Collection, processing and storage of urinary samples
For quantitative comparisons it is necessary to measure the rate of excretion of putative biomarkers. Thus, a 24-h urine collection would be desirable. However, this is an inconvenient procedure that is characterized by low patient compliance and thus unreliable results.15 An alternative is to collect a spot urine sample and normalize the biomarker concentration by the creatinine concentration. For this, the first morning urine may be used, although this has the potential drawback of bladder and bacterial contamination due to a long residence-time in the bladder.16 Thus, from the perspective of practicality and sterility, ideal initial conditions for urine collection may be the second morning urine. Studies on proteinuria have shown that the method of collecting random urine samples is valid and that the rate of protein excretion correlates well with that found in 24-h urine.15
Once the urine is collected, another potential problem is proteolysis,17 which can be dealt with through the use of protease inhibitors in the collection vessel.17 However, some agents that are commonly used in the laboratory such as leupeptin and phenyl methyl sulphonyl fluoride (PMSF) are expensive. Hence, it will be important to determine the lowest cost set of protease inhibitors that are compatible with the protection of the collected human samples from degradation. Alternatively, it is possible that protease inhibitors may not be needed for studies involving spot urine collections that are processed or frozen immediately.
Subsequently, before the urinary exosomes are isolated, procedures can be carried out to remove tubular casts, cells and abundant proteins that may obscure lower abundant proteins. Examples of the latter include the Tamm–Horsfall protein6,18 and albumin when there is proteinuria.19 Casts and intact cells can be removed by low-speed centrifugation, albumin can be removed by albumin removal devices that use affinity ligands and, finally, Tamm–Horsfall protein can be removed with the use of reducing agents such as dithiothreitol.6 Although the removal of Tamm–Horsfall protein is necessary for the analysis of exosomes, it was recently shown that this protein itself may have pathophysiological significance.20 Hence, it could be possible that a simultaneous analysis of exosomes and other non-exosome proteins such as Tamm–Horsfall may provide the most complete pathophysiological information.
At least four different isolation protocols for urinary proteomics have been described.21 Due to the variability in protein chemical and physical properties, different methods will isolate different subpopulations of urinary proteins,21 each of which may be appropriate for different clinical questions. We have used a protocol that employs ultracentrifugation to precipitate exosomes from urine.6 This approach is potentially most valuable for the detection of defects in renal tubular function. It has been noted that this isolation protocol will enhance the detection of hydrophobic proteins in the apical endosomal pathway, whereas other isolation protocols, such as the acetone precipitation technique, will increase the identification of hydrophilic proteins that are derived from sources other than exosomes.21
Another point that merits emphasis is that ultracentrifugation will be an impractical method to use for large-scale studies and for the ultimate clinical application of urinary proteomics because of the time and cost involved. Possible alternative methods for isolation of exosomes include filtration, adsorption, evaporation, dialysis, and/or immuno-isolation.
Storage of samples may be required either directly after the initial urine collection or after the actual exosome isolation. In addition to storage of samples, shipping of samples may also be required, especially if different laboratories collaborate in urinary proteomics projects. Prior to the undertaking of biomarker discovery studies in urine it would be important to develop standardized methods for storing and shipping of samples.
Freezing of samples can potentially cause loss of exosomes due either to aggregation of vesicles or adsorption of exosomes to the surface of the collection vessel. Both problems could hypothetically be dealt with by adjusting sample pH or by adding a surfactant prior to freezing to minimize attraction between the vesicles. With regard to the degradation of frozen proteins over time, evidence from previous studies suggests that there is considerable interprotein variation in the degree of degradation.22,23 Furthermore, these studies concluded that if urinary proteins can be measured within 4 weeks after sampling, storage at 4°C appears to give the best results. It may be desirable to add biocides such as sodium azide or thymol. For longer time-periods, a storage temperature of −70°C or −80°C may be optimal. In both studies, storage at −20°C appeared least favourable both due to a higher degradation rate and the formation of a calcium-phosphate precipitate that was observed at this specific temperature.22,23 The adjustment of pH prior to freezing did not appear to affect protein stability.23
Quantification of urinary protein excretion and determination of its normal variability
A crucial step in urinary proteomics is the translation from biomarker discovery in a research setting to the diagnostic application in a clinical setting. This translation requires that a differentially expressed protein or group of proteins provides sufficient sensitivity, specificity and positive predictive value to be applied as a diagnostic test. As was discussed in the working paradigm, this translation step should occur in the validation phase (Fig. 3), where antibody-based assays of protein abundance rather than quantitative mass spectrometry is likely to be the most cost-effective technique. However, before differences in protein abundance can be interpreted, two questions should be addressed. The first question is how to quantify urinary protein excretion rates and the second question is how to establish the normal variability in excretion of a given urinary protein.
Theoretically, the best choice for quantification of protein excretion is to carry out a timed urine collection and express the excretion rate in conventional rate units such as nmol/h. However, as discussed previously, patient compliance is a frequent problem with timed urine collections and most investigators have adopted approaches that can be applied to spot urines. A frequently used approach is to normalize by the excretion rate of creatinine, inulin or other filterable but non-reabsorbable markers, thereby eliminating the time term in the variable.7 If creatinine is used, it must be recognized that creatinine excretion rate is not invariant.24
In the discovery phase, validation phase and clinical application phase, sample measurements in patients must be related to normal reference samples to draw conclusions. Thus, it is important to define a method for obtaining normal reference samples. To approach this problem, it would be useful to determine the intrinsic variability in the urinary proteome of normal human subjects. Potentially, the possible effects of age, gender, circadian rhythm, high- and low-salt diets and water loading and restriction need to be investigated. The sensitivity, specificity and thus the clinical value of a candidate biomarker can be defined only relative to a carefully chosen control.