Expression of the beta 2-subunit and apical localization of Na+-K+-ATPase in metanephric kidney

Christopher R. Burrow1, Olivier Devuyst2, Xiaohong Li1, Laura Gatti1, and Patricia D. Wilson1

1 Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029; and 2 Division of Nephrology, St. Luc Academic Hospital, University of Louvain Medical School, B-1200 Brussels, Belgium


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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During kidney organogenesis, the Na+-K+-ATPase pump is not restricted to the basolateral plasma membrane of the renal epithelial cell but is instead either localized to the apical and lateral membrane sites of the early nephron or expressed in a nonpolarized distribution in the newly formed collecting ducts. The importance of Na+-K+-ATPase beta -subunit expression in the translocation of the Na+-K+-ATPase to the plasma membrane raises the question as to which beta -subunit isoform is expressed during kidney organogenesis. Immunocytochemical, Western analysis and RNase protection studies showed that both beta 2-subunit protein and beta 2 mRNA are expressed in the early gestation to midgestation human metanephric kidney. In contrast, although beta 1 mRNA abundance is equivalent to that of the beta 2-subunit in the metanephric kidney, the beta 1-subunit protein was not detected in early to midgestation metanephric kidney samples. Immunocytochemical analysis revealed that both alpha 1- and beta 2-subunits were present in the apical epithelial plasma membranes of distal nephron segments of early stage nephrons, maturing loops of Henle, and collecting ducts during kidney development. We also detected a significant increase in alpha 1 and beta 1 mRNA after birth with a marked reduction in beta 2 mRNA abundance associated with an increase in alpha 1- and beta 1-subunit proteins and loss of beta 2 protein expression. These studies support the conclusion that the expression of the beta 2-subunit in the fetal kidney may be an important mechanism controlling polarization of the Na+-K+-ATPase pump in the epithelia of the developing nephron during kidney organogenesis.

sodium-potassium adenosinetriphosphatase; renal epithelium; sodium transport; nephron


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE Na+-K+-ATPase pump is a heterodimeric membrane protein composed of alpha - and beta -subunits, which catalyzes ATP-dependent sodium/potassium exchange essential for the maintenance of cellular volume and ionic homeostasis (36). The activity of Na+-K+-ATPase depends on the assembly of the 100-kDa alpha -catalytic subunit with a 40- to 60-kDa beta -subunit prior to transport of functional pumps to the plasma membrane (17). Assembly of alpha -beta heterodimers is required for intracellular transport of the Na+-K+-ATPase pump to the plasma membrane (41), and beta -subunit structure influences the function of the alpha -subunit in the catalysis of Na+/K+ exchange (15, 30, 31). There are three unlinked alpha -isoform genes (alpha 1, alpha 2, alpha 3) (37, 48, 55) and two beta -subunit genes (beta 1 and beta 2) (21, 40, 56) in the mammalian genome that exhibit cell- and tissue-specific patterns of expression (59). This diversity of isoforms potentially allows the expression of six different Na+-K+-ATPase heterodimers, which may result in functional differences in the catalysis of Na+/K+ exchange (7, 8) and fulfill the physiological requirements for specific Na+-K+-ATPase ion pumps in particular cell types.

In polarized reabsorptive epithelial cells in the intestine and kidney, the expressed alpha 1-beta 1 heterodimeric Na+-K+-ATPase (18) is mostly localized to the basolateral membrane domain as required for its function in the vectorial transport of sodium across the tubule (33, 34, 44). The molecular mechanism for targeting and retention of the Na+-K+-ATPase to the basolateral membrane of kidney epithelia has been extensively studied in cell model systems (46) and found to depend on basolateral membrane-specific vesicle transport pathways, apical-to-basolateral membrane transcytosis, and retention of pumps by stable binding to localized ankyrin sites in the cytoskeleton of the basolateral membrane (47). In one renal epithelial cell culture model system, it appears that the alpha -subunit may contribute the dominant sorting signal for basolateral membrane localization (45). In the retinal pigment epithelium, apical distribution of the ankyrin/fodrin cytoskeleton results in apical membrane localization of the Na+-K+-ATPase alpha 1-beta 1 heterodimers (25, 52) consistent with the model that epithelial membrane localization of Na+-K+-ATPase is dependent on ankyrin binding and retention in an apical multiprotein membrane cytoskeletal complex rather than on the function of an apical membrane vesicle sorting pathway.

The possibility that developmental specification of beta -subunit isoform expression may play a role in the plasma membrane localization of Na+-K+-ATPase in polarized epithelial cells or in neurons has not yet been extensively studied. In the choroid plexus, which has apical membrane localization of the Na+-K+-ATPase (49) associated with apical membrane ankyrin (39), there is expression of both the beta 1- and beta 2-isoforms (62, 65) suggesting that beta 2-isoform expression may also influence membrane localization of the Na+-K+-ATPase in epithelia. Although these studies suggest that expression of the beta 2-subunit in the choroid plexus may be a potential mechanism for directing apical membrane localization of the Na+-K+-ATPase in this tissue, both beta 1- and beta 2-subunits can be detected by immunolocalization studies in the apical plasma membranes of this epithelium using isoform-specific antisera (22, 39). In contrast, the exclusive expression of the beta 1-subunit without the beta 2-subunit (21) in the adult kidney might be important for the restricted localization of the Na+-K+-ATPase to the basolateral membranes of the tubular epithelial cells, but this question has not been addressed with appropriate studies in a suitable experimental model system.

The Na+-K+-ATPase beta 2-subunit is expressed in the central nervous system (CNS) glial cells, pinealocytes, and retinal photoreceptors (3, 54), where it assembles with the alpha 2-subunit (21) and can direct the assembly of functional alpha 1-beta 2, alpha 2-beta 2, and alpha 3-beta 2 Na+-K+-ATPase pumps (7, 8, 53). beta 2 -/- Homozygous knockout mice die from CNS lesions by postnatal day 18 (38). These results appear to be indicative of lethal region-specific failures of cellular ionic homeostasis and establish the essential function of the beta 2-subunit in CNS Na+-K+-ATPase catalytic activity but do not address its potential role in plasma membrane targeting.

During human kidney organogenesis (26), the Na+-K+-ATPase alpha -subunit has been shown to be partly localized to the apical membrane domains of renal epithelial cells in the developing nephron. Apical localization of the Na+-K+-ATPase has also been detected in a subset of collecting duct epithelia during kidney development in other mammals (4, 28, 43) in contrast to the largely basolateral membrane localization in the normal adult nephron (33, 34). In the adult kidney, the predominant Na+-K+-ATPase subunit genes expressed are alpha 1 (18) and beta 1 (11). Low-level transcriptional expression of alpha 2- and alpha 3-subunit genes and a truncated alpha 1 splice variant (11, 42) have also been reported in the rat kidney confirming previous reports of potential alpha -subunit diversity in the kidney (5, 27, 58). However, two independent studies have demonstrated only full-length alpha 1-subunit Na+-K+-ATPase protein in the adult kidney by Western blot (21, 61), and transcriptional expression of the beta -subunit is highly selective for the beta 1 gene with only trace amounts of beta 2 mRNA detectable (11). The absence of beta 2-subunit protein in the adult mammalian kidney has also been documented in earlier studies (21). Previous studies have demonstrated stable alpha 1 and beta 1 mRNA levels during kidney development (48) but have not analyzed transcriptional regulation of the beta 2-subunit gene or analyzed which beta -isoform is expressed in the apical membrane of the maturing nephron during kidney development.

We reasoned that beta 2-to-beta 1 isoform switching in kidney development might be an important control mechanism for relocalization of the Na+-K+-ATPase from the apical to the basolateral membrane. The precedent of developmental alterations in the ratio of beta 1/beta 2 expression is well established in the cerebellum (3, 38). In addition, in autosomal dominant polycystic kidney disease, aberrant apical distribution of Na+-K+-ATPase is associated with persistent expression of the Na+-K+-ATPase beta 2-subunit after birth (P. D. Wilson, unpublished observations).

In this study, we demonstrate expression of the Na+-K+-ATPase beta 2-subunit in newly formed nephrons in human fetal kidneys associated with apical membrane localization of the Na+-K+-ATPase alpha 1- and beta 2-subunits. These results support the hypothesis that beta 2-subunit expression during kidney development, and in the adult choroid plexus, may result in apical membrane localization of the Na+-K+-ATPase through altered handling of the alpha 1/beta 2 complex by the cellular mechanisms that direct nearly exclusive basolateral membrane localization of the alpha 1/beta 1 Na+-K+-ATPase.


    MATERIALS AND METHODS
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Tissues and cell culture. Human fetal (Anatomic Gift Foundation, Woodbine, GA) and normal adult human kidneys (National Disease Research Interchange, Philadelphia, PA) were procured under sterile conditions, flushed with neutral salts solution (Collins or UW), clamped, and stored in salts solution over ice for a maximum of 24 h prior to use. Parallel samples were frozen immediately in liquid nitrogen and stored at -80°C until use. All tissue samples were divided into three portions and used for 1) microdissection and tissue culture, 2) protein or RNA extraction, and 3) fixed for pathology, immunostaining, or in situ hybridization using 4% paraformaldehyde in diethyl pyrocarbonate-treated PBS at 4°C for 4 h. Microdissected human fetal collecting ducts were prepared for culture using previously described methods (63, 64) and immortalized with the retrovirus pZipneoTA58U19 (19). We packaged this vector in the amphotropic packaging cell line psi-CRIP (12), which we have used previously to derive immortalized human renal epithelial cell lines from the adult kidney (50). This vector transduces a temperature-sensitive allele of the SV40 large T antigen; after transfection and G418 selection, clonal cell lines were derived by limit dilution and selected on the basis of showing expression of the beta 2-subunit of the Na+-K+-ATPase. This led to the identification of the clones HFCT.6D and HFCT.6E used in the experiments shown in Fig. 6. The cell lines were cultured at 33°C in 25-cm2 flasks coated with type I (rat tail) collagen (Collaborative Research, Lexington, MA) until subconfluent and then shifted to 37°C for 7-10 days until harvest. Following aspiration of the cell culture media, T25 confluent monolayers were washed for 5 min with PBS (pH 7.4) at room temperature, scraped, and centrifuged for 8,000 g for 90 s. The cell pellet was flash frozen in liquid nitrogen and then stored at -80°C until use.

Antibodies. Polyclonal isoform-specific antibodies were prepared in rabbits using synthetic peptides specific for the Na+-K+-ATPase alpha 1-, beta 1-, and beta 2-isoforms (Immunodynamics). The alpha 1 immunizing peptide sequence was C<UNL>KGVGRDKYEPAAVS</UNL> (alpha  residues are marked with underscore), corresponding to amino acids 3-16 of human alpha 1 predicted sequence (PIR: locus A24414, accession no. A24414). The alpha 1 sequence is 93 residues NH2-terminal to the first transmembrane domain and therefore cytoplasmic; this alpha 1 sequence is 50% conserved in alpha 2 but entirely different (0/14 matches) from the alpha 3 sequence. The beta 1 immunizing peptide sequence was C<UNL>KFIWNSEKKEFLGR</UNL>, corresponding to amino acids 14-27 of the human beta 1 predicted sequence (GenBank: locus HSU16799, accession no. U16799). The beta 1 peptide sequence is identical in sheep, rat, dog, pig, and mouse. This peptide is part of the NH2-terminal cytoplasmic portion of the protein. A BLAST search using this peptide sequence resulted in significant matches only with other beta 1-subunits. The homologous beta 2 sequence to the beta 1 immunizing peptide is E<UNL>F</UNL>V<UNL>WN</UNL>PRTHQ<UNL>F</UNL>M<UNL>GR</UNL> and is 43% identical (6/14 residues, conserved residues are marked with underscore). The beta 2 immunizing peptide sequence was C<UNL>PKTENLNVIVNVSD</UNL>, homologous to amino acids 85-98 of the human predicted beta 2 sequence (GenBank: locus HUMATPBII, accession no. M81181). In contrast to the beta 1 peptide, the beta 2 peptide starts 20 amino acid residues COOH-terminal to the transmembrane domain and is extracellular. A BLAST search with the beta 2 sequence identifies only other beta 2 sequences; only 2 of 14 residues of this sequence are identical with the homologous beta 1 sequence. The beta 2 and beta 1 antibodies were purified from 15 ml crude antisera using specific peptide-derivatized 4-ml thiosepharose affinity chromatography columns. The alpha V anti-alpha Na+-K+-ATPase subunit used in the immunoprecipitation and immunolocalization experiments was a gift from D. M. Fambrough, which was raised against immunopurified chicken Na+-K+-ATPase (29, 35).

This monoclonal antibody (which recognizes a cytoplasmic epitope of the Na+-K+-ATPase alpha -subunit shared by all isoforms) and the alpha 6F anti-alpha monoclonal antibody (60) used in immunolocalization studies are also available from the Developmental Studies Hybridoma Bank (http://www.uiowa.edu/~dshbwww/info.html). The isoform-specific SpETB2 is anti-human beta 2 rabbit polyclonal antiserum (a gift from P. Martin-Vasallo) and was raised against a human beta 2-subunit fusion protein (amino acid residues 54-290) expressed in Escherichia coli. The characterization of the SpETB2 antiserum has been published (22).

Western immunoblot analysis and immunoprecipitation. Membrane extracts were prepared from human fetal (12-24 wk gestational age) and normal adult kidneys according to the method described by Jørgensen (32). After washing in ice-cold PBS, pH 7.4, the kidneys were finely minced in ice-cold homogenization buffer (300 mM sucrose, 25 mM HEPES made to pH 7.0 with 1 M Tris) containing the protease inhibitors 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Boehringer), 1 mM benzamidine (Sigma), 10 µg/ml leupeptin (Boehringer), 1 µg/ml pepstatin A (Boehringer), 1 µg/ml aprotinin (Boehringer), and 1 µg/ml chymostatin (Boehringer), and then homogenization was performed in the cold using a Potter apparatus. The homogenate was centrifuged at 1,000 g for 20 min at 4°C to remove nuclei and cell debris. The supernatant was further centrifuged at 80,000 g for 30 min at 4°C. The pellet (whole cell membranes) was suspended in the ice-cold homogenization buffer, and protein concentrations were determined with the BCA protein assay (Pierce), using BSA as standard. For Na+-K+-ATPase beta 1-subunit detection by Western blots (n = 5 fetal; n = 5 adult kidneys), detergent extraction of membranes was required, as follows: the 80,000 g pellet was resuspended in ice-cold detergent extraction buffer [20 mM Tris · HCl, 120 mM NaCl, 2 mM EDTA, 2 mM EGTA, and 0.1 mM dithiothreitol (DTT), pH 7.4] containing the protease inhibitors described above, incubated for 15 min on ice with either 1% octylglucoside (Pierce) or 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, Pierce), and then centrifuged at 100,000 g for 1 h at 4°C. For Na+-K+-ATPase beta 2-subunit detection by Western blots (n = 9 fetal; n = 9 adult kidneys), detergent extractions were made with the same protocol using either 1% SDS or 1% NP-40 as the detergent. For Na+-K+-ATPase alpha 1-subunit (n = 9 fetal; n = 9 adult kidneys), 1% SDS or 0.5% Triton X-100 (Boehringer) was used as the detergent. Protein concentrations were determined on the supernatant, which contained the solubilized membrane proteins, and the extracts were used immediately. All extracts were solubilized for SDS-PAGE by heating either at 95°C for 2 min (for beta 1 and beta 2 Na+-K+-ATPase isoforms) or at 60°C for 12 min (alpha 1 Na+-K+-ATPase) in sample buffer [1.5% SDS, 10 mM Tris · HCl, pH 6.8, 0.6% DTT, and 6% (vol/vol) glycerol]. Proteins (20 µg/lane) were separated by electrophoresis through 0.1 × 9 × 6-cm 12% acrylamide slabs and transferred to nitrocellulose. Membranes were blocked for 30 min at room temperature in blotting buffer (50 mM NaPO4, 150 mM NaCl, and 0.05% Tween 20, pH 7.4) containing 5% nonfat dry milk, followed by incubation with the primary antisera and affinity-purified antibodies (anti-alpha 1, anti-beta 1, or anti-beta 2) in the blotting buffer containing 2% BSA at 4°C for 18 h. The membranes were then washed in several changes of blotting buffer, incubated for 30-60 min with peroxidase-labeled goat anti-rabbit IgG (Kirkegaard & Perry), washed again, and visualized after 1-min incubation with enhanced chemiluminescence (Amersham) at room temperature.

For immunoprecipitations, whole cell extracts were prepared from frozen cell pellets of cell lines HFCT.6D and HFCT.6E after lysis in 500 µl cold lysis buffer [10 mM Tris, pH 7.2, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM vanadate, 0.5% NP-40, and 1% Triton X-100 containing the protease inhibitors 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Boehringer, Indianapolis, IN), 1 mM benzamidine (Sigma, St. Louis, MO), 10 µg/ml leupeptin (Boehringer), 10 µg/ml pepstatin A (Boehringer), 1 µg/ml aprotinin (Boehringer), and 1 µg/ml chymostatin (Boehringer)]. After 30-min incubation on ice, the suspension was dispersed by aspiration using an 18-gauge needle and then centrifuged at 14,000 rpm for 15 min at 4°C. Cell lysates (200 µg protein) were incubated for 1 h at room temperature with 25 µl of Protein A/G Plus agarose (Santa Cruz Biotechnology), and 10 µl of the anti-alpha Na+-K+-ATPase subunit monoclonal antibody alpha V in a total volume of 400 µl. The pellet was collected at 8,000 rpm for 2 min and washed three times with RIPA buffer (Ca2+- and Mg2+-free PBS, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1% phenylmethylsulfonyl fluoride, 1 µg/ml aprotonin, and 1 mM vanadate), and the pellet was resuspended in 40 µl 1× electrophoresis sample buffer [10 mM Tris, pH 6.8, 6% glycerol, 4.0% (vol/vol) beta -mercaptoethanol, 1.5% SDS], boiled for 3 min, centrifuged at 8,000 rpm for 2 min and analyzed by Western blot following SDS-PAGE using an 8% polyacrylamide gel.

Immunocytochemistry. For light microscopy, paraffin-embedded tissue sections on glass slides were first dewaxed and rehydrated through a graded series of ethanols. 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 min at room temperature in a humidified atmosphere. Tissue sections were incubated for 45 min at room temperature in a humidified chamber with the following primary antibodies: anti-chicken Na+-K+-ATPase alpha -subunit monoclonals (alpha V, and 6F12) from Dr. D. M. Fambrough (Johns Hopkins University, Baltimore, MD) (n = 8 independent fetal kidney blocks); or polyclonal anti-alpha 1 (n = 27 independent fetal kidney blocks), anti-beta 2 (n = 28 independent fetal kidney blocks) antisera raised in rabbits against isoform-specific peptides as described above. Affinity-purified anti-beta 2 antibody was used to confirm beta 2 immunolocalization (n = 10 independent fetal kidney blocks used). Primary antibodies were diluted in PBS containing 2% BSA (1:100 to 1:500); washed three times in PBS-Tween 20 (0.02%); incubated for 45 min with biotinylated goat anti-rabbit IgG (Vector Laboratories), washed twice for 5 min each in PBS-Tween and once for 5 min in PBS, incubated for 45 min with avidin-biotin peroxidase (Vectastain Elite, Vector Laboratories), and washed for 5 min in PBS followed by two washes of 5 min each in Tris-buffered saline. Color development was carried out for 10-45 min using aminoethylcarbazole as substrate. Sections were mounted in Aquamount (Polysciences) and viewed under a Nikon FXA-Microphot equipped with Nomarski optics.

Preparation of riboprobes. The alpha 1, beta 1, and beta 2 PCR products were subcloned into the vector pBSIIKS(-); sequence and insert orientation were verified by DNA sequencing. Antisense probes were prepared from Xho I-restricted plasmids transcribed in vitro with T7 RNA polymerase in the presence of [32P]UTP (Amersham) as follows: RNA polymerase concentration 2.5 U/µl in 40 mM Tris-Cl (pH 8.0), 25 mM NaCl, 8 mM MgCl2, 2 mM spermidine-HCl3, 10 mM DTT, 400 µM ATP, 400 µM CTP, 400 µM GTP, 12.5 µM [alpha -32P]UTP (400 Ci/mmol), and 2 U/µl placental RNase inhibitor (Boehringer) in a reaction volume of 20 µl for 30 min at 37°C followed by a 15-min 37°C incubation with 0.5 U/µl RNase-free DNase (Boehringer). The riboprobes were purified with phenol/CHCl3/isoamyl alcohol extraction and precipitated three times in 75% ethanol plus 0.5 M NH4OAC prior to use.

RNase protection assays. For hybridizations, 20 µg of total RNA (or control tRNA) was resuspended in 40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, and 80% formamide in the presence of 2 × 105 cpm antisense probe and 2 × 105 cpm internal control 18S riboprobe (Ambion) in 30 µl for 16 h in a 45°C bath (for alpha 1, n = 6 fetal kidney, n = 7 adult kidney RNA samples; for beta 1, n = 6 fetal kidney, n = 7 adult kidney RNA samples; for beta 2, n = 7 fetal kidney, n = 7 adult kidney RNA samples). A volume of 350 µl of 10 mM Tris-Cl (pH 7.5), 300 mM NaCl, 5 mM EDTA containing 40 µg/ml RNase A (Boehringer), 0.2 µg/ml RNase T1 (Boehringer) was then added to the hybridization mixture, and RNA digestion was performed at 30°C for 30 min followed by proteinase K digestion in 0.5% SDS, phenol/CHCl3/isoamyl alcohol extraction and ethanol precipitation. The assay products were fractionated using a 6% urea-PAGE system, and dried gels were examined by autoradiography with an intensifying screen for 12-72 h. The alpha 1 undigested probe length was 496 bp, beta 1 was 506 bp, and beta 2 was 612 bp.


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ABSTRACT
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MATERIALS AND METHODS
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Characterization of anti-alpha 1 and anti-beta 2 isoform-specific Na+-K+-ATPase antisera. The rabbit polyclonal anti-alpha 1 raised against the NH2-terminal peptide <UNL>KGVGRDKYEPAAVS</UNL> was used for Western immunoblot analysis of solubilized membrane proteins isolated from the normal adult human kidney and compared with the alpha V anti-alpha 1 monoclonal antibody raised against the chicken Na+-K+-ATPase alpha 1-subunit (gift of D. M. Fambrough, Johns Hopkins University). As shown in Fig. 1A, the rabbit polyclonal anti-alpha 1 Na+-K+-ATPase antiserum detected the same 100-kDa band expressed in the adult human kidney (left two lanes) as was detected by the control anti-alpha 1 Na+-K+-ATPase monoclonal antibody (right two lanes). This 100-kDa band was not detected by the rabbit preimmune control Western immunoblot (data not shown). The rabbit polyclonal anti-alpha 1 Na+-K+-ATPase antiserum also detected basolateral expression of the alpha 1-subunit in the renal tubules of adult rat kidneys (see Fig. 5L) and human kidneys (data not shown). The characterization of the rabbit polyclonal antiserum raised against the beta 1 peptide <UNL>KFIWNSEKKEFLGR</UNL> demonstrated specific recognition of a 42-kDa protein in the adult kidney not recognized by the preimmune antiserum control which was specifically competed by the addition of the immunizing peptide. This 42-kDa protein was also detected by the anti-beta 1 antiserum following affinity purification over a beta 1-peptide-containing column (data not shown, available on request). The rabbit polyclonal antiserum raised against the beta 2 peptide <UNL>PKTENLNVIVNVSD</UNL> detected a 50-kDa protein (Fig. 1B, lanes 1 and 2) by Western immunoblotting of solubilized membrane proteins from the human fetal kidney not detected by preimmune antiserum (Fig. 1B, lane 4); confirmation that this 50-kDa protein is the beta 2-subunit was obtained using affinity-purified anti-beta 2 antiserum (Fig. 1B, lane 3) which also detected the 50-kDa beta 2-subunit and a minor band at 30-35 kDa. The observed molecular mass of the beta 2-subunit was in the range of 46-51 kDa reported for the beta 2-subunit in the choroid plexus (65) and brain (3). The difference between the predicted molecular mass of the beta 2 gene product of 33.2 kDa and the observed molecular mass of 45-50 kDa is likely due to N-linked glycosylation (21); the 30- to 35-kDa band detected in Fig. 1B, lane 3, with the affinity-purified antibody may reflect the presence of some nonglycosylated beta 2-subunit core protein in the fetal kidney solubilized membrane extracts. Further characterization of the beta 2-subunit anti-peptide antiserum showed that it detects apical plasma membrane beta 2-subunit protein in human beta 2-subunit-expressing transfected MDCK cells but not in control MDCK cells (data not shown; D. M. Fambrough, personal communication); The specificity of the beta 2 antiserum and lack of cross-reactivity with the beta 1-subunit is demonstrated by the lack of detection of the beta 1-subunit in the adult kidney by immunocytochemistry (not shown), or Western immunoblotting (Fig. 2). We have also shown that the immunizing beta 2 peptide will specifically compete the immunodetection of the beta 2-subunit detected by Western blotting (data not shown). Our beta 2 anti-peptide antiserum detects the same protein on Western immunoblots of fetal collecting duct cell lines cell extracts (shown in Fig. 6A), as does the previously characterized beta 2-isoform-specific SpETB2 rabbit polyclonal antiserum raised against a human beta 2 fusion protein expressed in E. coli (22).


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Fig. 1.   Characterization of alpha 1 and beta 2 isoform-specific rabbit polyclonal antisera. A: alpha 1 Na+-K+-ATPase Western immunoblot of solubilized membranes. Lanes 1 and 2, membrane extracts prepared from 17-yr-old and 20-yr-old human kidneys, respectively, stained with anti-peptide alpha 1-subunit anti-peptide antiserum 1300 (at dilution of 1:12,500); lanes 3 and 4, same membrane extracts blotted with anti-chicken alpha 1 monoclonal antibody alpha V at dilution of 1:10,000 (gift of D. M. Fambrough). B: beta 2 Na+-K+-ATPase Western immunoblot of solubilized membranes prepared from 18-wk fetal kidney: lane 1, 1% SDS extract, 1:5,000 dilution of unpurified beta 2 antiserum immunoblot; lane 2, 1% NP-40 extract, 1:5,000 dilution of unpurified beta 2 antiserum immunoblot; lane 3, 1% SDS extract, 1:1,000 beta 2 peptide column affinity-purified antiserum immunoblot; lane 4, 1% SDS extract, beta 2 preimmune antiserum blot.



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Fig. 2.   Developmental regulation of Na+-K+-ATPase subunit expression. A: Western immunoblot analysis of Na+-K+-ATPase alpha 1-subunit expression (all membranes solubilized with 1% SDS): lanes 1-3, fetal kidney; lanes 4-6, adult kidney; lane 1, 18-wk fetal kidney; lane 2, 20-wk fetal kidney; lane 3, 24-wk fetal kidney; lane 4, 17 yr; lane 5, 20 yr; and lane 6, 34 yr. B: Western immunoblot analysis of Na+-K+-ATPase beta 1-subunit expression (all membranes solubilized with 0.5% CHAPS, antiserum 1303, 1:5,000 dilution): lanes 1-3, fetal kidney; lanes 4-6, adult kidney; lane 1, 16 wk; lane 2, 19 wk; lane 3, 23 wk; lane 4, 2 yr; lane 5, 16 yr; and lane 6, 31 yr. C: Western immunoblot analysis of Na+-K+-ATPase beta 2-subunit expression (all membranes solubilized with 1% SDS): lanes 1-3, fetal kidney; lanes 4-6, adult kidney; lane 1, 18 wk fetal kidney; lane 2, 20-wk fetal kidney; lane 3, 24-wk fetal kidney; lane 4, 17 yr; lane 5, 20 yr; and lane 6, 34 yr.

The Na+-K+-ATPase beta 2-subunit substitutes for the beta 1-isoform during human kidney organogenesis. Immunoblot analysis of alpha 1 expression in human fetal kidney at 18, 20, and 24 wk of gestation demonstrated a 100- to 110-kDa band (lanes 1-3, Fig. 2A) with increased expression in the adult kidney as detected in membrane extracts prepared from kidney tissue obtained from 17-, 20-, and 34-yr-old donors (lanes 4-6, respectively, Fig. 2A). In contrast, Western immunoblot studies demonstrated no beta 1 protein in fetal kidney at 16, 19, and 23 wk gestation (lanes 1-3, Fig. 2B), whereas the 42-kDa beta 1-subunit was detected in kidney tissue membrane extracts prepared from 2, 16, and 31-yr-old donor kidneys (lanes 4-6, Fig. 2B) (the 90- to 95-kDa and 140-kDa bands detected are antigenically unrelated to the beta 1-subunit). Expression of the 50-kDa beta 2-subunit protein in fetal kidneys was demonstrated at 18, 20, and 24 wk of gestation (lanes 1-3, Fig. 2C) with absent expression evident in the adult kidney obtained from 17-, 20-, and 34-yr-old donors (lanes 4-6, Fig. 2C). On longer exposure of this Western blot, faint expression of the beta 2-subunit was seen in two of three adult kidneys. During kidney development, the level of beta 2-subunit expression appeared to be constant from 15-24 wk by Western blot (data not shown). We have not yet determined when beta 1-subunit expression replaces beta 2, but downregulation of beta 2 mRNA to undetectable levels has been observed in a newborn kidney (see Fig. 3C, lane 3).


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Fig. 3.   RNase protection analysis of Na+-K+-ATPase isoform expression. A: RNase protection analysis of beta 2 mRNA. Lane 1, 12-wk human fetal kidney RNA; lane 2, human adult kidney RNA (52 yr old); lane 3, undigested probe. Asterisk identifies the 18S ribosomal RNA protectomer control band; arrow points to the beta 2 protectomer, which identifies beta 2 mRNA. B: RNase protection analysis of alpha 1 (left) and beta 1 (right) mRNA expression. Lane 1, 13-wk RNA; lane 2, 16-wk RNA; lane 3, 20-wk RNA; lane 4, 24-wk RNA; lane 5, newborn kidney RNA; lane 6, 2-yr-old RNA; and lane 7, 20-yr-old kidney RNA. Arrows point to the alpha 1 and beta 1 protectomers; asterisk indicates the 18S ribosomal RNA protectomer control band. C: RNase protection assays of Na+-K+-ATPase subunit isoform mRNA expression (20 µg total RNA per hybridization). Lanes 1-3, newborn kidney RNA; lanes 4-6, 2-yr-old kidney RNA; lanes 1 and 4, alpha 1-subunit mRNA; lanes 2 and 5, beta 1-subunit expression; lanes 3 and 6, beta 2-subunit expression; lanes 7-9, undigested probe; lane 7, alpha 1 probe; lane 8, beta 1 probe; lane 9, beta 2 probe; lanes 10-12, hybridization controls (20 µg tRNA instead of total RNA); lane 10, alpha 1 probe tRNA control; lane 11, beta 1 probe tRNA control; and lane 12, beta 2 probe tRNA control.

A reduction in beta 2 mRNA abundance in association with beta 1 translational activation results in beta 2/beta 1-isoform switching during maturation of the nephron. Quantitative analysis of Na+-K+-ATPase isoform mRNA abundance by RNase protection analysis supported the conclusion that beta 2 mRNA levels are significantly increased in the fetal kidney compared with the adult kidney (lane 1 vs. 2, Fig. 3A). This pattern of predominantly fetal kidney expression of the beta 2 mRNA is reversed for the alpha 1- (Fig. 3B, left) and beta 1-subunits (Fig. 3B, right), which show marked induction during kidney maturation in the fetus and after birth. As shown in Fig. 3C, some of the increase in abundance of the alpha 1- and beta 1-subunits mRNA occurs postnatally (lane 1, newborn kidney alpha 1 mRNA, vs. lane 4, alpha 1 mRNA detected in sample prepared from 2-yr-old donor kidney; lane 2, newborn kidney beta 1 mRNA, vs. lane 5, beta 1 mRNA detected in sample prepared from 2-yr-old donor kidney). Although we found variable expression of beta 2 mRNA by RNase protection and RT-PCR with beta 2-specific primers (data not shown) in some adult kidney samples, the beta 2 mRNA abundance was lower than that detected in fetal kidney and a small fraction of the beta 1 mRNA abundance in the adult (data not shown). The finding of low-level expression of beta 2 mRNA in the adult rat kidney has been previously reported by others (11). We next examined the relative abundance of alpha 1, beta 1, and beta 2 mRNA during kidney development from 13 to 24 wk gestational age (lane 1, 13 wk; lane 2, 16 wk; lane 3, 20 wk; lane 4, 24 wk in Fig. 4, A-C). In these experiments, we found that the level of beta 1 and beta 2 mRNA was nearly equal during kidney development with the alpha 1 mRNA being slightly more abundant than the beta -isoform mRNAs after 20 wk of gestation (compare lanes 3 and 4 in Fig. 4, A-C). The reduction in beta 2 mRNA abundance in the adult kidney suggests that transcriptional repression of the beta 2 promoter or a developmental reduction in beta 2 mRNA stability during nephron maturation is a potential control mechanism for switching off beta 2 protein expression in the adult kidney. In contrast, the beta 1-subunit mRNA is easily detectable in all stages of kidney organogenesis (Figs. 3, B and C, and 4B) despite the absence of detectable beta 1-subunit protein by immunoblot studies in the fetal kidney (Fig. 2B). We have not determined whether alterations in beta 1 mRNA polyadenylation during kidney development may control beta 1 protein expression as has been shown during early Xenopus laevis development (9). There is also a definite increase in alpha 1 and beta 1 mRNA abundance between fetal, newborn, and adult stages (Fig. 3, B and C), supporting a potential role for either transcriptional activation of the alpha 1- and beta 1-subunit promoters or an increase in alpha 1 and beta 1 mRNA stability in the developmental regulation of renal Na+-K+-ATPase expression.


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Fig. 4.   RNase protection analysis of Na+-K+-ATPase subunit mRNA in kidney development. A: RNase protection assays of Na+-K+-ATPase alpha 1-subunit isoform mRNA expression (20 µg total RNA per hybridization). Lane 1, 13-wk fetal kidney; lane 2, 16-wk fetal kidney; lane 3, 20-wk fetal kidney; lane 4, 24-wk fetal kidney; lane 5, undigested alpha 1 probe; and lane 6, 20 µg tRNA hybridization control. B: RNase protection assays of Na+-K+-ATPase beta 1-subunit isoform mRNA expression (20 µg total RNA per hybridization). Lane 1, 13-wk fetal kidney; lane 2, 16-wk fetal kidney; lane 3, 20-wk fetal kidney; lane 4, 24-wk fetal kidney; lane 5, undigested beta 1 probe; and lane 6, 20 µg tRNA hybridization control. C: RNase protection assays of Na+-K+-ATPase beta 2-subunit isoform mRNA expression (20 µg total RNA per hybridization). Lane 1, 13-wk fetal kidney; lane 2, 16-wk fetal kidney; lane 3, 20-wk fetal kidney; lane 4, 24-wk fetal kidney; lane 5, undigested beta 2 probe; and lane 6, 20 µg tRNA hybridization control. M, marker lanes.

The alpha 1- and beta 2-subunits are expressed in apical plasma membranes of epithelial cells in the maturing nephron during nephrogenesis. Following induction of the mesenchymal cells of the metanephric blastema by the ureteric bud, a morphogenetic program is initiated that, in association with epithelial cell fate specification and differentiation, results in the formation of nephrons (16). In this developmental pathway, the acquisition of epithelial membrane polarity is required to generate a tubular epithelium with basolateral Na+-K+-ATPase essential for reabsorption of water and solute from the tubular lumen. In previous reports, it has been noted that some of the alpha -subunit Na+-K+-ATPase is located in the apical membrane of fetal collecting ducts (43). During human kidney organogenesis, nephron formation occurs principally during the second trimester, and we therefore sought to analyze alpha 1- and beta 2-subunit membrane localization in the primitive renal vesicle, early nephron, and maturing nephron in 12-24 wk metanephric kidneys to determine the first stage of nephron development associated with activation of Na+-K+-ATPase protein expression.

The earliest epithelial structures formed in the developing kidney are the renal vesicle, comma-shaped bodies, and S-shaped bodies. None of these early epithelial structures were found to express detectable alpha 1 or beta 2 Na+-K+-ATPase; in addition, there was no expression of either subunit in the undifferentiated blastemal cell layer just under the capsule of the 12-16 wk fetal kidney (data not shown). However, immunolocalization of the beta 2-subunit to the apical membrane was seen in the distal nephron and developing loop of Henle as early as the stage III-IV developing nephron as shown in Fig. 5, A and B [this section was made above the level of the glomerulus; the nephron staging system used was developed by Dorup and Maunsbach (14)]. The immunocytochemical staining utilized the beta 2 peptide affinity-purified antiserum used for Western immunoblotting in Fig. 1B, lane 3. The early proximal tubular portion of these early nephrons did not express the beta 2-subunit, nor was glomerular expression detected. However, loop of Henle staining with localization of the beta 2-subunit to the apical membrane was clearly shown in Fig. 5E. In Fig. 5, C and D, the immunocytochemical staining with the beta 2 antiserum (Fig. 5C) is compared with the preimmune control (Fig. 5D), which shows an absence of background staining in the same tissue sample.


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Fig. 5.   Immunolocalization of beta 2- and alpha 1-subunit isoforms in human metanephric kidney. A and B: beta 2 Na+-K+-ATPase expression (affinity-purified anti-beta 2 antibody) localized to the apical membrane of the distal proximal tubule and loop of Henle shown by arrowheads in B in a stage IV nephron (24 wk gestational age). Note expression in distal portion of proximal tubule (distal P.T.), loop of Henle, and distal tubule, but no expression in early proximal tubule. C: beta 2-subunit expression in a stage IV nephron in distal segments shown by arrows (15 wk). D: beta 2 preimmune antiserum shows lack of staining in multiple cross-sectional tubular profiles (arrowheads) in same fixed tissue sample as used in C. G, glomerulus. E: beta 2-subunit expression in a developing loop of Henle (arrowheads) and in a cross-sectional profile of a tubule (arrows show outer margin of this tubule segment) with no staining in glomerulus (G) (12 wk). F: alpha 1-subunit expression in same nephron as shown in E showing intense staining in a cross-sectional profile of the same tubule as in E (small arrows show basal plasma membranes of the epithelial cells at outer margin of this tubule segment). Less intense apical staining was seen in loop of Henle (arrowheads in F and in serial section stained with beta 2-subunit antiserum in E). G: alpha 1-subunit expression in early nephron shows apical and lateral staining of a distal tubular cross-sectional profile (arrowhead) with expression absent in glomerulus (G); note predominantly apical staining in tubule just above arrowhead (13 wk). H: alpha 1-subunit expression throughout a newly forming loop of Henle (arrows) was seen in apical membrane (arrowhead) (13 wk). I: alpha 1 preimmune control shows an absence of staining in proximal tubule (arrowhead), distal tubule segments (arrow), and glomerulus (G) (15 wk). J: beta 2-subunit expression in medulla of metanephric kidney. Large collecting ducts (arrows) show apical membrane staining as well as lesser amounts of basolateral membrane staining (19 wk, affinity-purified anti-beta 2 antibody). K: alpha 1-subunit expression in medulla of metanephric kidney. Large collecting ducts (arrows) show apical and basolateral membrane localization of alpha 1-subunit in contrast to the more restricted apical localization of the alpha 1-subunit in an adjacent tubule (arrowhead). L: alpha 1-subunit expression in medulla of adult rat kidney. Cross-sectional tubular profiles show basolateral staining (arrowheads) for alpha 1 Na+-K+-ATPase, but no detectable staining was seen in apical membranes. For each photomicrograph, 20 µm is indicated by bar at bottom right (final magnification ranges from ×284 to ×563). All sections were stained either with alpha 1 or beta 2 immune serum or preimmune controls and visualized with Nomarski optics. Gestational age of metanephric kidneys is given in weeks.

We have studied the expression of the Na+-K+-ATPase alpha 1-isoform in the stage IV developing nephron. In contrast to the apical plasma membrane distribution of the beta 2-subunit, both apical and lateral membrane alpha 1-subunit localization were detected in the early loop of Henle and distal tubule (Fig. 5, F-H). Some cross-sectional profiles show more prominent lateral alpha 1 expression in early nephrons (arrowhead, Fig. 5G) than in others, although this is less consistent than apical membrane localization. We have recently confirmed the presence of some alpha 1-subunit in basolateral plasma membranes in early nephrons (stage III-IV) using the alpha V and the alpha 6F monoclonal antibodies (data not shown). We next analyzed whether apical membrane localization of both the alpha 1- and beta 2-subunits could be demonstrated in the same tubule segment of a developing nephron using serial sections. As can be seen in Fig. 5, E and F, apical membrane localization of the beta 2- and alpha 1-subunits was found in the same nephron segment of an early nephron in serial sections prepared from a 12-wk human fetal kidney (arrows). In Fig. 5, E and F, the arrows precisely mark the basal cell membranes on the circumference of this tubule, which did not have detectable basolateral alpha 1- or beta 2-subunits by immunohistochemistry. The uniform apical membrane staining of the beta 2-subunit was also seen throughout the loop of Henle (Fig. 5E). Although this same loop of Henle (marked by arrowheads in Fig. 5, E and F) in serial section did show faint apical alpha 1 expression in the serial section, alpha 1 expression in an early loop of Henle was more clearly demonstrated in another early nephron (arrowhead in Fig. 5H); some of these profiles clearly also showed some alpha 1-subunit localized to the lateral membrane (as was shown in Fig. 5G as well). The alpha 1 preimmune control antiserum produced no staining (shown in Fig. 5I). In further studies examining expression of Na+-K+-ATPase isoforms in the human fetal kidney medulla, we found apical as well as basolateral membrane localization of the beta 2- (Fig. 5J) and alpha 1-isoforms (Fig. 5K) in maturing collecting ducts (arrows in Figs. 5, J and K). In contrast, exclusive basolateral alpha 1 expression (Fig. 5L, arrowheads) without apical membrane alpha 1 localization was detected in the adult rat kidney using this same polyclonal alpha 1 antiserum.

The alpha 1- and beta 2-subunits assemble into an Na+-K+-ATPase holoenzyme protein complex in the apical plasma membranes of human fetal collecting duct cells. Although it is well established that the alpha 1- and beta 2-subunits can assemble into a functional of Na+-K+-ATPase holoenzyme complex (7, 8, 53), the formal possibility exists that during fetal kidney development, the beta 2-subunit might be targeted to the plasma membrane independently from the alpha 1-subunit. To confirm assembly of the alpha 1- and beta 2-subunits in the fetal kidney collecting duct epithelia, we performed immunoprecipitation experiments using the alpha V anti-alpha -Na+-K+-ATPase monoclonal antibody followed by beta 2 detection with immunoblotting with either our anti-beta 2 peptide antiserum or with the SpETB2 anti-human beta 2-isoform-specific antibody [gift of Dr. P. Martin-Vasallo (23)], which has identical specificity to our anti-peptide beta 2 antiserum (Fig. 6A). For these experiments, we established the immortalized human fetal kidney collecting duct cell line HFCT.6D using the retroviral vector pZipneoTA58U19 (see MATERIALS AND METHODS), which we have previously employed for the immortalization of human adult kidney renal epithelial cell lines (50). HFCT.6D expresses the Na+-K+-ATPase alpha 1- and beta 2-subunits in a pattern consistent with apical membrane (data not shown). In contrast, we have not detected the typical "chicken-wire" pattern indicative of basolateral membrane localization of the Na+-K+-ATPase in this cell line. As shown in Fig. 6B, the identical beta 2 doublet band was detected in the immunoprecipitate by immunoblotting with the SpETB2 antiserum (lane 1) as was detected in the cell lysate (lane 2) of the HFCT.6D cell line. With omission of the alpha V monoclonal antibody (lane 3), no beta 2-subunit was recovered after precipitation with protein A/G agarose beads alone excluding nonspecific binding of the beta 2-subunit to the agarose matrix of the beads. We have also confirmed that our anti-peptide beta 2 antiserum detects the identical beta 2-subunit following immunoprecipitation with the alpha V anti-alpha -Na+-K+-ATPase monoclonal antibody in other epithelial cell lines that express both the alpha 1- and beta 2-subunits (data not shown). In addition, it is evident that the beta 2-subunit is heavily glycosylated in two independent HFCT cell lines (HFCT.6D and HFCT.6E, see Fig. 6); since full glycosylation of Na+-K+-ATPase beta -subunits depends on assembly with the alpha -subunit in the endoplasmic reticulum (1, 20), this strengthens the conclusion that the alpha 1/beta 2 complexes detected in these experiments are associated with the plasma membrane.


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Fig. 6.   Detection of alpha 1beta 2 Na+-K+-ATPase complexes in fetal collecting duct cells (HFCT). A: beta 2-subunit detection by Western immunoblot of cell lysate prepared from the HFCT.6E cell line with anti-SpETB2 (1:1,000 dilution) in lane 1 and anti-beta 2 peptide rabbit polyclonal antiserum 1305 (1:1,000 dilution) (lane 2) confirming identical specificity of these two beta 2-subunit isoform-specific antisera. B: HFCT.6D cell lysate was immunoprecipitated with the anti-alpha 1 Na+-K+-ATPase monoclonal antibody alpha V and immunoblotted with the SpETB2 beta 2-subunit antiserum (1:2,000 dilution) (lane 1). Lane 2, SpETB2 blot of 20 µg of HFCT cell lysate; lane 3, SpETB2 immunoblot of precipitates prepared from HFCT cell lysate with omission of the alpha V monoclonal antibody. Identical beta 2 doublet band present in the cell lysate (lane 2) is recovered in the immunoprecipitate (lane 1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although the alpha -subunit of the Na+-K+-ATPase is the catalytic subunit for Na+/K+ exchange at the cell membrane, elucidation of the function of the beta -subunit has revealed its essential role in the translocation of the alpha -subunit to the plasma membrane (41) and provided insights into its function in the regulation of the kinetics of Na+/K+ exchange by the alpha -subunit (30). The alpha -subunit also plays a role in localization of the Na+-K+-ATPase to the basolateral membrane of renal epithelial cells mediated by stable association with ankyrin sites (13) associated with the fodrin cytoskeleton (46, 47) in the kidney. It also appears that the NH2-terminal half of the alpha -subunit is important in sorting for basolateral membrane localization of newly synthesized pumps (45). In the choroid plexus (2, 39) and retinal pigment epithelium (25), an extensive series of studies has demonstrated the potential importance of binding of the Na+-K+-ATPase alpha -subunit to an apical membrane ankyrin-cytoskeletal complex as a principal mechanism for polarization of the sodium pump to the apical membrane domains in these epithelial cell types. By comparison, relatively little is known about the potential role of the beta -subunit in the regulation of cell membrane polarity of the Na+-K+-ATPase, although beta -subunit signal sequence(s) might be important for basolateral membrane vesicle transport of the Na+-K+-ATPase in renal epithelial cells. Alternatively, beta -isoform structure might influence binding of the alpha -subunit to the ankyrin cytoskeletal complex and influence membrane localization of the Na+-K+-ATPase through this mechanism.

The acquisition of basolateral membrane polarization of Na+-K+-ATPase necessary for tubular reabsorption in the adult kidney occurs well after nephron formation since apical, as well as basolateral, membrane localization of the alpha -subunit is identified throughout at least the 12-24 wk gestational age range in human kidney development (this study) and has been consistently seen in other studies of mammalian kidney development (4, 28, 43). It is also important to recognize that even in the adult kidney, biochemical and immunohistochemical studies have consistently demonstrated the presence of small amounts of apical membrane Na+-K+-ATPase in the kidney (33, 34, 44). Therefore, the developmentally acquired molecular mechanisms that control cell polarization do not result in the complete loss of apical membrane Na+-K+-ATPase. In this study we have shown that beta 2-subunit is induced in the stage III-IV early nephrons as the loop of Henle is first formed where both the alpha 1- and beta 2-isoforms are found in the apical membrane domains. We also made a surprising observation that there is little, if any, apparent expression of the beta 1-subunit protein during early kidney development detected by immunoblotting using our beta 1-subunit isoform-specific antiserum. This result was unexpected, because throughout nephrogenesis, there is a similar abundance of beta 1 and beta 2 mRNA by RNase protection analysis of total fetal kidney RNA. Although we cannot completely exclude that the absence of detectable beta 1 protein in the fetal kidney might somehow derive from antigen masking, we consider this unlikely, as we have employed multiple alternate detergent extractions (including octylglucoside, CHAPS, NP-40, or SDS in a range of concentrations) and have consistently failed to detect fetal kidney beta 1 protein by immunoblotting. It is also possible that a low level of beta 1-subunit expression is present during kidney organogenesis which is below the limit of sensitivity of our Western blot assay. However, the lack of detectable expression of fetal kidney beta 1 protein expression in our studies supports the conclusion that selective translational silencing of the beta 1 mRNA may occur during kidney development. A similar lack of beta 1 protein expression due to developmental translational silencing was recently demonstrated in the early development of the X. laevis embryo (9). Postnatal increases in the levels of the beta 1-subunit have also been observed previously, although the regulatory basis for this had not been defined (57). Recently, the potential importance of posttranscriptional upregulation of beta 1-subunit expression has also been identified as an important mechanism of postnatal increases in the basolateral membrane Na+-K+-ATPase in the newborn guinea pig renal cortex (24). The loss of beta 2 expression and the activation of beta 1 protein biosynthesis apparently occurs late in gestation and may be required for the acquisition of basolateral polarization of the Na+-K+-ATPase alpha -subunit in the kidney tubule. These data therefore suggest that basolateral membrane polarization of the Na+-K+-ATPase during maturation of the renal tubule may be regulated at least in part at the level of beta -subunit isoform expression and support the conclusion that the molecular structure of the beta -subunit may influence the sorting of the Na+-K+-ATPase into either specific basolateral or apical vesicle transport pathways.

The hypothesis that the beta 2-subunit contains a signal for apical membrane localization of the Na+-K+-ATPase has recently been tested in MDCK cells. These studies have shown that transfection of the human beta 2-subunit into MDCK cells results in the apical localization of beta 2-subunits (Ref. 51; and D. M. Fambrough, personal communication). In contrast, MDCK cell transfection of the beta 1-subunit results in the assembly of alpha 1-beta 1 heterodimers with basolateral membrane localization (17). This supports the conclusion that sequence differences between the beta 1- and beta 2-subunit influence molecular mechanisms involved in vesicle transport pathways or binding to components of the cytoskeleton important for the establishment and maintenance of membrane protein polarization in epithelia. The existence of a specific basolateral vesicle transport pathway in renal epithelial cells has been established (10, 46) and may in part be directly or indirectly (through an effect on the alpha -subunit) dependent on beta 1 amino acid sequences not found in the beta 2-subunit. The low homology between the beta 1- and beta 2-subunits in the cytoplasmic NH2 terminus (30% identity residues 1-30 of beta 2 vs. beta 1), which would be on the outer surface of a transport vesicle, suggests the potential importance of this sequence in regulating apical versus basolateral delivery of the Na+-K+-ATPase in renal epithelia cells. The expression of the beta 2-subunit in the choroid plexus, which also demonstrates apical membrane localization of the apical Na+-K+-ATPase, suggests that developmental selection of beta -subunit isoform expression may be a general mechanism for the regulation of membrane polarization of the Na+-K+-ATPase in epithelial cells. A difficulty with this model is that the apical membranes of the choroid plexus epithelium contain both beta 1- and beta 2-subunits, as shown by immunolocalization studies using isoform-specific antisera (22). However, if higher order Na+-K+-ATPase multimers [i.e., (alpha 1beta 1)2] exist in cells (see Ref. 36), (alpha 1)2(beta 1beta 2) heteromultimers could also potentially be formed and be preferentially retained in the apical membrane domains.

Our finding of apical localization of Na+-K+-ATPase during development is not likely related to a failure of formation of intercellular tight junctions, as ultrastructural studies have demonstrated that even the epithelia of the early renal vesicle have already acquired apical tight junctions (14). Our results, therefore, suggest that the membrane localization of the Na+-K+-ATPase may in part be developmentally controlled at the level of beta -isoform expression and not only dependent on the binding of the alpha -subunit to the ankyrin-fodrin cytoskeletal components. As a partial validation of this model, it has recently been established that beta 2-subunit expression in MDCK cells results in its apical membrane localization under experimental conditions associated with a normally polarized basolateral ankyrin-fodrin network.

It is also important to emphasize that in the early nephron the alpha 1-isoform is also found in the basolateral membranes of fetal tubule segments including the proximal tubule and the loop of Henle, where we have not been able to detect expression of the beta 2-subunit (see Fig. 5G) which is consistently apical in these segments. Although this might suggest the possibility that the alpha 1-subunit may reach the basolateral plasma membrane without association with the beta 2-subunit, we think that immunolocalization studies alone are insufficient evidence for reaching this conclusion given the uniform requirement for beta -subunit association with the alpha -subunit for its stabilization and insertion into the plasma membrane. In contrast, we find that although beta 2-subunit is preferentially localized to the apical membrane of the maturing collecting ducts (Fig. 5J), there is also some basolateral membrane beta 2-subunit localization. For the alpha 1-subunit, there is clearly both apical membrane as well as basolateral localization in the maturing collecting duct (Fig. 5K), as previously seen in the rat (4) and rabbit postnatal kidney (43). In sum, there exist in the fetal kidney collecting ducts, as well as in more proximal segments of the tubule in the early nephron, apparent differences in the relative staining intensity of the alpha 1- and beta 2-subunits in the basolateral membrane (alpha 1 greater than beta 2), which might indicate different subunit stoichiometries in the apical versus basolateral membrane in tubulogenesis.

A conservative interpretation of our results is that during tubulogenesis, alpha 1/beta 2 Na+-K+-ATPase heterodimers are not exclusively directed or retained in the apical membrane in all nephron segments, but may also be localized to the basolateral membranes as found in the developing collecting duct system. These results suggest that the beta 2-subunit does not have a functionally dominant and exclusive apical membrane targeting sequence active in all nephron segments during organogenesis despite the fact that the beta 2-subunit shows preferential localization to the apical membrane of transfected MDCK cells. Our findings are consistent with a model that alpha 1/beta 2 heterodimeric Na+-K+-ATPase pumps may partially escape the in vivo cellular control mechanisms that ensure basolateral membrane localization, with the result that in certain tubule segments, their localization is largely apical, whereas in others (such as the collecting duct) there is essentially a nonpolarized distribution of pumps throughout the plasma membrane. In sum, this model predicts that the alpha 1/beta 1 Na+-K+-ATPase pump is subject to a much more stringent regulatory mechanism, which results in highly selective basolateral membrane localization with a far lower level of apical membrane Na+-K+-ATPase than is possible with the more relaxed control mechanisms governing polarization of the alpha 1/beta 2 Na+-K+-ATPase.

The functional role of apical Na+-K+-ATPase during tubulogenesis remains to be established. Expression of apical Na+-K+-ATPase in the loop of Henle during its formation in the early nephron appears to precede formation of a functional glomerular tuft and supports the possible importance of basal-to-luminal sodium transport in the establishment of a lumen in the distal nephron. This model proposes that the requirement for apical membrane Na+-K+-ATPase during tubulogenesis is similar to the function of the apical membrane Na+-K+-ATPase in trophectodermal cells in the developing blastocyst (6). The developmental regulatory mechanisms directing the reprogramming of renal tubular epithelial cells for postnatal life are aimed at assuring basolateral membrane localization of the Na+-K+-ATPase. Our data support the potential importance of analysis of the molecular basis of transcriptional repression of the beta 2 Na+-K+-ATPase gene and translational activation of beta 1 mRNA expression in establishing basolateral membrane localization of the Na+-K+-ATPase in the renal epithelial cells of the developing nephron during kidney organogenesis and in early postnatal life.


    ACKNOWLEDGEMENTS

Katherine Thornton, Samantha Wilson, Rebecca Zausmer, Lillian Kang, and Lida Zhen are gratefully acknowledged for technical assistance in these studies. We thank Dr. Douglas Fambrough for the gift of the alpha V anti-chicken alpha -subunit Na+-K+-ATPase monoclonal antibody, Dr. Pablo Martin-Vasallo for the anti-beta 2 Na+-K+-ATPase antibody SpETB2, and Dr. Alicia McDonough for many helpful suggestions.


    FOOTNOTES

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-44833 (to P. D. Wilson).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. R. Burrow, Box 1243, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY, 10029-6574 (E-mail: chris_burrow{at}smtplink.mssm.edu).

Received 12 January 1998; accepted in final form 27 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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