1 Renal Research, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut 06516; 2 Division of Nephrology, Oregon Health Sciences University, Portland, Oregon 97201-2940; and 3 Physiologisches Institute, Freiburg D-79014, Germany
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ABSTRACT |
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We sought to assess whether the
distal convoluted tubule (DCT) segment of the rabbit nephron expresses
a functional epithelial sodium channel. First, the transepithelial
voltage (Vte, lumen vs. bath) was measured in
isolated perfused DCT segments (assessed separately in the upstream
half and the downstream half of the DCT). Vte
was zero and not affected by amiloride or barium in the upstream DCT.
Vte was sometimes negative in the downstream DCT
and depolarized by amiloride and hyperpolarized by barium, suggesting
inclusion of connecting tubule (CNT) cells. To determine expression of
epithelial sodium channel (ENaC) mRNA subunits by the upstream DCT,
rabbit -,
-, and
-ENaC cDNA fragments were cloned and primers
were selected for single-nephron RT-PCR analysis. Although
-ENaC was
expressed by the DCT,
- and
-ENaC were not detected in the DCT.
In contrast, the CNT, CCD, and outer medullary collecting duct (OMCD)
expressed all three subunits. Nedd4 was also not detected in the DCT
but was expressed by the CNT, CCD, and OMCD. When upstream DCT
fragments were grown to confluent monolayers in primary culture, the
epithelia exhibited negative voltages and high transepithelial
resistances and expressed mRNA for all three ENaC subunits as well as
for Nedd4. The absence of a negative voltage and failure to detect
transcript for
- and
-ENaC and Nedd4 in the native rabbit DCT
suggest that the sodium channel is not a significant pathway for sodium
absorption by this segment. The phenotype conversion observed when DCT
cells are grown in culture does not rule out the possibility that there may be conditions in which the DCT in the intact kidney expresses sodium channel activity. The results are consistent with the notion that DCT sodium transport is predominantly, if not exclusively, electroneutral.
amiloride; epithelial sodium channel ; Nedd4; cloning; cell culture
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INTRODUCTION |
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THE PRESENCE OF AMILORIDE-sensitive sodium channels in the mammalian renal distal tubule1 and the cortical collecting duct (CCD) is well established. Although it is clear that the late2 portion of the distal tubule exhibits both amiloride-sensitive net sodium absorption and a lumen-negative transepithelial voltage (Vte) (4, 11, 19), the expression of sodium channel activity in the early3 portion of the distal tubule is controversial. Data from at least two groups (5, 8, 29) that assessed the function of the early distal tubule [predominantly distal convoluted tubule (DCT) epithelium] in the intact rat kidney suggest that electrogenic sodium transport is not the primary mechanism for sodium absorption. These studies demonstrated the primary site of action of the thiazide class of diuretics is in the early distal segment, whereas the late distal segment is the primary site of action of the potassium-sparing diuretic amiloride. Unfortunately, the high degree of cellular heterogeneity and the short length of the distal tubule do not permit ruling out an effect of amiloride in DCT cells nor ruling out an effect of thiazides in initial collecting tubule (ICT) cells.
Studies using a cell line derived from immunodissected mouse DCT+thick ascending limb (TAL) cells (21) and having features of DCT cells have demonstrated amiloride-sensitive transport in these MDCT cells in culture (10). This flux cannot be localized to the apical or the basolateral membrane, however, because MDCT cells do not form tight junctions and do not develop a Vte or resistance (Rte). These studies do not establish whether an amiloride-sensitive sodium channel is a feature of the DCT of the intact kidney.
The cloning of the epithelial sodium channel (ENaC) in the rat
(2) has enabled investigators to probe for mRNA expression in kidney and to perform immunolocalization studies. Duc and co-workers (7) concluded from studies using both in situ
hybridization methods and immunocytochemistry that all three subunits
of ENaC, -,
-, and
-, are expressed along the rat distal
nephron extending from the DCT to the outer medullary collecting duct
(OMCD). Ciampollillo and co-workers (3) used in situ PCR
methods to localize
-ENaC to the region of the nephron extending
from the medullary TAL (mTAL; including the DCT) to the inner medullary
collecting duct (IMCD) (3). Positive identification of the
DCT in histological sections undergoing these procedures is often not
possible, however, and additional markers to co-localize DCT cells were
not used in these studies.
In vitro studies of the isolated and perfused DCT of the rabbit kidney report luminal negative voltages (15, 24, 25, 31) and amiloride effects (24, 25). We found that the luminal negative Vte was low; however, it varied over a wide range. It is possible that this variability is caused by inadvertent inclusion of CNT cells in the perfused structure.
ENaC regulation by a cell-expressed, developmentally downregulated neuronal precursor (Nedd4) (26, 27) has been identified as an important mechanism by which the level of sodium channel expression in the apical membrane is controlled. In an immunohistochemical study (28), Nedd4 expression was detected primarily in the distal portions of the nephron but was notably absent from the DCT segment. Thus it is possible that both ENaC and Nedd4 are not expressed in the DCT.
The purpose of the present study is to examine whether the DCT segment
of the rabbit nephron expresses the functional sodium channel. To do
this we assessed amiloride-sensitive Vte in
isolated perfused DCT subsegments and determined -,
-, and
-ENaC mRNA expression by using the single-dissected-nephron RT-PCR
technique. Second, to determine whether ENaC expression correlates with
Nedd4 expression, we determined the distribution of Nedd4 mRNA along the nephron. Third, we determined expression of ENaC and Nedd4 mRNA by
DCT cells grown in primary culture and compared this with native tissue.
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METHODS |
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Dissection of nephron segments. New Zealand White rabbits (1-2.5 kg) were anesthetized with ketamine (50 mg/kg im) and pentobarbital sodium (50 mg/kg iv), kidneys removed via a flank incision and chilled in ice-cold dissecting solution (pH 7.4; in mM: 135 NaCl, 1 Na2HPO4, 1.5 MgSO4, 2 CaCl2, 5 KCl, 5 glucose, 5 HEPES). The rabbit was euthanized by overdose with pentobarbital sodium. RNAse-free media, beakers, and instruments were used when tubules were to be used for RT-PCR experiments. Transverse slices (1 mm) of the kidney were transferred to a dissection dish that was placed on the stage of a dissecting microscope (transmitted light, model SZH, Olympus) fitted with a water-chilled chamber. Sections were transilluminated, and tubules were dissected by hand at ×60 magnification by using sharpened forceps.4 Guidelines for dissection in the heterogeneous distal portion of the nephron were taken from Morel (16).
The DCT was identified by locating and dissecting the structure consisting of a glomerulus with associated upstream TAL and downstream DCT and CNT segments. The procedure for isolating and harvesting the DCT portion is described as follows. The nephron is cut within the DCT, downstream of the transition from TAL to DCT. This transition usually occurs close to the glomerulus, and the abrupt change in tubule diameter is not visible (when the transition is visible, the DCT is cut downstream to this site). Within the DCT, the nephron generally makes a hairpin turn and returns to contact its own glomerulus. Before the nephron makes contact with the afferent arteriole (6), it undergoes a visible transition from DCT to CNT (DCT length is ~0.5 mm from TAL transition to CNT transition). To harvest the DCT for RT-PCR and primary culture experiments, the tubule is cut at the hairpin turn, yielding ~300 µm of DCT tissue/dissected nephron. This fragment of DCT (see Perfusion of isolated DCT segments in vitro) does not contain TAL cells or CNT cells. Proximal convoluted tubules (PCT) cortical thick ascending limbs (cTAL) and cortical collecting ducts (CCD) were dissected from the rays in the cortex. Proximal straight tubules (P3), mTAL, and OMCD were dissected from the outer medulla. Glomeruli (G) and connecting tubules (CNT) were dissected from the labyrinth and arcades. IMCD were dissected from the inner medulla. For these segments it was usually possible to obtain single nephron fragments at least 1 mm in length. To minimize contamination of specific nephron samples with other cells, small bundles of tissue were separated first from kidney slices and then transferred to a new dish for final dissection in fresh medium.Perfusion of isolated DCT segments in vitro. For in vitro perfusion of the DCT, the entire hairpin structure (see Dissection of nephron segments) is utilized, and the segment harvested extends from the TAL-DCT transition to the DCT-CNT transition (visual criteria used to exclude TAL and CNT cells). Subsequently, cutting once at the top of the hairpin turn yields two fragments, the "upstream" DCT and the "downstream" DCT.
Individual tubules are then transferred to a bath chamber (Hampel) mounted on the stage of an inverted microscope (Zeiss IM35). The microscope is fixed to a vibration isolation table (Micro-G). The bath contains control perfusion solution (continuously flowing), with its temperature maintained at 37°C. Each end of the tubule is aspirated into a glass tubule-holding pipette fitted into a tubule perfusion system (Luigs and Neumann) according to the method of Burg (1) and modified by Greger and Hampel (12). A third pipette concentric to one of the holding pipettes is now advanced into the lumen of the tubule (perfusion end) and permits perfusion of the lumen. Fluid is collected continuously at the other end (collection end). An outermost pipette with a dielectric liquid (Sylgard) in the tip is advanced over the tubule-holding pipette and the first part of the tubule to make an electrical seal and prevent short-circuiting the Vte. Vte is measured at the perfusion end via the perfusion pipette that is connected to an electrometer (WPI, KS-700) via a salt bridge. The perfusion pipette is fitted with a fluid exchange pipette, permitting multiple reloading with different solutions during one experiment and exposure of the apical membrane to more than two solutions. Fluid changes in the lumen are accomplished by switching a hydrostatic pressure head from one channel to anther via a pneumatically activated three-way valve (Altex). Solutions bathing the luminal and basolateral aspects of the tubules were identical under control conditions (in mM: 150 Na, 4 K, 141 Cl, 1.5 Ca, 1 Mg, 1.5 lactate, 5 acetate, 5 glucose, 5 PIPES). Luminal perfusates containing amiloride (10Cloning of rabbit ENaC cDNA
fragments (-,
-, and
-).
Total RNA was isolated from 1-g samples of kidney by using TRIzol
reagent (GIBCO-BRL). Poly-A RNA was isolated subsequently from total
RNA by using oligo(dT)-cellulose (Boehringer Mannheim). One microgram
polyA-RNA was primed with oligo(dT), and first-strand cDNA was
synthesized by using Superscript II reverse transcriptase (GIBCO-BRL)
in a final volume of 20 µl.
Cloning of rabbit Nedd4 cDNA fragment. RNA preparation from rabbit kidney and cDNA synthesis were carried out as described above in the cloning of ENaC cDNA fragments. Exact-match primers were selected from regions of nucleotide identity between mouse (D85414) and rat (U50842) sequences. The sense primer was GATGAAAATCGTTTGACAAGAGATGATTTC; the antisense primer was GAAGTTTCATTTCAAATTTGTTTGG. PCR was performed as above except that XL DNA polymerase (PerkinElmer) was used, and extension was at 68°C for 2 min.
Northern blot of kidney cortex, outer medulla, and inner medulla. Cortex, outer medulla, and inner medulla were dissected from 1- to 2-mm transverse slices of rabbit kidney, and polyA-RNA was extracted as described above. Ten micrograms polyA-RNA were loaded per lane and transferred to a nylon membrane. Blots were prehybridized for at least 4 h in Church Gilbert [0.5 M sodium phosphate (pH 7.2), 1 mM EDTA, 7% SDS, 1% bovine serum albumin, 100 µg/ml salmon sperm DNA] at 68°C and then hybridized (106 cpm/ml, where cpm is counts/min) overnight by using random prime-labeled probes. Blots were rinsed once and then washed for 30 min in 2× standard sodium citrate (SSC; composition: 0.15M NaCl, 0.15 sodium citrate, pH 7) and 0.5% SDS at room temperature. Final wash at 68°C was done for 30 min in 0.5× SSC and 0.1 SDS, with periodic monitoring by a Geiger counter for low background counts. Blots were exposed to film from several hours up to several days.
Three blots were prepared and probed separately withSingle-nephron RT-PCR analysis.
For single-nephron RT/PCR experiments, a total of 4 mm of tubule
(2-12 nephron fragments; see Dissection of nephron
segments) was transferred directly into a 0.5-ml microfuge tube
containing 10 µl of lysis solution (2% Triton X-100, 5 mM
dithiothreitol, 2.2 U/µl RNAsin, diethylpyrocarbonate-treated water).
Tubule fragments were transferred by adsorbing to glass beads (0.5 mm
diameter) held by forceps. All samples were frozen at 70°C until
the time of the experiment.
Primary culture of DCT explants. DCT cells were grown on a transparent collagen-coated, permeable membrane mounted on a plastic cylinder (Transwell-col, Costar, 6-mm-diameter cell culture insert). This cell culture insert was placed in a 24-well tissue culture plate with 1 ml of culture medium (RPMI-1640, 5% fetal bovine serum, 2 g/l NaHCO3, 10 mM HEPES buffer, 100 U penicillin g/ml, 0.1 mg/ml streptomycin, 5 µg/ml transferrin, 5 µg/ml insulin, 50 nM dexamethasone, 5 pM triiodothyronine) in the bath, and 150 µl of medium inside the insert. The plates were preequilibrated at 37°C with 5% CO2.
Dissected DCT segments were temporarily transferred to a fresh dissecting dish on ice containing culture medium. About five to eight DCT segments were deposited onto each cell culture insert by using a micropipettor. Plates were left undisturbed for 2-3 days, then observed daily for tubule attachment and cellular outgrowth. Cells were fed about three times per week. On reaching visual confluence (10-15 days), Vte and resistance (Rte) were determined (EVOM, WPI). An insert without cells was used as a blank to correct for baseline Vte and Rte. Values for Vte (mV) are apical vs. bath; Rte is in ![]() |
RESULTS |
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In vitro perfusion of DCT subsegments.
The portion of DCT extending from the junction with the cTAL to the
junction with the CNT was dissected, and all cTAL and CNT were cut away
by using visual criteria (see METHODS). The DCT was cut
into upstream and downstream subsegments. Figure
1 shows that the
Vte of all upstream DCT segments was zero when they were perfused with control solution. Addition of either amiloride or barium to the luminal aspect of the upstream DCT had no effect on
the Vte. When the downstream DCT was studied,
some tubules exhibited a lumen negative Vte. In
each case, a lumen negative Vte was abolished
with luminal amiloride or increased with application of luminal barium.
The effects of both agents were rapid and readily reversible.
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ENaC cloning. Cloning of rabbit ENaC subunits permitted selection of primers for the single-nephron RT-PCR experiments described below. All primers used in the present study are listed in METHODS.
Table 1 shows the percent similarity of the cloned rabbit ENaC subunit amino acid sequence to rat and human forms. The alignment was performed by using MegAlign (DNA Star, Clustal method, gap penalty of 10 for multiple alignments, and PAM250 residue weight table).
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Nedd4 cloning. Cloning of rabbit Nedd4 permitted selection of primers for the single-nephron RT-PCR experiments described below. All primers used in the present study are listed in METHODS. The percent similarities of rabbit Nedd4 cDNA fragment to human, rat, and mouse sequence are 88.8, 78.4, and 80.6, respectively. The accession number for the rabbit Nedd4 sequence is AF229024.
Northen blot of ENaC in cortex, outer medulla, and
inner medulla.
Figure 2 shows three blots of rabbit
kidney cortex, outer medulla, and inner medulla polyA-RNA. The
left side of the top panel shows one blot probed
with -ENaC. A single prominent band of ~3.7 kb was detected in
cortex, outer medulla, and inner medulla. The amount of message was
similar in each of the three regions. The same blot was washed and
subsequently probed with GAPDH. A band of ~1.6 kb was detected for
GAPDH and was of similar intensity in cortex, outer medulla, and inner
medulla, suggesting equal loading of the lanes. Faint higher bands in
GAPDH panels represent residual ENaC signal that did not wash off. The
middle panel shows that
-ENaC is expressed in cortex and
outer medulla but not in inner medulla. The single band detected runs
at ~3.1 kb and was more intense in cortex than in outer medulla.
Probing with GAPDH confirmed even loading of the lanes. The
bottom panel shows that
-ENaC is expressed in cortex and
outer medulla but not inner medulla. Two bands were detected at ~4.0
and 2.8 kb. The larger transcript was more prominent than the lower
transcript, and the cortex expresses both at a higher level than outer
medulla. Probing with GAPDH confirmed even loading of the lanes with
polyA-RNA.
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Single-nephron RT-PCR.
Figure 3 shows an ethidium
bromide-stained gel from a single experiment employing primers for the
OCRL gene. Raw data for the IMCD are not shown on this figure because
of physical constraints of the gel box. The IMCD was tested in separate
experiments and was also positive. A band of the expected size is
visible for each of the nephron segments tested (including the IMCD;
data not shown). This constitutes the positive control for cDNA
synthesis (see METHODS) in each sample tube.
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Primary DCT culture.
"Upstream" DCT explants were grown until visually confluent, and
subsequently the Vte and
Rte were monitored daily until stable values
were recorded for 2-3 consecutive days (maximum total culture time
of explants is 21 days). Lumen negative Vte and
Rte of confluent DCT monolayers were 13.7 ± 2.28 mV (n = 33) and 932 ± 86.3
/cm2 (n = 33), respectively. Luminal
sodium concentration was lowered (mean decrease 27.2 ± 2.66 mM,
n = 33), reflecting net sodium absorption, and luminal
potassium concentration was increased (mean increase 8.2 ± 1.22 mM, n = 33), reflecting net potassium secretion over a
24 h period.
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DISCUSSION |
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The final processing of glomerular filtrate by distal nephron segments is critical in the regulation of renal sodium excretion. More than 85% of sodium filtered is absorbed before tubule fluid emerges from the loop of Henle and enters the distal tubule. Regulated absorption of sodium by the DCT, CNT, CCD, OMCD, and IMCD is responsible for providing overall sodium homeostasis by the kidney and results in the excretion of only ~1% of the filtered amount. The DCT segment of the mammalian kidney plays an important role in this process; however, the precise mechanisms contributing to sodium absorption by the DCT have not been elucidated fully. In this study we sought to investigate whether amiloride-sensitive epithelial sodium channels contribute to sodium transport by the DCT segment of the rabbit kidney.
The present data suggest that, in the native DCT of the rabbit, sodium
absorption does not likely occur via epithelial sodium channels. In
support of this finding, when DCT are perfused in vitro, the voltage is
zero and not affected by amiloride. Also, mRNA for the - and
-ENaC subunits was not detectable in the native DCT segment. In
addition, Nedd4, which has been associated with ENaC trafficking from
the apical membrane, does not appear to be expressed in the native DCT.
In contrast to native tissue, DCT segments grown in primary culture
exhibited a different phenotype. Thus monolayers generated from
cultured DCT explants developed a lumen negative voltage, expressed all
three ENaC subunits, and in addition expressed Nedd4. Taken together,
these findings are consistent with amiloride-sensitive sodium transport
occurring primarily in the connecting tubule and collecting duct system but not in the rabbit DCT. Furthermore, it appears that under the
present culture conditions, DCT cells dedifferentiate and do not
provide a suitable model for the DCT.
In vitro perfusion of DCT segments. The rabbit was chosen for performance of these studies because the transitions from TAL to DCT and DCT to CNT occur abruptly, and the DCT can be dissected readily (see METHODS). In the rat kidney, a second type of DCT cell has been identified that appears at mid-DCT (18), which would decrease the likelihood of obtaining a homogeneous segment for this type of perfusion study.
In a previous preliminary study (31), we had observed that the voltage of perfused rabbit DCT segments was lumen negative. However, although most of the values were low or near zero, there was a large degree of scatter in the data. Also, because the DCT segment of the nephron is short (~0.5 mm in length), tubules were cut as close to the transitions as possible to maximize segment lengths for perfusion. In the present study we sought to reexamine the Vte of the true DCT. Upstream DCT segments (see METHODS) uniformly had voltages near zero (control; C in Fig. 1) and lacked response to luminal amiloride and barium. In contrast, some downstream DCT segments exhibited a barium- and amiloride-sensitive lumen negative voltage. It is possible that, when the nephron was cut at the DCT-CNT junction, in some instances CNT cells were included in the perfused structure. It may be that these cells account for the barium- and amiloride-sensitive lumen negative voltage noted in some of the downstream DCT segments. The effects can be accounted for by the presence of apical sodium channels and potassium channels in some of the cells being perfused. We conclude that rigorous criteria must be applied during dissection to separate DCT and CNT cells. Furthermore, we believe that some CNT cells may have been included in a number of the DCT segments perfused in our previous study (31). The present data suggest that the native rabbit DCT likely does not express significant amiloride-sensitive sodium channel activity. In all of the subsequent experiments performed in the present study, when the DCT was investigated, the upstream DCT was used exclusively. The present results also provide a basis for the interpretation of previous Vte data obtained by using the isolated perfused tubule technique. It is possible that, in those studies (15, 24, 25, 31), inadvertent inclusion of CNT cells in the perfused structure could have contributed to the lumen negative Vte that was observed.ENaC Northern analysis.
Northern analysis of rabbit kidney revealed expression of all three
ENaC subunits. - and
-ENaC were expressed as single prominent
bands whereas two transcripts for
-ENaC were detected. Although the
transcript size for
-ENaC is similar to that of the rat (3.7 kb),
transcript sizes for
- and
-subunits differ somewhat from the
results reported in the rat (2). In rabbit, the
-subunit is larger (3.1 vs. 2.2 kb), and two
-subunit transcripts (2.8 and 4 kb) are expressed instead of one (3.2 kb).
ENaC and Nedd4 mRNA
expression by tubule segments.
ENaC was expressed predominantly by distal nephron segments; however,
the distribution was not uniform for the individual ENaC subunits.
-ENaC was detected in all nephron segments downstream of the thin
ascending limb of Henle's loop. The only segments where
-ENaC could
not be detected were the proximal tubule and the glomerulus.
- and
-ENaC were expressed consistently only in the CNT, CCD, and OMCD. On
the basis of previous studies both in vivo and in vitro (4, 5, 8,
11, 19, 29), amiloride-sensitive sodium transport and lumen
negative voltages are found in these segments of the nephron. Thus the
present data are consistent with a fully functional sodium channel
present in these nephron segments and confirm that all three subunits
are expressed.
DCT grown in culture.
Our results in the DCT grown in vitro under cell culture conditions
contrast starkly with the results obtained in the native tubules.
Primary cultures of DCT fragments express -,
-, and
-ENaC mRNA
as well as Nedd4 mRNA (Figs. 4 and 7). In addition, the monolayers
exhibit a lumen negative voltage, absorb sodium, and secrete potassium.
All of these features are typical of more distal nephron segments,
including the connecting tubule and CCD.
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ACKNOWLEDGEMENTS |
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Portions of this data have been published previously in abstract form (30, 33, 34). This research was supported by a Merit Review award from the Department of Veterans Affairs.
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FOOTNOTES |
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1 The distal tubule is defined as that portion of the nephron extending from the macula densa to the confluence of the nephron with another nephron to form the collecting duct. The distal tubule is a cytologically heterogeneous nephron segment and contains a portion of the thick ascending limb (TAL), the distal convoluted tubule (DCT), the connecting tubule (CNT) and the initial collecting tubule (ICT).
2 The term "late" refers to the final 50% of the distal tubule. The late distal tubule is composed primarily of ICT and a portion of the CNT.
3 The term "early" refers to the initial 50% of the distal tubule. The early distal tubule is composed primarily of DCT and a portion of the CNT.
4 We have also used a method of collagenase digestion (22) to aid in dissection of rabbit kidney nephron segments. Results from experiments using this method do not differ from results using hand-dissected segments. Collagenase treatment can only be utilized for molecular experiments and not for in vitro microperfusion studies.
Address for reprint requests and other correspondence: H. Velázquez, VA Connecticut Healthcare System, Research Office 151, West Haven, CT 06516 (E-mail: heino.velazquez{at}yale.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 29 June 2000; accepted in final form 10 November 2000.
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