1 Kidney Institute, Departments of 2 Biochemistry and Molecular Biology and 3 Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160
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ABSTRACT |
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We used an unambiguous in vitro method to determine if inner
medullary collecting ducts (IMCD) have intrinsic capacities to absorb
and secrete solutes and fluid in an isotonic medium. IMCD1, IMCD2, and IMCD3 were dissected from kidneys of
young Sprague-Dawley rats. 8-Bromo-3',5'-cyclic monophosphate
(8-BrcAMP) stimulated lumen formation and progressive dilation in all
IMCD subsegments; lumen formation was greatest in IMCD1.
Benzamil potentiated the rate of lumen expansion in response to
8-BrcAMP. Fluid entered tubule lumens by transcellular secretion rather
than simple translocation of intracellular fluid. Secreted lumen
solutes were osmometrically active. Inhibition of protein kinase A with
H-89 and Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate
blocked fluid secretion. The rate of lumen expansion was reduced by the
selective addition of ouabain, barium, diphenyl-2-carboxylate,
bumetanide, glybenclamide, or DIDS, or reduction of extracellular
Cl. We conclude that IMCD absorb and secrete electrolytes
and fluid in vitro and that secretion is accelerated by cAMP. We
suggest that salt and fluid secretion by the terminal portions of the renal collecting system may have a role in modulating the composition and volume of the final urine.
kidney; chloride transport; cystic fibrosis transmembrane conductance regulator; anion transport; fluid secretion; salt secretion
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INTRODUCTION |
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IT IS WIDELY APPRECIATED
THAT net renal NaCl excretion is the difference between the
amount of NaCl filtered by glomeruli and the amount reabsorbed by the
contiguous nephron segments. This classic view supposes that tubular
NaCl reabsorption is exquisitely regulated by individual tubular
segments to reclaim all but a tiny fraction of the filtered NaCl
(1). The principal reabsorptive transporters thought to
determine salt balance include a variety of Na+-dependent
mechanisms in the proximal tubules, a
Na+-K+-2Cl cotransporter in the
ascending limb of Henle's loop, a NaCl cotransporter in the distal
convoluted tubule (NCCT) and an electrogenic Na+ channel
(ENaC) in the collecting duct system.
The collecting ducts are in an especially strategic location at the
terminal end of the tubular system where they could have a commanding
role in the day-to-day regulation of NaCl balance. Recent evidence,
based on the molecular identification of major classes of transport
proteins, indicates that collecting duct cells are armed with ENaC
(1, 14, 54), cyclic-nucleotide-gated nonselective
cation channels (CNG; Refs. 26, 46), cystic fibrosis transmembrane
conductance regulator (CFTR) Cl channels (18, 30,
47), K+ channels (20, 38, 50), and
aquaporins (11, 23, 31). These transport pathways could
contribute to net absorption or net secretion of solutes and fluid,
depending on the physiological needs of the animal. However, in situ
micropuncture (8, 15, 43) and retrograde catheterization
studies (3, 41) of inner medullary collecting ducts (IMCD)
have contributed to a rather confused understanding of net NaCl
transport in these segments under normal conditions and their role in
mediating natriuresis after acute loading with salt and water.
Unfortunately, direct in vitro perfusion of solitary IMCD has not
clarified the issue because the observed net NaCl transport rates were
so low as to be at the limit of quantification (36).
These quantitative issues not withstanding, it is
interesting to consider that a finer control of net NaCl excretion
might be in order were tubular NaCl reabsorption to operate in concert with a variable amount of regulated NaCl secretion in the collecting ducts. The identification of functionally active CFTR in collecting duct cells (18, 30, 47) raises the possibility that
cAMP-dependent NaCl secretion might have a role, and a few studies have
found evidence consistent with net secretion. Sonnenberg
(41) used a retrograde microcatheterization method to show
in rats acutely expanded with Ringer solution that Na+ and
fluid were added to the tubular fluid of IMCD. Some investigators (15, 43) believe that the evidence for net secretion of
solutes in that study was not compelling and suggested that the results were compromised by the collection of an admixture of fluid from deep
and superficial nephrons that emptied into the IMCD. In support of the
observation by Sonnenberg (41), Cl secretion
in the IMCD has been observed by a number of laboratories; however, the
relationship between Cl
secretion and fluid secretion in
this segment is unclear. Rocha and Kudo (34) showed that
in isolated perfused IMCD segments, dibutyryl-cAMP, a permeable cAMP
analog, increased the unidirectional isotopic flux of Na+
and Cl
from the bath to the lumen. Unfortunately, these
results have not been confirmed in other laboratories. On the other
hand, Wall et. al. (48) demonstrated net Cl
secretion by isolated IMCD segments. Fluid secretion was also observed;
however, the net volume flux did not reach statistical significance.
More recently, NaCl and fluid secretion have been demonstrated in
cultured polarized monolayers of IMCD cells (17). Moreover, a short-circuit current method was used to show that primary
cultures of rat collecting duct cells derived from the renal papilla
secreted Cl
in response to interleukin-1
(17), and Kizer et. al. (22) observed in a
continuous murine cell line derived from the initial IMCD (mIMCD-K2)
that cAMP and arginine vasopressin stimulated Cl
secretion that was sensitive to several transport inhibitors. Evidence
for CFTR-type Cl
channels that were inhibited by the
apical application of CFTR inhibitors was found in both rat and mouse
IMCD cells (18, 47). Consequently, mammalian IMCD have
molecular transporters to promote both NaCl absorption and secretion;
however, clear-cut evidence of an intrinsic process that couples net
solute and fluid secretion has not been convincingly demonstrated in
intact collecting duct segments.
In the present study, we used a direct and unambiguous method to detect and quantify net fluid secretion in isolated segments of the rat IMCD. We used this method to 1) determine whether isolated IMCD from the rat kidney have an intrinsic capacity for sustained cAMP-dependent fluid secretion; 2) determine whether there is heterogeneity in the response to cAMP by different subsegments of the IMCD; and 3) characterize some of the cellular mechanisms contributing to fluid secretion.
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METHODS |
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Tissue preparation.
Anesthesia was induced in male Sprague-Dawley rats, weighing
90-200 g (3-5 wk of age), by inhalation of isoflurane and
100% O2. The left kidney was rapidly removed, and a
transverse 0.5- to 1.0-mm slice containing the renal papilla was
obtained. The animals were killed by exsangination following guidelines
established by the institutional animal care committee. The renal
cortex was removed, and the medulla was placed in a 1:1 mixture of DMEM
and Ham's F-12 (DME/F-12; JRH Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenite (ITS), 100 IU/ml penicillin G, and 0.1 mg/ml streptomycin (P/S) at room temperature and
equilibrated with 5% CO2 and room air. Microdissection was done by methods previously established in this laboratory
(12). IMCD, conforming to standard nomenclature
(5, 29), were dissected at 25°C with fine-tipped forceps
and an American Optical (no. 570, magnification ×7 to ×42) from the
outer third (IMCD1), the middle third (IMCD2),
or the terminal third of the medulla (IMCD3). In
preliminary studies, we found that the IMCD1 had the
greatest capacity for fluid secretion; thus unless specified otherwise, experiments were conducted in IMCD1. Individual IMCD or
translucent tissue bundles containing one to three collecting ducts
were isolated and attached to six-well culture plates (CoStar,
Cambridge, MA) and coated with a thin layer of
poly-L-lysine to secure them throughout the study
(35). After preparation of the tissue (1 h), tubules were briefly examined to confirm that IMCD lumens had collapsed. The
tubules were bathed in 5 ml of DME/F-12 containing ITS + P/S (308 mosmol/kgH2O); unless indicated otherwise, the incubation medium contained 5% FBS. At this time (zero time), agonist and/or inhibitors were added to the medium and the culture plate containing the tubules was gradually warmed to 37°C in a humidified incubator containing an atmosphere of 5% CO2-room air. Indomethacin
(10 µM) was added to all media to inhibit cyclooxygenase and the
formation of PGE2, an adenylate cyclase agonist. Plates
were removed from the incubator, and lumen diameters were measured at
1, 6, and 24 h unless indicated otherwise.
Measurement of tubule lumen diameter. In initial experiments, culture plates containing IMCD were placed on the stage of a Nikon inverted microscope and lumen diameters were measured by video analysis, using a previously described method (49). The video image of the tubule was recorded on videotape, and the IMCD lumen (inner diameter) and the entire IMCD (outer diameter) were measured at ten locations over a tubule length of 90-100 µm using video analysis (JAVA; Jandel, Corte Madera, CA). The mean diameter was used to calculate volume and was expressed as nanoliter per millimeter tubule length. This method of measurement enabled us to precisely monitor changes in collecting duct lumen and cell volume during transepithelial fluid secretion; however, this approach was not suitable for the rapid analysis of relatively large numbers of tubules in which several agonists and inhibitors might be evaluated. Thus in most experiments we monitored changes in lumen diameter using a lense micrometer in the microscope ocular, an approach similar to that used by Kudo et. al. (24). Each data point represented the largest diameter in a dilated segment of tubule greater that 500 µm in length. This routine enabled us to rapidly measure changes in lumen diameter in several tubules under different experimental conditions and gave results that were in line with the more laborious video-based method. Unless noted otherwise, volumes were determined from the lumen diameter measured with an ocular micrometer.
Assuming cylindrical tubule geometry, lumen volume was determined fromAnimal preparation. Several pretreatment conditions were examined in preliminary experiments. The capacity of IMCD to secrete fluid could be demonstrated most consistently in collecting ducts removed from animals that were diuretic before they were killed. Unless stated otherwise, animals were given free access to water containing 3% sucrose for 17 h to increase urine flow and reduce medullary osmolality (19). The NaCl in ordinary rat chow was supplemented by including 0.225% NaCl in the drinking water to ensure that the animals were natriuretic. Urine osmolality measured with a vapor pressure osmometer was 293 ± 64 mosmol/kgH2O (means ± SD). In a separate group, rats receiving normal drinking water ad libitum were infused intraperitoneally with isotonic saline (3-5% of body wt) 2 h before death; 1 h before death they were given furosemide intraperitoneally (0.5 mg/kg body wt) to induce diuresis. Urine osmolality in six of these animals averaged 454 ± 206 mosmol/kgH2O.
Materials.
Chemicals were purchased from Sigma (St. Louis, MO) unless indicated
otherwise. H-89, Sp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Sp-cAMP[S]), and Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMP[S]) were purchased from
Calbiochem (La Jolla, CA); diphenyl-2-carboxylate (DPC) was purchased
from Fluka. Agonists and inhibitors were stored at 20°C as stock
solutions; 8-bromo-3',5'-cyclic monophosphate (8-BrcAMP), Sp-cAMP[S], Rp-cAMP[S], and othrovanadate were dissolved in
aqueous solutions; DPC, bumetanide, DIDS, glybenclamide, and benzamil were dissolved in DMSO, and indomethacin and H-89 were dissolved in
ethanol. Appropriate solvent controls were included in each study.
Statistics. Data are presented as means ± SE. Instat (GraphPad, San Diego, CA), a statistical package, was used for data analysis. Where appropriate, Student's t-test or one-way ANOVA and the Student-Newman-Keuls multiple comparison posttest were used to determine statistical significance. Data groups containing heterogeneous variances indicated by Bartlett's test were analyzed using the nonparametric tests, Mann-Whitney U test or the Kruskal-Wallis nonparametric ANOVA, and Dunn's posttest. P < 0.05 was taken to indicate statistical significance.
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RESULTS |
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The study of vectorial fluid transport in isolated IMCD has been problematic. Thus our initial studies were designed to establish optimum conditions for the determination of solute and fluid transport in freshly microdissected IMCD.
Preliminary Studies
cAMP induces lumen expansion in intact rat IMCD.
We examined the effect of 8-BrcAMP, a classic secretagog, on intact
collecting ducts isolated from rat kidneys to determine whether
mammalian IMCD have the capacity for cAMP-dependent salt and fluid
secretion. Collecting ducts from the outermost one-third of the inner
medulla (IMCD1) were isolated as individual segments or
studied within small translucent bundles of medullary tubules. We found
that 20-30 IMCD from this region of the medulla could be easily
dissected in 1 h. Individual IMCD and small tubule bundles containing
several IMCD were incubated in DME/F12, ITS + 5% FBS at 37° C. During the dissection period, IMCD lumens collapsed as fluid was
reabsorbed. We measured lumen diameters 1, 6, and 24 h after the
tubules were placed in the incubator.
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Segmental differences in fluid secretion.
There is morphological and functional heterogeniety along the IMCD
(6, 29, 33, 34, 36, 37, 53). To determine whether
IMCD1, IMCD2 and IMCD3 responded
differently to cAMP, the three subsegments of the IMCD from the same
kidneys were incubated in media containing benzamil, indomethacin, and
8-BrcAMP (Fig. 1). During the 6-h
incubation, 12 of 13 IMCD1 developed lumens, with an
average secretion rate of 0.030 ± 0.007 nl · h1 · mm
1
(P < 0.05). Lumens also formed in 4 of 12 IMCD2 and in 5 of 14 IMCD3, reflecting average
rates of secretion of 0.007 ± 0.006 and 0.004 ± 0.005 nl · h
1 · mm
1,
respectively. On the basis of this initial study, it appeared that
IMCD1 had the greatest intrinsic capacity for
cAMP-dependent solute and fluid secretion. Thus we focused the
remainder of our investigation on fluid and electrolyte transport by
the IMCD1.
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Nature of the accumulated fluid.
The appearance of the lumens during the accumulation of tubule fluid
was distinctive, and the cellular margins were clearly defined (Fig.
2). No lumens could be detected at the
beginning of the experiments (zero time, Fig. 2, A and
C). Maximally dilated lumens developed after 24-h treatment
with 8-BrcAMP to 0.334 and 0.621 nl/mm tubule length (B and
D, respectively). Because the final lumen volumes were equal
to or greater than the initial cell volumes in the lumen-collapsed
tubules, the lumen contents could not have derived exclusively from an
intracellular source. Thus transepithelial movement of fluid into the
lumen must have occurred to account for lumen expansion.
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Effect of serum proteins on fluid secretion.
In preliminary experiments to optimize experimental conditions, we
examined the effect of increasing concentrations of 8-BrcAMP in either
serum-free medium or a medium supplemented with 5% fetal bovine serum
(Fig. 4). In the absence of serum, a
maximal rate of fluid secretion was obtained with the addition of 500 µM 8-BrcAMP. By contrast, IMCD1 incubated in 5% FBS
achieved a greater maximal rate of fluid secretion at a concentration
of only 200 µM. To determine whether this enhancement of secretory
response by FBS was due to the addition of serum proteins or
unidentified secretagogs present in serum, we compared the effect of
5% FBS to medium containing dialyzed BSA with the same final protein
content (0.3%). In 24 IMCD1, the rate of fluid secretion
in medium containing 200 µM 8-BrcAMP and 0.3% BSA was 0.033 ± 0.005 nl · h1 · cm2. This was
not significantly different from the rate of secretion in medium
containing 200 µM 8-BrcAMP and 5% FBS (0.038 ± 0.005 nl · h
1 · cm2,
n = 20). Thus it appeared that FBS and dialyzed BSA
both increased the magnitude of fluid secretion stimulated by cAMP to
the same extent, indicating that serum proteins are important for
maintaining cell transport functions.
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Effect of preceding water and NaCl intake on fluid secretion by
microdissected IMCD1.
In 275 IMCD1 isolated from 27 rats maintained on a high
intake of water and NaCl for 17 h, 29.1% of the tubules formed
lumens after 1 h of incubation in 5% FBS plus 200 µM 8-BrcAMP
(Table 2). The average lumen diameter was
2.4 ± 0.3 µm. By 6 h, 88.7% of the IMCD1 had
developed lumens with an average diameter of 15.8 ± 0.5 µm.
After 24-h incubation, 94.4% of the tubules had developed lumens, and
the average lumen diameter had increased to 20.9 ± 0.5 µm. When
diuresis was induced by acute volume expansion together with furosemide
administration (Table 2), the tubule lumens did not expand as rapidly
as was seen in the longer term high-water/NaCl intake group, although
the maximal level of expansion at 24 h was nearly identical.
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Studies to Evaluate Cellular Mechanisms of Solute and Fluid Secretion in IMCD1
Fluid secretion by IMCD1 depends on cAMP.
H-89, an inhibitor of protein kinase A (PKA), was used to determine
whether the effect of cAMP on fluid secretion by IMCD1 involved the activation of this protein kinase. IMCD1 from
two rats were incubated in either control media (n = 23), 200 µM 8-BrcAMP (n = 14) or 10 µM H-89 plus
8-BrcAMP (n = 18) (Fig.
5). 8-BrcAMP added to the medium-induced
fluid secretion at a rate (0.042 ± 0.007 nl · h1 · mm
1) that was
greater than that observed in control medium (0.010 ± 0.003 nl · h
1 · mm
1,
P < 0.01). H-89 eliminated the effect of 8-BrcAMP on
fluid accumulation by the IMCD (0.000 ± 0.002 nl · h
1 · mm
1,
P < 0.001). The preincubation of IMCD for 1 h in
500 µM Rp-cAMP[S], a competitive inhibitor of cAMP, decreased the
rate of 8-BrcAMP-stimulated fluid secretion from 0.071 ± 0.008 (n = 22) to 0.042 ± 0.004 nl · h
1 · mm
1,
(n = 20, P < 0.05).
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Effects of ouabain, orthovanadate, and barium on fluid secretion by
IMCD1.
To determine whether the accumulation of fluid was dependent on active
electrolyte transport, ouabain, an inhibitor of
Na+-K+-ATPase, and barium, a broad specificity
K+ channel inhibitor, were added separately to
IMCD1 stimulated with 8-BrcAMP (Table
3). Ouabain (1 mM) diminished
cAMP-induced fluid secretion by 45.8%. Rodent
Na+-K+-ATPase is relatively resistant to
ouabain, and higher concentrations than this are required for complete
inhibition (18). We observed that 5 mM ouabain completely
blocked luminal fluid accumulation (data not shown); however, the cells
appeared swollen, and we could not be certain that the inhibition of
secretion was a selective result of
Na+-K+-ATPase inhibition as opposed to
nonspecfic changes secondary to cell injury. In a separate group of
IMCD1, orthovanadate (10 µM), a potent but nonspecific
inhibitor of the Na+-K+- ATPase
(9), plus 1 mM ouabain inhibited the cAMP-stimulated fluid
secretion by 74.2%, more than ouabain alone, and there was no apparent
effect on cell volume (Table 3). Barium reduced cAMP-dependent secretion by intact IMCD1 by 85.4% (P < 0.05) (Table 3).
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Effect of anion transport inhibitors on fluid secretion by
IMCD1.
We determined whether fluid secretion was coupled to anion secretion
through apical CFTR Cl channels by incubating
IMCD1 in the presence of DPC (1 mM), or glybenclamide (50 µM), inhibitors of CFTR Cl
channels (Fig.
6). DPC reduced the rate of
cAMP-dependent secretion from 0.045 ± 0.009 (n = 12) to
0.001 ± 0.001 nl · h
1 · mm
1
(n = 11, P < 0.001). Glybenclamide, a
compound that binds to sulfonylurea receptors and inhibits ATP-binding
cassette proteins such as CFTR and the ATP-sensitive K+
channel (40), reduced fluid secretion stimulated by
8-BrcAMP from 0.077 ± 0.010 (n = 11) to
0.009 ± 0.003 nl · h
1 · mm
1
(n = 13, P < 0.001).
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Effect of basolateral Cl entry mechanisms on fluid
secretion by IMCD1.
Cl
/HCO
cotransport have been shown to have an impact on anion secretion in
cultured mouse (22), but not in rat IMCD cell preparations (55). We examined the independent effects of bumetanide
(Na+-K+-2Cl
cotransport
inhibitor) and DIDS (an inhibitor of
Cl
/HCO
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Effect of reduced extracellular Cl on fluid secretion
by IMCD1.
To examine the role of Cl
more directly, we substituted
equimolar cyclamate for all Cl
in the isotonic Ringer
medium used previously in our laboratory (9, 12, 35, 49).
FBS (5%) and ITS were added to the control and low-Cl
solutions; thus the final chloride content of the cyclamate medium was
~5 mM. In the control Ringer medium (Cl
, 119 mM), the
rates of 8-BrcAMP-stimulated fluid secretion were lower than we had
observed previously in the more complex DME/F-12 medium. To improve the
sensitivity of the measurements of fluid secretion, we extended the
incubation to 15 h. In control Ringer, the mean secretion rate was
0.012 ± 0.004 nl · h
1 · mm
1
(n = 9). By contrast, in the low-Cl
medium, cAMP-induced fluid secretion was completely inhibited (
0.001 ± 0.001 nl · h
1 · mm
1,
n = 8, P < 0.05).
Evidence for benzamil-insensitive fluid absorption.
To determine whether there is a component of fluid absorption
insensitive to benzamil, we incubated 13 IMCD1 in medium
containing benzamil (10 µM) and added 200 µM 8-BrcAMP to stimulate
fluid secretion. Changes in lumen volume were measured over a 3-h
interval (Fig. 8). The average rate of
fluid secretion in this initial 3-h period was 0.035 ± 0.009 nl · h1 · mm
1. After 3 h in 8-BrcAMP and benzamil, Cl
secretion was inhibited by
the addition of DIDS and bumetanide to the external medium. During the
subsequent 3-h period, the lumens of the IMCD1 treated with
the Cl
inhibitors nearly collapsed (fluid reabsorption,
0.017 ± 0.006 nl · h
1 · mm
1), whereas the
control tubules continued to accumulate tubule fluid at a rate of
0.042 ± 0.22 nl · h
1 · mm
1 (Fig. 8).
This experiment implicates a benzamil-insensitive absorptive process in
the IMCD1.
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DISCUSSION |
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We have used a straightforward in vitro method to quantify the rate of transepithelial fluid transport by rat IMCD. The central observation was that collapsed, nonperfused IMCD incubated in media supplemented with permeable analogs of cAMP progressively expanded with fluid derived from the extracellular medium, thereby establishing that this segment of the collecting duct has an intrinsic capacity to secrete fluid that may be coupled to active NaCl secretion.
One advantage of this method is that net fluid movement could be measured in several tubule segments thereby permitting the direct evaluation of different experimental conditions on IMCD isolated from the same animal. IMCD1 reliably secreted fluid on the addition of 8-BrcAMP to a greater extent than either IMCD2 or IMCD3. It is important to note in this respect that in preliminary studies (data not shown) we observed that fluid was also secreted into cortical collecting ducts and outer medullary collecting ducts, although we have insufficient measurements to determine whether the rates of secretion are different from those observed in IMCD1. It is conceivable, therefore, that under certain conditions solutes and fluid can be added to the urine concomitantly at several loci along the collecting duct system.
Net fluid secretion leading to lumen dilation was enhanced by the administration of benzamil to inhibit Na+ absorption, by preconditioning animals with a high-fluid and NaCl intake before dissecting the collecting ducts, and by including serum proteins in the incubation media. Under optimum conditions, nearly 95% of the IMCD1 secreted fluid in response to cAMP (Table 2).
The basal rate of fluid secretion varied among IMCD1 taken from the same kidney, but in paired studies, 8-BrcAMP consistently increased the rate of fluid secretion above baseline (Tables 1 and 3, Figs. 5, 6, and 7). The wide-ranging baseline and agonist-dependent rates of transport are not an unusual finding in studies that use microdissected tubule segments and may reflect alterations in transport consequent to radical changes in tissue osmolality, composition, and oxygenation of the environment surrounding the tubule, and the removal of endogenous hormones and mediators. It is widely acknowledged that the intrinsic transport processes in microdissected tubules are generally well preserved for several hours in vitro although the absolute rates of transport may be quantitatively different than in the in situ state (12). Thus the present study highlights a relatively unappreciated function of the IMCD that may have physiological and pathophysiological relevance.
Net fluid transport by IMCD reflects the arithmetic difference between the individual fluxes of secreted and absorbed solutes. In the present study, we have used transport inhibitors to evaluate the contributions of specific classes of ion transporters in the promotion of net fluid secretion. The reduction in sodium absorption by benzamil clearly augmented the rate of net solute and fluid secretion (Table 1). In IMCD1, fluid and solute absorption were reduced by benzamil (Table 1) but were not completely eliminated by it (Fig. 8). A residual level of net fluid absorption after inhibition of ENaC and nonspecific cation transport with benzamil, and chloride secretion with DIDS and bumetanide (Fig. 8) is consistent with a benzamil-resistant solute absorptive mechanism. A few studies have demonstrated a thiazide-sensitive electroneutral NaCl transport pathway in cortical regions of the CD that may contribute to salt absorption (45, 51); however, exploration of this absorptive pathway was beyond the scope of the present study. If there is a component of solute absorption in IMCD1 that is insensitive to benzamil then maximal rates of cAMP-dependent solute and fluid secretion may not have been achieved in the present study.
Previous studies designed to quantify net fluid transport have failed to demonstrate significant fluid secretion by isolated perfused IMCD (36, 42, 48). Given the low rates of transport, quantification of net fluid fluxes using volume markers is relatively imprecise. Moreover, conditions for optimum net ion transport may not have been met in the previous in vitro studies. We found that nutrients in DME/F-12 and serum proteins increased the secretion of fluid by IMCD. We also observed that the hydration state of the animal before death was important for the subsequent demonstration of solute and fluid secretion in dissected tubules. The increase in urine flow and reduction in urine osmolality for several hours before the death of the animal may have helped to maintain optimum metabolism of the tissue during microdissection (Table 2).
Solute and fluid secretion by IMCD. In the present study, 8-BrcAMP stimulated solute and fluid accumulation in all segments of the rat IMCD; however, secretion was greatest in the IMCD1 (Fig. 1). This axial transport heterogeneity is in keeping with studies that have demonstrated morphological and functional heterogeneity along the IMCD (6, 29, 33, 34, 36, 37, 53). In rats, the initial IMCD1 contains ~90% principal cells and 10% intercalated cells (29, 54). Chloride channels have been demonstrated in both cell types (39). By contrast, most of the IMCD2 and the entire IMCD3 are made up of a distinct cell type called the IMCD cell (6, 29). IMCD1, the segment in which we found cAMP-stimulated net fluid secretion to be the greatest, expresses electrogenic ion transport evinced by a lumen-negative potential (24, 34, 42).
Net addition of NaCl to the tubular fluid of IMCD in situ was demonstrated in retrograde microcatheterization experiments in acutely volume-expanded rats (41). Na+ and fluid were added to IMCD accessible by this technique in amounts approximating 10% of the filtered loads. Low rates of net Na+, ClCellular mechanism of fluid secretion by IMCD1.
8-BrcAMP and Sp-cAMP[S], permeable analogs of cAMP, and forskolin,
which directly activates adenylyl cyclase, stimulated fluid secretion
by the IMCD1. Inhibition of cAMP-stimulated fluid secretion by H-89 (Fig. 5) and a reduction in fluid secretion by Rp-cAMP[S] confirmed that cAMP-dependent PKA was instrumental in the mediation of
the nucleotide effect. The rat IMCD1 appeared to have many features in common with other epithelia in which the net secretion of
electrolyte and fluid is stimulated by cAMP (25, 32, 40). In most secretory tissues, solute and fluid secretion depend on the
establishment and maintenance of electrochemical gradients for
Na+ and Cl generated by
Na+-K+-ATPase, operating in conjunction with
basolateral K+ channels and
Na+-K+-2Cl
cotransporters and
Cl
/HCO
channel inhibitors DPC and glybenclamide (Fig.
6). The effects of these inhibitors, together with the elimination of
net fluid secretion on reducing the medium chloride concentration,
place the IMCD1 in the company of lung and intestinal
epithelia that are noted for bidirectional transport of NaCl under the
control of cAMP (25). The cell model in Fig.
9 illustrates a hypothetical organization
of transporters involved in NaCl and fluid secretion in the
IMCD1.
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Possible role of NaCl and fluid secretion in IMCD.
NaCl secretion appears to be a conserved transport mechanism in the
evolution (4) and embryologic development of the
metanephric kidney (16). In aglomerular marine fish, urine
is formed by tubular salt and fluid secretion (7).
Beyenbach and Frömter (4) demonstrated
cAMP-stimulated Cl secretion in the proximal renal
tubules of the glomerular spiny dogfish shark. CFTR has been
demonstrated in many structures involved in the regulation of NaCl
balance in nonmammalian species, i.e., shark rectal gland
(32) and salt glands of marine Aves (10), and
has been shown to be present in the epithelial cells of the distal
nephron as early in evolution as the amphibians (27). In
nephrogenesis, the early appearance of CFTR in conjunction with tubule
lumen formation before the onset of glomerular filtration has suggested
a role for chloride-mediated fluid secretion in tubule morphogenesis
(16, 52). Thus salt-driven fluid secretion in adult kidney
could be a residual mechanism crucially employed in an earlier
embryonic state as a means of urine formation in relatively immature
tubule segments. Because ontogeny appears to recapitulate phylogeny in
kidney evolution and development, a mechanism for secretory urine
formation may conceivably have some basis in heritage from early
pronephric and mesonephric species and developmental forms in which
glomerular filtration was underdeveloped.
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ACKNOWLEDGEMENTS |
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We are grateful to Thomas DuBose, Jr., for reading the manuscript and for helpful discussions.
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FOOTNOTES |
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This work was supported by grants from the Department of Health and Human Services (J. J. Grantham) P01-DK-53763, P50-DK-57301, and a National Research Service Award (D. P. Wallace) F32 DK-09929-01. Portions of this study were published in abstract (J Am Soc Nephrol 10: 26A, 1999).
Address for reprint requests and other correspondence: J. J. Grantham, Dept. of Medicine and Biochemistry and Molecular Biology, Univ. of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas 66160-7382 (E-mail: jgrantha{at}kumc.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 27 October 2000; accepted in final form 13 February 2001.
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