Characterization of PKD Protein-Positive Exosome-Like Vesicles
Marie C. Hogan*,
Luca Manganelli*,,
John R. Woollard*,
Anatoliy I. Masyuk,
Tatyana V. Masyuk,
Rachaneekorn Tammachote*,
Bing Q. Huang,
Alexey A. Leontovich,
Thomas G. Beito||,
Benjamin J. Madden¶,
M. Cristine Charlesworth¶,
Vicente E. Torres*,
Nicholas F. LaRusso,
Peter C. Harris* and
Christopher J. Ward*
* Division of Nephrology and Hypertension, Center for Basic Research in Digestive Diseases, Bioinformatics Core Facility, Division of Biomedical Informatics, || Mayo Ab Core, and ¶ Mayo Proteomics Core, Mayo Clinic, Rochester, Minnesota; and Department of Nephrology, University of Modena and Reggio Emilia, Modena, Italy
Correspondence: Dr. Christopher J. Ward, Division of Nephrology & Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: 507-266-3050; Fax: 507-266-9315; E-mail: ward.christopher{at}mayo.edu
Received for publication June 2, 2008.
Accepted for publication September 5, 2008.
Proteins associated with autosomal dominant and autosomal recessivepolycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin)localize to various subcellular compartments, but their functionalsite is thought to be on primary cilia. PC1+ vesicles surroundcilia in Pkhd1del2/del2 mice, which led us to analyze thesestructures in detail. We subfractionated urinary exosome-likevesicles (ELVs) and isolated a subpopulation abundant in polycystin-1,fibrocystin (in their cleaved forms), and polycystin-2. Thisremoved Tamm-Horsfall protein, the major contaminant, and subfractionatedELVs into at least three different populations, demarcated bythe presence of aquaporin-2, polycystin-1, and podocin. Proteomicanalysis of PKD ELVs identified 552 proteins (232 not yet inurinary proteomic databases), many of which have been implicatedin signaling, including the molecule Smoothened. We also detectedtwo other protein products of genes involved in cystic disease:Cystin, the product of the mouse cpk locus, and ADP-ribosylationfactor-like 6, the product of the human Bardet-Biedl syndromegene (BBS3). Our proteomic analysis confirmed that cleavageof polycystin-1 and fibrocystin occurs in vivo, in manners consistentwith cleavage at the GPS site in polycystin-1 and the proproteinconvertase site in fibrocystin. In vitro, these PKD ELVs preferentiallyinteracted with primary cilia of kidney and biliary epithelialcells in a rapid and highly specific manner. These data suggestthat PKD proteins are shed in membrane particles in the urine,and these particles interact with primary cilia.
Autosomal dominant polycystic kidney disease (ADPKD) is themost common hereditary renal disease, affecting between 1:400to 1:1000 individuals.1,2 There are two genetic loci, PKD1 andPKD2, producing the proteins polycystin-1 (PC1)3–5 andpolycystin-2 (PC2),6 respectively. Autosomal recessive polycystickidney disease (ARPKD), the most common cause of hereditarychildhood PKD, is caused by mutations to PKHD1, which encodesfibrocystin/polyductin (FCP).7,8 These three PKD proteins havebeen localized to primary cilia,9,10 where the PC1/PC2 complexacts as a flow sensor on the cilium.11 The role of FCP is lessclear, but it complexes with PC2.12,13
Another site of PC1 expression is in urinary exosomes, smallvesicles (50 to 100 nm in diameter) present in normal urine,that have been analyzed as a source of biomarkers for variousrenal diseases.14,15 Urinary exosomes are thought to be endproducts of the multivesicular body (MVB)-sorting pathway inwhich membrane proteins are uniquely packaged into intraluminalvesicles (ILVs) within the MVB, some of which are secreted asexosomes when MVBs fuse with the apical plasma membrane.
MVBs and exosomes have been shown to have a role in left/right(L/R) axis determination in the embryonic node. These MVBs,termed nodal vesicular parcels (NVPs), are released from thefloor of the node and swept by nodal flow to the left side,where they interact with the "picket-fence" immotile cilia.16Symmetry breaking is dependent on a PC2 Ca2+-dependent flux.17,18
Transmission electron microscopy studies of dilated bile ductsfound in ARPKD mouse model Pkhd1del2/del2 showed PC1+ exosome-likevesicles surrounding cholangiocyte primary cilia, whereas onlyoccasional single ELVs were found attached to WT cilia.19 Theobservations of abundant PC1 in ELVs and of abnormal ELV accumulationin FCP-deficient mice led us to examine whether these may havea functional role in the urinary and biliary systems, analogousto the NVP in the node.
To determine which PKD proteins are present in urinary ELVsand their relative size with respect to recombinant protein,we prepared a crude preparation. Western analysis compared thisELV preparation with exogenously expressed full-length proteinsin PEAK cells (human embryo kidney cells) using antibodies tothe LRR region of PC1 (7e12),20 the N-terminus of FCP and tothe C-terminus of PC2.21 Strong signals were detected usingjust 2 µg of total ELV protein (compared with 10 to 50µg of kidney membrane preparation used in previous studiesof renal cells to detect PC120), with the product sizes consistentwith the predicted and recombinant glycosylated molecular weightof two of the proteins: PC2, 130 kD; and FCP, 500 to 550 kD(Figure 1, B and C). However, the PC1 in ELVs was appreciablylarger than the recombinant PC1 (Figure 1A). To confirm thatPC1 was specifically detected, we deglycosylated both ELV andrecombinant PC1. In this case, both the recombinant and ELVPC1 co-migrated at approximately 340 kD (predicted 325 kD),confirming identity and showing that ELV PC1 has extensive N-linkedglycosylation.
Figure 1. Western analysis shows urinary ELVs are enriched for PC1, PC2, and FCP. (A) Comparison of ELV (Ex) (lane 1) and exogenously expressed PC1 (rPC1; 5 µg membrane protein (lane 2) and after PNGase treatment (lanes 3 and 4), detected with PC1 mAb (7e12). ELV PC1 seems to be heavily glycosylated and similar in size to unGPS cleaved PC1 (550 kD), but after deglycosylation is similar in size (340 kD) to the GPS cleaved rPC1. (B) Detection of PC2 in ELVs (Ex; lane 5) and rPC2 (lane 6) with the PC2 antisera YCC2 shows a 130-kD product in both—a probable dimer is also seen with exogenously expressed PC2 (lane 6). (C) A similar sized product (550 kD) is detected in ELVs (lane 7) and rFCP (lane 8) with the FCP mAb11.
Purification of PKD-ELVs
To obtain significant quantities of urinary ELVs, we developeda scaled-up modification of the procedures of Pisitkun et al.14and Zhou et al.15 The material generated by this protocol isrich in the most abundant urinary protein, Tamm-Horsfall protein(THP; also known as uromodulin). Electron microscopic studiesshowed that THP formed long higher order fibrils that were contaminatingthe exosomal preparations (Figure 2D). Initially, we attemptedto remove the THP using sucrose gradients in normal water, butthe THP and PKD-ELVs co-banded. We therefore increased the densityof the gradient using heavy water. The crude ELVs were fractionatedby density ultracentrifugation on a D2O 5 to 30% sucrose gradient(Figure 2, A through D). Western analysis of fractions fromthese gradients showed that the bulk of the THP pelleted andthat fractions 8 and 9 were enriched for PC1. The peak refractiveindex for the PKD-ELVs was = 1.3530 (SD ± 0.0019; n= 6), representing a density of approximately 1.17 kg/L in D2O.By using this gradient, we were able to remove >99% of theTHP from the ELVs and to isolate 100 µg of PC1-enrichedELVs per liter of urine, <1% of the initial protein in thecrude ELV preparation (Figure 3, A and B). The stem cell markerCD133 (prominin) also co-sediments with PC1, PC2, and FCP. Thiscontrasted with other proteins previously detected in urinaryELVs that were found in different fractions: Aquaporin 2, infractions 1 through 5, and the steroid-resistant nephrotic syndromeprotein, podocin (NPHS2), that was enriched in fractions 10through 14 (Supplemental Figure 1).
Figure 2. Purification of human urinary ELVs on a 5 to 30% sucrose gradient in D2O. (A) Refractive index profile (in red) ranging linearly from = 1.33 to 1.37 and protein concentration of the fractions (in blue) in µg/ml. Most of the protein is in the first three and last two fractions. (B) Distribution of PC1, PC2, FCP, and CD133, which co-band in fractions 7, 8, and 9. THP is present in the pellet and the last three fractions. (C) SEM of PKD-ELVs bound to poly-l-lysine coverslips from fraction 9 (bar = 1 µm) and (D) ELVs and fibrils of THP from fraction 12 (same scale as in C). (E through I) ImmunoEM shows that PC1, PC2, FCP, and CD133 are co-localized on ELVs. (E) Control (no primary antibody) on negatively stained ELVs. (F) PC2 (10 nm gold) and CD133 (5 nm gold), (G) PC2 (10 nm gold) and PC1 (5 nm gold), (H) PC2 (10 nm gold) and FCP (5 nm gold), and (I) a large multilaminate ELV, PC2 (10 nm gold), and FCP (5 nm gold) suggesting a MVB origin for PKD-ELVs.
Figure 3. Coomassie stained 4 to 12% SDS-PAGE gels of urinary ELVs. (A) A total of 30 µg of crude ELVs prepared by ultracentrifuging urine at 150,000 x g for 1 h. Note the large amount of THP centered at approximately 85 kD. (B) A total of 30 µg of pure PKD-ELVs; note the lack of a prominent THP band. (C) A total of 30 µg of pure PKD-ELVs that have been deglycosylated with PNGase F; note the prominent PNGase F band at 36 kD. The 35 gel slices selected for the proteomic analysis are shown.
The morphology of the PKD-ELVs was similar to a "deflated football"with concave and convex sides. Diameters ranged from 66.1 to187.0 nm, with a mean of 107 nm (SD ±22.1; n = 56; Figure 2,E through I), overlapping in range with the measurements reportedby Pisitkun et al.14 Immunoelectron microscopic analysis ofthe PKD-ELVs showed that PC1, FCP, and CD133 all co-localizewith PC2.
Proteomic Analysis of the PKD-ELVs
To characterize the full range of proteins in the PKD-ELVs,we undertook a proteomic analysis of this fraction. Becausemany ELV proteins are extensively glycosylated, we analyzedELVs with and without peptide:N-glycosidase F treatment to removeN-linked sugars. ELVs were prepared from four healthy adults(two male and two female), and the PC1+ peak fractions werepooled. These were then run on a 4 to 12% PAGE gel that wassectioned into 35 separate slices (Figure 3, B and C). To enrichfor genuine PKD-ELV proteins, we analyzed in detail only thosewith at least three unique peptides and 10% coverage in bothglycosylated and unglycosylated samples, a total of 552 proteins(Supplemental Database 1).
Presence of PC1, PC2, and FCP and Interacting Partners
Pisitkun et al.14 showed that PC1 was present in a relativelycrude preparation of urinary exosomes by both proteomics andWestern blotting. We focused on a subfraction of ELVs in theurine and were able to show that PC2 and FCP are also present,because the PKD-ELVs are more concentrated, permitting detailedanalysis of their contents by mass spectrometry. Ranking proteinsby the total number of unique peptides (in the deglycosylateddata set), FCP (PKHD1_HUMAN; 80 peptides, coverage 30%) is oneof the most highly represented, ranked second; PC1 (PKD1_HUMAN;42 peptides, coverage 13%), also ranked highly at 15th, andPC2 (PKD2_HUMAN; 21 peptides, coverage 25%) ranked 90th. Thisreinforces that they are abundant in ELVs; however, when thisproteome is ranked as a function of the percentage protein coverageby peptides, FCP is 376th, PC2 is 416th, and PC1 is 528th (Table 1).
Table 1. PC1-interacting proteins found in PKD-ELVsa
We surveyed the literature for described PC1-, PC2-, and FCP-interactingproteins defined by yeast two-hybrid and other analysis andranked these by percentage coverage by unique peptides (Table 1).The PC2-interacting proteins CD2AP (CD2AP_HUMAN; rank 126, 39peptides, 60% coverage)22 and -actinin-1 (ACTN1_HUMAN; rank511, five peptides, 15% coverage)23 were present. The Na/K-transportingATPase -1 chain precursor (EC 3.6.3.9; AT1A1_HUMAN; rank 226,46 peptides, 46% coverage), a described PC1-interacting protein,was also present.24 G(i)1 subunit (GNAI1_HUMAN; rank 54, 11peptides, 71% coverage), G(i)2 subunit (GNAI2_HUMAN; rank 81,19 peptides, 67% coverage), and G(q) subunit (GNAQ_HUMAN; rank187, 8 peptides, 52% coverage) were also observed (Table 1).These subunits have been shown to interact with 74 amino acids(aa) in the cytoplasmic C-terminal tail of mouse PC1.25,26
In Vivo Cleavage of PC1 and FCP Assessed by Peptide Distribution
Both FCP and PC1 are thought to be cleaved within their extracellulardomains,27–29 but many of these data were derived fromoverexpressing full-length cDNAs in cell lines. Proteomic analysisof the PKD-ELVs allowed assessment of cleavage of the in vivoproducts without the need for antibodies to different regionsof the proteins. Cleavage data for PC1 and FCP were derivedfrom the distribution of peptides on the gel in the glycosylatedrun (the deglycosylated run had some smearing probably as aresult of incomplete deglycosylation; Figure 3). A total of32 peptides to PC1 were in gel slice 4 (approximately 500 kD);these peptides all were N-terminal to the G protein–coupledreceptor proteolytic site (GPS) site at aa3048; the most C-terminalpeptide ended at aa2865 (Figure 4A, Supplemental Figure 2).There was one peptide in gel slice 8 and four in gel slice 10(approximately 150 kD; Figure 3B), all of which mapped C-terminalto the GPS site, the most N-terminal peptide started at aa3190(Figure 4A, Supplemental Figure 3). Hence, these data are consistentwith a GPS cleavage event at 3048..3049,28,29 an N-terminalfragment of approximately 500 kD and a C-terminal fragment ofapproximately 150 kD with a cleavage point occurring betweenaa2865 and aa3190. No other slices contained PC1 peptides.
Figure 4. Cleavage of PC1 and FCP and comparison of PKD-ELV proteome with other urinary proteomes. (A) The PC1 protein with the GPS cleavage site at aa 3048..3049. Peptides found in gel slice 4 (approximately 500 to 550 kD) are represented by black bars; peptides found in gel slices 8 and 10 (approximately 150 kD) are represented by red bars (Supplemental Figure 2). (B) The FCP protein with the proprotein convertase consensus at 3615..3620. Peptides found in gel slices 3 to 4 (approximately 550 kD) are represented by black bars, whereas the two peptides seen in gel slice 18 (approximately 55 to 60 kD) are represented by red bars (Supplemental Figure 3). No other gel slices yielded peptides from PC1 and FCP in the glycosylated run. (C) Comparison Venn diagram of peptides found in a total urine proteome (Adachi),14 crude urinary ELVs (Pisitkun),33 with PKD-ELVs (our data, Hogan). Numbers represent the number of shared proteins in the respective overlapping circles. The individual sets of proteins are available in Supplemental Database 2.
In FCP, we identified 41 peptides in the glycosylated run. Inslices 3 and 4 (approximately 500 to 550 kD), there were 39peptides, the farthest C-terminal ending at aa3609. In gel slice18 (approximately 55 to 60 kD), there were two peptides, themost N-terminal ending starting at aa3661 (Figure 4B, SupplementalFigure 3). This supports the work of Kaimori et al.,27 who postulateda cleavage event at the proprotein convertase site (RAKRKR 3615..3620).The unglycosylated mass of this C-terminal fragment was 49.8kD, and there are three potential N-linked glycosylation sitesin the extracellular region of the fragment. This is powerfulindependent evidence of extracellular cleavage of these twoproteins.
Presence of Proteins Derived from Genes Involved in Cystic Disease
More than 25 proteins causing Bardet-Biedl syndrome, nephronophthisis,Meckel-Gruber syndrome, Senior-Løken syndrome, Joubertsyndrome, and orofaciodigital syndrome and animal models ofsyndromic PKD have been associated with the cilium and basalbody. Interestingly, only two of these proteins are observedin PKD-ELVs, the protein cystin (CYS1_HUMAN; rank 160, fourpeptides, 56% coverage), which is the product of the cpk locusin mice,10,30 and the product of the Bardet-Biedl syndrome 3locus, ADP-ribosylation factor-like protein 6 (ARL6_HUMAN; rank341, five peptides, 33% coverage).31,32
Comparison with Other Urinary Proteomes
We compared the PKD-ELV proteome with two others: The Pisitkunproteome of crude ELVs and the Adachi proteome of entire urine.33The data showed that PKD-ELVs had 232 unique proteins not foundin the Adachi or Pisitkun sets; 191 proteins were shared betweenthe Pisitkun and PKD-ELVs data set, and 245 were shared withthe total urine proteome of Adachi; 116 proteins were sharedamong all three sets. This clearly shows that the PKD-ELVs representa novel protein set (Figure 4C, Supplemental Database 1b).
In Vivo Interaction of PKD-ELVs and Primary Cilia in Human and Mouse ARPKD
Transmission electron microscopy analysis of kidney from a patientwith severe ARPKD (T36M, V1627F) showed that the primary ciliawere surrounded by small vesicles, some of which were closelyapplied to the shaft of the cilium (Figure 5, A and B). Observationsmade in the Pkhd1del2/del2 mouse indicated that primary ciliain the mouse biliary tree were also associated with small vesicles,some of which were attached to the cilium (Figure 5D).19 Toquantify this phenomenon, we counted the number of adherentELVs per micrometer on wild-type (WT) and Pkhd1del2/del2 biliarycilia. In WT mice (Figure 5C), we found 1.45 ELVs/µm (SD±1.32; n = 5 cilia; 6.5 µm total length), and inPkhd1del2/del2 biliary cilia, we found 31.1 ELVs/µm (SD±5.9; n = 4 cilia; 5.45 µm total length; P <0.02, two-sample test, normal approximation). Staining Pkhd1del2/del2biliary epithelia with our anti-PC1 mAb, 7e12, showed that theseprimary cilium-interacting vesicles were PC1+ (Figure 6, A andB) and that similar structures were present in MVBs as ILVsin normal rat biliary epithelium (Figure 6, F and G). PC1 waslocalized to the primary cilium in several studies.10,11,34We show that the apical membrane and much of the shaft of theWT primary cilium is negative for PC1 but is surrounded by intenselyPC1+ ELVs (PKD-ELVs; Figure 6, C and D). We also show electronmicroscopic micrographs showing PKD-ELVs that seem to be adheringto cilia (Figures 5; 6, A and B; 7; and 8). This may accountfor the PC1 staining seen on cilia by light immunofluorescencemicroscopy, which can be punctate in nature. We also observeda PKD-ELV emerging from an intracellular vesicle near the baseof the cilium (Figure 6E). These results suggest that ELVs interactwith primary cilia in vivo and that in Pkhd1 mutants, ELVs accumulateon the primary cilium. Furthermore, we observed a 1- to 2-µmMVB interacting with a primary cilium in a patient with ARPKD(Figure 5B). This event may be analogous to the smash eventobserved in the embryonic node by Tanaka et al.16 Importantly,in all cases, there seemed to be two bilipid membranes at thesite of attachment, suggesting an adhesive rather than a buddingevent (Figure 5).
Figure 5.In vivo analysis of ELV–ciliary interactions in human ARPKD and the Pkhd1del2/del2 mouse. (A and B) Transmission electron microscopy of primary cilia from the collecting duct of a neonate with mutation-proven ARPKD. (A) 120 nm diameter ELV adhering to the shaft of a primary cilium (bar = 100 nm). (B) A large MVB (1 to 2 µm) interacting with a primary cilium (bar = 500 nm). (Insert) Enlarged view of the adhesion site. Note the presence of two membranes at the site of attachment in both A and B. (C) ELVs interacting with a WT mouse biliary primary cilium (bar = 100 nm). (D) Accumulating ELVs on a biliary cholangiocyte primary cilium of a Pkhd1del2/del2 mouse (bar = 100 nm). Note (arrows) the interaction seems to be an adhesive rather than a budding event.
Figure 6. Location of PKD ELVs in wild-type rat biliary epithelium and Pkhd1del2/del2 mouse biliary epithelium: (A and B) PC1 staining in Pkhd1del2/del2 mouse biliary cilia using the PC1 monoclonal antibody 7e12 and nanogold with gold-on-gold enhancement. (A) An ELV adherent to the shaft of Pkhd1del2/del2 mouse biliary primary cilium; no primary antibody, control (bar = 100 nm). (B) A cluster of PKD-ELVs interacting with a Pkhd1del2/del2 mouse biliary primary cilium (black arrows) (bar = 100 nm). This shows that the vesicles/exosomes adherent to the primary cilium in the Pkhd1del2/del2 mouse are PC1 positive. (C through G). TEM showing rat biliary epithelium stained with anti-PC1 as above; (C) wild-type rat biliary primary cilium stained with 7e12 showing apical membrane microvilli and a primary cilium shaft. Both are negative for PC1; however, the primary cilium is associated with nearby PC1 positive cup shaped ELVs (PKD-ELVs) (black asterisk) (scale bar = 500 nm). (D) An enlarged view of these (x2). (E) A PKD-ELV (white asterisk in C) extruding from an intracellular vesicle (perhaps an MVB) close to the base of the primary cilium (x3). (F and G) MVB origin of PC1+ ELVs. (F) Normal rat biliary epithelia showing an unstained MVB, no primary antibody (bar = 100 nm); (G) MVB showing ILVs (white arrows) positive for PC1 (bar = 100 nm). The bulk of PC1 staining is therefore restricted to the MVB and PKD-ELV.
Figure 7. Biotinylated PKD-ELVs interact with primary cilia in IMCD3 cells. Biotinylated PKD-ELVs were applied to polarized IMCD3 cells for 1, 2, 5, and 10 min, then washed three times in Hanks’ and fixed in 2.5% glutaraldehyde PBS. The ELVs were detected with 1.4 nm of Nanogold, enhanced using the autometallographic deposition of gold on gold, and then coated with carbon to a thickness of 15 nm. (Left) Standard SEMs of the IMCD (using secondary electrons). (Right) Images generated by backscattered electrons showing the distribution of gold and therefore exosomal protein. Control IMCDs were not treated with ELVs. Bar = 500 nm. Magnification, x15,000.
Figure 8. ELVs fuse with a subpopulation of primary cilia in BUs (primary biliary epithelial cells) and IMCD3 cells. (A, C, E, and G) secondary electrons; (B, D, F, and H) backscattered electrons. (A and B) Magnified view of a non–ELV-treated primary cilium. (C and D) Magnified view of a primary cilium treated with ELVs for 1 min. Note the raised areas on the cilium, suggesting that the ELVs are interacting with the cilium (examples arrowed). (E and F) Magnified primary cilia at 2 min, note that one cilium (arrowed) has no gold associated with it. (G and H) Magnified, SEM of an IMCD3 primary cilium treated with PKD-ELVs for 1 min. Note the raised blebs on the tip and shaft of this primary cilium (examples arrowed). Bars = 500 nm. Magnifications: x60,000 in A through D; x15,000 in E and F; x80,000 in G and H.
In Vitro Analysis of ELV–Cilium Interactions
To test the interaction between ELVs and cilia, we surface-biotinylatedurinary ELVs and purified them on a D2O sucrose gradient. Weused polarized inner medullary collecting duct (IMCD3) cellsand sections of WT rat biliary tree biliary units (BUs) as targetsfor interaction. Target cells were incubated for 1, 2, 5, and10 min with biotinylated ELVs. The interaction was then stoppedusing 2.5% glutaraldehyde, and the ELVs were visualized with1.4 nm of Nanogold and gold enhancement.
These studies showed that ELVs could interact with cilia in<1 min and that they were then cleared rapidly so that by10 min few cilia had gold on their surface (Figure 7). The findingswere consistent in IMCD cells (Figure 7) and BUs (Figure 8,A through F). At high magnifications, the gold staining appearsin patches of 50 to 100 nm on the cilium (Figure 8, C and D,and G and H). Here, some of the gold patches are raised abovethe level of the shaft of the cilium, as though the interactingELVs are adhering and perhaps fusing with the cilium. The rapidclearance of ELVs from the primary cilium, within approximately10 min, suggests that they can integrate with the retrogrademotors in the cilium35 or detach or are degraded.
In the biliary epithelia, there seem to be two populations ofcilia: one capable of interacting with ELVs and another thatlacks this ability. These act as an internal control for specificity(Figure 8, E and F), where BUs have been treated with PKD-ELVsfor 2 min. One cilium is heavily decorated with gold detectedon backscatter, and one is not labeled.
Building on the work of Pisitkun et al.,14 we developed newmethods to resolve at least three different subpopulations ofELVs by density in human urine, enriched respectively for aquaporin2, POD, and PC1/PC2/FCP/CD133 (the last group, we termed PKD-ELVs;Supplemental Figure 1). We characterized an extensive proteomefrom the PKD-ELVs and showed that both PC1 and FCP are cleavedin vivo. Finally, following up on our observations that micewith a targeted defect in the Pkhd1 gene accumulate vesicleson primary cilia of cholangiocytes in dilated bile ducts, weshowed that biotinylated PKD-ELVs can interact with the ciliaof biliary and kidney epithelial cells.19
We characterized PKD-ELVs in detail and showed them to be abundantin PC1, PC2, and FCP by Western blot analysis, immunoelectronmicroscopy, and proteomics. PC1 and FCP are difficult to detectin normal adult kidneys, so the finding of a fraction wherePC1, PC2, and FCP are abundant suggests functional relevance.The abundance of these proteins in the PKD-ELVs enabled us toask questions about the in vivo processing of these proteins.We demonstrated that PC1 is highly glycosylated and that PC1and FCP are cleaved in vivo, events that have previously beenclearly visualized only in exogenous expression systems.27–29Analysis of the distribution of tryptic PC1 and FCP peptideswith reference to the molecular weight of the parent protein(the molecular weight of the proteins in that particular gelslice) is consistent with PC1 cleavage at the GPS site and cleavageof FCP at the proprotein convertase site (Supplemental Figures2 and 3). Although these observations prove that cleavage occursin vivo, they do not show exactly where the cleavage occurs.
PKD-ELVs contain many proteins found in exosomes from othersources, and our comparative analysis revealed considerableoverlap with the proteome extracted from urinary exosomes byPisitkun et al.14and with that isolated directly from urine33;however, we detected 232 proteins that were not present in theseprevious proteomes,14,33 showing the unique nature of thesevesicles (Figure 4, Supplemental Database 1a) and adding tothe total urinary proteome identified to date. The PKD-ELV proteomeis enriched in proteins involved in the biogenesis of ILVs,including all known members of the human ESCRT-III complex,and supports an MVB origin. This origin is further supportedby the finding of PC1+ ILVs in rat cholangiocyte MVBs (Figure 6,F and G) and the presence of occasional multilaminate polycystin-positivestructures in human urine (Figure 2I); however these ELVs containonly low levels of the exosomal marker CD63 and are rich inCD133. Initially, CD133 was thought to be an apical marker;however, recent studies confirmed that it also co-localizeswith CD63 in intracellular vesicles.36,37 Further characterizationwill be required to determine the origin of these vesicles thatwe have thus termed exosome-like vesicles.36 Although much ofour data support an MVB origin, budding from the apical membraneor even cilia cannot be ruled out.
Observations in the Pkhd1del2/del2 mice showed that the primarycilia of the biliary tree are decorated with PC1+ structuresthat we assume are ELVs, and we therefore applied biotinylatedELVs to BU and IMCD3 cells. This resulted in the transfer ofbiotinylated protein to the primary cilia, whereas microvilliof the same cells showed minimal labeling. Furthermore, theinteraction seemed specific because only a subpopulation ofprimary cilia could take up the label in BU (Figure 8, E andF). The ELV–cilium interaction probably depends on oneor more ELV membrane proteins (the PKD-ELV proteome containsat least 96 of these). Adhesion candidate molecules are likelyto be long: PC1 and FCP are predicted to have extracellulardomains of at least 100 nm, approximately the size of an ELV.This size prediction is based on crystallography studies showingthat the PKD1 domain is 4.4 nm long and is repeated 16 timesin the extracellular region of PC1 (i.e., total length approximately70.4 nm).38 There are also three other large domains in theextracellular portion of PC1: REJ (approximately 1000 aminoacids), a C-type lectin (approximately 107 aa), and LRR domain(approximately 145 aa), yielding an overall length of approximately100 nm.4,5 These would significantly increase the functionaladhesive area of ELVs (by approximately nine-fold). BecauseFCP contains multiple domains similar to those involved in hostinvasion (PA14 and PBH domains8,39,40), it is possible thatthis molecule behaves as an invasin and the accumulation ofELVs on the shaft of primary cilia in FCP (PKHD1) mutants mayrepresent a failure of the invasion process, but FCP itselfmay not be responsible for the initial adhesion event.
Tanaka et al.16 showed that, in the node, MVBs (termed NVPs)are released from the floor of the node and swept by nodal flowto the left side of the node, where they interact with the picketfence cilia. Symmetry breaking is associated with a PC2-dependentCa2+ flux on the left side of the node.17,18 We hypothesizedthat a similar process may be found in adult epithelia and thatthe rate of ELV interaction with cilia may be a proxy for flow.11Conversely, it is widely known that ciliary bending independentof exosomes causes an increase in intracellular calcium41; theexosome mechanism may not be a proxy for flow per se but actsas a separate and distinct flow-dependent mechanism. PKD-ELVscontain a wide range of signaling molecules: at least 48 GTPasesand their associated proteins and four seven-membrane-spanningproteins (Smoothened, RAIG-1, RAIG-2, and RAIG-3; Table 2),with implications for cAMP signaling. cAMP has been shown tobe a critical signaling molecule in both ADPKD and ARPKD, andtherapies have been developed on the basis of lowering its levelin the collecting duct and biliary epithelium.42,43 Smoothenedis present with its associated Gi proteins, but Hedgehog(s)or Patched(s) was not detectable in PKD-ELVs. If cilium–ELVinteractions involve a fusion event, then we predict that activeSmoothened will be delivered to the cilium with profound biologicconsequences.44
Table 2. Seven-transmembrane–spanning proteins found in PKD-ELVs
In conclusion, PKD-ELVs and their interaction with primary ciliarepresent a new dimension to the biology of polycystic kidneyand liver disease. This interaction unifies two disparate localizationsfor PC1 and raises the possibility of a novel form of "urocrine"and perhaps "bilocrine" signaling, fostering communication alongthe length of the nephron/biliary tree.
Isolation of Urinary ELVs
Urinary ELVs were isolated following a modification of the methodof Zhou et al.14,15 The first void of the day was collected,and one tablet of Complete proteinase inhibitor cocktail (Hoffmann-LaRoche Inc., Nutley, New Jersey) was added. The urine was chilledand centrifuged at 15,000 x g for 15 min in an SLC-6000 rotorto remove cellular debris, filtered through an 8-µm nylonfilter, and then centrifuged at 150,000 x g in a Sorvall T-647.5rotor for 1 h. The pellet was resuspended in 1 ml of PBS 1xComplete at concentration of 2 mg/ml protein. This materialwas mainly composed of THP and some ELVs and was referred toas crude ELVs.
For removal of the THP from the preparation, 0.5 to 1.0 ml ofcrude ELVs were layered on top of a 5 to 30% sucrose gradientwith D2O as the solvent, buffered to pH 6.0 with 20 mM MES (theaverage pH of urine), generated using a BioComp Gradient MasterStation (BioComp Instruments, Inc., Fredericton, NB, Canada).The gradient was centrifuged at 200,000 x g for 24 h in a SorvallTH-641 rotor, and then the fractions were harvested by taking14 fractions, 6-mm in length, from the 12-ml (Sorvall 06752)tube using a BioComp Gradient Master station. Fractions werediluted five- to 10-fold in PBS and centrifuged at 150,000 xg for 1 h in a Sorvall Surespin 630 rotor to recover PKD-ELVs.These were stored in 100 µl of 0.25 M Sucrose 20 mM HEPES(pH 7.4) with added Complete at –80°C.
Proteins were run on MOPS gels in normal water, stained anddestained, and digested with trypsin again in normal water,diluting residual D2O to background levels before proteomicsand reversing any potential exchange reactions. The total timeexposed to normal water was at least 5 d. The slowest protonexchange in denatured proteins is in the order of a few secondsat pH 7.0 at 20°C.45 For this reason, the proteins werenot accidentally labeled with deuterium because back-exchangewas complete.
Use of Amino Reactive Biotinylation Reagents
For labeling ELVs with biotin, crude ELVs were dialyzed fourtimes in a 10-kD cutoff dialysis cassette (Pierce, Rockford,IL) against 1 L of PBS (each dialysis was 20 min in duration).This removed free amines (urea) and small peptides. EZ-LinkSulfo-NHS-LC-Biotin [sulfosuccinimidyl-6-(biotinamido) hexanoate;Pierce] was added to a concentration of 2 mM to the ELVs andincubated on ice at 4°C for 30 min. The reaction was terminatedby addition of glycine to a concentration of 20 mM. The ELVswere then loaded onto a 5 to 30% sucrose D2O gradient. The efficacyof labeling was then assayed by running 0.1 µg of ELVson a 4 to 12% SDS-PAGE gel and Western blotting with 1:10,000streptavidin–horseradish peroxidase. This showed thatthe ELVs were biotinylated and the proteins were not degraded,the controls being negative (data not shown).
Isolation of Intrahepatic Bile Duct Segments, BUs
BUs were microdissected from normal rat liver using a proteinase-freemodification of the protocol described by Roberts et al.46 Briefly,rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally),then the portal vein was exposed and cannulated and the liverwas perfused with ice-cold saline. Subsequently, 2 to 3 ml ofliquid trypan blue agar was injected into the portal vein tofacilitate identification of portal tracts. The liver was surgicallyremoved and immersed in ice-cold preoxygenated HEPES-bufferedsaline (pH 7.4). After mechanical removal of the hepatic capsuleand surface hepatocytes, intrahepatic bile ducts were exposedand microdissected using a Zeiss Stemi SV11 dissection microscope(Carl Zeiss MicroImaging, Inc., Thornwood, NY). The BUs werecut into 1.0- to 1.5-mm segments and transferred to a culturechamber. Viability was assessed by trypan blue exclusion; onlyBUs without evidence of trypan blue uptake were used. ViableBUs were then cut longitudinally and treated with ELVs at aprotein concentration of 40 µg/ml in Hanks’ bufferedsaline for 1 to 10 min, then washed twice in Hanks’ andfixed in 2.5% glutaraldehyde for at least 1 h. BUs were thenquenched in 100 mM glycine in PBS twice. Blocking of nonspecificsites was achieved with 0.5% BSA and 0.1% gelatin in PBS for1 h. The BUs were then probed with a 1:100 dilution of 1.4 nmof Nanogold streptavidin for 30 min in the blocking reagent,then washed three times with PBS. Gold enhancement was donewith a gold enhancement kit (Nanoprobes, Incorporated, Yaphank,NY). BUs were washed with water four to five times and dehydratedthrough ethanols 50-70-95-100-100-100%, each step for 3 to 5min, then critical-point drying and carbon coating were performed.Carbon coating was for 15 to 20 nm thickness. Specimens wereviewed with a Hitachi 4700 scanning electron microscope withsecondary and backscattering beam (Hitachi America, Ltd., Brisbane,CA).
Participants
We obtained urine samples from normal human volunteers and apatient with ARPKD. Approval from the Mayo Institutional ReviewBoard was obtained, and informed consent from each participantwas given. Institutional Review Board approval and informedconsent were obtained for the use of human tissue that wouldotherwise be considered surgical waste. Tissue was obtainedfor research after nephrectomy in a patient with ARPKD.
Mice and Rats
All procedures were conducted under protocols approved by theInstitutional Animal Care and Use Committee at Mayo Clinic inaccordance with the Animal Welfare Act and the Department ofHealth and Human Services.
C.J.W. was funded by National Institutes of Health grant RO1DK65056.
We thank Dr. Yiqiang Cai for the gift of Ab YCC2 to PC2, Dr.Thomas Hiesberger for the human FCP cDNA construct, ChristopherJ. Mason for the Bioinformatics searches, Jon E. Charlesworthfor assistance with electron microscopy, and Dr. David H. Coombsfor assistance with the Biocomp Gradient Master station.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Supplemental information for this article is available onlineat http://www.jasn.org/.
Torres V, Holley K, Offord K. Epidemiology. In:
Problems in Diagnosis and Management of Polycystic Kidney Disease, edited by Grantham JJ, Kansas City: PKR Foundation Kansas City, 1985
, pp 349
–369
Wilson PD: Polycystic kidney disease.
N Engl J Med 350
: 151
–164, 2004[Free Full Text]
The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. The European Polycystic Kidney Disease Consortium.
Cell 77
: 881
–894, 1994[CrossRef][Medline]
Polycystic kidney disease: The complete structure of the PKD1 gene and its protein. The International Polycystic Kidney Disease Consortium.
Cell 81
: 289
–298, 1995[CrossRef][Medline]
Hughes J, Ward CJ, Peral B, Aspinwall R, Clark K, Millan JL, Gamble V, Harris PC: The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains.
Nat Genet 10
: 151
–160, 1995[Medline]
Mochizuki T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJ, Somlo S: PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.
Science 272
: 1339
–1342, 1996[Abstract]
Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly V, Cunningham JM, Bacallao R, Ishibashi M, Milliner DS, Torres VE, Harris PC: The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.
Nat Genet 30
: 259
–269, 2002[CrossRef][Medline]
Onuchic LF, Furu L, Nagasawa Y, Hou X, Eggermann T, Ren Z, Bergmann C, Senderek J, Esquivel E, Zeltner R, Rudnik-Schoneborn S, Mrug M, Sweeney W, Avner ED, Zerres K, Guay-Woodford LM, Somlo S, Germino GG: PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.
Am J Hum Genet 70
: 1305
–1317, 2002[CrossRef][Medline]
Ward CJ, Yuan D, Masyuk TV, Wang X, Punyashthiti R, Whelan S, Bacallao R, Torra R, LaRusso NF, Torres VE, Harris PC: Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.
Hum Mol Genet 12
: 2703
–2710, 2003[Abstract/Free Full Text]
Yoder BK, Hou X, Guay-Woodford LM: The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia.
J Am Soc Nephrol 13
: 2508
–2516, 2002[Abstract/Free Full Text]
Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J: Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.
Nat Genet 33
: 129
–137, 2003[CrossRef][Medline]
Wang S, Zhang J, Nauli SM, Li X, Starremans PG, Luo Y, Roberts KA, Zhou J: Fibrocystin/polyductin, found in the same protein complex with polycystin-2, regulates calcium responses in kidney epithelia.
Mol Cell Biol 27
: 3241
–3252, 2007[Abstract/Free Full Text]
Wu Y, Dai X-Q, Li Q, Chen CX, Mai W, Hussain Z, Long W, Montalbetti N, Li G, Glynne R, Wang S, Cantiello HF, Wu G, Chen X-Z: Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin.
Hum Mol Genet 15
: 3280
–3292, 2006[Abstract/Free Full Text]
Pisitkun T, Shen R-F, Knepper MA: Identification and proteomic profiling of exosomes in human urine.
Proc Natl Acad Sci U S A 101
: 13368
–13373, 2004[Abstract/Free Full Text]
Zhou H, Yuen PST, Pisitkun T, Gonzales PA, Yasuda H, Dear JW, Gross P, Knepper MA, Star RA: Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.
Kidney Int 69
: 1471
–1476, 2006[Medline]
Tanaka Y, Okada Y, Hirokawa N: FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left-right determination.
Nature 435
: 172
–177, 2005[CrossRef][Medline]
Pennekamp P, Karcher C, Fischer A, Schweickert A, Skryabin B, Horst J, Blum M, Dworniczak B: The ion channel polycystin-2 is required for left-right axis determination in mice.
Curr Biol 12
: 938
–943, 2002[CrossRef][Medline]
McGrath J, Somlo S, Makova S, Tian X, Brueckner M: Two populations of node monocilia initiate left-right asymmetry in the mouse.
Cell 114
: 61
–73, 2003[CrossRef][Medline]
Woollard JR, Punyashtiti R, Richardson S, Masyuk TV, Whelan S, Huang BQ, Lager DJ, vanDeursen J, Torres VE, Gattone VH, LaRusso NF, Harris PC, Ward CJ: A mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation.
Kidney Int 72
: 328
–336, 2007[CrossRef][Medline]
Ong AC, Harris PC, Davies DR, Pritchard L, Rossetti S, Biddolph S, Vaux DJ, Migone N, Ward CJ: Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue.
Kidney Int 56
: 1324
–1333, 1999[CrossRef][Medline]
Cai Y, Maeda Y, Cedzich A, Torres VE, Wu G, Hayashi T, Mochizuki T, Park JH, Witzgall R, Somlo S: Identification and characterization of polycystin-2, the PKD2 gene product.
J Biol Chem 274
: 28557
–28565, 1999[Abstract/Free Full Text]
Lehtonen S, Ora A, Olkkonen VM, Geng L, Zerial M, Somlo S, Lehtonen E: In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.
J Biol Chem 275
: 32888
–32893, 2000[Abstract/Free Full Text]
Li Q, Montalbetti N, Shen PY, Dai X-Q, Cheeseman CI, Karpinski E, Wu G, Cantiello HF, Chen X-Z: Alpha-actinin associates with polycystin-2 and regulates its channel activity.
Hum Mol Genet 14
: 1587
–1603, 2005[Abstract/Free Full Text]
Zatti A, Chauvet V, Rajendran V, Kimura T, Pagel P, Caplan MJ: The C-terminal tail of the polycystin-1 protein interacts with the Na,K-ATPase alpha-subunit.
Mol Biol Cell 16
: 5087
–5093, 2005[Abstract/Free Full Text]
Parnell SC, Magenheimer BS, Maser RL, Rankin CA, Smine A, Okamoto T, Calvet JP: The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro.
Biochem Biophys Res Commun 251
: 625
–631, 1998[CrossRef][Medline]
Parnell SC, Magenheimer BS, Maser RL, Zien CA, Frischauf A-M, Calvet JP: Polycystin-1 activation of c-Jun N-terminal kinase and AP-1 is mediated by heterotrimeric G proteins.
J Biol Chem 277
: 19566
–19572, 2002[Abstract/Free Full Text]
Kaimori J-Y, Nagasawa Y, Menezes LF, Garcia-Gonzalez MA, Deng J, Imai E, Onuchic LF, Guay-Woodford LM, Germino GG: Polyductin undergoes notch-like processing and regulated release from primary cilia.
Hum Mol Genet 16
: 942
–956, 2007[Abstract/Free Full Text]
Ponting CP, Hofmann K, Bork P: A latrophilin/CL-1-like GPS domain in polycystin-1.
Curr Biol 9
: R585
–R588, 1999[CrossRef][Medline]
Qian F, Boletta A, Bhunia AK, Xu H, Liu L, Ahrabi AK, Watnick TJ, Zhou F, Germino GG: Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1-associated mutations.
Proc Natl Acad Sci U S A 99
: 16981
–16986, 2002[Abstract/Free Full Text]
Hou X, Mrug M, Yoder BK, Lefkowitz EJ, Kremmidiotis G, D'Eustachio P, Beier DR, Guay-Woodford LM: Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease.
J Clin Invest 109
: 533
–540, 2002[CrossRef][Medline]
Chiang AP, Nishimura D, Searby C, Elbedour K, Carmi R, Ferguson AL, Secrist J, Braun T, Casavant T, Stone EM, Sheffield VC: Comparative genomic analysis identifies an ADP-ribosylation factor-like gene as the cause of Bardet-Biedl syndrome (BBS3).
Am J Hum Genet 75
: 475
–484, 2004[CrossRef][Medline]
Fan Y, Esmail MA, Ansley SJ, Blacque OE, Boroevich K, Ross AJ, Moore SJ, Badano JL, May-Simera H, Compton DS, Green JS, Lewis RA, van Haelst MM, Parfrey PS, Baillie DL, Beales PL, Katsanis N, Davidson WS, Leroux MR: Mutations in a member of the Ras superfamily of small GTP-binding proteins causes Bardet-Biedl syndrome.
Nat Genet 36
: 989
–993, 2004[CrossRef][Medline]
Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M: The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins.
Genome Biol 7
: R80
, 2006[CrossRef][Medline]
Sohara E, Luo Y, Zhang J, Manning DK, Beier DR, Zhou J: Nek8 regulates the expression and localization of polycystin-1 and polycystin-2.
J Am Soc Nephrol 19
: 469
–476, 2008[Abstract/Free Full Text]
Follit JA, Tuft RA, Fogarty KE, Pazour GJ: The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.
Mol Biol Cell 17
: 3781
–3792, 2006[Abstract/Free Full Text]
Marzesco A-M, Janich P, Wilsch-Brauninger M, Dubreuil V, Langenfeld K, Corbeil D, Huttner WB: Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells.
J Cell Sci 118
: 2849
–2858, 2005[Abstract/Free Full Text]
Fonseca AV, Bauer N, Corbeil D: The stem cell marker CD133 meets the endosomal compartment: New insights into the cell division of hematopoietic stem cells.
Blood Cells Mol Dis 41
: 194
–195, 2008[CrossRef][Medline]
Jing H, Takagi J, Liu J-h, Lindgren S, Zhang R-g, Joachimiak A, Wang J-h, Springer TA: Archaeal surface layer proteins contain beta propeller PKD, and beta helix domains and are related to metazoan cell surface proteins.
Structure 10
: 1453
–1464, 2002[Medline]
Rigden DJ, Mello LV, Galperin MY: The PA14 domain, a conserved all-beta domain in bacterial toxins, enzymes, adhesins and signaling molecules.
Trends Biochem Sci 29
: 335
–339, 2004[CrossRef][Medline]
de Groot PW, Klis FM: The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity.
Mol Microbiol 68
: 535
–537, 2008[CrossRef][Medline]
Praetorius HA, Frokiaer J, Nielsen S, Spring KR: Bending the primary cilium opens Ca2+-sensitive intermediate-conductance K+ channels in MDCK cells.
J Membr Biol 191
: 193
–200, 2003[CrossRef][Medline]
Wang X, Ii VG, Harris PC, Torres VE: Effectiveness of vasopressin V2 receptor antagonists OPC-31260 and OPC-41061 on polycystic kidney disease development in the PCK rat.
J Am Soc Nephrol 16
: 846
–851, 2005[Abstract/Free Full Text]
Masyuk TV, Masyuk AI, Torres VE, Harris PC, Larusso NF: Octreotide inhibits hepatic cystogenesis in a rodent model of polycystic liver disease by reducing cholangiocyte adenosine 3',5'-cyclic monophosphate.
Gastroenterology 132
: 1104
–1116, 2007[CrossRef][Medline]
Rohatgi R, Milenkovic L, Scott MP: Patched1 regulates hedgehog signaling at the primary cilium.
Science 317
: 372
–376, 2007[Abstract/Free Full Text]
Krishna MM, Hoang L, Lin Y, Englander SW: Hydrogen exchange methods to study protein folding.
Methods 34
: 51
–64, 2004[CrossRef][Medline]
Roberts SK, Kuntz SM, Gores GJ, LaRusso NF: Regulation of bicarbonate-dependent ductular bile secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts.
Proc Natl Acad Sci U S A 90
: 9080
–9084, 1993[Abstract/Free Full Text]
This article has been cited by other articles:
A. Kurbegovic, O. Cote, M. Couillard, C. J. Ward, P. C. Harris, and M. Trudel Pkd1 transgenic mice: adult model of polycystic kidney disease with extrarenal and renal phenotypes
Hum. Mol. Genet.,
January 19, 2010;
(2010)
ddp588v2.
[Abstract][Full Text][PDF]
P. C. Harris 2008 Homer W. Smith Award: Insights into the Pathogenesis of Polycystic Kidney Disease from Gene Discovery
J. Am. Soc. Nephrol.,
June 1, 2009;
20(6):
1188 - 1198.
[Abstract][Full Text][PDF]
M. A. Knepper Common Sense Approaches to Urinary Biomarker Study Design
J. Am. Soc. Nephrol.,
June 1, 2009;
20(6):
1175 - 1178.
[Full Text][PDF]
Top
Abstract
Introduction
= 1.3530 (SD ± 0.0019; n = 6), representing a density of approximately 1.17 kg/L in D2O. By using this gradient, we were able to remove >99% of the THP from the ELVs and to isolate 100 µg of PC1-enriched ELVs per liter of urine, <1% of the initial protein in the crude ELV preparation (Figure 3, A and B). The stem cell marker CD133 (prominin) also co-sediments with PC1, PC2, and FCP. This contrasted with other proteins previously detected in urinary ELVs that were found in different fractions: Aquaporin 2, in fractions 1 through 5, and the steroid-resistant nephrotic syndrome protein, podocin (NPHS2), that was enriched in fractions 10 through 14 (Supplemental Figure 1). 
-actinin-1 (ACTN1_HUMAN; rank 511, five peptides, 15% coverage)23 were present. The Na/K-transporting ATPase 
