title: Strategy and rationale for urine collection protocols employed in the NEPTUNE study.
abstract: BACKGROUND
  Glomerular 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  The 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.
  
  
  BACKGROUND
  ClinicalTrials.gov Identifier NCT01209000.
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA. hogan.marie@mayo.edu.
authors.forename: Marie C
authors.initials: MC
authors.lastname: Hogan
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA. Lieske.John@mayo.edu.
authors.forename: John C
authors.initials: JC
authors.lastname: Lieske
authors.affiliation: Department of Internal Medicine - Nephrology, University of Michigan Health System, Ann Arbor, MI, USA. NEPTUNE-Study@umich.edu.
authors.forename: Chrysta C
authors.initials: CC
authors.lastname: Lienczewski
authors.affiliation: Cardiovascular Research, Mayo Clinic, Rochester, MN, USA. Nesbitt.Lisa2@mayo.edu.
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Nesbitt
authors.affiliation: Pediatric Nephrology, University of Michigan, Ann Arbor, MI, USA. larysa@med.umich.edu.
authors.forename: Larysa T
authors.initials: LT
authors.lastname: Wickman
authors.affiliation: Nephrology and Hypertension Research, Mayo Clinic, Rochester, MN, USA. Heyer.Christina@mayo.edu.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Heyer
authors.affiliation: Nephrology and Hypertension Research, Mayo Clinic, Rochester, MN, USA. Harris.Peter@mayo.edu.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Harris
authors.affiliation: Department of Internal Medicine, Division of Nephrology and Hypertension, University of Kansas Medical Center, Kansas City, KS, USA. cward6@kumc.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ward
authors.affiliation: Nephrology and Hypertension Research, Mayo Clinic, Rochester, MN, USA. Sundsbak.Jamie@mayo.edu.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Sundsbak
authors.affiliation: Oncology, Novartis Pharma AG, Basel, Switzerland. luca_manganelli@virgilio.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Manganelli
authors.affiliation: Department of Internal Medicine - Nephrology, University of Michigan Health System, Ann Arbor, MI, USA. wenjunj@umich.edu.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Ju
authors.affiliation: Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. jeffreyk@mail.nih.gov.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Kopp
authors.affiliation: Division of Nephrology and Kidney Research Institute, University of Washington, Seattle, WA, USA. nelsonpj@uw.edu.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Nelson
authors.affiliation: Division of Nephrology and Hypertension, Harbor-UCLA Medical Center, Torrance, CA, USA. sadler@labiomed.org.
authors.forename: Sharon G
authors.initials: SG
authors.lastname: Adler
authors.affiliation: Department of Medicine, University Health Network and University of Toronto, Toronto, ON, Canada. heather.reich@uhn.ca.
authors.forename: Heather N
authors.initials: HN
authors.lastname: Reich
authors.affiliation: Renal-Electrolyte and Hypertension Division, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. lholzman@mail.med.upenn.edu.
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Holzmann
authors.affiliation: Department of Internal Medicine - Nephrology, University of Michigan Health System, Ann Arbor, MI, USA. kretzler@umich.edu.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kretzler
authors.affiliation: Department of Internal Medicine - Nephrology, University of Michigan Health System, Ann Arbor, MI, USA. markusbi@umich.edu.
authors.forename: Markus
authors.initials: M
authors.lastname: Bitzer
mesh_terms: D018070:Biological Specimen Banks / Q000458:organization & administration*; D015415:Biomarkers / Q000097:blood; D005260:Female; D006801:Humans; D008297:Male; D009404:Nephrotic Syndrome / Q000175:diagnosis* / Q000652:urine*; D011507:Proteinuria / Q000175:diagnosis / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014021:Tissue Preservation / Q000379:methods*; D014481:United States; D059349:Urine Specimen Collection / Q000379:methods*
chemical_list: D015415:Biomarkers