Abstract
Introduction: Accurate and non-invasive monitoring of renal allografts post-transplant is essential for early detection of acute rejection (AR) and to impact the long-term survival of the transplant. We present the development and validation of a noninvasive, spot urine-based diagnostic assay, based on measurements of six urinary DNA, protein, and metabolic biomarkers. Materials and Methods: Male and female, adult and pediatric, patients receiving a kidney from related, unrelated living donors, or unrelated deceased donors were included. Markers were selected based on legacy urine genomic, metabolomic, and proteomic studies conducted by our group. For microwell-based measurement of cfDNA, we developed a 5’ biotinylated oligonucleotide complementary chemiluminescent immunoprobe for the measurement of specific target cfDNA fragments, as previously described. Streptavidin-HRP (R&D Systems) and SuperSignal ELISA Femto Substrate (Thermo Fisher Scientific) were used for luminescent detection and quantitation. CXCL10, clusterin, and methylated cfDNA (m-cfDNA) were measured using custom-developed assays. Total protein was measured using the Pierce Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scientific). Urinary creatinine was used to normalize the five other biomarkers for urinary concentration and hydration status and was measured using the Creatinine Assay Kit (BioAssay Systems). Results: The performance of this assay for detecting kidney injury in both native kidneys and renal allografts is presented on a cohort of 601 distinct urine samples. The urinary composite score enables diagnosis of AR, with a receiver-operator characteristic (ROC) curve area-under-the-curve (AUC) of 0.99. In addition, we demonstrate the clinical utility of this assay for predicting AR, prior to a rise in the serum creatinine, enabling earlier detection of rejection than currently possible by standard of care tests.Conclusion: This noninvasive, sensitive, and quantitative approach is a robust and informative method for the rapid and routine monitoring of renal allografts.
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