Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051
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ABSTRACT |
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Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized.
aquaporin-1 water channel; UT-A2 urea transporter; ClC-K1 chloride channel; reverse transcription-polymerase chain reaction in single tubules; immunocytochemistry; differential interference contrast optics; countercurrent multiplier
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INTRODUCTION |
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THE CELLULAR STRUCTURE and function and the architectural arrangement of the thin limbs of Henle's loop are apparently critically important in the generation of the osmotic gradient in the mammalian inner medulla, although the mechanistic details of this process are still far from clear (8, 17). Within the rat inner medulla, the descending thin limb (DTL) contains two types of epithelia and the ascending thin limb (ATL) contains one type of epithelium based on ultrastructural studies (7, 6, 18). The DTL enters the inner medulla as a type 2 epithelium and gradually changes to a type 3 epithelium at variable depths between the base and the tip of the papilla, whereas the ATL consists entirely of type 4 epithelium (6, 7, 18). In addition, the epithelia of DTL and ATL freshly teased from rat renal papillary tissue have distinctively different appearances when viewed with Nomarski differential interference contrast (DIC) optics (see RESULTS) (1). From a functional point of view, the inner medullary DTL has a high water permeability and relatively high urea permeability, whereas the inner medullary ATL has a very low water permeability but a substantial sodium chloride permeability (17). Recent molecular studies have contributed to an understanding of these functional differences. The constitutively active water channel aquaporin-1 (AQP1) is found in both the luminal and basolateral membranes of the DTL and accounts for the observed water permeability (9, 12, 13), whereas no water channels are found in the ATL (17). The urea transporter UT-A2, whose activity is enhanced by water deprivation, is also found in the DTL but not the ATL (19). In contrast, a chloride channel ClC-K1 is expressed in the basolateral membrane and possibly in the luminal membrane of the ATL, but not in the DTL (22, 23, 24). The expression of ClC-K1 is also increased by dehydration (22, 24).
However, in contrast to the prevailing view that each thin limb consists of a continuous DTL- or ATL-type of epithelium, in a previous functional study we found thin limbs that apparently consisted of segments of DTL-type cells interspersed between segments of ATL-type cells as viewed with DIC optics (1). In the present study, we further characterized these segments using visual observations, immunocytochemistry, and RT-PCR. The results indicate that these mixed-type thin limb segments are numerous and are located distinctly above the tip of Henle's loop. Moreover, those segments containing DTL-type cells, as viewed with DIC microscopy, express AQP1 and UT-A2. Neighboring segments of these same thin limbs that contain ATL-type cells, as viewed with DIC microscopy, do not express AQP1 or UT-A2. Furthermore, ATL-type regions express ATL-specific ClC-K1, whereas DTL-type regions do not.
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METHODS |
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Animals. Young male Munich-Wistar rats (average wt, 90 g), male Sprague-Dawley rats (average wt, 90 g), male ICR mice (average wt, 30 g), and New Zealand White rabbits (average wt, 2 kg) were purchased from Harlan Sprague Dawley (Indianapolis, IN). The animals were anesthetized with pentobarbital sodium (0.2 ml/100 g body wt, rats and mice; ~3 ml/kg body wt, rabbits).
Isolation of thin limbs of Henle's loops. The kidneys were removed and placed in an ice-cold buffer consisting of 280 mM sucrose and 10 mM HEPES. The buffer had a pH adjusted to 7.4 with Tris base and was gassed with 100% O2. Slices of the medulla were transferred to a dissecting dish on the base of a stereomicroscope at room temperature and were viewed with light reflected from a mirror below the dish. Individual thin limb segments were teased out beginning near the border of the inner medulla and the inner stripe of the outer medulla and continuing toward the tip of the papilla. All dissections were performed in the absence of enzymatic agents. Identification of the segments of thin limbs was made initially entirely on structural grounds (location and cell appearance with light microscopy) (see RESULTS). The DTL can generally be differentiated from the ATL by the appearance of the luminal border, even under a stereomicroscope in the dissecting dish. However, the differences in the appearance of the cells of each segment, which are quite striking, were used to confirm the identification of individual DTL and ATL segments while they were being viewed at ×400 magnification with an Olympus IX70 inverted compound microscope equipped with DIC optics (see RESULTS).
To estimate the number of mixed tubule segments in the inner medulla, we divided the inner medulla from 6 kidneys from 4 Munich-Wistar rats into ~15 sections of equal size. From random sections, all the exposed thin limb segments longer than 500 µm were dissected free. These were then divided into three groups: pure descending segments, pure ascending segments, and mixed-type segments. The number in each group, as a percentage of the total number of tubules isolated from each rat, was then determined.
Immunocytochemical analyses of AQP1 and CLC-K1 protein in isolated
thin limbs. A layer of Cell-Tak adhesive (Becton-Dickinson, Bedford, MA) was applied to a glass microscope slide, which was then
immersed in HEPES-sucrose in a petri dish. Immediately following isolation from the medulla, thin limbs were transferred by Pasteur pipette into the solution and were positioned onto the Cell-Tak with
glass fibers. Tubules were then fixed in 4% paraformaldehyde for 10 min, permeabilized with 100% methanol for 2 min at 20°C, and washed once for 10 min with 5% BSA in PBS.
Immunocytochemistry was conducted using affinity-purified polyclonal antibodies raised against the COOH-terminal region of either AQP1 (21) (antibody provided by Dr. John Regan, University of Arizona) or ClC-K1 (antibody provided by Dr. James Wade, University of Maryland). Primary antibody or nonimmune rabbit or chicken serum was applied overnight at 4°C. Rhodamine-conjugated goat anti-rabbit or FITC-conjugated rabbit anti-chicken immunoglobulins (1:500) were applied for 20 min at room temperature. The tubules were then washed in PBS (3 periods of 5 min each) and mounted with Daco mounting medium (Carpenteria, CA). Fluorescence of tubules labeled with rhodamine- or FITC-conjugated antibodies was viewed with a Leica-TCS confocal microscope with a ×10 or ×20 objective. Multiple frames were made of tubules larger than the field of view, and these were combined into composite images using Adobe Photoshop software.
DIC images of all tubules for comparison with the fluorescent images were obtained during the paraformaldehyde fixation period using an Olympus IX70 inverted compound microscope equipped with a ×20 objective (LCPlanFl, 0.40 NA). Images were photographed on Fuji Super HGII film (ASA 200). Negatives were digitized and multiple frames were combined into a composite image using Adobe Photoshop software.
RT-PCR detection of AQP1 and UT-A2 in isolated thin limbs.
RT-PCR was carried out as described by Moriyama et al. (11), with some
modifications. Pure DTL, pure ATL, and mixed-type thin limb segments
were dissected from the inner medulla in HEPES-sucrose solution
containing 10 mM vanadyl ribonucleoside complex (VRC, Life
Technologies). Tubule segments were then washed in a solution of
HEPES-sucrose without VRC. Pure DTLs and ATLs and descending and
ascending segments of mixed-type thin limbs were identified with an
Olympus IX70 compound microscope using DIC optics as indicated above
(see RESULTS). In mixed-type thin limbs, descending regions were isolated from ascending regions with a dissecting needle, and
~50 µm of the intervening regions were discarded so that each isolated region was pure. Tubule lengths were then determined with an
ocular micrometer. Each tubule segment, in a volume of 2 µl
HEPES-sucrose solution, was transferred into a PCR tube containing 4 µl of a solution consisting of 6.4 U of RNase inhibitor
(Boehringer-Mannheim), 5 mM dithiothreitol, and 2% Triton X-100 in
diethyl pyrocarbonate-treated water. After incubation at
room temperature for 5 min, the tube was immersed in liquid nitrogen
for 5 s and stored at 20°C prior to conducting the RT-PCR reaction.
Reverse transcription components were added to a final volume of 20 µl. These included 40 U of RNase inhibitor, 1 mM dNTP mix, 1.5 µg oligo(dT)12-18 primer and 9 µg random primer (Life Technologies), and 200 U of Superscript II RNA reverse transcriptase (Life Technologies) in proprietary buffer. Tubes were mixed briefly by tapping. They were then incubated first at room temperature for 10 min and then at 42°C for 50 min. The reaction was terminated by heating at 70°C for 15 min. PCR primers were synthesized by Integrated DNA Technologies (Coralville, IA). The AQP1 sense primer was 5'-ATG-GCC-AGC-GAG-TTC-AAG-AAG-AA-3', and the antisense primer was 5'-GGC-CCC-ACC-CAG-AAA-ATC-CAG-TG-3' (10). These primers produced a predicted amplification product of 647 bp. The UT-A2 sense primer was 5'-ATG-GAA-GCT-AGA-CTA-CAT-CTT-3', and the antisense primer was 5'-TCC-GTG-TGA-CTG-TTC-TCC-3' (20). This primer pair is specific for UT-A2 and does not amplify other reported rat UT isoforms. The UT-A2 primers produced a predicted amplification product of 353 bp. The cyclophilin sense primer was 5'-GGG-GAG-AAA-GGA-TTT-GGC-TA-3', and the antisense primer was 5'-ACA-TGC-TTG-CCA-TCC-AGC-C-3' (5). These primers produced a predicted amplification product of 257 bp. The RT reaction was added to the PCR reaction components to a final volume of 100 µl. This solution included PCR buffer (Life Technologies), 1.5 mM of MgCl2, 200 pmol of each primer, 200 µM dNTP mix, and 5 U of Taq polymerase (Life Technologies). PCR was conducted in a Hybaid thermocycler by heating to 94°C for 3 min, followed by 30 cycles at 94°C for 1 min, 51°C for 2 min, 72°C for 1.5 min. The final extension was carried out at 72°C for 10 min. PCR products were ethanol-precipitated overnight, size-fractionated on 1.5% agarose gel, and stained with ethidium bromide. PCR products were sequenced by the DNA Sequencing Service of the Arizona Research Laboratories, Division of Biotechnology, University of Arizona.
Chemicals and reagents. Chemicals and reagents, other than those for which the source is mentioned specifically above, were obtained from standard sources. They were all of the highest purity available.
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RESULTS |
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Structural identification of DTL-type and ATL-type segments of
single thin limbs of Henle's loop in the inner medulla. Rat inner
medullary DTL were differentiated from ATL on the basis of structural
characteristics when viewed with a compound microscope equipped with
Nomarski DIC optics (1). The cells of the DTL with their nuclei
appearing to bulge into the lumen create a distinct luminal outline
(Fig. 1). The cells of the ATL appear
flatter than those of the DTL and exhibit dominating, round nuclei
(Fig. 1). A distinct luminal outline is absent in the ATL (Fig. 1). These structural characteristics closely resemble those previously reported for the chinchilla thin limbs of the inner medulla (2). When
first beginning to tease out thin limbs (1), we made certain that the
cells we were defining as "DTL-type" based on the work of Chou
and Knepper (2) were seen at the beginning of the DTL (following the
straight portion of the proximal tubule) and that the cells we were
defining as "ATL-type" were seen at the end of the ATL where it
joined the thick segment of the ascending limb. In this way, we thought
that we could be absolutely certain that segments with these cell types
truly represented either DTL or ATL. The reason for this is that we
were interested in the papillary thin limbs (those from juxtamedullary
nephrons), and it was rarely possible to tease out a whole DTL or ATL.
However, as we continued teasing out tubules using DIC optics, we
frequently observed individual, straight thin limbs that consisted of
alternating segments of DTL-type cells and ATL-type cells (mixed-type
thin limbs) (Fig. 2). The DTL-type and
ATL-type segments of each individual mixed-type thin limb were variable
in length, and multiple segments of each DTL-type and ATL-type portion
were commonly present in a single, straight thin limb (Fig. 2).
Mixed-type thin limbs were evident at positions ranging from the outer
portion of the inner medulla to near the tip of the papilla where the
longest loops extend. However, they were always located distinctly
above the bend of the loops where another specialized epithelium has
been reported (4). These mixed-type thin limbs were present in the inner medulla not only of male Munich-Wistar rats but also of male
Sprague-Dawley rats, male ICR mice, and male New Zealand White rabbits
(Fig. 2). We also examined thin limbs from the inner medulla of female
Munich-Wistar rats and New Zealand White rabbits and found no evidence
of sexual dimorphism.
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The proportion of pure DTL, pure ATL, or mixed-type thin limbs in the
inner medulla of Munich-Wistar rats was estimated by counting in vitro
the exposed thin limbs from random sections as described in
METHODS. Thin limbs that exhibited a continuous or
uninterrupted DTL or ATL appearance of greater than 500 µm were
judged to be pure DTL or pure ATL, since the DTL-type and ATL-type
segments of mixed-type thin limbs are typically less than 500 µm in
length (although many of them are 200-300 µm in length) (e.g.,
Fig. 2). However, since the entire thin limb was not completely
isolated, we cannot be certain that the segments that we counted as
"pure" were absolutely free of mixed portions. On the basis of
these criteria, we found that ~55% of 120 thin limbs were mixed-type
and the remaining 45% were pure DTL or pure ATL in nearly equal
proportion (Fig. 3).
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Expression of mRNA for AQP1 and UT-A2 in DTL-type and ATL-type
segments of single thin limbs of Henle's loop in the inner medulla. We attempted to determine whether the DTL-type and
ATL-type regions of mixed-type thin limbs each expressed molecular
characteristics distinctly expressed by either pure DTL or pure ATL. To
do this, we used RT-PCR to assess the presence of mRNA that codes for
the proteins AQP1 and UT-A2 in segments from single isolated thin limbs. AQP1 (14, 16) and UT-A2 (19) are expressed in the DTL but not in
the ATL. We first established that the primer pairs for AQP1 and UT-A2
amplified the predicted products in isolated pure DTLs and ATLs from
Munich-Wistar rat kidneys. We isolated thin limbs that were greater
than 1 mm in length and consisted entirely of either DTL or ATL type
cells. We then removed short fragments from each of these pure-type
thin limbs and carried out RT-PCR on the individual thin limb
fragments. Approximate normalization of starting tissue was established
by amplifying mRNA for the gene encoding the cyclosporin binding
protein, cyclophilin. The gene for cyclophilin is expressed in both DTL
and ATL (Fig. 4). The RT-PCR analyses show
that AQP1 and UT-A2 are present in pure DTL but not in pure ATL (Fig.
4). These results were comparable in four separate experiments.
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We then amplified mRNA for AQP1 and UT-A2 in DTL-type and in ATL-type segments isolated from mixed-type thin limbs of Munich-Wistar rats. A DTL-type segment was isolated from a mixed-type thin limb and placed into a PCR reaction tube. An immediately adjacent ATL-type segment was then isolated and placed into a second reaction tube. Expression of AQP1 and UT-A2 from each of these mixed thin limb segments followed a pattern identical to that seen for pure DTL and pure ATL. The RT-PCR analyses show that AQP1 and UT-A2 are present in DTL-type segments and not in ATL-type segments (Fig. 4). These results were comparable in four separate experiments.
For all RT-PCR reactions, no products were detected when the reverse transcriptase enzyme was omitted from the reverse transcription reaction, indicating that all PCR products were derived from reverse-transcribed mRNA (Fig. 4). All primer pairs produced amplified products that consisted of the appropriate sizes. Selected PCR products from each primer pair were sequenced and determined to have an identical sequence to that predicted for the amplified regions.
Expression of AQP1 and ClC-K1 protein in DTL-type and ATL-type
segments of single thin limbs of Henle's loop in the inner medulla. Expression of AQP1 and CLC-K1 protein in pure DTL and pure
ATL was assessed in intact, isolated rat thin limbs with immunocytochemistry. AQP1 was expressed in pure DTLs, whereas CLC-K1
was expressed in pure ATLs (Fig. 5). In
some segments, staining was not entirely homogeneous along the length
of the tubule. This may be due in part to the fact that portions of the tubule may be raised or lowered relative to other portions, since the
layer of Cell-Tak may not be entirely level. In addition, there
infrequently appear to be from one-to-several DTL-type cells within a
predominantly ATL-type region, or from one-to-several ATL-type cells
within a predominantly DTL-type region. Fluorescence of tubules
prepared in the absence of primary antibody was equivalent to
background levels.
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Expression of AQP1 and ClC-K1 protein in distinct regions of mixed-type
isolated thin limbs was also assessed with immunocytochemistry. The
precise location of DTL-type and ATL-type regions along the length of
mixed thin limbs was determined from structural studies with DIC
microphotography. These structurally identified regions were then
compared with confocal immunofluorescence images to determine whether
AQP1 and CLC-K1 were differentially expressed within these regions.
AQP1 was expressed in DTL-type, but not ATL-type, regions of mixed-type
thin limbs of the inner medulla (Fig. 6).
ClC-K1 was expressed in ATL-type, but not DTL-type, regions of mixed
thin limbs of the inner medulla (Fig. 7).
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DISCUSSION |
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In the present study, we showed that a significant portion of the thin limbs of Henle's loop in the inner medulla of rat, mouse, and rabbit kidneys consist of alternating and repeating segments of DTL-type cells and ATL-type cells. These are found in straight portions of the thin limbs distinctly above the bend of the loop.
We established the identity of specific thin limb segments by means of several criteria. Initially, we identified DTL-type and ATL-type segments on the basis of the structural characteristics observed in vitro with DIC microscopy (1, 2). Thin limbs that were pure, i.e., consisted only of DTL-type or ATL-type cells, appeared to be structurally identical to the DTL-type or ATL-type segments of mixed-type thin limbs on the basis of low-magnification (×400) DIC microscopy. Transmission electron microscopic studies are required to determine whether the ultrastructure of the cells in the segments from mixed-type thin limbs is identical to that of the cells in pure DTL and ATL.
We next confirmed that these unique segments selectively expressed the appropriate molecular markers that are specific for DTL and ATL. Using RT-PCR, we established that the DTL-type regions of mixed-type thin limbs expressed genes for AQP1 and UT-A2 that are present in pure DTL. Using immunocytochemistry, we showed that the protein encoded by AQP1 exhibited this same restricted distribution and was located in the DTL-type segments of mixed-type thin limbs and in pure DTL. Likewise, we found that ATL-type regions of mixed-type thin limbs and pure ATL did not express AQP1 or UT-A2 but did express ClC-K1.
It is important to emphasize that the mixed-type thin limbs make up a significant proportion of the total population of thin limbs in the inner medulla. A random evaluation of the number of the different types of thin limbs present in the inner medulla indicated that at least 50% were mixed-type, consisting of both ATL-type and DTL-type segments. We were not able to determine what proportion of these mixed-type thin limbs lay on the descending or ascending side of the loop of Henle. Although this count cannot be considered a rigorous analysis of frequency, the prevalence of these mixed-type thin limbs makes it very likely that they play a significant role in papillary function.
The presence of a large number of mixed-type thin limbs in Munich-Wistar rat kidneys, along with the presence of mixed-type thin limbs in the kidneys of other species, suggests that they may serve a common and important function in mammals. At present, we can only speculate on their possible significance. In chinchillas and rats (and, we assume, other mammalian species), the water permeability of the DTL is markedly higher than the water permeability of the ATL (2, 4, 15). High water permeability of the mouse DTL is due to the existence of AQP1 (3) in the basolateral and apical membranes. If we assume that this is true for the rat also, then the DTL-type segments of the mixed-type thin limbs should have significantly higher water permeability than the ATL-type segments of the mixed thin limbs. The overall effect would make mixed-type thin limbs less permeable to water than pure DTL but more permeable to water than pure ATL. The reverse pattern should hold for urea and chloride permeabilities. Thus mixed-type thin limbs might have a significant influence on the generation of the osmotic gradient within the inner medulla that is required for the concentrating mechanism to function. However, actual integrative function will be critically dependent on the number, length, position within the papilla, and overall architectural arrangement of the segments of each type.
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ACKNOWLEDGEMENTS |
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This study was supported in part by NIH Research Grant DK-16294; Training Grants HL-07249, NS-07309, and GM-08400; Grant ES-06694 for the Southwest Environmental Health Sciences Center; and NSF Grant STI 96-20092.
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FOOTNOTES |
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We thank Dr. John Regan of the University of Arizona for providing us with AQP1 antibodies and Dr. James B. Wade of the University of Maryland for providing us with ClC-K1 antibodies.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. H. Dantzler, Dept. of Physiology, College of Medicine, Univ. of Arizona, Tucson, AZ 85724-5051 (E-mail: dantzler{at}u.arizona.edu).
Received 29 June 1999; accepted in final form 6 October 1999.
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