Kidney Holger

Involvement of Endothelium-derived Relaxing Factor

in the Pressure Control ^of

Renin Secretion from Isolated Perfused Kidney

HolgerScholzand Armin Kurtz

Institut furPhysiologie der Universitdt Regensburg, D( W) -8400Regensburg, Germany

Abstract

Using isolatedratkidneys perfusedatcontrolledpressure, we examinedapotentialroleofendothelium-derived relaxingfac- tor (EDRF) in the pressure control ofrenin secretion. We found that stimulation of EDRF release by acetylcholine (1

,tmol/liter)

increased meanperfusateflow rates from15.0±0.5 to 18.0±0.5 ml/min per g and average renin secretion rates from3.5±0.5to16.0±2.0ngangiotensinI/hper min per g at a perfusion pressure of100 mmHg(mean±SEM,n =6). Those effects ofacetylcholineweresignificantly reduced during inhibi- tionofEDRFformation with NG-nitro-L-arginine (100,umol/

liter),but they were notaffected withthecyclooxygenase inhib- itorindomethacin (10 ,mol/liter).

Loweringoftheperfusion pressurefrom 100 mmHg to 40 mmHgresultedin anincreaseofaverage reninsecretionrates from 3.5±0.5to79±12 ng AngI/h per min per g undercontrol conditions (n=8), andto171±20ngAngI/hper min per g in thepresenceof 10,mol/literacetylcholine(n=3).Theriseof renin secretion in response toa reduction ofthe renalartery pressurewasmarkedlyattenuated with inhibitors ofEDRFfor- mation suchasNG-nitro-L-arginine (100 ,umol/liter)andre- latedcompounds. During inhibition ofEDRFformation,addi- tion ofsodium nitroprusside (10 ,umol/liter)increased mean perfusateflowratesfrom 12.0±0.5to23.0±2.0ml/minper g and averagerenin secretionratesfrom 2.0±0.5to18.0±1.5ng AngI/hper minperg at100 mmHg(n= 5).Lowering of the perfusionpressurefrom100mmHgto40mmHgunderthose conditionsincreasedaveragereninsecretionrates to220±14ng AngI/hper min per g(n=5).

Taken together, our findings suggest that EDRF and re- lated activators of soluble guanylate cyclase stimulate renin secretion from isolatedkidneys, predominantlyatlowerperfu- sion pressure. Moreover, pressurecontrol of renin secretion appearstorequirethetonicalstimulationbyintrarenalEDRF.

(J.Clin.Invest.1993.91:1088-1094.) Keywords:juxtaglomer- ularcells*nitro-L-arginine*acetylcholine* sodiumnitroprus- side*guanylate cyclase

Introduction

Sincethefirstdemonstrationby Goldblattandco-workers( ), ithasbeen wellestablished that theintrarenalperfusionpres- AddressCorrespondencetoHolgerScholz, M.D., Institut furPhysiolo- gie I,Universitat Regensburg, Postfach 101042, D(W)-8400 Regens- burg, Germany.

Receivedfor publication 11 March 1992 andin revisedform 15 October1992.

sureisamainphysiological control parameter for reninsecre- tion from the kidneys. Reninthatis released fromthejuxtaglo- merular

(JG)'

cells byregulatedexocytosis indirectly increases theblood pressure, andthis effect is mediatedby thevasocon- stricting potency of angiotensin11(2). Thus,pressure control of renin secretion isorganized in form ofanegative feedback loopwith the amount ofrenin released fromthekidneyscorre- lating inverselywith the intrarenal arterial pressure(2, 3).

Therenin producingJG cells arelocated inthe wall of the afferentarteriolesand a boardofindirect evidence suggeststhat alsothis so-called "baroreceptor" is installed withinthe renal vasculature (4-6). The subcellular signaling pathways, how- ever, along which the renal artery pressure modulates renin secretion are not yet understood. One hypothesis existing aboutthis baroreceptor mechanism suggestsadirect effect of theintrarenal pressure onJG cells thatismediatedby a pres- sure-dependent stretch of the JG cell membrane (6). Experi- mentalattempts to provesuchadirect pressure effectonrenin secretion from JG cells have produced controversial results.

Whileonegroupdemonstrated changes of renin secretionfrom isolated perfused rabbit afferent arterioles in response to changes of the perfusion pressure (7), others did not report pressure related reninsecretion in asimilarpreparation (8).

Nonetheless, thesedifferent findingsmay give some hints for the possiblebaroreceptor function; apressuredependence of reninsecretion, namely, wasonly found withafree-flow sys- tem allowing concomitant changes of flow in response to changesof theperfusionpressure(7),but notwithastop-flow systempermitting modulationof thehydrostaticpressureonly (8). Those results could indicate that baroreceptor function requires flow throughthe afferentarterioles and therefore di- rectingattentiontowardsapossibleroleofendothelialcellsin the pressurecontrol of renin secretion.

Endothelialcells infacthaveproperties makingthemsuit- ableformechanotransduction.Forinstancetheyareequipped with ionchannelsthat becomeactivatedduring increasedshear stress(9, 1

0).

Moreover,theyreleasevasoactiveautacoidssuch asendothelium-derived relaxingfactor(EDRF),which iscon- sideredtobenitric oxide(NO) (11),inaflow-dependentfash- ion (12, 13). Recent evidence suggeststhattheactivation of shearstress-sensitive potassiumchannels iscausallyinvolved in the releaseofEDRF( 14). Furthermore, ithas beendemon- stratedthatendothelialcellscanmodulaterenin secretionfrom isolated JG cellsinprimaryculture(15).

Nonetheless,thephysiologicalroleofthevascularendothe- lium in thecontrol of reninsecretion isnotknown.Also the

1.Abbreviations usedinthis paper: Ang, angiotensin; ANP, atrialnatri-

ureticpeptide; D-NNA, NG-nitro-D-arginine;EDRF,endothelium-de-

rivedrelaxation factor; GFR, glomerular filtrationrate;JG,juxtaglo- merular(cells); L-NAME, nitro-L-argininemethylester; L-NNA, NG- nitro-L-arginine;NMMA, NG-monomethylarginine;NO,nitricoxide;

RSR, reninsecretionrate;SNP, sodiumnitroprusside.

1088 H.Scholz andA.Kurtz J.Clin.Invest.

©TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/93/03/1088/07 $2.00 Volume91, March 1993,1088-1094

effect of EDRF on renin secretion is notunequivocal. There areindirectobservations, suggestingeither astimulatory (15) or aninhibitoryeffect of EDRF on renin release in vitro(16).

Inview of this background, itappearedreasonable to us to examineapossible physiological role of EDRF in theregula- tion of renin secretion, in particular, in the control of renin release by the renalperfusion pressure. Tothisend, we have systematically examined to what extent stimulation or inhibi- tion of EDRF formation modulates the pressuredependence of renin secretion from isolated perfusedratkidneys.Weused the model of isolated rat kidneys perfused atcontrolledpressure because we have previously found that theamount of renin released from this preparation correlates inversely with the renal artery pressureand also thebasic mechanisms ofmyo- genic autoregulation are preserved under those condi- tions( 17 ).

Methods

Isolatedperfusedratkidney.MaleSIVstrainrats(250-350gbody wt) havingfreeaccess tocommercialpelletchow and tapwater wereob- tained from the local animal house and usedthroughout. Kidneyper- fusionwasperformedinarecyclingsystemaccordingtothetechnique of Schurek and Alt (18)with minormodificationsasdescribedprevi- ously (19). Inbrief,theanimalswereanesthetized with 150 mg/kg of 5-ethyl-(1'-methyl-propyl)-2-thio-barbituric acid (Inactin®; BYK Gulden, Konstanz, Germany). Volume lossduring the preparation

wassubstitutedbyintermittentinjectionsofphysiologicsaline(- 2.5 mltotally)viaacatheterwhichwasinserted into thejugularvein. After opening oftheabdominalcavity byamidline incisiontherightkidney was exposed and placed in a thermostatically controlled metal chamber. Therightureter wascannulated withasmallpolypropylene tube( PP-10) thatwasconnectedto alarger polyethylenecatheter( PE- 50;Labotrade,Schonenbuch,Switzerland). After intravenousheparin injection (2 U/gLiquemin®;Roche, Basel,Switzerland) the aorta was clampeddistaltotherightrenal artery and thelarge vesselsbranching off the abdominal aorta wereligated.Adouble-barreled cannulawas inserted into theabdominalaortaandplaced closely to theorigin of the right renal artery. Afterligation ofthe aortaproximal to the right renal arterytheaortic clampwasquicklyremoved andperfusionwasstarted insitu with aninitial flowrate of 8 ml/min. The right kidneywas excised andperfusion at constantpressure (100 mmHg)wasestab- lished. To this end, the renal artery pressurewasmonitored through the inner part oftheperfusion cannula (Statham transducer P 10 EZ:

Gould, Spectramed Ltd., Coventry,UK) and the pressuresignal was usedforfeedbackcontrolofaperistalticpump.Theperfusion circuit wasclosed bydraining the renal venous effluent via a metal cannula back into a reservoir (200-220 ml). The basic perfusion medium, which was taken from the thermocontrolled (370C)reservoir, con- sistedofamodified Krebs-Henseleit solution containing all physiologic aminoacids exceptL-argininein concentrations between 0.2 and 2.0 mM,and8.7 mM glucose, 0.3mM pyruvate,2.0 mM L-lactate, 1.0 mMa-ketoglutarate, 1.0mM L-malate, 6.0mMurea, and 1 mU 100 mlvasopressin 8-lysine.Theperfusatewassupplemented with 6g/ 100 mlBSA and withfreshlywashed human red blood cells ( 10±2% hemat- ocrit). Ampicillin (3mg/100 ml)andflucloxacillin (3 mg/100ml) wereadded toinhibit possible bacterial contamination of the medium.

Toimprovethefunctional preservation of the preparation, the perfus- ate wascontinuously dialysed against a 25-fold volume of the same composition but without containing erythrocytes and albumin. For oxygenationof theperfusionmedium the dialysate was gassed with a 95%oxygen/5%carbon dioxide mixture. Perfusate flow rate was ob- tained from the revolutions of the peristaltic pump that was calibrated before and after each experiment. Renal flow rate and perfusion pres- sure werecontinuously monitored by a potentiometric recorder (Kipp

&Zonen,Delft, Netherlands).Stock solutions of thedrugstobetested

(see below)weredissolved infreshly prepared perfusateandinfused

into the arterial limb oftheperfusioncircuitdirectlybefore thekidneys (peristalticpump2132Microperpex®; LKB, Bomma, Sweden)at 1%

of therateofperfusateflow. For determination ofperfusaterenin activ- ity (pRA)aliquots (100,tl)weredrawn from the arterial limb of the circulation and the renalvenouseffluent, respectively.Thesamples werecentrifuged(4"C)at 1,500gfor 15 min(SorvallRT6000;Sor- vall) and the supernatantswerestoredat-20'C untilassayedfor renin activity.

Determination ofreninactivity. Perfusatesampleswereincubated for 1.5hat370C withplasmaofbilaterally nephrectomizedmalerats asrenin substrate(20).Thegenerated angiotensinIwasdeterminedby radioimmunoassay(Medipro AG,Teufen, Switzerland).

Reninrelease.Inapreviousstudyperformedwiththesameexperi- mentalmodel,wehavefound that renin isnotinactivatedduringits passagethrough isolatedperfusedratkidneys ( 19). Therefore,renin secretory rates werecalculated from the arteriovenous differences of perfusatereninactivityand thecorrespondingrenal flowrates.

Chemicals.Acetylcholine, L-arginine, NG-nitro-L-arginine,nitro-L- argininemethylester,sodium nitroprusside, 8-bromo-cGMP, ratsyn- thetic atrial natriureticpeptide,indomethacin,andisoproterenolwere

purchased from Sigma International. N0-monomethylarginine and N0-nitro-D-argininewereobtained from Calbiochem(Lucerne,Swit- zerland)andfrom Serva(Heidelberg, Germany), respectively.

Presentationofresults. Graphsshowingrenalperfusateflow and reninsecretionrates are exactredrawingsfromoriginaltraces.Allex-

perimentswereperformed accordingtostandardprotocolsand each pointrepresents themeanofnexperiments.Perfusatesamplesfor deter- mination of reninactivitywerecollectedin2-min intervals and this is indicated(0)inFigs.2-6.Asterisks indicate the first valuebeing signifi-

cant vs.controls.

Statisticalanalysis.Thesamekidneyswerefirsttakenascontrols andwerethen usedfor theexperimentalprotocols. Levelsofsignifi-

cance werecalculatedusing pairedStudent'sttest.P<0.05wasconsid- eredsignificant.

Results

Basalperfusateflowratesthroughtheisolatedratkidneysper- fused at 100 mmHg were 15.0±0.5 ml/min per g (mean±SEM; n = 15) and basal renin secretion rates were 3.5±0.5ngAngiotensinI (AngI)/hpermin per g(n= 15).

To obtain first evidence whetherEDRFmodulates renin secretion from wholekidneysat all, the hormoneacetylcho- line,which isawell-knownactivatorof endothelialEDRFre- lease(21), wasadded totheperfusate. Acetylcholine used in theconcentration rangebetween 10 nmol/literand 1 ,umol/

liter causedadose dependent increase ofperfusate flowrate and also led to a graded stimulation of renin secretion at a

perfusionpressureof100mmHg(Fig. 1).Ataconcentration of 1

gmol/liter

acetylcholine, renin secretion rates were in- creasedfromthe basal levelof 3.5±0.5to 16.0±2.0ngAngI/h perminper g(n = 6).Inparallel, urine flowratesincreased from 71±14,l/min per gunder controlconditionsto maxi- mally 159±30,ul/minper gwith1 ,imol/liter acetylcholine(n

=6).

NG-nitro-L-arginine(L-NNA; 100

pmol/liter),

an inhibi- torofEDRF formation (22), significantly reducedthe renal vascularrelaxation and abolishedthe enhancement of renin secretionproducedbyacetylcholine(1 tmol/liter). Withinthe samekidneysthe vasodilatingand reninstimulatoryeffectof acetylcholinecould bepartiallyrestoredbyremovingL-NNA from the perfusion medium and byadding 1 mmol/liter of L-arginine (Fig. 2).

EDRFis consideredto actinitstargetcells byactivatingthe solubleguanylate cyclase and thereby increasingthe cellular Endothelium-derivedRelaxingFactorandReninSecretionfromIsolatedKidney 1089

20[

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2 14-

LL

20

2

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15x

10 _

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'.' 5to

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0 10-8 10-7 10-6

Acetylcholine (M)

Figure1.Perfusate flowratesand reninsecretionratesinisolatedrat kidneysperfusedataconstantpressureof 100 mmHg in thepresence

ofgradedconcentrationsofacetylcholine.Data^aremeans±SEM of six kidneys.Asterisksindicate P^<0.05vs.control.

cyclic GMP levels (23). We therefore assessed the effect of atrialnatriureticpeptide(ANP),which isanactivatorofpartic- ulateguanylatecyclase(24)onreninsecretion fromour prepa-

rations. As shown in Table I, ANP ( 100 pmol/liter, and 1

nmol/ liter) ledtoadosedependentincreaseofreninsecretion

ratestomaximalvalues of11.0±3.0ngAngI/hperminper g

(n = 5). At bothconcentrations used, ANP did notchange perfusateflowthrough the kidneys. Also, the membraneperme-

ablecyclic GMP analogue 8-bromo-cyclicGMP(100

,mol/

liter)significantly increased renin secretionratesto 12.0±2.5

ngAngI/hperminper g(n = 3). Forcomparison, addition of 10 nmol/liter isoproterenol, which stimulates renin release throughacyclic AMPdependentmechanism(2), resultedin renin secretion rates of55.0±5.0 ngAngI/h per min per g

(n= 5).

Sinceacetylcholinenotonly enhancesEDRFformationin endothelialcells but also the release ofprostacyclin(25),which inturn might stimulate renin secretion (26), we used indo- methacintoinhibitprostacyclin synthesis.AsshowninFig. 3, indomethacin(10Ismol/ liter) hadnosignificantinfluencenei- theronbasal flownoronreninsecretionrates. Inparticular, indomethacindidnotaffect thevasorelaxingandrenin stimula- toryeffects ofacetylcholine(Fig. 3).

Apossiblerole ofendogenousEDRF releaseinthekidneys

onpressure-controlledreninsecretionwasassessedbyinvesti- gating theeffects of three different inhibitorsofEDRFforma- tion, namely NG-nitro-L-arginine (L-NNA; 100 ,umol/liter), nitro-L-argininemethylester (L-NAME; 100 ,umol/liter) and

NG-monomethylarginine(NMMA;200pomol/liter) onrenin secretion. As shown previously, stepwise reductions of the renalarterypressureintherangebetween 100mmHgand40 mmHgresultinexponentially increasingreninsecretionrates in thispreparation ( 17).Asastandard protocoltostimulate

pressure related reninreleaseasingle pressurestepfrom 100 mmHgto40mmHg^wasthereforeperformed.Undercontrol conditionsthismaneuverledtoanincreaseofreninsecretion from 3.5±0.5to79±12ngAngI/hperminper g(n= 8)(Fig.

4).InthepresenceofL-NNA(100tLmol/liter),perfusateflow ratessignificantly decreased from 15.0±0.5 to 12.0±0.5 ml/

min per g(n = 8) (Table II) and also renin secretion rates tendedtodecreaseat100mmHg(Fig. 4).Moreclearly,therise ofrenin secretion inresponsetoareductionoftheperfusion

pressureto40 mmHg wassignificantly attenuatedinthe pres- enceof 100

,gmol/liter

L-NNA(13±3vs.79±12ngAngl/hper min per g). About 50% ofthe normal pressure response of reninsecretioncould be restored byremovingL-NNA fromthe perfusateandbyadding 1 mmol/literofL-arginine(Fig. 4).

To test for the specificity ofthe effects obtainedwith L- NNA, we also investigated the influence of

N0-nitro-D-argi-

nine (D-NNA), a stereoisomer without inhibitory effect on EDRFformation(27).D-NNA(100

,gmol/liter)

didnot alter renal flow ratesand even slightly improved thepressure re- sponseof renin secretion, while L-NNA (100,umol/liter)al- mostblunted the rise of renin release in the same kidneys (Fig.

5). Forcontrol, weexamined as towhether L-NNA didalso affect the renin stimulatory potency ofisoproterenol, which activates renin secretion via cyclic AMP formation (2). As shown in Fig. 5, isoproterenol usedat a concentration of

10

nmol/liter, even in the presence of L-NNA and alsoduring D-NNAadministration,significantly increased renin secretion rates. Similar to L-NNA, L-NAME, and NMMA, two other inhibitors ofEDRFformation,alsosignificantlyreducedrenin releaseat arenalarterypressureof 40 mmHg (TableII).On a molarbasis, L-NNA and NMMA were morepotent than L- NAME indecreasing renin secretionat aperfusionpressure of 40 mmHg(Table II). Like L-NNA, L-NAME, and NMMA alsosignificantlyreducedperfusateflowrates to 13.0±0.5and 12.5±0.5 ml/minper g (n ⁼ 3), respectively(Table II). All three inhibitors of EDRF formation did not significantly changeurineflowrates(TableII).

Assumingthatafall of the renalarterypressurerequires the releaseofEDRFtobecomeaneffectivestimulus for reninre- lease, wetestedastowhetherpressurerelatedrenin secretion couldbereestablishedbymimickingNO releaseduringinhib- ited EDRFformation. Tothisend,weexaminedpressure-de- pendentreninsecretionin thecombinedpresenceofanEDRF inhibitor(L-NNA)andofanexogenousnitrate(sodiumnitro- prusside

[SNP]

) thatmimicks the actionof EDRF.Asshown inFig.6, theaddition ofSNP(10

jimol

^/liter)in the presenceof L-NNA (100,umol/liter)ledto apromptincreaseofperfusate flowratesfrom 12.0±0.5to23.0±2.0 ml/minperg(n=5) and to anincreaseofreninsecretionratesfrom2.0±0.5to18.0±1.5

ng

AngI/h

per min per g(n ⁼ 5) at 100mmHg. When the

Nitro-L-Arginine 100pM L-Arginine1mM Acetylcholine

A

@ 0E.

~

_L E

pM

12-

8-1

B-,c 30- .s- 20-

x:I 10-

oo

_ 0+0--v °0-§0 cr ^'

0 '0-o-oo

5min

Figure2.Perfusateflowrates(A)andreninsecretionrates(B)in isolatedratkidneysperfused at 100mmHginthe absenceandpres- enceof1,umol /literacetylcholineand100,mol /literNG-nitro-L-ar- ginine.Eachpointrepresentsmean±SE of three kidneys. Asterisks indicate the first values being significantly different from control P

<0.05.

1090 H.Scholz andA. Kurtz oRSR

*Flowrate

TableI.EffectsofANP, 8-bromo-cGMP, and IsoproterenolonReninSecretionRates(RSR) in IsolatedKidneys PerfusedatConstant Pressure of 100mmHg

Control ANP ANP 8-bromo-cGMP Isoproterenol

100pmol/tlier ^Inmol/liler 100,gmol/liter 10nmol/liter

RSR ngAngI/h perminperg 3.5±0.5 5.5±1.0 11.0±3.0* 12.0±2.5* 55.0±5.0*

(5) (3) (5) (3) (5)

The numberofexperimentsis given in parentheses. Dataareexpressedas±SEM.AsterisksindicateP <0.05vs.controls.

perfusionpressurewasthen reducedto40mmHgin the com- binedpresenceof L-NNA( 100,gmol/liter)and SNP10 ,umol/

liter),reninsecretionratesincreasedto220± 14ngAngI/hper min per g (Fig. 6).

Finally,weexamined whetherthe pressuredependenceof renin secretion was altered under conditions of stimulated EDRF release. To thisend,reninsecretion rates in the absence and in the presence ofacetylcholine were determined in the same kidneys at perfusion pressures of 140, 100, and 40 mmHg. AsshowninFig. 7,acetylcholine usedat a concentra- tion of 10

timol/liter

significantlyaltered therelationshipbe- tweenperfusionpressure and reninsecretion, inaway thatit weakly increased renin release at high pressure and potently enhancedrenin secretionatlowperfusionpressure: At a renal artery pressure of 140 mmHg, acetylcholine (10,gmol/liter) didnotsignificantly affect renin secretionrates, whereas asig- nificantincrease of renin release from 3.5±0.5to 17.0±2.5ng AngI/hpermin per g (n=3)wasobtainedat 100 mmHg(Fig.

7). When the perfusion pressure was reduced to40 mmHg, reninsecretionratesincreasedtomaximal values of 171±20ng AngI/h per min per g (n= 3) in thepresenceof acetylcholine

(Fig. 7).

Discussion

Theaimof thisstudy was toinvestigateapossible involvement ofEDRF in the pressure control of reninsecretion from the kidneys, whichis commonly referredto astheintrarenal barore- ceptor mechanism of renin secretion. As an experimental model, we used an isolated rat kidney preparation perfused withamedium containingredblood cells, which is suitable to

Acetylcholine1pM Indomethocin 10pM

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cm

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20- I10-0.

c r -x c _5

studytheregulation ofrenin secretion at the organ level(17, 19). Inparticular, this model displays an inverserelationship between perfusion pressure and renin release (Fig. 7, reference 17) in a fashion similar to the in vivo situation (28).Moreover, wehaverecentlydemonstrated that the basic mechanismsof myogenic autoregulation of flow are preserved ( 17) and also characteristic signaltransduction mechanisms suchasangio- tensin II induced inhibition orisoproterenol related stimula- tion of renin release are operatingunder those experimental conditions( 17, 19).

Wefound thatacetylcholine, which activates the formation of endothelialautacoids suchasEDRFalso in renal vascular beds (21, 29), caused a dosedependent decrease in vascular resistance and an increase in renin secretion rates (Fig. 1).

Since these effects of acetylcholine were reversiblysuppressed in the presenceof an established inhibitor of EDRFformation (Fig. 2), it appears most likely that the vasorelaxing andthe renin stimulatory action of acetylcholine were bothmediated by EDRF. In the same kidney preparations inhibition ofprosta- glandin synthesis with indomethacin did not affect neither renal vascularrelaxation nor the stimulation of renin release causedby acetylcholine (Fig. 3) and apossible involvement of prostacyclin in theactions of acetylcholine becomestherefore less likely (30). A vasodilating effectofacetylcholine in the isolatedperfused rat kidney is in keeping with previousstudies (31,32) as well as with a more general conceptindicatinga role of the vascularendothelium, particularly of EDRF, in the va- sorelaxant action ofacetylcholine (33). Stimulation ofrenin

A- 201

E 70

50

ir

-g

5

c 70-

U1 50

m:z

5min.

Figure 3.Effect of indomethacin ( 10 ,umol/liter) onrenalflowrates (A) andrenin secretionrates(B)in the absenceandpresenceoface- tylcholine (1 4smol/liter).Data aremeans±SEMoffive kidneys.As- terisks indicatethefirst valuesbeingsignificantly differentvs.controls P<0.05.

Nitro-L-Arginine

100pM L-ArgininelmM

40 40 40mmHg

04 + 4,4 *

ll "4t{e-'*4

5 min

Figure 4. Perfusate flow rates(A) andrenin secretionrates(B)in isolatedratkidneys perfusedat 100 mmHg and 40 mmHg inthe presence of 100,umol/literNG-nitro-L-arginineor 1 mmol/literL-ar- ginine.Data aremeans±SEMof eight kidneys. Asterisks indicatethe firstvaluesbeingsignificantly different fromcontrols P<0.05.

Endothelium-derivedRelaxingFactorandReninSecretionfrom IsolatedKidneY 1091

Table11.EffectsofL-NNA, L-NAME, andNMMA onRSR,PerfusateFlow, andUrineFlowinIsolatedPerfusedRatKidneys

Control L-NNA L-NAME NMMA

100Mmol/liter 100jmol/l1iter 200Mmol/liter

RSR ngAngI/hpermin per g

at40 mmHg 79±12(8) 13±3*(8) 31±6*(3) 10±2*(3)

Flow rate ml/min per g

at100 mmHg 15.0±0.5(8) 12.0±0.5*(8) 13.0±0.5*(3) 12.5±0.5*(3)

Urine flowml/min per g

at 100 mmHg 71±14(8) 68±5(8) 77±10(3) 75±7(3)

The numberofexperimentsisgiveninparentheses.Dataaremeans±SEM.Asterisks indicateP<0.05vs.control.

releasebyacetylcholine has previously been reported for dogs (34) and turtles(35).Astimulatoryeffect of EDRFonrenin secretion is also supported by our finding thatinhibition of EDRFsynthesis ledto adecreaseofbasal renin release(Fig.2).

This observation is in good accordance with a recent study reporting that renin secretion from isolated rat kidneys per- fusedat a constantrenalartery pressureof 80 mmHgwassignif- icantly reduced with nitro-L-arginine, an inhibitorofEDRF formation (36). A similar findingwas made in anesthetized dogs inwhichblockade of NO synthase causedadecrease of plasma reninactivity (37).A stimulatory effect ofEDRF on renin secretion is also supported byourrecent cell coculture study with isolatedjuxtaglomerularcells and vascular endothe- lialcells(15), butit isatvariance with another in vitro study performedonkidney slicesandsuggestinganinhibitoryaction ofEDRF onrenin release(16).

Inviewofthe latterdiscrepancy,itmustbeconsideredthat acetylcholine induced stimulation of renin secretion as ob- servedinthisstudywaspossiblyrelatedtochangesof flowor was mediated by the macula densa mechanism rather than being caused bya direct stimulatory effect ofEDRF on JG cells.However,sinceacetylcholineincreased renin releasepre- dominantly at lowerpressure values without affecting renal flow rates inthis pressure range (Fig. 7), the stimulation of

F-IsoproterenollOnM- Nitro-D-Arginineo100pM Nitro-L-Arginine 1001M

40 40 4OmmHg

As,,, 201 Ci 51

B 90A

070-^30-I

,=~~

^{__ _i o<}

;10 /

S min

Figure5.Perfusate flowrates(A)andrenin secretionrates(B)inan isolatedratkidney perfusedat100 and 40mmHgin the presenceof 100 ,umol/literN-Gnitro->Darginineor 100 tLmol/literN'-nitro-L-ar- ginine.Inthe presenceof bothdrugs,reninsecretionwasstimulated by 10nmol/liter isoproterenol.Thecombination ofexperiments shown inthisfigurewasdonewithtwokidneys,whichproducedvery similar results.Originalredrawingfrom therecordingsofoneof these experimentsis shown.

reninsecretion in response to acetylcholine was probably not mediated by parallelchangesof flow.Moreover,acetylcholine alsosignificantlyincreasedurine flowrates at aperfusionpres- sure of100mmHg, and iftherewas aneffecton glomerular filtration rate (GFR)at all,oneshould therefore expectthat acetylcholine enhanced rather than decreasedGFR (32).Asa consequence, the tubularsodiumchloridedelivery should ifat allincrease, therebycausing inhibition rather thanastimula- tion of renin secretion viathe maculadensamechanism (38).

AmajorpathwayalongwhichEDRF exertsitscellular ef- fects is via stimulation ofsoluble guanylate cyclase activity leadingto arise ofintracellularcGMP levels (23). Theroleof cGMP in the controlof renin secretion isnotyetunequivocally understood. Membrane permeable cGMP analogues such as 8-bromo-cGMPhavebeenfound eithertoinhibitrenin release fromkidney slices(39) and from cultured JG cells (39, 40)or tohavenoeffectonreninsecretion fromkidneyslices (41) and isolated perfusedkidneys(42). AlsoexperimentswithANP, an activator ofparticulate guanylate cyclase (24), have produced divergentresults.Thus,ANPhasbeenfoundtoinhibit(39)or tostimulate (41) renin secretion in vitro fromkidneyslices and to decrease renin release from cultured JG cells(43) and in vivo upon systemic application (44). Intrarenal infusion of ANPintoconsciousdogs (45),aswell asapplicationofANP into isolatedperfusedratkidneys(46), however, has beenob- servedtostimulate renin release. Thefindingsobtained inthis studywouldsuggestthatanincrease of renalcGMP levels hasa stimulatoryrather thananinhibitoryeffectonreninsecretion.

Whetherthis effect is dueto adirect action ofcGMP in JG cells

SodiumNitroprussido10pM Figure6. Perfusate flow Nitro-L-Arginine 100PM rates (A)and renin se- 40 40 4OmmHg cretionrates (B) in iso-

20 lated ratkidneys per-

c-i 151+ fusedat

100

and40

GE 10] mmHg in the absence

s- 1 and presence of 100

B. 200- Amol/liter

160- N-nitro-L-arginine^and

120 in the combined pres-

z 1 ] | ~~~~~~~enceof 100,umol/liter

80 N-nitro-L-arginineand

o> 40 10Mmol/liter SNP.u

0- Dataaremeans±SEM

5min offivekidneys.Aster- isks indicate the firstvaluesbeingsignificantlydifferent vs.controls P<0.05.

1092 H. Scholz and A. Kurtz

Figure 7.Relationship

200- between renin secretion

ratesandperfusion

m 150 pressureinisolatedper-

:S \fused ratkidneys in the

Ex

\absence andin the pres-

c100- \ ence of 10IOmol/liter

50 acetylcholine. Reninse-

c< 50- i \*cretionratesweredeter-

c mined 6 minafter pres-

0 surestepsfrom100to

0 20 40 60 80 100 120 140 140or40mmHg,which Perfusion Pressure(mmHg) wereperformed firstin

the absence andthenin the presenceofacetylcholine in thesamekidneys.Dataare means±SE of threekidneys. AsterisksindicateP<0.05 vs.absence ofacetylcholine.o,Control;*,acetylcholine(10

AsM).

orindirectly mediated by other cellscannot bedistinguished from ourexperiments with whole kidneys. It is also possible that thestimulatoryeffect ofEDRF onrenin secretionwasnot relatedtocGMPformation because thereisaccumulatingevi- denceforbiologic effects of EDRF/NO thatare notlinkedto cGMPformation (47,48).

Irrespective ofthe molecularmechanismbywhichEDRF enhancesrenin secretion from wholekidneys,ourfindingsalso showthat the enhancementof renin release from thekidneysin response to areductionoftheperfusionpressure was almost blunted in the presence ofdrugs (Fig. 4, Table II), thatare commonlyconsideredtoinhibitEDRFformation (13, 22, 49, 50). Thefollowinglines of evidencesupportthespecific action of these inhibitors ofEDRFsynthesisonrenin release: Firstly, theeffects of L-NNA and related compounds could partially be reversed by theaddition ofhigher concentrations of L-arginine (Figs. 2 and 4) which isan established maneuver to restore inhibitedEDRFformation (49). Secondly,nitro-D-arginine,a stereoisomer ofnitroarginine, which ispresumednot toblock EDRFsynthesis(27), alsodid not attenuatethepressurede- pendenceof reninsecretion(Fig. 5).Thirdly,thestimulatory effect ofisoproterenol,whichdirectly^enhancesrenin secretion byincreasingcellularcAMPlevels(2)wasnotinfluenceddur- inginhibition of EDRF formation (Fig. 5). Finally, substitu- tion ofEDRFbyan artificial analogue, suchasSNP reestab- lished thepressure dependenceof renin secretion,evenin the presenceofinhibitors ofEDRFsynthesis(Fig. 6).

Takentogether,weinfer fromourfindingsthat EDRFisa stimulatory signal for renin secretion in thekidneys, and we suggest that tonicallyreleased EDRF accounts for the rise of renin secretion caused byafallof theperfusionpressure.

Thequestion arisesas towhether thetypicalinverserela- tionshipbetween renin secretion and renalarterypressure re- sultsfromapressure-dependentformation of EDRF, inaway thatEDRFreleaseis low athighpressure andhighat lowpres- sure.Anotherpossibilitywould bethat thereleaseofEDRFis less pressure dependent, but that the stimulatory effect of EDRFonrenin secretion iscounterbalanced by a second pres- surerelatedprocess. Weattemptedtodistinguishbetween both possibilities by assaying cGMPinthe venous renaleffluentas anindirectmeasureforrenal EDRFactivity. However,cGMP concentrationsin theperfusatewerebelow thedetection limit oftheradioimmunoassayused(<25fmol/ml),thusallowing

noinformation about pressure dependent formation of EDRF inthekidneys.

Nonetheless, wethink that there are two lines of indirect evidence suggestingthat the effect ofEDRF on renin secretion from JG cells rather than the formation of EDRF itselfwas controlledbytheperfusionpressure.Thus, acetylcholinepro- ducedapotent EDRFdependent vasodilation at 100 mmHg (Fig. 2), indicating that the release of higher quantities of EDRF canprincipally occur in this pressure range. At the same timethere was only a relatively weak EDRF dependent stimula- tionof renin secretion at100 mmHg, but a powerful increase of renin release withdecreasing perfusionpressure(Fig.7).Simi- larly, mimicking the effect of EDRF on soluble guanylate cy- clase activity with SNPmarkedly decreased vascular resistance but onlyweakly stimulated renin secretion at 100mmHg(Fig.

6). Similar to acetylcholine thestimulatory effect of SNP on renin secretion became enhanced by lowering the perfusion pressure to40 mmHg (Fig. 6).

Takentogether, ourfindingsarecompatiblewith the con- cept that thebaroreceptormechanismcontrollingrenin secre- tion consists of at least twocomponents. A tonical stimulatory one provided by the continuous release of EDRF from the vascularendothelium orfrom other cellularstructuresof the kidney. And asecondinhibitoryone, that isdirectlyrelated to the perfusion pressure and that is enough potent to neutralize thestimulatoryinfluenceof EDRFathigherpressurevalues.

Acknowledgments

We wish to thank Marlies Hamann andKarl-HeinzGotzfordoingthe artwork and HanneloreTrommerforprovidingexpertsecretarial help.

This studywas in partfinancially supported by grants from the Swiss National Science Foundation(31-26381.89)and the Deutsche Forschungsgemeinschaft (Ku859/2-1).

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