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BASIC SCIENCE |
*Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts; Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York, New York; and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
Correspondence to Dr. Jing Zhou, Harvard Institutes of Medicine, Room 522, Brigham and Women’s Hospital and Harvard Medical School, 4 Blackfan Circle, Boston, MA 02115. Phone: 617-525-5860; Fax: 617-525-5861; E-mail: zhou{at}rics.bwh.harvard.edu
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Abstract |
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Introduction |
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ARPKD is caused by mutations in PKHD1. Sixty-three mutations have been identified to date, including frameshift, nonsense, missense, and out-of-frame splicing alterations (11–13). The longest isoform of the PKHD1 transcripts among human subjects is 16,235 bp (12). Several shorter transcripts of PKHD1 were also suggested (13,14). The open reading frame in the human gene is 12,222 bp, encoding a large 4074-amino acid protein with a calculated molecular mass of 447 kD, designated fibrocystin/polyductin/tigmin (FPC) (12–14). FPC was proposed to be a novel single-transmembrane protein with a short 192-amino acid carboxyl-terminal intracellular domain and a very large amino-terminal extracellular portion containing domains such as TIG, TIG-like, TMEM2, and DKFZ homologs and to belong to a protein superfamily with adhesive functions (12–14). The longest open reading frame of the mouse ortholog of PKHD1 encodes a protein of 4059 amino acids; the mouse and human protein sequences are 73% identical overall and 55% identical in the carboxyl-terminal tail. Study of the expression pattern in mouse metanephros by in situ hybridization demonstrated that Pkhd1 transcripts are not expressed in metanephric mesenchyme but are strongly expressed in the branching ureteric bud, which later develops into the adult collecting ducts (15,16). In postnatal kidney tissue, strong Pkhd1 expression was observed in collecting ducts, with lower levels in proximal and distal tubules (15).
The primary cilium, which is found on many types of mammalian epithelial cells, is a specialized sensory organelle extending from the cell surface (17–19). It is assembled as an extension of a basal body, which itself originates from one of a pair of centrioles (20). In most kidney tubular epithelial cells except intercalated cells, a single primary cilium protrudes into the tubular lumen (21). The primary cilium in kidney tubules contains a central axoneme with a 9+0 arrangement of microtubules (22). IFT88/Polaris, the protein product encoded by Tg737, whose mutation causes PKD in mice, is localized in the peri-basal body region and primary cilium and is required for ciliogenesis in Chlamydomonas (23), in Caenorhabditis elegans (24), and in mice (23,25). Recent evidence demonstrated that cilia are able to sense fluid flow (26) and that this sensory process is mediated by polycystin 1 (PC1) and PC2, the proteins responsible for autosomal dominant PKD (27). Whether FPC is expressed and functions in cilia is not known. Therefore, as the first step in elucidating the pathogenesis of ARPKD, we generated two antipeptide antibodies (803 and 804) against FPC and characterized them with immunoblotting and immuno-light microscopy and immuno-electron microscopy. We determined that FPC in kidney tubular cells is localized to the primary cilia, with concentration in the peri-basal body region.
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Generation and Purification of Polyclonal Antibodies against Human FPC
To generate antibodies against human FPC, we selected one peptide sequence located in the intracellular domain (amino acids 3912 to 3925, KRRESQGPKKEDTV) and one located in the extracellular domain (amino acids 488 to 502, REKHQIRVRAQRLPE). Each peptide was synthesized and injected into two rabbits (Research Genetics, Invitrogen). Antibodies from 10-wk blood samples were affinity-purified and designated 803 and 804.
Cell Culture and Transient Transfection
Human embryonic kidney (HEK) 293T cells were cultured in 1x Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY) containing 10% FBS (Invitrogen) and were transiently transfected with FuGENE 6 transfection reagent (Roche, Welwyn Garden City, UK) after overnight culture, according to the manufacturer’s instructions. Dolichos biflorus agglutinin (DBA)-positive mouse embryonic kidney (MEK) cells (Pkd1null/+, Pkd1null/null, and Pkd1del34/del34) (28,29) were derived from embryonic day 15.5 kidneys from wild-type and Pkd1 mutant mice bearing temperature-sensitive simian virus large T antigen (27). Cells were cultured at 33°C in SV40 epithelium medium, containing 1x DMEM, 5% FBS, 0.75 µg/L IFN-
, 1 g/L insulin, 0.67 mg/L sodium selenite, 0.55 g/L transferrin, 0.2 g/L ethanolamine, 36 ng/ml hydrocortisone, 0.1 µM 2,3,5-triido-L-thyronine, 0.3 mg/ml glutamine, 0.1 mM citrate, and appropriate amounts of antibiotics. All of the cell culture supplements were obtained from Invitrogen except for IFN-, hydrocortisone, and 2,3,5-triido-L-thyronine (Sigma Chemical Co., St. Louis, MO). These cells have been passaged >50 times with no apparent phenotypic changes. Human kidney DBA-positive renal cortical tubule epithelial cells (30) were cultured in DMEM/15% FBS at 37°C; canine kidney MDCK cells were cultured in DMEM/10% FBS at 37°C. These studies were completely in compliance with human research guidelines, and institutional review board approval was obtained.
Western Blotting
Two days after transfection, cultured HEK 293T cells were harvested and solubilized with RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.6) containing appropriate proteinase inhibitors (complete EDTA-free tablets; Roche) and 10 mM PMSF. The cell lysates were frozen and thawed for three rounds before being passed through a 27-gauge syringe 10 times. Cells were then centrifuged at 10,000 x g for 10 min at +4°C. Twenty microliters of the supernatant were used for immunoblotting analysis.
The protein samples were subjected to electrophoresis in a 12% acrylamide Laemmli resolving gel (mini-gel electrophoresis system; Bio-Rad Laboratories, Richmond, CA) and transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). After being blocked with 5% nonfat dry milk (Bio-Rad Laboratories) in PBS for 1 h, the filter was incubated with rabbit antibody to FPC, with or without peptide, for another 1 h. After three washes with PBS/0.2% Tween 20 (Bio-Rad Laboratories), the filters were incubated with donkey anti-rabbit Ig conjugated with horseradish peroxidase-linked antibody (1:10,000; Amersham Biosciences). After thorough washing, the bound antibodies were detected with the ECL Western blotting analysis system (Amersham Pharmacia Biotech), according to the manufacturer’s instructions. For Myc tag detection, the filter was stripped with Restore Western blot stripping buffer (Pierce Chemical Co., Rockford, IL), reblotted with mAb to Myc (1:5000; Invitrogen), and subjected to the procedures described above. For peptide blocking experiments, 803 and 804 were incubated with FPC peptides (200-fold molar excess) for at least 1 h at room temperature before being added to the filter.
Immuno-Light-Microscopic Analysis of Kidney Cells and Kidney Tissues
The methods for immunostaining in kidney cells and kidney tissues have been described in detail (27). In brief, cells were cultured as described above for at least 2 d (to approximate subconfluence), fixed with 3% paraformaldehyde/2% sucrose, and permeabilized with 0.5% Triton X-100 (Sigma). Permeabilized cells were incubated with the antibody against FPC (1:250 for 803 and 804) for 1 h at room temperature and then with the FITC-labeled goat anti-rabbit mAb (1:1000). For double-labeling, the antibody against FPC and mAb to acetylated 
-tubulin or -tubulin (1:10,000) were concomitantly incubated. After thorough washing, both Texas Red-labeled (1:1000) and FITC-labeled secondary antibodies (1:1000) were added for 1 h at room temperature. Before mounting with Prolong antifade medium (Molecular Probes, Eugene, OR), the cells were incubated for 5 min with 4'-6-diamidino-2-phenylindole (1:5000 in 1x PBS). For tissue staining, human and mouse kidney tissues were fixed with 4% paraformaldehyde for 24 h and then transferred overnight to 30% sucrose in 0.2 M phosphate buffer containing Na2HPO4 and NaH2PO4. The tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA), sectioned at 5 to 7 µm, and fixed in acetone at -20°C for 5 min. Other procedures for staining of the kidney tissue were the same as for the cells except without Triton X-100. A Zeiss Axioskop2 Plus fluorescence microscope (Carl Zeiss, Inc., Thornburg, NY) was used to observe the signal, and the Spot camera system (Diagnostic Instruments) was used for photography.
Immunoprecipitation of Endogenous FPC with 803 and 804
DBA-positive renal cortical tubule epithelial cells and MDCK cells were harvested and solubilized as described above for HEK 293T cells. Normal human kidney samples were homogenized in RIPA buffer with a mortar and pestle and then a tissue homogenizer. Cell lysates and tissue homogenates were centrifuged at 10,000 x g for 20 min at +4°C. The supernatants were precleared with protein A-agarose beads (Invitrogen) for 1 h at +4°C before the addition of 803 or 804. After endogenous FPC was immunoprecipitated overnight, protein A-agarose beads were added and incubated at +4°C for 3 h, followed by centrifugation at 10,000 x g for 1 min at +4°C. The beads were washed twice with RIPA lysis buffer. Finally, 50 µl of sample buffer was added and the samples were analyzed with Western blotting. The procedures were the same as described above except that the samples were electrophoresed on a 5% acrylamide Laemmli resolving gel.
Immuno-Electron Microscopy
For detection of FPC in mouse cells by immuno-electron microscopy, MEK cells were fixed in freshly prepared 4% formaldehyde/0.1% glutaraldehyde/PBS for 10 min. After thorough washing, the cells were incubated with 803 (1:250) in 0.5% Nonidet P-40/PBS for 1 h, followed by incubation with 10-nm gold-conjugated secondary antibody (1:40; Molecular Probes) for another 1 h. Cells were then postfixed for 1 h in 2.5% glutaraldehyde (electron microscopy grade; Electron Microscopy Services, Washington, PA) in PBS, followed by 1 h in 1% OsO4 in PBS at room temperature. Cells were washed three times in PBS, dehydrated to 95% ethanol, stained en bloc with 4% uranyl acetate in 95% ethanol for 20 min, washed twice with 95% ethanol, and then embedded in Araldite (SPI-Pon Araldite kit; SPI Supplies, West Chester, PA). Thin sections were cut parallel to the surface of the cell monolayer, stained with uranyl acetate and lead citrate, and examined with a Philips CM10 electron microscope (Philips Medical Systems, Eindhoven, The Netherlands).
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Detection of FPC in the Primary Cilia and in the Basal Body Area in Human Kidney Tubules
To determine the specificity of 803 in tissue staining, we performed immunostaining with preimmune serum, 803, and 803 plus its immunogen. Because we hypothesized a ciliary localization of FPC, we also double-labeled tissue sections with an antibody to a ciliary marker, acetylated -tubulin. Whereas the acetylated -tubulin-specific antibody delineated the cilia very well, preimmune serum did not yield obvious signals on tissue sections (Figure 2, A to C). Double-labeling with 803 and the acetylated -tubulin-specific mAb demonstrated that FPC was located primarily in the basal body area, at the base of the cilium, in collecting duct cells (Figure 2, F to H). The reactivity of 803 with tissue sections was blocked with preincubation with its FPC peptide, whereas staining for acetylated -tubulin, a ciliary marker, remained (Figure 2, K to M). To confirm that FPC was localized in the peri-basal body region, we double-labeled the cells with 803 and a mAb to -tubulin, a basal body marker. We observed that FPC colocalized with -tubulin in collecting duct cells (Figure 2, F' to H').
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-tubulin (Figure 2, P to R). We did not detect specific signals on cilia or in the basal body area (Figure 2, P' to R') in a kidney from a patient with ARPKD. The immunostaining data for human fetal and ARPKD kidneys were confirmed with 804. Like 803, 804 recognized FPC at the base of the cilia (Figure 3, a to c) and detected no specific signal in a kidney from a patient with ARPKD (Figure 3, a' to c').
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-tubulin-specific mAb demonstrated that FPC was located mainly in the basal body area in mouse kidney tubules (Figure 4, F to H). The antibody 803 preabsorbed with its peptide failed to stain the basal bodies, whereas acetylated -tubulin staining was not affected (Figure 4, K to M). To identify a cell line that expresses FPC, we examined FPC expression in embryonic day 15.5 MEK epithelial cells positive for DBA, a collecting duct marker (27). After 2 to 4 d of culture at 33°C, the primary cilium in each cell was visible. Preimmune serum did not demonstrate any staining on mouse cells (Figure 5, A to C). Double-labeling with 803 and either -tubulin-specific or -tubulin-specific mAb demonstrated that FPC was clearly situated at the base of the cilium and colocalized with -tubulin (Figure 5, E to G and E' to G'). The staining signals for FPC in basal bodies could be blocked with preincubation of 803 with its peptide (Figure 5, I to K). Similar results were obtained when MDCK cells were stained with 803 (Figure 6, A to C).
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-tubulin. Interestingly, 804 stained the whole cilium in some cells (Figure 6, m to o). The specificity and staining pattern of 804 were also tested in MDCK cells. Again, FPC was observed in the basal body area (Figure 6, e to g and e' to g') and the ciliary shaft (Figure 6, e'' to g''). Preimmune serum did not yield any staining (Figure 6, a to c). The staining of FPC in primary cilia could be blocked with preincubation of 804 with its FPC peptide (Figure 6, i to k).
FPC Expression in Pkd1 Mutant Cells
As one way to study the relationship between FPC and PC1, we compared the FPC expression pattern in cells with a targeted Pkd1 mutation (Pkd1null/+, Pkd1null/null, or Pkd1del34/del34) with that in wild-type cells. The cells were derived from DBA-positive embryonic day 15.5 MEK specimens from the same litter, with or without a targeted mutation in the mouse Pkd1 gene (27). We observed that FPC was expressed in the basal bodies and the centrioles of all three kinds of mutant cells (Figure 7, E to G, I to K, and M to O). Neither mislocalization nor a difference in staining intensity was observed among the four groups (Figure 7).
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Discussion |
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The localization pattern observed for FPC is strikingly similar to that previously observed for PC1, a large integral membrane glycoprotein (31–33) that functions in the cilium (27), and for components of the intraflagellar transport (IFT) system, which moves proteins into the cilium and along the ciliary shaft (34). For example, both kinesin II and dynein 1b/2, the motors for anterograde and retrograde IFT, respectively, are located primarily at the base of the cilium, with minor localization to the ciliary shaft (35–37). Similarly, the IFT proteins IFT88/Polaris (25) and IFT52 (37), which are subunits of the IFT particles that carry ciliary proteins as cargo (34), are located primarily in a pool surrounding the basal bodies, with smaller amounts actually engaged in transport along the ciliary shaft. Importantly, defects in the IFT system, specifically in kinesin II (38) and IFT88/Polaris (23), cause structural defects in primary cilia, resulting in cystic kidneys in mice. Immunocytochemical and immuno-electron-microscopic studies cannot distinguish between components of the IFT system and their cargo, which is thought to include membrane proteins (34). However, because FPC is predicted to be a transmembrane protein (12,13), it is likely that its primary localization to the peri-basal body region reflects its presence in post-Golgi vesicles that are targeted to the base of the cilium, where they dock with the plasma membrane before the protein is moved onto the ciliary membrane. Although such membrane vesicles would not be preserved after the standard permeabilization treatment used for immunolocalization, they are readily apparent in specimens prepared for conventional transmission electron microscopy (39). Similar accumulation and docking of rhodopsin-containing vesicles occur at the base of the mammalian photoreceptor-connecting cilium before fusion of the vesicles with the plasma membrane and transport of rhodopsin, via the connecting cilium, to the photoreceptor outer segment (40). Further studies will be necessary to determine the conditions affecting the relative amounts of FPC in the peri-basal body region versus the ciliary shaft.
The localization of FPC to the basal body region and in some cases the ciliary shaft suggests that FPC has a structural or functional role in the primary cilium. Structural analysis of the FPC molecule indicates that its intracellular domain contains several potential phosphorylation sites for protein kinase A and protein kinase C in humans and mice, whereas the extracellular part of the molecule contains several different functional domains, suggesting roles in cellular signaling (12–14). The primary cilium is a sensory organelle that relays signals to the cell body, including those that control cell differentiation, proliferation, and apoptosis (19), and a classic function of the centrosome/basal body is to nucleate or bind microtubules (41,42), thus establishing cell polarity. Therefore, FPC may play a role in transducing extracellular signals to the cell, to control the cell cycle and cell differentiation.
Ciliary length is one parameter for evaluation of the roles of molecules located in the cilium; e.g., the Tg737orpk mutation in mice causes shorter than normal cilia in kidney tubules (23). We did not observe shorter cilia in Pkd1-knockout mice, but we did discover that the Pkd1 mutant cells failed to initiate Ca2+ responses to physiologic fluid flow, indicating that PC1 is a functional component of the cilium (27,43). Considering the similar expression patterns and somewhat similar molecular structures for FPC and PC1, we hypothesize that FPC plays a sensory role in kidney epithelia, as does PC1. It would be interesting to examine the ciliary length in kidney tubules among patients with ARPKD and to test the role of FPC as a mechanosensor.
PKD involves sequential steps from cyst formation and enlargement to fluid secretion and accumulation within the cysts (44). During this complicated process, abnormal cellular proliferation, differentiation, and/or apoptosis can occur. It is generally thought that cystogenesis results from increased proliferation and de-differentiation, but how PKHD1 mutations among patients with ARPKD initiate these changes is not known. It is tempting to speculate that FPC interacts with other proteins associated with the basal body/cilium and that its dysfunction results in disruption of the protein complex and leads to cystogenesis. Because the major autosomal dominant PKD protein PC1 is also associated with the basal bodies of primary cilia, we tested the effects of PC1 mutations on FPC expression. We observed no obvious differences in FPC expression levels and patterns between wild-type MEK epithelial cells and cells with various Pkd1 mutations (Pkd1null/+, Pkd1null/null, or Pkd1del34/del34). Therefore, disruption of PC1 does not seem to physically disrupt the normal localization of FPC, suggesting that FPC localization is independent of the PC1 protein complex. This is consistent with the differences in clinical presentations for patients with ARPKD versus autosomal dominant PKD. Further studies are required to determine whether the FPC pathway interacts with the polycystin signaling pathway.
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-helix 1 repeats. Am J Hum Genet 70: 1305–1317, 2002[CrossRef][Medline]
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