J Am Soc Nephrol 11: 814–827, 2000 Cellular and Subcellular Distribution of Polycystin-2, the Product of the PKD2

LUKAS FOGGENSTEINER,† A. PAUL BEVAN,* RUTH THOMAS,* NICHOLAS COLEMAN,‡ CATHERINE BOULTER,§ JOHN BRADLEY,† OXANA IBRAGHIMOV-BESKROVNAYA,ʈ KATHERINE KLINGER,ʈ and RICHARD SANDFORD* Departments of *Medical Genetics, †Medicine, and ‡Pathology, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom; §Department of Genetics, University of Cambridge, Cambridge, United Kingdom; and ʈGenzyme Genetics, Framingham, Massachusetts.

Abstract. Mutations in the PKD1 and PKD2 account for and developmentally regulated expression of polycytin-2, with 85 and 15% of cases of autosomal dominant polycystic highest levels in the kidney in the thick ascending limbs of the disease, respectively. Polycystin-2, the product of the PKD2 loop of Henle and the distal convoluted tubule. In contrast, gene, is predicted to be an integral membrane protein with polycystin-1 expression, while localizing to the same tubular homology to a family of voltage-activated Ca2ϩ channels. In segments, was highest in the collecting ducts. Immunohisto- vitro studies suggest that it may interact with polycystin-1, the chemical staining and immunofluorescence microscopy local- PKD1 gene product, via coiled-coil domains present in their ized polycystin-2 to the basolateral plasma membrane of kid- C-terminal domains. In this study, the cellular and subcellular ney tubular epithelial cells compared with the junctional distribution of polycystin-2 is defined and compared with localization of polycystin-1. Differences in the developmental, polycystin-1. A panel of rabbit polyclonal antisera was raised cellular, and subcellular expression of polycystin-1 and poly- against polycystin-2 and shown to recognize a single band cystin-2 suggest that they may be able to function indepen- consistent with polycystin-2 in multiple tissues and cell lines dently of each other in addition to a potential in vivo interaction by immunoprecipitation and Western blotting. Immunostaining via their C-termini. of and murine renal tissues demonstrated widespread

Autosomal dominant polycystic kidney disease (ADPKD) is mutations in PKD2 cause a milder phenotype than those in one of the most common inherited disorders of , with a PKD1 with a slower decline in renal function, the clinical prevalence of approximately 1 in 800 in all ethnic groups (1). features are indistinguishable between the two groups (6). It is characterized by progressive bilateral renal cyst formation, Targeted mutations in the murine pkd1 and pkd2 genes also which results in a gradual decline in renal function such that produce a cystic phenotype, suggesting that the protein prod- half of affected individuals require renal replacement therapy ucts of these two genes, polycystin-1 and polycystin-2, may by their sixth decade (2). Eight percent of all individuals in interact directly or form separate parts of a common cellular renal replacement programs have a diagnosis of ADPKD. pathway (7,8). Cysts are also seen in the liver and more rarely in the pancreas. Polycystin-1 is composed of a novel array of structural and Other more variable features of the disease include cardiovas- functional domains that suggest it is an integral plasma mem- cular and connective tissue abnormalities such as hypertension, brane glycoprotein involved in cell-cell or cell-matrix interac- mitral valve prolapse, berry aneurysms of the cerebral circu- tions (9,10). Its precise function is still unknown. A consensus lation, and abdominal wall hernias (3). derived from the many published reports of the cellular and Eighty-five percent of cases of ADPKD are due to mutations subcellular localization of polycystin-1 defines it as a devel- in PKD1 with the remainder occurring in PKD2 (4). A few opmentally regulated integral membrane protein expressed at ADPKD families appear unlinked to either PKD1 or PKD2, highest levels in fetal tissues (11–15). In adult tissues it is suggesting the presence of a rare third , PKD3 (5). While predominantly localized to the distal part of the nephron and collecting ducts and other epithelial structures such as bile and pancreatic ducts (13,15). Received June 4, 1999. Accepted September 21, 1999. Polycystin-2 is predicted to be an integral membrane protein Correspondence to Dr. Richard Sandford, Department of Medical Genetics, with intracellular N- and C-termini (16). It has significant Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Hills homology to the pore-forming domains of a number of voltage- Road, Cambridge CB2 2XY, United Kingdom. Phone: ϩ44 1223 762616; Fax: ϩ44 1223 331206; E-mail: [email protected] activated cation channels, suggesting that it may function as an 1046-6673/1105-0814 subunit (16,17). A new member of the polycystin Journal of the American Society of Nephrology family, polycystin-L/polycystin-2L, has recently been de- Copyright © 2000 by the American Society of Nephrology scribed (17,18). It has extensive homology to polycystin-2 but J Am Soc Nephrol 11: 814–827, 2000 Expression Pattern of Polycystin-2 815

is not thought to be involved in the pathogenesis of ADPKD. peptide coupled to NHS-Sepharose 4 Fast Flow (Pharmacia, Uppsala, Its role in other renal cystic diseases remains to be determined. Sweden), according to the manufacturer’s instructions, to generate The predicted in vivo interaction between polycystin-1 and antibodies PKD2-NP and PKD2-CP, respectively. Nucleotides 2134 polycystin-2 has been demonstrated in vitro, suggesting that to 2973 of PKD2, which encode the predicted C-terminal cytoplasmic they may form subunits of a large membrane-associated com- region of polycystin-2, were cloned into pGEX-4T (Pharmacia) to produce a glutathione S-transferase (GST)-PKD2 bacterial fusion plex (19,20). They should therefore have extensive overlap in protein. Polyclonal antiserum was raised in New Zealand White cellular and subcellular localization. Staining in the mouse rabbits and affinity-purified against immobilized GST (Pierce, Rock- using an anti-polycystin-2 antibody recognizing the C-terminal ford, IL) to remove anti-GST antibodies and against the immunizing region of the protein has demonstrated a distinctive, develop- fusion protein to produce antibody PKD2-CFP. mentally regulated expression pattern (21). In the mature kid- Human and murine tissue lysates were prepared by homogenizing ney, strongest staining in a basolateral distribution was seen in fresh tissue in lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% the medullary thick ascending limbs of the loop of Henle and Triton X-100, 1 mM ethylenediaminetetra-acetic acid, and Com- the distal convoluted tubule, with weaker staining of the prox- plete™ protease inhibitors) (Boehringer Mannheim, Mannheim, Ger- imal tubules and cortical and medullary collecting ducts (21). many). For immunoprecipitation, human kidney lysates, prepared as Polycystin-2 expression in humans has been reported to be above or in vitro-translated flPKD2, were incubated with antibodies more widespread than in mouse with coordinate expression coupled to protein A-Sepharose (Pharmacia). Sodium dodecyl sulfate- polyacrylamide gel electrophoresis was carried out under reducing or with polycystin-1 (22). Staining in the kidney was seen in most nonreducing conditions in 7.5% gels. nephron segments with highest levels seen in medullary col- For Western blotting, were transferred onto nitrocellulose lecting ducts. membranes and blocked in 5% milk protein before incubation with the In this report we describe the generation of a panel of primary antibody overnight at 4°C. An anti-rabbit Ig/ polycystin-2-specific antibodies and their use to define poly- peroxidase conjugate was used for detection. For detection of poly- cystin-2 expression in human and mouse. Identical staining cystin-2 in Western blots following immunoprecipitation, antibody patterns in human and mouse define polycystin-2 as a devel- PKD2-CFP was biotinylated with biotin-NHS (Vector Laboratories, opmentally regulated integral membrane protein that localizes Burlingame, CA) according to the manufacturer’s instructions and to the basolateral plasma membranes of renal tubular epithelial detected after blocking with avidin/biotin blocking reagents (Vector Laboratories) using streptavidin- horseradish peroxidase. All immu- cells. Differences between the temporal and spatial expression 35 of polycystin-1 and polycystin-2 suggest that while they may noblots were visualized by chemiluminescence. S-Methionine-la- beled polycystin-2 was visualized after polyacrylamide gel electro- interact and form a large membrane-associated complex, they phoresis by autoradiography. Gels were fixed in 10% acetic acid/25% may also function independently of each other. isopropanol for 30 min and soaked in AMPLIFY™ (Amersham) for 20 min before vacuum drying and autoradiography. Materials and Methods Construction and in Vitro Translation of a PKD2 Subcellular Fractionation cDNA Fresh human kidney tissue was obtained from nephrectomy spec- PKD2 cDNA clones were identified from an adult human liver imens and minced at scissor-point in ice-cold 5 mM Tris-HCl buffer, cDNA library constructed in pCMV-SPORT (Clontech, Palo Alto, pH 7.4, containing 0.25 M sucrose, 1 mM benzamidine, 1 mM

CA). No clones were full-length, so clone K1-1 (a kind gift from S. phenylmethylsulfonyl fluoride, 1 mM MgCl2, and 2 mM NaF before Somlo, Yale University School of Medicine, New Haven, CT), which homogenization. The plasma membrane fraction was prepared as contained the 5Ј 1.4-kb segment of PKD2, was used to generate a described previously (23) with total particulate and cytosol fractions full-length construct in pcDNA3 (Invitrogen, San Diego, CA). The generated after a 200,000 ϫ g centrifugation step. All preparative EcoRI site in the 3Ј untranslated region of PKD2 (nucleotide position procedures were performed at 4°C. Fractions were analyzed by West- 4844) was removed, and the 1.4-kb EcoRI insert of K1-1 was ligated ern blotting under nonreducing conditions using antibody PKD2-CFP into the remaining EcoRI site at nucleotide position 1395. Restriction as described above. analysis and complete sequencing confirmed the integrity of the final full-length PKD2 clone, flPKD2. In vitro translation of the flPKD2 construct and a luciferase control Immunohistochemistry ␮ incorporating 35S-methionine (Amersham, Buckinghamshire, United All tissue samples examined were 5- m sections of formalin-fixed, Kingdom) was performed using the TNT Coupled Reticulocyte Ly- paraffin-embedded tissues. A standard two-stage indirect immunoper- sate System (Promega, Madison, WI) according to the manufacturer’s oxidase staining protocol was used for all tissues (Vectastain ABC protocol. System; Vector Laboratories) as described previously (15). As con- trols, sections were stained with antibody diluent alone (5% goat serum in Tris-buffered saline), preimmune serum, and antibody pre- Antibody Preparation, Western Blotting, and absorbed with the immunizing peptide. Incubations with tissue sec- Immunoprecipitation tions were carried out at 4°C overnight in all cases, and subsequent Peptides NH2-MVNSSRVQPQQPDCys-COOH and NH2-CEG- steps were carried out at room temperature. Staining was visualized MEGAGGNGSSNVH-COOH corresponding to amino acids 1 to 12 with either diaminobenzidine (brown) or VIP (purple) (Vector Labo- and 953 to 968 of polycystin-2 were synthesized and conjugated to ratories), and sections were counterstained with Gill’s hematoxylin. keyhole limpet hemocyanin (Immune Systems Limited, Paignton, Antibody PKD2-NFP was used at a dilution of 1:50. Antibodies to United Kingdom). Polyclonal antisera were raised in New Zealand polycystin-1, band 3 (AE1) and the epithelial ␤ White rabbits and affinity-purified against 10 mg of the immunizing subunit were used as described previously (15,24,25). Band 3 anti- 816 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 814–827, 2000

Figure 1. Characterization of polycystin-2 antibodies. (a) Specific recognition of the glutathione S-transferase (GST)-polycystin-2 fusion protein by PKD2-CFP and PKD2-CP. The GST-polycystin-2 fusion protein (approximately 60 kD) was run on a 10% polyacrylamide gel and probed with PKD2-CFP (lane 1), PKD2-CFP preabsorbed with the immunizing fusion protein (lane 2), PKD2-CP (lane 3), and PKD2-CP preabsorbed with the immunizing peptide (lane 4). Both antibodies specifically recognized the fusion protein, which contains the C-terminal cytoplasmic domain of polycystin-2. (b) Immunoprecipitation of in vitro-translated full-length PKD2 construct with PKD2-NP. In vitro- translated, 35S-methionine-labeled polycystin-2 and luciferase were immunoprecipitated using antibody PKD2-NP. The lysates and immuno- precipitation reactions were run on a 7.5% polyacrylamide gel in reducing conditions and visualized by autoradiography. The in vitro-translated PKD2 product ran at approximately 110 kD under reducing conditions (lane 5). No band of similar size was seen in the in vitro-translated luciferase control reaction (lane 6). No protein was immunoprecipitated from the luciferase control (lane 7), whereas the 110-kD band was immunoprecipitated from the PKD2 cDNA reaction (lane 8). This could be abolished by preabsorption of the antibody with the immunizing peptide (lane 9), and no band was detected using the preimmune serum (lane 10). (c) Immunoprecipitation of polycystin-2 from human kidney lysate with PKD2-NP and PKD2-CP. Human adult kidney lysates were run on 7.5% polyacrylamide gels under nonreducing conditions and probed with PKD2-CFP preimmune serum (lane 11), PKD2-CFP (lane 12), and PKD2-CFP preabsorbed with immunizing fusion protein (lane 13). A single specific band of approximately 140 kD corresponding to polycystin-2 was seen and was abolished by preabsorption of PKD2-CFP with the immunizing fusion protein. Immunoprecipitations from human kidney lysates were run under identical conditions and probed with biotinylated PKD2-CFP. Immunoprecipitations were performed with PKD2-NP (lane 14), PKD2-CP (lane 15), PKD2-NP preimmune serum (lane 16), and PKD2-CP preimmune serum (lane 17). Both PKD2-NP and PKD2-CP, but not preimmune serum, identified a single immunoreactive band of the same molecular mass. J Am Soc Nephrol 11: 814–827, 2000 Expression Pattern of Polycystin-2 817

Table 1. Properties of anti-polycystin-2 antibodies

Species and Type Western Antibody of Antibody Immunohistochemistry Immunofluorescence Blot Immunoprecipitation

PKD2-NP Rabbit polyclonal ϩϩ ϩϩ Ϫ ϩϩ PKD2-CP Rabbit polyclonal ϩ/Ϫϩ Ϫϩϩ PKD2-CFP Rabbit polyclonal ϪϩϩϩNot done

Confocal Immunofluorescence Microscopy Sections of fresh-frozen human kidney (5 ␮m) were fixed in 1% 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 Laborato- ries, Abingdon, United Kingdom), anti-ZO-1 antibody (catalog no. 1520; Chemicon, Temecula, CA), and tetramethylrhodamine isothio- cyanate-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 -conjugated goat anti-mouse Ig (Jack- son). 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.

Results Antibody Production and Characterization Antibodies to polycystin-2 were raised in rabbits against N-terminal and C-terminal keyhole limpet hemocyanin-conju- gated peptides and a C-terminal GST-cytoplasmic tail fusion protein. Both of the C-terminal antibodies, PKD2-CP and PKD2-CFP, recognized the C-terminal GST fusion protein (molecular mass, approximately 60 kD) by Western blot anal- ysis (Figure 1a). Specificity was confirmed by preincubation of Figure 2. Western blotting of polycystin-2 from tissue and cell ly- the antibodies with the appropriate immunizing peptide or sates. Tissue and cell lysates were prepared as described in Materials fusion protein, which completely abolished recognition of the and Methods and run on 7.5% polyacrylamide gels in nonreducing GST fusion protein. PKD2-NP was shown to specifically rec- conditions. (a) Western blotting of cell line and tissue lysates probed ognize the 110-kD 35S-methionine-labeled in vitro-translated with PKD2-CFP. Polycystin-2 was detected in normal adult human flPKD2 by immunoprecipitation using in vitro translated lu- kidney (lane 1), human fetal kidney (lane 2), human fetal liver (lane ciferase as a control (Figure 1b). Only PKD2-CFP recognized 4), adult mouse kidney (lane 6), COS-1 cells (lane 7), and Madin- Darby canine kidney (MDCK) cells (lane 8), but not in fetal human a band consistent with polycystin-2 by Western blotting under brain (lane 3) or adult mouse brain (lane 5). (b) Western blotting of nonreducing conditions. Recognition of this band was abol- human adult kidney homogenate fractions. Polycystin-2 was detected ished by preincubation of PKD2-CFP with the immunizing by PKD2-CFP under nonreducing conditions in total adult human GST fusion protein, and no signal was detected by the preim- kidney homogenate (lane 9). After centrifugation it was absent from mune serum (Figure 1c). Therefore, to further determine the the cytosol (lane 10) and present in the total particulate fraction (lane specificity of the anti-peptide antibodies PKD2-NP and PKD2- 11). It is also seen after isolation of the plasma membrane fraction CP, each was used for immunoprecipitation from human kid- (lane 12). ney lysates (Figure 1c). Biotin-labeled antibody PKD2-CFP was used to detect the immunoprecipitated proteins. Both antibodies specifically immunoprecipitated a band of identical size. This had the same molecular mass as the band detected by body BRIC155 (kindly provided by Michael Tanner, University of antibody PKD2-CFP in a Western blot of normal human kid- Bristol, United Kingdom) was used to identify type A intercalated cells in collecting tubules and collecting duct (CD), and the epithelial ney. Therefore, all antibodies recognized a single band consis- sodium channel ␤ subunit antibody (kindly provided by Dr. Cecilia tent with polycystin-2 either by immunoprecipitation or West- Canessa, Yale University, New Haven, CT) was used to identify distal ern blotting (Table 1). convoluted tubule (DCT), cortical collecting ducts, and outer medul- Using lysates from multiple human and murine tissues and lary collecting ducts. cultured cell lines, antibody PKD2-CFP (1:5000 dilution) rec- Figure 3. Polycystin-2 expression in adult human kidney. Anti-polycystin-1 antibody FP-LRR and anti-polycystin-2 antibody PKD2-NP were used to stain paraffin-embedded human adult kidney. In adult kidney, polycystin-2 expression was seen in the thick ascending limb of the loop of Henle (L), distal tubules (DT) including the distal convoluted tubule (DCT) and cortical collecting tubules (CT), with weak staining of the collecting duct (CD). No staining was seen in the proximal convoluted tubule (PCT) or glomerulus (G). (a) Normal adult kidney cortex stained with anti-PKD2-NP preimmune serum (ϫ200, VIP). (b) Normal adult kidney cortex stained with anti-PKD2-NP preabsorbed with the immunizing peptide (ϫ200, VIP). (c) Antibody PKD2-CP produced weak staining of distal tubules only in an identical pattern to PKD2-NP (ϫ200, diaminobenzid