Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery. Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery. Exosomes are classically formed from the inward invagination and pinching-off of the late endosomal membrane. This results in the formation of a multivesicular body (MVB) laden with small lipid-bilayered vesicles (∼40–100 nm in diameter), each of which contains a sample of the parent cell's cytoplasm.1.Stoorvogel W. Kleijmeer M.J. Geuze H.J. et al.The biogenesis and functions of exosomes.Traffic. 2002; 3: 321-330Crossref PubMed Scopus (590) Google Scholar Fusion of the MVB with the cell membrane results in the release of these ‘exosomes’ from the cell, and their delivery into the blood, urine, or other body fluids. Exosome-like vesicles including ‘shedding microvesicles’2.Cocucci E. Racchetti G. Meldolesi J. Shedding microvesicles: artifacts no more.Trends Cell Biol. 2009; 19: 43-51Abstract Full Text Full Text PDF PubMed Scopus (1285) Google Scholar may also be formed by the budding-off of the cell's plasma membrane, and although more heterogeneous in size,2.Cocucci E. Racchetti G. Meldolesi J. Shedding microvesicles: artifacts no more.Trends Cell Biol. 2009; 19: 43-51Abstract Full Text Full Text PDF PubMed Scopus (1285) Google Scholar, 3.Marzesco A.M. Janich P. Wilsch-Bräuninger M. et al.Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells.J Cell Sci. 2005; 118: 2849-2858Crossref PubMed Scopus (333) Google Scholar may also contain a snapshot of the parent cell's RNA. Although the majority of microvesicles isolated from urine are thought to be exosomes,4.Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1439) Google Scholar both exosomes and other microvesicles do co-isolate during ultracentrifugation and ultrafiltration isolation techniques and will, therefore, be collectively referred to as microvesicles here. Recent pioneering oncology research has shown that exosomes carry mRNA and/or miRNA5.Skog J. Würdinger T. van Rijn S. et al.Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3251) Google Scholar, 6.Valadi H. Ekström K. Bossios A. et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (7531) Google Scholar, 7.Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1743) Google Scholar that may encode tumor markers, potentially circumventing the need for biopsies and highlighting the enormous diagnostic potential of exosome biology.5.Skog J. Würdinger T. van Rijn S. et al.Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3251) Google Scholar Comprehensive studies have been conducted on the proteomic analysis of urinary microvesicles, revealing that they contain a variety of cell-specific proteins/transporters from the kidney and the urogenital tract.4.Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1439) Google Scholar, 8.Gonzales P.A. Pisitkun T. Hoffert J.D. et al.Large-scale proteomics and phosphoproteomics of urinary exosomes.J Am Soc Nephrol. 2009; 20: 363-379Crossref PubMed Scopus (491) Google Scholar It was further shown that urinary microvesicles are very stable, highlighting their potential use as a reliable urinary marker.9.Zhou H. Yuen P.S. Pisitkun T. et al.Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.Kidney Int. 2006; 69: 1471-1476Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar However, there have been no in-depth studies analyzing the nature of nucleic acids within the microvesicles or their reliability as a source of nucleic acid biomarkers for renal function. Whole urine is known to contain nucleic acids derived from whole cells and free DNA.10.Cristaudo A. Vivaldi A. Guglielmi G. et al.A simple method to reveal possible RAS mutations in DNA of urinary sediment cells.J Environ Pathol Toxicol Oncol. 1997; 16: 201-204PubMed Google Scholar, 11.Botezatu I. Serdyuk O. Potapova G. et al.Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism.Clin Chem. 2000; 46: 1078-1084PubMed Google Scholar However, such extraneous nucleic acids may not be a reliable source of biomarkers as they may be derived from apoptotic cells, the transcriptional profile of which may not be representative of a functioning cell. The investigation of new biomarkers for renal disease is currently an important and pressing issue, with renal disease affecting up to 1 in 10 of the US population.12.Coresh J. Selvin E. Stevens L.A. et al.Prevalence of chronic kidney disease in the United States.JAMA. 2007; 298: 2038-2047Crossref PubMed Scopus (3662) Google Scholar Urinary microvesicles may provide a unique means to analyze the transcriptional profile of the kidney as they are derived from functioning cells. The isolation of microvesicles usually calls for ultracentrifugation, but recent studies have shown that they may also be rapidly isolated using filtration concentrators,13.Cheruvanky A. Zhou H. Pisitkun T. et al.Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.Am J Physiol Renal Physiol. 2007; 292: F1657-F1661Crossref PubMed Scopus (267) Google Scholar increasing the potential for their use in routine diagnostic analysis. Currently, it is not known whether the use of filtration concentrators would yield intact microvesicles for RNA extraction. Here we conduct an in-depth analysis of the nucleic acids associated with urinary microvesicles, including (i) the potential for extraneous nucleic acid contamination during urinary microvesicle isolation, (ii) the non-invasive identification of renal related transcripts from various regions of the nephron and collecting duct by reverse transcriptase-PCR (RT-PCR), (iii) the application of microvesicle derived RNA analysis in renal pathophysiology, (iv) analysis of RNA integrity in microvesicles versus whole cells in urine, and (v) the nucleic acid analysis of isolated microvesicles using filtration concentrators versus ultracentrifugation. These studies increase our understanding of urinary microvesicles and support their potential as a novel source of new and much needed biomarkers for renal disease analysis. Figure 1a shows transmission electron microscope images of MVBs present in rat renal tissue. This shows that exosomes can indeed be released from various regions of the nephron, as well as from both intercalated and principal cells of the collecting duct. Using transmission electron microscopy we also examined the pellet isolated by differential centrifugation to show that the pellet was indeed rich in microvesicles (Figure 1b). To ensure that the RNA was coming from microvesicles and not large membrane blebs, a Percoll gradient was used to separate the pelleted microvesicles based on density (Figure 1c) and RNA was extracted from each fraction. Results revealed that the RNA obtained was indeed coming from microvesicles within the same density range as that previously described for urinary exosomes.14.Keller S. Rupp C. Stoeck A. et al.CD24 is a marker of exosomes secreted into urine and amniotic fluid.Kidney Int. 2007; 72: 1095-1102Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar Using differential centrifugation in combination with RNase and DNase digestion of the microvesicle pellet, we determined whether extraneous nucleic acids may co-isolate with urinary microvesicles. Results revealed that extraneous DNA (that is, free DNA) contaminates isolated urinary microvesicles (Figure 2a) and serum-derived microvesicles (see Supplementary Figure S1d). The level of extraneous DNA varied between subjects but was consistently shown to be predominantly in the range of ∼25–1500 nucleotides (nt). In contrast, no obvious extraneous RNA could be detected (Figure 2b), consistent with the presence of ribonucleases in urine.15.Sugiyama R.H. Blank A. Dekker C.A. Multiple ribonucleases of human urine.Biochemistry. 1981; 20: 2268-2274Crossref PubMed Scopus (29) Google Scholar Analysis of the nucleic acids within microvesicles revealed some striking features, including the presence of prominent 18S and 28S rRNA peaks similar to those seen for RNA isolated from rat kidney tissue (Figure 2c). Such rRNA was not prevalent in previously reported serum-derived or cell culture-derived microvesicles, although they may be detectable depending on isolation technique as shown in the serum RNA isolation profile in Supplementary Figure S1d. Both urinary microvesicles and kidney tissue also contained small RNAs when miRNA isolation methods were used (Figure 2d) as previously reported.16.Zhou H. Cleary R.C. Bogaert Y.E. et al.Combination of microRNA192 and microRNA27b from Urinary Exosomes Differentiate between Renal Tubular Damage and Glomerular Injury. SA-PO2505] American Society of Nephrology, Philadelphia PA2008Google Scholar Various extraction kits are available for the isolation of RNA and removal of DNA. A comparative analysis of RNeasy Qiagen kits and the acid phenol/chloroform-based mirVana kit is shown in Supplementary Figure S1. Download .jpg (.04 MB) Help with files Supplementary Figure 1 To determine the nucleic acid content within urinary microvesicles, the pellets were subjected to RNase and DNase digestion to remove extraneous contamination, followed by RNase and/or DNase digestion of intra-microvesicular nucleic acids during column-based nucleic acid isolation. On-column RNase digestion almost completely abolished the nucleic acid profile (Figure 2e), suggesting that RNA represents the most abundant nucleic acid within microvesicles. Further, on-column digestion with DNase revealed that the remaining peak could be further decreased, suggesting that there may be some DNase digestible material within microvesicles (Figure 2f). To further determine whether microvesicles contain mRNA transcripts encoding markers from various nephron and collecting duct segments, RNA isolated from urinary microvesicles of four human controls (23–32 years of age) was subjected to RT-PCR. Both glyceraldehyde 3-phosphate dehydrogenase and β-actin genes were identified in all samples (Figure 3a). Next we examined 15 transcripts characteristic of various regions of the nephron and collecting duct (Figure 3b). These included proteins and receptors implicated in various renal diseases: podocin from the glomerulus, cubilin from the proximal tubule, and aquaporin 2 from the collecting duct. Genes from all regions examined could be identified, consistent with the results obtained in Figure 1a. This shows that microvesicles containing mRNA are released from all regions of the nephron and the collecting duct and are, therefore, a novel non-invasive source of potential nucleic acid biomarkers for renal disease. To test the hypothesis that microvesicles may be used to non-invasively examine renal genes in disease, the V-ATPase B1 subunit knockout mouse model of renal acidosis was used.17.Finberg K.E. Wagner C.A. Bailey M.A. et al.The B1-subunit of the H(+) ATPase is required for maximal urinary acidification.Proc Natl Acad Sci USA. 2005; 102: 13616-13621Crossref PubMed Scopus (106) Google ScholarFigure 4 shows that expression of the V-ATPase B1 subunit and aquaporin 2 mRNA can be examined non-invasively in the microvesicles using RT-PCR (Figure 4a). In addition, real-time PCR was used to quantitatively examine the expression of the V-ATPase B2 subunit. The renal expression of the V-ATPase B2 subunit is the same in V-ATPase B1 -/- animals as in V-ATPase B1 +/+ mice (Figure 4b) despite the loss of the B1 subunit, confirming the results obtained for the corresponding kidneys as well as previously reported data.18.Păunescu T.G. Russo L.M. Da Silva N. et al.Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice.Am J Physiol Renal Physiol. 2007; 293: F1915-F1926Crossref PubMed Scopus (56) Google Scholar Although mRNA transcripts can be isolated from urinary microvesicles, which are devoid of extraneous DNA and RNA, what advantage does this mRNA source have over RNA derived from cells and cell debris in the urine? To examine this, we utilized a ‘whole’ urine RNA extraction kit (see Materials and methods) and compared the RNA profile with that obtained from microvesicles in the same sample. A large amount of nucleic acid could be isolated using the ZR urine RNA isolation kit (Figure 5a), the majority of which was DNA (Figure 5a). The profile appeared broad and lacked 18S and 28S rRNA peaks in comparison with that normally obtained from tissue or from within microvesicles (see Figure 2c), indicating considerable degradation. In contrast, RNA from microvesicles isolated from the same urine sample had clearly visible 18S and 28S rRNA peaks, indicating good quality RNA (Figure 5b). Analysis of the nucleic acid profile of the various pellets obtained during microvesicle differential centrifugation revealed that pellets from the 300 g (Figure 5c) and 17,000 g (Figure 5d) spins exhibited a nucleic acid profile similar to those obtained from the ZR urine RNA isolation kit and were, indeed, made up of a large proportion of DNA (Figure 5c and d, respectively) along with degraded RNA. This showed that the RNA isolated from urinary cells is less stable than the RNA isolated from urinary microvesicles. Although urinary microvesicles seem like a promising source of renal biomarkers, their isolation by ultracentrifugation is both time consuming and requires elaborate equipment. We, therefore, investigated whether filtration concentrators, previously used to isolate urinary microvesicles for protein analysis,13.Cheruvanky A. Zhou H. Pisitkun T. et al.Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.Am J Physiol Renal Physiol. 2007; 292: F1657-F1661Crossref PubMed Scopus (267) Google Scholar could yield viable microvesicles for RNA extraction. To test this, we processed urine samples up to the 0.8-μm filter step (see Materials and methods) and compared them with samples processed using a 100 kDa MWCO filtration concentrator (Millipore, Bedford, MA, USA) versus ultracentrifugation both with RNase digestion, and with and without DNase digestion to remove extra-microvesicular nucleic acid contamination. Results revealed that the ultracentrifugation method and the filtration concentration method yielded similar RNA concentrations from 75 ml urine samples (ultracentrifugation 410±28 pg/μl, filtration concentrator 381±47 pg/μl mean ± s.d., not statistically significant) with minimal degradation (Figure 6a), suggesting that the use of filtration concentrators may be a reliable method for the isolation of urinary microvesicles for RNA analysis. Finally, we examined whether the urine pre-processing steps could be replaced with just a 0.8-μm filtration step (see Materials and methods). Results using ultracentrifugation (Figure 6b) and filtration concentrators (Figure 6c) revealed that the 0.8 μm filtration could indeed replace the urine pre-processing steps, further decreasing the isolation time. Currently, renal biomarkers are limited to urinary protein analysis and changes in the glomerular filtration rate. Biomarkers at the nucleic acid level are understudied, in part because this requires the invasive and expensive procedure of organ biopsy. However, urinary microvesicles now offer a novel means to obtain this information without the need for invasive and expensive biopsy procedures, potentially taking renal biomarker discovery to a new level. Analysis of extraneous nucleic acid contamination during urinary microvesicle isolation revealed that there was the potential for DNA contamination. This could be easily removed by (i) digestion of the microvesicle pellet with DNase or (ii) the use of genomic DNA elimination kits such as the RNeasy Plus Micro kit. External DNase digestion should now be part of the standard procedure when isolating microvesicles for nucleic acid analysis. This also appears to remove DNase-susceptible ‘apoptotic DNA ladder’ like material, which may contaminate serum microvesicle pellets, as shown in the Supplementary Data. The finding that there was no detectable extraneous RNA contamination was not surprising, because urine contains ribonucleases.15.Sugiyama R.H. Blank A. Dekker C.A. Multiple ribonucleases of human urine.Biochemistry. 1981; 20: 2268-2274Crossref PubMed Scopus (29) Google Scholar The fact that microvesicles can resist RNase and DNase digestion and still protect the nucleic acids contained within them is quite remarkable, and adds further support to the previously reported stable nature of urinary exosomes.9.Zhou H. Yuen P.S. Pisitkun T. et al.Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.Kidney Int. 2006; 69: 1471-1476Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar Download .doc (.1 MB) Help with doc files Supplementary Data A potential advantage of carrying out extra-microvesicular rather than on-column DNase digestion is that it leaves the nucleic acids within exosomes untouched. This is important, particularly in cases where potential DNase digestible material may be captured from the cytoplasm of a cell and may itself be a source of biomarkers for non-coding sequences, which are now believed to have a potential role in cell regulation.19.Mattick J.S. Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms.Bioessays. 2003; 25: 930-939Crossref PubMed Scopus (416) Google Scholar, 20.Mattick J.S. Makunin Non-coding RNA.Human Mol Genet. 2006; 15: R17-R29Crossref PubMed Scopus (1563) Google Scholar, 21.Frith M.C. Pheasant M. Mattick J.S. The amazing complexity of the human transcriptome.Eur J Hum Genet. 2005; 13: 894-897Crossref PubMed Scopus (128) Google Scholar Other studies have also suggested that mitochondrial DNA is present in exosomes isolated from astrocytes and glioblastoma cell cultures,22.Guescini M. Genedani S. Stocchi V. et al.Astrocytes and glioblastoma cells release exosomes carrying mtDNA.J Neural Transm. 2010; 117: 1-4Crossref PubMed Scopus (390) Google Scholar suggesting that cancer-related microvesicles may contain mtDNA. Overall, our studies suggest that microvesicles can be a reliable source of living cell cytoplasm-derived nucleic acids for biomarker discovery, and are devoid of extraneous nucleic acids when processed correctly. Although urine-derived microvesicles contain an RNA profile similar to whole tissue including prominent 18S and 28S rRNAs, this rRNA material appears less prevalent in microvesicles isolated from the serum5.Skog J. Würdinger T. van Rijn S. et al.Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3251) Google Scholar, 6.Valadi H. Ekström K. Bossios A. et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (7531) Google Scholar, 7.Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1743) Google Scholar and saliva.23.Palanisamy V. Sharma S. Deshpande A. et al.Nanostructural and transcriptomic analyses of human saliva derived exosomes.PLoS One. 2010; 15: e8577Crossref Scopus (217) Google Scholar It is not known whether this is due to differences in microvesicular; (i) yield, (ii) stability, or (iii) origin (that is, whether the RNA profile may be different in shedding microvesicles versus those derived from MVBs) in the various body fluids. A comparison of RNA sources in urine revealed that rRNA peaks appeared better preserved in microvesicles versus cells in urine, suggesting that microvesicles are a reliable source of stable nucleic acids and that they protect their inner content which is extremely important for downstream RNA analysis. Unlike whole cells, microvesicles are quite resistant to freeze–thawing, and nucleic acids can be extracted from the urinary exosomes following freeze–thawing (Russo et al., unpublished data). This suggests that in frozen archived samples, microvesicles may also be a more reliable source of RNA for longitudinal studies than whole cells from urine. Further, it is not known how release of cells from their physiological setting (that is, loss of cell–cell and cell–substrate interaction, and local stimuli) affects gene expression in whole cells found in urine. Urinary microvesicles may also be considered as a unique source of RNA not only because of their stable and non-invasive nature, but also because their RNA represents a snapshot of the whole urinary system. This is unlike the RNA obtained from renal biopsy, which represents a small sample from only one of the two kidneys. The presence of mRNA transcripts encoding renal genes from various regions of the nephron and the collecting duct was also confirmed. These transcripts were contained within microvesicles and were confirmed as mRNA-derived, because a poly-A tail-specific RNA amplification technique was used. Many of the genes analyzed are disrupted in various renal diseases, including podocin in glomerular diseases such as steroid-resistant nephrotic syndrome,24.Boute N. Gribouval O. Roselli S. et al.NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.Nature Genet. 2000; 24: 349-354Crossref PubMed Scopus (1134) Google Scholar cubilin25.Kozyraki R. Kristiansen M. Silahtaroglu A. et al.The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region.Blood. 1998; 91: 3593-3600Crossref PubMed Google Scholar associated with proteinuria in Imerslund–Gräsbeck syndrome, and aquaporin 2 associated with diabetes insipidus.26.Nielsen S. Chou C.-L. Marples D. et al.Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.Proc Nat Acad Sci. 1995; 92: 1013-1017Crossref PubMed Scopus (844) Google Scholar To further highlight the use of microvesicular RNA analysis in renal pathophysiology, we used both RT-PCR for the mRNA detection of genes as well as real-time PCR for the relative quantitation of renal genes expressed in the V-ATPase B1 knockout mouse. The analysis of mRNA in urinary microvesicles paralleled that obtained in renal tissue from these mice18.Păunescu T.G. Russo L.M. Da Silva N. et al.Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice.Am J Physiol Renal Physiol. 2007; 293: F1915-F1926Crossref PubMed Scopus (56) Google Scholar (and this study) and adds support for the use of urinary microvesicles for pathophysiological analysis. The use of filtration concentrators to isolate urinary microvesicles indicates that elaborate ultracentrifugation steps may not be required for the isolation of microvesicles for nucleic acid analysis. The use of a 100 kDa MWCO membrane aided in the removal of DNase I (∼39 kDa) and RNase A (∼13.7 kDa) from the sample following extraneous nucleic acid digestion steps. This rapid isolation technique, which reduces 70 min centrifugation steps down to 4 min steps (potentially reducing more than 3.5 h of ultracentrifugation to <30 min in a bench-top centrifuge), is extremely important for future studies into biomarker discovery and has the potential to move exosome biology into clinical laboratories as a routine diagnostic procedure. In summary, we have shown that urinary microvesiclesmay (i) be complexed with extraneous DNA, highlighting the importance of DNA removal from the sample before nucleic acid analysis; (ii) contain mainly RNA, including prominent 18S and 28S rRNA similar to that seen in tissue-derived RNA; (iii) contain mRNA transcripts representing markers from all regions of the nephron and the collecting duct, suggesting that the mRNA contained within them is stable for RT-PCR analysis; (iv) contain RNA that is more stable than RNA extracted from whole urine; and (v) be rapidly isolated using filtration concentrators without significant loss of RNA integrity, providing a rapid means to isolate microvesicles without the need for elaborate ultracentrifugation. These findings pave the way for the use of urinary microvesicles as a novel source of new and much needed nucleic acid biomarkers for renal disease. Human urine was obtained under the approved instititional review board guidelines of the Massachusetts General Hospital. Urine pre-processing by the ‘normal method’ included centrifugation of the urine at 300 g for 10 min at 4 °C, centrifugation of the supernatant at 17,000 g for 20 min at 4 °C, and filtration of the supernatant through a 0.8 μm filter (cellulose nitrate membrane filter unit; Nalgene, Rochester, NY, USA). Alternatively, urine pre-processing using the ‘0.8μm method’ included filtration of the urine directly through the 0.8 μm filter without any pre-centrifugation steps. For analysis of extraneous nucleic acids ∼25 ml of duplicate urine samples was used, and ∼75 ml duplicates were used for the analysis of intra-microvesicular nucleic acids. For comparison of the nucleic acid extraction kits ∼75 ml of duplicate urine samples was used. For comparison of the RNA from urinary cells extracted using the ZR isolation kit, 300 and 17,000 g pellets, and the corresponding RNA from microvesicles, ∼75 ml of duplicate urine samples was used. For analysis of filtration concentrators versus ultracentrifugation, ∼75 ml of duplicate urine samples was used. For RT-PCR analysis 200 ml urine samples were used. Urine samples were collected over a 24