed by using AP-pNPP Liquid Substrate System for ELISA (Sigma-Aldrich, St. Louis, MO). Absorption was measured at 405 nm with a SpectraMax 190 microplate reader (Molecular Devices).…”

“…15 In an attempt to evaluate clinically relevant de novo, renal-specific, non-HLA antibody responses, we utilized protein arrays and customized informatics for studying non-HLA antibody responses in stable grafts 16,17 and acute rejection 11,18 to interrogate previously unidentified non-HLA antibody CAI. To investigate the non-HLA pathogenicity in CAI, we excluded patients with signs of acute rejection, purely focusing on non-HLA antibody responses in the absence of any interval acute rejection episodes.…”