Angiotensin II binding sites in individual segments of the rat nephron

Angiotensin II binding sites in individual

segments of the rat nephron.

S K Mujais, … , S Kauffman, A I Katz

J Clin Invest.

1986;

77(1)

:315-318.

https://doi.org/10.1172/JCI112293

.

The sites of action of angiotensin II along the nephron are not well defined and both

proximal and distal effects are suggested. Using a microassay that permits measurement of

hormone binding in discrete tubule segments, we determined the binding sites of

125I-angiotensin II along the nephron of Sprague-Dawley rats. Specific binding in proximal

convoluted tubule (PCT) (at 25 degrees C, pH 7.4) was linearly related to tubule length and

saturable, with an apparent maximal binding capacity of approximately 300 amol X cm-1.

Binding specificity was verified in competition experiments that revealed significant (P less

than 0.001) and comparable competition for radioligand binding by angiotensin II and

angiotensin precursor, metabolite, and analogues, whereas unrelated peptides of similar

size (bradykinin, ACTH [1-10]) were without effect. The profile of specific angiotensin II

binding along the nephron was: PCT, 216 +/- 13; pars recta, 86 +/- 14; medullary thick

ascending limb of Henle's loop, 46 8; cortical thick ascending limb of Henle's loop, 77

+/-8; distal convoluted tubule, 49 +/- 10; cortical collecting tubule, 15 +/- 1; medullary collecting

tubule, 32 +/- 7 amol X cm-1. These results indicate the presence of specific angiotensin II

binding sites in all tubule segments studied, but binding capacity was highest in the

proximal convoluted tubule, in agreement with transport studies that localize the effects of

[…]

Research Article

Rapid

Publication

Angiotensin

11 Binding Sites

in Individual Segments

of

the Rat Nephron

SalimK. Mujais, Susan Kauffman, and Adrian1.Katz

DepartmentofMedicine, UniversityofIllinoisatChicago, Chicago, Illinois60612;andDepartmentofMedicine, TheUniversityof

Chicago Pritzker School ofMedicine, Chicago,Illinois60637

Abstract

The sites of action of angiotensin II along the nephron are not welldefined and both proximal and distal effects are suggested. Usingamicroassay that permits measurement of hormone bind-ing in discrete tubule segments, we determined the bindbind-ing sites of

'25I-angiotensin

IIalong the nephron ofSprague-Dawley rats. Specific binding in proximal convoluted tubule (PCT) (at 250C, pH 7.4) was linearly related to tubule length and saturable, with

an apparent maximal binding capacity of -300 amol cm-'. Binding specificity was verified in competition experiments that revealedsignificant (P < 0.001) and comparable competition for radioligand binding by angiotensin II and angiotensin precursor, metabolite, andanalogues, whereas unrelated peptides of similar size (bradykinin,ACITH11-101) were without effect. The profile ofspecific angiotensin II binding along the nephron was: PCT, 216±13; pars recta, 86±14; medullary thick ascending limb of Henle's loop, 46±8; cortical thick ascending limb of Henle's loop,77±8; distal convoluted tubule, 49±10; cortical collecting tubule, 15±1; medullary collecting tubule, 32±7 amol

cm-'.

These results indicate the presence of specific angiotensin II binding sites in all tubule segments studied, but binding capacity was highest in the proximal convoluted tubule, in agreement with transport studies thatlocalizethe effects of the hormone inthis segment.

Introduction

AngiotensinII

(All)'

influences both renalhemodynamics (1) andtubular transport processes(2),but its effectsonthe latter

areincompletelydefined. Althoughclearance studiesprovided

circumstantialevidence foratubulareffectof Allatboth

prox-imalanddistal nephron sites,thenatureof thisinteraction

re-mainscontroversial. In micropuncture studiesin therat(3, 4)

the

findings

were

again

variable,

ranging

from inhibition(3)to

stimulation oftransport (4), and including a dose-dependent bimodal effect (5)thatprobablyexplainsthedivergent findings.

Theearlynegative findingsinisolatedperfused rabbit

proximal

Addressreprintrequests to Dr.Mujais, Section of Nephrology,

Depart-mentofMedicine, Northwestern University Medical School, 303 East

ChicagoAve.,Chicago,IL6061 1.

Receivedforpublication 13 August 1985.

1.Abbreviations used inthis paper:All,angiotensinII; PCT,proximal

convolutedtubule(s); PR,pars recta.

tubules (6) have been ascribed to degradation of All by serum angiotensinases present in the bathing medium, since recent studies utilizing artificial bath solutions described an electro-neutral stimulation of fluid transport (7).

Although these reports suggest a dose-dependent bimodal effect of angiotensinIIatmultiple sites along the nephron, they donotdefine the exact tubular sites of action of the hormone. Binding sites for All have been identified in the glomerular tuft (8), renomedullary interstitial cells in tissue culture (9), renal tubular cellfragments (10-13), and bovine kidney proximal tu-bulecell culture (14). However, these studies were performed

utilizingheterogenouspreparations and thus do not identify the targetnephronsegment(s)affectedby the hormone. The present study was undertaken to determine the binding sites of All along the ratnephron using a microassay that permits measurement of hormonebindingindiscretenephron segments.

Methods

Tubule microdissection. Kidneys were obtained from 68 Sprague-Dawley

ratsweighing 180-200gandfed regularlaboratory chow adlib. until study. Procedures for tissue preparation and tubule microdissection have beenpreviously described (15) and will be presentedbriefly.Under

pen-tobarbital anesthesia theleftkidneywasperfused withacold collagenase-containing solution (NaCl, 137 mM; KCI, 5 mM; MgSO4, 0.8 mM; Na2HPO4, 0.33 mM; KH2PO4, 0.44 mM;MgCl2,ImM; CaCI2, ImM;

Tris-HCl, 10mM; collagenase,type I(Sigma Chemical Co., St.Louis,

MO), 400 U/ml; and bovineserum albumin, 0.05%; pH 7.4). Thin pyramidswere cutalong thecorticopapillaryaxisandincubated for7 mininthe samemediumat 35°C, after which theywere rinsed with

coldmicrodissection solution(NaCl, 100 mM;MgCl2,4mM;Tris-HCI,

50mM; dithiothreitol, 2mM; bovineserum albumin, 0.5%;pH 7.4). Segments from differentpartsof thenephron weredissected freehand understereomicroscopic observation,transferredto asunken bacterio-logic slide, and photographedtodetermine theirlength.

25I-Angiotensin

II binding assay. Experiments were done using

proximalconvoluted tubulestodetermineoptimaltemperature, hormone

concentration,andincubation time for thebindingassay.The

compo-sition of the incubation solution usedwasidenticaltothatused in mi-crodissection above,exceptfor theaddition ofradioligandandunlabeled

hormone. Theangiotensinaseinhibitor dithiothreitolwasaddedtoboth the microdissection and the incubation solutionstominimizeAll

deg-radation.

Severaltubules fromthesamenephronsegment(averagetotallength,

3-5 mm)were pooled ineach sampleandplaced on small piecesof aluminum foil in wellsofan aluminumplaque. Themicrodissection solution was thenaspirated andreplaced by2 ul ofcold incubation mediumusinganautomatic Hamiltondispenser.Incubationwasstarted by immersingtheplaquesinawaterbathat25°C,andwascarriedout

for30 min withoutagitation.Incubationwas_{stopped by}

cooling

onice.

Theincubation mediumwasaspiratedandthe tubuleswerewashedfour times with2glofacoldstopsolution

containing

50mMMgCl2,300 mMNaCI,and50 mMTris, pH7.4. After the finalrinsing,the solution

J.Clin. Invest.

©TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/86/01/0315/04 $1.00

wascompletely removed, the pieces of aluminum foil containing thl tubules were transferred to counting vials, and the radioactivity was counted inagamma counter (Beckman Biogamma, Bria, CA).

To allow assessment of the binding of Alltoother cellcomponents including luminal membranes, osmotic shockwasperformed in separate experiments before thebindingassay asfollows: After transfer of the tubules to the aluminum wells, the microdissection solutionwasaspirated andreplaced with 2

_IAl

ofdistilledwater. Atthe endof 5 min the hypotonic solution was removed, the incubation solutionwasadded, and

experi-mentswere carried out as described above.

Todetermine the specificity of All binding, PCTwereincubated in the presence of 1,000-fold excess unlabeled All, All analogues

(Sar'-Ala8-angiotensin II,Sar1-Thr8-angiotensinII), angiotensin I, angiotensin III, and unrelated peptide hormones of similar molecular weight (bra-dykinin, ACTH [1-10] fragment). Angiotensin converting enzyme in-hibitor was added to the incubation medium (final concentration, ImM) whendisplacement by angiotensin I was performed.

Calculations. 125I-angiotensinIIbinding was determined in each an-imal inquadruplicate, each sampleconsistingof 3-5mmoftubule,and

alltubulesofaparticular segment fromasingleanimal representasingle datapoint in the figures.Inaddition,blanks(incubatedwithouttubules)

were used for each assay condition. Adherence of the labeled hormone

toaluminum foilwasnegligible. Bindingisexpressedasattomoles12511

angiotensinIIbound per centimeter oftubuleusingthefollowingformula: bound 1251-angiotensinII=(X-X0) (L.SRA)-,where Xis the mea-suredradioactivity(counts/20min) ofthesample; X0is themeancounts

of the blanks;Lis thelength of the tubule in cm; and SRA is thespecific

radioactivity of '25I-angiotensin II incountsper 20 min per attomole. Specific bindingwas calculated from the difference between the total binding (measured in the presence of'25I-AII alone)andthenonspecific

binding (measured in the presence of1,000-foldexcessunlabeledAll).

Counts per minute of total binding ranged between seven times the backgroundcountsinproximal convoluted tubules(PCT)andtwoto threetimes thebackgroundcountsincorticalcollectingtubules(CCT).

Examples of actual counts per 20 min for total andnonspecific binding,

respectively, were 5,900 and 3,100 in PCT, and 2,050 and1,600in CCT; backgroundcountsaveraged820/20min.

Toverify theappropriateness ofnormalizing bindingper unitlength of tubule,weexamined inpreliminary experimentstherelationship

be-tweenthetwovariables. PCT segments of variable totallength (2.2-11.0 mm)wereused. Bindingwasfoundtobehighlycorrelatedwith tubule length (r=0.93,P<0.001),andthereforewasexpressed inallsubsequent experiments in amol *cm-'.

Materials.

(Tyrosyl-'251)-(5-L-isoleucine)-AII

(specific activity, 1,880

uCi/,ug)

waspurchased fromNewEngland Nuclear,Boston, MA.All, Des-Asp-'-angiotensin I, Val4-angiotensinIII,Sar'-Ala8-angiotensinII,

Sar'-Thr8-angiotensinII,angiotensin convertingenzyme inhibitor

(Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro),bradykinin,and ACTH[ 1-10]

frag-mentwerepurchased from Sigma Chemical Company.

Statistical methods. Statistical analysiswasdoneusing thepaired

Student'st test.Valuesareexpressedasmean±SEM.

Results

The temporalprofile of binding in PCT at different temperatures isdepicted in Fig. 1. At4°Cbinding was minimal throughout

the period of observation. This finding allowed us to consider that rapid cooling of the tubules on ice can be equated with termination ofthe incubation period. At 25°C, total and specific

bindingincreased rapidly, reached a peak at 30min,andshowed

aslowdecline thereafter. At 37°C, total binding also peaked at 30 min.Incontrast to the findings at 25°C, however, nonspecific

binding continued to increase with time. Specific binding de-creased significantly after 15 min, a finding consistent with a limited stability of the angiotensin receptor or of the

receptor-ligand complex at thistemperature.

3TC

(n=5)

25 C

(n=4)

120

m_-0_E

c

4±

~~-

-*1-10 2 _4 50 60

-4

10 20 30 40 50 60 10 20 30 40 50 60

Time Time

Figure1.Timecourseof'251-angiotensinIIbindingin PCTat

differ-enttemperatures. Specific bindingwashighestat250C.*,total;A,

nonspecific;o,specific.

The effects ofincreasing concentrations of labeled All on

bindingin PCT are shown in Fig. 2. A progressive increment in

total, specific, and nonspecific binding was observed with

in-creases in free ligand until a concentration of 8-9 nM was reached. Beyondthis level, no further increase in specific binding

wasobserved, while total and nonspecific binding increased more

slowlyandin tandem. The apparent maximal binding capacity of the PCT estimated from this plot was -_{300 amol}-cm-'.

Theprofile ofspecific

125I-angiotensin

IIbindingalong the

ratnephron is shown in Fig. 3. For these studies, a radioligand

concentration of 5-6 nM was chosen because it was sufficient

to allow reproducible measurements in all nephron segments.

Specific bindingwashighest in the PCT, followed by the pars

recta(PR) and the cortical and medullary portions of the thick

ascending limb of Henle's loop. Very high specific binding

was also identified in dissected glomeruli (302±13 amol

-glomerulus-').

Nonspecific binding was also highest in the PCT

(69amol-_{cm-')followed byPR(30 amol}-cm-'),and itranged

between 6 and 19 amol-

cm-'

in the more distal segments of the nephron. Disruption of proximal tubule cell integrity by short exposure to a hypotonic medium significantly increased totalbinding(radioligand concentration, 3.5 nM) from 90±10

E

0

oi~~~~~e

E

co

C

C)

C 0

0

c.

C~~~~~.

123 4 567 8 10 12 14 16 125_{1-Angiotensin} II

_(nM)

Figure2. Bindingof

"'25-angiotensin

IIinPCTatvarying ligand

c

m

un E

= 0

c E

U)

N6

200

100

9 riT ;

PCT PR MAL CALDCTCCT MCT

Figure3. Profile of'25I-angiotensinIIbinding in seven nephron seg-ments.Significant specific binding (P <0.001 vs. zero) was identified in all nephron segments studied. Number ofanimals is given in bars.

to301±32amol-cm-' (P < 0.001), specific binding from 74±9

to278±25 amol-cm-' (P < 0.001), with a more modest

incr.e-ment in nonspecific binding from 24±1 to47±3 amol-cm-' (P

<0.001).

Competitionexperimentsweredonetodetermine the spec-ificity of the All binding measured in PCT (Fig. 4). At a

radi-oligandconcentration of 3.5 nM, andcompetitorconcentration of 3.5 uM, significantcompetition(P<0.001)wasdisplayedby all analogues, precursor, and metabolite ofangiotensinIIstudied. While their affinity for the AIIbinding site was generally com-parable, that of Sar'-Thr8-AII wasslightly higher than that of

Sarl-Ala8-AII (P<0.025), andangiotensin III wasbetterthan angiotensin I (P< 0.01) in this respect. Unrelated peptidesof similar molecularweight (bradykinin, ACTH[1-10] fragment)

caused no significant competition with the radioligand for its

bindingsites.

Discussion

Thisstudy documents for the first time the

profile

ofAllbinding

sites alongthe rat nephron. Specific bindingwas identified in

mostsegments butwashighestin theproximaltubule. Binding

in PCT was dependentontubulelength andligandconcentration andwassaturable,withanapparent maximalbinding capacity

of -300 amol-

cm-'.

Specific bindingwasunstable at

physio-logic

temperatuc'^

(370C),whilenonspecific binding progressively

increasedwith time.Furthermore,beyond 15 min ofincubation,

C:

*a 120

C.

- 100

=Co *o,- 80

C)

, _{° 60}

0

c 40

In 20

LI Total 0] Sar Ala8AII 3 Bradykinin

0 A IIiSarThr8AII L ACTH(1-10)

C AIII EI A I

p - <.001 - X N. S ->

Figure 4.Competition ofpeptides with '25I-AIIforbindingsitesin the PCT.Significantcompetition (P<0.001)wasdisplayedby

angioten-sinII precursor,metabolite,andsynthetic analogues,butnotbyeither

bradykininorACTH[1-10] fragment.Numberof animals isgivenin

bars.

nonspecific bindingtended to behigherat370C comparedwith 250C. Similar temperature-related changes in binding

charac-teristics were reported by Kitamura et al. (16), who studied

an-giotensin IIbinding to rat glomeruli.

Our results indicate that All binding was highest in the

prox-imal convoluted tubule. A high degree of binding to this segment is consistent with the findings ofstudies using cortical membrane

preparations (10-13), and cultures of proximal tubule cells ( 14). Medullary receptorsfor angiotensin II have not been well char-acterized before. On the basis of autoradiographic studies, Geh-lert et al. (17) and Mendelsohn et al. (18) have suggested the

presenceof binding sites in the medulla of rat and dog kidney. However, no specific tubular segment localization was attempted. The present studyis the first to identify and quantitate All bind-ing in specific medullary tubule segments. Bindbind-ing in the thick ascendinglimbof Henle's loop was higher in the cortical than in the medullary portion, and was intermediate between that measuredin proximal and collecting tubules.

Theprofile of AII binding sites defined in this report is also consistent with the known sites of direct tubular actions of the hormone. Several studies have shown an effect of All on fluid transport in the PCT (2, 7). The peptide also increases phos-pholipid turnover in microdissected PCTs as evidenced by en-hanced 32P labeling of phosphatidylcholine and phosphatidyl-inositol (19). Evidence for distal effects of All is less direct (2), butidentification of specific binding sites in distal segments in this paper suggests that the alterations reported in clearance studies may, at least in part, reflect direct tubular effects (2).

When intact tubulesegments are used, binding is presumed

to occur predominantly to basolateral membranes of tubular cells, which are directly exposed to the ligand. Studies using renal membranepreparations have, however, described both lu-minal and basolateral membrane binding of All (10-13). To explore luminal binding sites in isolated tubules, the cells were permeabilized and disrupted by incubation in a hypotonic me-dium. This procedure ledtoasignificant increase in specific All

binding,witha moremodestincrement innonspecific binding. This increase inspecific binding after cell disruption may reflect exposure of additionalbinding sites likely locatedatthe luminal membrane, as theradioligand gains access to these sites as well. Luminalbindingof angiotensin IImay be a prelude toits deg-radation by brush border enzymes in the PCT,amajor site of metabolismofthefiltered peptide (20), whereas bindingto ba-solateral membrane receptorsprobably precedesitsbiologiceffect

on the renal cell. Results ofsegmental distribution described aboveindicatingpreferential bindingtothe PCT and thediluting

segment supportthis view:thebest known effects ofAll onthe

renaltubule takeplace in thesesameregionsof thenephron (2).

In summary, in this studywehave determined theprofile

of Allbindingsites alongthe ratnephron. Bindingwashighest

in thePCT,asegment knownto beatarget site for the effects ofthe hormoneontransport andmetabolism,butwasalso

iden-tifiedinsegmentspreviously unsuspected ofbeingsitesofaction of thispeptide. The roleofangiotensinIIinthefunctionof the latterrequires further study.

A _b.l +

This work has beensupported bygrantsfromtheChicagoHeart

Asso-ciation and the NationalKidney Foundation of Illinois(Dr.

Mujais),

andbygrants AM 13601 andAM 19250 fromthe NationalInstitutes ofHealth(Dr.Katz).

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