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Am J Physiol Renal Physiol 281: F531-F545, 2001;
0363-6127/01 $5.00
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Vol. 281, Issue 3, F531-F545, September 2001

Immunocytochemical localization of Na-K-ATPase alpha - and gamma -subunits in rat kidney

Randall K. Wetzel and Kathleen J. Sweadner

Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The gamma -subunit of the Na-K-ATPase is a single-span membrane protein that alters the kinetic properties of the enzyme. It is expressed in the kidney, but our initial observations indicated that it is not present in all nephron segments (Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. J Biol Chem 274: 33183-33185, 1999). Here we used triple-label confocal immunofluorescence microscopy in rat kidney with antibodies to Na-K-ATPase alpha 1- and gamma -subunits and nephron segment-specific markers. Na-K-ATPase alpha 1-subunit stain was low but unambiguous in proximal segments, moderate in macula densa, connecting tubules, and cortical collecting ducts, high in thick ascending limb and distal convoluted tubules, and nearly undetectable in glomeruli, descending and ascending thin limb, and medullary collecting ducts. The gamma -subunit colocalized at staining levels similar to alpha 1-subunit in basolateral membranes in all segments except cortical thick ascending limb and cortical collecting ducts, which had alpha 1-subunit but no detectable gamma -subunit stain. Selective gamma -subunit expression may contribute to the variations in Na-K-ATPase properties in different renal segments.

immunofluorescence; confocal microscopy; nephron; colocalization; sodium pump


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENAL CONTROL OF NA+ and K+ balance is complex and entails ensembles of apical and basolateral transporters that play specialized roles in different segments of the nephron (23, 36). One of the most physiologically important transporters is the Na-K-ATPase, which is crucial for the absorptive, secretory, and concentrating capacity of the kidney (14). In the nephron, the Na-K-ATPase has been localized on the basolateral surface of most tubules and is directly responsible for sodium reabsorption and for maintaining ion gradients that are used in the redistribution of water, other ions, and solutes. The Na-K-ATPase is composed of two subunits, alpha  (112 kDa) and beta  (32 kDa plus glycosylation). There are four known alpha -subunit isoforms (alpha 1, alpha 2, alpha 3, and alpha 4) and three known beta -subunit isoforms (beta 1, beta 2, and beta 3) (for review, see Ref. 8), but thus far, alpha 1- and beta 1-subunits are the only isoforms generally accepted to be expressed as proteins in adult kidney tubules (8, 16) or detected by quantitative PCR (25). In addition, the Na-K-ATPase has a third nonobligatory subunit, gamma , that is expressed predominantly in the kidney (35). The gamma -subunit belongs to the FXYD gene family of small single-span membrane proteins that function as ion transport regulators or channels (43). Expression of the gamma -subunit was shown to alter the voltage sensitivity and interaction with extracellular K+ and Na+ of Na-K-ATPase in Xenopus oocytes (7), and transfection and coexpression of gamma -subunit with alpha - and beta -subunits in a rat kidney cell line stably decreased the apparent Na+ and K+ affinity of the Na-K-ATPase measured in vitro (4) and increased the affinity for ATP (reviewed in Ref. 46; Ref. 48). Recently, the difference in apparent Na+ affinity has been ascribed to an increase in K+ competition for Na+ activation of the pump (39).

The luminal, interstitial, and intracellular concentrations of Na+, K+, and other ions vary along the length of the nephron, creating microenvironments that may require adjustment of pump functional properties. Because the renal Na-K-ATPase operates well below its maximal velocity and close to its Michaelis-Menten constant, small changes in Na-K-ATPase apparent affinity for Na+ or K+ can have major consequences for epithelial transport (21). There are segment-specific differences in Na+ apparent affinity (6, 10, 20, 22) that cannot be explained by the intrinsic properties of the enzyme's known alpha 1- and beta 1-subunit isoforms.

The distribution of Na-K-ATPase alpha -subunit in the kidney has been examined extensively in previous studies, using both immunohistochemistry (27, 30, 37, 41) and Western blots (31, 50). In addition, Na-K-ATPase mRNA has been localized in the nephron using in situ hybridization (11, 17) and by segment-specific quantification of mRNA (25, 50). Although there are few minor differences, these studies all indicate that the highest expression levels of Na-K-ATPase are in the medullary and cortical thick ascending limb (mTAL and cTAL, respectively) and distal convoluted tubule (DCT). There are lower levels in the proximal convoluted tubule (PCT) and cortical collecting duct (CCD), and very low levels of expression in glomeruli, descending or ascending thin limb of Henle (DTL and ATL, respectively), and outer and inner medullary collecting duct (OMCD and IMCD, respectively). These data correlate with studies that have examined the amount of Na-K-ATPase hydrolytic activity in isolated nephron segments (14, 28), indicating that the highest activity is in the TAL and DCT, moderate activity is in the PCT and CCD, and very low activity is in the proximal straight tubule (PST), DTL, and ATL.

The above studies examined either whole Na-K-ATPase or the alpha - or beta -subunits. The distribution of gamma -subunit is less well studied. Mercer et al. (35) localized alpha - and gamma -subunits in the sheep kidney cortex using immunofluorescence and found the strongest stain in DCT, connecting tubule (CNT), and principal cells of the collecting duct and the weaker stain in proximal segments. Furthermore, they noted that alpha - and gamma -subunits were always colocalized and were either present or absent together. Hayward et al. (25) identified gamma -subunit transcripts in PCT and PST. Initial experiments with our anti-gamma -subunit antibodies, however, indicated that some nephron segments appeared to express alpha - and beta -subunits, but not gamma -subunits (4). Therefore, we have used confocal immunofluorescence microscopy to examine the expression of gamma  in rat kidney sections double or triple stained with antibodies to Na-K-ATPase alpha 1-, beta 1-, and gamma -subunits, combined with antibodies to known nephron segment-specific markers to specifically identify the gamma -subunit-expressing segments.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies. Table 1 lists the antibodies used. Monoclonal anti-gamma -subunit antibody McG-11H is an IgG isolated from a BALB/c mouse injected with the synthetic peptide CGGSKKHRQVNEDEL (corresponding to the COOH-terminal 15 amino acids of the rat gamma -subunit) bound to keyhole limpet hemocyanin. Polyclonal anti-gamma -subunit antibody RCT-G1 was isolated from a rabbit injected with the same peptide and has been previously described (4). The specificities of antibodies McK1 (anti-alpha 1-subunits) and Ball757 (anti-beta 1-subunits) have also been previously described (3, 5). Antibodies directed against known markers of specific nephron segments were also used. Anti-aquaporin-1 (AQP1) specifically labels PCT, PST, and DTL (51), anti-rTSC labels DCT (38), anti-Tamm-Horsfall labels the TAL (42), anti-neuronal nitric oxide synthase (nNOS) labels macula densa cells (49), and anti-calbindin brightly labels CNT and lightly labels DCT (45).

                              
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  		Table 1.   Antibodies used for immunofluorescence

Immunocytochemistry. Adult male CD rats were anesthetized just to the point of cessation of respiration with ether and then immediately perfused with 150 ml of PBS (0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2) followed by perfusion with 300 ml of 2% paraformaldehyde in periodate-lysine buffer (PLP fixative) (32). The kidneys were removed, bisected, and postfixed by immersion in fresh PLP for an additional 2 h with gentle agitation at room temperature. They were rinsed in several changes of PBS for several hours at room temperature and then immersed in 30% sucrose in PBS overnight at 4°C. They were embedded in TBS tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC) in aluminum boats, frozen on liquid nitrogen, and stored at -20°C. Cryostat sections (8-10 µm) were picked up on ProbeOn Plus positively charged microscope slides (Fisher Scientific, Pittsburgh, PA) and stored at -20°C until use. Immediately before staining, slides were brought to room temperature and a PAP pen (Kiyota International, Elk Grove, IL) was used to draw a hydrophobic ring around the sections.

For double immunofluorescence with mouse and rabbit antibodies, slides were rinsed in PBS for 10 min and then laid flat in a dark moist box for all subsequent incubations. The sections were covered (~50 µl per section) with 1% SDS in PBS for 5 min. This SDS treatment has been shown to enhance immunostaining to Na-K-ATPase and other antigens in the kidney (9). The slides were rinsed thoroughly in PBS (3 × 10 min) and then similarly incubated with 1% normal goat serum and 0.5% NEN blocking reagent (New England Nuclear, Boston, MA) in PBS with 0.3% Triton X-100 (PBSt) for 1 h at room temperature. This blocking solution was removed with an aspirator, and a mixture of one mouse antibody and one rabbit antibody at the appropriate dilution (see Table 1) in PBSt was immediately applied to the sections and incubated overnight at 4°C. The slides were rinsed in PBS for 10 min, high-salt PBS (450 mM NaCl in 100 mM phosphate buffer, pH 7.2) for 5 min, and then normal PBS two more times for 10 min each. They were then incubated in a mixture of Cy3-conjugated goat anti-mouse IgG (1:300; Accurate, Westbury, NY) and FITC-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA) in PBSt for 2 h. Finally, they were rinsed in normal and high-salt PBS and coverslipped in Vectashield fluorescence mounting medium (Vector Laboratories, Burlingame, CA).

For triple-label immunofluorescence involving two different mouse antibodies and a rabbit antibody, tyramide signal amplification (TSA) was used for one antibody. Slides were rinsed in PBS for 10 min, immersed in 0.25% H2O2 in PBS for 60 min to quench endogenous peroxidase activity, rinsed in PBS for 5 min, and incubated with SDS and blocking solutions as described above. The sections were then incubated with the anti-alpha 1-mouse monoclonal antibody McK1 at a dilution of 1:750 in PBSt. This high dilution of McK1 was detectable only with tyramide amplification and not with directly conjugated fluorescent secondary antibodies. The slides were rinsed in normal and high-salt PBS as described above, incubated in biotinylated horse anti-mouse IgG (1:500; Vector, Burlingame, CA) in PBSt for 2 h, rinsed in normal and high-salt PBS, incubated in streptavidin-horseradish peroxidase (1:100; NEN) in PBSt, rinsed in normal and high-salt PBS, and then incubated in Cy3-tyramide reagent (1:100) in NEN amplification diluent (NEN) for 5-7 min. The slides were rinsed in several changes of PBS for 30 min and then incubated with a second mouse antibody and a rabbit antibody in PBSt overnight at 4°C. They were then rinsed in normal and high-salt PBS and incubated in Cy5-conjugated goat anti-mouse IgG (1:300) and FITC-conjugated goat anti-rabbit IgG (1:200; Jackson) in PBSt for 2 h. The slides were then rinsed and coverslipped as above. It should be noted that when tyramide amplification is used with high dilutions of McK1, stain is not as uniform and crisp as usual, and stain of segments like PCT that have less Na-K-ATPase is harder to detect than usual. Figures 1, 2, 11, and 12 show conventional staining with McK1, which clearly reveals the light stain seen in proximal segments.


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  		Fig. 1.   Confocal images of kidney cortex double labeled with alpha 1- and gamma -subunit antibodies. The 2 bottom images are of alpha 1- (red, Cy3) and gamma -subunits (green, FITC) individually, whereas the top image shows the 2 colors merged. alpha 1- and gamma -Subunits were colocalized (yellow in the merged image) in dimly labeled presumptive proximal convoluted tubule (PCT; asterisk) and in brightly labeled presumptive distal convoluted tubule (DCT; thick arrow), whereas glomeruli (G) were unlabeled. Some vertically oriented presumptive cortical thick ascending limb (cTAL) contained bright alpha 1-subunit stain but little or no gamma -subunit stain (thin arrow). Similarly, presumptive cortical collecting duct (CCD) had alpha 1-subunit stain but no gamma -subunit stain (arrowhead). Scale bar, 50 µm.



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  		Fig. 2.   Kidney double labeled with alpha 1- (a, d, g, j, m), gamma -subunit (b, e, h, k, n) antibodies, and 2-color merged images (c, f, i, l, o) at the level of the cortex (a-c), cortex/medulla border (d-f), medulla outer stripe (OS)/inner stripe (IS) border (g-i), medulla inner stripe (j-l), and outer medulla/inner medulla (OM/IM) border (m-o). alpha 1- and gamma -subunits colocalized in all levels of the kidney except in vertically oriented tubules in the cortex that contained bright alpha 1- but no gamma -subunit stain (red in the merged image). Tubules in the OM contained bright alpha 1- and gamma -subunit stain, whereas tubules in the IM contained little or no stain. Scale bar, 50 µm.

For triple-label immunofluorescence involving the goat anti-Tamm-Horsfall antibody, the slides were rinsed, incubated in SDS and blocking solutions, and then incubated with McK1 (mouse), RCT-G1 (rabbit) and anti-Tamm-Horsfall (goat) primary antibodies in PBSt as described above for regular double-label immunofluorescence. However, because unbound anti-goat secondary antibody would bind to the goat anti-mouse and goat anti-rabbit secondary antibodies, sections were incubated first in Cy3-conjugated donkey anti-goat IgG (1:1,000; Jackson) in PBSt for 2 h, rinsed thoroughly to remove any unbound secondary antibodies, then incubated in the Cy5-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit antibodies in PBSt.

Slides were examined and images were collected on a Nikon TE300 fluorescence microscope equipped with a Bio-Rad MRC 1024 scanning laser confocal system, version 3.2.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stain for the Na-K-ATPase alpha 1-subunit in different nephron segments was consistent with previous reports. Figure 1 illustrates a section from the rat renal cortex double labeled with mouse monoclonal alpha 1- and rabbit polyclonal gamma -subunit antibodies, showing each antibody separately and in a combined image. Glomeruli were unstained. Yellow in the combined image shows locations of coexpression that can be seen in both lightly labeled presumptive PCT (asterisk) and brightly labeled presumptive DCT (thick arrow). Expression of alpha 1- without gamma -subunits was very obvious in the heavily stained straight tubules, which from their shape, location, and cell morphology are likely to be cTAL (thin arrow); further evidence is shown below. Closer inspection also shows alpha 1- without gamma -subunits in thin-celled segments with a patchy distribution of stain (arrowhead). As will also be shown below, the stained cells are the principal cells of the CCD.

Figure 2 shows representative sections from all levels of the kidney. alpha 1- (red) and gamma -subunits (green) were colocalized (yellow) on the basolateral surfaces of the most prominently stained tubules throughout the kidney. Figure 2, a-c, shows the same images as in Fig. 1 but reduced to scale. This is superficial cortex, marked by the presence of three glomeruli. Figure 2, d-f, shows the transition to the outer stripe of the outer medulla, where DCT, cTAL, and proximal tubules coexist. The lightly stained proximal tubules all had gamma -subunits, whereas the brightly stained tubules sometimes did and sometimes did not. Figure 2, g-i, shows the transition from outer stripe to inner stripe, and j-l show inner stripe, where mTAL stain predominates. At this location, gamma -subunits always colocalized with alpha 1-subunits. Figure 2, m-o, shows the transition from outer medulla to inner medulla, where there was little stain detected for either subunit.

Stain for the beta 1-subunit coincided with that for alpha 1-subunit, as shown in Fig. 3. This is a triple-label experiment in which the alpha 1-subunit antibody was diluted to the point that tyramide amplification was the only way to detect it. This method resulted in spotty stain appearance in regions of low antigen concentration (red), but it allowed two antibodies from the same species (mouse in this case) to be used on the same section. The other mouse antibody (anti-gamma -subunit) was stained by conventional directly conjugated secondary antibody (blue). beta 1-Subunit was stained by a rabbit antibody, and in the combined image it can be seen that alpha 1-, beta 1-, and gamma -subunit stain coincided (pink) in both lightly stained regions (presumptive PCT) and heavily stained regions (DCT). In presumptive cTAL, only alpha 1- and beta 1-subunits were seen (orange-yellow).


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  		Fig. 3.   Kidney cortex triple labeled with Na-K-ATPase alpha 1- [red, tyramide signal amplification (TSA)-Cy3], beta 1- (green, FITC), and gamma -subunit (blue, Cy5) antibodies. alpha 1- and beta 1-subunits were always colocalized, and were usually colocalized with gamma -subunits. However, the TAL contained alpha 1- and beta 1-subunit stain, but not gamma -subunit (yellow in the merged image). Scale bar, 50 µm.

To identify specific tubules within the nephron, sections were triple labeled with alpha 1-, gamma -subunits, and a segment-specific antibody (Figs. 4-10). Because most of the marker antibodies were generated in either mouse or rabbit, we used tyramide amplification to stain for alpha 1-subunits using a dilution of McK1 that was not detectable with directly conjugated secondary antibodies and then stained for the marker and gamma -subunits using either mouse marker antibody with the rabbit polyclonal anti-gamma -subunit, RCT-G1, or rabbit marker antibodies with the mouse monoclonal anti-gamma -subunit, McG-11H. Elimination of any one of the three primary antibodies eliminated all stain for that specific antibody without affecting the stain from the other two (not shown). One antibody (anti-Tamm-Horsfall) was generated in goat, and therefore we used conventional triple-label immunofluorescence with three directly conjugated secondary antibodies.


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  		Fig. 4.   Aquaporin-1 (AQP1), a marker of proximal tubule. Kidney cortex was triple labeled with alpha 1- (red, TSA-Cy3), AQP1 (green, FITC), and gamma -subunit (blue, Cy5) antibodies. Proximal segments containing AQP1 stain were very lightly labeled with alpha 1- and gamma -subunits. Conversely, distal segments that were not labeled by the AQP1 antibody were brightly stained by alpha 1- and gamma -subunits. Scale bar, 50 µm.



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  		Fig. 5.   Kidney OM triple labeled with alpha 1 (red, TSA-Cy3)-, AQP1 (green, FITC), and gamma -subunit (blue, Cy5) antibodies. Proximal segments containing AQP1 stain were very lightly labeled with alpha 1- and gamma -subunits, which were always colocalized. Distal segments, presumably mTAL, were brightly labeled by alpha 1- and gamma -subunit antibodies but were devoid of AQP1 stain. The dashed line indicates the border between the OS and the IS of the OM. Scale bar, 50 µm.



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  		Fig. 6.   Kidney medulla triple labeled with alpha 1 (red, TSA-Cy3)-, AQP1 (green, FITC), and gamma -subunit (blue, Cy5) antibodies. Proximal segments in the OM and IM containing AQP1 stain showed little or no alpha 1- or gamma -subunit stain. Ascending segments were brightly stained with alpha 1- and gamma -subunit antibodies in the OM but were very lightly stained in the IM. The dashed line indicates the border between the IS of the OM and IM. Scale bar, 50 µm.



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  		Fig. 7.   Tamm-Horsfall antigen, a marker of TAL. Kidney was triple labeled with Tamm-Horsfall (red, Cy3), gamma  (green, FITC)-, and alpha 1-subunit (blue, Cy5) antibodies. Tamm-Horsfall stain was bright in TAL and much lighter in DCT. Although the DCT was brightly stained by both alpha 1- and gamma -subunit antibodies, the cTAL was only stained by alpha 1- and not by gamma -subunits. Scale bar, 50 µm.



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  		Fig. 8.   Neuronal nitric oxide synthase (nNOS), a marker of macula densa. High-magnification image of kidney cortex triple labeled with alpha 1 (red, TSA-Cy3)-, nNOS (green, FITC), and gamma -subunit (blue, Cy5) antibodies. The cells of the macula densa contained bright cytoplasmic nNOS stain, as well as alpha 1- and gamma -subunit stain on their basolateral surface. The adjacent portion of the cTAL was brightly stained by the alpha 1-subunit antibody, but not by gamma -subunit. Scale bar, 25 µm.



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  		Fig. 9.   Thiazide-sensitive Na+-Cl- cotransporter (rTSC), a marker of DCT. Kidney cortex was triple labeled with alpha 1 (red, TSA-Cy3)-, rTSC (green, FITC), and gamma -subunit (blue, Cy5) antibodies. DCT that were apically labeled with the rTSC antibody were also basolaterally labeled with alpha 1- and gamma -subunits. Presumptive cTAL contained only alpha 1-subunit stain (arrow), whereas presumptive connecting tubule (CNT) contained patchy alpha 1- and gamma -subunit stain but not rTSC (arrowhead). Scale bar, 50 µm.



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  		Fig. 10.   Calbindin, a marker of CNT. Kidney cortex was triple labeled with alpha 1 (red, TSA-Cy3)-, gamma -subunit (green, FITC), and calbindin (blue, Cy5) antibodies. Bright calbindin stain was seen in CNT, whereas lighter stain was seen in DCT and collecting ducts. DCT containing light calbindin stain were also brightly stained by alpha 1- and gamma -subunits. CNT were brightly labeled by alpha 1- and gamma -subunit antibodies. Calbindin-stained CCD were lightly labeled by alpha 1-subunit, but contained little or no gamma -subunit stain. A macula densa can also be seen in this image (arrowhead). Scale bar, 50 µm.

To identify proximal and descending segments, we used an AQP1 antibody. AQP1 stain was localized at the apical surface in PCT, PST, and DTL. Figures 4, 5, and 6 show AQP1-stained segments at three different levels in the kidney. The most superficial cortex is shown in Fig. 4, where the AQP1-stained PCT (green) all showed basolateral stain for gamma  (blue)- as well as alpha 1-subunits (red). The DCT were brightly stained for alpha 1- and gamma -subunits (bright purple in the combined image) but unstained for AQP1. Figure 5 shows the boundary between outer medullary outer stripe (OS) and inner stripe (IS). The brightly AQP1-stained segments (PST and short-loop DTL, depending on diameter) had barely detectable stain for alpha 1- or gamma -subunits. The bright purple alpha 1- and gamma -subunit stain was presumably in mTAL, judging from the absence of AQP1. Figure 6 shows the boundary between outer medulla and the inner medulla. Bright AQP1 stain of DTL was uniform across the boundary, and contained little detectable stain for either alpha 1- or gamma -subunits, and the purple stain for alpha 1- and gamma -subunits in mTAL stopped abruptly at the boundary.

To identify the TAL, we used an antibody against Tamm-Horsfall antigen that is known to brightly label both the mTAL and cTAL and to lightly label the DCT (42). Figure 7 shows that cTAL that contained bright Tamm-Horsfall and alpha 1-subunit stain were devoid of gamma -subunit stain and were purple in the combined image. In contrast, DCT, identified by light Tamm-Horsfall stain, contained bright alpha 1- and gamma -subunit stain and were aqua in the combined image.

The cells of the macula densa, located in the juxtaglomerular apparatus in the cTAL, are known to express nNOS (49). Using an antibody against nNOS, we were able to clearly distinguish between cTAL, which contain macula densa, and mTAL, which do not, and also to clearly identify the transition from cTAL to DCT, which occurs immediately after the cTAL passes the juxtaglomerular apparatus (not illustrated). A juxtaglomerular apparatus is seen in Fig. 8. The cytoplasm of the cells of the macula densa was stained with nNOS antibody, and the cells had light to moderate alpha 1- and gamma -subunit stain on their basolateral surface. However, the remaining portion of the adjacent cTAL contained only alpha 1-subunit stain and not gamma -subunit stain. In the combined image, the macula densa is largely green, whereas the surrounding cTAL is red, and adjacent PCT are light purple.

The thiazide-sensitive Na+-Cl- cotransporter (rTSC) antibody is a known marker of DCT (38). The apical surface of the DCT was clearly labeled with anti-rTSC, whereas the basolateral surface was very brightly stained with both alpha 1- and gamma -subunit antibodies (Fig. 9). In the combined image, these are the tubules with blue-purple basolateral surfaces and green apical stain. Two other kinds of brightly stained tubules can be seen: those with alpha 1- but no gamma -subunits or rTSC, which are presumably cTAL (arrow), and those with alpha 1- and gamma -subunit stain but only faint rTSC stain (arrowhead). The patchy alpha 1- and gamma -subunit stain of these last tubules suggests connecting or collecting tubules with intercalated cells, and these are identified more specifically in the next figure.

The calbindin antibody is known to brightly stain CNT and also to lightly stain late DCT and collecting duct principal cells (45). Many features can be seen in Fig. 10. cTAL, stained only for alpha 1-subunits, is seen as long vertical tubules (red). DCT was stained with only alpha 1- and gamma -subunit antibodies (yellow). The CNT was brightly stained by the calbindin antibody and by both alpha 1- and gamma -subunit antibodies (purple to white in the three-color image). This stain was patchy, consistent with Na-K-ATPase stain in principal cells. The CCD was lightly but clearly stained by the calbindin antibody, and the principal cells were stained for alpha 1- but not gamma -subunits. Interestingly, a macula densa with prominent gamma -subunit stain is also visible in this figure (arrowhead), with bright alpha 1-subunit stain in the adjacent cTAL cells.

The collecting duct was stained with anti-V-type ATPase antibody (H+-ATPase), which stains intercalated cells from the cortex through the IMCD (1). For these experiments, conventional double labeling was used to enhance our ability to detect alpha 1-subunit at low levels, because tyramide amplification at very high antibody dilutions gives less uniform stain of alpha 1-subunits. Figure 11 shows superficial cortex H+-ATPase-stained intercalated cells in CCD alternating with alpha 1-stained principal cells (arrowheads). In the bottom panels, it can be seen that such alpha 1-stained cells do not have stain for gamma -subunits (red in the combined image), in contrast to DCT (solid yellow) and presumptive CNT (patchy yellow). Figure 1 also shows a presumptive CCD with patchy stain of alpha 1- but not gamma -subunits running vertically next to presumptive cTAL.


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  		Fig. 11.   H+-ATPase as a marker of intercalated cells. Kidney cortex was double labeled with either alpha 1-subunit (red, Cy3) and H+-ATPase (green, FITC) antibodies (top) or alpha 1 (red, Cy3)- and gamma -subunit (green, FITC) antibodies (bottom). H+-ATPase was seen in intercalated cells and alpha 1-subunit stain was seen in principal cells of the CCD (arrowheads, top), but the CCD was not stained by the gamma -subunit antibody (arrowheads, bottom). Scale bar, 100 µm.

Figure 12 shows the boundary between outer and inner medulla. Here, H+-ATPase stain of medullary collecting duct continued unbroken across the boundary, whereas alpha 1- and gamma -subunit stain of mTAL stopped abruptly. Under the conditions used, stain for either alpha 1- or gamma -subunits was undetectable in OMCD or IMCD.


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  		Fig. 12.   Kidney medulla double labeled with either alpha 1 (red, Cy3)-subunit and H+-ATPase (green, FITC) antibodies (top) or alpha 1 (red, Cy3)- and gamma -subunit (green, FITC) antibodies (bottom). H+-ATPase was seen in intercalated cells of the medulary collecting ducts (MCD) (arrowheads, top). No alpha 1- or gamma -subunit stain was seen in the MCD (arrowheads, bottom). Scale bar, 100 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, Na-K-ATPase alpha 1-subunit expression was highest in the TAL and DCT, intermediate in the CNT and CCD, and low in the PCT. The gamma -subunit was colocalized with the alpha 1-subunit and was stained at levels similar to alpha 1-subunit in all tubules except cTAL and CCD, which had no detectable gamma -subunits. Figure 13 diagrams the results. Stain for Na-K-ATPase has been detected in the medullary collecting duct by others with more sensitive detection (27, 40, 41). For present purposes, we can only say that the levels of expression must be very low.


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  		Fig. 13.   Summary diagram of the Na-K-ATPase alpha 1- and gamma -subunit expression patterns along the length of the nephron. The thickness of each line represents the apparent level of expression. The distributions of the various markers are also shown. Macula densa, which consists of a small patch of cells in the cortical thick ascending limb at the juxtaglomerular apparatus at the glomerulus, is indicated on the diagram. These cells were positive for gamma -subunits at the basolateral surface. Drawn in the style of Piepenhagen et al. (37).

Mercer et al. (35) previously examined the expression of alpha - and gamma -subunits in the sheep kidney cortex using immunofluorescence. The strongest stain was seen in DCT, CNT, and principal cells of CCD, and the weaker stain was seen in proximal segments. Furthermore, they noted that alpha - and gamma -subunits were always colocalized and were either present or absent together. This contradicts our results, and the reason for the discrepancy is not known. Although it is possible that there is a species difference between rat and sheep, preliminary studies in our lab have shown that the pattern of gamma -subunit expression in mouse kidney is similar to that in rat kidney. It is possible that cTAL was simply not examined in the sheep study. Hayward et al. (25) examined the levels of alpha - and gamma -subunit mRNA in isolated proximal segments of the rat kidney. Similar amounts of alpha - and gamma -subunit transcripts were found in the PCT and in the PST, but other more distal segments were not examined.

While this paper was under review, Pu et al. (39) also reported gamma -subunit localization in the rat kidney, with an antibody that detects both splice forms like ours, and with splice-specific antibodies. Their results differ from ours in two important respects, in that they reported finding gamma -subunit expression in cTAL and in CCD. We hypothesize that this is due mainly to differences in the identification of nephron segments. In their Fig. 7A, for example, a gamma -subunit stained segment is labeled cTAL, which according to our data may be more likely to be a piece of DCT cut in cross section. Next to it, under the "g" is a segment that we would guess is authentic cTAL: unstained for gamma -subunit except for the surface facing the glomerulus, which appears to be a macula densa. They further concluded that gamma -subunit was present in cTAL on the basis of colocalization with Tamm-Horsfall antigen; clear colocalization of gamma b-subunit with Tamm-Horsfall antigen can be seen in Fig. 8H, but we have seen such stain in TAL only in the deepest cortex and in the OS of the outer medulla. Pu et al. (39) identified gamma -subunit stain in CCD on the basis of colocalization with AQP2, but in rodents AQP2 is also found in CNT (13), where we observed gamma . On the whole, though, the results are similar. Minor discrepancies could also be due to the fact that different fixation protocols were used.

Functional implications. It is interesting to note that alpha 1-, beta 1-, and gamma -subunits were coexpressed throughout the nephron except in the cTAL and CCD. In fact, although the expression of alpha 1-, beta 1-, and gamma -subunits is very high in the DCT, the adjacent cTAL is seemingly devoid of gamma -subunits despite equally high-alpha 1- and -beta 1-subunit expression. We have previously shown that transfection and coexpression of gamma -subunit with alpha - and beta -subunits in a rat kidney cell line reduced the Na-K-ATPase apparent affinity for sodium and potassium in vitro (4). Therefore, one would expect that Na-K-ATPase in segments that do not express gamma -subunits would have a higher ion affinity than those that do. The Doucet and Feraille laboratories have examined ion affinities in isolated segments from the rabbit and rat nephron. Their results indicate that Na-K-ATPase affinity for sodium is higher in the CCD than in the PCT, mTAL, or cTAL (6, 10, 19, 22) and that affinity in cTAL is higher than in PCT (6). Affinity for potassium is similar in PCT, mTAL, and CCD (15). The higher sodium affinity in the cTAL and CCD than in more proximal segments is consistent with the hypothesis that segments that don't express gamma -subunits have higher Na+ affinity than those that do.

If gamma -subunit expression decreases Na-K-ATPase apparent affinity for Na+ in vivo as it does in vitro (4), then one would expect Na-K-ATPase in the cTAL to have a higher affinity for Na+. As much as 30% of the filtered load of Na+ is reabsorbed in the TAL, yet this portion of the nephron is impermeable to water (24). Consequently, the concentration of Na+ in the lumen decreases along the length of the TAL such that the lowest concentration in the entire nephron is in the cTAL. It is possible, therefore, that the conditions in the cTAL require Na-K-ATPase pumps with higher Na+ affinity (no gamma -subunit). The lower affinity expected (but not yet measured in isolated segments) in the gamma -subunit-expressing DCT and CNT may reflect the primary role of these segments to make adjustments to luminal content. We speculate that if gamma -subunit expression can be regulated, it is here and in the collecting duct that changes will be seen.

Expression of gamma -subunit in a human embryonic kidney cell line has also been shown to increase Na-K-ATPase affinity for ATP in vitro (39, 47, 48). This raises an intriguing apparent contradiction. The concentration of ATP in the cTAL has been reported to be 25-45% lower than in the mTAL or DCT (44). If gamma -subunit expression increases Na-K-ATPase affinity for ATP in vivo as this in vitro experiment suggests, then the Na-K-ATPase in the cTAL may be less active because of its lower affinity for ATP.

Splice variants of gamma -subunit. The renal Na-K-ATPase gamma -subunit has recently been shown to exist as two variants, the gamma a- and gamma b-subunits, with different NH2-terminal sequences (29, 43). Because we have data indicating that expression of these variants may result in similar Na+ but different K+ affinities (2), it is important to examine the distribution of these two gamma -subunit variants in the nephron. The monoclonal and polyclonal anti-gamma -subunit antibodies used here are both directed against a portion of the COOH-terminus of gamma -subunit that is identical in the splice variants. We have obtained data indicating coexpression of gamma a- and gamma b-subunits in mTAL in the IS, but preferential expression of gamma a-subunit in PCT and PST and preferential expression of gamma b-subunit in DCT, CNT, and in mTAL in the OS, again differ in part from the results reported by others (39).

Hypomagnesemia. Hypomagnesemia is a condition of magnesium wasting that occurs when the kidney is unable to reabsorb sufficient Mg2+ from the renal ultrafiltrate (for review, see Ref. 12). This disease is characterized by low serum Mg2+ levels, but different forms of hypomagnesemia involve shifts in other serum and urine electrolytes. Normally, the bulk of Mg2+ reabsorption (65-75%) in the kidney takes place via a passive or paracellular pathway in the cTAL. Significant amounts of Mg2+ (5-10%) are also reabsorbed via an active transcellular pathway in the DCT. Not surprisingly, the different forms of hypomagnesemia have been linked to mutations in genes that encode ion channels and tight junction proteins in the cTAL and DCT (34). Recently, Meij et al. (33) have identified a mutation in the Na-K-ATPase gamma -subunit gene that is responsible for the disorder known as isolated dominant renal hypomagnesemia. This dominant negative mutation is caused by a single residue substitution in the transmembrane domain of gamma -subunit (Gly to Arg) that prevents proper membrane insertion or routing of gamma -subunit (33). Because we have shown in the present study that gamma -subunit is not expressed in cTAL, it is possible that this gamma -subunit mutation is affecting active Mg2+ reabsorption in the DCT by preventing insertion of alpha -beta -gamma -subunit pumps and possibly of other membrane proteins, by accumulating misfolded protein in the endoplasmic reticulum. That the mutation does not have a more obvious effect on Na+ excretion may be due to the presence of one good copy of the gene and the existence of a threshold for pathology that is reached only in the cells with the very highest gamma -subunit expression levels, as seen here, in DCT. It is notable that gamma -subunit is also implicated in preimplantation blastocoel formation in mice (26), and yet humans with the mutation are clearly viable. A knockout of the gamma -subunit gene would be more informative.

In conclusion, expression of Na-K-ATPase with and without the regulatory gamma -subunit has been described in identified renal segments. The segments without gamma -subunit, the cTAL, and the CCD, are those that have been shown to have the Na-K-ATPase with the highest endogenous affinity for Na+. Further work on the physiological role of gamma -subunit would be timely.


    ACKNOWLEDGEMENTS

We thank W. J. Ball, Jr., S. Nielsen, S. C. Hebert, and D. Brown for their generosity with antibody markers. We also thank D. Brown and E. Arystarkhova for helpful discussions.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant 5R01- HL-36271 to K. J. Sweadner.

Address for reprint requests and other correspondence: K. J. Sweadner, 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129 (sweadner{at}helix.mgh.harvard.edu).

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.

Received 16 February 2001; accepted in final form 14 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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