1Department of Pediatrics, University of Utah, Salt Lake City, Utah; 2Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown; and 3Department of Medicine, Harvard Medical School, Boston, Massachusetts
Submitted 10 February 2004 ; accepted in final form 13 December 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
collecting duct; enhanced green fluorescent protein
The renal collecting duct plays a critical role in the regulation of extracellular volume, osmolality, and pH. It is composed of two structurally and functionally distinct cell types: principal cells and intercalated cells (33). While principal cells are involved in the maintenance of sodium and water balance, intercalated cells play an important role in acid-base homeostasis (1, 7, 34, 44). Two types of intercalated cells have been identified. Type A intercalated cells (A-IC), which express the V-ATPase in their apical pole, are involved in proton secretion. Type B intercalated cells (B-IC), which exhibit apical, bipolar, or basolateral V-ATPase localization, secrete either protons (B-IC with apical V-ATPase) or HCO3 (B-IC with basolateral V-ATPase) (2, 911, 48). Type B intercalated cells also have been identified by their lack of expression of the basolateral Cl/HCO3 exchanger AE1 (1). Other reports have labeled the cells with apical V-ATPase and without AE1 expression as "non-A-non-B cells" (29). In the present study, for simplicity, all V-ATPase-positive and AE1-negative intercalated cells are referred to as B-IC. Genetic (27, 45) and acquired (38) alterations of the V-ATPase are associated with disorders of acid-base balance. Such examples indicate that the proper expression and regulation of the V-ATPase in intercalated cells is essential to the maintenance of acid-base balance. However, the regulation of V-ATPase in intercalated cells in health and disease remains incompletely understood.
The epididymis is the site where spermatozoa undergo their final maturation and are then stored (3, 18, 26, 40). As sperm travel along the epididymis, they are maintained immotile and the enzymes involved in acrosomal initiation are prevented from inducing premature activation. An initial step in sperm activation occurs after ejaculation, when spermatozoa are mixed with prostatic fluid, which is rich in HCO3 and higher in pH than the epididymal fluid. Therefore, acidification of the luminal fluid of the epididymis is a critical factor in the establishment of an optimum environment for sperm maturation and storage. Similar to the kidney, the epididymal epithelium is a complex structure composed of at least three cell types: narrow/clear, principal, and basal cells. Narrow and clear cells express high levels of the V-ATPase in their apical plasma membrane and in subapical vesicles (4, 12, 14). While narrow cells are found in the initial segments of the epididymis, clear cells are present in the caput, corpus, and cauda epididymidis. These cells resemble type A intercalated cells. They are responsible for the bulk of proton secretion and are therefore key players in luminal acidification (4, 12, 14).
The characterization of intercalated cell and narrow/clear cell development and function has been limited considerably by the complexity of the kidney and epididymis and by the cellular heterogeneity of the epithelial tubules that form these organs. The understanding of specific gene and protein expression in these cells would therefore benefit greatly from techniques allowing selective isolation and purification of these cell types from the kidney and epididymis. The purpose of this study was to create a transgenic mouse that expresses enhanced green fluorescent protein (EGFP) specifically in kidney intercalated cells and in epididymal narrow/clear cells to allow direct visualization of these cells, which would facilitate their study in the intact animal and would allow their selective isolation using fluorescence-assisted techniques.
Because both intercalated and narrow/clear cells express high levels of the V-ATPase, an EGFP transgene was designed using the promoter of one of its subunits. The V-ATPase is a multisubunit complex composed of two domains: the V1 and V0 domains (46, 49). The V1 domain (catalytic domain) is involved in the hydrolysis of ATP and is composed of eight different subunits (AH). The V0 domain (membrane domain) is responsible for proton translocation across the plasma membrane and consists of five different subunits. Interestingly, while many V-ATPase subunits are expressed in several cell types, some of these subunits, including the B subunit, have more than one isoform with cell-specific expression patterns. For example, while the B2 isoform is present in a variety of tissues, the B1 isoform is expressed much more abundantly in a limited number of cell types, including kidney intercalated cells (36), connecting tubule cells (17), and epididymal narrow/clear cells (5). Therefore, in the present study, the promoter of the ATP6V1B1 gene encoding the V-ATPase B1 subunit was used to drive cell-specific expression of an EGFP transgene. Characterization of these transgenic mice using quantitative RT-PCR demonstrated specific expression in the kidney, the male reproductive tract, and the lung. Immunofluorescence microscopy demonstrated specific expression in intercalated cells of the collecting duct and connecting tubule, connecting tubule cells, and narrow/clear cells in the epididymis. In addition, nonciliated airway cells also expressed EGFP and the B1 V-ATPase subunit.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of the human V-ATPase B1-subunit promoter EGFP transgene. The EGFP coding region from the pEGFP-1 vector (Clontech, La Jolla, CA) was ligated into the vector containing 6.5 bp of the 5'-flanking region of the ATP6V1B1 gene. A polyadenylation signal from the SV40 virus early region with an added ClaI site was ligated into a site downstream from the EGFP coding region. This product was designated as pB1EGFPpA. Each plasmid intermediate and the final plasmid product were analyzed by performing PCR and restriction mapping to confirm the proposed structure. All ligation junctions were sequenced to confirm that the construct had the desired structure. The key elements are the 6.5-kb B1 promoter, 0.7-kb EGFP coding region, and 0.3-kb SV40 early region polyadenylation signal with unique ClaI sites upstream from the promoter and downstream from the polyadenylation sequence.
Generation and breeding of transgenic mice. The B1-EGFP transgene was separated from the vector by agarose gel purification after digestion of pB1EGFPpA with ClaI. The DNA was isolated by electroelution, concentrated using an Elutip-D column (Schleicher & Schuell, Keene, NH), and resuspended in injection buffer consisting of 10 mM Tris, pH 7.4, and 0.1 mM EDTA. Transgenic mice were created by the University of Utah transgenic mouse core facility using standard procedures (21). Briefly, the pronucleus of single-cell C57BL6 x CBA F1 embryos was microinjected with the purified transgene, and the resulting embryos were implanted into pseudopregnant females. The resulting pups were genotyped by performing PCR of tail DNA. The transgenic founders were each bred with C57BL/6 x CBA F1 mice. The resulting F1 and F2 animals were analyzed for expression of the transgene. All procedures were performed according to protocols approved by the University of Utah Institutional Animal Care and Use Committee.
PCR genotyping. DNA was isolated from a tail biopsy specimen. Transgene was detected by performing PCR amplification of 100 ng of mouse genomic DNA using 25-µl reactions containing 0.05 U/µl Taq DNA polymerase (Invitrogen, Carlsbad, CA), 200 µM each of dATP, dCTP, dGTP, and dTTP, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris·HCl, pH 8.4, 0.4 µM forward primer, and 0.4 µM reverse primer. The thermocycling programs for genotyping consisted of a cycle at 94°C for 60 s followed by 3035 cycles of 94°C for 20 s, 5760°C for 20 s, and 72°C for 50 s, and a final extension at 72°C for 5 min (Table 1). The cycle number and annealing temperature were primer set dependent (Table 1). The cycle number was limited to nonsaturating conditions. Nontransgenic tail DNA was spiked with transgene DNA to simulate 1100 copies per cell equivalent of transgene DNA. The oligonucleotide primers B1EGFPF and B1EGFPR were used to detect the B1-EGFP transgene (Table 1). As a control for DNA integrity, the oligonucleotide primers AQP2GF and AQP2GR (Table 1) were used to detect the endogenous AQP2 gene (GenBank accession no. NT_039350).
|
RNA was then prepared for three offspring from each founder, and reverse transcription reactions were performed in the presence and absence of reverse transcriptase to verify that transgene expression resulted from amplification of cDNA rather than from residual genomic DNA (data not shown). Real-time PCR was performed with a panel of mouse organ cDNA in parallel with dilutions of reference cDNA as a standard curve. Expression was calculated from the standard curves and then expressed in arbitrary units of EGFP or the B1 subunit relative to GAPDH.
Fluorescence microscopy. Transgenic mouse tissues were prepared according to the following procedures. Transgenic mice were anesthetized by administering inhaled halothane and fixed using cardiac perfusion with 2% paraformaldehyde (PFA) in PBS (10 mM sodium phosphate buffer containing 0.9% NaCl, pH 7.4; 2% PFA) in PBS at room temperature. The kidneys, male reproductive tract, and lungs were removed and fixed by immersion in 2% PFA in PBS for 2 h at room temperature and overnight at 4°C. The tissues were processed using two methods.
Transgenic kidneys were embedded in 3% agarose in PBS and cut into 150- to 200-µm sections with an oscillating microtome (OTS-3000; Electron Microscopy Sciences, Fort Washington, PA). Sections were viewed using fluorescence confocal microscopy (MRC 1024 confocal system; Bio-Rad, Hercules, CA). Images were captured digitally using Lasersharp version 3.2 software (Bio-Rad). A z-series of 11 sections was collected every 1, 1.5, 3, 5, or 10 µm with a x2.5 lens objective. A montage for each z-series was digitally created using Lasersharp version 3.2 software.
Transgenic kidneys, epididymis, and lung were sectioned with a cryomicrotome and immunostained according to the following procedure (13). Mice were fixed using cardiac perfusion with PLP fixative containing 4% PFA, 10 mM sodium periodate, 75 mM lysine, and 5% sucrose. The tissues were cryoprotected by immersion in 30% sucrose in PBS for 4 h, mounted in Tissue-Tek (Miles, Elkhart, IN), and frozen at 30°C in a Reichert Frigocut cryostat (Reichert Jung, Derry, NH). Sections were cut at 4 µm and placed onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were hydrated for 5 min in PBS and treated for 4 min with 1% sodium dodecyl sulfate (SDS) in PBS. After the sections were washed three times with PBS for 5 min each, nonspecific staining was blocked in a solution of 1% BSA in PBS for 15 min. The sections were incubated at room temperature with an affinity-purified rabbit antibody to AQP2 (47), a polyclonal rabbit antiserum to the V-ATPase B1 subunit (5, 36), or an affinity-purified chicken antibody to the V-ATPase E subunit (6). These primary antibodies were detected using goat anti-rabbit or anti-chicken IgG conjugated to CY3. Some kidney sections were double-immunostained for the V-ATPase B1 subunit and for calbindin 28K using a mouse anti-calbindin antibody (Calbindin D-28K clone CB-955; Sigma). In these sections, the V-ATPase was detected using goat anti-rabbit AMCA (blue), and calbindin was detected with donkey anti-mouse CY3. Some epididymal sections were stained using a polyclonal rabbit anti-rat CAII antibodies (provided by William S. Sly, Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO), followed by goat anti-rabbit CY3. Lung sections were stained with a monoclonal anti--tubulin antibody (T5168; Sigma) and detected with donkey anti-mouse AMCA or donkey anti-mouse CY3. Some lung sections were stained with 4,6-diamidino-2-phenylindole (DAPI) stain for nuclei. The slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) diluted 1:1 in Tris·HCl buffer (1.5 M, pH 8.9). Images were captured with a Hamamatsu Orca digital camera (Bridgewater, NJ) mounted on a Nikon Eclipse 800 microscope. Pseudocolor images were merged using IPLab Spectrum software (Scanalytics, Fairfax, VA). Cy3 appears red, AMCA and DAPI appear blue, and EGFP is green.
DNA sequencing. Plasmids containing the RT-PCR products, transgene, and transgene intermediates were sequenced by the University of Utah DNA Sequencing Facility. They use the dye primer system for universal primers and the dye terminator system for other primers. The products were analyzed using the ABI Prism 377 or 3700 DNA analyzers (Applied Biosystems, Foster City, CA). The results were analyzed using Sequencher (Gene Codes, Ann Arbor, MI) and Omiga (Oxford Molecular).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Qualitative real-time RT-PCR analysis of kidney and male reproductive tract revealed expression of EGFP in three different lines of transgenic mice. Quantitative RT-PCR analysis was performed on organ panels prepared from three animals each for lines 1 and 3. A limited panel of epithelial and nonepithelial organs was chosen. Note that the male reproductive tract preparation included a combination of epididymis and vas deferens. Representative organ panels for lines 1 and 3 are shown in Fig. 3, A and B. EGFP is expressed in the kidney, male reproductive tract, and lung, where the B1 subunit also is expressed. The expression of EGFP and the B1 subunit is below the threshold for detection in the other organs. EGFP is expressed at high levels in the kidney, male reproductive tract, and lung in both transgenic lines of mice. This expression parallels that of the endogenous B1-subunit gene. These results indicate that 6.5 kb of the B1-subunit promoter are sufficient to drive selective expression in kidney, male reproductive tract, and lung.
|
|
|
|
Connecting segments, identified by their calbindin-positive staining (2, 19), also show EGFP expression (Fig. 6). In this segment, most cells express the B1 subunit of the V-ATPase. Connecting tubule (CNT) cells, which express calbindin, show lower expression of both the V-ATPase B1 subunit and EGFP compared with intercalated cells, which are negative for calbindin but express high levels of the V-ATPase B1 subunit and EGFP. Thus the level of EGFP expression parallels the level of V-ATPase B1-subunit expression.
|
|
|
|
Immunofluorescence microscopy of lung airway epithelia. The localization of EGFP in the lung was examined. As shown in Fig. 11, EGFP was expressed in airway epithelial cells. Double-labeling for tubulin showed that EGFP is abundant in nonciliated cells, negative for tubulin, and not detectable in ciliated cells. The B1 subunit of the V-ATPase was then localized in epithelial cells of lung airways, and its distribution was compared with the EGFP expression pattern. Some EGFP-expressing airway epithelial cells were brightly stained for the B1 subunit. Other EGFP-positive cells showed less intense B1 staining. Furthermore, apical tubulin staining was absent from EGFP-expressing cells (Fig. 12D). We conclude, therefore, that only nonciliated cells express EGFP and that the level of V-ATPase B1-subunit expression in these nonciliated cells is variable but is often much greater than in the adjacent ciliated cells.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The study of subsets of specialized cells, including kidney intercalated cells and epididymal narrow/clear cells, has thus far been limited by the complexity of the organs in which they are found. The tubules that compose these organs contain various cell types, which all present specific characteristics. Our B1-EGFP transgenic mouse should prove useful for a variety of fluorescence microscopy-assisted techniques aimed at isolating intercalated cells and clear cells as well as collecting ducts. For example, we are now in a position to isolate these cells using fluorescence-activated cell sorting (FACS) of tissue digests either to culture them in vitro or to examine and characterize their gene and protein expression patterns. Also, it is now possible to microdissect specifically collecting ducts from collagenase-treated kidneys using fluorescence microscopy. Alternatively, laser capture microdissection, complemented with fluorescence microscopy, can now be used to isolate intercalated or clear cells from kidney or epididymis sections. With any one of these cell isolation methods, gene expression is determined using quantitative RT-PCR, microarray analysis, or immunoblotting under a variety of physiological and pathophysiological conditions. In this way, interference due to the contribution of neighboring cells is considerably (if not completely) reduced and the collecting duct, intercalated cell, and clear cell-specific gene expression profiles can be determined more precisely.
EGFP can also be used as a vital marker for the appearance of kidney intercalated cells and epididymal clear cells during development. Intercalated cells and epididymal clear cells appear progressively during pre- and postnatal development, respectively (5, 28, 30). By examining EGFP fluorescence, the onset of expression is easily determined and intercalated or clear cells can be isolated at each particular stage of development for examination of their gene and protein expression profiles.
These novel mice also are extremely useful for characterizing the regulation of the V-ATPase B1-subunit expression itself. EGFP is a vital marker that could be used as a reporter gene for expression of this subunit. Comparison of the proximal 10 kb of human and mouse V-ATPase B1 subunit promoters reveals that two regions of conservation are present in the promoter: a short proximal segment and a longer distal segment. On the basis of the current understanding of comparative genomics, these regions probably represent regulatory elements that may participate in the cell type-specific expression of the ATP6V1B1 gene (32). Clearly, further studies are required to elucidate the cell-specific regulatory elements and the transcription factors involved in the regulation of the V-ATPase B1-subunit expression in kidney intercalated cells and epididymal narrow and clear cells. Mating of these mice with other genetically modified mice that have the potential to alter development will allow for rapid and easy screening of the factors involved in the establishment of the intercalated cell and narrow/clear cell phenotype and/or in the expression of the V-ATPase B1-subunit or other genes in these cells under physiological and pathophysiological conditions.
A final point is that the expression of EGFP in nonciliated cells of the airway was an unexpected finding. A review of the literature indicates that the pH of airway surface liquid is relatively acidic (25). Although glandular epithelium secretes HCO3-rich fluid, the surface epithelium acidifies airway surface liquid (24). Furthermore, the acidification is inhibited by bafilomycin A1, suggesting involvement of vacuolar H+-ATPase (24). Airway pH may affect ciliary beat frequency (15), mucous rheology (22), and smooth muscle tone (39). Thus airway pH may be important for normal airway physiology. In addition, abnormal airway surface layer pH also may be important in diseases such as asthma (23) and cystic fibrosis (37). Further studies of V-ATPase are required to understand its role in lung physiology and pathophysiology.
In summary, the B1-EGFP-transgenic mice clearly exhibit specific cell expression of EGFP in the kidney and epididymis as well as in lung airway cells. Our studies demonstrate that cell-specific expression in these organs is conferred by 6.5 kb of the V-ATPase B1-subunit promoter. These mice represent a powerful new model for the study of the factors responsible for the establishment, maintenance, and modulation of these cell phenotypes.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Alper SL, Natale J, Gluck S, Lodish HF, and Brown D. Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase. Proc Natl Acad Sci USA 86: 54295433, 1989.[Abstract]
3. Bedford JM. Maturation, transport, and fate of spermatozoa in the epididymis. In: Handbook of Physiology: Male Reproductive System, edited by Hamilton DW and Greep RO. Bethesda, MD: Am Physiol Soc, 1975, sect. 7, vol. 5, chapt. 14, p. 303317.
4. Breton S, Smith PJ, Lui B, and Brown D. Acidification of the male reproductive tract by a proton pumping H+-ATPase. Nat Med 2: 470472, 1996.[CrossRef][ISI][Medline]
5. Breton S, Tyszkowski R, Sabolic I, and Brown D. Postnatal development of H+ATPase (proton-pump)-rich cells in rat epididymis. Histochem Cell Biol 111: 97105, 1999.[CrossRef][ISI][Medline]
6. Breton S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, and Brown D. The B1 subunit of the H+ATPase is a PDZ domain-binding protein: colocalization with NHE-RF in renal B-intercalated cells. J Biol Chem 275: 1821918224, 2000.
7. Brown D and Breton S. Mitochondria-rich, proton-secreting epithelial cells. J Exp Biol 199: 23452358, 1996.
8. Brown D and Breton S. H+V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways. J Exp Biol 203: 137145, 2000.[Abstract]
9. Brown D, Gluck S, and Hartwig J. Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H+ATPase. J Cell Biol 105: 16371648, 1987.[Abstract]
10. Brown D, Hirsch S, and Gluck S. An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations. Nature 331: 622624, 1988.[CrossRef][ISI][Medline]
11. Brown D, Hirsch S, and Gluck S. Localization of a proton-pumping ATPase in rat kidney. J Clin Invest 82: 21142126, 1988.[ISI][Medline]
12. Brown D, Lui B, Gluck S, and Sabolic I. A plasma membrane proton ATPase in specialized cells of rat epididymis. Am J Physiol Cell Physiol 263: C913C916, 1992.
13. Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, and Alper S. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261267, 1996.[ISI][Medline]
14. Brown D, Smith PJ, and Breton S. Role of V-ATPase-rich cells in acidification of the male reproductive tract. J Exp Biol 200: 257262, 1997.
15. Clary-Meinesz C, Mouroux J, Cosson J, Huitorel P, and Blaive B. Influence of external pH on ciliary beat frequency in human bronchi and bronchioles. Eur Respir J 11: 330333, 1998.
16. García-Villalba P, Denkers ND, Wittwer CT, Hoff C, Nelson RD, and Mauch TJ. Real-time PCR quantification of AT1 and AT2 angiotensin receptor mRNA expression in the developing rat kidney. Nephron Exp Nephrol 94: e154e159, 2003.[CrossRef][ISI][Medline]
17. Gekle M, Wunsch S, Oberleithner H, and Silbernagl S. Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties. Pflügers Arch 428: 157162, 1994.[CrossRef][ISI][Medline]
18. Hinton BT and Palladino MA. Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech 30: 6781, 1995.[CrossRef][ISI][Medline]
19. Hoenderop JGJ, Hartog A, Stuiver M, Doucet A, Willems PHGM, and Bindels RJM. Localization of the epithelial Ca2+ channel in rabbit kidney and intestine. J Am Soc Nephrol 11: 11711178, 2000.
20. Hogan B, Beddington R, Costantini F, and Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1994.
21. Hogan B, Beddington R, Costantini F, and Lacy E. Production of transgenic mice. In: Manipulating the Mouse Embryo: A Laboratory Manual (2nd ed.), edited by Hogan B, Beddington R, Costantini F, and Lacy E. Cold Spring Harbor, NY: Cold Spring Laboratory, 1994, p. 217252.
22. Holma B. Influence of buffer capacity and pH-dependent rheological properties of respiratory mucus on health effects due to acidic pollution. Sci Total Environ 41: 101123, 1985.[CrossRef][ISI][Medline]
23. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TA, and Gaston B. Endogenous airway acidification: implications for asthma pathophysiology. Am J Respir Crit Care Med 161: 694699, 2000.
24. Inglis SK, Wilson SM, and Olver RE. Secretion of acid and base equivalents by intact distal airways. Am J Physiol Lung Cell Mol Physiol 284: L855L862, 2003.
25. Jayaraman S, Song Y, and Verkman AS. Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea. Am J Physiol Cell Physiol 281: C1504C1511, 2001.
26. Jones RC and Murdoch RN. Regulation of the motility and metabolism of spermatozoa for storage in the epididymis of eutherian and marsupial mammals. Reprod Fertil Dev 8: 553568, 1996.[ISI][Medline]
27. Karet FE, Finberg KE, Nelson RD, Nayir A, Mocan H, Sanjad SA, Rodriguez-Soriano J, Santos F, Cremers CWRJ, Di Pietro A, Hoffbrand BI, Winiarski J, Bakkaloglu A, Ozen S, Dusunsel R, Goodyer P, Hulton SA, Wu DK, Skvorak AB, Morton CC, Cunningham MJ, Jha V, and Lifton RP. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 21: 8490, 1999.[CrossRef][ISI][Medline]
28. Kim J, Cha JH, Tisher CC, and Madsen KM. Role of apoptotic and nonapoptotic cell death in removal of intercalated cells from developing rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 270: F575F592, 1996.
29. Kim J, Kim YH, Cha JH, Tisher CC, and Madsen KM. Intercalated cell subtypes in connecting tubule and cortical collecting duct of rat and mouse. J Am Soc Nephrol 10: 112, 1999.
30. Kim J, Tisher CC, and Madsen KM. Differentiation of intercalated cells in developing rat kidney: an immunohistochemical study. Am J Physiol Renal Fluid Electrolyte Physiol 266: F977F990, 1994.
31. Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, and Moorman AFM. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307: 6369, 2002.[CrossRef][ISI][Medline]
32. Loots GG, Locksley RM, Blankespoor CM, Wang ZE, Miller W, Rubin EM, and Frazer KA. Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons. Science 288: 136140, 2000.
33. Madsen KM and Tisher CC. Structural-functional relationship along the distal nephron. Am J Physiol Renal Fluid Electrolyte Physiol 250: F1F15, 1986.[Abstract]
34. Madsen KM, Verlander JW, and Tisher CC. Relationship between structure and function in distal tubule and collecting duct. J Electron Microsc Tech 9: 187208, 1988.[CrossRef][ISI][Medline]
35. Morrison TB, Weis JJ, and Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24: 954962, 1998.[ISI][Medline]
36. Nelson RD, Guo XL, Masood K, Brown D, Kalkbrenner M, and Gluck S. Selectively amplified expression of an isoform of the vacuolar H+-ATPase 56-kilodalton subunit in renal intercalated cells. Proc Natl Acad Sci USA 89: 35413545, 1992.
37. Poschet J, Perkett E, and Deretic V. Hyperacidification in cystic fibrosis: links with lung disease and new prospects for treatment. Trends Mol Med 8: 512519, 2002.[CrossRef][ISI][Medline]
38. Purcell H, Bastani B, Harris KP, Hemken P, Klahr S, and Gluck S. Cellular distribution of H+-ATPase following acute unilateral ureteral obstruction in rats. Am J Physiol Renal Fluid Electrolyte Physiol 261: F365F376, 1991.
39. Ricciardolo FL, Rado V, Fabbri LM, Sterk PJ, Di Maria GU, and Geppetti P. Bronchoconstriction induced by citric acid inhalation in guinea pigs: role of tachykinins, bradykinin, and nitric oxide. Am J Respir Crit Care Med 159: 557562, 1999.
40. Robaire B and Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 52: 226236, 1995.[Abstract]
41. Rodriguez CM, Kirby JL, and Hinton BT. The development of the epididymis. In: The Epididymis: From Molecules to Clinical Practice: A Comprehensive Survey of the Efferent Ducts, the Epididymis, and the Vas Deferens, edited by Robaire B and Hinton BT. New York: Kluwer Academic/Plenum, 2002, p. 119130.
42. Saboli I, Wuarin F, Shi LB, Verkman AS, Ausiello DA, Gluck S, and Brown D. Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase. J Cell Biol 119: 111122, 1992.[Abstract]
43. Sakurai H and Nigam SK. Molecular and cellular mechanisms of kidney development. In: The Kidney: Physiology & Pathophysiology (3rd ed.), edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, vol. 1, p. 685702.
44. Schuster VL. Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55: 267288, 1993.[CrossRef][ISI][Medline]
45. Smith AN, Finberg KE, Wagner CA, Lifton RP, Devonald MAJ, Su Y, and Karet FE. Molecular cloning and characterization of Atp6n1b: a novel fourth murine vacuolar H+-ATPase a-subunit gene. J Biol Chem 276: 4238242388, 2001.
46. Smith AN, Lovering RC, Futai M, Takeda J, Brown D, and Karet FE. Revised nomenclature for mammalian vacuolar-type H+ ATPase subunit genes. Mol Cell 12: 801803, 2003.[CrossRef][ISI][Medline]
47. Stevens AL, Breton S, Gustafson CE, Bouley R, Nelson RD, Kohan DE, and Brown D. Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens. Am J Physiol Cell Physiol 278: C791C802, 2000.
48. Teng-umnuay P, Verlander JW, Yuan W, Tisher CC, and Madsen KM. Identification of distinct subpopulations of intercalated cells in the mouse collecting duct. J Am Soc Nephrol 7: 260274, 1996.[Abstract]
49. Wagner CA, Finberg KE, Breton S, Marshansky V, Brown D, and Geibel JP. Renal vacuolar ATPase. Physiol Rev 84: 12631314, 2004.
50. Wittwer CT, Herrmann MG, Moss AA, and Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22: 130138, 1997.[ISI][Medline]
51. Zharkikh L, Zhu X, Stricklett PK, Kohan DE, Chipman G, Breton S, Brown D, and Nelson RD. Renal principal cell-specific expression of green fluorescent protein in transgenic mice. Am J Physiol Renal Physiol 283: F1351F1364, 2002.