Apical Plasma Membrane Mispolarization of NaK-ATPase in Polycystic Kidney Disease Epithelia Is Associated with Aberrant Expression of the β2 Isoform
2000; Elsevier BV; Volume: 156; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64726-8
ISSN1525-2191
AutoresPatricia D. Wilson, Olivier Devuyst, Xiaohong Li, Laura Gatti, D Falkenstein, Shawn Robinson, Douglas M. Fambrough, Christopher R. Burrow,
Tópico(s)Renal and related cancers
ResumoAutosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical 22Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of α1, α3, β1, and β2 subunits of NaK-ATPase. High levels of expression of α1 and β1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas β2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against α1 and β1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle's loop. In ADPKD kidneys, α1 and β2 subunits were localized to the apical epithelial cell membranes, whereas β1 was distributed throughout the cytoplasm and predominantly in the endoplasmic reticulum, but was not seen associated with cystic epithelial cell membranes or in cell membrane fractions. Polarizing, renal-derived epithelial Madin Darby canine kidney cells, stably expressing normal or N-terminally truncated chicken β1 subunits, showed selective accumulation in the basolateral Madin Darby canine kidney cell surface, whereas c-myc epitope-tagged chicken β2 or human β2 subunits accumulated selectively in the apical cell surface. Similarly, human ADPKD epithelial cell lines, which endogenously expressed α1 and β2 NaK-ATPase subunits, showed colocalization at the apical cell surface and coassociation by immunoprecipitation analysis. These results are consistent with a model in which the additional transcription and translation of the β2 subunit of NaK-ATPase may result in the apical mislocalization of NaK-ATPase in ADPKD cystic epithelia. Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical 22Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of α1, α3, β1, and β2 subunits of NaK-ATPase. High levels of expression of α1 and β1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas β2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against α1 and β1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle's loop. In ADPKD kidneys, α1 and β2 subunits were localized to the apical epithelial cell membranes, whereas β1 was distributed throughout the cytoplasm and predominantly in the endoplasmic reticulum, but was not seen associated with cystic epithelial cell membranes or in cell membrane fractions. Polarizing, renal-derived epithelial Madin Darby canine kidney cells, stably expressing normal or N-terminally truncated chicken β1 subunits, showed selective accumulation in the basolateral Madin Darby canine kidney cell surface, whereas c-myc epitope-tagged chicken β2 or human β2 subunits accumulated selectively in the apical cell surface. Similarly, human ADPKD epithelial cell lines, which endogenously expressed α1 and β2 NaK-ATPase subunits, showed colocalization at the apical cell surface and coassociation by immunoprecipitation analysis. These results are consistent with a model in which the additional transcription and translation of the β2 subunit of NaK-ATPase may result in the apical mislocalization of NaK-ATPase in ADPKD cystic epithelia. Autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal genetic disease inherited as a dominant trait in humans, with a prevalence of 1:1000 recently shown to be associated with mutations in the PKD1 or PKD2 genes in 85% and 10% of cases, respectively.1Dalgaard OZ Polycystic Disease of the Kidneys. Little, Brown, Boston1963: 1223-1258Google Scholar, 2Gabow PA Ikle DW Holmes JH Polycystic kidney disease: progressive analysis of nonazotemic patients and family members.Ann Intern Med. 1984; 101: 238-247Crossref PubMed Scopus (137) Google Scholar, 3Mochizuki TG Wu T Hayashi S Xenophontos L Veldhuisen B Saris J Reynolds D Cai Y Gabow P Pierides A Kimberling W Breuning MH Deltas CC Peters DJM Somlo S PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1198) Google Scholar, 4The European Polycystic Kidney Disease Consortium The polycystic kidney disease 1 gene encodes for a 14 kb transcript and lies within a duplicated region on chromosome 16.Cell. 1994; 77: 882-894Google Scholar ADPKD is characterized by massive cystic enlargement of renal tubules as a result of increased epithelial cell proliferation, aberrant fluid secretion, mispolarization of several membrane proteins, and extracellular matrix abnormalities.5Du J Wilson PD Abnormal polarized location of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD.Am J Physiol. 1995; 269: C487-C495PubMed Google Scholar, 6Wilson PD Pathogenesis of polycystic kidney disease: altered cellular function.in: Watson ML Torres VE In Polycystic Kidney Disease. Oxford Medical Publications, Oxford, UK1996: 125-163Google Scholar, 7Wilson PD Du J Norman JT Autocrine, endocrine and paracrine regulation of growth abnormalities in autosomal dominant kidney disease.Eur J Cell Biol. 1993; 61: 131-138PubMed Google Scholar, 8Wilson PD Sherwood AC Palla K Du J Watson R Norman JT Reversed polarity of Na+-K+-ATPase: mislocation to apical plasma membrane.Am J Physiol. 1991; 260: F420-F430PubMed Google Scholar Despite the recent cloning of the complete complementary DNAs (cDNAs) for PKD1 and PKD2, the function of the predicted encoded proteins remains obscure.9Hughes J Ward CJ Peral B Aspinwall R Clark K San Millan JL Gamble V Harris PC The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains.Nat Genet. 1995; 10: 151-160Crossref PubMed Scopus (785) Google Scholar, 10The International Polycystic Kidney Disease Consortium Polycystic kidney disease: the complete structure of the PKD-1 gene and its protein.Cell. 1995; 81: 289-298Abstract Full Text PDF PubMed Scopus (627) Google Scholar However, studies showing similarities between the expression and plasma membrane distribution of several proteins in ADPKD and fetal epithelia have suggested a potential role for the PKD1 protein (polycystin 1) in the regulation of renal development, epithelial polarization and differentiation, and matrix adhesion.11Wilson PD Burrow CR Autosomal dominant polycystic kidney disease: cellular and molecular mechanisms of cyst formation.Adv Nephrol. 1992; 21: 125-142PubMed Google Scholar, 12Wilson PD Falkenstein D The pathology of human renal cystic disease. In Tubulo-interstitial and cystic disease of the kidney.Curr Top Pathol. 1995; 88: 1-50Crossref PubMed Scopus (60) Google Scholar, 13Wilson PD Hreniuk D Gabow PA Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia.J Cell Physiol. 1992; 150: 360-369Crossref PubMed Scopus (83) Google Scholar This is further supported by recent studies showing expression of high levels of PKD1–encoded messenger RNA (mRNA) and protein in the ureteric bud structures of the developing kidney.14Ward CJ Turley H Ong AC Comley M Biddolph S Chetty R Ratcliffe PJ Gatter K Harris PC Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult and polycystic kidney.Proc Natl Acad Sci USA. 1996; 93: 1524-1528Crossref PubMed Scopus (218) Google Scholar, 15Wilson PD Epithelial cell polarity and disease.Am J Physiol. 1997; 272: F1-F9PubMed Google Scholar The kidney is an organ that functions to reabsorb essential fluid and ions, and this is facilitated by the strictly polarized distribution of numerous transporters, enzymes, and antigens distributed along the 10 distinct segments of the nephron in an epithelial cell type-specific fashion. The polarization of membrane proteins is a critical component in the differentiation of renal tubule epithelial cells and is largely established in the human metanephric kidney before birth. NaK-ATPase is an important, complex membrane protein of all cells including polarized renal epithelial cells. It exchanges sodium for potassium and functions as the ubiquitous electrogenic sodium pump in all animal cells and is responsible for the active transport of sodium ions out of cells. NaK-ATPase is very highly expressed in renal tubule epithelia, where it is restricted to the basolateral plasma membranes. This segregated distribution is associated with vectorial (apical-to-basal) transport of sodium and is a critical feature in the generation of sodium ion gradients, which in turn regulate other Na-coupled ion movements as well as osmotic fluid reabsorption. A major and pathophysiologically significant abnormality in ADPKD cystic epithelia is the mispolarization of a fully functional NaK-ATPase to the apical membranes of cyst epithelia. This in turn is associated with basal-to-apical sodium ion transport leading to luminal fluid accumulation.8Wilson PD Sherwood AC Palla K Du J Watson R Norman JT Reversed polarity of Na+-K+-ATPase: mislocation to apical plasma membrane.Am J Physiol. 1991; 260: F420-F430PubMed Google Scholar, 12Wilson PD Falkenstein D The pathology of human renal cystic disease. In Tubulo-interstitial and cystic disease of the kidney.Curr Top Pathol. 1995; 88: 1-50Crossref PubMed Scopus (60) Google Scholar, 16Mangoo-Karim R Uchic ME Grant M Shumate WA Calvet JP Park CH Grantham JJ Renal epithelial fluid secretion and cyst growth: the role of cyclic AMP.FASEB J. 1989; 3: 2629-2632Crossref PubMed Scopus (105) Google Scholar Some but not all other membrane and membrane-associated proteins show aberrant polarization in ADPKD cystic epithelia, including the epidermal growth factor receptor (EGFR), calpactin, ankyrin, fodrin, and E-cadherin.5Du J Wilson PD Abnormal polarized location of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD.Am J Physiol. 1995; 269: C487-C495PubMed Google Scholar, 6Wilson PD Pathogenesis of polycystic kidney disease: altered cellular function.in: Watson ML Torres VE In Polycystic Kidney Disease. Oxford Medical Publications, Oxford, UK1996: 125-163Google Scholar In its mature form, NaK-ATPase is a heterodimeric, complex, integral membrane protein composed of two α and two β subunits. NaK-ATPase α subunits are multimembrane-spanning, nonglycosylated proteins of 98 to 100 kd, and they contain the catalytically significant phosphorylation, ATP, and ouabain-binding sites. NaK-ATPase β subunits, by contrast, span the membrane only once, are variably glycosylated on their exterior aspect, accordingly range in molecular mass from 35 kd to 55 kd, and are absolutely required for the functioning of the assembled NaK-ATPase complex. At least three different isoforms of NaK-ATPase α subunits (α1, α2, α3) and three of NaK-ATPase β subunits (β1, β2, β3) have been cloned from a variety of species, organs, and developmental stages.17Good PJ Richter K Dawid IB A nervous system-specific isotype of the beta subunit of Na(+), K(+)-ATPase expressed during early development of Xenopus laevis.Proc Natl Acad Sci USA. 1990; 87: 9088-9092Crossref PubMed Scopus (92) Google Scholar, 18Kawakami K Nojima H Ohta T Nagano K Molecular cloning and sequence analysis of human NaK-ATPase beta-subunit.Nucleic Acids Res. 1986; 14: 2833-2844Crossref PubMed Scopus (102) Google Scholar, 19Kawakami K Ohta T Nojima H Nagano K Primary structure of the alpha-subunit of human Na,K-ATPase deduced from cDNA sequence.J Biochem (Tokyo). 1986; 100: 389-397Crossref PubMed Scopus (99) Google Scholar, 20Ovchinnikov Y Monastyrskaya GS Broude NE Ushkaryov Y Melkov AM Smirnov Y Malyshev IV Allikmets RL Kostina MB Dulubova IB Family of human Na+, K+-ATPase genes: structure of the gene for the catalytic subunit (alpha III-form) and its relationship with structural features of the protein.FEBS Lett. 1988; 233: 87-94Crossref PubMed Scopus (62) Google Scholar, 21Shull MM Lingrel JB Multiple genes encode the human Na+,K+-ATPase catalytic subunit.Proc Natl Acad Sci USA. 1987; 84: 4039-4043Crossref PubMed Scopus (121) Google Scholar Although the functional implications of these different isoforms remain obscure, the α1 and β1 subunits are the most ubiquitous and are characteristic of normal kidneys, whereas α3 expression is characteristic in brain, and α2 and α3 are frequently expressed in fetal tissues.22Emanuel JR Garetz S Stone L Levenson R Differential expression of Na+,K+-ATPase alpha- and beta-subunit mRNAs in rat tissues and cell lines.Proc Natl Acad Sci USA. 1987; 84: 9030-9034Crossref PubMed Scopus (87) Google Scholar, 23Shyjan AW Cena V Klein DC Levenson R Differential expression and enzymatic properties of the Na+,K(+)-ATPase alpha 3 isoenzyme in rat pineal glands.Proc Natl Acad Sci USA. 1990; 87: 1178-1182Crossref PubMed Scopus (101) Google Scholar Of particular interest, the NaK-ATPase β2 (β-adhesion molecule on glia) subunit is not only tissue restricted and developmentally regulated with high levels in human fetal liver and rat brain but it also functions as a neural adhesion molecule on glia and promotes neurite outgrowth, suggesting a role in normal differentiation of neurons in vivo.24Gloor S Antonicek H Sweadner KJ Pagliusi S Frank R Moos M Schachner M The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase.J Cell Biol. 1990; 110: 165-174Crossref PubMed Scopus (313) Google Scholar, 25Magyar JP Bartch U Wang ZQ Howells N Aguzzi A Wagner EF Schachner M Degeneration of neural cells in the central nervous system of mice deficient in the gene for the adhesion molecule on glia, the β2 subunit of murine Na,K-ATPase.J Cell Biol. 1994; 127: 835-845Crossref PubMed Scopus (90) Google Scholar, 26Martin-Vasallo P Dackowski W Emanuel JR Levenson R Identification of a putative isoform of the Na,K-ATPase beta subunit: primary structure and tissue-specific expression.J Biol Chem. 1989; 264: 4613-4618Abstract Full Text PDF PubMed Google Scholar, 27Muller HG Gloor S Schachner M Functional characterization of beta isoforms of murine Na,K-ATPase: The adhesion molecule on glia (AMOG/beta 2), but not beta 1, promotes neurite outgrowth.J Biol Chem. 1993; 268: 26260-26267PubMed Google Scholar The specific mechanisms underlying the sorting and polarized delivery of NaK-ATPase in renal epithelia are not fully understood. Early studies suggested direct targeting of NaK-ATPase to basolateral membranes of a polarized renal epithelial cell line, Madin Darby canine kidney (MDCK) cells, whereas others implicated indirect targeting followed by stabilization with the insoluble components of the membrane cytoskeleton, ankyrin, and fodrin.28Caplan MJ Anderson HC Palade GE Jamieson JD Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes.Cell. 1986; 46: 623-631Abstract Full Text PDF PubMed Scopus (132) Google Scholar, 29Hammerton RW Krzeminski KA Mays RW Ryan TA Wollner DA Nelson WJ Mechanism for regulating cell surface distribution of Na+,K(+)-ATPase in polarized epithelial cells.Science. 1991; 254: 847-850Crossref PubMed Scopus (240) Google Scholar More recent studies implicate both specific basolateral sorting signals in targeting of NaK-ATPase, the lipid environment in the trans-Golgi network, and complex formation with ankyrin, fodrin, and E-cadherin at the membrane.30Gundersen D Orlowski J Rodriguez-Boulan E Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.J Cell Biol. 1991; 112: 863-872Crossref PubMed Scopus (156) Google Scholar, 31Mays RW Siemers KA Fritz BA Lowe AW Van Meer G Nelson WJ Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.J Cell Biol. 1995; 130: 1105-1115Crossref PubMed Scopus (195) Google Scholar, 32Zurzolo C Rodriguez-Boulan E Delivery of Na+,K+-ATPase in polarized epithelial cells.J Cell Biol. 1993; 260: 550-552Google Scholar In the kidney epithelial cell line LLC-PK1, the N-terminal region has been implicated in sorting of HK-ATPase or NaK-ATPase,33Gottardi CJ Caplan MJ An ion-transporting ATPase encodes multiple apical localization signals.J Cell Biol. 1993; 121: 283-293Crossref PubMed Scopus (121) Google Scholar but interactions between the β subunits and a region in the C terminal of NaK-ATPase are also necessary for targeting.34Fambrough DM Lemas MV Hamrick M Emerick M Renaud KJ Inman EM Hwang B Takeyasu K Analysis of subunit assembly of the Na-K-ATPase.Am J Physiol. 1994; 266: C579-C589PubMed Google Scholar, 35Jaunin P Jaisser F Beggah AT Takeyasu K Mangeat P Rossier BC Horisberger JD Geering K Role of the transmembrane and extracytoplasmic domain of β subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.J Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar, 36Matter K Hunziker W Mellman I Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants.Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar Further studies show that proteins or protein complexes may contain multiple sorting determinants (eg, 36). The current studies have been undertaken to examine the mechanisms underlying the important mispolarization of NaK-ATPase in ADPKD epithelia. Our previous studies suggested that NaK-ATPase is specifically mistargeted to the apical plasma membranes of cystic epithelia in human ADPKD, that it is fully catalytically active and binds 125I-labeled ouabain, and that the mispolarization leads to basal-to-apical net transport of Na+ ions in membrane-grown ADPKD epithelia in vitro.8Wilson PD Sherwood AC Palla K Du J Watson R Norman JT Reversed polarity of Na+-K+-ATPase: mislocation to apical plasma membrane.Am J Physiol. 1991; 260: F420-F430PubMed Google Scholar, 12Wilson PD Falkenstein D The pathology of human renal cystic disease. In Tubulo-interstitial and cystic disease of the kidney.Curr Top Pathol. 1995; 88: 1-50Crossref PubMed Scopus (60) Google Scholar In this study we examine NaK-ATPase isoform expression and distribution in vivo and in vitro and suggest that revertant or persistent expression of the fetal kidney β2 isoform is a potentially important mechanism for the apical mispolarization in ADPKD. ADPKD kidneys and age-matched normal human kidneys were procured under sterile conditions, flushed with neutral salts solution (Collins or University of Wisconsin Solution), clamped, and stored in salts solution over ice for a maximum of 24 hours before primary culture. Parallel samples were frozen immediately in liquid nitrogen and stored at −80°C until use. ADPKD kidneys were designated as either end stage or early stage. End-stage kidneys (n = 12) contained no normal renal tubules, were greatly enlarged with multiple cysts, and were procured surgically during nephrectomies caused by the loss of renal function. Early-stage ADPKD kidneys (n = 6) contained both normal and cystic tubules, functioned without requirement for dialysis or transplant, and were procured from pretransplant cadavers. Normal kidneys (n = 18) were age-matched to the ADPKD samples (28–55 years) and were procured from transplant-prepared cadavers without warm ischemia. Additional samples of fetal kidneys (16–19-week gestation), collected in a similar manner, were used for control studies. All tissue samples were divided into three portions and used for microdissection and tissue culture and protein or RNA extraction, or they were fixed for pathology, immunostaining, or in situ hybridization, using 4% paraformaldehyde in diethyl pyrocarbonate–treated phosphate-buffered saline (PBS) at 4°C for 4 hours. Primary monolayer cultures of renal tubule segment-specific and ADPKD cyst epithelia were derived from normal adult proximal tubules, thick ascending limb of Henle, and collecting tubules and from ADPKD cyst-lining epithelia as previously described.37Wilson PD Monolayer cultures of microdissected renal tubule epithelial segments.J Tissue Cult Methods. 1991; 13: 137-142Crossref Scopus (16) Google Scholar, 38Wilson PD Dillingham MA Breckon R Anderson RJ Defined human renal tubular epithelia in culture: growth, characterization and hormonal response.Am J Physiol. 1985; 248: 436-443Google Scholar, 39Wilson PD Schrier RW Breckon RD Anderson RJ A new method for studying human polycystic kidney disease epithelia in culture.Kidney Int. 1986; 30: 371-378Crossref PubMed Scopus (91) Google Scholar Cells were grown in segment-selective media in 25 cm2 flasks coated with type I (rat tail) collagen (Collaborative Research, Lexington, MA), dissociated with collagenase (Life Technologies, Inc., Grand Island, NY), and seeded at confluent density on translucent permeable membrane supports (Transwel-COL, Costar, Cambridge, MA). For immunocytochemistry, confluent cell cultures on membranes were washed with PBS at 4°C for 3 minutes, fixed for 5 minutes in 4. paraformaldehyde in PBS, pH 7.4 at 4°C, and washed three times for 3 minutes each in cold PBS before immunostaining. MDCK cells were obtained from the American Type Culture Collection (Gaithersburg, MD), and a subclone, A4, was used to generate MDCK stable cell lines. Standard growth medium consisted of high-glucose Dulbecco's modified essential medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin. For use in transfection, cDNAs were cloned into expression plasmid pCB6. cDNAs encoding chicken NaK-ATPase α1 and β2 subunits have been described.40Lemas MV Fambrough DM Sequence analysis of DNA encoding an avian Na(+),K(+)-ATPase beta 2-subunit.Biochim Biophys Acta. 1993; 1149: 339-342Crossref PubMed Scopus (19) Google Scholar, 41Takeyasu K Tamkun MM Siegel NR Fambrough DM Expression of hybrid (Na+ + K+)-ATPase molecules after transfection of mouse Ltk-cells with DNA encoding the beta-subunit of an avian brain sodium pump.J Biol Chem. 1987; 262: 10733-10740Abstract Full Text PDF PubMed Google Scholar The chicken β2 subunit cDNA was modified to encode a c-myc epitope11Wilson PD Burrow CR Autosomal dominant polycystic kidney disease: cellular and molecular mechanisms of cyst formation.Adv Nephrol. 1992; 21: 125-142PubMed Google Scholar at the β2 subunit C terminus, which is in the βsubunit ectodomain. DNA encoding the human NaK-ATPase β2 subunit was cloned from a human retinal cDNA library in λ gt10, kindly provided by Jeremy Nathans (The Johns Hopkins University School of Medicine, Baltimore, MD). A probe for the human β2 subunit cDNA clones was generated by polymerase chain reaction (PCR), using primers based on the nucleotide sequence encoding the human β2 subunit.33Gottardi CJ Caplan MJ An ion-transporting ATPase encodes multiple apical localization signals.J Cell Biol. 1993; 121: 283-293Crossref PubMed Scopus (121) Google Scholar This probe was used to isolate a full-length clone (∼1.5 kb) from the library. Identity of the clone was confirmed by nucleotide sequencing. The 5′ end of the clone began 80 bp upstream from the reported nucleotide sequence of human β2 subunit cDNA (GenBank accession number M81181) and ended in the 3′ untranslated region. MDCK cells were transfected in 60-mm tissue culture dishes. Lipofectin (Life Technologies) was used to introduce the plasmids into the MDCK cells. Clones of cells resistant to G418 (400 μg/ml) were isolated and screened for expression of exogenous NaK-ATPase β subunits. For screening, the cells were plated on cover slips at high density and induced for 48 h in 10 mmol/L butyrate. Induced cells were then screened for expression of the exogenous β subunit by immunofluorescence microscopy. For light microscopy, paraffin-embedded tissue sections on glass slides were first dewaxed and rehydrated through a graded series of ethanols. Cells and tissues were then incubated in 0.3. H2O2 in methanol to block endogenous peroxidase activity, followed by incubation with 10% normal goat serum in PBS for 20 minutes at room temperature in a humidified atmosphere. Cells and sections were incubated for 45 minutes at room temperature in a humidified chamber with the following primary antibodies: polyclonal anti-NaK-ATPase α subunit raised in rabbits against the whole molecule purified from dog, by courtesy of WJ Nelson; and antichicken NaK-ATPase α subunit monoclonal antibodies (mAbs) (α5; 2F, F12 available through the Development Studies Hybridoma Bank, Iowa City, IA); or polyclonal anti-α1 (#1300), α3 (#1301), β1 (#1303), and β2 (#1305) raised in rabbits against isozyme-specific peptides and affinity purified on a peptide column (Immuno Dynamics, La Jolla, CA). All anti-α1 antibodies recognized a single 98-kd band and showed identical staining patterns in immunocytochemistry of kidney tissues. Primary antibodies were diluted in PBS containing 2% bovine serum albumin (BSA) (1:100–1:500), washed three times in PBS-Tween 20 (0.02%), incubated for 45 minutes with biotinylated goat anti-rabbit immunoglobulin G (IgG; Vector Laboratories, Burlingame, CA), washed twice for 5 minutes each in PBS-Tween and once for 5 minutes in PBS, incubated for 45 minutes with avidin-biotin peroxidase (Vectastain Elite, Vector Laboratories), and washed for 5 minutes in PBS, followed by two washes of 5 minutes each in Tris-buffered saline. Color development was carried out for 10 to 45 minutes, using aminoethylcarbazole as substrate. Sections and cells were mounted in Aquamount (Polysciences, Niles, IL) and viewed under a Nikon FXA-Microphot equipped with Nomarski optics. For electron microscopy and enzyme and immunocytochemistry, tissue sections and cells grown on membranes were fixed with 2. paraformaldehyde or 1% glutaraldehyde in PBS and incubated either for immunoperoxidase localization of antigens or in reaction media for localization of NaK-ATPase enzymatic reaction products after postcoupling with lead.42Ernst SA Palacios J Siegel GJ Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus.J Histochem Cytochem. 1986; 34: 189-195Crossref PubMed Scopus (66) Google Scholar Pre-embedding immunoperoxidase reactions were carried out by 1) incubation for 45 minutes at room temperature in 1:50 to 1:500 dilutions of α1, β1, and β2 peptide antibodies in PBS with BSA; 2) washing in three changes of PBS for 5 minutes each at 4°C followed by 45 minutes at room temperature in peroxidase-conjugated anti-rabbit IgG; 3) two washes of 5 minutes each at 4°C in PBS and one wash in Tris-buffered saline; and 4) incubation in diaminobenzidine to allow visualization of the electron-dense peroxidase reaction product. Tissue sections and cells on membranes were then dehydrated and embedded in Araldite (Taab) resin, ultrathin sections were cut on an ultramicrotome (Sorvall), and sections were viewed, with and without uranyl acetate counterstain, using a JEOL electron microscope. For immunofluorescence microscopy, cells were first fixed in a 2. formaldehyde solution in PBS. Antibodies were diluted to 5 μg/ml in a Hanks' balanced salt solution containing 2% horse serum and 20 mM Tris-Cl, pH 7.5. For basolateral labeling of polarized MDCK cells, 0.25% saponin was added to permeabilize the surface membrane. Apical membrane labeling was performed in the absence of saponin. Three primary antibodies were used: mAb 9E10 to the c-myc epitope43Evans GI Lewis GK Ramsay G Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.Mol Cell Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2170) Google Scholar that was engineered to be a C-terminal epitope on the chicken NaK-ATPase β2 subunit, mAb b24 to the chicken NaK-ATPase β1 subunit,44Fambrough DM Bayne EK Multiple forms of (Na+K+)-ATPase in the chicken.J Biol Chem. 1983; 258: 3926-3935Abstract Full Text PDF PubMed Google Scholar and the polyclonal antipeptide antibody (#1305) to the human NaK-ATPase β2 subunit. All three antibodies recognized extracellular epitopes on their target β subunits. Primary antibody solution was applied to fixed cells for one hour at room temperature. Cells were rinsed and then incubated in an fluorescein isothiocyanate-labeled secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG; Kirkegaard and Perry, Gaithersburg, MD). Cells were viewed and photographed with a Zeiss Axioskop epifluorescence microscope and Kodak Tmax 400 film. An additional set of MDCK cells stably transfected with β2, as well as ADPKD epithel
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