For clinical and research purposes, it is important that urine destined for isolation of exosomes is collected and stored in a standardized way. There are three important steps that should be addressed to standardize the collection and storage of urine: (1) addition of protease inhibitors and preservative during urine collection, (2) storage of urine at stable temperatures, for short-term and long-term use, and (3) extensively vortexing urine samples after thawing [12, 22] (see more at http://​www.​niddk.​nih.​gov/​research-funding/​at-niddk/​labs-branches/​kidney-disease-branch/​renal-diagnostics-therapeutics-unit/​sample-collection-storage-exosome-analysis/​Pages/​default.​aspx). Without the use of protease inhibitors, various key proteins often detected in urinary exosomes can degrade. The typical cocktail of protease inhibitors and preservative includes phenylmethanesulfonyl fluoride (PMSF), leupeptin, and sodium azide. From our own experiences, storage of urine at −80 °C provides a more stable condition than storing samples at −20 °C. With extensive vortexing after thawing, samples stored at −80 °C have the highest recovery of exosomes—up to 100 % when compared to freshly processed urine. In contrast, urine stored at −20 °C has only 87 % exosome recovery after extensive vortexing. Nanoparticle tracking analysis of urinary exosomes has recently confirmed a storage condition of −80 °C with the addition of protease inhibitors is the best [22]. Urine collected in the morning, both first and second urine of the day, has similar exosome contents and can be used interchangeably for experimental research purposes [12].

After the discovery of potential renal disease-related biomarkers in urinary exosomes, researchers have worked to identify a variety of methods for faster and more efficient ways to collect urinary exosomes for clinical applications. This exploration began because the first method of isolation, ultracentrifugation, requires long processing times and access to expensive equipment. Despite the applications of novel techniques, including membrane filtration and exosome precipitation methods, ultracentrifugation methods show the highest exosome purity. Table 5.1 shows a comparison of urinary exosome isolation techniques.

Ultracentrifugation was the original method of reproducibly isolating urinary exosomes [2]. This technique requires collecting urine in the collection solution (protease inhibitors and sodium azide), spinning the urine at a low speed (17,000×g) to remove whole cells, large membrane fragments, and cellular debris, followed by spinning the supernatant at a high speed (200,000×g) for 1 h to pellet the exosomes (for more details please check http://​www.​niddk.​nih.​gov/​research-funding/​at-niddk/​labs-branches/​kidney-disease-branch/​renal-diagnostics-therapeutics-unit/​exosome-preparation/​Pages/​default.​aspx). This initial low-density exosome pellet commonly includes some contamination of highly abundant urinary proteins, including albumin and uromodulin, also known as Tamm-Horsfall protein (THP) [2, 23]. THP forms double-helical fibrils by a disulfide cross-link zona pellucida (ZP) domain, which entraps exosomes and is co-isolated with the low-density pellet. Adding the reducing agent dithiothreitol (DTT) with a subsequent high-speed spin reduces the presence of THP in the low-density pellet [2]. However, this method does not completely decontaminate the sample of all THP and other abundant proteins. Alternatively, a more purified exosome pellet can be obtained using an additional step of double-cushion ultracentrifugation [23] or the use of heavy water and a sucrose gradient in ultracentrifugation [24] to separate the exosomes and contaminating proteins based on density, whereas ultracentrifugation followed by size exclusion chromatography (UC-SEC) can be used to separate contaminating proteins from exosomes based on molecular weight [25]. However, these methods are still time-consuming and labor intensive and require expensive equipment.

In 2007, Cheruvanky et al. [3] used commercially available nanomembrane concentrators to filter exosomes from urine of healthy volunteers and proteinuric patients with focal segmental glomerulosclerosis (FSGS). The exosome isolation time was reduced from 4 h using standard ultracentrifugation to 0.5–2 h using nanomembrane filters [3]. Using this system, the authors were able to detect various proteins typically found in urinary exosomes, even from patients who had high quantities of contaminating proteins, demonstrating that this method could be used for both clinical and routine experimental applications. Other microfiltration methods have also been shown to provide an efficient way of isolating urinary exosomes with reduced contamination of highly abundant urinary proteins as compared to nanomembrane filtration and ultracentrifugation techniques [26]. Alternatively, precipitation methods have been used to isolate urinary exosomes. In 2012, Alvarez et al. [27] showed that a commercially available exosome precipitation kit called ExoQuick-TC could be used to isolate urinary exosomes, although a modified protocol provided improved results. This modified exosome precipitation method was shown to provide a more efficient yield of miRNA and mRNA than protein compared to sucrose cushion ultracentrifugation.

A challenge that researchers in the urinary exosome field face is defining the methods of exosome normalization. Without standardized normalization protocols, biomarker discovery studies from urinary exosomes and the subsequent comparisons between patient-to-patient samples will be less reliable. There are several methods of normalization, including time normalization, creatinine normalization, and protein normalization.

The quantitative measurement of urinary biomarkers in terms of excretion rate is the optimal method for normalization and has been used for many years for analysis of classical urinary biomarkers such as total protein and albumin (e.g., expressed in g/day or µg/min). Assessments of urinary biomarkers based only on concentrations are unsatisfactory because normal physiological variations in water excretion can dilute or concentrate urinary proteins making this measurement unreliable for both intra- and interpersonal comparisons. Time normalization, the collection of urine from patients within the same bracket of time, is thus the most accurate method of measuring urine exosomal protein excretion rates and is the most reliable method of comparing patient exosome products side-by-side. An even more accurate time normalization would be the collection of all urine from the same patient over 24 h, but this approach has several practical limitations.

As there are difficulties acquiring time-normalized urine samples, and this method also rules out using urine samples from a biobank because these samples are typically spot urine samples, a more common method of normalization, creatinine normalization, is used in a clinical setting. Creatinine is typically excreted in urine at a steady rate. The average 24-h urine creatinine excretion rate for the general population is approximately 1,000 mg/day per 1.73 m² body surface area. Given the amount of creatinine in the spot urine, an estimated rate of exosome excretion can be assigned. However, this method has limitations; it does not take into account actual difference in individual creatinine excretion rates or renal diseases that make creatinine excretion rates unstable, such as acute kidney disease.

Normalization based on the amount of a particular protein in urine has been proposed, including the uses of THP, exosomal markers (e.g., ALIX or TSG101), or other biomarker proteins that when measured together provide a concentration ratio that correlates with disease state [6, 28]. However, further studies are needed to confirm the validity of these normalization methods. An alternative method of normalization that has recently been utilized is counting exosome number. Using a new nanoparticle tracking analysis system, urinary exosomes in whole urine were counted, sized, and analyzed [22]. This new method of analysis may ultimately be less time-consuming and provide a more standardized normalization procedure, yet comes with the disadvantage of requiring expensive equipment.