Sulfonylurea-Sensitive K+ Transport is Involved in Cl- Secretion and Cyst Growth by Cultured ADPKD Cells
Lawrence P. Sullivan*,
Darren P. Wallace*,
Tony Gover,
Paul A. Welling,
Tamio Yamaguchi*,
Robin Maser*,
Jason W. Eppler* and
Jared J. Grantham*
*Departments of Molecular and Integrative Physiology, Biochemistry, and Molecular Biology and Medicine, University of Kansas Medical Center, Kansas City, Kansas; and Department of Physiology, University of Maryland, Baltimore, Maryland.
Correspondence to Dr. Lawrence P. Sullivan, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd. Kansas City KS 66160. Phone: 913-588-7412; Fax: 913-588-7430; E-mail: lsulliva{at}kumc.edu
ABSTRACT. Transepithelial chloride and fluid secretion by manytypes of epithelia involves activation of a conductive K+ pathwaythat serves to support the electrochemical driving force forCl- secretion. This study sought to determine if such a pathwayis involved in Cl- and fluid secretion by the cystic epitheliain autosomal dominant polycystic kidney disease (ADPKD). Primarycultures of cells derived from the cysts of patients with ADPKDwere used. Confluent monolayers of these cells, mounted in Ussingchambers, were stimulated to secrete Cl- by application of theadenylyl cyclase agonist, forskolin. The effects of variousK+ channel blockers on the increase in short-circuit current(Isc) generated by active Cl- secretion were determined. Charybdotoxin,an inhibitor of Ca2+-sensitive K+ channels exerted no effect.Similarly, the chromanole 293B, an inhibitor of cAMP-inducedK+ conductance, exerted no effect on cAMP-dependent anion secretion.Glibenclamide, an inhibitor of ATP-sensitive K+ channels andthe cystic fibrosis transmembrane conductance regulator (CFTR),modestly inhibited the forskolin-stimulated current when appliedto the apical surface of the monolayers, suggesting a relativelyweak effect on CFTR. Basolateral application of glibenclamideinhibited Isc to a greater extent. This latter effect may bedue to inhibition of a K+-conductive transport step. Glibenclamideexerted little effect on the Isc of nonstimulated monolayers.Cyst growth in ADPKD is driven by cell proliferation and Cl-and fluid secretion. The effect of glibenclamide on the growthof cysts formed within a collagen gel by cultured ADPKD cellswas tested. Addition of glibenclamide to the media bathing thecysts inhibited their growth. Glibenclamide also blocked theformation of cysts when it was added to the media at the timethe cells were seeded within the collagen gel. Glibenclamidewas also found to inhibit the proliferation of ADPKD cells.RT-PCR analysis demonstrated that the ATP-sensitive K+ channel,Kir 6.2, is expressed in cultured ADPKD cells and in normalhuman kidney. These results suggest that ATP-sensitive K+ channelblockers should be investigated as possible therapeutic agentsto inhibit cyst growth in ADPKD.
In autosomal dominant polycystic kidney disease (ADPKD), smallgroups of abnormal, proliferating, tubular cells form cyststhat eventually pinch off of the parent nephron. These cystscontinue to grow by proliferation and by accumulation of fluidin the lumen, contributing to the morbidity of this hereditarydisease. The fluid accumulation is the result of fluid secretionby the abnormal cells. Research into the mechanisms involvedin the process of secretion has shown that the fluid secretionis driven by mechanisms that are similar to those found in othersecretory epithelia (Figure 1). Na-K-ATPase, located in thebasolateral membrane, and K+ channels in either or both theapical and basolateral membranes establish the transmembraneelectrochemical gradients that drive Cl- uptake across the basolateralmembrane and Cl- exit across the apical membrane via the cysticfibrosis transmembrane conductance regulator (CFTR) and purinergic-regulatedCl- pathways (1,2). Na+ appears to be driven into the lumenvia the paracellular pathway by the lumen-negative transepithelialelectrical gradient.
Figure 1. Transport mechanisms involved in fluid secretion by autosomal dominant polycystic kidney disease (ADPKD) cells. The Na-K-ATPase mechanism in the basolateral membrane establishes the transmembrane gradients driving the secondary Na-K-2Cl cotransport mechanism, which carries Cl- into the cell. The exit of Cl- across the apical membrane via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is driven by the electrochemical gradient for Cl- across that membrane. That gradient is presumably established primarily by the transmembrane K+ chemical gradient in conjunction with K+ channels that may be present in one or both membranes.
In many epithelia that secrete salt and water, stimulation ofsecretion is accompanied by activation of basolateral K+ channelsthat assist in maintaining the electrochemical gradient drivingCl- exit across the apical membrane (Figure 1). The nature ofthe K+ channel or channels involved in that secretion apparentlyvaries among the types of these epithelia. In a cultured cellline derived from a human colon carcinoma, T84, calcium-activatedK+ channels are upregulated when secretion is stimulated andinhibition or further stimulation of those channels inhibitor stimulate Cl- secretion (3,4). In some intestinal cells,a cyclic-AMP–activated K+ channel may play a similar role(5).
We have initiated a study to determine the nature of the transmembraneK+ pathways that participate in Cl- and fluid secretion by autosomaldominant polycystic kidney disease (ADPKD) cells. Neither calcium-activatednor cyclic-AMP–activated K+ channels appear to play arole in Cl- and fluid secretion. We have uncovered evidencethat an ATP-sensitive K+ pathway is active when secretion isstimulated by cAMP agonists, and we have found that the mRNAfor the KATP channel, Kir 6.2, is expressed in ADPKD cells.Glibenclamide, an inhibitor of that channel, blocked Cl- secretionby cultured cystic epithelia, inhibited growth and formationof cysts derived from cultured ADPKD cells, and reduced proliferationof these cells.
Cell Culture
We harvested epithelial cells from the renal cysts of ADPKDpatients and grew primary cultures from these cells. This procedurehas been described in detail elsewhere (6,7). Steps were takento reduce the contamination of fibroblasts during the dissectionperiod and during the separation of the epithelial cells fromthe connective tissue (8). ADPKD cells were maintained in a1:1 mixture of Dulbecco modified Eagle’s medium and HamF12 (DME/F12; JRH Biosciences, Lenexa, KS) supplemented with5% fetal bovine serum (FBS; HyClone, Logan, UT), 110 IU/ml penicillinG, 0.1 mg/ml streptomycin (P/S; Sigma Chemical, St. Louis MO),and 5 µg/ml insulin, 5 µg/ml transferrin, and 5ng/ml sodium selenite (ITS; Biomedical Products, Bedford, MA)on plastic until they were harvested by trypsinization.
Electrical Measurements
ADPKD cells were plated on permeable supports (Snapwell; diameter,12-mm) coated with a mixture of types I and III collagen. Whencell layers were confluent, the supports were mounted in Ussingchambers (Harvard Apparatus, Holliston, MA). Both sides of themonolayer were bathed in a Ringer solution containing 147 mMNa+, 119 mM Cl-, 20 mM HCO3-, 5 mM K+, 2.5 mM HPO42+, 1.2 mMSO42+, 1.2 mM Mg+, 6 mM alanine, 5 mM acetate, 5 mM glucose,4 mM lactate, 1 mM citrate, 0.5 mM butyric acid, and 14 mM raffinose.The medium in each half-chamber was circulated by a bubble-liftmethod with a gas mixture of 95% O2–5% CO2 and maintainedat 37°C.
The transepithelial voltage was measured using glass capillarytubes containing a plug of 5% agar in 3 M KCl. Ag-AgCl electrodeswere inserted into the 3 M KCl solution filling the capillaryabove the plug. A coil of platinum wire (30 gauge) served asa current electrode. The transepithelial potential (Vte), theshort-circuit current (Isc), and the transepithelial resistancewere measured with a dual epithelial voltage-clamp apparatus(Warner Instrument) as described previously in detail (9). Measurementswere made at 5-min intervals. In each control and experimentalperiod, the measurements were continued until they stabilized.The average of the last two measurements in each period wasused for comparison between pairs of monolayers and among groups.
Measurement of Cyst Growth
The methods for inducing cultured epithelial cells to form cystshave been described previously (10,11). Briefly, ADPKD cellswere suspended in ice-cold type I collagen (Vitrogen; CollagenCorp, Palo Alto, CA), and 0.1 ml of the suspension, containing1000 cells, was added to wells of a 96-well plate (Falcon; growtharea of each well, 0.32 cm2). Warming the plate to 37°Ccaused polymerization of the collagen, trapping the cells withina collagen matrix. The gel was then covered with DME-F12 mediacontaining 25 ng/ml epithelial growth factor (EGF) and 5 µMforskolin. These two agents induce cyst formation and growth(11). After 10 to 12 d of incubation, the plate was placed onthe stage of an inverted microscope and, using x40 magnification,the number of cysts in each well was counted and the diameterof each cyst was measured using an ocular scale in which onedivision = 25 µm at x40. Cysts in the gel with a diametergreater than 125 µm could be clearly identified by theirthree-dimensional appearance and the presence of a lumen. Objectsin the gel with a smaller diameter were not counted nor measured.The wells were then divided into groups of three. The mediumin the control wells was replaced with medium of the same composition(EGF and forskolin), and the medium of the experimental wellswas replaced with one containing EGF, forskolin, and glibenclamide.The measurements were repeated after an additional 4 to 6 dof incubation. In a separate group of experiments, the effectof glibenclamide on cyst formation was determined by addingit to the media on day zero.
Cell Proliferation Measurements
The method of measuring cell proliferation has been describedpreviously (12). Briefly, the Promega Cell titer 96 MTT Assaymethod, as reported by Rankin et al., was used to measure theoptical density (OD) of a proliferation-dependent reaction product(13). This method was validated for ADPKD cells (12). Approximately4000 cells were seeded into individual chambers of a 96-wellplate. The cells were incubated initially in DME/F12 mediumsupplemented only with penicillin, streptomycin, ITS, and 1%FBS. After 24 h, ITS was removed and the FBS was reduced to0.002% to arrest growth. Wells were divided into groups of foureach. The effect of glibenclamide on control cells and on cellsstimulated to proliferate with 10 µM forskolin and 25ng/ml EGF was determined after 24 to 48 h of incubation.
Identification of K+ Channel mRNA
Total RNA was isolated from primary cultures of ADPKD cyst epithelialcells by the method of Chomczynski and Sacchi (14). First strandcDNA was generated from total RNA (approximately 1 µg)using oligo dT (15mer) and SuperScript reverse transcriptase(RT, Invitrogen 200U). RT reactions were carried out at 42°Cfor 50 min in 15 µl of 20 mM Tris-HCl, 50 mM KCl, 2.5mM MgCl2, 0.1 mg/ml BSA, 0.5 mM dNTPs, and 10 mM DTT. Afterthe RT reaction, RNase H (Boehringer Mannheim) was added toeach reaction tube (0.1 U/µl) and incubated at 37°Cfor 20 min. The negative control reactions (RT-) were handledin an identical manner, except reverse transcriptase was excluded.
PCR were carried out in a 50-µl reaction volume containing1 µl of the kidney-RT reaction solution, 10 mM Tris aminomethane-HCl(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dNTPs,50 pM of the 5' and 3' primers, and 1 unit AmpliTaq DNA polymerase.After the addition of the enzyme, the reaction was raised to94°C for 1 min and then sequentially cycled 18 to 32 timesfor 1-min durations at each of the following temperatures: 60°C(annealing), 72°C (extending), and 94°C (denaturing)using an MJ Research, Inc. Thermal cycler. Oligonucleotidescorresponding to bp 567–590 (sense strand) and 1075–1053(antisense strand) of the human Kir 6.2 and bp 567–590(sense strand) and 1072–1050 (antisense strand) of Kir6.1 were used in parallel reactions. PCR products were resolvedby agarose gel (1.2%) electrophoresis and ethidium bromide staining.In some cases, PCR products were gel purified and subclonedinto pCRScript (Stratagene) for sequencing. Sequencing was performedwith an automated Abi Prism TM dye terminator cycle sequencer.
Effect of K+ Conductance Inhibitors on Cl- Secretion
Charybdotoxin (CBTX) inhibits the activity of Ca2+-sensitiveK+ channels and attenuates cAMP stimulated Cl- secretion bythe intestinal cell line, T84 (3). However, CBTX in doses rangingfrom 1 to 100 nM did not affect the forskolin-stimulated Iscin ADPKD monolayers (Figure 2A). A cAMP-sensitive K+ conductanceis also known to be activated in intestinal cells when Cl- secretionis stimulated by agents that raise cellular cAMP levels, andthis conductance is inhibited by the chromanole 293B (5,15).However this inhibitor, used in doses from 1 to 200 µM,also had no effect on the forskolin-stimulated Isc (Figure 2B).
Figure 2. Effect of two K+ channel blockers on the increase in short-circuit current (Isc) in ADPKD monolayers. Values are mean ± SEM. n = 4 ADPKD monolayers for each of the two series of experiments. After control measurements were made, 10 µM forskolin was added to the basolateral surface and the steady-state Isc was recorded. The blockers were then added, and the Isc was again recorded. (A) Effect of 100 nM charybdotoxin. (B) Effect of 200 µM 293B.
Effect of Glibenclamide on Isc
ATP-sensitive K+ channels are known to be present in renal tubularepithelial cell membranes (16–19), so an inhibitor ofthis type of K+ conductance, the sulfonylurea compound glibenclamide,was tested and found to be an effective inhibitor of the forskolin-stimulatedIsc. Glibenclamide is known to combine with a sulfonylurea receptor(SUR), which then inhibits an associated ATP-sensitive K+ channel(20). CFTR, which is present in the apical membrane of ADPKDcells (21), also serves as a SUR, and glibenclamide is knownto be a modest inhibitor of CFTR-mediated Cl- secretion (22).Thus we tested the effect of glibenclamide, applied to the apicalsurface of ADPKD monolayers, on the forskolin-stimulated Iscand compared that to the effect obtained from basolateral applicationof the drug. The comparison is presented in Figure 3. A significanteffect of apical application of the drug was obtained at a concentrationof 100 µM (10.9 ± 2.1% inhibition) whereas basolateralapplication of 50 µM reduced the forskolin-stimulatedIsc by 16.5 ± 2.7%, 100 µM reduced it further to47.1 ± 5.0%, and 200 µM abolished the stimulatedIsc. The effects of basolateral application of 100 µMglibenclamide on Isc, the transepithelial potential difference(Vte), and the transepithelial resistance (Rte) are detailedin Table 1. Forskolin stimulated Isc and reduced Rte but didnot affect Vte. The subsequent application of 100 µM glibenclamidereduced the Isc and Vte but did not change Rte.
Figure 3. Comparison of effects of apical versus basolateral application of glibenclamide on the forskolin-stimulated Isc in ADPKD monolayers. Values are means ± SEM. n = 6 for each series. *P < 0.01.
In other secretory epithelia, the colon for instance, K+ channelsare activated when Cl- secretion is induced (5). To investigatethe possibility that a K+ conductance is activated by the inductionof secretion by ADPKD monolayers, the following experiment wasperformed. Glibenclamide (100 µM) was applied to the basolateralsurface of one of a pair of monolayers, and forskolin was thenapplied to both. The effect of glibenclamide on Isc in the absenceof forskolin was measured, and its effect on the subsequentforskolin stimulation was determined. The results of five suchexperiments are presented in Figure 4. The control values forthe paired monolayers did not differ (control bar in Figure 4A:4.5 ± 0.2 versus control bar in Figure 4B: 4.7 ±0.3 µA/cm2). The steady-state Isc after the applicationof glibenclamide averaged 14% less than the control Isc (4.0± 0.3 µA/cm2) for those monolayers, but this wasstatistically insignificant (Figure 4B: P > 0.05). The subsequentapplication of forskolin to both monolayers resulted in a 141%increase in Isc in the control monolayer (to 10.8 ± 0.3µA/cm2, Figure 4A) and a 100% increase in the monolayerthat received the glibenclamide (to 8.1 ± 0.3 µA/cm2,Figure 4B). The data suggest that a glibenclamide-sensitiveK+ conductance is relatively inactive in the absence of stimulatedsecretion but is much more active when secretion is stimulatedby adenylyl cyclase agonists.
Figure 4. Effect of glibenclamide (GBC) on the resting Isc and on the subsequent response to forskolin. Values are means ± SEM. n = 5 pairs of monolayers. Drug concentrations were 100 µM GBC applied to the basolateral surface and 10 µM forskolin (FSK). (A) After obtaining the control readings of Isc, forskolin alone was added to the control monolayers. (B) GBC was added to the experimental monolayers after the control readings of Isc were obtained, and forskolin was then applied. Statistical comparison was made with one-way ANOVA and the Bonferroni post-test. Control values for the paired monolayers did not differ. *Comparison of the control and GBC values for the experimental monolayers: NS. Comparison of the forskolin values for the paired monolayers: P < 0.001.
Comparison of the Effects of Barium and Glibenclamide on Isc
The effect of barium, a relatively nonspecific potassium channelblocker, was tested to compare its effect to that of glibenclamide.Barium (1 mM) was added to the bathing medium after the effectof forskolin was established; when the Isc reached a steadystate, 200 µM glibenclamide was added. In one group ofexperiments, the two blockers were added to the medium bathingthe apical surface of the monolayer; in a second group, theywere added to the basolateral medium. The results are presentedin Table 2. Basolateral application of barium was much moreeffective in reducing the forskolin-stimulated Isc (Fsk Isc- Control Isc) than apical application (121% versus 17%). Thesubsequent addition of glibenclamide caused further inhibitionin both groups of monolayers. In the apical group, glibenclamidereduced the forskolin-stimulated Isc by 26.5 ± 2.3%,a value not different from the 31.9 ± 5.2% obtained byapical application of glibenclamide alone (Figure 3) (unpairedt test, P = 0.361). This suggests that the two blockers inhibitdifferent pathways in the apical membrane of the monolayers.
Table 2. Comparison of apical versus basolateral effects of barium on forskolin stimulated Isca
Effect of Glibenclamide on Expansion of Cultured Microcysts
When ADPKD cells are seeded within a hydrated collagen gel matrix,spherical microcysts form as the cells proliferate and transepithelialfluid secretion fills the lumen (11). The effect of glibenclamideon the forskolin-stimulated Isc suggested the possibility thatthe drug might slow the expansion of these microcysts. To testthis, ADPKD cells were grown within a collagen gel covered witha medium containing 5 µM forskolin and 25 ng/ml EGF. After10 to 12 d of culture, the number of cysts formed were countedand their diameters were measured. Glibenclamide was added tothe culture medium, and the measurements were repeated 3 to8 d later. In the initial experiment (Figure 5A), measurementof cyst growth after 3 d of exposure to 50 µM glibenclamideindicated that glibenclamide significantly inhibited that expansion.Repeat measurements after an additional 4 d of exposure indicatedthat both the 25 and 50 µM doses significantly blockedfurther expansion of the cysts. The effect of lower doses ofglibenclamide (Figure 5B) was tested in a subsequent experiment,and the results indicated that doses of 1 and 10 µM wereineffective. However the results obtained with 25 µM glibenclamideconfirmed the results of the earlier experiment.
Figure 5. Effect of GBC on cultured cyst growth. Cells were seeded within a collagen matrix and incubated in 5 µM forskolin and 25 ng/ml epithelial growth factor (EGF). The results of two different experiments are illustrated. Values are means ± SEM. The number of cysts measured for each condition is indicated in each bar. Nonparametric ANOVA and Dunn post-test were used to compare values. (A) The cysts were allowed to grow for 10 d; after the control measurements were made, GBC (25 or 50 µM) was added. The measurements were repeated 3 and 7 d later. *P < 0.05. (B) The experiment was repeated to test the effect of lower doses of GBC. The cysts were allowed to grow for 12 d before the control measurements and the subsequent addition of 1, 10, and 25 µM GBC. The measurements were repeated 8 d later. *P < 0.05.
Effect of Glibenclamide on Cyst Formation
Glibenclamide is known to affect proliferation of a varietyof cells (23–25). Thus we tested its effect on the proliferationof cells required for cyst formation. Three groups of four wellsseeded with ADPKD cells were used. Glibenclamide was added totwo of the groups at the time the cells were seeded in collagenmatrix. Measurements were made 19 d later. The results are presentedin Table 3. The average number of cysts and their diameter inthe wells treated with 25 µM glibenclamide were less thanhalf of that of the control group. Only two cysts were foundin the four wells treated with 50 µM glibenclamide.
Table 3. Effect of glibenclamide on cyst formationa
Effect of Glibenclamide on Cell Proliferation
The effect of glibenclamide on cyst formation indicated thatcell proliferation was inhibited. This was confirmed by a moredirect means of measuring proliferation (see Materials and Methods).In basal medium, 25 µM glibenclamide exerted no effect(Figure 6), but 50 µM glibenclamide significantly inhibitedproliferation 58%. The combination of forskolin and EGF in theabsence of glibenclamide stimulated proliferation 45%. In thepresence of these agonists, 25 µM glibenclamide againexerted no significant effect but 50 µM glibenclamideinhibited proliferation 63%.
Figure 6. The effect of GBC on cell proliferation in the absence and in the presence of forskolin (Fsk) and EGF. OD, optical density. Values are mean ± SEM. n = 4. Comparisons were made using one-way ANOVA and the Bonferroni post-test. *Comparison with control: P < 0.05. +Comparison with the effect of Fsk + EGF: P < 0.05.
Search for a Glibenclamide-Sensitive K+ Channel
Glibenclamide is known to inhibit a class of ATP-sensitive K+channels. To determine if such a channel is present in ADPKDcells, mRNA was isolated from cultured cells. Specific primersfrom the KATP subunit Kir 6.2 corresponding to bp 1075–1053and bp 567 to 590 were used in PCR with first strand cDNA (RT+)or mRNA (RT-) from the cyst cells and from normal human kidneycells (Fig. 7). A PCR product of the expected size (508 bp)was generated with RT+ samples from both normal kidney and ADPKDcyst cell RNA. The identity of the Kir 6.2 PCR product was confirmedby subcloning and sequencing. Although we found Kir 6.1 in thehuman kidney, the Kir 6.1–specific primers failed to amplifya product from cyst cDNA (not shown). These results indicatethat Kir 6.2 is expressed in cultured ADPKD cyst epithelia.
Figure 7. PCR with first strand cDNA (RT+) or mRNA (RT-) from human kidney and ADPKD cysts. ADPKD cells were cultured in the presence or absence of forskolin. See text for details.
This study provides indirect functional evidence that K+ channelsparticipate in the secretion of chloride induced by adenylylcyclase agents in ADPKD cystic cells as they do in other secretoryepithelia. However, the data obtained with the use of specificblockers of K+-conductive pathways suggest that the K+ conductanceinvolved in cystic tissue differs from that in intestinal tissue.In the latter tissue, a Ca+2-sensitive K+ conductance and acAMP-sensitive K+ conductance have been shown to play a rolein the secretion of Cl- induced by adenylyl cyclase agonistsor cAMP (5). In that tissue, charybdotoxin, a blocker of Ca2+-sensitiveK+ channels, and 293B, a blocker of the cAMP-sensitive K+ conductance,inhibit Cl- secretion. However, neither inhibitor exerted aneffect on the forskolin-induced increase in Isc in ADPKD monolayers(Figure 2).
We found that the sulfonylurea compound, glibenclamide, didexert an effect on Cl- secretion. Application of glibenclamideto the basolateral surface of the monolayers exerted a morepotent effect than application to the apical surface (Figure 3and Table 1). We also found that glibenclamide inhibited theformation and growth of cysts formed by cultured ADPKD cellsand inhibited proliferation of these cells. In a similar study,Hanaoka and Guggino (26) found that glibenclamide (100 µM)inhibited the growth of ADPKD microcysts stimulated by Sp-cAMP.
The response to glibenclamide may be complex. The Cl- channel,CFTR, is known to be inhibited by glibenclamide (22). This mayaccount for the modest effect of apical application of the drug.CFTR has many of the structural features of sulfonylurea receptors(SUR), which are associated with ATP-sensitive K+ channels (27).Coexpression of ROMK2, an inwardly rectifying ATP-sensitiverenal K+ channel with CFTR in Xenopus oocytes significantlyenhanced the sensitivity of ROMK2 to glibenclamide (28). Similarly,coinjection of Kir 1.1a (ROMK1) and CFTR cRNA into Xenopus oocytesled to the expression of K+-selective channels with sulfonylureasensitivity and ATP-gating properties (19). Other binding cassetteproteins, particularly the SUR2 forms, may also associate withROMK1 or ROMK2 in vivo (29). It is possible that CFTR may controlsuch channels in the apical membranes of ADPKD cells in a mannersimilar to its control of the epithelial Na channel (ENaC).Thus the apical effect of glibenclamide on the Isc could bedue to inhibition of the Cl- channel function of CFTR, inhibitionof the SUR effect of CFTR on ATP-sensitive K+ channels, and/oran effect on another SUR associated with ATP-sensitive K+ channels.
To investigate the apical effect of glibenclamide further, wetested the effect on the forskolin-stimulated Isc of the nonspecificK+ channel blocker, barium, and the combination of barium withglibenclamide on the apical surface of the monolayers (Table 2).Barium is known to inhibit ATP-sensitive K+ channels (30,31).Barium alone exerted a modest effect on the Isc, and the subsequentaddition of glibenclamide yielded an effect that was not differentin magnitude from the effect of glibenclamide alone, suggestingthat the combined effects of the two blockers were additive.If glibenclamide were inhibiting an ATP-sensitive K+ conductanceon the apical surface, one would expect that barium would alsoinhibit that conductance and that the combined effects of thetwo agents would not be additive. The most likely conclusionis that glibenclamide inhibits the Cl- channel function of CFTR.Barium was as effective in inhibiting the forskolin-stimulatedIsc as glibenclamide when it was applied to the basolateralsurfaces of the monolayers, and the effects of the two agentson this surface were not additive.
It is likely that the more potent basolateral effect of glibenclamideis due to inhibition of a K+ conductance in the basolateralmembrane. We have identified the presence of mRNA for the ATP-sensitiveK+ channel, Kir 6.2, in both normal human kidney and in culturedADPKD cells. This channel, when associated with a SUR, is knownto be blocked by glibenclamide (19). It appears that this isthe first report of the presence of Kir 6.2 in renal epithelialcells. The location of this channel within the kidney and itsorientation to the apical or basolateral membrane of tubularcells has not yet been determined. Kir 6.1 appears to be thepredominant ATP-sensitive K+ channel in the rabbit proximaltubule (31), and we detected its presence in the human kidneytissue. We did not detect the presence of mRNA for Kir 6.1 inthe human ADPKD cystic cells.
Glibenclamide exerted little or no effect on the unstimulatedIsc of ADPKD monolayers but inhibited the response to forskolin(Figure 4). Tsuchiya, Welling, and coworkers have shown thatthe activity of ATP-sensitive K+ channels in the proximal tubuleparallel the activity of the Na-K-ATPase pump (17–18).In the unstimulated monolayers used in our study, the cellularlevels of ATP may have been high, inhibiting the activity ofthese channels. The increased activity of the Na-K-2Cl cotransporter,after stimulation of Cl- secretion by forskolin, would increasethe cellular concentration of Na+, leading to an increase inthe activity of the Na-K pump and reducing the level of ATP.This would increase the activity of the ATP-sensitive K+ channeland make it more susceptible to inhibition by glibenclamide.
To further examine the effect of glibenclamide on ADPKD cells,we measured the rate of growth of cysts formed by cultured cellsseeded within a collagen matrix. Glibenclamide in a dose rangeof 25 to 50 µM inhibited the growth of established cysts(Figure 5); when it was applied at these doses to the cellsat the time they were seeded, the drug inhibited the formationof the cysts (Table 3).
Cyst growth depends not only on fluid secretion but also oncell proliferation. K+ conductances have been shown to be involvedin proliferation of a number of cell types, and glibenclamidehas been shown to inhibit or stimulate that proliferation (23–25).The results of these studies do indicate that, when glibenclamideis present in sufficient concentrations to affect chloride transport(Figure 3) and to inhibit cell proliferation (Figure 6), itis a powerful inhibitor of cyst formation (Table 3) and cystgrowth (Figure 5).
Glibenclamide did not appear to have a toxic effect on the ADPKDcells. In the studies on the monolayers, tissue resistance wasnot affected (Table 1). In the experiments on cyst growth, glibenclamidedid not reduce the size or the number of cysts (Figure 5).
The results of this study may have therapeutic implicationsfor the treatment of ADPKD. Theoretically, inhibition of fluidsecretion would diminish the rate of cyst enlargement. However,most of the macroscopically enlarged cysts in ADPKD are blindsacs with no access to glomerular filtrate (32). Thus applicationof an inhibitor of CFTR, located in the apical membrane of cystcells (21), would be problematic. On the other hand the inhibitionof K+ conductance in the basolateral membrane, accessible bydrugs in the blood, would offer a way to diminish cAMP-activatedfluid transport. To the extent that K+ channel blocking agentsalso inhibited cell proliferation, compounds patterned afterglibenclamide might be found that would attack both the cellproliferation and fluid secretion arms of cystogenesis.
In conclusion, possible K+ conductance pathways that might contributeto the process of Cl- secretion by ADPKD cells were investigated.Specific inhibitors of Ca2+-activated K+ channels and cAMP-sensitiveK+ channels were without effect. However glibenclamide, an inhibitorof ATP-sensitive K+ channels did inhibit Cl- secretion. Whenapplied to the apical membranes of monolayers composed of ADPKDcells, it modestly inhibited Cl- secretion, presumably by inhibitingthe action of CFTR. Application to the basolateral surface produceda stronger inhibition. We also found that glibenclamide inhibitedthe growth of cultured ADPKD microcysts, blocked the formationof these cysts, and inhibited ADPKD cell proliferation. A searchfor ATP-sensitive K+ channels in cultured ADPKD cells indicatedthat Kir 6.2 is expressed in cultured ADPKD cyst epithelia andin normal human kidney. These results suggest that ATP-sensitiveK+ channel blockers should be investigated as possible therapeuticagents to inhibit cyst growth in ADPKD.
Acknowledgments
This work was supported by grants from the Department of Healthand Human Services PO1-DK-53763, P50-DK-57301 (JJ Grantham),RO1-DK-54231 (PA Welling), and a National Research Service AwardF32-DK-09929–01 (DP Wallace). PA Welling is an EstablishedInvestigator of the American Heart Association.
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Received for publication November 2, 2001.
Accepted for publication July 10, 2002.
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Abstract
Introduction
