BASIC RESEARCH www.jasn.org Human Urinary Exosomes as Innate Immune Effectors † † ‡ Thomas F. Hiemstra,* Philip D. Charles, Tannia Gracia, Svenja S. Hester,§ † ‡ | Laurent Gatto, Rafia Al-Lamki,* R. Andres Floto,* Ya Su, Jeremy N. Skepper, † ‡ Kathryn S. Lilley, and Fiona E. Karet Frankl *Department of Medicine, †Cambridge Centre for Proteome Research and Cambridge Systems Biology Centre, Department of Biochemistry, ‡Department of Medical Genetics, and |Multi-Imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, United Kingdom; and §Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom ABSTRACT Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm–Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract. J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013101066 Exosomes form as intraluminal vesicles of multi- every cell type along the nephron.13,14 The array of vesicular bodies (MVBs), contain membrane and functions ascribed to exosomes in other tissues has cytoplasmic proteins, have a cytoplasmic-side in- kindled recent interest in the functional significance ward membrane orientation, and are released intact of urinary exosomes. Hogan et al.13 suggested inter- into the extracellular space (Figure 1A). First de- action of exosome-like vesicles with primary cilia of scribed in maturing ovine reticulocytes,1 exosomes renal epithelial cells, and Street and coworkers15 are released by many cell types2 and have been con- showed in vitro uptake of exosomes by a renal cortical ventionally regarded as a vehicle for shedding ob- solete protein. However, emerging evidence has revealed a variety of exosomal functions, including Received October 11, 2013. Accepted January 6, 2014. 3 the intercellular transfer of membrane receptors Published online ahead of print. Publication date available at 4–6 7 and RNA, induction of immunity, antigen pre- www.jasn.org. sentation,8 modulation of bone mineralization,9 Correspondence: Prof. Fiona E. Karet Frankl, Department of 10 and antiapoptotic responses. Medical Genetics, University of Cambridge, Cambridge Institute Nanovesicles were first shown in human urine by for Medical Research, Cambridge Biomedical Campus Box 139, Kanno and colleagues11 and subsequently, were Hills Road, Cambridge CB2 0XY, UK, or Prof. Kathryn S. Lilley, Cambridge Centre for Proteomics and Cambridge Systems Bi- 12 shown to represent exosomes. Consistent with a ology Centre, Department of Biochemistry, University of Cambridge, renal tubular epithelial origin, renal tubular epithelial Cambridge CB2 1QR, UK. Email: [email protected] or fek1000@ cells contain MVBs at the apical surface, and urine cam.ac.uk exosomes contain apical membrane proteins from Copyright © 2014 by the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc, 2014 ISSN : 1046-6673/2509-ccc 1 BASIC RESEARCH www.jasn.org Published reports of the urinary exoso- mal proteome have limited value in illumi- nating the potential functions of urinary exosomes for several reasons. First, protein identification by mass spectrometry (MS) has, until recently, yielded results with unacceptably low reproducibility and high false-positive rates,22,23 and previous re- ports are not free of these limitations. Sec- ond, studies have often aimed to maximize the number of protein identifications and hence, biomarker candidates rather than applying or reporting rigorous protein identification thresholds. Third, most have relied on pooled samples from up to six donors and have not reported interin- dividual variability or reproducibility. Here, we sought, for the first time, to ascribe functionality to human urinary exosomes. We initially performed rigorous, conservative tandem MS analysis of sepa- rate human urinary exosomal samples to allow enrichment scoring24 to elucidation of urine exosomal function. RESULTS Exosomal samples were obtained from 10 healthy volunteers (five men and five women) ages 23–36 years. Nine subjects were Caucasian, and one man was Mauri- Figure 1. Vesicles isolated from human urine are consistent with exosomes. (A) tian. Samples prepared from 455628 ml sec- – Exosomes are derived from the endocytic pathway (1 4) forming through invagination ond morning void contained 0.5460.18 mg of the limiting membrane of the MVB (3). They are released into the urinary space from protein/ml urine. Exosomal integrity was renal tubular epithelial cells through fusion of the MVB with the apical plasma mem- fi fi fi con rmed by the electron microscopy brane (4). (B and C) Exosomal isolation was con rmed by the identi cation by negative 6 stain EM of nanovesicles (black arrows; characteristic mean 50-nm size distribution. (D) (EM) demonstration of 54.5 14-nm ves- fi Uromodulin (white streaks in B and dark in C; open arrows in B and C) cofractionated icles (Figure 1, B and D), the identi ca- with exosomes but was confirmed to be extraexosomal (5 nm gold-labeled; white tion of known exosomal markers TSG101, arrows in C). (E) Western blot confirmed the presence of known exosomal constituents enolase-1, CD63, podocin, and aquaporin-2 in vesicle preparations but did not confirm them in precipitated protein from exosome- by Western blot and immunogold EM depleted urine. (F) Immuno-EM with 5 (TSG101 and CD63) or 15 nm (AQP2) gold (Figure 1, E and F), and the confirmation particle-labeled antibodies showed vesicular residency of known exosomal con- of a cytoplasmic side inward membrane ori- stituents (arrows). Vesicles were nonpermeabilized; thus, positive staining with an anti- entation (Figure 1F). CD63 antibody directed against an extracellular epitope indicated the cytoplasmic We applied a number of methodological side inward membrane orientation characteristic of exosomes. EDUP, exosome-depleted approaches to overcome limitations of pre- urine protein; HKM, human kidney membrane; MW, molecular weight; TSG101, tumour susceptibility gene 101. vious studies. (1) Samples were not pooled but analyzed separately. (2)MSwasper- formed without and with a uromodulin ex- collecting duct cell line, leading to speculation that exosomes clusion list25 on a high-sensitivity instrument. (3) Data were may provide intrarenal proximal-to-distal transapical renal tu- analyzed using stringent and novel bioinformatics ap- bular epithelial signaling through RNA transfer. However, most proaches26 (Supplemental Material). (4) All ambiguous pep- studies on urine exosomes to date have focused on biomarker tides were excluded unless matched only to products of a discovery, resulting in the publication of several urine exosome single gene. From 50 mg exosomal protein per subject, we protein compendia.12,13,16–21 identified 601 unique proteins by MS, with a median 2 Journal of the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc,2014 www.jasn.org BASIC RESEARCH 2 2 (interquartile range [IQR]) P value of 1.17310 13 (4.08310 4) suggesting a potential role in the targeting of RNA to exo- (Supplemental Table 1). Importantly, the use of a fixed uromodulin somes,4 or involved in innate immunity and the response to exclusion list25 and a posterior error-derived protein type I infection. Uromodulin was present in all 10 samples, consis- error estimator termed espresso (Supplemental Material) un- tent with previous reports, but confirmed as extraexosomal by masked proteins that would not otherwise have been evident immunogold EM (Figure 1D). All MS data have been depos- (Figure 2A). The complete proteome included the known exo- ited with the ProteomeXchange consortium repository somal markers TSG101, CD14, and CD59. However, 307 (51%) (http://proteomecentral.proteomexchange.org; reference had not previously been identified in exosomes from any source PXD000117). compared with the EXOCARTAdatabase.27 There was only min- Using enrichment scoring (ES),24 we found, in addition to imal overlap with the soluble urinary proteome (Supplemental the expected enrichment for cytoskeletal proteins (ES=10.45) Figure 1). and proteins involved in the endocytic pathway and vesicle As previously reported for exosomes,12 the cellular origin of formation (ES=8.06), a very significant enrichment for pro- the majority of identified proteins was in vesicles or the endo- teins involved in immunity and host defense (ES=3.27, cytic vesicular pathway, cytoplasm, plasma membrane, and P,0.001). The MS-derived type