Strategy and rationale for urine collection protocols employed in the NEPTUNE study

Abstract

BackgroundGlomerular diseases are potentially fatal, requiring aggressive interventions and close monitoring. Urine is a readily-accessible body fluid enriched in molecular signatures from the kidney and therefore particularly suited for routine clinical analysis as well as development of non-invasive biomarkers for glomerular diseases.MethodsThe Nephrotic Syndrome Study Network (NEPTUNE; ClinicalTrials.gov Identifier NCT01209000) is a North American multicenter collaborative consortium established to develop a translational research infrastructure for nephrotic syndrome. This includes standardized urine collections across all participating centers for the purpose of discovering non-invasive biomarkers for patients with nephrotic syndrome due to minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Here we describe the organization and methods of urine procurement and banking procedures in NEPTUNE.ResultsWe discuss the rationale for urine collection and storage conditions, and demonstrate the performance of three experimental analytes (neutrophil gelatinase-associated lipocalin [NGAL], retinol binding globulin, and alpha-1 microglobulin) under these conditions with and without urine preservatives (thymol, toluene, and boric acid). We also demonstrate the quality of RNA and protein collected from the urine cellular pellet and exosomes.ConclusionsThe urine collection protocol in NEPTUNE allows robust detection of a wide range of proteins and RNAs from urine supernatant and pellets collected longitudinally from each patient over 5 years. Combined with the detailed clinical and histopathologic data, this provides a unique resource for exploration and validation of new or accepted markers of glomerular diseases.Trial registrationClinicalTrials.gov Identifier NCT01209000Electronic supplementary materialThe online version of this article (doi:10.1186/s12882-015-0185-3) contains supplementary material, which is available to authorized users.

Renal matrix Gla protein expression increases progressively with CKD and predicts renal outcome

“…Socio-demographic data and medical history were recorded, and blood and urine specimens were collected at baseline and each follow-up visit for up to 5 years (Hogan et al, 2015). eGFR (mL/min/1.73m 2 ) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula for participants ≥ 18 years old and the modified CKiD-Schwartz formula for children < 18 years old.…”

The relatively poor correlation between random and 24-hour urine protein excretion in patients with biopsy-proven glomerular diseases

“…All urine samples were processed per the published NEPTUNE study protocol 30 . Whole urine aliquots frozen to −70°C were used for the current measurements.…”

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

“…The urine EV task force of the International Society for Extracellular Vesicles is in the process of recruiting uEV researchers to perform collaborative studies of rigor and reproducibility to address the outlined knowledge gaps attributed to EVs. One strategy to address this may be to assess contamination after collection and discard contaminated samples (Hogan et al, 2015), however this is not practical for all applications. Ultimately the task force recognizes that functional analysis of uEVs is very early in its evolution and identification of strategies to address the above challenges should be a research priority.…”