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Indomethacin secretion in the isolated perfused

proximal straight rabbit tubule. Evidence for

two parallel transport mechanisms.

D de Zeeuw, … , H R Jacobson, D C Brater

J Clin Invest.

1988;

81(5)

:1585-1592.

https://doi.org/10.1172/JCI113492

.

We studied indomethacin as a probe of anion transport across the isolated perfused

proximal straight tubule of the rabbit and discovered that a substantial component of

transport may occur by anion exchange at the basolateral membrane. Various perturbations

involving direct or indirect dissipation of the cellular sodium gradient (ouabain, sodium- or

potassium-free solutions, cooling to 18 degrees C) resulted in only a 50% inhibition of

indomethacin transport, which raised the question of a co-existent alternative pathway for

secretion. Similarly, the anion exchange inhibitor, 4,4'-diisothiocyanostilbene (DIDS),

diminished indomethacin secretion by only 50%. Cooling followed by DIDS or the reverse

sequence resulted in additive inhibition such that the combination abolished active

secretion of indomethacin. We conclude that active secretion of indomethacin by the

proximal straight tubule appears to be in part sodium gradient dependent; the remainder

may be driven by an anion exchanger on the basolateral membrane.

Research Article

Find the latest version:

(2)

Indomethacin

Secretion in the Isolated Perfused Proximal Straight Rabbit Tubule

Evidence for Two Parallel Transport Mechanisms

Dickde Zeeuw,* Harry R. Jacobson, and D.CraigBratert

Departments ofInternalMedicine andPharmacology, University of TexasHealthScience Center atDallas,Dallas, Texas75235; *University ofGroningen, Groningen, TheNetherlands;and

tIndiana

UniversitySchool ofMedicine,Indianapolis, Indiana46202

Abstract

Westudied indomethacin as a probe of anion transport across theisolated perfusedproximal straight tubule of the rabbit and

discovered that a substantial component of transport may occurby anion exchangeatthe basolateral membrane.Various

perturbations involving director indirect dissipation of the cel-lularsodiumgradient (ouabain, sodium- or potassium-free

so-lutions, cooling to 18°C) resulted in only a 50% inhibition of

indomethacintransport, which raised the question of a co-ex-istent alternative pathway for secretion. Similarly, the anion exchangeinhibitor, 4,4'-diisothiocyanostilbene(DIDS),

dimin-ished indomethacin secretionbyonly50%. Cooling followedby

DIDS or the reverse sequence resulted in additive inhibition such that thecombination abolishedactive secretionof indo-methacin.

We conclude thatactive secretionof indomethacin by the

proximal straighttubule appears to be in partsodiumgradient dependent; the remaindermay bedriven byananion exchanger

on the basolateral membrane.

Introduction

Thesecretion ofawide

variety

of both endogenousand exoge-nousorganic anions is one of theexcretory functions ofthe kidney (1, 2). The latter include numerous drugs. There is

generalagreement that thesite of action oforganic anion se-cretion ispredominantlytheproximal straightrenaltubule,in

particular the S2segment

(3).

In contrast, the mechanism of

secretion is much debated. Different carrier-mediated and

passive

transport processeshavebeen

suggested

to be present across the basolateral aswell as the luminal epithelial cell

membrane.Anactive

sodium-coupled

aniontransport process appearstobeaprimarycandidate for the transfer of the anion

from bloodtocell (4,

5),

whereas

passive

diffusion

and/or

an anionexchangemechanismhave beensuggestedtoplayarole in transport acrossthe luminal membrane (6). However, re-cent reviewson renal aniontransport byM0llerand Sheikh(1)

This workwaspresented in partattheannualmeetingof the American Society ofNephrologyin 1985.

AddresscorrespondencetoDr.Brater,ClinicalPharmacologySec tion, WishardMemorial Hospital,WestOutpatient Bldg,Room316, 1001 W. 10thSt.,Indianapolis,IN46202. Addressreprintrequeststo

Dr. deZeeuw, Dept. ofMedicine, Div. ofNephrology, University

Hospital, 59Oostersingel,TheNetherlands.

Receivedforpublication10July1987 andinrevisedform20

No-vember 1987.

andGrantham(2)illustratethat many other transport systems mayalso be involved in the secretion of organic anions.

Theabovementioned characteristics of the transport sys-tem have derived, for the most part, from studies using

p-aminohippurate

(PAH)'

as a probe. Although the choice of PAHas a prototype seems logical (low protein binding, high

secretionrate, nointracellular metabolism,easy chemical

de-termination), extrapolation ofPAH studies to a general con-ceptof anion secretory mechanisms may be incorrect for sev-eral reasons. First,to allow quantitation, PAH transport mea-surements are oftenperformed at high concentrations of the probe. Thus, the transport mechanism(s) operative at such

concentrationsmay not be applicable to drugs that are present

in blood at much lower

(pharmacological)

levels. Second, thereisevidence that theaniontransport system may be

di-vided intosubsystems, each with specificity for certain anions

(1).

Moreover, the relative contribution of these multiple

mechanismsto drugsecretionmay be dose dependent. Hence,

data withPAH may notextrapolatewell toother compounds. For these reasons we chose the isolatedperfusedrabbit proxi-malstraighttubule(PST)to study the processes involved in the renal secretion of another organic anion, the NSAID

indo-methacin, which is highly bound to plasma proteins and is

actively secreted. Moreover, weperformedthesestudies using pharmacological concentrations ofthe drug. In doing so, we

found evidence fortwoparalleltransporters.

Methods

Isolatedperfusedtubule. Thetechnique ofperfusingthe isolated PST was used as previously described by Burg (7, 8). Briefly, slices were cut from kidneys of 1.5-2.0-kgNewZealandWhite rabbits.A PSTwas

dissected free hand in cooled(40C)ultrafiltrate-like solution(in milli-molar: 105, NaCl; 5, KCI;25, NaHCO3;2.3, Na2HPO4; 1, MgSO4;

10,

sodium acetate; 5, alanine; 8.3, glucose; and 1.8,

CaCl2;

anditwas

bubbled with 95%

02/5%

CO2toreachapH of 7.40). Onlycortical nephron segments of the PSTwereused. Tubuleswerebathed in the ultrafiltrate-like solution with5vol%of filtered fetal calfserum(Gibco, GrandIsland, NY). Thebath wasexchanged continuouslyat aflow rate of - 0.5ml/min. The tubulewassealedintoacollectionpipette using SylgardNo. 184(DowComing Corp., Midland, MI).Inone set

ofexperimentsasodium-freesolutionwasused. For this purpose the sodium ionwasreplaced byN-methyl-D-glucamine (SigmaChemical Co.,St.Louis, MO)(inmillimolar: 140,N-methyl-D-glucamine Cl; 5,

potassiumacetate;30, N-methyl-D-glucamine HCO3 (made from N-methyl-D-glucamineClbybubblingwith 20%CO2);2.3,KH2P04; 1, magnesium acetate; 8.3, glucose; 5,alanine; 1.8,calciumacetate.Fetal calfserumwasexhaustivelydialyzed againstdouble-distilledwater to

remove thesodium,andwasthen addedtothebathingsolution.Less

1.Abbreviationsused in thispaper:DIDS,4,4'-diisothiocyanostilbene;

Jindo, indomethacin flux; J, volume flux; PAH,p-aminohippurate;

PST,proximal straighttubule.

J.Clin.Invest.

©TheAmerican

Society

for Clinical

Investigation,

Inc.

(3)

than 10-7 M of sodium, measured by atomic absorption, could be detected inthis sodium-free bathing solution. The potassium-free

me-dium used in anothersetof experiments was prepared as ultrafiltrate-likesolutionsubstituting5mMKCIby 5 mM NaCl.Inthis protocol, dialyzedfetal calf serum was usedtoprepare thebathing solution. In all experiments,indomethacin(Merck, Sharp & Dohme Research Labo-ratories,WestPoint, PA)aswellasthecompetitive inhibitors sodium PAH (Eastman-Kodak Co., Rochester, NY), probenecid (Sigma Chemical Co.), ouabain(ICN, Irvine, CA), and 4,4'-diisothiocyanos-tilbene(DIDS) (lot04065,Polysciences, Inc., Warrington,PA)were

preparedin the bathingsolution to the desired concentration.All pi-pettesused in theperfusion set-upweresiliconized.

Transepithelial voltage (PD, inmillivolts)wasmeasuredbymeans

of anagarose Ringer's solution-calomel electrode series. Exhaustively dialyzed, tritiated inulinwasaddedtotheperfusateas a marker for volumeflux

(J4).

Indomethacin transport measurement. A 45-minequilibration pe-riodpreceded the actualperfusion experiments. Each indomethacin flux

(Jind.)

measurement(uptothree pertubule)wasbracketed bya

measurementofJu,and a 15-minequilibration separated each pertur-bation. Jo, measurements were made with a50-nl constantvolume pipette,and a 110 nl constant volume pipette was used for indometh-acin (seebelow). Only one collection for indomethacinmeasurement

wasmade per perturbation to allow for multiple perturbations within the timespanof one experiment inatubule. The accuracy of sucha

singleJindo measurementwastested infour tubules byperformingthree consecutive periods of indomethacin collections (1 5-min equilibra-tion,J,, Jindo, andJ.).Thus, theinterval between these Jindo

measure-mentswas comparablewith thatusedin theactualexperiments.The

difference among these repetitive sampleswas4.2±3.5% (mean±SD). Allindomethacin sampleswereimmediately transferred to a glass tube (seebelow) and storedat4VC in the dark.

Transfer and measurement ofthe indomethacin sample. The tech-nique used to transfer thecollected indomethacin sample has been described elsewhere(8).Briefly,during collection and transfer, evapo-rationof the sample was prevented byuseof gamma terpinene, an oil thatdidnotinterfere with the indomethacinmeasurement.The 110-nl indomethacin sample was immediately transferred to a small glass tubecontaining 640 nlwater(pH=7.4)containing internal standard (phenylbutazone).

Sampleswereimmediately assayed forindomethacin by HPLC as previously described (9).Asemi-microbore column(10Mm)(Bondpak C18; Waters Assoc., Div. ofMillipore Corp., Milford, MA) allowed small sampleinjections and increased the sensitivity of the measure-mentsby increasing the concentration of drug in the detector. The mobile phaseconsisted of water and methanol (45:55). The flow rate was 130

gl/min,

pumped with a modified Waters solvent delivery system(model 6000 A; Waters Assoc.).Amicroflow cell (1.9Mll)was used in theultraviolet (UV) detector (model 440; Waters Assoc.). Di-rectlyafter the UV detector, 4NNaOH was added to the mobile phase throughaT-connector at aflow rate of 1.3

3,l/min,

thus maintaining an optimal pH of 12.75 in a reaction loop (130

Al,

640C).Thus, indo-methacin was hydrolyzed to a reaction product that could be measured

atlow concentrations by fluorescence detection using an excitation wavelength of 295 nm and emission wavelength of 376 nm (model 650-10 S; Perkin Elmer Corp., Instrument Div., Norwalk, CT). The intraassay variability of 10-7 and10-6Mindomethacin standards was 5 and3%, respectively. Day-to-day coefficients of variation were 6 and 2%, respectively. Recovery ofindomethacin for collected and trans-ferred samples was 99±12%.

Calculations. Jo, was determined by the following formula:

J,

=(Vi - V)/L,where

J,

is rate ofnet volume flux in nl *mm-'*min-', Viis perfusionratein nanoliters, Vt,is collection rate in nanoliters, and L is tubularlength in millimeters. The twoJ.sperperiod that bracketed the

JAndocollection wereaveraged. Jindowasdeterminedbythefollowing

formula:Jindo =[ Vi (perfusate indo)-

Vt,

(collectate

indo,)]/L,

where Jindo is rate of indomethacin secretion in fmol *mm- min-', and

indo,

is measured concentration ofindomethacin in micromolar.

Sinceperfusate

indo,

iszero,thisequation simplifiesto:Jindo = [-Vo (collectate

indoj)]/L.

Unless notedotherwisemean±SD are given. Statistical analysis wascarriedoutusinga(paired) ranktest(Wilcoxon).

Results

Indomethacinsecretion rate. The effect of various concentra-tions of indomethacin onJvand PD is shown in Table I. The average tubularlengthwas2.5±0.6 mm. PD and

J,

were mea-suredduring a control period and compared with a period with different bath concentrations of indomethacin ranging from 10-6 to 3 X l0-s M. In 12 of thesetubules, multiple (up to threepertubule) indomethacin concentrations were tested in random order. Table I shows that indomethacin did not signif-icantly affect PD orJo atthe drug concentrationstested, al-though Jvtended to decrease at the highest bath concentra-tions.

Jindo rates were measured in 22 PST segments (length,

2.5±0.6 mm) at a bath concentrationof 10-6 M. The

indo-methacin concentrationin thecollectedperfusatewas2.9±1.3 X 10-6 M, indicating bath-to-lumen transport against a chemical gradient; calculated Jindo averaged 16.8±8.8

fmol*mm-'*min-'. Mean PD and Jv were -1.4±0.4mVand

0.39±0.15 nl *mm-'*min-', respectively. Values forJindodid notcorrelatewithPD orJ,

In 9 of these22 tubules and in an additional 8 PST seg-ments, Jindo was measured at higher bath concentrations of indomethacin (5 X 10-6,

10-',

1.6 X

10-5,

and 3 X 10-5 M).

AJndo

increased withincreasingbath concentrations of thedrug

(Fig. 1). If one assumes only one indomethacin transport site atthebasolateral membrane, nointracellular metabolism of indomethacin, andan unrestricted one-waytransfer of

indo-methacinacrosstheluminalmembrane,Michaelis-Menten

ki-neticscan beapplied tothecurvilinear relationship between

Jindo and bathdrug concentration. The inset ofFig. 1 shows such a curve transformation. Although this derivation gives theimpression that indomethacin secretion at leastpartially

follows Michaelis-Menten kinetics, the deviation from a

straight line issuchthatparallelorsequentialtransport

mecha-nismsshould not beexcluded.

Effect

of other organic anions on Jindo. To test whether the

observedindomethacin secretion followedasimilar

transcellu-lar pathwayasdescribed forother organicanions, the effect of twoprototypic substrates fortheorganic acidtransportsystem

(PAH and probenecid) on Jindo was evaluated (Fig. 2).

TableL. Effect of Varying Indomethacin Bath Concentrations on PDandWaterReabsorption(Jo)in PSTSegments

PD J.

[Indomethacin]b,h Control Indo Control Indo mV ni.mm-'.min-'

106 M (n=7) -1.4±0.3 .-1.6±0.2 0.48±0.16 0.44±0.15

5X10-6M (n=7) -1.2±0.4 -1.2±0.3 0.40±0.13 0.35±0.18

10-'M (n= 11) -1.3±0.4 -1.4±0.5 0.39±0.12 0.34±0.15

1.6X 10-5M

(n=6) -1.5±0.4 -1.6±0.3 0.40±0.10 0.33±0.11 3X 10-5M (n=6) -1.5±0.4 -1.6±0.4 0.40±0.10 0.35±0.17

(4)

-1ZLI

,-E (7)

E~~

100 ,,'

E

-6 80 C* 2

tCC

c 60 | 15

401 M9)8 X

9-,t

A'~~~~~~~~~~~mQlx

~ ~ ~~~~~~~~r=-0Q921

=22S fmol.mm-1. mind1

20 (22)/ 3 Km=18x1046M

;

Ka~~~~1-6

k-5 2;- 15M

0

to-6

10-5

2.i0-5

3.;0-5

M

[ Indomethacin]

Figure 1. Effect (mean±SEM) of varying indomethacinbath

concen-trationsonbath-to-lumentransport

(J1d.)

inthePST.Theinset showsacurvetransformation (Hanes plot). The values in

parenthe-sesrepresentthenumber of tubules studiedat thegiven

indometha-cinconcentration.

First, the effect ofbath concentrations of PAH ranging

from 6 X

1-'

to 4X 10-3 M onindomethacin (10-6M bath

concentration) secretionwasmeasured in sevenPST. Aftera

controlmeasurement,Jindowas measuredafteraddingPAHto

the bathatthedesired

concentration,

starting with either the highestor the lowest PAHconcentration, followedbyanother two PAH

concentrations

in random order, and a recovery

period.

AttheseconcentrationsPAHhad no

significant

effect

on PD

(-1.7±0.3

mV atthe lowest to -1.8±0.1 mV atthe

highest bath concentrations). In contrast, J, decreased by 33±16% of controlatthehighestPAHconcentrations.Atthe

end ofthe

experiment

Jo recovered to 82±10% of control

values.With increasing bath concentrations ofPAH,Jindo de-creased reciprocally from a control value of 19.2±9.4

fmol

-mm-' min'

to < 17% of control

Jindo

atthe

highest

PAHconcentration used(4X 10-3 M)

(Fig. 2).

Atthe end of

theexperiment,

Jind0

recoveredto 103±30% of control values.

n'

a \II

dda*\~~~~\

U'~~~\

I Np

I 0~~~'

I-~~~~~~

10-4

1Inhibitor]

10-3 10-2M

Figure 2. Effect ofvaryingbathconcentrations ofprobenecidand

PAHonindomethacin(bath concentration, 10-6 M)transportrate

(Jind.)in thePST,expressedaspercentchange comparedwith control.

In an additional five experiments a similar dose-dependent

inhibition of indomethacin (10-6 Mbath) secretion was

ob-served by adding varying concentrations ofprobenecid to the bath (10-6to

10-4

M). Thehighestprobenecid bath

concen-trations hadnoeffecton PD (-1.6±0.1 vs. -1.8±0.2mV),nor on

Jh,

(0.46±0.14 vs. 0.47±0.14 nl

mm-'

-min-').

Fig. 2

shows that

10-6

M probenecid had no significant effect on

control

Jind0

(18.4±10.5 fmol-

mm-'

*

min'),

whereas 10-4M

nearlycompletely inhibited indomethacin secretion.Inall

ex-periments, completerecovery(92±5%)tocontrolJindovalues was observed 30 minafter withdrawing probenecid from the

bathing solution. Apparently, both organic anionscompetefor indomethacin bath-to-lumen transport, and both PAH and

probenecidare able to nearlycompletelyabolish indometha-cin secretion. Although the

inhibition profile

showsasimilar

pattern, probenecid is a much more potent inhibitorthan

PAH: -100 times less probenecid than PAH is needed for half-maximal inhibition of

Jindow

Temperature dependency of

Jj,,Mis

The effect of cooling to

180C

on Jindo is depicted in Fig. 3. In thefirst setof

experi-ments (n = 12), bath temperaturewas lowered from

380

to

180C.

Asalsoshown byothers(10, 11), coolingto

180C

was

sufficient to totally inhibit net volume reabsorption in these

PSTsegments,which isreflected by the decrease ofcontrol

J"

from 0.39±0.17 to -0.02±0.04 nl

mm-' min-'.

Similarly, cooling inhibited indomethacin secretion from 24.0±12.0 to

10.5±2.9 fmol-

mm-'

*

min'.

However, as can be seeninFig.

3, in none ofthe tested tubules wasindomethacintransport

completely abolished by

cooling,

the mean inhibition

being

48±15%.Toascertain whether the inhibition of

AJnd0

wasstable

with time, the cooling periodwasextendedto 60min infour of

the above

experiments.

No furtherdecrease in Jindo was

ob-served

comparing

15 min

cooling

(51±6%) with 40-min (51±7%) and with

1-h

cooling (54±6%).Infour ofthecooling

experiments,the

bathing

solutionwasreheatedto

370C,

lead-ingto arecoveryof

Jindo

to82±10% of control.Inthe remain-ing four experiments, probenecid

(10-4

M)was added to the

bath

during

continued

cooling.

Thisresulted in afurther de-crease of

JAndo

to < 15% of

control,

such that

negligible

amounts of

indomethacin

could bedetectedin the collected

perfusate

of three ofthefour PSTsegments.

To further explore the temperaturedependency of indo-methacin transport asrelated to active fluid

reabsorption

in

thePST,anadditional setof

perfusion

experimentswas

per-formed (n = 5). After control

Jindo

andJo measurements at

380C,

the bath temperaturewaslowered in four consecutive

steps

(330, 250, 180,

and

120C, respectively).

Ineach of these

C00

so- Figure3.Effect of

cool-100 t t 50 \ ing(180C)andrecovery

80 tocontrol bath

temper-o

E \ ature

(380C)

on

bath-ak

60

30

to-lumen indomethacin

50 V E

trantransport

(Jindo)

in the

*~~~~50

~~~~~PST,

expressedas

per-40 centchange compared

with control(left)and

0i .-. . asabsolute fluxrates

38C 1"C 3eC 3 18°C 380C (right). The shaded area (right)indicates the mean±SD ofthecalculated fluxrate,assumingtheindomethacin concentration in the lumenequalsthat in thebathingsolution

(10-6

M).

Indomethacin Secretion

by

Proximal

Straight

Tubule 1587

L-

(5)

-periods

Jindo

and

Jv

weremeasured.Jo,decreased from 0.45±15 to 0.30±0.08 (330C),to0.11±0.04(250C), andto0.00±0.03

nl mm-'

min'

at 18'Cbath temperature. No further de-crease of

Jv

was observed at

12'C

(0.00±0.01). Whereas a

maximumdecrease of

Jv

wasreachedatthe bath temperature

of 18'C, Jindo inhibition atthis temperature was 53±14% of

control,consistent withprior experiments.Byfurther

decreas-ing bath temperature to

12'C,

Jindo decreased to 31±6% of control values.Fig.4 shows Arrheniusplotsof bothJvand .Jndo

calculatedfrom all tested bath temperatures.AssumingthatJv solely reflects active sodium transport across the basolateral

membraneby

Na',K+-ATPase,

one can calculate the

activa-tionenergy of this enzymetobe between 15.5 and 22.6 kcal/ mol(12).Theprofileof the ArrheniusplotforJindois difficult to interpret without

knowledge

of the

carrier(s)

that is (are) involved. However,the dissimilaritybetween the

Jv

andJindo

plots suggeststhatindomethacin secretionisnot

solely

linked to anenergy-dependenttransport process.

Sodium

dependency of

Judo

To more

clearly distinguish

between changes in indomethacin transport and changes in

active sodiumtransport in the PST,aseries ofperturbations

were used.First, the effect of adding 10-5 M ouabain tothe

bathingsolutionwastested in five tubules. After 10min

oua-bain had completely inhibitedwaterreabsorptioninthese

tu-bules(control

Ju,

0.40±0.20to-0.04±0.03 nl mm-'*

min').

Jindo (control, 36.7±12.4 fmol*

mm'

*

min-),

however, was

inhibited by only 47±18%

(Fig.

5,

left).

Toinhibit active

so-diumtransportbyadifferent

mechanism,

wealso substituted

N-methyl-D-glucamine for sodiumin thebathandperfusate.

In three experiments with this sodium-free medium, J,, de-creased from 0.33±0.10

(baseline)

to -0.01±0.05 n1 mm-' min-'.

Again, Jindo

(control, 47.1±13.4 fmol mm-'*min-')wasinhibited submaximallyby 36±3% (Fig. 5,

right).

In the lastseries of experiments (n = 3), bathing and

perfusion

solutionwere substituted by potassium-free media.

Jo,

was

totally

inhibited

(from

0.27±0.03 to 0.02±0.01 nl *mm-'*min-) by thismaneuver,whereasJindoremained at alevel40±4%ofcontrol(34.7±13.1 fmol-

ml-'

min-').

Like

109I indo 1.4

1.3

1.2

11

1.0'

0.9s

0.8

0.7

380 330 25' 18' 12'C

togJv

-0.3

-0.5

-0.7'

-0.9.

-1.1

-1.3.

-1.5

-1.7.

380 33* 25' 18' 12'C

32 33 34 35 36 32 33

341

35 36 (temperature)-lx104('K1)

Figure 4. Arrheniusplotsofbath-to-lumenindomethacin (bath

con-centration, 10-6M)transport(left)andlumen-to-bath net water

reabsorption (Jv,right)inthe PST measured at different bath tem-peratures.

100

80

60

40

20

C

b

Io ouaboi

r-Figure 5. Effect of ouabain (bathconcentration, 10- M) (left)andreplacementof the bathand lumenNa' by N-methyl-D-glucamine(right)on indomethacin (bath

concentra-tion, 10-6 M) transport in the PST.The shadedareas repre-sentthe mean±SD changeof indomethacintransportduring zeroNa' cooling (1 8C).

the resultsduring18'Ccooling, the above perturbations

com-pletely inhibited sodium transport (estimated from

J),

whereas adistinct component of indomethacin transport

re-mained uninhibited. These data raised thequestionof an in-domethacin transport componentindependent of

Na'

trans-port.

Effect of

DIDSon

Jindo.

Since it has been suggested that

anion exchange may play a role in transport of anions in the PST (13-16), weevaluated the effects ofan anion-exchange

inhibitor (DIDS) on

JiAnd.

To find anoptimal dose of DIDS,

dose-responseexperimentswerefirst conducted. Fig.6 shows the effect onJindo of adding DIDS to the bathing solution of

fivePST segments.Consecutive increases inthe doseof DIDS

incrementally decreased Jindo to a maximum inhibition of 56±6%of control (23.4±2.5 fmol-

mm-'

*

min-').

Theplateau wasreachedwith

I0-5

MDIDS. Atlower bath concentrations,

DIDS had littleeffectonJ.; onlyat

10-4

MdidDIDS tendto

inhibit!,,,

from 0.37±0.14to0.25±0.13

nl-mm-'

*min-'.

Infour PSTsegments theeffect of

10-4

MDIDSfollowed bysuperimposed

cooling

of the bathing solutionto 18'C was

evaluated. DIDS marginally reduced

Jv

from 0.38±0.05 to

0.30±0.07 nl mm-I

min-';

cooling further decreased

Jv

to

-0.03±0.01

nlo

mm-'* min-'.

DIDS inhibited indomethacin

transport to 54±6% of control values (30.4±4.5

fmol-mm-'*

min',

Fig. 7, top). Superimposedcooling fur-therdecreased Jindoto 14±3% of control. The effect ofadding

DIDS to acooled tubulewasevaluated inanother fourPST segments. Cooling decreased

J,

from 0.40±0.20 to

-0.03±0.04 nl mm-'1

min-',

whereas superimposed DIDS did not further alter J,, significantly (-0.01±0.05

nl *

mm'

*

min').

Cooling inhibitedJindoto45±4.5%of

con-100- 25

.~80- .20

60-

'15

~0 10

20 5

0., 0.

-10-6 10-5 10-4M 10-6 10-5 10-4M

[DIOS]

Figure6.Inhibitory effectof increasingbath concentrations of DIDS

onindomethacin(bath concentration, 10-6M) transport in thePST, expressedasthepercent change compared with control(left)and as

absoluteflux rates (right).

(6)

c

20

E 151

E

E

10-R

51

E

0

E

so

c_

_%n

38C 38C 180C

DIDS DIDS

36-

30-24

18-12

6

380C 380C 18C

0ols DIOS

Figure7. Effectof cooling (18C) and superimposedDIDS (bath

concentration, I0- M)onbath-to-lumenindomethacin(bath

con-centration, 10-6M)transportinthePST(top),and the effectof

DIDS andsuperimposed coolingonindomethacintransport (bot-tom).Theplotsonthe leftrepresentthepercentchange compared

with control; the plotsonthe rightrepresentthe absoluteJind.rates.

trol(23.9±3.1 fmol*mm-'

min-'),

whereasadding DIDSto the bath further inhibitedJindoto 14±3% ofcontrol, avalue

statisticallynotdifferentfromzero(Fig. 7, bottom).

Discussion

The present study documents the active secretion of the

or-ganic anion indomethacin across the rabbit renal PST, and addressessomeof theunderlying mechanism(s)forthis

trans-port. Ingeneral, indomethacin behaves like otherorganic

anionstested forsecretionbythePST. At the low (pharmaco-logic) bath concentrations used in thisstudy, indomethacin

wassecreted into the lumen of the PST againstaconcentration

gradient. The process appears tobe saturable at higher bath concentrations ofthe drug; further, secretion is completely inhibited by probenecid and PAH, suggesting atranscellular routefor indomethacintransport.Therelatively high

concen-trationsofcompetitorsneeded for inhibition indicate that in-domethacin has one of the highest affinities for the carrier

quantifiedtodate(2).Variousexperimental approachesinthe

presentstudy revealed dependence ofindomethacinsecretion

on the sodium gradient. Interestingly, however, only about

half of the indomethacin transport appeared susceptible to perturbationsofthisgradient, leavingaconsiderableamount ofdrugsecretion viaindependent mechanism(s). The remain-ing portion of Jindowaseliminatedbytheanion-exchange in-hibitor DIDS, raising the important question of whether a

basolateral anionexchangeprocessaccountsfor theother half of indomethacin secretion.

Beforeinterpretingourobservationsin thecontext of the currentconcepts of anion transport bythe PST, oneshould

address the possible biasintroduced by the study design and

thechoice of indomethacin as thestudied compound. Three

issues may have confounded our experiments: first, protein

binding of indomethacin; second, cellular metabolism of

in-domethacin; and lastly, the absenceofinformation on

indo-methacintransportprocesses across the luminal membrane. As far as protein binding is concerned, indomethacin is

highlybound toalbumin, with various estimates ranging from

90to >99% (17, 18). For reasonsofstandardization and

via-bility ofthestudiedtubule, we added 5%fetalcalfserum to the

bath.Inthis setting, protein binding of indomethacin(1o-6M) was 65±3% (ultrafiltration technique using 14-mm MPS YMT

filters [Amicon Corp., Danvers, MA]). The free amount of drug increased with higher bath concentrations of

indometha-cin (percent binding decreased to 50±4% at 1O-4 M indo-methacin). Ifoneassumes that only the unbound fraction of

the drug in the bathing solution is available for secretion,the

actual

Vm.

and Km arelower than thevalues we calculated.

However,correctingthe dose-response curvefor free

concen-trations of indomethacin did not affect theinterpretation of Fig. 1.Theassumption that freeasopposedto totaldrug

con-centration ismost relevantremains

speculative,

sinceno

con-clusivedata areavailableas towhether thetransporteris able

to

"strip"

theanion from

albumin,

aprocesswhich is theoreti-cally dependentupontheaffinity of the anion for thetransport system(19).Futurestudies should address this issue.

NeitherPAH, probenecid,cooling (11 C), or ouabain

af-fected the protein binding of indomethacin (61±3, 65±4,

62±3, and 61±5%, respectively). Hence, interpretation of these data wouldnotbeaffected byprotein binding.However,

DIDS

(10-4

M) increased the amount of free indomethacin

(48±4% bound), possibly by competition forthealbumin binding site. Not

accounting

for this effectwould have served

onlyto underestimate the

inhibitory

effectsofDIDS on indo-methacin transport, since (if only free drug is secreted) the higher free concentration of indomethacin inthe bathduring DIDScompared with control wouldhave resulted in ahigher

Jindo.

Interestingly,

review of the literature

concerning

trans-port ofPAH and other anions by the PST reveals no data

concerning protein binding

norchangesinbindingcausedby inhibitorsin thebathing solution.Althoughprotein binding of

PAH is claimedtobeverylow(- 25%)(20), largevariations

have been reported (21). As faras the current data are

con-cerned,our

analysis

is in themostconservative fashion. Any unaccounted for effects of

binding

wouldonlyserveto

amplify

our

findings.

Very little information is available concerning renal

me-tabolismofindomethacin.Inhuman

pharmacokinetic

studies, half of the clearance of indomethacin is accounted for by

0-demethylation,

and a smaller

portion

is

N-deacylated

(22).

Mostof theremainder of the active drugaswellastheinactive metabolites are

glucuronidated

and excreted via

kidney

and

bile. Whether

glucuronidation

occurs in the

kidney

is un-known. However, since only very lowconcentrations or no

glucuronide conjugates

are measured in serum, and

large

amounts have beendetectedin the

urine,

glucuronidation

may well occur in the kidney(23). Indeed, we observed an extra

peak

(nexttothe solvent

front)

in

chromatograms

ofthe col-lected

perfusate

that wasnotpresent in the

bathing

solution.

Wehave not yet been ableto

conclusively

establishthis

peak

to be indomethacin

glucuronide (or

another

metabolite).

This

may be due to the extreme

instability

ofthe

glucuronide,

100-- 80

I.-8 60-- 40

20

O- 100-- 80100-- 80-3 60

4-.'.

(7)

which can readilydissociate to indomethacin and glucuronic

acid (23). Relative to the indomethacin peak, the extrapeak was small (- 15%), and it did not appear to change signifi-cantly during the various perturbations used in our studies.

Therefore, althoughwe were not able toquantifytubular

me-tabolism(if it exists),we feltit reasonable to assume that

ob-served changesinindomethacin secretion were nottheresult

of changesinPSTintracellular metabolism.

Alloftheconclusions concerningindomethacin secretion

inthe presentstudyare restrictedtothe basolateralmembrane, basedontheassumptionthat indomethacinmovement across

the luminal membrane is driven by passive diffusion (6) or

alternatively by an anion exchange mechanism (16). Subse-quent studies should characterize the transport step at this

barriermorecarefully.

Howdo our data onindomethacintransport contribute to currentconcepts ofanion transport mechanisms in the PST? Todate twomodelshavebeenproposed. The first andoldest theory is thatananion, andinparticularthe prototypePAH,is

secretedacrossthebasolateralmembraneviasecondary active

transport dependent upon

Na'-K'

ATPase; anion then

dif-fusesacross the luminal membrane drivenbythehigh

intra-cellular concentration (6). Many studies indifferent species, using different experimental

techniques

anddesigns,have

ad-dressedthesodium dependency of this active basolateral anion

transport. Somefound PAH transport todecreasealmost

to-tally

using

either ouabain or sodium-free

bathing

solutions

(3-5, 24-28). In the present study we consistently found a

submaximal inhibition of Jindoby directlyorindirectly

remov-ing the sodium

gradient.

Thereasonfor

persistence

of

Jindo

in

these

settings

comparedto observationswith otheranionsis unclear.Inaddition, it is uncertainas towhether our data are

unique

toindomethacinorrepresentageneralaspectof anion

transport. Because ofoursensitive analytical techniques, we wereableto use a

relatively

low concentration of anion (10-6 M) compared with other studies (2

10'

M). So doingmay have revealedtheexistence ofa nonsodium gradient-related carrierthat is driven by anion orintracellular counter-anion concentrationsrather thanby the

Na'

gradientacross the

ba-solateral membrane. Because of the higher anion

concentra-tions used by other

investigators,

such pathways may have

been"swamped" and thereby undetected. In pastreviewson

the anion transport system, authors have suggested that the sodium gradient itselfwas not sufficient to accountfor the

entire PAH

gradient

acrossthe basolateralmembrane (1, 2).

This

suggestion

is supportedbythe resultsofthe present study,

inthat we were not able to completelyinhibit

JAndo

by

dissipa-tion ofthesodiumgradient.

In addition to sodium-dependent anion transport, Dantzler(16) has indirectly offered evidence that a

4-aceta-mido-4'-isothyocyanostilbene-2,2'-disulfonate (SITS)-inhibit-ablecarrier mechanismmay be present on the basolateral (as well as on the luminal) side of the tubule. Although experi-mentshavesuggested,to date none have clearly demonstrated

theexistence ofsuch acarrierindependent from

sodium-de-pendent mechanisms. Thepresent study offersdata that are

consistent

withasecondcarrier(anion exchange?)at the

baso-lateral membrane. The discontinuous shape of the dose-re-sponse curve,and the inabilitytocompletely inhibitJindoby

interference with the sodium gradient (whereas water

reab-sorption

is abolished), together with theclear dissociation of

the Arrhenius

plots

of

J,

and

Jindo, strongly

suggestthe

pres-enceofasecond carrier(29). Moreover, probenecidand PAH caninhibitthe transport processcompletely,thusmakingthe

possibility of paracellularindomethacinmovement as an

ex-planation for theuninhibitedportionof Jindohighlyunlikely.

The inhibitory capacity ofDIDS itselfonJindo, and the fact thatthesame inhibition can be achieved whenasodium

gra-dient is absent

(cooling),

points againtothe existence oftwo separate transport mechanismsonthe basolateral membrane. In thiscontext itis important to discuss our reasons for

using coolingtoshow sodium dependency during the DIDS

experiments rather than ouabain or

Na'-free

solutions. Though the latter two maneuvers are morecommonlyusedto

dissipatethesodium gradient, theyaffected the tubule

prepara-tion inourexperimentsso as topreclude performanceofmore than oneperturbation. Perhapsindomethacin hada noxious

effectsuchthat

superimposed

ouabainor a

Na'-free

environ-mentadverselyaffected

viability.

Cooling,

ontheotherhand, didnotaffect the

stability

of the

preparation,

as shown

by

the similarity of the Jindomeasurementsduring coolingaswellas the recovery dataafter

reheating

(Fig. 3).

These

practical

justi-fications foruseof

cooling notwithstanding,

this

perturbation

seems a valid probe of energy-dependent sodium transport.

Shaferet al. haveshownthe

similarity

of

cooling

andouabain

on thePST (11).Inaddition,our data showequal abolishment of Jv by

cooling

aswithouabain and

Na'-free

solutions. More-over,inhibition of Jindowassimilar with all threemaneuvers. Thus, though additivity

of

effects of DIDS plus ouabain or DIDSplus

Na'-free

solutions wouldrepresentideal confirma-tionof the DIDS plus cooling experiments,wearguethat the interpretation of the additive studies is notobviatedbytheir absence.

Intheory, DIDS could simply beacompetitive inhibitor of

theaniontransport system as wasalsosuggested by Koshieret al.(30). However, the fact that DIDS only minimally affected J,and the fact that DIDS could inhibitJindo

by

at most

50%,

makesthis unlikely. Itmight be arguedthat albumin in the

bathing solutionmay have bluntedtheeffect ofDIDS(15, 16,

30). Thisseemsunlikely, since the final concentration of

albu-min in the bath (0.1%) was below that which reversed the

inhibition ofPAH transportcaused by DIDS and SITS (15,

30). Furthermore, concentrations of DIDSwere used an order

ofmagnitude higher than those which caused maximal effects

onindomethacintransport(Fig. 6). Thus,evenifsomeof the

DIDS were bound to albumin, it is likely that enough free

concentrationwasavailabletocause amaximal effect. Although the specific effect ofDIDS on the PST is not

known, its documented effectonanionexchange in the eryth-rocyte may in theory be extrapolated to the PST. If so, the second carrier forindomethacin transport on the basolateral membrane could be an anion exchanger. In this respect the second conceptof aniontransport across theproximaltubule proposed byUllrich (31) is of interest.Several groups, using a

varietyof experimentalpreparations(kidney slices, membrane

vesicles,in vivomicroperfusion), foundPAH transport across thebasolateralmembrane to be clearly independent of a

so-dium gradient

(13,

31-34).Therefore,Ullrich et al.(14,31, 36) andalsoDantzler et al.(35) explaintheeffects of perturbations

aimed at dissipatingthe sodium gradient to be the result of

secondary changesin thetubularcellsrather than their

exert-inga specific effect on the PAH carrier. Ullrich argues for a countertransport of anion (PAH) vs. OH- at the basolateral membrane

(14).

Kasheretal. supported the model of an anion

(8)

exchange

process for PAHtransportacrossbasolateral mem-branevescicles

(37). However, they

concluded thistransport was

Na' dependent.

Asfaras the

DIDS-inhibitable

compo-nentof Jindointhe present

study,

ourdataareconsistent with

the

theory

of basolateral anion

exchange.

The

question

still

remains, however,

asto

why

we werenotabletocompletely

inhibitindomethacin secretion with either DIDSorby totally

inhibiting

sodium transport

(as

measured

by

water

reabsorp-tion).

As

such,

ourdata remainmostconsistent withtwo

trans-port

pathways

forindomethacin. Wecannotexclude the

possi-bility

that these

phenomena

are

peculiar

to use of the

tech-nique

of the isolated

perfused

tubule.

However,

one canargue

similarly

that data obtainedfromisolated membranes is less

physiological. Moreover,

in support of the concept oftwo aniontransport

pathways,

Lee etal.

(38)

observedin the

iso-lated

perfused

rat

kidney

thatfurosemide secretionwas best characterized

by

the presence ofbotha

high affinity,

low

ca-pacity

system, aswellasalow

affinity, high capacity

system.

Further studiesare

clearly

neededtoresolvesuchissues.

In

conclusion,

the present

study

shows that the NSAID indomethacin is

transcellularly

secretedinto the lumen of the

PST

by

ananiontransport mechanism likethatdescribed for

PAH. The transportis characterized

by

a low transportrate and

high affinity

for the carrier. Half of the indomethacin secretion is blocked

by

maximal inhibition of sodium

trans-portinthe

PST,

whereas the remaindercanbe abolished

by

the anion

exchange

inhibitor DIDS. The datasuggestthatat

pharmacological

concentrations,

indomethacin movement

across the basolateral membrane of the PST is the result of

sodium indomethacin

co-transport

aswell as of

indometha-cin-anion

exchange.

Ratherthan

explaining

themechanism of

"active"

basolateral anion

transport

aseither

totally

sodium

gradient

dependent

or as

totally

driven via anion

counter-transport,

we feel that ourdata aremost consistent with the simultaneous existence of both

transport

mechanismson the basolateral membrane of the rabbitPST.

Acknowledaments

Theauthors wishtothank Ms. Rebecca Aricheta for her skillful tech-nical assistance.In addition, wegratefully acknowledgethesupport

andadvice ofDr.MichelBaum,Dr. MatthewD.Breyer,andDr.Juha

P.Kokko.

This workwassupported byNationalInstitutes of Healthgrants

AM-27069 (to Dr.Brater)and AM-23091(toDr.Jacobson).Thestudy wascompletedduringasabbaticalyearofDr.deZeeuw,whichwas

supportedinpartbytheUniversityofGroningen.

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8. de Zeeuw, D., D. C. Brater, and H. R. Jacobson. 1987. Micro-HPLC and transfertechniques for directly measuring drug (indometh-acin) transportacross the isolated perfused renal proximal straight tubule. J.Pharmacol. Methods. In press.

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References

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