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

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. Trial registration ClinicalTrials.gov Identifier NCT01209000 Electronic supplementary material The online version of this article (doi:10.1186/s12882-015-0185-3) contains supplementary material, which is available to authorized users.


Controls Patients
Patients vs Controls

Urine Collections
Timed urine collections are as reported in the original study protocol Version 2.5 for this study. Cell pellets were spun at 1000 g for 12 minutes. Samples are barcoded, the label providing a unique aliquot identifier number, study number assignment, participant ID number, visit number, aliquot type and aliquot number.
Procedure for spot urine processing: Procedure for 24-hour urine sample processing:

Urinalysis
This is site specific, but typically the data collection would include the opportunity to obtain color, appearance, specific gravity, pH, leukocyte esterase, nitrite, protein, glucose, ketones, urobilin, bilirubin, and blood.

Exosome Isolation and Storage
Fresh urine (270 mL) was obtained from seven individuals and prespun at 3000 g to remove cells and debris, then ultracentrifuged at 150,000 g for 1hour to pellet a mixture of Tamm Horsfall protein (THP) and exosome like vesicles (ELVs), termed "crude exosomes." The exosomes were frozen at -80ºC until use. For raw urine storage studies, we employed 400 mL fresh urine collected with complete protease inhibitor and 0.1% sodium azide and created 10 x 20 mL aliquots each. The visit zero urine was immediately centrifuged for 1 hr, then filtered to obtain crude exosomes; we resuspended the exosome pellets in 100uL 0.25M sucrose. The other aliqouts were stored at RT, 4ºC and -80ºC then were retrieved for exosome isolation and analysis by Western using 10 µL of exosome preparation loaded in each lane. For the long-term storage study, we took 6 samples of frozen exosomes that were frozen for >1 year after isolation from 50 mL of whole urine. After centrifugation at 4000 g for 15 min to remove cell pellet, the supernatant was centrifuged at 40,000 g for 2 hours to obtain the exosomes. The exosomes were frozen at -80ºC until needed. For the thaw process, 50 mL frozen urine was thawed at 4ºC overnight, then vigorously vortexed and exosomes extracted per our standard ultracentrifugation protocols. The exosomal proteins PODXL, PKHD1, PKD1 were examined under similar conditions. Western analysis compared freshly extracted exosomes to those extracted from urine at RT for 24 hours or stored at 4°C x 24 hours, without protease inhibitors and sodium azide, since this preservative is used in our working protocol.
Western Blotting: Cell pellets were centrifuged at 4000 g for 20 minutes. Antibodies used in these studies Manufacturer's instructions were followed with the exception of DNase treatment.

DNA Isolation From Urine or Buccal Samples:
First morning void urine samples were spun at 4,000 g x 20 minutes to collect a cell pellet, which was resuspended in 1 mL cell lysis solution, transferred to a 15 mL falcon tube, and Proteinase K (5 µL reconstituted in 50mM Tris-Hcl pH 8; 10mM CaCl 2 ) was added.
Samples were incubated at 55'C overnight, then continued with standard Puregene DNA (Qiagen) isolation protocol, then cooled to RT by placing on ice for 2 min. Total volume was then divided into four 1.5 mL tubes (500 uL each

26.B.1. UPDATES TO SPOT URINE PROCESSING PROCEDURES:
Effective 11/2012 1. Research Coordinators (RCs) will no longer fill sets of cryovials with equal volumes of sample. Effective 11/2012 please fill each cryovial with the maximum volume (1.6 mL) in sequential order (based on the aliquot number indicated on barcoded label) until all corresponding urine sample has been aliquotted. Discard remaining empty cryovials on-site.
2. When transferring urine from the original collection container or the processing tube for freezer storage, it is imperative that these containers are not overfilled. The following procedures take into account liquid expansion when samples freeze.

a) Filling cryovials with biospecimens:
 Digital pipettors: Transfer a total of 1.6 mls of sample into the cryovials.
 Transfer pipettes (disposable, plastic): Note the two ridges below the cap of the cryovial. Use the lower ridge as a reference point for filling cryovials. Please see Figure 1 below.
When transferring urine samples from the centrifuge tubes into cryovials for freezer storage, it is imperative that these containers are not overfilled. The following directions take into account urine volume expansion when frozen.
The small, lowest ridge on the cryovial should be used for reference. Fill cryovial to this point, or with 1.6 mLs of sample.

Procedure Overview
This procedure describes the process for preparing one whole urine sample type (U), two urine supernatant sample types (S, Q) and 2 urine pellet sample types(AP-E and AP-Q) from a spot (random) urine sample for NEPTUNE storage at -80° C. Shipping details are provided in Appendix N of the NEPTUNE Manual of Procedures.
Spot urine is collected in the same fashion for both pediatric and adult study participants. Spot urine samples are obtained at each study visit, including the baseline [V2] and the Biopsy [V3] visits, and all follow-up visits.

26.C.1. UPDATES TO 24-HOUR URINE SAMPLE PROCESSING PROCEDURES
Effective 11/2012 1. Research Coordinators (RCs) will no longer fill sets of centrifuge tubes or cryovials with equal volumes of sample. Effective 11/2012 please fill each tube/cryovial with the maximum volume (40.0 mL and 4.5 mL, respectively) in sequential order (based on the aliquot number indicated on barcoded label) until all corresponding urine sample has been aliquotted. Discard remaining empty cryovials on-site.
2. When transferring urine from the original collection container or the processing tube for freezer storage, it is imperative that these containers are not overfilled. The following procedures take into account liquid expansion when samples freeze.
When transferring 24-hour samples into the 50 and 5 mL storage tubes, it is imperative that the tubes are not overfilled. The following directions take into account urine volume expansion when frozen.

Procedure Overview
This procedure describes the process for preparing aliquots of the 24-hour urine sample for NEPTUNE storage at -80° C. Shipping details are provided in Appendix N of the NEPTUNE Manual of Procedures.
Required Supplies: Included in kit:  2 x 50 mL orange top centrifuge tubes  5 x 5.0 mL cryovials (Pre-labeled as follows)  1 pre-labeled, empty 50 mL centrifuge tube 1 labeled as follows:  1 pre-labeled, 50 mL centrifuge tube containing 6 L of Protease Inhibitor 2  5 pre-labeled 5.0 mL cryovials 3 numbered as follows:  Empty 24-hour urine container for tare Provided by site:  24-hour urine sample from study participant  Scale  Pipettes and tips or disposable pipettes (10 ml, 5 ml, 1 ml sizes) or plastic transfer pipettes  Gloves, goggles, and lab coat