The Effect of Caffeine on Renal Epithelial Cells from Patients with Autosomal Dominant Polycystic Kidney Disease
Franck A. Belibi,
Darren P. Wallace,
Tamio Yamaguchi,
Marcy Christensen,
Gail Reif and
Jared J. Grantham
Kidney Institute, Department of Medicine, University of Kansas Medical Center, Kansas City, Kansas.
Correspondence to Dr. Jared J. Grantham, University Distinguished Professor, Director, Kidney Institute, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7382. Phone: 913-588-6074; Fax 913-588-7606; E-mail: jgrantha{at}kumc.edu
ABSTRACT. Autosomal dominant polycystic kidney disease (ADPKD)is a hereditary disorder characterized by the progressive enlargementof cysts derived from tubules. Tubule cell proliferation andchloride-dependent fluid accumulation, mechanisms underlyingcyst expansion, are accelerated by adenosine 3':5'-cyclic monophosphate(cAMP). This study examined the extent to which caffeine maystimulate the production of cAMP by cyst epithelial cells, therebyadversely increasing proliferation and fluid secretion. Muralepithelial cells from ADPKD cysts and normal human kidney cortexcells (HKC) were cultured, and cAMP levels were determined inresponse to caffeine and receptor-mediated agonists linked toadenylyl cyclase. Caffeine, a methylxanthine, slightly increasedbasal levels of cAMP, as did other nonselective phosphodiesterase(PDE) inhibitors, 1-methyl-3- isobutyl xanthine and theophyllineand rolipram, a specific PDE IV inhibitor. More importantly,clinically relevant concentrations of caffeine (10 to 50 µM)potentiated the effects of desmopressin (DDAVP), prostaglandinE2 (PGE2), and isoproterenol to increase cAMP levels in bothADPKD and HKC cells. By contrast, at concentrations that augmentedthe DDAVP response, caffeine attenuated cAMP accumulation byadenosine, implicating an action apart from the inhibition ofPDE. Caffeine enhanced the effect of DDAVP to stimulate transepithelialshort-circuit current of polarized ADPKD monolayers, reflectingan increase in chloride secretion. Caffeine potentiated theeffect of DDAVP and PGE2 to increase the levels of phosphorylatedextracellular signal-regulated kinase (P-ERK). By contrast,P-ERK levels in HKC cells were not raised by increased intracellularconcentrations of cAMP. It is concluded that PDE inhibitionby caffeine increases the accumulation of cAMP, and throughthis mechanism activates the ERK pathway to cellular proliferationand increases transepithelial fluid secretion in ADPKD cysticepithelium. Caffeine is, therefore, a risk factor for the promotionof cyst enlargement in patients with ADPKD.
Autosomal dominant polycystic kidney disease (ADPKD) is themost common potentially lethal hereditary renal disorder inadults (1). The intrarenal production of adenosine 3':5'-cyclicmonophosphate (cAMP) has a major role in the pathogenesis ofthe disease because this second messenger stimulates transepithelialsecretion and accumulation of cyst fluid as well as cell proliferation(2–4). Receptor-mediated agents that increase the activityof adenylyl cyclase (arginine vasopressin [AVP] prostaglandinE2 [PGE2], -adrenergic agonists) and inhibitors of phosphodiesterase(caffeine, theophylline) consequently have the potential toaccelerate the progression of ADPKD (4–7). However, nodirect studies of renal cells derived from human ADPKD cystshave examined the hypothesis that caffeine in concentrationsthat are clinically relevant has the potential to adverselyaffect renal cyst enlargement by promoting increased intracellularlevels of cAMP.
In the current study, we examined cells cultured from muralcysts of patients with ADPKD and cells cultured from normalhuman renal cortex (HKC) for the effect on the accumulationof cAMP of caffeine alone and in combination with adenylyl cyclaseagonists of physiologic interest. The results are interpretedto support the view that caffeine has the potential to enhancethe progression of ADPKD by increasing the production of cAMPin renal epithelial cells.
Cell Culture
Cells were retrieved from the renal cysts of three patientswith ADPKD who underwent elective nephrectomy and from the normalrenal cortex (HKC) of a nephrectomy specimen removed for adenocarcinoma.The cells were placed in primary culture as described previously(4,8,9). The protocols were approved by the Human Subjects Committeeat the University of Kansas Medical Center. Cells maintainedin liquid nitrogen were thawed and directly seeded onto collagen-coatedTranswell plates (Costar, Cambridge, MA) containing a 1:1 mixtureof Dulbecco’s modified Eagle’s medium and Hams F12(DMEM/F12) supplemented with 5% fetal bovine serum (FBS; Hyclone,Logan, UT), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and5 µg/ml insulin, 5 µg/ml transferin, and 5 ng/mlsodium selenite (ITS; Collaborative Biomedical Products, Bedford,MA). Media were replaced every 2 d.
Measurements of Intracellular cAMP
3 x 104 cells were seeded into individual chambers of collagen-coatedTranswell plates in DMEM/F12 containing 5% FBS, ITS, and penicillin/streptomycin.Near confluence, the medium was changed to 1% FBS to reducethe rate of growth. Twenty-four hours later, cells were rinsedin fresh DMEM/F12 containing 1% FBS for 15 min and in an isotonicRinger’s solution for 15 min. The rinsing procedure ensuredthe removal of autocrine cAMP agonists that may have accumulatedin the conditioned medium. Stock solutions of Desmopressin (DDAVP),PGE2, isoproterenol, and adenosine were added directly to theincubation medium. Stock solutions of the PDE inhibitors caffeine,rolipram, 1-methyl-3-isobutyl xanthine (IBMX), and theophyllinewere made in DMSO, and 1:100 to 1:1000 dilutions of this wereadded directly to the incubation medium. Equivalent concentrationsof DMSO were added to control medium. For the highest concentration(1000 µM), caffeine was not completely soluble in thestock solution, so a well-mixed slurry was added directly tothe medium. PGE2, isoproterenol, adenosine, caffeine, IBMX,and theophylline were purchased from Sigma (St. Louis, MO).DDAVP was purchased from Rhone Poulenc Rorer Pharmaceuticals(Collegeville, PA). Rolipram was purchased from Calbiochem (LaJolla, CA).
Intracellular cAMP was extracted into 80% methanol, reconstitutedin 0.05 mol/L sodium acetate, and quantified by an enzyme-immunoassaysystem (Amersham Pharmacia Biotech, Buckinghamshire, UK). Inparallel experiments, cell protein content was measured witha BCA protein assay kit (Pierce, Rockford, IL). cAMP contentwas initially expressed in pmol/cm2 and converted to pmol/mgprotein using a conversion factor determined in the parallelexperiments.
Bioelectric Measurements
Monolayers of ADPKD cells, grown on Snapwell membranes (Costar),were mounted in modified Ussing chambers as described previously(8,10). The chambers contained 5 ml of medium and were gassedand stirred with a slow stream of 5% CO2/95% O2. The mediumused in these experiments was an isotonic Ringer’s solutionthat contained no serum or serum components. The transepithelialelectrical potential difference (Vte), the short-circuit current(Isc), and monolayer resistance (Rte) were determined. The monolayerswere maintained in the short-circuited state, and the open-circuitvoltage was determined at 1- to 5-min intervals. After a periodof equilibration, measurements were made in sequential 5-minperiods. Isc was recorded 15 min after the addition of agonistsor a combination of agonists with caffeine to both basolateraland apical solutions.
Antibodies
Anti-ERK1 (C-16), ERK2 (C-14), and phospho-ERK (E-4) were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit,-mouse, or -rat IgG–conjugated horseradish peroxidase(HRP) antibodies (Santa Cruz Biotechnology) were used as secondaryantibody.
Western Blot Analyses
105 cells were seeded in 100-mm-diameter plastic dishes in DMEM/F12with 5% FBS, ITS, and penicillin/streptomycin. Near confluence,FBS was reduced to 1% for 24 h. Before incubation with agonists,cells were washed in 1% FBS for 15 min and in Ringer’smedium for 15 min. Caffeine, DDAVP, and PGE2 were added in Ringer’smedium and incubated for 15 min. Cells were lysed in 500 µlof ice-cold lysis buffer (TLB; 20 mmol/L Tris [pH 7.4], 137mmol/L NaCl, 25 mmol/L -glycerophosphate, 2 mmol/L ethylenediaminetetraacetic acid [EDTA], 1 mmol/L sodium orthovanadate, 2 mmol/LNaHPO4, 1% Triton X-100, 10% glycerol, 1 mmol/L phenylmethylsulfonylfluoride [PMSF], 5 µg/ml aprotinin, 5 µg/ml leupeptin,2 mmol/L benzamidine, and 0.5 mmol/L dithiothreitol). Insolublecell lysate was removed by centrifugation. Aliquots of solublecellular protein were quantified. Cell lysate (20 µg ofprotein) was then heated (95 to 100°C) in sodium dodecylsulfate (SDS) sample buffer, separated by 10% SDS–polyacrylamidegel electrophoresis (PAGE), and transferred to nitrocellulosemembranes (Hybond ECL, Amersham). After transfer, the membraneswere blocked with 5% milk in TBS-T (20 mmol/L Tris-HCl, 137mmol/L NaCl, and 0.05% Tween 20 [pH 8.0]) for 1 h at room temperatureor overnight at 4°C. Blocked membranes were incubated withprimary antibody in 5% milk in TBS-T for 2 h at room temperatureor overnight at 4°C. Membranes were then washed three timeswith TBS-T and incubated with secondary antibody with 5% milkin TBS-T for 1 h. The membranes were washed three times withTBS-T, and proteins were visualized using an enhanced chemiluminescencesystem (ECL, Amersham Life Science, Arlington Heights, IL).Intensity of the blots was detected and quantified by a Fluor-SMax multi-imager system (Bio-Rad, Hercules, CA).
Effects of Methylxanthines on cAMP Content of Cells from ADPKD Kidneys
In these experiments, cells were rinsed carefully before addingphosphodiesterase (PDE) inhibitors to reduce endogenous activationof adenylyl cyclase by autocrine or paracrine substances producedby the cells. We determined the relative potency of caffeine,theophylline, rolipram, and IBMX to increase cAMP accumulationin the cells in the absence of exogenous adenylyl cyclase agonists.The increase in intracellular cAMP caused by these PDE inhibitorswas concentration-dependent (Figure 1). Theophylline, a weakerinhibitor, caused a small increase in cAMP levels, but the valuesdid not reach statistical significance. This initial study suggeststhat, despite extensive washing before adding PDE, a small amountof endogenous activated adenylyl cyclase remained in these cells.
Figure 1. Concentration-dependent effects of phosphodiesterase (PDE) inhibitors on basal adenosine 3':5'-cyclic monophosphate (cAMP) levels in autosomal dominant polycystic kidney disease (ADPKD) cells. Cells were extensively rinsed before addition of PDE inhibitors for 15 min. Horizontal dashed line indicates basal cAMP level (2.32 ± 0.24 pmol/mg). cAMP is expressed in relation to total protein content. Values are mean ± SE; n = 6. ADPKD cells are from two different patients. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the baseline value.
We next determined the effect on cAMP accumulation in renalcells after treatment with receptor-mediated agonists. DDAVP,a V2 receptor agonist, PGE2, a renal autocoid, and isoproterenol,a -adrenergic receptor agonist caused concentration-dependentincreases in cAMP (Figure 2). Each of these agonists increasedcAMP to levels several times greater than seen with the highestconcentration of caffeine alone (Figure 1; 1000 µM). Todetermine the extent to which caffeine may potentiate receptor-mediatedincreases in cAMP, sub-maximal concentrations of DDAVP (20 mU/ml),PGE2 (12.5 ng/ml), and isoproternol (1 nM) were evaluated inconjunction with the methylxanthine. Concentrations of caffeinethat slightly increased cAMP levels when added alone strikinglypotentiated the effects of DDAVP (Figure 3). This synergisticinteraction was illustrated most clearly at the highest concentrationof caffeine used in the study (1000 µM). DDAVP alone increasedcAMP to 31.3 ± 13.6 pmol/mg (n = 12). By contrast, theaddition of caffeine, which alone increased basal cAMP to 2.9± 0.5 pmol/mg of protein (n = 12), further increasedcAMP levels to 81.6 ± 21.4 pmol/mg (n = 12). Thus, thecaffeine effect was more than additive to that of DDAVP alone,indicating a synergistic mechanism of action.
Figure 2. Effect of receptor-mediated agonists on cAMP levels in ADPKD cells. Cells were rinsed extensively before addition of agonists for 15 min. ** P < 0.01 and *** P < 0.001 indicate difference from the baseline value; n = 4.
Figure 3. Potentiation of desmopressin (DDAVP)–induced cAMP accumulation by caffeine in ADPKD cells. Cells were rinsed extensively before adding agonists for 15 min. Values are mean ± SE (n = 12) from three different ADPKD patients. * P < 0.05 and *** P < 0.001 compared with the baseline value.
To determine if caffeine potentiation of DDAVP-induced cAMPaccumulation was unique to ADPKD cells, we perfomed the sameexperiment using HKC lacking mutations in polycystins (Figure 4).Interestingly, 10 and 100 µM caffeine, diminishedcAMP to 2.7 ± 0.8 and 1.7 ± 0.3 pmol/mg, respectively,below the baseline level of 4.6 ± 0.9 pmol/mg. On theother hand, the highest concentration of caffeine (1000 µM)increased the cAMP level to 9.6 ± 1.9 pmol/mg above thebaseline level. In the HKC cells, DDAVP (20 mU/ml) increasedcAMP from the baseline to 81.9 ± 7.2 pmol/mg, and thiswas increased further by incremental concentrations of caffeine(Figure 4). Thus, as with ADPKD cells, caffeine potentiatedthe effect of DDAVP to stimulate the accumulation of cAMP, althoughin contrast to ADPKD cells, low concentrations of the methylxanthinealone appeared to inhibit the accumulation of the cyclic nucleotide.
Figure 4. Potentiation of DDAVP-induced cAMP accumulation in human kidney cortex cells (HKC) cells by caffeine. Cells were rinsed extensively before adding agonists for 15 min. Values are mean ± SE; n = 4. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the baseline value.
It may be expected that plasma levels of caffeine in the renalmedulla may fall within the range of 10 to 100 µM in individualsingesting moderate amounts of coffee (11–14). We determined,therefore, the extent to which representative adenylyl cyclaseagonists may be potentiated by clinically relevant concentrationsof caffeine (Figure 5). The modest concentrations of caffeine(50 and 100 µM) clearly augmented the effect of a submaximalconcentration of DDAVP, but the increases in cAMP levels causedby the methylxanthine in conjunction with PGE2 and isoproterenolappeared to be simply additive. This set of experiments demonstratesthat caffeine has the capacity to potentiate the action of certaincAMP agonists in ADPKD cells.
Figure 5. Potentiation by caffeine of DDAVP–, prostaglandin E2 (PGE2)–, and isoproterenol–induced cAMP accumulation in ADPKD cells. ADPKD cells were rinsed extensively before adding agonists for 15 min. Values are mean ± SE; n = 4. *** P < 0.001 compared with the baseline value.
The effects of caffeine, IBMX, and theophylline on the generationof cAMP are complicated by the fact that they also inhibit A1and A2 adenosine receptors (11,15–17). Thus under conditionsin which adenosine may be generated by renal epithelial cells,these methylxanthines may decrease (block A2 receptors) or increase(block A1 receptors) basal levels of cAMP. To explore the potentialinterference of adenosine receptor blockade in relation to thePDE inhibitor effect, we determined the effect of rolipram,a type IV PDE inhibitor that does not interact with adenosinereceptors, on cAMP levels in ADPKD cells Rolipram alone increasedcAMP levels from 2.7 ± 0.2 to 4.1 ± 0.5 (n = 4)pmol/mg of protein. Adenosine (10 µM) alone strongly increasedcAMP levels, and rolipram (100 µM) potentiated this effect(Figure 6). These findings indicate that in ADPKD cells an effectof methylxanthines on adenosine receptors is not a requirementfor them to inhibit PDE.
Figure 6. Potentiation of adenosine-induced cAMP stimulation in ADPKD cells by rolipram. ADPKD cells were rinsed extensively before adding agonists for 15 min. Values are mean ± SE; n = 4. *** P < 0.001 compared with the baseline value.
To evaluate caffeine as an adenosine antagonist, cAMP levelswere measured in ADPKD cells after adding adenosine alone orin combination with increasing concentrations of the methylxanthine.In contrast to rolipram, caffeine did not potentiate the effectof adenosine. Rather, the methylxanthine at concentrations greaterthan 10 µM diminished the effect of adenosine to stimulatecAMP accumulation (Figure 7). These results are interpretedto indicate that caffeine antagonized but did not completelyblock the effect of adenosine to stimulate cAMP production,probably through the inhibition of A2A receptors (15).
Figure 7. Attenuation of adenosine-induced cAMP stimulation in ADPKD cells by caffeine. ADPKD cells were rinsed extensively before adding caffeine and adenosine for 15 min. Values are mean ± SE; n = 4. *** P < 0.001 compared with the baseline value.
Bioelectric Effect of Caffeine on ADPKD Cells
It has previously been shown in ADPKD that the increase in Iscin response to cAMP agonists reflects an increase in transepithelialchloride secretion (2,3). Twenty-five confluent ADPKD monolayersfrom three subjects developed stable baseline Vte (-1.6 ±0.1 mV), Rte (472 ± 44 ohm/cm2), and Isc (2.6 ±0.2 µA/cm2). Fifteen minutes after adding 10 and 100 µMcaffeine, Isc increased 0.1 ± 0.04 µA/cm2 and 0.4± 0.03 µA/cm2, respectively (Figure 8). DDAVP aloneraised Isc by 1.1 ± 0.2 µA/cm2. Adding DDAVP andcaffeine simultaneously increased the Isc to levels greaterthan when either agent was added alone.
Figure 8. Effect of caffeine on DDAVP-induced Cl- secretion in ADPKD cells. ADPKD monolayers were rinsed and incubated in Ringer’s for 15 to 30 min before adding DDAVP, caffeine, or DDAVP plus caffeine. In some monolayers (white columns), caffeine was added alone. Values are mean ± SE; n = 5. *** P < 0.001 compared with the baseline value.
Effect of Caffeine, DDAVP, and PGE2 on Phosphorylated ERK in ADPKD Cells
Previous studies have demonstrated that cAMP activates ERK,a signaling kinase that promotes increased cellular proliferation(4). Western blot studies were done to determine the extentto which caffeine increased the expression of P-ERK in ADPKDcells and potentiated the effects of cAMP agonists (Figure 9).Caffeine (100 µM) alone increased P-ERK intensity by 49%;DDAVP and PGE2 increased P-ERK intensity by 44.6% and 63.6%,respectively. In combination with caffeine, DDAVP and PGE2 increasedP-ERK intensity by an additional 180 and 185%, respectively.ERK expression was not changed by the increased levels of intracellularcAMP, indicating that the increased levels of P-ERK were dueto the activation of ERK. By contrast, in HKC cells P-ERK levelswere not stimulated by DDAVP or caffeine or a combination ofthese agents (data not shown), confirming previous observationsthat cAMP does not activate ERK in renal cells lacking polycystinmutations.
Figure 9. Potentiation of DDAVP– and PGE2–induced ERK activation by caffeine in ADPKD cells. ADPKD cells were rinsed extensively before adding agonists for 15 min. The Western blot is representative of experiments using cells from three different ADPKD kidneys. In the graph, phosphorlylated p44/p42 ERK is expressed as % of control intensity. Quantification of Western blot relative intensities, mean ± SE, n = 3. ** P < 0.01 compared with the baseline value of 100.
cAMP has an important role in determining the rate at whichrenal cysts enlarge in ADPKD (1–3). Perhaps the most importanteffect of this second messenger is the stimulation of muralcell proliferation, the process that increases the surface areaof cysts and increases their volume capacity (4,18). In contrastto normal renal tubules in which cell growth is inhibited bycAMP (6,19), the nucleotide stimulates the proliferation ofADPKD cells by activating the MAP kinase pathway (4). Thus,mutations in either PKD1 or PKD2 may radically alter the phenotypicresponse of renal epithelial cells to the growth effects ofcAMP (1). Evidence derived from other cellular systems indicatesthat the mitogenic action of cAMP may be cell-specific (20,21).
cAMP also stimulates the transepithelial secretion of NaCl andwater, thereby adding fluid to the cavity created by the proliferatingmural cells (2,3). Thus, common agonists that promote physiologicactions by regulating the adenylyl cyclase pathway, e.g., AVP,PGE2, and -adrenergic compounds, have the potential to acceleratethe growth of cysts in polycystic kidney disorders.
In classic studies, caffeine was used to inhibit PDE and therebyraise intracellular levels of cAMP (22–24). Caffeine wasused for many years as a reagent to block PDE in biochemicalstudies until more potent (1-methyl-3-isobutyl xanthine, theophylline)and specific (rolipram) inhibitors became available. In thecurrent study, we found that caffeine, IBMX, and rolipram, andto a lesser extent, theophylline, increased basal intracellularlevels of cAMP in renal epithelial cells derived from patientswith ADPKD (Figure 1). This finding indicates that PDE inhibitionprevents the metabolism of cAMP and suggests that there is alow level of adenylyl cyclase activity in ADPKD cells in theabsence of exogenous agonists. More importantly, the PDE inhibitors,including caffeine, potentiated the effects of submaximal concentrationsof receptor-mediated agonists of physiologic importance to elevateintracellular levels of cAMP (Figures 2, 3, and 6). Thus, byinhibiting PDE and reducing the metabolism of cAMP, amountsof caffeine too low to affect cAMP levels alone in resting renalcells can potentiate the actions of common adenylyl cyclaseagonists to stimulate the growth of cysts in ADPKD.
In a recent study, levels of extracellular adenosine triphosphate(ATP) in the sub-micromolar range were found to activate specificpurinergic receptors and stimulate chloride secretion in humanADPKD cells through an intracellular calcium signaling mechanism(25). In the current study, we determined that adenosine, thebackbone of ATP and a major renal autocoid, strikingly increasedthe accumulation of cAMP by ADPKD cells (Figure 6). This effecton cAMP levels was potentiated by rolipram, a selective inhibitorof PDE (5), supporting the view that adenosine signaling inADPKD cells involves the activation of adenylyl cyclase.
The interaction of methylxanthines with adenosine is complicatedby the fact that, in addition to inhibiting PDE, they may blockA1 and A2 receptors, thereby influencing the generation of cAMP(5). Binding of adenosine to A1 receptors blocks adenylyl cyclasethrough the activation of a Gi inhibitory mechanism. By contrast,binding to the A2 subtype activates adenylyl cyclase througha Gs stimulatory protein (26–28). Rolipram, which lacksadenosine receptor affinity and primarily blocks PDE, elevatedbasal levels of cAMP (Figure 1) in ADPKD cells and stronglypotentiated the effect of adenosine to increase intracellularlevels of the nucleotide (Figure 6). By contrast, caffeine didnot potentiate the action of adenosine. Rather, this methylxanthinedecreased the levels of intracellular cAMP generated by adenosinein a concentration-dependent manner (Figure 7). These experimentsare most economically interpreted to indicate that caffeineinhibition of A2 receptor activation in ADPKD cells obscuresany effect of the methylxanthine to block A1 receptors.
We examined the extent to which cAMP generated by caffeine’spotentiation of receptor-linked physiologic agonists affectedmechanisms that have been shown to be important in the promotionof cyst enlargement. Net transepithelial chloride secretioncoupled to the movement of sodium chloride and fluid into thecyst cavity are stimulated by a host of receptor-mediated agonistsincluding vasopressin, one of the most important. The V2 receptoragonist, DDAVP, increased intracellular levels of cAMP in ADPKDcells (Figures 2 through 5), and this effect was increased furtherby concentrations of caffeine in a clinically relevant range(Figure 8). Although the combination of caffeine and DDAVP wasnot synergistic, the additive effects of these agents to increasecAMP levels would promote the secretion of NaCl and fluid intorenal cysts. It is interesting to note that caffeine has alsobeen shown to stimulate chloride transport directly, independentlyof cAMP (29). The extent to which this mode of chloride transportmay be operative in ADPKD cells was not explored in the currentstudy.
cAMP is mitogenic in mural cells from ADPKD cysts but not incells cultured from normal renal cortex (4,18). In ADPKD, thenucleotide activates the classic MAP kinase pathway distal toRas in a way that complements the action of receptor-tyrosine-kinaseagonists such as epidermal growth factor (EGF) (4,20,21). Inthe current study, we evaluated the effect of caffeine on theactivation of ERK, an important intermediary in the MAP kinasepathway. Caffeine alone slightly increased P-ERK levels in ADPKDcells and greatly potentiated the effect of DDAVP and PGE2 onP-ERK expression (Figure 9). P-ERK is thought to be a key intermediatein the pathway leading to cell proliferation in ADPKD cells(4,20,21). The evidence in this study is therefore consistentwith the view that caffeine, by inhibiting PDE, promotes theaccumulation of cAMP in response to endogenous adenylyl cyclaseagonists, thereby increasing cellular proliferation and transepithelialfluid secretion in renal cysts.
The clinical relevance of the current studies depends on theextent to which cultured renal epithelial cells reflect thefunction of the in situ precursor cells. It is important tonote in this regard that the stimulation of transepithelialfluid secretion by cAMP agonists has been demonstrated in intactrenal cysts in situ and in vitro in studies of cells culturedfrom cysts (8,30,31). Experiments utilizing a murine model ofpolycystic kidney disease have also shown that renal and urinelevels of cAMP are abnormally increased (32). Previous studieshave shown that cAMP activates ERK in cultured ADPKD cells butnot cells from normal kidneys (4). In a rat model of ADPKD,activation of ERK has been demonstrated in whole kidney extractsby Western blot and in individual cysts by immunocytochemistry(33). Activated ERK has also been detected in the epithelialcells lining ADPKD cysts (34). Thus, the data from cell culturestudies, though indirect, support a role for cAMP in fluid secretionand cell proliferation in ADPKD.
The oral administration of caffeine to Han:SPRD rats with ADPKDfor 5 mo did not alter renal morphology or size appreciably;however, the methylxanthine elevated mean arterial BP significantly(7). GFR was 33% lower in the treated than in the untreatedcystic animals, although this reduced level did not reach statisticalsignificance, probably owing to the small number of animalsin the treated group. An examination of the effect of caffeineon GFR and fibrosis markers in larger numbers of Han:SPRD animalswould be interesting in this regard.
The extent to which caffeine may elevate renal tissue levelsof cAMP depends on the amount that is ingested and the rateof metabolism by the liver (35–37). Pharmacokinetic studiesindicate that the ingestion of 1 to 3 cups of coffee leads toplasma caffeine levels between 10 to 50 µM in 30 to 90min. Correspondingly higher levels of caffeine would be expectedwith greater ingested amounts of caffeine. Given the availabilityof coffee shops and huge containers of carbonated beveragescontaining caffeine, it would not be unexpected to find usageto exceed 1000 mg per day in the more avid drinkers.
The peak levels of caffeine that may be achieved in renal tissueafter dietary ingestion are unknown. The extent to which caffeinemay be secreted into the urine by the organic anion transportmechanism is unknown as is the extent to which the methylxanthinemay be accumulated in the interstitium of the renal medullaby the counter-current multiplier mechanism. It is interestingto note that the Olympic Committee has established a urine caffeineeligibility limit of 62 µM.
In view of the knowledge that cAMP increases cyst growth andthat caffeine is used widely by coffee, tea, and carbonatedbeverage drinkers, the Polycystic Kidney Foundation has recommendedthat patients with PKD reduce or eliminate the use of thesesubstances. The evidence presented in the current study supportsthat recommendation.
Acknowledgments
This study was supported by grants from the National Institutesof Health (P01 DK53763, P50 DK57301) and the Polycystic KidneyFoundation. We thank James Calvet, PhD, and Lawrence Sullivan,PhD, for helpful discussions.
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Received for publication April 1, 2002.
Accepted for publication May 14, 2002.
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Top
Abstract
Introduction
-adrenergic agonists) and inhibitors of phosphodiesterase (caffeine, theophylline) consequently have the potential to accelerate the progression of ADPKD (4–7). However, no direct studies of renal cells derived from human ADPKD cysts have examined the hypothesis that caffeine in concentrations that are clinically relevant has the potential to adversely affect renal cyst enlargement by promoting increased intracellular levels of cAMP. 
