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*
Department of Medical Genetics, University of Cambridge, Addenbrooke's
Hospital, Cambridge, United Kingdom
Department of Medicine, University of Cambridge, Addenbrooke's Hospital,
Cambridge, United Kingdom
Department of Pathology, University of Cambridge, Addenbrooke's Hospital,
Cambridge, United Kingdom
§
Department of Genetics, University of Cambridge, Cambridge, United
Kingdom
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Genzyme Genetics, Framingham, Massachusetts.
Correspondence to Dr. Richard Sandford, Department of Medical Genetics, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, United Kingdom. Phone: +44 1223 762616; Fax: +44 1223 331206; E-mail: rns13{at}cam.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eighty-five percent of cases of ADPKD are due to mutations in PKD1 with the remainder occurring in PKD2 (4). A few ADPKD families appear unlinked to either PKD1 or PKD2, suggesting the presence of a rare third locus, PKD3 (5). While mutations in PKD2 cause a milder phenotype than those in PKD1 with a slower decline in renal function, the clinical features are indistinguishable between the two groups (6). Targeted mutations in the murine pkd1 and pkd2 genes also produce a cystic phenotype, suggesting that the protein products of these two genes, polycystin-1 and polycystin-2, may interact directly or form separate parts of a common cellular pathway (7,8).
Polycystin-1 is composed of a novel array of structural and functional domains that suggest it is an integral plasma membrane glycoprotein involved in cell-cell or cell-matrix interactions (9,10). Its precise function is still unknown. A consensus derived from the many published reports of the cellular and subcellular localization of polycystin-1 defines it as a developmentally regulated integral membrane protein expressed at highest levels in fetal tissues (11,12,13,14,15). In adult tissues it is predominantly localized to the distal part of the nephron and collecting ducts and other epithelial structures such as bile and pancreatic ducts (13,15).
Polycystin-2 is predicted to be an integral membrane protein with intracellular N- and C-termini (16). It has significant homology to the pore-forming domains of a number of voltage-activated cation channels, suggesting that it may function as an ion channel subunit (16,17). A new member of the polycystin family, polycystin-L/polycystin-2L, has recently been described (17,18). It has extensive homology to polycystin-2 but is not thought to be involved in the pathogenesis of ADPKD. Its role in other renal cystic diseases remains to be determined.
The predicted in vivo interaction between polycystin-1 and polycystin-2 has been demonstrated in vitro, suggesting that they may form subunits of a large membrane-associated complex (19,20). They should therefore have extensive overlap in cellular and subcellular localization. Staining in the mouse using an anti-polycystin-2 antibody recognizing the C-terminal region of the protein has demonstrated a distinctive, developmentally regulated expression pattern (21). In the mature kidney, strongest staining in a basolateral distribution was seen in the medullary thick ascending limbs of the loop of Henle and the distal convoluted tubule, with weaker staining of the proximal tubules and cortical and medullary collecting ducts (21). Polycystin-2 expression in humans has been reported to be more widespread than in mouse with coordinate expression with polycystin-1 (22). Staining in the kidney was seen in most nephron segments with highest levels seen in medullary collecting ducts.
In this report we describe the generation of a panel of polycystin-2-specific antibodies and their use to define polycystin-2 expression in human and mouse. Identical staining patterns in human and mouse define polycystin-2 as a developmentally regulated integral membrane protein that localizes to the basolateral plasma membranes of renal tubular epithelial cells. Differences between the temporal and spatial expression of polycystin-1 and polycystin-2 suggest that while they may interact and form a large membrane-associated complex, they may also function independently of each other.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro translation of the flPKD2 construct and a luciferase control incorporating 35S-methionine (Amersham, Buckinghamshire, United Kingdom) was performed using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's protocol.
Antibody Preparation, Western Blotting, and Immunoprecipitation
Peptides NH2-MVNSSRVQPQQPDCys-COOH and NH2-CEGMEGAGGNGSSNVH-COOH
corresponding to amino acids 1 to 12 and 953 to 968 of polycystin-2 were
synthesized and conjugated to keyhole limpet hemocyanin (Immune Systems
Limited, Paignton, United Kingdom). Polyclonal antisera were raised in New
Zealand White rabbits and affinity-purified against 10 mg of the immunizing
peptide coupled to NHS-Sepharose 4 Fast Flow (Pharmacia, Uppsala, Sweden),
according to the manufacturer's instructions, to generate antibodies PKD2-NP
and PKD2-CP, respectively. Nucleotides 2134 to 2973 of PKD2, which
encode the predicted C-terminal cytoplasmic region of polycystin-2, were
cloned into pGEX-4T (Pharmacia) to produce a glutathione
S-transferase (GST)-PKD2 bacterial fusion protein. Polyclonal
antiserum was raised in New Zealand White rabbits and affinity-purified
against immobilized GST (Pierce, Rockford, IL) to remove anti-GST antibodies
and against the immunizing fusion protein to produce antibody PKD2-CFP.
Human and murine tissue lysates were prepared by homogenizing fresh tissue in lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1 mM ethylenediaminetetra-acetic acid, and CompleteTM protease inhibitors) (Boehringer Mannheim, Mannheim, Germany). For immunoprecipitation, human kidney lysates, prepared as above or in vitro-translated flPKD2, were incubated with antibodies coupled to protein A-Sepharose (Pharmacia). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out under reducing or nonreducing conditions in 7.5% gels.
For Western blotting, proteins were transferred onto nitrocellulose membranes and blocked in 5% milk protein before incubation with the primary antibody overnight at 4°C. An anti-rabbit Ig/horseradish peroxidase conjugate was used for detection. For detection of polycystin-2 in Western blots following immunoprecipitation, antibody PKD2-CFP was biotinylated with biotin-NHS (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions and detected after blocking with avidin/biotin blocking reagents (Vector Laboratories) using streptavidin- horseradish peroxidase. All immunoblots were visualized by chemiluminescence. 35S-Methionine-labeled polycystin-2 was visualized after polyacrylamide gel electrophoresis by autoradiography. Gels were fixed in 10% acetic acid/25% isopropanol for 30 min and soaked in AMPLIFYTM (Amersham) for 20 min before vacuum drying and autoradiography.
Subcellular Fractionation
Fresh human kidney tissue was obtained from nephrectomy specimens and
minced at scissor-point in ice-cold 5 mM Tris-HCl buffer, pH 7.4, containing
0.25 M sucrose, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM
MgCl2, and 2 mM NaF before homogenization. The plasma membrane
fraction was prepared as described previously
(23) with total particulate
and cytosol fractions generated after a 200,000 x g
centrifugation step. All preparative procedures were performed at 4°C.
Fractions were analyzed by Western blotting under nonreducing conditions using
antibody PKD2-CFP as described above.
Immunohistochemistry
All tissue samples examined were 5-µm sections of formalin-fixed,
paraffin-embedded tissues. A standard two-stage indirect immunoperoxidase
staining protocol was used for all tissues (Vectastain ABC System; Vector
Laboratories) as described previously
(15). As controls, sections
were stained with antibody diluent alone (5% goat serum in Tris-buffered
saline), preimmune serum, and antibody pre-absorbed with the immunizing
peptide. Incubations with tissue sections were carried out at 4°C
overnight in all cases, and subsequent steps were carried out at room
temperature. Staining was visualized with either diaminobenzidine (brown) or
VIP (purple) (Vector Laboratories), and sections were counterstained with
Gill's hematoxylin. Antibody PKD2-NFP was used at a dilution of 1:50.
Antibodies to polycystin-1, band 3 (AE1) and the epithelial sodium channel
ß subunit were used as described previously
(15,24,25).
Band 3 antibody BRIC155 (kindly provided by Michael Tanner, University of
Bristol, United Kingdom) was used to identify type A intercalated cells in
collecting tubules and collecting duct (CD), and the epithelial sodium channel
ß subunit antibody (kindly provided by Dr. Cecilia Canessa, Yale
University, New Haven, CT) was used to identify distal convoluted tubule
(DCT), cortical collecting ducts, and outer medullary collecting ducts.
Confocal Immunofluorescence Microscopy
Sections of fresh-frozen human kidney (5 µm) were fixed in 1%
formaldehyde for 1 min. Primary and secondary antibodies were applied for 2 h
at room temperature. Dual staining was performed using anti-E-cadherin
antibody (catalog no. BTA 1; R&D Laboratories, Abingdon, United Kingdom),
anti-ZO-1 antibody (catalog no. 1520; Chemicon, Temecula, CA), and
tetramethylrhodamine isothiocyanate-conjugated Arachis hypogaea
lectin (catalog no. L-7381; Sigma, Poole, United Kingdom). Secondary reagents
were FITC-conjugated goat anti-rabbit Ig (Jackson, Baltimore, MD) and
tetram-ethylrhodamine isothiocyanate-conjugated goat anti-mouse Ig (Jackson).
Six washes of 15 min each in phosphate-buffered saline were carried out at
room temperature after application of primary and secondary reagents. Sections
were examined using a Bio-Rad MRC 1000 confocal microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using lysates from multiple human and murine tissues and cultured cell lines, antibody PKD2-CFP (1:5000 dilution) recognized a single band under nonreducing conditions by Western blotting (Figure 2a). The band migrated at approximately 140 kD when compared with molecular weight markers run under reducing conditions. Bands of identical size were seen in human adult and fetal kidney, human fetal liver, and murine adult kidney. No signal was detected in human fetal and murine adult brain.
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Subcellular Fractionation
By Western blotting, polycystin-2 was shown to be present in adult human
whole kidney lysate. After fractionation, immunoreactivity was absent from the
cytosolic fraction and present in the total particulate and plasma membrane
fractions consistent with its proposed plasma membrane localization
(Figure 2b).
Polycystin-2 Expression in Cultured Cells
Polycystin-2 expression was determined in several cell lines including
Madin-Darby canine kidney (MDCK) cells, human umbilical vein endothelial
cells, and Cos-7 cells. Western blotting of cell lysates confirmed the
presence of a specific polycystin-2 band in MDCK and Cos-7 cells
(Figure 2a). No consistent
pattern of subcellular distribution was observed by confocal
immunofluorescence microscopy between different cell lines. A punctate
cytoplasmic pattern was seen in the CD-derived MDCK cells, while a
Golgi/endoplasmic reticulum staining pattern was seen in Cos-7 cells. No
staining was seen in human umbilical vein endothelial cells.
Polycystin-2 Expression in Normal Human Adult Kidney
Antibody PKD2-NP consistently gave the strongest staining in
paraffin-embedded tissues. No staining was observed with preimmune serum, and
all tissue staining was abolished by preabsorption with the immunizing peptide
(Figure 3, a and b). Staining
was unaffected by antigen retrieval techniques such as microwaving or trypsin
digestion. Localization was identical to that obtained with antibody PKD2-CP
(Figure 3c). However, staining
with this antibody produced a weak signal at high concentrations and it was
therefore not used for routine immunohistochemistry. No tissue staining was
observed with antibody PKD2-CFP (Table
1). In sections from six normal human adult kidneys obtained from
tumor resection specimens or autopsy, strong expression of polycystin-2 was
seen in the medullary thick ascending limbs of the loop of Henle and cortical
distal tubules, with very weak expression in outer medullary CD
(Figure 3, d, e, and k). The
proximal convoluted tubule (PCT), interstitium, glomeruli, and vasculature
were consistently negative. Staining was markedly accentuated on the
basolateral aspect of tubular epithelial cells and extended into the basal
cytoplasm (Figure 3, e and
f).
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Polycystin-1 expression was determined in the same tissues. A great proportion of cortical distal tubules was positive compared to polycystin-2, and expression was strongest in cortical and medullary CD (Figure 3, d, g, and l). Adjacent 5-µm sections of normal adult human kidney were stained with anti-FP-LRR and anti-PKD2-NP antibodies to determine the degree of colocalization of polycystin-1 and polycystin-2 expression (Figure 3, d and g). Precise colocalization could be seen in only a subset of cortical tubules. All tubules that expressed polycystin-2 expressed polycystin-1, while many other tubules expressed polycystin-1 alone. To determine the identity of the subset of tubules that were polycystin-2-positive, antibodies to the epithelial sodium channel ß subunit and band 3 (anion exchanger-1) were used (24,25). These antibodies recognize the DCT, cortical collecting ducts and outer medullary collecting ducts, and type A intercalated cells in collecting tubules and CD, respectively (26,27). Staining of adjacent sections for polycystin-2 and band 3 demonstrated marked differences in expression (Figure 3, h and i). Except for a minority of cortical tubules, the staining pattern was nonoverlapping. More extensive overlap with the epithelial sodium channel ß subunit was seen. Polycystin-2 expression was therefore mainly localized to the thick ascending limbs and the DCT.
Polycystin-2 Expression in Adult Mouse Kidney
Antibody PKD2-NP was also used to stain paraffin-embedded murine tissues.
No staining was seen using preimmune serum, and it was abolished by
preabsorption with the immunizing peptide
(Figure 4a). In normal adult
mouse kidney, the highest levels of expression of polycystin-2 were also seen
in the medullary thick ascending limbs of the loop of Henle and cortical
distal tubules, with weaker expression in inner medullary CD and urothelium
(Figure 4, b through f). Staining was also markedly accentuated on the basolateral aspect of tubular
epithelial cells extending into the basal cytoplasm
(Figure 4d). Very weak basal
staining of the PCT could also be determined in some sections
(Figure 4d). The interstitium,
glomeruli, and vasculature were consistently negative. Therefore, the pattern
of polycystin-2 expression in adult kidney was identical between human and
mouse.
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Polycystin-2 Expression in Fetal Kidney and Other Normal Tissues
The pattern of tubular staining seen in adult human kidney was also
observed in fetal kidney at a variety of gestational ages ranging from 22 to
40 wk (Figure 5). At 17 wk,
polycystin-2 expression in the kidney was barely detectable in ureteric bud
and primitive tubules (Figure
5a). No staining of commaand S-shaped bodies was seen. In later
gestation, staining intensity increased in primitive tubules eventually
adopting the adult pattern of distribution by 30 wk
(Figure 5, b and c). The same
tissues stained with anti-FP-LRR showed stronger expression of polycystin-1 at
all stages in ureteric bud-derived structures with predominant cortical
tubular and medullary CD expression (Figure
5, d through f).
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The same pattern of expression was also seen in the developing mouse kidney (Figure 5, g through i). No staining was seen in the ureteric bud or metanephric blastema at day 11.5 post coitus, but by day 14.5 post coitus weak staining of ureteric bud and primitive tubules was seen (Figure 5h). Comma- and S-shaped bodies remained negative. By term, strong basolateral staining of inner cortical distal tubules was seen while the nephrogenic zone remained negative. Medullary staining was present in thick ascending limbs in a pattern identical to adult tissues (Figure 4). CD were positive only when seen in adult tissues (Figure 4f). Polycystin-1 expression was detected at higher levels at days 11.5 and 14.5 in ureteric bud and primitive tubules with apical accentuation, while comma- and S-shaped bodies were again negative (Figure 5i).
Extrarenal expression of polycystin-2 was seen in the human fetus and adult (Figure 6). Expression was observed in a variety of epithelial cell types. For example, staining of epithelial cells of the developing bronchial glands of the lung was seen at 14 wk, with strong expression in the epithelium of the trachea and in the chondrocytes adjacent to developing bronchi. Expression was also seen in squamous epithelial cells of the esophagus at 20 wk (Figure 6a). Nonepithelial sites of expression included fetal adrenal cells at 14 wk (Figure 6b), parathyroid gland at 22 wk (Figure 6c), as well as cardiac myocytes and the mesenchymal cells of developing cardiac valves (Figure 6d). Very weak staining of pancreatic duct epithelial cells and islets was seen at 22 wk gestation with stronger staining in adult tissues (Figure 6, e and f). Weak bile duct staining was only seen in adult tissues (Figure 6g). No staining of hepatocytes or the exocrine pancreas was seen. Other sites of expression included weak staining of colonic mucosal epithelial cells (Figure 6h). Adult skin, spleen, lung, breast, esophagus, and cardiac muscle were negative.
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Polycystin-2 Expression in Human Cystic Tissues
Polycystin-2 expression was examined in sections of ADPKD kidney and liver
(Figure 7). Information about
linkage to either PKD1 or PKD2 was not available in these cases. In sections
from six different ADPKD tissues, the majority of cysts, approximately 80%,
stained positively (Figure 7, a through
c). Sections from multicystic dysplastic kidneys also demonstrated
cystic epithelial staining (Figure
7d).
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Subcellular Localization of Polycystin-1 and Polycystin-2
Confocal Microscopy. PKD-NP and PKD2-CP gave identical patterns of
staining in normal adult kidney frozen sections. Preimmune serum alone
produced no staining, and staining with PKD2-CP and PKD-NP could be abolished
by preincubation with the relevant immunizing peptide
(Figure 8a). In sections of
renal cortex, strong staining was seen in distal nephron segments
(Figure 8b). No staining was
seen in the PCT, glomeruli, or vascular structures. The distal tubular
distribution of polycystin-2 was confirmed by costaining with Arachus
hypogaea lectin, which is specific for the apical surface of distal
tubular and collecting duct epithelial cells
(Figure 8b). Costaining with
ZO-1 (which identifies the zonula occludens) and E-cadherin (which identifies
the lateral cell membrane) was used to orientate the cell surfaces
(Figure 8, c and d). High power
images of the majority of individual tubular epithelial cells showed
polycystin-2 staining in a pattern identical to that observed in paraffin
sections and consistent with a basolateral membrane distribution
(Figure 8c). Occasional
cytoplasmic staining pattern was seen in some cells. Polycystin-1 staining in
identically prepared frozen sections from the same kidney tissue, with
anti-FP-LRR and anti-FP-46 to 1, demonstrated predominant localization to the
lateral aspect of the basolateral membrane and precise colocalization with
E-cadherin (Figure 8, e and f). The nuclear staining observed with anti-FP-LRR has previously been reported as
nonspecific (15).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two recent reports describing the distribution of polycystin-2 have provided some conflicting data. In humans, its expression pattern has been reported to be identical and coordinate with polycystin-1, providing support for a direct interaction between these proteins, while in mouse clear differences were found in its developmental and tissue expression (21,22). We therefore compared expression in human and mouse using the same antibody for tissue localization.
Our observations of polycystin-2 expression in human and mouse, using an N-terminal antibody, show striking similarity with the report of its cellular and subcellular localization using a C-terminal antibody in murine tissues (8,21). Polycystin-2 expression was highest in the thick ascending limb of the loop of Henle and the DCT in normal adult human and murine kidney. Much weaker staining was seen in medullary CD. This contrasts with the predominant CD expression pattern previously reported by some investigators for polycystin-1 and more recently noted by Ong et al. (13,15,22). Expression levels have also been reported to be consistently higher during development with maximal expression coinciding with the period of tubule differentiation and maturation (13,28). Again, this contrasts with polycystin-2, which maintains high levels of tubular expression in the adult kidney.
To investigate this further, we undertook staining of serial tissue sections from human adult renal cortex and medulla with antibodies to polycystin-1 and polycystin-2 (Figure 3). This demonstrated intriguing differences in the expression pattern between the two proteins. In the cortex, overlap of expression occurred only in a subset of tubules, with polycystin-1 expression being more extensive including the cortical CD (Figure 3g). The polycystin-2-positive cortical tubules were identified as being predominantly DCT by staining of adjacent sections with the segment-specific markers band 3 and the epithelial Na channel ß subunit (25,26). Consistent with this, in the medulla polycystin-1 was strongly expressed in CD and only weakly expressed in the thick ascending limbs of the loop of Henle, while polycystin-2 had the reciprocal pattern of expression (Figure 3, k and l). Therefore, polycystin-1 and polycystin-2 stain the same nephron segments but have a reciprocal gradient of expression. These differences in the spatial pattern of expression of the polycystins were also seen in the developing human fetal kidney, where temporal differences of expression also occurred. While renal expression levels of both proteins increased during gestation, polycstin-1 expression appears stronger during nephrogenesis than polycystin-2, an observation apparent in both human and murine tissues (Figure 5). In addition, polycystin-1 expression appears to decline in adult compared to fetal kidney, while polycystin-2 expression is maximal in the mature kidney. This suggests that polycystin-1 and polycystin-2 may have complementary but differing roles in the development and maintenance of tubular epithelial cell function.
While extensive overlap in extrarenal tissue expression does occur between polycystin-1 and polycystin-2, discordant expression was seen in some tissues such as pancreatic islet cells. This also suggests that both proteins may have independent tissue-specific functions in addition to a functional connection in kidney, liver, and pancreatic epithelial cells.
Our study of the precise temporal, cellular, and subcellular distribution of polycystin-2 was designed to investigate whether it was expressed coordinately with polycystin-1, both temporally and spatially, and whether they had an identical subcellular distribution. Although not confirming a direct interaction between polycystin-1 and polycystin-2 in vivo, their colocalization to the same cell and subcellular location would be expected if they formed a membrane-associated complex. The predominant localization of polycystin-1 to the lateral compartment of the basolateral membrane in normal adult human kidney strongly suggests a role in cell-cell adhesion or signaling (Figure 8). Its previously reported localization to all membrane surfaces in fetal kidney epithelial cells may reflect the state of differentiation and maturation of the kidney (13,14). Terminal differentiation of tubular epithelial cells therefore results in the restricted pattern of subcellular distribution. Identical changes in the subcellular distribution of the neural-cell adhesion molecule L1 have also been reported during nephrogenesis (29). In contrast to this, immunohistochemistry and confocal microscopy both localize polycystin-2 to the basolateral membrane of renal tubular epithelial cells (Figures 3,4, and 8). Although there is some overlap in the membrane localization of the two proteins, the differences suggest that polycystin-2 may function independently of polycystin-1. Future studies of polycystin function will have to overcome the technical difficulties of analyzing such large proteins and demonstrate that these proteins associate in vivo. However, the identification of other interacting molecules, especially extracellular ligands of polycystin-1 and the development of in vitro expression systems for functional studies, will provide the most important steps in determining the role of these molecules in normal cellular function.
The primary structure of polycystin-1 suggests that it is involved in cell-cell or cell-matrix interactions, and its unique composition of novel and recognizable protein domains suggests that it may have a variety of extracellular and intracellular protein ligands and more than one basic function (9,30). The demonstration of potential interactions of polycystin-1, interactions with heterotrimeric G proteins, regulators of G protein signaling, and the activation of AP-1 and Wnt signaling also support this hypothesis (19,20,31,32,33). In contrast, the homology of polycystin-2 with the subunits of voltage-activated calcium channels and the trp family of agonist-activated capacitative calcium channels suggests that it functions as part of an ion channel (16,17,18). Although some in vitro evidence favors the polycystins acting together at the cell surface, different temporal and spatial patterns of localization now suggest that they may have independent functions involving multiple signaling pathways that converge on a single effector molecule such as AP-1 (34). This transcription factor is involved in a wide range of cellular functions and may regulate the full maturation of developing tubular epithelial cells, which when perturbed may result in cyst formation.
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