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Am J Physiol Renal Physiol 279: F874-F883, 2000;
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Vol. 279, Issue 5, F874-F883, November 2000

Expression of aquaporins in the renal connecting tubule

Richard A. Coleman, Daniel C. Wu, Jie Liu, and James B. Wade

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The renal connecting tubule (CNT) is a distinct segment that occurs between the distal convoluted tubule (DCT) and the cortical collecting duct. On the basis of its characterization in rabbit it is widely believed that connecting tubule cells have a low permeability to water and do not respond to vasopressin. Here we utilize segment-specific markers and specific aquaporin antibodies to characterize expression of water channels in CNT of the rat by immunocytochemistry. Colocalization of aquaporin 2 (AQP2), AQP3, and AQP4 with Na+, Ca2+ exchanger (NCX), a transporter characteristic of the connecting tubule, gave heterogeneous labeling. There was aquaporin labeling in many but not all regions labeled by NCX. Colocalization of AQP2 with AQP3 and with AQP4 showed that AQP3 and AQP4 labeling were always accompanied by AQP2. Immunogold labeling and electron microscopy showed that NCX-labeled cells with AQP2 labeling had the morphology of CNT cells, whereas NCX-labeled cells without AQP2 labeling were DCT cells. The latter regions were identified as the late region of the DCT known as DCT2. Additionally, regions of CNT lacking AQP2 labeling could be identified in Brattleboro rats not treated with vasopressin but not in such animals chronically treated with deamino-Cys1,D-Arg8-vasopressin (dDAVP). Quantitative analysis of labeling was consistent with expression of AQP2 over a longer region of CNT after dDAVP exposure.

kidney; urinary concentrating mechanism; water channel; collecting duct; distal tubule


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MANY TRANSPORT AND MORPHOLOGICAL studies have developed evidence that the renal tubular regions downstream from the thick ascending limb are structurally and functionally heterogeneous (26). Three specific distal regions have been defined structurally in the cortex: the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD). In the case of midcortical and deep nephrons, DCTs of multiple nephrons join to CNTs that ascend as arcades through the cortical labyrinth before becoming CCDs and descending back through medullary rays toward the renal medulla. Immunocytochemical studies have established that CNTs display high levels of Na+, Ca2+ exchanger (NCX) activity (4, 27). In the rat, the final portion of the DCT also displays high NCX such that the DCT can be subdivided into a region, called DCT1, that expresses the thiazide-sensitive NaCl cotransporter (NCC) only and a region, called DCT2, that expresses both NCC and NCX (25). The CNT can be distinguished from the DCT2 by immunocytochemistry because, whereas both express NCX, the CNT does not express NCC.

Little functional data are available for the CNT region because it is inaccessible to micropuncture and very difficult to study by in vitro tubule perfusion because of its branched nature. On the basis of available microperfusion data for rabbit arcades (11), it was long believed that all connecting tubule segments display a low water permeability that is insensitive to vasopressin exposure. Furthermore, arcades from rabbits that were microdissected with care to exclude CCD segments displayed only a very modest vasopressin-sensitive adenylate cyclase activity (22). Thus the connecting tubule became widely viewed as a segment unresponsive to vasopressin on the basis of data for rabbit, a species with a rather poor concentrating ability. The role of this region in other animals has been little studied, but connecting tubule segments dissected from the mouse and rat have been shown to display a high level of adenylate cyclase activation in response to vasopressin (21).

A central action of vasopressin in mammalian concentrating ability is the insertion of aquaporin 2 (AQP2) water channels into the apical plasma membrane (23). Whereas it is generally believed that this response is limited to the principal cells of the renal collecting duct, cells of the rat arcade region have been reported to express AQP2 and the V2 receptor, also (16). It has been uncertain, however, whether the cells expressing AQP2 at this site are morphologically CNT cells or represent CCD principal cells that are known to occur in a portion of the rat CNT (6,13). In the present work we have utilized immunolocalization and electron microscopy to characterize AQP2-expressing cells in the rat CNT. We find that AQP3 and AQP4, as well as AQP2, can occur in CNT cells. In addition, there appears to be a region of CNT that does not express AQP2 in the absence of vasopressin but can be induced to express AQP2 by long-term exposure to vasopressin.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kidneys from five male Spraque-Dawley and eight male Brattleboro(di/di) rats were perfusion-fixed for 5 min with paraformaldehyde (2%) in PBS, pH 7.4. For electron microscopy studies, glutaraldehyde (0.01%) was added to the perfusion fixative to improve structural preservation. The kidneys were then perfused with cryoprotectant and prepared for immunolocalizations as previously described in detail (31). Confocal microscopy used a Zeiss 410 LSM microscope.

For light microscopy, combinations of primary antibodies were used in colocalization experiments: AQP2, 3, or 4 raised in rabbit, previously characterized (8, 23, 29), kindly provided by Mark Knepper, and NCX (27), kindly provided by Robert Reilly. Rabbit antibodies to AQP3 and 4 were colocalized with AQP2 raised in guinea pig (GP7) to the same AQP2 peptide used for the rabbit antibody. AQP2 was also colocalized with NCC by using antibody raised in guinea pig (GP16) to an NCC fusion protein kindly provided by David Ellison. This fusion protein and antibody raised to it have been previously described (3), as well as NCX and NCC [L573, described previously (15)]. In each case, one of the antibodies was raised in rabbit and the other in guinea pig. These antibodies, mixed in pairs as listed above, were used to localize their respective antigens. Primary antibodies were diluted to 10 µg/ml with incubation medium (50 ml of PBS, 0.05 g of BSA, 200 µl of 5% NaN3) and applied to cryostat sections,12 µm thick on glass coverslips, overnight. After this incubation, sections were washed in high-salt wash (50 ml of PBS, 0.5 g of BSA, 1.13 g of NaCl, 200 µl of 5% NaN3) and incubated for 2 h at 4°C with a mixture of fluorescein-labeled donkey anti-rabbit IgG and Texas red-labeled donkey anti-guinea pig IgG (Jackson Immuno Research) for 2 h. For triple labeling, chicken anti-AQP2 (LC54, to the peptide CVELHSPQSLPRGSKA), guinea pig anti-NCX and rabbit anti-NCC (L573) were localized with appropriate species-specific antibodies coupled to Alexa 488 and 568 (Molecular Probes, Eugene, OR) and Cy-5 (Jackson Immuno Research).

For electron microscopy, AQP2 and NCX were colocalized in 8- to10-µm cryosections on glass coverslips by using rabbit anti-AQP2 primary antibody followed by goat anti-rabbit secondary tagged with 6-nm colloidal gold, and guinea pig anti-NCX primary antibody followed by donkey anti-guinea pig secondary tagged with Texas red. To distinguish DCT segments from CNT, NCX, and the thiazide-sensitive NaCl cotransporter (NCC; also known as TSC), they were colocalized in the cryosections, by using guinea pig anti-NCX primary antibody followed by Texas red-labeled donkey anti-guinea pig secondary, and rabbit anti-NCC primary antibody followed by FITC-labeled donkey anti-rabbit secondary.

Tissue sections for electron microscopy were incubated at 4°C with the primary antibodies for 19 h, rinsed in five changes of high-salt wash over 6 h, incubated in the secondary antibodies for 19 h and rinsed in five changes of high-salt wash over 24 h. Micrographs were taken of these sections before postfixation for later identification of the labeled tubules in the embedded cryosections.

After antibody treatments the sections were fixed in 2% glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 10 min at room temperature, rinsed well in 0.1 M cacodylate, fixed in 1% osmium tetroxide for 30 min at 4°C, and dehydrated and embedded on the coverslip in a thin layer of epoxy resin. Because Texas red fluorescence survives postfixation and epoxy embedding, NCX-containing tubules could be identified after embedding. The embedding plastic was scored around the area of interest; the section of plastic was removed from the coverslip with 7% hydrofluoric acid, glued to a blank, and thin sectioned. Ultrastructural identification of cell types in NCX-containing tubule segments and comparison with AQP2 localization allowed us to determine the cell types that contain AQP2 in the CNT.

For quantitative immunolabeling experiments, Brattleboro rats were infused with vehicle or deamino-Cys1, D-Arg8-vasopressin (dDAVP) for 7 days as previously described (14). Kidneys from the two groups were treated identically throughout. Cross sections were cut near the center of each kidney with a consistent section plane orientation. Although this method tends to bias the results on the basis of absolute tubule area, relative tubule areas in the vehicle- and dDAVP-treated samples should be appropriately sampled.

Coded tissue samples were evaluated by using digital images of the cortex and the Metamorph image-analysis system (Universal Imaging) on the basis of pairwise double labeling: AQP2 + NCX and NCC + NCX. In each case the area of colabeling was measured as well as the area labeled by each antibody alone. Area determinations were made by calibrating the pixel size in micrographs standardized for magnification and area. A calibration reticle was used to accurately determine the magnification of the micrograph; then, knowing the pixel resolution of the image, the dimension of a pixel could be calculated. To determine tubule area, Metamorph would count the number of pixels of the selected color and multiply them by the area of a pixel.

Whereas AQP2 and NCC are located apically and NCX is located at the basal surface, at the magnification and pixel size used, fluorescence tended to fill the cell and those tubules with overlapping labeling, showing a uniform mixing of color to produce yellow that the Metamorph program could readily distinguish and quantitate. In addition, a high-gain image was taken for each area sampled to normalize for tissue area in the field. The image was processed by thresholding such that all tissue was rendered white and the background was black. As before, Metamorph was then used to count the number of pixels for the selected color (white), and this value was multiplied by the area of a pixel to give the total area of tissue in the image. In this way, values for the area of labeled tubules could be normalized as percentage of the total tissue area in the image to eliminate variation due to differences in nontubular area in the field. Statistical assessment of quantitative differences was carried out by using a repeated-measures analysis (ANOVA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Light microscopic colocalization of NCX and AQPs. In the rat kidney cortex a distinct heterogeneity of labeling for NCX and AQP2 was observed, with some NCX-positive segments showing strong labeling with AQP2 and others showing no detectable AQP2 labeling (Fig. 1). A similar labeling pattern was observed with AQP3 and AQP4 (data not shown). To determine the distribution of AQP3 and AQP4 with respect to AQP2, sections were colabeled with AQP2 antibody and, respectively, AQP3 (Fig. 2) or AQP4 (Fig. 3). AQP2 labeling always corresponded to AQP3 and AQP4 labeling, establishing a correspondence between expression of AQP2 and basolateral expression of AQP3 and AQP4. Note in Figs. 2 and 3 the intense band of basolateral labeling characteristic of CNTs, due to their highly elaborated basal membrane (arrows, Fig. 2B), compared with the relatively modest basal surface of CCDs (arrows, Fig. 2B inset). This, together with the absence of AQP2-positive/NCX-negative cells in the CNT, indicates that the aquaporin labeling occurs in CNT cells and does not result from the presence of principal cells within regions of CNT in the rat. As previously described for CCD (24), AQP2 is not restricted to the apical membrane but also occurs in the basolateral membrane of CNT cells to a variable extent (Figs. 2 and 3).


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  		Fig. 1.   Colocalization of aquaporin 2 (AQP2; A) and Na+, Ca2+ exchanger (NCX; B) in rat connecting tubule. AQP2 is predominately apical, whereas NCX is predominately basal. Tubule cross sections having labels for both AQP2 and NCX are from connecting tubules, whereas those displaying labels for only NCX (* in B) are from the most distal part of the distal convoluted tubule (DCT2). Bar, 25 µm.



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  		Fig. 2.   Colocalization of AQP2 (A) and AQP3 (B). These tubules, which are typical of AQP2-containing renal connecting tubules (CNT), show that the 2 channels occur in the same cells. Arrows in B indicate strong basal labels for AQP3 in CNT cells. Principal cells of the cortical collecting ducts (CCDs) have relatively modest basal labeling for AQP3 (arrows, inset), correlated with a much less elaborated basal membrane in the CCD principal cells compared with the CNT cells. Bar, 25 µm.



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  		Fig. 3.   Colocalization of AQP2 (A) and AQP4 (B). The tubule in this section is typical of the CNT tubules that contain AQP2 and shows that these two aquaporins label the same cells. Bar, 25 µm.

Electron microscopic localization of NCX and AQP2. Electron microscopy was carried out to confirm the identification of AQP2 in CNT cells. Specimens embedded in epoxy resin on coverslips were examined with fluorescence microscopy to identify tubules labeled with antibody to NCX. Regions with several such tubules were removed from the coverslip and sectioned for electron microscopy. The thin sections were compared with fluorescence micrographs taken of the same areas to determine which tubules contained NCX labeling. These and nearby tubules were examined for colloidal gold labeling of AQP2. Tubules devoid of labeling for NCX but with AQP2 gold labeling represented collecting ducts. NCX-labeled cells with AQP2 labeling were different from CCD principal cells, having few microvilli, a moderate number of mitochondria, and extensive infoldings of the basal membrane (Fig. 4A). These are characteristics of the CNT cell. The CNT cells displayed AQP2 labeling of cytoplasmic vesicles and their apical membrane (Fig. 4B).


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  		Fig. 4.   A: electron micrograph of CNT cell, identified by the presence of both AQP2 (colloidal gold label) and NCX (Texas red label detected by fluorescence light microscopy of plastic-embedded specimen before thin sectioning). This cell has the typical CNT cell structure, with numerous infoldings of the basal membrane and fewer mitochondria than in the DCT. Bar, 1 µm. B: higher magnification of CNT cell. The AQP2 localization pattern on the apical membrane and in cytoplasmic vesicles is similar to that typical of collecting duct principal cells. Bar, 0.5 µm.

As expected from the light microscopic localizations, we also observed NCX-labeled tubules that failed to label with AQP2 antibody. As shown in Fig. 5 by electron microscopy, these cells have few microvilli and long, rather straight mitochondria that are regularly spaced in deep interdigitations of the basal membrane. This ultrastructure is characteristic of distal tubule cells. This finding indicates that the NCX-positive tubules that fail to express AQP2 represent DCT2 regions.


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  		Fig. 5.   Electron micrograph of cell containing NCX, detected by fluorescence microscopy of plastic-embedded specimen, but no detectable AQP2. Note the deep infoldings of the basal membrane and the numerous long, regularly spaced mitochondria oriented perpendicular to the base of the cell that are typical of DCT cells. Bar, 1 µm.

Immunolocalization of AQP2 with respect to NCC and NCX. To confirm that the DCT2 fails to label with AQP2, we carried out immunolabeling with antibodies to NCC. When NCX and NCC were localized in the same sections (Fig. 6), three categories of labeled tubule segments could be identified: 1) segments containing only NCX (CNT regions); 2) segments containing only NCC (DCT1 regions); and 3) segments containing both NCX and NCC in the same cells (DCT2 regions). As expected, regions showing the transition from DCT1 to DCT2 (diagonal lines, Fig. 6) were also observed.


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  		Fig. 6.   Colocalization of NCX (A) and thiazide-sensitive NaCl cotransporter (NCC, B) in rat kidney cortex. NCC is found only in the DCT, whereas NCX is found in both the CNT and the most distal portion of the DCT. Segments containing both transporters (DCT2) appear to correspond to the segments that contain NCX but not AQP2 (Fig. 1). Segments containing only NCC (DCT1) are the proximal portion of the DCT. The line marks the boundary between the DCT1 and the DCT2. Bar, 25 µm.

To be able to characterize AQP2 labeling in each of the regions, we carried out triple immunolocalizations of AQP2, NCX, and NCC by using antibodies raised in three different species. To determine whether this localization might vary with vasopressin exposure, this was carried out on tissue from Brattleboro rats treated for 7 days with vasopressin and vehicle-treated control animals. The localization for such a control animal is shown in Fig. 7A, such that AQP2 labeling is shown in green, NCX in red, and NCC in blue. As expected, there are tubules with NCC only (blue only) representing DCT1, and NCC + NCX-expressing regions (blue + red) representing DCT2. These regions lack AQP2 labeling. There are also regions showing only AQP2 (green only) that represent CCD, as expected. However, two types of CNT regions could be identified. In some there was labeling with AQP2 (green) as well as NCX (red), but in some there was no detectable labeling of AQP2 (Fig. 7A). There are also occasional cells even within the region expressing AQP2 that express NCX but not AQP2 (arrowheads, Fig. 7A). This suggests that in animals lacking vasopressin there are regions of CNT that do not express AQP2. These regions were not found in animals exposed to long-term dDAVP (Fig. 8). Figure 9 shows a section from a dDAVP-treated animal through the transition from DCT2 (on the left) to CNT (on the right). Figure 9A shows that this region is labeled by NCX throughout its length. Figure 9B shows that NCC labeling is restricted to the left-hand portion and ends abruptly (arrows). The right-hand portion of the region is strongly labeled by AQP2 (C), with apical labeling extending up to the NCC labeling (lower arrow). There also appears to be a faint cytoplasmic labeling of AQP2 in DCT2 cells at the left near the transition.


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  		Fig. 7.   A: a triple-labeled section of cortex from a vehicle-treated Brattleboro rat. This micrograph demonstrates the distribution of AQP2 (green) with respect to NCX (red) and NCC (blue). There are 2 types of CNTs in these animals, one containing AQP2 and NCX (bottom) and others (*) labeled only by NCX. B: the AQP2 component of the labeling in the same section, confirming the absence of AQP2 labeling in these NCX-containing tubules (*). Arrowheads in A and B indicate individual CNT cells having only NCX labeling but found in CNT tubule segments double labeled for AQP2 and NCX. Bar, 100 µm.



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  		Fig. 8.   A: a triple-labeled section of cortex from a deamino-Cys1, D-Arg8-vasopressin (dDAVP)-treated Brattleboro rat. Fluorescence colors indicate primary antibodies as described in Fig. 7. Unlike the vehicle control kidney (Fig. 7), no region or individual cells labeled only for NCX can be detected. B: AQP2 component of the labeling in the same section showing, compared with A, that AQP2 is found in association with NCX in all segments in which NCC is not associated with NCX. Thus under these conditions, there is no class of tubules or cells in the rat kidney cortex that contain NCX only. Bar, 100 µm.



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  		Fig. 9.   Triple-labeled section from a dDAVP-treated Brattleboro rat. This shows the transition from a region of DCT2 on the left to a CNT on the right. A: labeling by NCX is throughout the region. B: labeling by NCC is prominent in the left-hand region and ends abruptly at the arrow. C: AQP2 labeling of the apical membrane begins just to the right of the arrow. There is a faint cytoplasmic labeling of AQP2 in DCT2 cells near the transition. Bar, 25 µm.

Quantitative assessment of immunolabeling. To test the possibility that AQP2 labeling extends over a larger portion of the CNT with dDAVP exposure, we carried out a quantitative evaluation of labeling. Examples of typical micrographs used for this evaluation are shown in Fig. 10. Coded tissue samples were evaluated from vehicle-infused Brattleboro rats and animals infused for 7 days with dDAVP. As described in METHODS, quantitation was carried out by using random digital images of the cortex and pairwise double labeling: AQP2 + NCX and NCC + NCX. In each case the area of colabeling was measured as well as the area labeled by each antibody individually. Whereas AQP2 and NCC are located apically and NCX at the basal surface, at the low magnification used, tubules with overlapping labeling showed a uniform overlap of mixed color (yellow) that the Metamorph program could readily distinguish and quantitatively measure. In addition, a high-gain image was taken for each area sampled to normalize for tissue area in the field (in this way values could be normalized to eliminate variation due to differences in nontubular area in the field).


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  		Fig. 10.   Typical micrograph pair used for regional quantitation, showing AQP2 localization (A) and NCX localization in the same microscope field (B). A second micrograph, of NCX localization, with contrast similar to that shown for AQP2, was taken and used, as A, for analysis of the area labeled by the antibodies. B: a typical high-gain image used to determine the total area of tissue in the field (see METHODS for details). Bar, 100 µm.

The result of this quantitation is shown in Table 1. The fraction (%) of tubule area labeled by one or more antibodies is listed for each of the distal regions of interest. There is a significant difference in the fractional area only for the AQP2-labeled CNT, which is increased by ~60% by dDAVP treatment. This increase does not seem to be due to altered expression by the adjacent DCT2 and CCD segments because their areas are not significantly changed by dDAVP treatment. In this regard, the CCDs serve as an internal standard for fluorescence flair, which would tend to increase the apparent area of measured tubules. The increased cellular content of AQP2 that is expected in dDAVP-treated CCD does not appear to cause a significantly increased tubular area measurement (Table 1, column CCD) and thus does not explain the increased area of AQP2-labeled CNT with dDAVP treatment.

                              
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  		Table 1.   Fractional labeling of distal regions

It is possible that an increase in tubular diameter or cell height might be responsible for the increase in area of the AQP2-labeled CNT. To test this we measured these parameters in cross-sectioned tubules from coded samples from these animals. Outer tubule diameters (dDAVP: 46.4 ± 6.1 µm vs. vehicle controls: 44.5 ± 7.4 µm) and cell height (dDAVP: 9.7 ± 1.3 µm vs. vehicle controls 13.7 ± 2.6 µm) were not significantly different (P = 0.45 for tubule diameters; P = 0.06 for cell height). Note that these cell height measurements would, if anything, tend toward a reduction in measured area with dDAVP treatment. Thus these findings are consistent with expression of AQP2 over a longer length of the CNT in animals chronically exposed to dDAVP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The distal region of the nephron between the end of the thick ascending limb and the CCD has an extremely complex morphology. Not only do four distinct cell types occur here but also there can be striking adaptive changes (12) and substantial axial differences in the appearance of these cells (13). The availability of probes allowing investigators to localize Na transporters and other markers has led to the realization that there are also important axial differences in protein expression (1). This region is the site of important transporters for calcium (10, 27) and potassium (1, 18, 20) as well as expressing elements participating in the aldosterone response (3, 5, 7, 14, 28, 30). The present work adds new evidence that this region can also participate in vasopressin-regulated water conservation.

Physiological significance of aquaporins in the CNT. It has generally been believed that collecting ducts are the exclusive site where vasopressin increases water permeability to achieve water retention by the kidney. However, this view ignores early micropuncture work indicating an effect on water absorption proximal to the collecting duct (9, 32). The present work, when taken together with previous evidence that AQP2 and V2 receptors occur in arcade segments (16), argues strongly that the CNT represents the site of this water reabsorption. Water reabsorption at this site would be driven by the luminal hypotonicity produced by the thick ascending limb and distal tubule. Loffing et al. (19) recently showed that the rabbit, a species with a high water intake, does not express detectable AQP 2 in the CNT. However, species like the rat with a highly developed concentrating ability may utilize this segment to achieve water reabsorption in a region of the kidney where the blood supply is plentiful. Quantitatively, reabsorption at such a site is likely to be physiologically important in producing a highly concentrated urine because more water is reabsorbed in the cortex, raising tubular fluid to isotonicity than is reabsorbed in the medulla (17).

Localization of aquaporins in CNT cells. Previous work by our laboratory had developed evidence that AQP2 could occur within the region of connecting tubule arcades that are interposed between DCTs and CCDs (16). However, identification of the cell type containing the AQP2 in the CNT has been problematic due to the presence of principal cells in the CNT of the rat. The present work provides evidence that the collecting duct aquaporins (AQP2, AQP3, and AQP4) can all occur in true CNT cells. At the light microscope level, it is clear that the cells labeled by NCX and the AQPs show dramatically greater basolateral amplification than principal cells in the CCD. In addition, our electron microscopic characterization showed that, of the NCX-containing cell types, the AQP2-containing cells had the ultrastructural appearance of CNT cells, whereas NCX-labeled cells that lacked AQP2 had the appearance of DCT cells.

Distal regions. A complex labeling pattern was observed with all three collecting duct aquaporins we localized. In each case, regions were observed where the AQPs colabeled with NCX but regions were also observed that showed NCX labeling without AQP labeling. Previous observations have shown that NCX-labeled tubules in rat (25) but not rabbit (2) have two distinct regions in series. There are an NCC-expressing region called by these authors the DCT2 (25) and a region that does not express NCC that is CNT. We were able to show that regions positive for NCC and NCX (DCT2 regions) do not label with AQP2, whereas regions positive for both AQP2 and NCX fail to label with NCC (i.e., are CNT). However, a careful analysis of the labeling in Brattleboro control rats unexpectedly showed that there are also regions in these animals that labeled with NCX but failed to label with AQP2 or NCC. However, in vasopressin-treated animals we found that even cells adjacent to the DCT2 express AQP2. This indicates that in the absence of vasopressin the CNT has an upstream region that lacks detectable AQP2 but that chronic vasopressin exposure can induce expression in this region. To test this possibility, we carried out quantitative measurements of tubular labeling and colabeling to determine whether the area of NCX-labeled tubules labeled by AQP2 was altered by chronic vasopressin exposure. These data showed a substantial increase in this category, whereas markers for other segments were unaltered. Although the amount of increase is small in absolute terms, it represents more area than the DCT2 and an increase of >60%. It is conceivable that this might occur by a selective hypertrophy or hyperplasia of the AQP2-expressing region of the CNT. However, from the observations discussed above and the fact that the total area of NCX labeling was unaltered, we consider it most likely that there is induction of expression in the upstream portion of the CNT. Previously quantitative ELISA measurements on microdissected CNT arcades had shown that the region expresses on average ~64% of the AQP2 expressed by a comparable length of CCD but that amount nearly doubled in animals exposed to antidiuretic hormone for 48 h through thirsting (16). In light of the present findings, it is likely that the lower level of AQP2 is in part due to the inclusion of CNT regions that lack AQP2 in the arcades assayed. It is likely that the increase in expression measured reflects not only an increase in AQP2 levels per cell but expression of AQP2 over a longer length of CNT.

Table 1 provides an estimate of the relative length of different distal regions based on marker expression. It is interesting to note that the amount of CNT actually appears to be slightly greater than the amount of DCT1 and nearly as great as the entire DCT. This indicates that the CNT may have a much more important functional role in water and electrolyte homeostasis than widely believed.

In conclusion, we have used localization of antibodies to the aquaporins and distal transporters to show that the aquaporins are expressed in CNT cells in the rat. In addition, we have developed evidence that the initial portion of the CNT lacks detectable levels of AQP2 in the absence of vasopressin exposure but chronic treatment with vasopressin appears to induce its expression throughout the CNT. This finding raises the possibility that some nephron segments may not be fixed with respect to transporter expression but may be able to increase the length that expresses the transporter under adaptive stresses.


    ACKNOWLEDGEMENTS

The authors are very grateful to Robert Reilly and Mark Knepper for providing antibodies and to David Ellison for providing the NCC fusion protein we used to raise antibody to that transporter in guinea pig. We also are very grateful to these individuals for extremely valuable discussions and encouragement.


    FOOTNOTES

The work reported here was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32839. The Confocal Microscope Facility used for the immunolocalizations was funded by National Science Foundation Grant BIR9318061.

Address for reprint requests and other correspondence: J. B. Wade, Dept. of Physiology, 655 W. Baltimore St., Univ. of Maryland, Baltimore, MD 21201.

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 4 February 2000; accepted in final form 23 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 279(5):F874-F883
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