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Proteomics of Urinary Vesicles Links Plakins andComplement to Polycystic Kidney Disease

Mahdi Salih,* Jeroen A. Demmers,† Karel Bezstarosti,† Wouter N. Leonhard,‡

Monique Losekoot,§ Cees van Kooten,| Ron T. Gansevoort,¶ Dorien J.M. Peters,‡

Robert Zietse,* and Ewout J. Hoorn,* on behalf of the DIPAK Consortium

*Department of Internal Medicine, Division of Nephrology & Transplantation, and †Proteomics Center, ErasmusMedical Center, Rotterdam, The Netherlands; ‡Departments of Human Genetics, §Clinical Genetics, and|Nephrology, Leiden University Medical Center, Leiden, The Netherlands; and ¶Department of Nephrology,University Medical Center Groningen, Groningen, The Netherlands

ABSTRACTNovel therapies in autosomal dominant polycystic kidney disease (ADPKD) signal the need for markers ofdisease progression or response to therapy. This study aimed to identify disease-associated proteins inurinary extracellular vesicles (uEVs), which include exosomes, in patients with ADPKD. We performedquantitative proteomics on uEVs from healthy controls and patients with ADPKDusing a labeled approachand then used a label-free approach with uEVs of different subjects (healthy controls versus patients withADPKD versus patients with non-ADPKD CKD). In both experiments, 30 proteins were consistently moreabundant (by two-fold or greater) in ADPKD-uEVs than in healthy- and CKD-uEVs. Of these proteins, weselected periplakin, envoplakin, villin-1, and complement C3 and C9 for confirmation because they werealso significantly overrepresented in pathway analysis and were previously implicated in ADPKD patho-genesis. Immunoblotting confirmed higher abundances of the selected proteins in uEVs from three in-dependent groups of patients with ADPKD. Whereas uEVs of young patients with ADPKD and preservedkidney function already had higher levels of complement, only uEVs of patients with advanced stages ofADPKD had increased levels of villin-1, periplakin, and envoplakin. Furthermore, all five proteins correlatedpositivelywith total kidney volume. Analysis in kidney tissue frommicewith kidney-specific, tamoxifen-induciblePkd1deletiondemonstrated higher expression inmore severe stages of the disease and correlationwith kidneyweight for eachproteinof interest. In summary, proteomicanalysis of uEVs identifiedplakins andcomplement asdisease-associated proteins in ADPKD. These proteins are new candidates for evaluation as biomarkers ortargets for therapy in ADPKD.

J Am Soc Nephrol 27: ccc–ccc, 2016. doi: 10.1681/ASN.2015090994

Autosomal dominant polycystic kidney disease(ADPKD) is the most common inherited kidneydisease, affecting approximately 4 in 10,000 individu-als.1 It is caused by mutations in the PKD1 or PKD2gene, encoding for polycystin-1 and polycystin-2 pro-teins.2 Both proteins are associated with primary ciliaand are thought to play a role in stretch-activated sig-naling. Loss of function of polycystins results in thedevelopment of fluid-filled cysts, ultimately leadingto disruption of the normal kidney parenchyma. Inthe last decade, urinary extracellular vesicles (uEVs,which also include the so-called exosomes)3 haveemerged as promising markers for kidney disease.4–6

These nanosized vesicles are released by direct shed-ding or by fusion of multivesicular bodies with theplasmamembrane.7 Their content comprises proteins

Received September 8, 2015. Accepted January 19, 2015.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Ewout J. Hoorn, Internal Medicine – Ne-phrology, Erasmus Medical Center, PO Box 2040, Room H-438,3000 CA Rotterdam, The Netherlands. Email: [email protected]

Copyright © 2016 by the American Society of Nephrology

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and nucleic acids, both of which have been explored as bio-markers.5 More specifically, uEVs appear to mirror the cellularmake-up of renal epithelial cells. For example, we previ-ously showed that aldosterone increased the sodium chloridecotransporter in both the kidney and uEVs.8 Twenty percent to60% of renal cysts in ADPKD remain connected with the parentnephron,9,10 so that a substantial portion of uEVs in ADPKDmay be derived from cyst epithelial cells. Studying uEVs inADPKD may address the pathophysiology of the disease be-cause uEVs contain polycystins and interact with primarycilia.11 We therefore hypothesized that studying uEVs in

ADPKD is more advantageous than study-ingwhole urine. Accordingly, the aimsof thisstudy were to (1) compare the proteome ofwhole urine with the proteome of uEVs and(2) identify disease-associated proteins inuEVs from patients with ADPKD.

RESULTS

Characteristics of ParticipantsuEVs were isolated in four groups ofpatients with ADPKD due to a PKD1 mu-tation in order to identify and confirmdisease-associated proteins (Figure 1, Sup-plemental Table 1, and Table 1). (1) In theidentification cohort, we used labeledproteomics to identify proteins withhigher or lower abundance in uEVs of pa-tients with ADPKD. We also analyzed theproteome of whole urine to compare itwith the uEV proteome. (2) In confirma-tion cohort 1, we used label-free proteomics,included different patients with ADPKD,and also included patients with non-ADPKD CKD. Patients with CKD werematched by age, sex, and eGFR, and this

group was used to exclude proteins that may be related toimpaired kidney function in general. The reason to use bothlabeled and label-free proteomics techniques is that the two ap-proaches complement each other in terms of quantitation(labeled) and sensitivity (label-free). (3) In confirmation cohort2, uEVs were isolated and compared with the uEVs of the pa-tients with CKD from the validation cohort. (4) Finally, confir-mation cohort 3 consisted of healthy control subjects, youngpatients with ADPKD who have preserved renal function(CKD stage 1) and those with more progressive disease(CKD stages 2–4).

Figure 1. Sample collection, processing, and analysis in the four study groups. (A andB) Identification and confirmation cohorts used for proteomic analysis. Quantitativeproteomics was performed using dimethyl labeling in the identification cohort andlabel-free methods in confirmation cohort 1 (see Concise Methods). (C and D) Con-firmation cohorts 2 and 3 were used for the immunoblotting analysis. *Patients fromvalidation cohort.

Table 1. Characteristics of patients and healthy persons

Cohort Participants Age (yr) Men (%) CKD Stages eGFR (ml/min per 1.73 m2) HtTKV UAlb

Identification cohort Healthy (n=6) 49.265.1 50 – NM – 2.261.2ADPKD (n=6) 48.765.5 50 2–3 47.564.2 11016346 3.061.4

Confirmation cohort 1 Healthy (n=6) 49.763.5 50 – NM – 0.560.1CKDa (n=6) 48.863.2 50 3 45.763.5 – 8.264.8ADPKD (n=6) 50.363.1 50 2–3 49.864.5 14816223 2.360.4

Confirmation cohort 2 CKD (n=4) 44.362.1 75 3 48.564.6 – 6.566.0ADPKD (n=5) 47.663.3 60 2–3 44.2610.9 12586355 6.062.8

Confirmation cohort 3 Healthy (n=4) 30.060.7 50 – NM – 6.361.8ADPKD (n=6) 27.862.0 50 1 109.864.3 622.3648.2 7.562.0ADPKD (n=11) 43.661.8 45 2–4 45.163.8 14756297.4 2.761.3

The eGFR was calculated by the four-variable Modification of Diet in Renal Disease formula. HtKTV, height-adjusted total kidney volume; UAlb, urinary albumin; –,not applicable; NM, not measured.aCKD due to hypertensive nephropathy in all patients.

2 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2016


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Qualitative Comparison of the Urinary Proteome:Whole Urine versus uEVsFigure 2A shows that 1048 proteins were identified in wholeurine and 1245 proteins were identified in uEVs, of which 527overlapped (see www.proteomexchange.org, identifierPXD003298, for a list of all proteins). The total number ofidentified proteins in urine was therefore 1766. Of interest,although whole urine still contained uEVs, 718 proteins wereidentified only in uEVs. This suggests that isolation of uEVsresults in a different set of proteins not found in whole urine.Figure 2B characterizes how the unique proteins distribute to the

different cellular components. Whole urine showed more cellsurface, plasma membrane, and extracellular proteins, whereasuEVs showed more cytoplasmic, cytoskeletal, endosomal, andmitochondrial proteins. Of the 517 unique proteins in wholeurine, the majority (389 proteins [75%]) had a molecularmass below the cutoff for glomerular filtration (,70 kDa).12

We also performed a qualitative comparison to identify over-represented pathways in whole urine and uEVs of patients withADPKD (Table 2).13 Using the Database for Annotation, Visu-alization, and Integrated Discovery (DAVID) annotation tool(National Institute of Allergy and Infectious Diseases, Bethesda,

Figure 2. Number of identified proteins and their cellular localization. (A) Venn diagram showing that uEVs contained a different setof unique proteins compared with whole urine. (B) Comparison of the cellular components to which the unique proteins identifiedwhole urine (n=521) and uEVs (n=718) belong. For comparison, whole urine is set at a relative abundance of 1. ER, endoplasmicreticulum.

J Am Soc Nephrol 27: ccc–ccc, 2016 Urinary Extracellular Vesicles in ADPKD 3

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MD),14 we identified that significantly over-represented path-ways in ADPKD-uEVs consisted of actin-related processes andimmune system processes, including complement activation.The latter finding was confirmed by an analysis using the KyotoEncyclopedia of Genes and Genomes (KEGG), which also in-dicated that "complement and coagulation cascades" (hsa04610)were over-represented in ADPKD (37 proteins; P=0.01).

Quantitative Proteomics of ADPKD-uEVsWe performed quantitative proteomics in the identificationcohort and confirmation cohort 1 in order to select proteins thatwere consistently higher or lower in uEVs of patients withADPKD and not related to CKD (Figure 1). Table 3 shows the 30proteins with consistently higher abundance in ADPKD-uEVsand the four proteins with lower abundance, including the ratiosfor themean ion intensities (ratioswere not calculated if proteinsin one of the control groups were absent or not very abundant).Among the identified proteins were the actin-modulating pro-tein villin-1, as well as plakins such as envoplakin and periplakin.Complement-related proteins, including complement C3 andC9, were also more abundant in ADPKD-uEVs.

Confirmation of Plakins and Complement in ADPKD-uEVsFive proteins thatweremore abundant in uEVs of patientswithADPKD were selected for confirmation and further charac-terization according to the pathway analysis and their possibleinvolvement in the pathophysiology of ADPKD.15–19 Theseproteins included villin-1, envoplakin, periplakin, C3, andC9 (Table 3). To confirm the quantitative proteomics results,we immunoblotted the five proteins using the same pooleduEV samples as were used for the proteomics studies (Figures1B and 3). Indeed, the abundance of all five proteins washigher for the ADPKD group than for the two other groups.

Similar abundances of CD9 suggested similar number of ves-icles in the three groups.20 The five proteins of interest weresubsequently analyzed in uEVs from a third group of patientswith ADPKD (Figure 1C) and compared with those from theCKD group (Figure 4). Again, this analysis confirmed thehigher abundance of the five selected proteins in ADPKD-uEVs, but now also on an individual basis. Because our iden-tification and confirmation cohorts 1 and 2 consisted of olderpatients who had ADPKD with CKD stages 2–3, we also ana-lyzed our proteins of interest in a third confirmation cohort(Figure 1D) consisting of younger patients with ADPKD andpreserved renal function and additional patients with APDKDwho had CKD stages 2–4 (Figure 5). Because CD9 declined withprogressive CKD, we analyzed the ratio between uEV proteinabundance and CD9. Villin-1, periplakin, and envoplakin wereincreased only in progressive CKD, whereas complement wasalready increased in uEVs from patients with ADPKD who hadpreserved renal function. In addition, all five proteins correlatedwith height-adjusted total kidney volume (Figure 5B).

Characterization of Plakins and Complement in uEVsWe further characterized the five proteins by density-basedfractionation using sucrose (Figure 6). This was done to analyzethe type of vesicles with which the proteins associate, using CD9and CD63 as markers for exosomes20 and NHE3 and AQP2 asmarkers for vesicles derived from the proximal tubule or collect-ing duct, respectively. An additional advantage of density-basedfractionation is that protein complexes and large protein aggre-gates may be coisolated during ultracentrifugation but do notfloat on a sucrose gradient.21 This analysis suggested the presenceof two populations of vesicles. More specifically, periplakin,envoplakin, villin-1, and C9 were detected in CD9- and CD63-positive fractions, suggesting their presence in exosomes.20 All sixproteins were also detected in the fractions representing denser

Table 2. Gene Ontology biologic process terms overrepresented in uEVs and whole urine of patients with ADPKD

Biologic Process GO Numbersa No. of Proteins

Urinary extracellular vesiclesActin filament-based process 0030029, 0030832, 0030036, 0008064, 0030833 60Lymphocyte-mediated immunity 0002449 25Activation of plasma proteins involved in acute inflammatory

response0002541 24

Adaptive immune response 0002250, 0002460 24Complement activation 0006956 23Oxidation reduction 0055114 81Positive regulation of hydrolase activity 0051345 22Regulation of protein polymerization 0032271 22

Whole urineInnate immune response 0045087, 0006955, 0050778, 0002252 107Programmed cell death 0012501, 0016265, 0008219 48Response to extracellular stimulus 0009991, 0031667 29Carbohydrate catabolic process 0016052 28Proteolysis 0006508 93

P#0.05 for all GO-terms as calculated by DAVID annotation tool.14 P#0.05 is considered strongly enriched. GO, Gene Ontology.aSeveral GO terms were combined if processes were similar and directly linked.



vesicles (fractions 2–4). The presence of AQP2 in these denservesicles (mainly in fraction 3) suggests that some of these vesiclesare derived from the collecting duct.22

Increased Plakins and Complement in ADPKD MouseModelsTo analyze whether the proteins of interest were also moreabundant in polycystic kidneys, we used three variants ofkidney-specific-tamoxifen-inducible Pkd1-deletion (iKsp-Pkd1del)mice (Supplemental Table 2).23 Pkd1 inactivation in these mice

was induced at postnatal day (P) 10, 18, or 40, which resultsin distinct PKD phenotypes. The P10 model rapidly developscysts primarily from distal tubules and collecting ducts,24

whereas the P40 model has a much slower progression, withcysts derived primarily from the proximal part of the nephronand to a lesser extent from distal tubules and collecting ducts.25

The P40 mice were euthanized after 117 or 140 days, resultingin a mild (normal blood urea) or severe (elevated blood urea)phenotype. In addition, P18- iKsp-Pkd1del mice make up anadult onset model with cysts from all different tubular

Table 3. Proteins more and less abundant in uEVs of patients with ADPKD

Group/Protein Group 1 (versus Healthy)a Group 2 (versus Healthy)a Group 2 (versus CKD)a

More abundantPlakinsDesmoplakin 4.4 NC NCEnvoplakinb,c 12.7 2.2 NCPeriplakinb 10.8 2.0 NC

ComplementComplement C3b 2.5 13.8 7.4Complement C5 3.9 NC 74.8Complement C4-B 3.1 5.9 4.3Complement C9b 7.5 10.2 5.8Complement factor B 13.7 20.3 8.5Complement C1q subcomponent subunit A 2.0 NC NC

GlycoproteinsProtein tweety homolog 3c 2.2 2.1 2.5Isoform 2 of solute carrier family 22 member 13c 5.4 2.2 N.C.V-set domain-containing T cell activation inhibitor 1 3.1 2.45 3.09Retinoic acid-induced protein 3c 6.6 2.15 3.45Pigment epithelium-derived factor 6.0 5.23 8.27Heparin cofactor 2 2.1 30.32 NCInter-a-trypsin inhibitor heavy chain H1 3.2 NC 21.69

MiscellaneousVillin-1b,c 3.0 2.4 11.8Tyrosine-protein phosphatase nonreceptor type 13c 2.6 2.36 NCSoluble of catechol O-methyltransferasec 2.1 4.10 2.98Protein crumbs homolog 3c 2.6 NC NCAconitate hydratase, mitochondrialc 3.0 3.05 NCApolipoprotein A-IVc 2.6 4.30 2.82Cysteine-rich C-terminal protein 1c 15.8 13.05 NCGlycogen phosphorylase, brain form 4.1 2.03 NCAngiotensinogen 10.5 11.70 9.95Calpain-5c 2.3 2.1 9.0EH domain-containing protein 4 2.6 2.2 15.1g-synuclein 2.2 3.4 NCProthrombin 6.0 3.96 2.82Plasminogen 23.6 4.84 2.77

Less abundantAnnexin A2 0.12 0.41 0.49Contactin-1 0.43 0.26 0.14Syndecan-4c 0.40 0.08 0.19Granulins OS 0.34 0.50 0.23

NC, not calculated; EH, EPS15 homology.aMean ion intensities were compared and are reported as ratios. No ratio was calculated (NC) if the protein was identified in patients with ADPKD but was absent inthe healthy persons or patients with non-ADPKD CKD (or identified at very low levels).bThese proteins were selected for confirmation and further characterization.cThese proteins were not identified in whole urine.




segments (W.N. Leonhard, D.J. Peters, unpublished observa-tions). The abundance of both periplakin and envoplakin wassignificantly higher in the P10, P18, and, to a lesser extent, P40severemodels (Figure 7A). Villin-1 was increased in P18 and P40.Complement C3d (the final breakdown product of activated C3)and C9 were increased in the P18model but decreased in P10. Ofnote, the lower abundances of C3d and C9 may be related to agerather than toADPKD(wild-typemice 4 and5were younger thanthe other wild-type mice but had the same age as the P10 mice)(Supplemental Table 2). No changes in C3 and C9 were foundin the P40 models. Furthermore, we found that protein abun-dance correlated positively with total kidney weight for villin-1,envoplakin, and complement C3d and C9 (Figure 7B).

DISCUSSION

uEVs are increasingly used to identify noninvasive markers ofdisease, including ADPKD. Here, we show higher abundancesof complement-related proteins (C3 and C9) and cytoskeletalproteins (villin-1 and plakins) in ADPKD-uEVs and suggestthat these proteins may be used as disease markers. Comple-ment C3 and C9 increased early in the disease, whereas thecytoskeletal proteins increased with more progressive disease.Proteomic analyses of uEVs should be interpreted criticallybecause many aspects of uEVs remain unclear.5 However, webelieve the strengths of the approach in this study were asfollows: (1) Two complementary proteomics approaches

were used to identify proteins of interest, (2) the proteins ofinterest were higher in four independent groups of patientswith ADPKD, (3) results were compared with non-ADPKDCKD-uEVs to exclude proteins related to kidney function de-cline in general, (4) the proteins of interest correlate to total kid-ney volume, and (5) the proteins of interest were also increased inmouse models of ADPKD.

The identificationof villin-1, plakins, and complement inuEVsofpatientswithADPKDmaybebiologicallyplausible.Villin-1 isanactin-modifying protein involved in cell morphology, actin re-organization, and cellmotility; in the kidney, it ismainly expressedin the brush border of the proximal tubules.26 Polycystin-1 isimplicated in the regulation of actin cytoskeleton organization,migration, and cell adhesion.27 Defects in polycystin-1 results incell-polarity defects28 and aberrant cell growth,29 which may ex-plain the increase of villin-1.

The desmosomal plaque consists of several transmembraneproteins belonging to the cadherin family (also called plakins30).Desmosomes form an adhesive junction at the basolateral mem-brane and are vital for stabilizing the epithelial sheet. Polycystin-1is associated with desmosomal proteins and is required for theestablishment of cell polarity.19,31 In ADPKD, polycystin-1 nolonger colocalizes with desmosomes, leading to mispolarizationof desmosomal proteins from the basolateral side to the apicaldomain.17 Thismay explainwhy we found higher abundances ofplakins in uEVs of patients with ADPKD.

Complement activation has previously been implicated inboth autosomal recessive polycystic kidney disease andADPKD.15,16,18 Gene expression analysis in cpk mice, a modelfor polycystic kidney disease, identified the innate immune re-sponse to be highly activated, specifically complement factorssuch asC3.16 In humans, cyst epithelial cells produce complementcomponents, including C3 and C9, as shown in immunohisto-chemical staining.15,18 Many of the complement components areabundantly present in human ADPKD kidney cyst fluid.32,33 Fur-thermore, inhibition of the complement system by rosmarinicacid in Pkd12/2 mice and Han:SPRD Cy/+ rats reduced cystgrowth,18 suggesting that the complement pathway is involvedin the pathogenesis of ADPKD. We identified nearly all comple-ment proteins in uEVs, both from the classic (C1q, C2, and C4)and alternative (complement factors B and D) pathways, alongwith inhibitors of this system (plasma protease C1 inhibitor, C4-binding protein, and complement factor B). Complement C9colocalized with the exosomal markers CD9 and CD63, suggest-ing its presence in exosomes. However, complement C3 wasmainly identified in a denser fraction, as was previously demon-strated.34Other studies also reported the presence of complementin uEVs34–36 and circulating EVs.37,38

It is unclear why complement is isolated in ultracentrifugedurine and whether complement is physically associated withuEVs. In theory, complement may be filtered from plasma andend up nonspecifically attached to uEVs. However, mostcomplementproteins are large (themajority exceeding70kDa)and therefore unlikely to be filtered. A more plausible expla-nation is the local production and excretion of complement by

Figure 3. Immunoblot confirmation of five candidate proteins.Immunoblot analysis of the five selected proteins using the samepooled urine as was used for quantitative proteomics in the val-idation group. The first three rows show results for isolated uEVs(pellet), while the last three rows show results for the supernatant(SN), which was used as negative control. Anti-complement C3antibody recognizes the C3 a chain (aC3) and its split product,iC3b. H, healthy persons; PKD, polycystic kidney disease.




renal epithelial cells,39 possibly to opsonize pathogens as a defensemechanism for urinary tract infections.40,41 The higher abun-dance of components of the complement system in uEVs ofpatients with ADPKDmay reflect increased production by renalcyst epithelial cells.

Recently, Hogan et al. also studied uEVsas a source of biomarkers for ADPKD.42 Inpatients with a PKD1 mutation, they foundpolycystin-1 and polycystin-2 to be decreasedand transmembraneprotein 2 (TMEM2) tobeincreased. The ratios between the twopolycystins and TMEM2 allowed differentia-tion between patients with ADPKD andhealthy persons. In our study, polycystinswerenot identified inourfirstquantitativepro-teomics experiment (identification cohort)and thereforewerenot selected for furthercon-firmation. In the second analysis (confirma-tion cohort 1), polycystin-2 was identifiedand was 83% lower in patients with ADPKDthan in healthy persons, but it was not identi-fied in the CKD group. Polycystin-1 was iden-tified only in the control group, and TMEM2was not identified at all.

We propose the following explanations forthese differences. First, to analyze the completespectrum of vesicles, we did not fractionateuEVs,whereasHogan et al. isolatedpolycystin-positive uEVs.42 This difference in isolationmethods may explain why polycystin-1 andpolycystin-2 were five- to seven-fold moreabundant in the study by Hogan et al.42 Sec-ond, our patients with ADPKD had a lowereGFR. In addition to the mutation, the de-creasing eGFR may explain why we observeda larger decrease in polycystin-2 and failed toidentify polycystin-1 in patients with ADPKD.

The reason to focusonuEVs is illustratedby the comparison of the urinary proteomeof whole urine with that of uEVs (Figure 2).The proteins identified in whole urine bymass spectrometry primarily consisted ofextracellular proteins with low molecularweight and were therefore most likelyplasma derived.12 Although whole urinestill contained uEVs, the isolation of uEVsyielded a set of 718 unique proteins thatwere primarily of intracellular origin.These differences underscore that the iso-lation of uEVs increases the identificationrate of low-abundant proteins that arelikely derived from renal epithelial cells.These proteins were not identified in wholeurine, probably because they were maskedby more abundant plasma-derived pro-

teins. To our knowledge, only one study has compared thesetwo proteomes, using less sensitive mass spectrometers (,100identified proteins in each fraction).43 Gradient fractionationshowed that all proteins were present in CD9+ and CD63+vesicles, compatible with exosomes, but also revealed their

Figure 4. Immunoblot analysis of proteins of interest in confirmation cohort 2. Immu-noblot analysis comparing the proteins of interest between individual patients with CKDand ADPKD. uEVs were isolated from individual spot urine samples of patients with CKDand ADPKD (confirmation cohort 2). Anti-complement C3 antibody recognizes the C3 a

chain (aC3) and its split product, iC3b. Error bars, SEM. *P,0.05.



presence in denser vesicles. These might be ectosomes from theglomerulus,34 but electron microscopy would be necessary toconfirm this. The distinction in vesicle subtype is relevant be-cause polycystin 1 and 2 are excreted in exosomes. This impliesthat selectively isolating CD9, CD63, or polycystin-positive ves-iclesmay be a useful strategy tomore specifically analyze disease-associated proteins.42

Our study has a number of limitations. Pooled urine was usedfor proteomics analysis to increase homogeneity, but this didnot allow statistical analysis of the proteomics results. This wasaddressed by analyzing the candidate proteins in individualpatients from a separate ADPDK group (Figures 4 and 5).

Although the inclusion of two proteomics studies reducedthe list of candidate proteins, we believe this approach ex-cluded proteins related to intersubject variation and non-ADPKD CKD. It is unlikely that uEV-markers will be usedas a diagnostic tool in ADPKD because the diagnosis can usu-ally be established by family history, ultrasonography, or com-puted tomography.2 Therefore, to be clinically useful, uEVmarkers should correlate with disease progression or responseto therapy. The proteins identified in this study should thereforebe considered candidate markers and require further evaluationin larger prospective studies using serial urine samples.44 Unlikethe complement proteins, villin-1 and the plakins appear

Figure 5. Immunoblot and total kidney volume analysis of proteins of interest in confirmation cohort 3. (A) Immunoblot analysiscomparing the proteins of interest in uEVs from individual healthy persons, young patients with ADPKD and preserved renal function,and patients with ADPKD and CKD stages 2–4. (B) Correlations of abundance of the uEV proteins of interest compared with height-adjusted total kidney volume (HtTKV). Spearman Rho S and P values are shown. Error bars, SEM. *P,0.05.



unsuitable for early monitoring of ADPKD. They may be eval-uated in more advanced stages of ADPKD, for example, for mon-itoring therapeutic response. Importantly, kidney injury unrelatedto ADPKDmay also increase complement in uEVs, although sev-eral studies have indicated that complement may play a more spe-cific role in the disease.15,18 These aspects should be considered infuture evaluation of the candidate proteins identified in this study.

In conclusion, we have demonstrated the advantage of uEVs toenrich the urinary proteome. We explored uEVs as potentialbiomarkers for ADPKDand identified several classes of proteins tobe specifically increased, including plakins and components of thecomplementsystem.Thesefindingswarrantfurther investigationoftheseproteinsaspotentialbiomarkers ina largerprospectivecohort.

CONCISE METHODS

Participants and Isolation of uEVsTheMedical Ethics Committee of the ErasmusMedical Center approved

this study (MEC-2012–313 andMEC-2013–370). Patients with ADPKD

were recruited from the ongoing Developing Interventions to Halt

Progression of ADPKD (DIPAK) 1 and DIPAK observational studies,

which include patients with ADPKD who have preserved renal function

as well as those with CKD stages 2–4.45 Genetic analysis was performed

for all patients. To increase homogeneity,we includedonly patientswith a

confirmed PKD1mutation (Supplemental Table 1). Two patients with a

negative PKD2 mutation and an ADPKD phenotype were also consid-

ered to have aPKD1mutation.46Each individual patientwasmatched for

age and sex to a healthy control (identification cohort) or matched for

age, sex, and eGFR to a patientwithCKD(confirmation cohorts 1 and2).

Additional inclusion criteria for the patients with CKDwere the absence

of ADPKD and minimal proteinuria (,1 g/10 mmol creatinine).

Previously, we successfully used spot urines for uEV analysis

(normalized by urinary creatinine) and prefer this over 24-hour urine

to limit protein degradation and the risk of incomplete collections.8

Therefore, second-morning spot urines were collected, a protease

inhibitor (cOmplete, Roche Diagnostics, Indianapolis, IN) was

added, and samples were immediately stored at 280°C until further

processing. uEVs were isolated using high-speed centrifugation and

ultracentrifugation (see Supplemental Data for complete protocol).8

Our protocol differed froma recent uEV study inADPKD,42which used

density gradient fraction to enrich for PKD-positive uEVs. We did not

Figure 5. Continued.





use this approach because we were interested in all uEVs. A recent study

compared different uEV isolation protocols and concluded they were

comparable.47 Dithiothreitol was used in our protocol because this dis-

rupts uromodulin,whichmay entrapuEVs,20 although itmay also cause

loss of certain uEV proteins. Obtained pellets were processed for mass

spectrometry or solubilized in Laemmli buffer for immunoblot analysis.

Supplemental Figure 2 shows a representative SDS-PAGE gel for three

uEV samples stained with Coomassie blue.

Mass SpectrometrySampleswereprepared formass spectrometry as describedpreviously.

Briefly, uEVs and acetone-precipitated whole urine were lysed and

trypsinized. Tryptic peptides were fractionated

by hydrophilic interaction liquid chromatogra-

phy, and each fraction was then analyzed by

liquid chromatography/mass spectrometry

(MS). For quantitative dimethyl labeling of

uEVs (identification cohort), desalting and re-

ductive dimethylation were performed on the

solid-phase extraction cartridge (as described else-

where48) before peptide fractionation by hydro-

philic interaction liquid chromatography. All

liquid chromatography/tandem MS (MS/MS)

analyses were performed on a Q Exactive mass

spectrometer (Thermo Fisher Scientific, Rockford,

IL). Each data collection cycle in the Q Exactive

consisted of one full MS scan (300–1750 m/z) fol-

lowed by 15 data-dependent tandem MS scans.

The proteomics data have been submitted to the

ProteomeXchange Consortium via the PRIDE

PRoteomics IDEntifications partner repository

with the dataset identifier PXD003298.49

BioinformaticsRaw data files were processed and analyzed using

Proteome Discoverer 1.4 (Thermo Fisher Scien-

tific) or MaxQuant.50 MS/MS spectra were

searched against theUniprot database (taxonomy:

Homo sapiens) with the following search param-

eters: 15-ppm precursor ion tolerance and 0.02-

Da fragment ion tolerance, fully tryptic digestion,

up to two missed cleavages allowed, posttransla-

tional static modifications of 57.02146 Da on

cysteine (carbamidomethyl), and dynamic modi-

fications of 15.99491 Da on methionine (oxida-

tion). For dimethylation-labeled uEV samples,

28.031 Da on lysine and the peptide N-terminus

(light) and 32.056 Da on lysine and the peptide

N-terminus (heavy) were added to the search pa-

rameters. The resulting data were analyzed using

the DAVID bioinformatics tool to determine

which Gene Ontology terms are overrepresented

relative to the complete set of identified proteins.

For this analysis, we excluded pathways that were

similarly enriched in healthy persons or the CKD

group. Gene Ontology terms were retrieved by the software tool for

rapid annotation of proteins (STRAP, version 1.5.0.0).51

Immunoblotting and Sucrose Gradient FractionationThe solubilized uEVpellet was preheated at 60°C for 15minutes. SDS-

PAGE was carried out on a 4%–20% gradient gel, and proteins were

transferred to Trans-Blot Turbo (Bio-Rad, Hercules, CA). The mem-

branes were blocked in 5% milk and were probed overnight at 4°C

with the antibody of choice. The following antibodies were used:

envoplakin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA),

periplakin (1:2000; Abcam, Inc., Cambridge, MA), complement

C3 (1:1000; Abcam, Inc.), complement C9 (1:1000; Abcam, Inc.),

Figure 6. Sucrose gradient fractionation. Sucrose gradient fractionation was per-formed to analyze the type of vesicles with which the identified proteins associate.Fraction 1 represents the most dense fraction. In addition to the six proteins of interest,we also analyzed CD9 and CD63 (markers for urinary exosomes) and NHE3 and AQP2(markers for proximal tubule and collecting duct). "Pellet" refers to a part of the pooledultracentrifugation pellet used for direct immunoblotting (positive control). Anti-complement C3 antibody recognizes the C3 a chain (aC3) and its split product, iC3b.




villin-1 (anti-human: 1:10,000 [Abcam, Inc.]; anti-mouse: 1:1000

[Cell Signaling Technology, Danvers, MA]), CD9 (1:200; Santa

Cruz Biotechnology) and CD63 (1:500; BD Biosciences, San Jose,

CA), NHE3 (1:10,000; Stressmarq), and AQP2 (1:1000; Stressmarq).

The antibodies against mouse C3dg, C3d, and C9 were generated by

one of the investigators (C.V.K.).52 After three washes (3310minutes

in TBS-Tween 20), membranes were incubated with the secondary

antibody in 5% milk (1:3000; Thermo Fisher Scientific) for 1 hour

and washed again. For visualization, blots were exposed to Pierce

enhanced chemiluminescent substrate and measured by Uvitec Alli-

ance 2.7 (Cambridge, United Kingdom). Chemiluminescence was

quantified using ImageQuant TL (Life Sciences, version 8.1),

Figure 7. Immunoblot analysis of proteins of interest in mouse model of ADPKD. (A) Immunoblot analysis comparing the proteins ofinterest in kidney homogenates of three inducible ADPKD mouse models. "P" indicates the postnatal day at which the Pkd1 gene wasinactivated with tamoxifen. The P40 mice were euthanized at two different time points, producing a mild (normal blood urea) or severe(elevated blood urea) phenotype. Each ADPKD group was compared with wild type. (B) Correlations between kidney weights andkidney abundances of villin-1, envoplakin, periplakin, and complements C3d and C9. Spearman Rho and P values are shown. *P,0.05.2KW/body wt, 2 kidney weight–to–body weight ratio.



backgroundwas subtracted, and ratiosweremeasured inExcel (Microsoft

Corp., Redmond, WA). For the sucrose gradient fractionation, we used

spot urine samples from four patients with ADPKD. After pooling the

samples and obtaining the uEV pellets, we ultracentrifuged uEVs over-

night on top of a 2.5–0.25-M sucrose gradient, corresponding to a density

of 1.32–1.03 g/m3. Each fraction was carefully removed, diluted in PBS,

and ultracentrifuged again to obtain the pellet. Fractions were solubilized

in Laemmli buffer for immunoblot analysis.

Mouse Models of ADPKDThe local animal experimental committee of the Leiden University

Medical Center and the Commission Biotechnology in Animals of the

DutchMinistry of Agriculture approved the animal experiments. The

generation of the iKsp-Pkd1del mice and tamoxifen administration to

these mice were described previously.23 On 3 consecutive days the

mice received a tamoxifen dosage of 6 mg/kg at P10–12, 150 mg/kg at

P18–20, or 200 mg/kg at P40-P42 (Supplemental Table 2). The P10

mice were euthanized at 33 days of age, at which point the mice have

relatively severe PKD. The P18 and the P40 mice were euthanized at

the onset of renal failure (defined as a blood urea level. 20 mmol/L,

as assessed by Reflotron technology; Kerkhof Medical Service). An

additional time point with mild PKD of the P40 mice (11 weeks after

tamoxifen) was included. Protein extraction from these kidneys was

performed as described previously.25

Statistical AnalysesImmunoblotting results were analyzed by t test or Mann–Whitney U

test, as appropriate. A P value #0.05 was considered to represent a

statistically significant difference. Gene ontology enrichment was cal-

culated by the DAVID bioinformatic tool, which applies the Fisher

exact test (P#0.05 is considered strongly enriched).

ACKNOWLEDGMENTS

WethankStevenA. vanderSchoot andNgaisahKlar for their technical

assistance.

E.J.H. is supported by grants from the Netherlands Organisation

for ScientificResearch (NWO,Veni 916.12.140) and theDutchKidney

Foundation (KSP-14OK19).

Figure 7. Continued.




TheDIPAKConsortium is an inter-university collaboration in The

Netherlands that is established to study ADPKD and to develop ra-

tional treatment strategies for this disease. The DIPAKConsortium is

sponsored by the Dutch Kidney Foundation (grant CP10.12). Prin-

cipal investigatorsare (inalphabeticalorder): J.P.H.Drenth(Department

of Gastroenterology and Hepatology, Radboudumc Nijmegen), J.W. de

Fijter (Department of Nephrology, Leiden University Medicla

Center [UMC]), R.T. Gansevoort (Department of Nephrology, UMC

Groningen), M. Losekoot (Department of Human Genetics, Leiden

UMC),E.Meijer (DepartmentofNephrology,UMCGroningen),D.J.M.

Peters (Department of Human Genetics, Leiden UMC), F.W. Visser

(Department ofNephrology, UMCGroningen), J.Wetzels (Department

ofNephrology,RadboudUMCNijmegen),andR.Zietse (Departmentof

Internal Medicine, Erasmus MC Rotterdam).

This work is also part of the project Proteins atWork, a program of

The Netherlands Proteomics Centre financed by The Netherlands

Organization for Scientific Research as part of the National Roadmap

Large-Scale Research Facilities of The Netherlands (project number

184.032.201).

DISCLOSURESNone.

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