Ubiquitous and Kidney-Specific Subunits of Vacuolar H+-ATPase Are Differentially Expressed during Nephrogenesis
François Jouret*,
Céline Auzanneau,
Huguette Debaix*,
Ge-Hong Sun Wada,
Chrystel Pretto,
Etienne Marbaix,
Fiona E. Karet,
Pierre J. Courtoy and
Olivier Devuyst*
* Division of Nephrology; ICP Cell Unit, Université catholique de Louvain, Brussels, Belgium; Doshisha University, Kyoto, Japan; and Department of Medical Genetics, Cambridge University, Cambridge, United Kingdom
Address correspondence to: Dr. Olivier Devuyst, Division of Nephrology, Université catholique de Louvain, 10 Avenue Hippocrate, Brussels, Belgium B-1200. Phone: +32-2-764-1855; Fax: +32-2-764-5455; E-mail; devuyst{at}nefr.ucl.ac.be
Received for publication November 12, 2004.
Accepted for publication July 26, 2005.
The vacuolar H+-ATPase (V-ATPase) is a ubiquitous multisubunitpump that is responsible for acidification of intracellularorganelles. In the kidney, a particular form of V-ATPase, madeof specific subunits isoforms, has been located at the plasmamembrane of intercalated cells (IC). Mutations in genes encodingIC-specific subunits cause infant distal renal tubular acidosis(dRTA), suggesting that the segmental distribution of thesesubunits is acquired at birth or during early infancy. However,the comparative ontogeny of the IC-specific versus the ubiquitoussubunits of V-ATPase and the mechanisms involved in their segmentalexpression remain unknown. Real-time reverse transcription–PCR,in situ hybridization, immunoblotting, immunostaining, and subcellularfractionation analyses characterized the expression and distributionof V-ATPase subunits, transcription factors, and differentiationmarkers during mouse nephrogenesis. Ubiquitous A, E1, B2, G1,and C1 subunits showed an early (embryonic day 13.5 [E13.5])and stable expression throughout nephrogenesis, followed bya slight increase around birth. The developmental pattern ofa1 was bimodal, with early induction, gradual decrease duringorganogenesis, and neonatal increase. These patterns contrastedwith the later (from E15.5) and progressive expression of IC-specifica4, B1, G3, and C2 subunits, after the induction of the forkheadtranscription factor Foxi1. From E15.5, Foxi1 mRNA was detectedin IC, where it co-distributed with B1 in late nephrogenesis.Immunostaining showed that the distribution of ubiquitous E1and B2 was acquired from E15.5, whereas a4 was located in ICduring late nephrogenesis. Subcellular fractionation showedthat in both fetal and mature (cortex and medulla) kidneys,E1 and a4 were located in endosomes. These data demonstratea differential expression and a coordinate regulation of IC-specificversus ubiquitous V-ATPase subunits during nephrogenesis. Theyprovide new insights into the complex regulation of V-ATPasesubunits, the maturation of IC along the nephron, and the pathophysiologyof hereditary dRTA.
The vacuolar-type proton ATPase (V-ATPase) is a ubiquitous multisubunitpump that is essential for the acidification of intracellularorganelles by coupling ATP hydrolysis to transmembrane protontransport (1). The V-ATPase has been located in a variety ofintracellular compartments, including clathrin-coated vesicles,endosomes, and lysosomes. Acidification of these intracellularorganelles is a prerequisite for a number of important intracellularprocesses, such as ligand-receptor dissociation, receptor recyclingand ligand degradation, storage, or intracellular targeting(2). In addition to its intracellular distribution, V-ATPasehas been located in the plasma membrane of specialized epithelialcells of the kidney and epididymis, as well as in the ruffledborder membrane of osteoclasts, where it functions in urinaryor semen acidification and in bone resorption, respectively(3).
The structure of V-ATPase includes at least 13 different subunitsthat form two functional domains, V0 and V1 (Figure 1) (3,4).According to quantitative amino acid analysis, the transmembraneV0 domain contains five different subunits that are organizedinto a complex that is responsible for proton translocation.The cytosolic V1 domain, which involves eight subunits, catalyzesATP hydrolysis and provides the energy necessary for activeproton transport. When located to the plasma membrane, the overallstructure of V-ATPase is very similar, but specific isoformsof V0 and V1 subunits are present (5).
Figure 1. Structural model of the vacuolar type H+-ATPase (V-ATPase). The transmembrane V0 domain of the mammalian V-ATPase is organized into a complex of a(c)4 to 5 c"d, whereas the cytosolic V1 domain has a stoichiometry of A3B3CDEFG2H2. The ubiquitous and the intercalated cell (IC)-specific alternate isoforms of V0 and V1 subunits are colored. In the plasma membrane of murine IC, a4, d2, B1, C2, and G3 substitute ubiquitous a1, d1, B2, C1, and G1, respectively. This model was modified from the yeast model to represent the mammalian V-ATPase complex (adapted with permission from reference 5). Note that the presence of up to three copies of each E and G per V-ATPase complex has been suggested from analysis of Coomassie staining (4) and that the existence of the c' subunit in mammals is uncertain (42).
The subunit isoforms of the V-ATPase are encoded by distinctgenes, with tissue-specific expression patterns. In the kidney,the V-ATPase is essentially distributed in two cell types thatare involved in acid–base homeostasis: the proximal tubulecells (PTC) and the intercalated cells (IC) of the collectingduct (CD) (5,6). In PTC, the V-ATPase has been located on theapical side, where it participates in HCO3– reabsorption(7). In addition, V-ATPase is responsible for correct vesicularacidification along the endocytic pathway, which is essentialfor receptor-mediated endocytosis and proper intracellular membranetargeting (8). In IC, a specialized form of V-ATPase, made oftissue-specific V0 and V1 subunits isoforms, mediates transepithelialproton transport, ensuring correct urinary acidification (7).
Despite the pivotal role of V-ATPase in acid–base homeostasis,the ontogeny of its subunits in the nephron remains unknown.During pre- and postnatal nephrogenesis, the expression andmaturation of several transporters implicated in acid–basehomeostasis is tightly regulated to compensate the acid-generatingprocess of growth (9,10). Term neonates are indeed characterizedby lower values of blood pH than adults, reflecting an incompletekidney maturation to reabsorb HCO3– (11). The targeteddisruption of the V0 c subunit in mouse leads to early fetallethality by lack of postimplantation development (12). By contrast,mutations in ATP6V0A4 and ATP6V1B1 genes, encoding IC-specificV0 a4 and V1 B1 subunits, respectively (13–15), have beenassociated with early-onset cases of distal renal tubular acidosis(dRTA), which suggests that the segmental distribution of IC-specificisoforms of V-ATPase could be acquired during early infancy.
In this study, we used real-time reverse transcription–PCR(RT-PCR) and in situ hybridization, coupled with immunoblottingand immunostaining analyses, to establish the comparative ontogenyof ubiquitous versus IC-specific subunits of V-ATPase duringmouse nephrogenesis. In addition, on the basis of the structuralmodel of V-ATPase (4), we assessed the relative mRNA expressionof alternate isoforms in the adult mouse kidney. Finally, thesubcellular distribution of V-ATPase subunits in the developingand mature kidney was compared with endosomal and lysosomalmarkers by analytical subcellular fractionation.
Mouse Kidney Samples
All samples were obtained from CD-1 mice (Iffa Credo, Brussels,Belgium), in accordance with National Institutes of Health guidelinesfor the care and use of laboratory animals and with the approvalof the Committee for Animal Rights of the Universitécatholique de Louvain. Fetuses were removed and microdissectedto isolate kidneys. Embryos from four different litters (average12 embryos per litter) were collected daily from embryonic day13.5 (E13.5) until day 5 after birth (newborn). Comparativestudies were performed with eight 12-wk-old male kidneys.
Real-Time RT-PCR
Mouse kidney samples were homogenized in Trizol (Invitrogen,Merelbeke, Belgium) to extract total RNA. Total RNA samples(2.7 µg) were treated with DNase I (Invitrogen) and reverse-transcribedinto cDNA using SuperScript II RNase H Reverse Transcriptase(Invitrogen). Specific primers for V-ATPase subunits, Foxi1,Pendrin (PDS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),cyclophilin, and hypoxanthine guanine phosphoribosyl transferase(HPRT1) were designed using Beacon Designer 2.0 (Premier BiosoftInternational, Palo Alto, CA) and are summarized in Table 1.Real-time PCR analyses were performed in duplicate with 200nM of both sense and antisense primers in a final volume of25 µl using 1 unit of Platinum TaqDNA Polymerase, 2 mMMgSO4, 400 µM dNTP, and SYBR Green I (Molecular Probe,Leiden, The Netherlands) diluted to 1:105. The PCR mixture contained10 nM fluorescein for initial well-to-well fluorescence normalization.PCR conditions were settled as incubation at 94°C for 3min followed by 40 cycles of 30 s at 95°C, 30 s at 61°C,and 1 min at 72°C. The melting temperature of PCR productwas checked at the end of each PCR by recording SYBR green fluorescenceincrease upon slowly renaturating DNA. For each assay, standardcurves were prepared by serial four-fold dilutions of mouseadult kidney cDNA. The efficiencies of the amplifications witheach primer set were calculated from the slope of the standardcurve [efficiency = (10–1/slope) – 1] and were closeto 100% (Table 1).
The relative mRNA expression of the various isoforms (A, a1,B2, E1, G1, C1, a4, B1, G3, and C2) was investigated in adultmale kidneys (n = 8), after normalization to HPRT1, cyclophilin,and GAPDH [Ratio = 2Ct (Reporter – Target Gene)]. Therelative changes in mRNA levels of these alternate isoformsduring ontogeny were determined by comparison with the adultmRNA level, after adjustment to GAPDH [Ratio = (Efficiencytargetgene)Ct(Adult – Sample)/(EfficiencyGAPDH)Ct (Adult –Sample)] (16). Real-time RT-PCR results were confirmed by usinganother set of primers for each of V-ATPase subunits.
In parallel with the relative quantification described above,we cloned DNA plasmids for each subunit and GAPDH to determinecopy numbers of V-ATPase subunits transcripts. The PCR productswere cloned into a pTZ57R vector (Fermentas GmbH, Vilnius, Lithuania)and transformed in JM 107 Competent Cells (MBI Fermentas). LB-brothcultures (150 ml) of single colonies were grown up overnightskating at 200 rpm at 37°C. Plasmid purification was performedwith the High Purity Plasmid Systems (Gentaur, Brussels, Belgium)according to the manufacturer’s recommendations. Standardcurves were generated from serial 10-fold dilutions of the purifiedplasmid that contained the appropriate cDNA, and regressionanalyses of the threshold cycle (Ct) values of standard dilutionseries were used to determine the amplification efficiency (datanot shown). The absolute amount of mRNA molecules of V-ATPasesubunits in mouse adult kidneys (n = 8) was determined by extrapolatingthe Ct values from the standard curves, according to the molecularweight of the plasmid (1.9 x 106 g/mol) and the Avogadro’snumber (1 mol = 6.022 x 1023 molecules), and then normalizedto the GAPDH level in each sample. Each experiment was run induplicate, with highly reproducible results.
In Situ Hybridization
Kidney samples were fixed in 4% formaldehyde (Boehringer Ingelheim,Heidelberg, Germany) in 0.1 M phosphate buffer (pH 7.4) beforeembedding in paraffin as described (17). Five-micrometer-thicksections were deparaffinized, rehydrated. and treated for 45min at 37°C with 1 mg/ml proteinase-K (Sigma, St. Louis,MO) in 100 mM Tris-HCl (pH 8.0) and 50 mM EDTA. Sections thenwere acetylated for 10 min in 0.25% acetic anhydride and 0.1M triethanolamine (pH 8.0) and prehybridized for 1 h at 52°C(Foxi1) or 56°C (B1) in the hybridization mixture that contained40% (vol/vol) formamide, 50% (wt/vol) dextran sulfate, 2% (vol/vol)Denhardt’s solution, and 2x SSC (1x SSC = 150 mM NaCland 15 mM sodium citrate) in a humidified chamber. A mixtureof two Foxi1- or B1-specific oligonucleotide probes (Foxi1 5'-AGGCTGGGTGACCTTCGAAGGCTGGATAAAGGGAACCGGG-3',5'-GAGTGGCTGTGAGCACAGCCTCTGGCACTGCTCGCTCCTCT-3'; B1 5'-AACATGGGTTCAAGCCATAAAGGAGAAGGCTGCAGCTGG-3',5'-TCCATTTCAGTTACATCTTTGGAGGCGGGGCTTCTATAAT-3') or of two controloligonucleotides with the complementary sequence was preparedby fluorescein-dUTP labeling of the 3' ends (Amersham Biosciences,Piscataway, NJ). The sections then were hybridized overnightat 52°C (Foxi1) or 56°C (B1) with the hybridizationmixture supplemented with 1.5 ng/ml labeled probes and washedwith decreasing concentrations of SSC (4x, 2x, and 1x). Allwashes were carried out at room temperature for 2 x 10 min.The detection of hybridized fluorescein-labeled probes was performedaccording to the immunostaining procedure, described below.
Antibodies
Immunoblotting and immunostaining analyses were performed withwell-characterized antibodies, including mAb against the E1subunit of the V-ATPase (a gift of Dr. S. Gluck, Universityof California, San Francisco, CA) (6); -actin (Sigma); fluorescein(Dako, Glostrup, Denmark); 1 Na+/K+-ATPase (Upstate BiotechnologyInc., Waltham, MA); rabbit polyclonal antibodies against a4subunit (13); B1, B2, G1, and G3 subunits (17); A subunit (WakoChemicals GmbH, Neuss, Germany); C1 subunit (Santa Cruz Biotechnology,Santa Cruz, CA); Rab5a (Santa Cruz Biotechnology); aquaporin-2(AQP2; Sigma); podocin (a gift from C. Antignac, Inserm U574,Necker Hospital, Paris, France) (18); sheep polyclonal antibodiesagainst megalin (a gift from P.J. Verroust, Saint-Antoine MedicalFaculty, Paris, France) (8); type II carbonic anhydrase (CAII;Serotec, Oxford, UK); goat polyclonal antibodies against cathepsinD (Santa Cruz Biotechnology); and chicken polyclonal antibodiesagainst pendrin (a gift from P. Kopp, Northwestern University,Chicago, IL).
Analytical Subcellular Fractionation
Kidneys from approximately 40 fetuses at E16.5 and dissectedcortex and medulla from five adult mice were minced in 0.25M sucrose and 3 mM imidazole buffer (pH 7.4) that containedComplete protease inhibitors (Roche, Vilvoorde, Belgium) andhomogenized in a Potter-Elvehjem tissue homogenizer (ThomasScientific, Swedesboro, NJ). A low-speed "nuclear" fractionwas pelletted at 700 x g for 10 min and extracted twice by resuspensionsedimentation. Pooled postnuclear supernatants were sedimentedfurther at 100,000 x g for 60 min in a 50Ti fixed-angle rotor(Beckman, Palo Alto, CA). The supernatant (S), which representsthe cytosolic fraction, was collected for further characterization.The high-speed pellet (MLP fraction of postnuclear particles)was resuspended in 1 ml of homogenization buffer, mixed with7 ml of 16% (vol/vol) Percoll (average final density 1.048 g/ml),layered over a 250-µl Percoll cushion, and centrifugedat 60,000 x g for 30 min in a 50Ti rotor into a self-generatinggradient, after which 10 fractions of approximately 750 µleach, excluding the packed cushion, were collected from thebottom. Each fraction was analyzed for density (refractometry),total protein content (Lowry assay), and amount of the indicatedantigens (immunoblotting).
Immunoblotting
Membrane extraction and immunoblotting were performed as describedpreviously (19). Briefly, kidney samples were homogenized inice-cold buffer [300 mM sucrose and 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid made to pH 7.0 with 1 M tris(hydroxymethyl)aminomethane(Tris)] that contained Complete protease inhibitors (Roche).The homogenate was centrifuged at 1000 x g for 15 min at 4°C,and the resulting supernatant was centrifuged at 100,000 x gfor 120 min at 4°C. The pellet was suspended in ice-coldhomogenization buffer, before determination of protein concentrationand storage at –80°C. After resolution by SDS-PAGEand blotting on nitrocellulose, the membranes were incubatedovernight at 4°C with primary antibodies, washed, incubatedfor 1 h at room temperature with appropriate peroxidase-labeledantibodies (Dako), washed again, and visualized with enhancedchemiluminescence. Normalization for -actin was obtained afterstripping the blots and reprobing with the anti–-actinantibody. Specificity of the immunoblot was determined by incubationwith nonimmune rabbit or mouse IgG (Vector Laboratories, Burlingame,CA) or control ascites fluid (Sigma). Densitometry analysiswas performed with a Canon CanoScan8000F using the NIH ImageV1.60 software. All immunoblots were performed at least in duplicate.
Immunostaining
After blocking endogenous peroxidase for 30 min with 0.3% hydrogenperoxide, sections were incubated with 10% normal goat serumfor 60 min, before incubation with the primary antibodies dilutedin PBS that contained 2% BSA for 60 min. After washing in 50mM Tris-HCl, sections were incubated successively with biotinylatedsecondary anti-IgG antibodies, avidin-biotin peroxidase, anddiaminobenzidine or aminoethylcarbazole (Vector Laboratories).For immunofluorescence studies, secondary antibodies were detectedwith Texas red–conjugated and FITC-conjugated avidin (VectorLaboratories). Sections were viewed under a Leica DMR coupledto a Leica DC300 digital camera (Leica, Heerbrugg, Switzerland).The specificity of immunostaining was tested by incubation (1)in the absence of primary antiserum and (2) with nonimmune rabbitserum or control rabbit or mouse IgG (Vector Laboratories).
Relative Expression of V-ATPase Subunits in Adult Mouse Kidney
The recently updated structure of the V-ATPase contains at least13 different subunits that form two functional domains, V0 andV1 (Figure 1). Before considering the expression of isoformsduring mouse nephrogenesis, we performed comparative real-timeRT-PCR quantification, with different reporter genes, to establishthe relative mRNA expression of ubiquitous and IC-specific subunitsin adult mouse kidney (Figure 2). These data were essentiallyconfirmed by absolute real-time RT-PCR using standard curvesof cloned DNA plasmids for the V-ATPase subunits and GAPDH (datanot shown). These studies revealed striking differences in termsof relative expression at the mRNA level that could not be explainedby the structural stoichiometry of the V-ATPase complex shownin Figure 1. For instance, the A subunit was 20-fold more abundantthan B2, although these subunits are considered to form theA3B3 catalytic core of the V1 domain. Moreover, comparativeanalysis of alternate isoforms inside ubiquitous (a1, B2, C1,and G1) or IC-specific (a4, B1, C2, and G3) pumps did not showa constant stoichiometry. These results suggest that the assemblyof the V-ATPase complex results from important posttranscriptionalmodifications and/or reflects significant differences in theturnover of its various components.
Figure 2. mRNA quantification of V-ATPase subunits. Real-time reverse transcription–PCR (RT-PCR) quantification of ubiquitous (A, a1, B2, E1, G1, and C1) and IC-specific (a4, B1, G3, and C2) subunits in mouse adult kidney (n = 8). The mRNA expression levels (mean ± SEM) were compared after adjustment to HPRT1 (x50), cyclophilin (x103), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; x2.103) as reporter genes. HPRT1, hypoxanthine guanine phosphoribosyl transferase.
Expression of V-ATPase Subunits mRNA in Developing Mouse Kidney
The differential expression patterns of ubiquitous and IC-specificV-ATPase subunits during mouse nephrogenesis were investigatedusing real-time RT-PCR analyses (Figure 3). The ubiquitous A,B2, E1, G1, and C1 subunits showed an early (E13.5) and stableexpression (approximately 40% of the adult level) throughoutnephrogenesis, reaching the adult level at birth (Figure 3A).Of note, the a1 isoform showed an earlier induction at E13.5(approximately 135%) followed by a gradual decrease during lateontogeny and a second increase around birth. This particulardevelopmental pattern is similar to that described for the endosomalkidney-specific chloride channel, ClC-5, and for the V0 d1 V-ATPasesubunit (19,20). In contrast, the corresponding IC-specificB1, G3, C2, and a4 subunits were barely detected at E15.5 andshowed a progressive induction during late ontogeny, like theanion transporter pendrin (Figure 3B). It must be noted thatthe forkhead transcription factor Foxi1, which is a member ofthe HFH/winged helix family, was detected from E15.5 (Figure 3B).These data show that the early expression of ubiquitoussubunits of V-ATPase definitively contrasts with the later andmore progressive appearance of Foxi1, pendrin, and IC-specificsubunits.
Figure 3. Expression of V-ATPase subunits in mouse developing kidney. Real-time RT-PCR quantification for mRNA expression of ubiquitous A, B2, E1, G1, C1, and a1 subunits (A) and IC-specific B1, G3, C2, and a4 isoforms, as well as Foxi1 and pendrin (pds) (B), during mouse nephrogenesis. The mRNA levels were first adjusted to GAPDH at every developmental stage, then normalized to the adult level set at 100% using the following formula: [Ratio = (Efficiencytarget)Ct (Adult – Sample)/(EfficiencyGAPDH)Ct (Adult – Sample)]. The early (embryonic day 13.5 [E13.5]) and stable expression of ubiquitous subunits during nephrogenesis clearly contrasts with the later and progressive appearance of IC-specific isoforms and pds, which follows the induction of the transcription factor Foxi1 at E15.5. Note that the a1 subunit is characterized by a bimodal developmental pattern.
Distribution of Forkhead Transcription Factor Foxi1 in Developing Mouse Kidney
The winged helix transcriptional activator Foxi1 has been locatedto the distal part of the nephron from E16.5 during mouse nephrogenesis(21) and recently implicated in IC differentiation along theCD (22). To investigate the putative link between Foxi1 andIC-specific V-ATPase isoforms expression, we performed in situhybridizations on mouse developing kidney with fluorescein-labeledoligonucleotides directed against Foxi1 or B1 (Figure 4). AtE15.5, Foxi1 was detected in some scattered cells of the medullaryCD, whereas no signal was found in glomeruli, PT, and differentiatingstructures in the cortex (Figure 4A). At later stages, Foxi1mRNA was identified in a subset of cells within distinct tubulesin cortex and medulla (Figure 4C). In these cells, Foxi1 co-distributedwith the IC-specific B1 V-ATPase subunit (Figure 4D). Altogether,the real-time RT-PCR and in situ hybridization data during mousenephrogenesis strongly support the role of Foxi1 in the differentiationof IC along the CD.
Figure 4. Distribution of Foxi1 and V-ATPase B1 subunit mRNA in mouse developing kidney. In situ hybridization for Foxi1 (A and C: antisense probe; B: sense); B1 V-ATPase subunit (D: antisense probe; E: sense) in the developing mouse E15.5 (A and B) and neonatal (C through E) kidney. At E15.5, Foxi1 mRNA is specifically detected in scattered cells within medullary collecting ducts (CD; A, arrowheads). In the newborn kidney, Foxi1 and B1 co-distribute in distinct cells within cortical CD (C and D, arrowheads on serial sections). No signal was detected when sense probes were used on consecutive sections (E). Bars = 50 µm in A and B; 20 µm in C through E.
Expression of V-ATPase Subunits in Developing Mouse Kidney
Well-characterized antibodies were used to evaluate the expressionof ubiquitous and IC-specific V-ATPase subunits in mature anddeveloping kidney (Figure 5). Postnuclear particles and high-speedsupernatant, resolved by differential centrifugation of adultkidney samples, were analyzed by immunoblotting. In the adultkidney, approximately 20% of V1 subunits, i.e., A, B2, E1 andG1, were detected in the cytosolic fraction, whereas the transmembranea4, as well as the V1 C1 subunit, was associated only with membranes(Figure 5A). During nephrogenesis (Figure 5B), ubiquitous V-ATPasesubunits (A, B2, E1, G1, and C1) were detected as early as E13.5and gradually increased from E16.5 until birth. In contrast,the IC-specific subunit isoforms (a4, B1, and G3) and the anionexchanger pendrin were detected only from E16.5 and increasedprogressively during late nephrogenesis.
Figure 5. Expression of V-ATPase subunits in mouse developing kidney. (A) Relative expression of V1 A, B2, E1, G1, and C1, and V0 a4 in membrane (MLP) and cytosolic (S) compartments of adult mouse kidney. Equal loads (20 µg) of postnuclear particles (MLP 37% of total homogenate proteins) and high-speed supernatant (S 29% of total homogenate proteins) were analyzed. Densitometry analyses show that approximately 20% of V1 subunits are not associated with membranes at steady state. The transmembrane V0 a4 subunit, as well as the V1 C1, are not detected in the cytosolic fraction. (B) Representative immunoblots for ubiquitous A, B2, E1, G1, C1, and IC-specific a4, B1, and G3 V-ATPase subunits and pds in fetal (from E13.5 to E18.5), newborn, and adult mouse kidneys. A total of 20 µg of protein was loaded in each lane. Blots were probed with antibodies against A (1:1000), B2 (1:1000), E1 (1:100), G1 (1:500), C1 (1:1000), a4 (1:5000), B1 (1:1000), and G3 (1:500) subunits; pendrin (1:2000); and, after stripping, -actin (1:10,000). Ubiquitous subunits are detected as early as E13.5 and gradually increase during late nephrogenesis, whereas the developmental pattern of IC-specific isoforms and pendrin is characterized by a later expression from E16.5.
Segmental Distribution of V-ATPase Subunits in Developing Mouse Kidney
The segmental distribution of ubiquitous and IC-specific subunitsin the developing mouse kidney was investigated using well-characterizedantibodies against E1, B2, and a4 (Figure 6). No immunostainingfor the IC-specific a4 subunit was detected at E14.5, by whichtime dispersed glomeruli and developing PT were observed inthe inner cortex (Figure 6A). At E15.5, the immunoreactive signalwas located in the apical area of scattered cells along thecortical (Figure 6B) and medullary CD (Figure 6C), whereas PT,ureteric buds, glomeruli, and differentiating structures remainedunlabeled. By contrast, at this fetal stage, the distributionof ubiquitous E1 and B2 was acquired in PTC (Figure 6, D andI), as well as the distribution of E1 in IC (Figure 6D, inset).From E16.5, apical staining for a4 was detected in scatteredIC (Figure 6, E, G, and H), clearly identified by co-distributionwith CAII (Figure 6, F and G) but negative staining for AQP2(Figure 6H). The distribution pattern of a4 in mouse developingkidney was restricted to IC (Figure 6J), similar to that observedin adult kidney (Figure 6K). The specificity of a4 stainingwas demonstrated with preimmune rabbit antibodies (Figure 6L).These results show that, in mouse developing kidney, the a4subunit is detected progressively during IC differentiation,whereas the distribution of ubiquitous E1 and B2 includes bothPTC and IC of the CD at an early stage.
Figure 6. Segmental distribution of E1 and a4 V-ATPase subunits in mouse developing kidney. Immunostaining for a4 (all panels except D, F, I, and L), E1 (D), B2 (I), type II carbonic anhydrase (F and G), aquaporin 2 (AQP2; H), megalin (I and J), and negative control (L) in developing mouse kidney at E14.5 (A), E15.5 (B through D), E16.5 (E through G and I), and E18.5 (H and J) and in adult mouse kidney (K and L). No specific staining for a4 is observed at E14.5 (A). From E15.5, a4 is detected in the apical area of scattered cells of cortical (B) and medullary (C) CD, whereas proximal tubule cells (PTC) remain unstained. At this stage, the ubiquitous E1 and B2 are already expressed in developing PTC (D and I) and in IC of the medullary and cortical (D, inset) CD. At E16.5, apical staining for a4 is still present in scattered IC of the CD (E, arrowheads), clearly identified by co-staining positive for type II carbonic anhydrase (CAII; F, arrowheads, and G) but negative for AQP2 (H). This segmental distribution restricted to IC in mouse fetal kidney (J) is similar to that observed in the adult kidney (K). No staining for a4 subunit was observed with preimmune rabbit serum (L). Magnification, x300 in A through F, K, and L; x450 in G through J.
Subcellular Fractionation in Percoll Gradients
In addition to its intracellular location where it mediatesvesicle acidification, the V-ATPase is located at the cell surfaceof renal IC. We investigated the subcellular distribution ofubiquitous E1 and IC-specific a4 subunits in the mature anddeveloping mouse kidney (Figure 7). Adult kidneys were dissectedinto cortex and medulla, and appropriate markers were analyzedby immunoblotting to validate the dissection (Figure 7A). Podocinand pendrin, as well as E1 and a4, were considerably enrichedin the cortex, whereas AQP2 was detected mostly in the medulla(Figure 7B). These results confirm that the two parts of adultkidneys were purified adequately, as indicated by an enrichmentratio >30 (podocin versus AQP2). Next, the subcellular distributionof E1 and a4 in fetal kidney and adult cortex and medulla wascompared (Figure 7C). Percoll gradients resolved a low-densitypeak (fractions 2 to 4) that comprised all (fetus) or the bulk(adult) of the early endosomal marker Rab5a from a high-densitypeak (fractions 9 and 10), including most of the lysosomal markercathepsin D. In the three tissue samples, both V-ATPase subunitspeaked in the low-density fractions, suggesting their majorassociation with endosomes and not with lysosomes.
Figure 7. Localization and subcellular distribution of V-ATPase subunits in fetal and adult (cortex and medulla) mouse kidneys. (A) Representative immunoblots for podocin, IC-specific a4 and ubiquitous E1 V-ATPase subunits, pendrin, -actin, Na+/K+-ATPase, and AQP2 in cortex and medulla of adult mouse kidney. Thirty micrograms of protein were loaded in each lane. Podocin (1:5000), a4 (1:5000), E1 (1:100), and pendrin (1:1000) are highly enriched in the dissected cortex and depleted in the dissected medulla; -actin (1:10,000) and Na+/K+-ATPase (1:5000) are equally expressed in both tissues; AQP2 (1:2000) is detected mostly in the dissected medulla. (B) The relative enrichment of these markers, based on densitometry. (C) Percoll gradients of total fetal (E16.5) and adult (cortex and medulla) mouse kidneys. Distributions after centrifugation are presented in comparison with the initial concentration (C/Ci) so that values >1 reflect organelle enrichment and values <1 reflect organelle depletion. For immunoblotting analyses, equal volumes were loaded in each lane. The gradients resolve a low-density peak (fractions 2 to 4), including early endosomes (Rab5a), from a high-density peak (fractions 9 and 10), enriched in lysosomes (cathepsin D). The two V-ATPase subunits peak in the low-density fractions, which suggests their major association with endosomes and not with lysosomes in both developing and adult (cortex and medulla) kidneys.
In this study, we showed that ubiquitous and IC-specific isoformsof V-ATPase subunits are differentially expressed in maturekidney and during mouse nephrogenesis. The early expressionof the ubiquitous A, B2, E1, G1, C1, and a1 subunits contrastswith the later and more progressive appearance of the IC-specificisoforms B1, G3, C2, and a4. The forkhead transcription factorFoxi1 mRNA was detected in IC from E15.5, with co-distributionwith the B1 subunit in late nephrogenesis. The segmental distributionof E1 and B2 was acquired in PTC at E15.5, whereas a4 co-distributedwith CAII in the IC during late nephrogenesis. Subcellular fractionationstudies indicated a preferential location of both ubiquitousand IC-specific subunits to endosomes in both developing andmature kidneys.
The subunit composition of the bovine V-ATPase, previously determinedby quantitative amino acid studies (1), was updated recentlyusing electron microscopy and single-molecule image analysis(4). These investigations led to a structural model of the mammalianV-ATPase, which seems to be similar to the F1F0-ATP synthasein yeast (4). As a preliminary to the ontogeny analysis, real-timeRT-PCR analyses in the adult kidney showed that the relativemRNA expression of V-ATPase subunits does not correspond tothe expected stoichiometry. These results were confirmed byabsolute mRNA quantification using standard curves from serialdilution of purified plasmids (data not shown). The ratio betweenthe two methods varied between 0.8 and 1.8 according to thesubunits, in agreement with previous comparative studies (23).This variability is attributed to distinct primer efficiencyand, overall, cumulative errors introduced by the additionalsteps of the absolute mRNA quantification (spectrophotometry,calculations of molecular weight) (24). Thus, the relative method,using primer sets of similar efficiency (Table 1) and threedistinct internal standards, was preferred to compare the abundanceof V-ATPase subunit transcripts in one single kidney sample.The comparative mRNA quantification of subunits demonstratesa variable expression from ubiquitous to IC-specific V-ATPasecomplexes. Although minimal and/or limited correlation betweenthe mRNA and protein levels is most often reported, the degreeof correlation could vary according to subcellular localizationor functional category (25). In the case of the renal V-ATPasecomplex, a first hypothesis could be significant variationsin the turnover of the subunits that compose the complex, aswell as a differential regulation of subunit mRNA stability(26). Because in vivo regulation of V-ATPase, which involvesthe reversible dissociation of V1 from V0 domain (27), doesnot require new protein synthesis but an efficient microtubularsystem (28), a second hypothesis could be posttranscriptionalmodifications during subunit synthesis and V-ATPase assembly.For instance, rearrangements during the formation of V0 andV1 domains might stabilize each of these subunits by tight interactionsand/or mutual chaperoning. Of interest, a selective posttranscriptionalcontrol of protein expression levels among loci of the V-ATPasecomplex has been shown to operate in Saccharomyces cerevisiae(29).
The molecular events that occur in renal tubular cells afterthe onset of glomerular filtration, which starts at E14 in mouse(30), remain incompletely elucidated. Recent evidence suggeststhat different steps of kidney organogenesis are reflected byclusters of differentially regulated genes, including transportersand ions-motive ATPases, under the control of specific transcriptionactivators (9,31). In rats (32) and humans (33), PT endocyticactivity is effective during nephrogenesis or immediately afterbirth. Both the receptor-mediated endocytic pathway and theapical targeting within PTC (8) depend on a correct vacuolaracidification, which is mediated by V-ATPase in PTC. Our real-timeRT-PCR and immunoblotting analyses show an early and stableexpression of ubiquitous isoforms of V-ATPase subunits duringnephrogenesis, which is in keeping with its essential role forearly mouse development (12). It is interesting that the endosomala1 subunit shows an early induction, a progressive decreaseduring late ontogeny, and a novel induction around birth. Sucha biphasic developmental pattern is also described for the renalchloride channel ClC-5 that co-locates with V-ATPase in PT endosomes(34), as well as for the V0 d1 subunit (20). ClC-5 is mutatedin Dent’s disease, an X-linked renal tubular disordercharacterized by a major and early defect in the PT endocyticpathway (35). Thus, besides the stable expression of ubiquitoussubunits, ClC-5 and a1 isoform, which are considered to targetcorrectly V-ATPase complex to appropriate intracellular compartments(36), both are expressed simultaneously in PTC as early as theglomerular filtration starts. These essential components ofthe endocytotic process, responsible for the reabsorption ofamino acids, vitamins, and minerals, may indeed be requiredearly for correct growth and development. Analytical subcellularfractionation clearly resolved the two V-ATPase subunits fromlysosomes, as assessed by the cathepsin D distribution, andrather pointed to a primary endosomal location, as shown byco-distribution with Rab5a. This low-density pattern is identicalto what we recently reported for ClC-5 (8). Thus, kidney endosomesseem to be well equipped with electrogenic proton pumps anda chloride conductance, both in the adult and the fetus.
The IC constitute approximately one third of the cells thatline the mouse CD (37). Although both - and -IC are characterizedby high CAII activity, they differ by the selective polarityof V-ATPase and type 1 anion exchanger, allowing them to participatein proton ( type) or bicarbonate ( type) urinary secretion (7).The differentiation and maturation of the various cell typeswithin the CD is poorly understood. Studies in the rat haveshown that V-ATPase and CAII are detected simultaneously inIC at the end of gestation (38). During the first weeks of life,IC continue their maturation but are partially removed fromspecific parts of the CD by apoptosis or luminal extrusion (39).Recently, the winged helix transcriptional factor Foxi1, previouslylocated in mouse fetal distal nephron (21), was shown to playan essential role in IC differentiation from epithelial precursorcells in the CD (22). In addition, Foxi1 is regarded as an upstreamregulator of the anion transporter pendrin during inner eardevelopment (40). Indeed, mice that lack Foxi1 showed a lackof differentiation of the distal nephron, leading to dRTA (22),as well as a sensorineural deafness resembling that of Pendredsyndrome (40). Our description of the developmental expressionof Foxi1, IC-specific V-ATPase isoforms, and pendrin providesadditional information on CD maturation in mouse. Real-timeRT-PCR showed that IC-specific subunits B1, G3, C2, and a4,as well as pendrin, are expressed later than the ubiquitousisoforms, with a progressive induction after Foxi1 appearance(E15.5). In situ hybridization showed that at E15.5, Foxi1 isexpressed in scattered cells of the medullary CD. At later stages,Foxi1 is identified in distinct cells within tubules in thecortex and outer medulla, in which it co-distributes with theIC-specific B1 subunit. In silico studies (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite)indicate that gene promoters of IC-specific markers, such astype 1 anion exchanger, AE4, pendrin, or IC-specific V-ATPasesubunits, exhibit Foxi1 DNA binding consensus sequence (21,22).Immunoblot studies showed a coordinate expression of the IC-specificsubunits and pendrin at approximately E16.5, with immunolocalizationof a4 and CAII in IC. Of note, subcellular fractionation showedthat, in both fetal (E16.5) and adult kidneys, a4 and E1 subunitsare located mostly in endosomes. This observation corroboratesa dynamic process that occurs in IC, which involves regulatedexocytic insertion of V-ATPase from a vesicular pool to theplasma membrane (41). Taken together, the mRNA and protein datasupport the case for a differential but coordinate regulationof IC-specific versus ubiquitous V-ATPase subunits during kidneydevelopment.
The comparative ontogeny of V-ATPase subunits helps us understandthe pathophysiology of inherited renal disorders such as Dent’sdisease and dRTA. Dent’s disease, which is due to inactivatingmutations in CLCN5 that presumably lead to impaired endosomalacidification in PTC, is characterized by low molecular weightproteinuria that can be detected within the first weeks of life(35). These clinical findings, which suggest that the molecularmechanisms that ensure endosomal acidification must be acquiredat birth (19), have their counterpart in our demonstration ofa parallel induction of the endosomal a1 isoform and ClC-5 thatculminates at birth. Given the essential role of V-ATPase inearly embryogenesis and its large tissue distribution, no mutationof any ubiquitous V-ATPase subunits has ever been reported,being definitively not compatible with life (12). In contrast,mutations in IC-specific isoforms have been associated withhuman dRTA (13–15). Some well-documented patients showclinical manifestations of dRTA during infancy, which suggeststhat the segmental expression of these IC-specific isoformsof V-ATPase must be acquired early, as indeed documented here.
In conclusion, our data demonstrate that ubiquitous and IC-specificisoforms of V-ATPase subunits are differentially expressed duringmouse nephrogenesis. These results provide new insights intothe segmental distribution of V-ATPase complexes in the kidneyand represent a useful tool to investigate further any newlydiscovered subunit isoforms. The deciphering of the complexmaturation of PTC and IC along the distal nephron also helpsus understand the pathophysiology of renal tubular disorders.
Acknowledgments
These investigations were supported by the Universitécatholique de Louvain, the Fonds National de la Recherche Scientifique(FNRS), the Forton Foundation, Concerted Research Actions, Inter-UniversityAttraction Poles, and the EuReGene integrated project of theEuropean Community (FP6). F.J. is a research fellow of the FNRS,and C.P. is a research fellow of the FRIA.
We are grateful to Y. Cnops, M. Leruth, L. Wenderickx, and P.Henriet for excellent technical help and discussion and to M.Futai, P. Gailly, A. Goffinet, N. Tajeddine, and M.-F. van denHove for material.
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Received for publication November 12, 2004.
Accepted for publication July 26, 2005.
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Top
Abstract
Introduction
Ct (Reporter – Target Gene)]. The relative changes in mRNA levels of these alternate isoforms during ontogeny were determined by comparison with the adult mRNA level, after adjustment to GAPDH [Ratio = (Efficiencytarget gene)
-actin (Sigma); fluorescein (Dako, Glostrup, Denmark); 
1 Na+/K+-ATPase (Upstate Biotechnology Inc., Waltham, MA); rabbit polyclonal antibodies against a4 subunit (13); B1, B2, G1, and G3 subunits (17); A subunit (Wako Chemicals GmbH, Neuss, Germany); C1 subunit (Santa Cruz Biotechnology, Santa Cruz, CA); Rab5a (Santa Cruz Biotechnology); aquaporin-2 (AQP2; Sigma); podocin (a gift from C. Antignac, Inserm U574, Necker Hospital, Paris, France) (18); sheep polyclonal antibodies against megalin (a gift from P.J. Verroust, Saint-Antoine Medical Faculty, Paris, France) (8); type II carbonic anhydrase (CAII; Serotec, Oxford, UK); goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology); and chicken polyclonal antibodies against pendrin (a gift from P. Kopp, Northwestern University, Chicago, IL). 
