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Am J Physiol Renal Physiol 275: F946-F954, 1998;
0363-6127/98 $5.00
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Vol. 275, Issue 6, F946-F954, December 1998

Concerted actions of IL-1beta inhibit Na+ absorption and stimulate anion secretion by IMCD cells

Russell F. Husted, Chong Zhang, and John B. Stokes

Laboratory of Epithelial Transport, Department of Internal Medicine, University of Iowa and Department of Veterans Affairs Medical Center, Iowa City, Iowa 52242

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Increasing evidence indicates that factors other than adrenocorticoid hormones can influence long-term regulation of Na+ transport by inner medullary collecting duct (IMCD) cells. We now report that, of 14 interleukins tested, only interleukin-1alpha (IL-1alpha ) and IL-1beta inhibited Na+ transport by primary cultures of rat IMCD. IL-1beta reduced both basal and mineralocorticoid (MC)-stimulated Na+ transport by 50-70%; its effect on glucocorticoid (GC)-stimulated Na+ transport was significantly less. IL-1beta continued to blunt MC stimulation of Na+ transport even after it had been removed from the medium for 24 h. The onset of action to inhibit Na+ transport was within 20 min. The acute effect from the basolateral surface was greater than that from the apical surface, but the effect from each surface was additive. In addition to its inhibitory effect on Na+ transport, chronic IL-1beta exposure increased both basal and cAMP-stimulated anion secretion rates. IL-1beta had no acute effect on anion secretion. Monolayers chronically treated with IL-1beta had an increased capacity to secrete fluid, as predicted from its effects on ion transport. Inhibitors of cyclooxygenase did not blunt the actions of IL-1beta . Furthermore, IL-1beta did not produce a rise in intracellular Ca2+. These results suggest novel signaling pathways induced by IL-1beta regulating Na+ and Cl- transport by the IMCD.

ion transport; amiloride; intracellular calcium; fluid transport; adenosine 3',5'-cyclic monophosphate; aldosterone

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

AS THE SEGMENT RESPONSIBLE for the final regulation of NaCl excretion, the inner medullary collecting duct (IMCD) has been the subject of many studies aimed at understanding how hormones, autacoids, and other factors modulate ion transport. One of the striking features of the isolated, perfused IMCD is that, in contrast to the cortical collecting duct (CCD), the magnitude of Na+ absorption following in vivo administration of adrenocorticoid hormones is rather small (19, 29, 33). The reason(s) for this in vivo resistance to aldosterone is not clear, but IMCD cells grown in primary culture respond to steroid hormones by increasing the rate of Na+ transport by two- to fourfold (12). We have previously reported that primary cultures of IMCD cells demonstrate a resistance to aldosterone when grown in the presence of fetal bovine serum (12).

In searching for an agent that might produce a resistance to aldosterone, we discovered that transform-ing growth factor-beta (TGF-beta ) exerts a potent effect on IMCD cells that lasts long after it has been removed from the culture medium (13). The effect of TGF-beta on Na+ transport is not acute but takes several hours to develop. This effect appears to involve the natriferic effects of adrenocorticoids, because TGF-beta has no effect on anion secretion but inhibits glucocorticoid (GC)- and mineralocorticoid (MC)-stimulated Na+ transport equally (13, 15). Surprisingly, several hormones that reportedly alter Na+ transport acutely in other preparations, such as atrial natriuretic factor and vasopressin, do not have an effect on Na+ transport by primary cultures of rat IMCD cells (13-15).

The molecular mechanisms involved in chronically regulating IMCD Na+ transport may be important in understanding some disorders of Na+ balance. We have recently shown that primary cultures of IMCD cells from prehypertensive Dahl salt-sensitive rats transport more Na+ than do IMCD cells from Dahl salt-resistant rats (16, 17). These results strongly imply that there are as yet unidentified factors that influence the responsiveness to aldosterone and can thus influence Na+ balance.

The purpose of the present experiments was to determine whether TGF-beta was responsible for the serum-induced resistance to aldosterone. The preliminary experiments suggested that other factor(s) participate in the serum effect. We thus directed our attention to a group of agents that have important biological actions and are known to influence Na+ excretion: the interleukins (3, 9, 20, 28, 42). The following is a report of a novel set of actions of interleukin-1beta (IL-1beta ) on Na+ and Cl- transport by IMCD cells.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell preparation. IMCD cells were isolated from 125- to 175-g Wistar rats of either sex using the hypotonic lysis isolation method described previously (11-13). Rats were anesthetized with methoxyflurane and decapitated. The kidneys were removed, then rinsed with PBS solution containing antibiotics. The inner medulla was dissected, minced, and incubated at 37°C in Krebs buffer containing 0.1% collagenase for 2-3 h. The IMCD cells were isolated by making the solution hypotonic with 2 vol of distilled water containing 10 µg/ml DNase. The cells were further subjected to two low-speed centrifugation steps, the second of which contained 10% bovine albumin. Cells were seeded at a density of 20 µg DNA/cm2 (~350,000 cells/cm2) onto collagen-coated polycarbonate filters glued to plastic cylinders or onto collagen-coated 12-mm Millicell PCF filters (Millipore, Bedford, MA).

Cells were grown for 3 days in a 1:1 mixture of DMEM and Ham's F-12 supplemented with 50 µg/ml gentamicin, 20 µg/ml norfloxacin, 5 nM triiodothyronine, 50 nM cortisol, 5 µg/ml transferrin, 5 µg/ml bovine insulin, 10 nM sodium selenite, and 1% wt/vol bovine serum albumin. The cells reached confluence by day 3 at which time cortisol, norfloxacin, and albumin were removed. Hormone or cytokine exposure was initiated on day 4 so that cells had 24 h of steroid-free incubation. On day 4, electrical parameters were measured, and when indicated, monolayers were assigned to a treatment group using a Latin square randomization based on short-circuit current (Isc).

MC treatment consisted of 100 nM aldosterone and 10 µM of the GC receptor (GR) antagonist RU-38486. GC treatment consisted of 100 nM dexamethasone and 10 µM of the MC receptor (MR) antagonist spironolactone (12, 30). Control monolayers received only the carrier (ethanol). Experiments were conducted on days 5 or 6.

Electrical measurements. Transmonolayer voltage, electrical resistance (RT), and Isc were initially measured in Ussing chambers constructed to accommodate the filter cups (Jim's Instruments, Iowa City, IA). Monolayers were bathed in DMEM and Ham's F-12 medium unless otherwise indicated. Measurements requiring longer than a few minutes were conducted in a separate set of chambers designed to accommodate Millicell filter bottom cups (18) and were bathed in a Krebs-Ringer bicarbonate solution consisting of (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 5 Na-HEPES, 5 H-HEPES, 1.5 Ca(NO3)2, 1 MgSO4, 1 Na2HPO4, and 5 D-glucose. For the studies on the acute effects of IL-1beta on Na+ transport, monolayers were bathed in the above solution where NaHCO3 had been replaced with NaCl (bicarbonate-free Ringer). Removal of HCO-3 greatly reduces the ability of the monolayer to secrete anions (18). The chambers were water-jacketed to 37°C, and electrical measurements were made with a University of Iowa voltage clamp (11, 12). HCO-3-containing solutions were gassed with 5% CO2 in air to maintain pH at 7.4. HCO-3-free solutions were gassed with 100% air. A positive Isc value represents a flow of positive charge from the luminal (apical) to the basolateral solution or a flow of a negative charge from the basolateral solution to the apical solution.

Protein and DNA measurements. Protein was measured by a fluorometric assay (36) using bovine albumin as standard. DNA was measured by the shift in fluorescence of Hoechst 33258 (22) using salmon testis DNA as standard.

Measurement of net fluid transport. Absorption or secretion of fluid was measured by placing 200 µl of medium on the apical surface and covering the medium with 140 µl of water-saturated mineral oil to reduce evaporation. All monolayers were incubated for 24 h. At the end of this time, the apical fluid was collected and measured gravimetrically. Preliminary data validated the accuracy of the volumetric measurement. Recovery of the solution was 99 ± 2%. This method is similar to that used by other investigators to measure fluid transport across epithelial monolayers in culture (32).

Intracellular calcium concentration. Cell Ca2+ concentration ([Ca2+]i) was determined in IMCD cells that had been grown on black polycarbonate filters with low intrinsic fluorescence as previously described for this laboratory (38). After we determined that the electrical characteristics were representative of the isolation, the filters were cut out of the plastic cups and transferred to a solution containing (in mM) 140 NaCl, 5 KCl, 5 Na-HEPES, 5 H-HEPES, 1 Na2HPO4, 1.5 CaCl2, 5 D-glucose, and 0.02 of the acetoxymethyl ester of fura 2 (fura 2-AM). The monolayers were incubated for 30 min at room temperature with gentle agitation. After the incubation, the monolayers were rinsed and placed in a specially designed chamber previously described in detail (43). This chamber permits continuous perfusion of the apical and basolateral solutions during [Ca2+]i determinations.

Fluorescence was measured using a microscope-based Deltascan photometric system (Photon Technology International, South Brunswick, NJ) with excitation wavelengths at 340 and 380 nm and emission wavelength at 510 ± 20 nm. The signal was processed on a personal computer using the equation and assumptions previously described (38).

Materials. Wistar rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). Unless otherwise specified, chemicals were purchased from Sigma (St. Louis, MO). TGF-beta 1, the interleukins, tumor necrosis factor-alpha (TNF-alpha ), interferon-gamma (IFN-gamma ), and latency-associated peptide (LAP) were purchased from R & D Systems (Minneapolis, MN); collagenase was from Boehringer Mannheim Diagnostics (Indianapolis, IN); fura 2-AM was from Molecular Probes (Eugene, OR); albumin was from Intergen (Purchase, NY); and gentamicin was from the University of Iowa Cancer Center. Culture media were provided by the University of Iowa Diabetes and Endocrinology Research Center. The turkey antiserum directed against TGF-beta was a generous gift from Dr. Kathy Flanders (National Institutes of Health); benzamil was a generous gift from Merck (West Point, PA); and RU-38486 was a generous gift from Roussel Uclaf, Romainville, France. Polycarbonate filters were purchased from Poretics (Livermore, CA). Plastic cylinders were purchased from ADAPS (Dunham, MA), and Millicell filters were purchased from Millipore (Bedford, MA). The interleukins were prepared in a stock solution of PBS and 0.1% albumin to a concentration of 10 µg/ml, divided into aliquots, and frozen. Stock solutions were thawed only once.

Statistics. Unless otherwise indicated, statistical analysis was conducted by ANOVA. When Bartlett's test indicated heterogeneous variances, analysis was conducted on log transformed values. Subsequent analysis between groups was performed using the Newman-Keuls test.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Neutralizing TGF-beta . In an attempt to reverse the inhibitory effect of serum on the ability of MC to stimulate Isc, we incubated IMCD monolayers with serum plus 2 µl/ml anti-TGF-beta antiserum (Ab477). Figure 1A shows that the antibody against TGF-beta did not have an effect on Isc when applied to control or MC-treated monolayers. The antibody did mitigate the effect of TGF-beta 1 in monolayers treated with a relatively small concentration (80 pM), suggesting that the antibody could act as a neutralizing agent in this system. Figure 1A also demonstrates that the antibody did not reverse the effect of fetal bovine serum to blunt the stimulatory effect of MC.


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  		Fig. 1.   Effect of agents that neutralize the action of transforming growth factor-beta (TGF-beta ) on short-circuit current (Isc). A: effect of neutralizing antiserum (2 µl/ml) against TGF-beta 1 (hatched bars) on monolayers treated with no steroid, mineralocorticoid (MC), MC with TGF-beta 1 (80 pM), and MC plus 10% fetal bovine serum. Antibody (Ab) reversed the effect of TGF-beta 1, but did not reverse the inhibitory effect of serum. Data are from 11-12 monolayers from 2 isolations. * Value greater than no steroid by ANOVA and Newman-Keuls (P < 0.01). # Value greater than paired condition not treated with antibody (P < 0.05). B: effect of latency-associated peptide (LAP, 1 µg/ml; hatched bars) on monolayers treated similarly to groups in A. LAP reversed the effect of 400 pM TGF-beta 1 (# P < 0.01) but had minimal effect on the group treated with MC + serum. Data are from 12-18 monolayers from 2-3 isolations.

In a further attempt to uncover evidence that TGF-beta might be the component of serum responsible for its effect on Isc, we incubated monolayers with LAP, a naturally occurring inactivator of TGF-beta (4). Figure 1B shows that 1 µg/ml LAP reversed the inhibitory effect of TGF-beta 1 but not the inhibitory effect of serum. These results suggest that there is another component(s) of serum that imparts to the IMCD the capacity to resist the natriferic action of aldosterone.

Examination of the interleukins. Because IL-1 has been shown to have an inhibitory effect on Na+ transport by IMCD cells (20, 21, 42), we elected to examine the effects of several of the interleukins and agents that might produce effects through similar mechanisms. Table 1 shows the effects of interleukins 1-13 as well as TNF-alpha , lipopolysaccharide (LPS), and IFN-gamma on Isc. There were no major effects on transmonolayer electrical resistance. Only IL-1alpha , IL-1beta , and LPS inhibited the Isc. Interestingly, IL-13 caused a modest increase in Isc.

                              
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  		Table 1.   Effect of cytokines on Na+ transport by MC-treated IMCD cells

Figure 2 shows the effect of concentration of IL-1alpha and IL-1beta on Isc in MC-treated monolayers. IL-1alpha was less potent, but at high concentrations (6 nM) both IL-1alpha and IL-1beta produced comparable inhibition. Because IL-1beta seemed to have the most potent inhibitory effect, we elected to study its effects in greater detail.


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  		Fig. 2.   Concentration effects of interleukin-1alpha (IL-1alpha ) and IL-1beta on Isc. Relative Isc is the fraction of Isc measured in MC-treated monolayers from the same isolation not exposed to IL-1. Control indicates the relative Isc of monolayers from the same isolation exposed to neither MC nor IL-1; n = 10-12 monolayers from 2 isolations.

Effects of IL-1beta . The effects of IL-1beta on Isc of monolayers treated with either no steroid, GC, or MC are shown in Fig. 3A. IL-1beta (600 pM) inhibited Isc in all groups. The effect was greatest in monolayers treated with MC.


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  		Fig. 3.   Effect of IL-1beta on Isc of inner medullary collecting duct (IMCD) monolayers treated with no steroid, glucocorticoid (GC), or MC. Hatched bars represent IL-1beta treatment (600 pM). A: effect on total Isc. * IL-1beta inhibited Isc in each of the treatment groups (P < 0.01). # IL-1beta inhibited Isc in the MC-treated group more than in the GC-treated group (P < 0.01); n = 36-52 monolayers from 6 isolations for each group. B: effect of GC, MC, and IL-1beta on benzamil (10 µM)-insensitive Isc. * Isc greater than groups not treated with IL-1beta (P < 0.01); n = 12 monolayers from 2 isolations for each group.

We have previously shown that the Isc of IMCD cells in primary culture consists of two separate active transport processes. The largest fraction of the Isc represents Na+ absorption and is characterized by its sensitivity to amiloride and analogs thereof (12). The amiloride-insensitive Isc is small and represents anion secretion (18, 43). We next sought to determine the extent to which the Isc in IL-1beta -treated monolayers represented Na+ transport by determining the sensitivity to benzamil, an analog of amiloride.

Figure 3B shows the magnitude of the benzamil-insensitive Isc for each of the treatment groups. There are three points. First, the magnitude of the residual current is relatively small, averaging ~1.2 µA/cm2 in monolayers not exposed to IL-1beta . Second, this residual Isc was not affected by GC or MC treatment. Third, in each group, IL-1beta caused a small but significant increase (0.5 µA/cm2) in Isc. The magnitude of the benzamil-insensitive Isc is sufficiently small that all conclusions regarding the effects of GC, MC, and IL-1beta on Isc are valid for the benzamil-sensitive Isc. Thus the values of Fig. 3A predominantly reflect Na+ absorption.

We next examined the effect of IL-1beta on transmonolayer resistance, protein content, and DNA content, parameters that might reflect toxic effects on the monolayers. Table 2 shows that in each case IL-1beta produced no reduction in the values; rather there were small increases in each parameter. The reason for these effects is not clear, but we infer from the direction of the changes that IL-1beta does not reduce Na+ transport by inflicting nonspecific cell damage.

                              
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  		Table 2.   Effect of IL-1beta on transmonolayer resistance, protein content, and DNA content

Time course of IL-1beta action. We asked two questions regarding the time course of IL-1beta on Na+ transport. The first was whether the effect was reversible. Figure 4 shows that, if the IMCD monolayers were exposed to MC and IL-1beta for 24 h, then removal of IL-1beta did not permit the MC to produce its usual stimulatory effect. (Studying longer recovery is not practical, because the monolayers begin to lose resistance and Isc after 6-7 days.) This "memory" effect is similar to that produced by TGF-beta 1 in IMCD monolayers (13).


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  		Fig. 4.   Lack of recovery of Isc following removal of IL-1beta from medium. Monolayers were randomized on day 4 (at which time there was no treatment) to 1 of 3 groups. Open squares, treatment with MC (without IL-1beta ) for the previous 24 h; solid squares, MC plus IL-1beta (600 pM) for the previous 24 h. Simultaneous exposure to IL-1beta and MC greatly blunted the increase in Isc produced by MC alone. There was no significant stimulation by MC following removal of IL-1beta from the medium (solid square followed by open square). Representative experiment shows 6 monolayers in each group.

The second question was related to how fast IL-1beta could initiate its inhibitory effect on Na+ absorption. Figure 5 shows the acute effects of IL-1beta on Isc in monolayers pretreated with MC for 24 h. The onset of the inhibitory effect was detectable within 20 min and reached a plateau within 1 h. Figure 5 also shows that IL-1beta was effective when added to the apical solution, the basolateral solution, or both. The effect from the basolateral solution was greater than apical solution and the effect from each surface was additive.


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  		Fig. 5.   Acute effect of IL-1beta on benzamil-sensitive Isc. All monolayers were treated with MC before adding IL-1beta (60 pM) to apical, basolateral, or both solutions as indicated. Isc was normalized to control monolayers not exposed to IL-1beta measured concurrently. Benzamil-insensitive Isc was determined 60 min after IL-1beta addition. Effects of adding IL-1beta to apical, basolateral, or both are different from each other (P < 0.05); n = 15-17 pairs of monolayers from 5-6 isolations in each group.

Potential mediators of the action of IL-1beta . Because previous results have suggested that PGE2 might play an important role in mediating the effects of IL-1beta on IMCD (3, 20, 28, 42), we investigated a possible role of cyclooxygenase metabolites in the action of IL-1beta . Figure 6 shows that coincubation of the monolayers with IL-1beta and the cyclooxygenase inhibitors aspirin or ibuprofen did not blunt the inhibitory effect of IL-1beta on Isc. We infer from these results that PGE2 (or another cyclooxygenase product) does not mediate the effects of chronic IL-1beta on these monolayers.


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  		Fig. 6.   Effects of inhibitors of cyclooxygenase on the chronic effect of IL-1beta on Isc. Monolayers were incubated with either MC (open bars) or MC and 600 pM IL-1beta (hatched bars). Monolayers were also incubated with aspirin (ASA, 500 µM) or ibuprofen (1 µM). Neither agent prevented the inhibitory effect of IL-1beta ; n = 24-48 monolayers from 4 isolations in each group. There was no interaction between the inhibitors (ASA and ibuprofen) and IL-1beta by 2-way ANOVA. * P < 0.01 compared with MC alone.

We also examined the possibility that the acute effect of IL-1beta was mediated by an acute increase in a cyclooxygenase product. Table 3 shows that ibuprofen did not impair the ability of IL-1beta to inhibit Isc within 60 min. Table 3 also shows that PGE2 did not have an acute effect on Isc. Taken together, these results make it highly unlikely that IL-1beta produces its acute or chronic inhibition of Isc by altering production of a cyclooxygenase product.

                              
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  		Table 3.   Acute effects of ibuprofen, IL-1beta , and PGE2 on the magnitude of Na+ transport by IMCD monolayers

Effects of IL-1beta on anion secretion. The results demonstrating that IL-1beta increased the benzamil-insensitive Isc (Fig. 3B) raised the possibility that IL-1beta stimulated anion secretion. We tested this idea by measuring the effects of cAMP agonists on the Isc in benzamil-treated monolayers (to inhibit Na+ transport). As shown in Fig. 7, monolayers exposed to IL-1beta for 24 h had a lower rate of Na+ transport (benzamil-sensitive Isc) and a greater peak and steady-state Isc response to cAMP agonists. This response to cAMP agonists is characteristic of anion secretion by IMCD cells in primary culture (18). Supportive evidence that this Isc represented anion secretion included 1) insensitivity to bumetanide (100 µM), 2) insensitivity to apical DIDS (100 µM), 3) dependency on HCO-3 for a maximum and sustained effect, and 4) inhibition by ouabain with a time course of 20-30 min. These features (data not shown) are characteristic of anion secretion by IMCD cells in culture (18).


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  		Fig. 7.   Effect of IL-1beta on anion secretion. All monolayers were treated with MC for 24 h. Chronic (24 h) exposure to IL-1beta (600 pM) reduced baseline Isc and benzamil-sensitive Isc. Chronic exposure also increased the peak and steady-state (benzamil insensitive) Isc stimulated by forskolin (F, 10 µM) and 3-isobutyl-1-methylxanthine (IBMX, 100 µM) (* P < 0.0001 by ANOVA). There was no effect of acute (bilateral) IL-1beta exposure (300 pM) on Isc stimulated by these cAMP agonists; n = 10-12 monolayers in each group from 2 isolations.

Figure 7 also demonstrates an important difference between acute and chronic IL-1beta . Monolayers exposed for 24 h had a greater rate of anion secretion in response to cAMP agonists than did monolayers exposed for only 20 min. The chronic treatment with IL-1beta produced a greater Isc before cAMP agonists as well as a greater peak and steady-state Isc in response to cAMP. A 20- to 30-min exposure to IL-1beta had no effect on the capacity for anion secretion, even though this length of exposure had a major effect on Na+ transport (Fig. 5).

We have previously reported that steroids do not affect the capacity for anion secretion by IMCD monolayers stimulated by cAMP agonists (15). We asked whether pretreatment with MC or GC might have an effect on the magnitude of anion secretion induced by IL-1beta . Figure 8A confirms that there was no effect of either GC or MC on cAMP-stimulated anion secretion. Furthermore, 24 h exposure to IL-1beta enhanced the cAMP-stimulated anion secretion irrespective of prior steroid exposure. In contrast to the differential effects of MC and GC on the action of IL-1beta on Na+ absorption, there seems to be no such effect on anion secretion.


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  		Fig. 8.   Modulation of cAMP-stimulated anion secretion. Anion currents measured in presence of benzamil (10 µM) and 10 min after exposure to cAMP agonists as in Fig. 7. A: monolayers treated with no steroids, GC, or MC, either with or without IL-1beta (600 pM) for 24 h. IL-1beta enhanced the anion secretion in each group (P < 0.001). There was no interaction between steroid treatment and IL-1beta treatment by 2-way ANOVA; n = 12-22 monolayers from 2-4 isolations. B: monolayers treated with ibuprofen (1 mM) or carrier (control) for 24 h. Ibuprofen did not prevent enhanced anion current by chronic IL-1beta (* P < 0.01 by ANOVA).

To test the possibility that cyclooxygenase products might play a role in the action of IL-1beta on anion secretion, we incubated monolayers with ibuprofen. There was no effect of this cyclooxygenase inhibitor (Fig. 8B) on anion secretion. There was also no effect of aspirin (data not shown). These results lead us to conclude that cyclooxygenase products do not play a role in the enhancement of anion secretion by IL-1beta .

Role of intracellular Ca2+. We measured [Ca2+]i, because others have reported a transient increase as a part of the intracellular signaling events produced by IL-1beta (2). In addition, an increase in [Ca2+]i has been associated with a reduction in Na+ transport in the CCD (31). IL-1beta had no effect on [Ca2+]i over the time course where it inhibited Na+ transport. We also examined the effect of cAMP agonists on [Ca2+]i, because of the possible role of cAMP in effecting changes in ion transport by the CCD (5). These agonists also had no effect (data not shown). We conclude that an increase in [Ca2+]i is not necessary to mediate either the IL-1beta -induced acute reduction in Na+ absorption or the cAMP-induced increase in anion secretion.

Actions of MC, IL-1beta , and cAMP on net fluid movement. The major driving force for fluid movement across epithelial cells is the osmotic gradient. The fact that the agents examined in this study could influence oppositely directed active ion transport suggested that they might produce oppositely directed net fluid transport. We tested predictions derived from the present results using a group of monolayers from an isolation where the RT values were greater than 650 Omega  · cm2. (Preliminary data indicated that monolayers with lower RT values were unable to generate reproducible fluid movement.)

The predictions were as follows. 1) Because the major effect of MC is to increase Na+ absorption, monolayers so treated should absorb fluid. 2) Because the major effect of cAMP agonists is to enhance anion secretion, monolayers treated with benzamil (to inhibit Na+ absorption) and cAMP should demonstrate net fluid secretion. 3) Finally, if IL-1beta can increase the capacity for anion secretion, monolayers treated with IL-1beta , cAMP, and benzamil should demonstrate more fluid secretion than monolayers not treated with IL-1beta .

Figure 9 shows that the predictions were generally correct. Monolayers treated with MC alone absorbed fluid. Monolayers exposed to cAMP agonists in the presence of benzamil had a small amount of fluid secretion. The magnitude of the fluid secretion was increased considerably with IL-1beta .


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  		Fig. 9.   Fluid absorption and secretion by IMCD monolayers in response to maneuvers designed to alter direction of active ion transport. Monolayers selected from an isolation with an electrical resistance >650 Omega  · cm2. Ordinate, change in volume of the apical solution over 24 h (which started at 200 µl). MC, mineralocorticoid agonist; benzamil, 10 µM; cAMP consisted of forskolin (10 µM), IBMX (100 µM), and 8-chlorophenylthio-cAMP (100 µM); IL-1beta , 600 pM. All agents were added (+) to basolateral solution except benzamil, which was added to apical solution; n = 3-4 monolayers in each group. * Different from other groups (P < 0.01). # Different from other groups (P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the process of attempting to uncover a factor(s) in fetal bovine serum that can prevent the stimulatory action of aldosterone on Na+ transport by the IMCD, we discovered that IL-1beta and IL-1alpha are potent inhibitors of Na+ transport. IL-1 is thus the second type of naturally occurring agent we have found capable of producing a long-term inhibition of Na+ transport by these cells. The other agent is TGF-beta (13). Both agents share the characteristic that even after they are removed from the medium, the monolayers remain resistant to the stimulating effect of steroid hormones on Na+ transport for at least 24 h (Fig. 4 and Ref. 13).

Even though both IL-1beta and TGF-beta inhibit Na+ transport, at least three features of their actions are substantially different. First, IL-1beta causes an inhibition of Na+ transport within 20 min (Fig. 5), whereas TGF-beta requires several hours (13). Second, chronic IL-1beta exposure increases the rate of basal and cAMP-stimulated anion secretion (Figs. 7 and 8), whereas TGF-beta does not (15). And third, the magnitude of the inhibition of Na+ transport by IL-1beta is significantly less in monolayers treated with GC than with MC (Fig. 3), whereas the magnitude of inhibition of Na+ transport by TGF-beta is similar for both steroids (13). To our knowledge, IL-1beta is the only agent that can produce both a reduction in Na+ absorption and an increase in the capacity for anion secretion (15). Neither of these agents can explain the serum-induced resistance to natriferic steroids. Antagonists to TGF-beta do not reverse the serum effect (Fig. 1, A and B), and the actions of IL-1beta are different from those produced by serum (data not shown). We therefore do not have a complete explanation of the serum effect in this preparation.

Inhibition of Na+ transport. We have expected that many vasoactive agents might exert acute effects on Na+ transport by IMCD. This expectation derives from the large number of hormones and cytokines that can acutely alter Na+ transport by the CCD (5, 35). The rabbit IMCD also seems to respond acutely to several vasoactive agents (41). It has therefore been somewhat surprising that relatively few agents are able to exert an acute inhibition of Na+ transport by rat IMCD cells in culture. Even agents that inhibit Na+ transport by rabbit IMCD cells in suspension are generally not effective in rat IMCD cells in culture (13-15). The reasons for these differences are not clear, but species differences in IMCD responsiveness might be a factor. For example, it is well appreciated that the responsiveness of the rat and rabbit CCD to a given agent can be quite different (6, 7).

The experiments testing whether IL-1beta receptors were present on the apical or the basolateral membrane produced another unexpected result. It appears that they are present on both surfaces. The fact that IL-1beta produced a smaller effect from the apical surface raised the possibility that this effect was secondary to a leakage across the monolayer with activation of receptors on the basolateral membrane. However, addition of IL-1beta to both surfaces produced a greater effect than addition to only one surface (Fig. 5). If the IL-1beta receptors were located only on the basolateral membrane, one would not expect an additional effect by adding IL-1beta to the apical surface. Whether the receptors are the same and are coupled to the same signaling system remains to be examined.

From the previous reports of the action of IL-1beta on IMCD cells, we suspected that the mediator of the acute inhibition might be PGE2 (20, 28, 42). However, acute inhibition of cyclooxygenase did not alter the ability of IL-1beta to inhibit Na+ transport, and acute exposure to PGE2 did not inhibit Na+ transport (Table 3). These results, together with the failure of chronic cyclooxygenase inhibition to affect the action of IL-1beta on Na+ transport (Fig. 6) lead us to conclude that cyclooxygenase products do not mediate the actions of IL-1beta . The mediators of these actions of IL-1beta remain to be elucidated.

Stimulation of anion secretion. The discovery that IMCD cells can secrete anions (18, 37) has prompted an evaluation of the physiological role of this process and the mechanisms involved in its regulation. There is an extensive literature on the cellular pathways involved in Cl- secretion by epithelial cells, and all models require an apical membrane anion (Cl-) channel, such as cystic fibrosis transmembrane conductance regulator, and a basolateral membrane transport pathway for anion entry (1, 27). Cl- is the primary anion that is secreted by IMCD, but it is possible that some HCO-3 is secreted as well (43). Studies on the regulation of anion secretion have focused primarily (if not exclusively) on the role of cAMP in stimulating secretion. This cAMP effect is acute (seconds) and can be produced by a variety of primary agonists (40).

There is no information (to our knowledge) regarding mechanisms whereby the maximum capacity for anion secretion can be modulated. We have discovered that maximal concentrations of cAMP agonists (i.e., forskolin and 3-isobutyl-1-methylxanthine) can produce different maximal rates of anion secretion depending on whether the monolayers were exposed to IL-1beta . This effect requires exposure to IL-1beta for more than 30 min (Fig. 7), suggesting that new protein synthesis is required. At the present time we have no information regarding which of the anion transport pathways in these IMCD cells (43) might be involved in this long-term regulation.

The consequences of the ability of IL-1beta to enhance the capacity for anion secretion are illustrated in the experiment in which we measured net fluid transport across the monolayer (Fig. 9). The important element of this series is that the IMCD is capable of secreting fluid and that exposure to IL-1beta enhances this capacity.

There are two direct implications of these results. First, it now seems possible that under the correct circumstances, the IMCD could secrete NaCl and water in vivo. We emphasize that under normal circumstances the osmotic gradient across the IMCD (with the hypertonic medullary interstitium) would favor water absorption, not secretion. However, even if the osmotic gradient were oriented so as to favor water absorption, the ion transport pathways could be oriented in such a fashion as to produce NaCl secretion. An example of a circumstance where such a situation might occur is that of high circulating concentrations of vasopressin. It has long been recognized that the ability to excrete (perhaps secrete) a NaCl load in this setting is exaggerated (34).

The second implication relates to the pathological state of renal cyst formation. It is generally recognized that renal cyst development requires both cell proliferation and the ability to secrete fluid (10). An enhanced capacity to secrete anions could play a potentially important role in the rate of cyst growth. Furthermore, to the extent that cyst formation is accompanied by an element of inflammation, the IL-1beta molecule itself would be a candidate for this process. IL-1beta might play a role in the proliferative process as well, given its ability to stimulate protein and DNA content in IMCD monolayers (Table 2).

Differential effects of steroids. It is now well established that steroids can stimulate electrogenic Na+ transport by collecting duct cells by acting via either MR or GR (23, 24). It is important to note that the majority of the adrenocorticoid effects (including aldosterone) on Na+ transport by cell lines (in contrast to primary cultures) appear to be produced via GR and not MR (8, 30, 39). The reasons for this situation are not clear, but probably relate to the loss of MR in the process of cell immortalization. Thus there is little information on the specific differences between the pathways activated by GC and MC in the process of stimulating Na+ transport.

Despite the paucity of information, there is good reason to believe that the pathways activated by GC and MC are not identical. On a molecular basis, transcription factors binding near hormone response elements interact differently with activated GR and MR (25). In addition, previous experiments from this laboratory have suggested different mechanisms for GC and MC in activating the Na+ pump (23). To these data, we can now add that the sensitivity to the effects of IL-1beta on Na+ transport depends on the specific type of steroid activation (Fig. 3A). Of course, we do not yet know the molecular mechanisms responsible for this difference. However, we speculate that factors active in modulating inflammation may also be important in modulating electrogenic Na+ transport.

Significance. There are three implications of these results. First, the novel phenomena produced by IL-1beta will provide important avenues for investigating intracellular regulation of ion transport. The fact that IL-1beta can inhibit Na+ transport acutely (Fig. 5) and that prolonged exposure to IL-1beta (24 h) produces a resistance to aldosterone for more than 24 h after IL-1beta is removed from the medium (Fig. 4) implies two separate mechanisms or levels of regulation of Na+ transport. The rapid effect probably involves activation of existing signaling pathways that are not dependent on an increase in intracellular Ca2+ or on the activity of cyclooxygenase (Fig. 6; Table 3). The more prolonged effect probably involves gene transcription.

The second implication involves a potential explanation for salt-losing nephropathy accompanying chronic inflammatory states such as interstitial nephritis. The actions of IL-1beta on the IMCD described here are consistent with a mechanism whereby IL-1beta produced locally by mononuclear inflammatory cells could contribute to the failure to conserve NaCl optimally. In this regard, the actions of IL-1beta may be a part of a concerted set of actions that effect an inability to activate normal Na+-conserving systems (26).

The third implication involves the pathogenesis of hypertension. We have recently discovered that IMCD monolayers from Dahl salt-sensitive rats absorb more Na+ than monolayers from Dahl salt-resistant rats (14, 16, 17). It seems possible that pathways involved in the long-term regulation of Na+ transport by IMCD cells might be altered or deranged in models of hypertension. Further study of the intracellular pathways whereby IL-1beta alters Na+ transport might provide important clues in understanding the pathogenesis of hypertension.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants DK-52617 and HL-55006 and by a grant from the Department of Veterans Affairs.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. B. Stokes, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242.

Received 7 May 1998; accepted in final form 3 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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