cAMP-dependent fluid secretion in rat inner medullary collecting ducts

Darren P. Wallace1,2, Lorraine A. Rome2, Lawrence P. Sullivan2, and Jared J. Grantham1,2,3

1 Kidney Institute, Departments of 2 Biochemistry and Molecular Biology and 3 Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta (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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INTRODUCTION
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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.

In experiments designed to examine the effect of reducing extracellular Cl-, tubules were incubated in a normal isotonic Ringer solution containing (in mM) 147 Na+, 119 Cl-, 20 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 6 alanine, 5 K+, 5 acetate, 5 glucose, 4 lactate, 2.5 HPO4-, 1.2 Mg2+, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 1.0 citrate, 0.5 butyric acid, 14 raffinose plus ITS and P/S (308 mosmol/kgH2O) equilibrated with 5% CO2-95% O2, or in a Ringer solution in which the molar equivalent of Cl- was replaced with cyclamate.

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 from pi R2 L, where R is inner tubule radius (10-4 cm) and L is unit tubule length (0.l cm). The outer diameter was used to determine total tubule volume (lumen and cell volumes). It was further assumed that length was constant as tubule lumen volume increased; thus elongation of the segment during lumen expansion would underestimate, to a minor extent, the true increase in tubule volume.

Animal 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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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.

In a preliminary experiment, we determined the effect of benzamil on lumen formation in IMCD1. Benzamil, an analog of amiloride, inhibits with greater affinity than amiloride both epithelial Na+ channels (ENaC) and cGMP-sensitive CNG channels in cultured IMCD cells (46). IMCD1 were randomly placed in either control media, media containing 200 µM 8-BrcAMP, 10 µM benzamil, or the combination of benzamil and 8-BrcAMP. In control medium, there was no detectable change in lumen volume after incubation of the tubules for 6 h, although sporadic lumens appeared after 24 h (Table 1). The addition of 8-BrcAMP to control medium had no effect at 1 and 6 h; however, after 24 h lumens clearly appeared in four of seven IMCD. It seemed reasonable to suppose that net salt and fluid secretion might be opposed by active Na+ reabsorption in these tubular segments and thereby contribute to the variable expression of tubule lumens. Indeed, we found benzamil treatment greatly potentiated the effect of 8-BrcAMP and led to more uniform fluid accumulation by the IMCD1 (Table 1).

                              
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Table 1.   Effects of 8-BrcAMP and benzamil on lumen volume in rat IMCD1

Rates of fluid secretion were calculated from the change in lumen volume over a 5-h interval (measurements at 1 and 6 h). The mean secretion rates for IMCD1 incubated in benzamil alone and in cAMP and benzamil were 0.009 ± 0.005 and 0.068 ± 0.005 nl · h-1 · mm-1 length, respectively (Table 1). In time course experiments, we found that secretion rates calculated from these two time points corresponded well with secretion rates determined from lumen volume changes measured over intervals as short as 2 h (data not shown).

On the basis of these findings, it appeared that cAMP stimulated net addition of salt and water in IMCD1 by a process opposed by a benzamil-sensitive absorptive mechanism. In subsequent experiments, we included benzamil in the incubation media to minimize this solute absorption.

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 · h-1 · 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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Fig. 1.   Segmental difference in cAMP-dependent fluid secretion. Inner medullary collecting ducts (IMCD)1 (n = 13), IMCD2 (n = 12), and IMCD3 (n = 14) were incubated in 200 µM 8-bromo-3',5'-cyclic monophosphate (8-BrcAMP) for 6 h. Benzamil (10 µM) and 10 µM indomethacin were added to inhibit Na+ absorption and ecosanoid synthesis, respectively. Lumens of IMCD1 accumulated fluid at a significantly faster rate than the 2 other segments, demonstrating segmental heterogeneity in the response to 8-BrcAMP. *P < 0.01, compared with IMCD2 and IMCD3.

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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Fig. 2.   Video images of 2 IMCD1. Lumens were collapsed at time 0 (A and C) and dilated with fluid in the presence of 200 µM 8-BrcAMP, 10 µM benzamil, and 10 µM indomethacin. After 24-h incubation (B and D), lumens were maximally dilated with fluid. Lumen volume and total volume were calculated from measurements of inner diameter (I.D.) and outer diameter (O.D.), respectively. Cell volume was determined by subtracting lumen volume from total volume.
Diameter, µm Volume, nl/mm Diameter, µm Volume, nl/mm
A C
Total 21.4 0.359 24.8 0.481
Lumen 0.0 0.000 0.0 0.000
Cell 0.359 0.481
B D
Total 33.8 0.894 38.6 1.172
Lumen 20.6 0.334 20.6 0.621
Cell 0.560 0.551


We determined whether the secreted fluid within the tubule lumens was osmotically active by sequentially bathing two IMCD1 with dilated lumens, secondary to cAMP-mediated fluid secretion, in media with increasing concentrations of either raffinose or NaCl (Fig. 3). Both osmolytes caused a rapid decline in lumen volume with each successive increase in bath osmolality. This was consistent with osmotic removal of water from the expanded tubule lumens, confirming that the bulk of the lumen contents was aqueous and contained osmotically active solutes. The greater degree of shrinkage of the tubule lumen in raffinose compared with equivalent concentrations of NaCl suggests that the epithelium of these segments may have a finite permeability to NaCl, thereby reducing the effective osmotic gradient with respect to the less permeant raffinose. Additional experiments outside the bounds of this study would be required, however, to evaluate this supposition.


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Fig. 3.   Fluid secreted into the lumen of IMCD1 is osmotically active. Two IMCD1 with dilated lumens, secondary to cAMP-dependent fluid secretion, were bathed in media containing increasing concentrations of raffinose () or NaCl (open circle ). Relative changes in lumen volume were determined from the volume in the test medium (Vtest) divided by the volume in isoosmotic medium (Viso).

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 · h-1 · 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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Fig. 4.   Effect of serum proteins on the fluid secretion response to 8-BrcAMP. Successive increments in 8-BrcAMP increased the rate of fluid secretion in IMCD1 in 5% fetal bovine serum (FBS) medium (10 to 15 tubules per group) and serum-free medium (11 to 29 tubules per group) *P < 0.05 compared with the control group (no 8-BrcAMP). Maximal rates of secretion were greater for tubules in 5% FBS (#P < 0.01) and the maximal rates of secretion were achieved at lower 8-BrcAMP concentration in FBS (200 µM) than in serum-free medium (500 µM). All media contained benzamil (10 µM) and indomethacin (10 µM).

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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Table 2.   Composite of the effect of 8-BrcAMP on fluid secretion by rat IMCD1

The rates of fluid secretion determined between 1 and 6 h in the high-water/NaCl intake and the acute volume expansion furosemide-treated groups were 0.045 ± 0.002 and 0.022 ± 0.003 nl · h-1 · mm-1, respectively. Consequently, the subsequent experiments to examine cellular mechanisms were done using animals that had been maintained on a high-water and NaCl intake for 17 h.

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 · h-1 · 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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Fig. 5.   Inhibition of protein kinase A (PKA) blocks cAMP-dependent fluid secretion by IMCD1. IMCD1 incubated in 200 µM 8-BrcAMP secreted fluid at a higher rate compared with the control group. Accumulation of fluid in the lumen of IMCD1 was completely blocked by 10 µM H-89. All media contained benzamil (10 µM) and indomethacin (10 µM). The number of IMCD1 is shown in parentheses below each bar. *P < 0.01 compared with control. #P < 0.001compared with 8-BrcAMP alone and not significant from 0.

Although not shown in Fig. 5, 200 µM Sp-cAMP[S], another permeable cAMP analog that activates PKA, also stimulated fluid secretion from a control baseline of 0.017 ± 0.005 (n = 14) to 0.047 ± 0.006 nl · h-1 · mm-1 (n = 7, P < 0.05). Forskolin, which directly activates adenylate cyclase, stimulated fluid secretion from 0.026 ± 0.006 (n = 14) to 0.047 ± 0.007 nl · h-1 · mm-1 (n = 18, P < 0.05). These experiments support the view that fluid secretion by IMCD1 depends in large measure on the cAMP signal transduction system.

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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Table 3.   Effect of ouabain, orthovanadate, and barium on fluid secretion by IMCD1

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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Fig. 6.   Effect of chloride channel inhibitors on cAMP-dependent fluid secretion by IMCD1. Diphenyl-2-carboxylate (DPC) and glybenclamide, inhibitors of Cl- channels including cystic fibrosis transmembrane conductance regulator (CFTR), eliminated fluid secretion induced by 200 µM 8-BrcAMP. All media contained benzamil (10 µM) and indomethacin (10 µM). The number of IMCD1 is shown in parentheses below each bar. *P < 0.001 compared with control. #P < 0.001 compared with 8-BrcAMP alone.

Effect of basolateral Cl- entry mechanisms on fluid secretion by IMCD1. Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and bumetanide-sensitive Na+-K+-2Cl- 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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange) on cAMP-induced fluid secretion by IMCD1 (Fig. 7). Both bumetanide and DIDS strongly inhibited the effects of exogenous cAMP on fluid secretion.


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Fig. 7.   Effect of basolateral Cl- entry inhibitors cAMP-dependent fluid secretion by IMCD1. Bumetanide (100 µM) and DIDS (50 µM) independently reduced the rate of fluid secretion by IMCD1 treated with 200 µM 8-BrcAMP. All media contained benzamil (10 µM) and indomethacin (10 µM). Number of IMCD1 is shown in parentheses below each bar. *P < 0.05 compared with control. #P < 0.05 compared with 8-BrcAMP alone.

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 · h-1 · 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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Fig. 8.   Benzamil-insensitive fluid absorption by the IMCD1. Sequential measurements of lumen volumes of IMCD1 (n = 13) incubated in 200 µM 8-BrcAMP + 10 µM benzamil were made. During the initial 3 h, the fluid secretion rate was 0.035 ± 0.01 nl · h-1 · mm-1. After 3 h, 100 µM bumetanide and 50 µM DIDS were added to the medium to block fluid secretion. IMCD1 absorbed fluid at a rate of -0.017 ± 0.006 nl · h-1 · mm-1, P < 0.001 compared with rate in the control period (0-3 h). In contrast, control IMCD1 (n = 11) that were isolated from the same kidney continued to secrete fluid throughout the 6-h incubation; rates of fluid secretion were 0.029 ± 0.015 and 0.047 ± 0.022 nl · h-1 · mm-1 for 0-3 and 3-6 h, respectively. All media contained indomethacin (10 µM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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+, Cl-, and fluid secretion were measured in in vitro perfused rat IMCD, stimulated with a permeable cAMP analog (34, 48). The results of the present study are in qualitative agreement with these earlier reports and support the view that rat IMCD have an intrinsic capacity to secrete NaCl and fluid. The extent to which the relatively low rates of stimulated secretion observed in this study are quantitatively similar to the in vivo state remains to be determined.

Cellular 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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (21, 25, 40). Inhibitors of electrolyte transport, including ouabain, orthovanadate, and barium (Table 3), and bumetanide and DIDS (Fig. 7) reduced the secretory response of IMCD1 to cAMP, as did CFTR Cl- 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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Fig. 9.   The proposed model for Na Cl--coupled fluid secretion by IMCD1 cells. Chloride enters the cell across the basolateral membrane via electrically neutral Na+-K+-2Cl- cotransport and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. These transporters utilize the basolateral transmembrane Na+ and Cl- electrochemical gradients to bring Cl- into the cell. CFTR, a cAMP-activated Cl- channel, provides a luminal pathway for Cl- efflux. A basolateral K+ conductance maintains the electrochemical gradient driving the efflux of Cl-. These pathways set up a lumen-negative transepithelial voltage that drives Na+ through the paracellular pathway. The net addition of solute establishes the osmotic force to drive the passive flow of water into the tubule lumen. NaCl secretion is opposed by absorptive mechanisms that include the epithelial Na channel, ENaC, and an uncharacterized benzamil-insensitive mechanism.

Other evidence leads us to think that Cl- transport has a commanding role in the promotion of net fluid secretion in IMCD. CFTR, a Cl- channel protein activated by PKA, has been detected in IMCD (30) as well as in cells cultured from these tubular segments (18, 47). In patch clamp studies of mIMCD-K2 cells, inhibitors of the CFTR Cl- channel (DPC, glybenclamide, and 5-nitro-2-[3-phenylpropylamino] benzoic acid) attenuated the secretory current stimulated by cAMP, whereas addition of DIDS did not inhibit the current (47). We observed that DPC and glybenclamide independently blocked cAMP-dependent fluid secretion, consistent with the coupling of transcellular Cl- secretion through CFTR channels with fluid transport. Glybenclamide also inhibits ATP-dependent K+ channels (40), and we cannot exclude this as an additional effect that inhibits cAMP-dependent fluid secretion by IMCD.

Both bumetanide and DIDS strongly inhibited cAMP-stimulated fluid secretion in IMCD, indicating that Cl- entry across the basolateral membrane may involve both Na+-K+-2Cl- cotransport and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. These findings are consonant with observations in a murine line of IMCD cells (22) but at variance with the results of studies utilizing primary cultures of rat IMCD cells (55). One reason for the discrepancy between the present results and those of Zhang et. al. (55) may be a consequence of the fact that we characterized transport in IMCD1 cells, whereas the cell culture method used by Zhang et. al. (55) included cells from IMCD2 and IMCD3 as well.

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.

It is also interesting to consider a possible role for salt-driven fluid secretion in IMCD in the potential regulation of extracellular fluid volume and composition. Salt consumption by mammals can vary widely. The capacity of the kidney to modulate fluctuations in salt load is generally thought to depend on precise alterations in the amounts of NaCl filtered by the glomeruli and the amounts of NaCl reabsorbed by downstream tubule segments. Normally, the proximal tubule reabsorbs ~60%, loop of Henle 25%, distal tubule 10%, and collecting duct 0-5% of the filtered load (1). For this serial regulatory process to work efficiently, each segment of the renal tubule must contribute to the reabsorption of all but a tiny fraction of the glomerular filtrate. It would seem that a higher degree of control might be obtained if most of the filtered salt was reabsorbed by segments of the nephron proximal to the collecting ducts, leaving net excretion under the control of the terminal segments of the renal tubule. A tightly regulated interplay between absorption and secretion in the collecting duct system would require fewer control mechanisms to formulate the amount of NaCl in the final urine.

cAMP stimulation of NaCl secretion by the IMCD may account for antidiuretic hormone (ADH)-induced natriuresis and chloruresis observed during hydropenia in the rat (28). It is conceivable that during dehydration, an increase in the circulating ADH level and elevation of cAMP in the collecting duct cells may stimulate these segments of the nephron to actively secrete NaCl. Therefore, ADH would not only be involved in the conservation of water by increasing the permeability to water of the collecting ducts but would also reduce plasma NaCl and extracellular fluid osmolality by activating salt secretion.

Apart from a potential role in tubulogenesis and salt balance, chloride-driven salt and fluid secretion has a clear role in the aberrant biogenesis of renal cysts. Cysts develop within all segments of mammalian nephrons, including the collecting duct system, and may be consequent to acquired or hereditary etiologies (2, 13). In autosomal dominant polycystic kidney disease, the most common inherited condition, the cysts are initially connected to the tubules of origin, but as they enlarge they become disconnected and function as isolated epithelial sacs within the parenchyma of the kidney. The accumulation of fluid within these renal cysts depends on the transcellular secretion of NaCl and fluid secretion involving mechanisms similar to those described in the present study (13, 44, 49). It would appear, therefore, that NaCl and fluid secretion by epithelial cells within cysts may be a normal process that becomes pathological by virtue of the fact that the fluid becomes entrapped within closed cysts rather than draining into a patent renal tubule segment.


    ACKNOWLEDGEMENTS

We are grateful to Thomas DuBose, Jr., for reading the manuscript and for helpful discussions.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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