👤 Scott Bringans

Diabetes-related chronic kidney disease (DKD) is a leading cause of end-stage kidney disease (ESKD), requiring costly dialysis or kidney transplantation. Existing standard- of-care tests for DKD have several limitations, and an alternative is Promarker®D, a validated plasma biomarker test system that predicts DKD in adults with diabetes up to 4 years before symptoms develop. To enable high-throughput application of PromarkerD, a novel CaptSureTM immunoassay version of the test was developed targeting plasma biomarkers Apolipoprotein A4 (ApoA4) and CD5 antigen-like (CD5L). The analytical performance of the assay was assessed, and clinical samples from 2 independent clinical cohorts (>1700 adults with type 2 diabetes [T2D]) were used for the development and external validation of the DKD predictive test. The PromarkerD test system combined ApoA4 and CD5L concentrations with clinical factors age and estimated glomerular filtration rate (eGFR) to calculate risk scores (0% to 100%) and classify study participants as either at low, moderate, or high risk for future kidney decline. PromarkerD demonstrated reliable analytical performance and provided a high discriminative capability in adults with T2D (receiver operating characteristic area under the curve [ROC-AUC]: 0.78 to 0.88) to predict 4-year kidney decline, defined as incident DKD (eGFR <60 mL/min/1.73 m2) or eGFR decline ≥40%, with sensitivity of 75.8% to 85.1% at the moderate-risk cutoff and specificity of >92% at the high-risk cutoff across the two cohorts. The next-generation PromarkerD test system offers a convenient yet highly effective tool for DKD risk assessment. By introducing PromarkerD to standard diabetes care, preventative treatment strategies may be implemented early before permanent kidney function loss occurs. Show less

To determine whether biomarkers for diabetic kidney disease (DKD) can be used to determine the prevalence, progression and/or incidence of diabetic retinopathy (DR) complicating type 2 diabetes. Proteomic biomarkers were measured in baseline fasting plasma from 958 Fremantle Diabetes Study Phase II participants whose baseline and, in those returning for follow-up (n = 764), Year 4 fundus photographs were graded for DR presence/severity. The performance of PromarkerD (three biomarkers and readily available clinical variables which identify prevalent DKD and predict incident DKD and estimated glomerular filtration rate decline ≥30% over four years) for detecting DR prevalence, progression and incidence was assessed using the area under the receiver operating curve (AUC). Logistic regression determined whether individual proteins were associated with DR outcomes after adjusting for the most parsimonious model. Plasma apolipoprotein A-IV (APOA4) was independently associated with moderate non-proliferative DR at baseline (OR (95% CI): 1.64 (1.01, 2.67), P = 0.047). Model discrimination was poor for all PromarkerD predicted probabilities against all DR outcomes (AUC ≤0.681). PromarkerD and its constituent biomarkers were not consistently associated with DR prevalence or temporal change. APOA4 was associated with prevalent DR, but not DR incidence or progression. Distinct pathophysiological mechanisms may underlie DKD and DR. Show less

PromarkerD is a proteomics derived test for predicting diabetic kidney disease that measures the concentrations of three plasma protein biomarkers, APOA4, CD5L and IBP3. Antibodies against these proteins were developed and applied to a multiplexed immunoaffinity capture mass spectrometry assay. In parallel, and facilitating current clinical laboratory workflows, a standard ELISA was also developed to measure each protein. The performance characteristics of the two technology platforms were compared using a cohort of 100 samples, with PromarkerD test scores demonstrating a high correlation (R = 0.97). These technologies illustrate the potential for large scale, high throughput clinical applications of proteomics now and into the future. Show less

PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application. A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories. Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer. An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients. Show less

The ability of current tests to predict chronic kidney disease (CKD) complicating diabetes is limited. This study investigated the prognostic utility of a novel blood test, PromarkerD, for predicting future renal function decline in individuals with type 2 diabetes from the CANagliflozin CardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline in 3568 CANVAS participants ( Show less

To validate the prognostic utility of a novel plasma biomarker panel, PromarkerD, for predicting renal decline in an independent cohort of people with type 2 diabetes. Models for predicting rapid estimated glomerular filtration rate (eGFR) decline defined as i) incident diabetic kidney disease (DKD), ii) eGFR decline ≥30% over four years, and iii) annual eGFR decline ≥5 mL/min/1.73 m During 4.2 ± 0.3 years of follow-up, 5-10% of participants experienced a rapid decline in eGFR. A consensus model comprising apolipoprotein A-IV (apoA4), CD5 antigen-like (CD5L), insulin-like growth factor-binding protein 3 (IGFBP3), age, serum HDL-cholesterol and eGFR showed the best performance for predicting incident DKD (AUC = 0.88 (95% CI 0.84-0.93)); calibration Chi-squared = 5.6, P = 0.78). At the optimal score cut-off, this model provided 86% sensitivity, 78% specificity, 30% positive predictive value and 98% negative predictive value for four-year risk of developing DKD. The combination of readily available clinical and laboratory features and the PromarkerD biomarkers (apoA4, CD5L, IGFBP3) proved an accurate prognostic test for future renal decline in an independent validation cohort of people with type 2 diabetes. Show less

To assess the ability of plasma apolipoprotein (apo) A-IV (apoA4), apo C-III, CD5 antigen-like (CD5L), complement C1q subcomponent subunit B (C1QB), complement factor H-related protein 2, and insulin-like growth factor binding protein 3 (IBP3) to predict rapid decline in estimated glomerular filtration rate (eGFR) in type 2 diabetes. Mass spectrometry was used to measure baseline biomarkers in 345 community-based patients (mean age 67.0 years, 51.9% males) from the Fremantle Diabetes Study Phase II (FDS2). Multiple logistic regression was used to determine clinical predictors of rapid eGFR decline trajectory defined by semiparametric group-based modeling over a 4-year follow-up period. The incremental benefit of each biomarker was then assessed. Similar analyses were performed for a ≥30% eGFR fall, incident chronic kidney disease (eGFR <60 mL/min/1.73 m Based on eGFR trajectory analysis, 35 participants (10.1%) were defined as "rapid decliners" (mean decrease 2.9 mL/min/1.73 m The current study has identified novel plasma biomarkers (apoA4, CD5L, C1QB, and IBP3) that may improve the prediction of rapid decline in renal function independently of recognized clinical risk factors in type 2 diabetes. Show less