Podium Presentation in Room 5 on Wednesday at 9:20 (Chair: Stephen Pennington)
Authors: Angela Mc Ardle1, Qin Fu1, Maryam Afkarian2* and Jennifer E Van Eyk1*
Introduction: People with reduced estimated glomerular filtration rate (eGFR) (<60 ml/min/1.73m2) are at risk of progressing to stage 5 chronic kidney disease (CKD) defined by end-stage kidney disease (ESRD). Individuals with type 2 diabetes are at particularly high risk and 30-40 % will develop diabetic kidney disease (DKD). Development of new therapies targeting this stage of DKD requires a detailed understanding of the mechanisms causing progression, particularly at this disease stage. Kidney impairment results in dysregulated and increased blood pressure and many patients with progressive DKD and also CKD are at risk of accelerated cardiovascular disease, a leading cause of death in the USA. Thus, disease management is focused on early detection and the timely initiation of treatment to improve outcome. A noninvasive urine test that could be used to; (i) identify individuals who will develop kidney failure and (ii) monitor disease progression, would be an extremely valuable tool for effective management of this disorder.
Mass spectrometry is recognized as a reproducible method for the identification of urine protein panels and for the translation of multiplexed assays into clinical practice. Hence, we propose to quantify the urine abundance of several proteins representing biologically relevant pathways associated with DKD progression from reduced eGFR to ESRD or those with a 50% drop in GFR over time.
Objectives: The objective of this study was to develop a targeted mass spectrometry assay to measure putative biomarkers of ESRD in patients with CKD and DKD.
Methods: Using skyline software (v.188.8.131.52), a parallel reaction monitoring assay was generated to target 19 proteins based on 38 proteotyic peptides. Representative isotopically labelled standard peptides were purchased and used to establish the PRM on a Qexactive Plus coupled to an Evosep One LC system. LLOD, LLOQ, inter and intra-day reproducibility, optimization of trypsin digest and urine sample prep were performed. To confirm the prognostic power of the protein panel the assays will be deployed in 282 urine samples from the chronic renal insufficiency cohort in a blinded and randomized study (n=282).
Results:19 proteins were targeted for method development. An in-silico and experimental workflow was devised to select 38 prototypic peptides for these target proteins. We compared protein extraction methods as well as a number of denaturing conditions. A protein extraction free workflow that uses sodium deoxycholate emerged as the most time- and cost-effective method. Using these conditions, it was possible to detect 8 proteins with 2 peptides and 6 proteins with 1 peptide whereas 5 proteins were below limit of detection. Results from analysis in CRIC samples are pending.
Conclusion: We have developed a PRM method to precisely quantify 14 proteins in human urine, which will be applied to urine samples from a case-control subset, nested in the Chronic Renal Insufficiency Cohort.
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