Renin and renin mRNA in proximal tubules of the rat kidney

Renin and renin mRNA in proximal tubules of

the rat kidney.

M Chen, … , J P Briggs, J Schnermann

J Clin Invest.

1994;

94(1)

:237-243.

https://doi.org/10.1172/JCI117312

.

The present study was undertaken to assess the presence of renin enzymatic activity and

renin mRNA in proximal tubules of rat kidneys, and to determine the effect of converting

enzyme inhibition (CEI) on proximal tubule renin gene expression. Proximal convoluted

tubules (PCT), proximal straight tubules (PST), outer medullary collecting ducts (OMCD),

and glomeruli (Gloms) were isolated by microdissection. Renin activity was measured in

sonicated segments by radioimmunoassay. Renin mRNA levels were assessed using a

quantitative PCR. Renin activity in PCT averaged 51 +/- 15 microGU/mm compared to 405

+/- 120 microGU/glomerulus. No measurable renin activity was found in PST and OMCD.

Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and

7.5. Renin mRNA was consistently detectable in cDNA prepared from PCT and PST,

although its abundance per mm tubule was about 1/500th that found in one glomerulus.

Renin mRNA was not detectable in OMCD. Tubular renin PCR product identity was

confirmed by restriction digestion. CEI administration increased glomerular renin activity

and renin mRNA, but not proximal tubular renin. The absence of a stimulatory effect of CEI

on proximal tubule renin gene expression suggests the operation of different intracellular

signals in control of renin synthesis in the proximal tubule than in the vascular compartment.

Research Article

Renin

and Renin

mRNA in

Proximal

Tubules of the Rat

Kidney

M.Chen, M. P.Harris, D.Rose,A.Smart,X.-R. He, M. Kretzler,J. P.Briggs, andJ.Schnermann

Departments of PhysiologyandInternal Medicine, The University of Michigan,AnnArbor, Michigan 48109

Abstract

The present study was undertakento assessthe presence of reninenzymatic activity and renin mRNA in proximal tu-bules of rat kidneys, and to determine the effect of con-verting enzyme inhibition (CEI) on proximal tubule renin geneexpression.Proximalconvolutedtubules(PCT), proxi-malstraight tubules (PST), outer medullary collecting ducts

(OMCD), andglomeruli (Gloms) were isolated by micro-dissection. Renin activity was measured in sonicated seg-ments by radioimmunoassay. Renin mRNA levels were as-sessedusingaquantitativePCR. ReninactivityinPCT

aver-aged 51± 15

IAGU/mm

compared to 405±120 ,uGU/

glomerulus. No measurable renin activitywasfound in PST and OMCD. Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and 7.5. Renin mRNA wasconsistently detectable in cDNA prepared from PCT and PST, although its abundance permmtubule was about1/500th that found inoneglomerulus. Renin mRNA was notdetectablein OMCD.Tubular renin PCR product identity was confirmed by restriction digestion. CEI admin-istration increased glomerular renin activity and renin mRNA, but not proximal tubular renin. The absence of a

stimulatory effect of CEI on proximal tubule renin gene expression suggests the operation of different intracellular

signalsincontrol of renin synthesis in the proximal tubule than in the vascular compartment. (J. Clin. Invest. 1994.

94:237-243.) Key words: Polymerase chain reaction * renin

synthesis*angiotensin convertingenzyme *outermedullary collecting duct-glomerulus

Introduction

Thereis evidence that the proximal tubule is a target site for the action of angiotensin H. The peptide has been shown to regulate the transport of NaHCO3 by affecting the activity of both the luminal Na/H exchanger and the basolateral Na/ 3HCO3 cotransporter (1, 2). These effects are mediated by angiotensin II receptors present in both membranes of the proxi-mal tubule cell (3).

AngiotensinHacting on the proximal tubule may be gener-ated inthe systemic circulation and delivered to its site of action

Address correspondence to Dr. J. P. Briggs, M.D., The University of

Michigan, Department of Internal Medicine, Division of Nephrology, 1150 W. Medical Center Drive, 1560 MSRBII,Ann Arbor, MI 48109-0676.

Receivedfor publication 20 October 1993 and in revised form 31 March 1994.

througheitherperitubularbloodorfiltered fluid. Alternatively, it ispossiblethatanautonomoustissue renin-angiotensin system generatesangiotensinH atthelevel of the proximal tubule(4),

andthereby permitsautocrineinteractions with itstargetcells. Arecentstudy showed that the concentration of angiotensinH in proximal tubular fluid is about 500- 1,000-fold higher than in plasma (5). This high concentration of angiotensinHin the proximal tubulesuggestsangiotensingenerationin the

immedi-atevicinityof theproximaltubule. It could be eithergenerated by glomerularepithelial cells,orby proximal tubule cells them-selves.

Immunocytochemical studies have shown the presence of

angiotensinogenin theproximal tubule(6),and

angiotensino-genmRNAatthissite has been detected by in situhybridization

methods, indicating that the renin substrate is, in fact, synthe-sizedbyproximal tubule cells (7, 8). There alsoappears tobe

an abundance of angiotensin converting enzyme in both the apical and basolateral membranes of the proximal tubule (9-12). Evidence insupportofarenin synthetic capacity in proxi-mal tubules, however, is less convincing. While positiverenin immunoreactivity was seen in apical cytoplasmic vesicles of proximal tubule cells (13-15), thepresence of renin mRNA could notbedemonstrated with in situ hybridization methods (14, 16, 17, 18). In addition, in a recentstudy using reverse transcription-PCR

(RT-PCR),1

norenin cDNAreaction prod-uct was detected in microdissected proximal segments of un-treated ratkidneys (19).

In the present experiments we applied a quantitative RT-PCR method developed inourlaboratorytoreassessthe ques-tion of presence and relative abundance of renin mRNA in renal proximal tubules, andtocompare mRNAlevelswithrenin enzyme activity in this segmentof the nephron. Furthermore, wedetermined the effect of converting enzyme inhibition (CEI) onproximal tubule mRNA in ordertoevaluatewhether glomer-ular and tubglomer-ular renin mRNA areregulated by similar mecha-nisms. Ourdata show the presence of renin transcriptsin both proximal convolutedandproximal straight tubules, but notouter medullary collecting ducts, while measurable epithelial renin activitywasonly seen in proximal convoluted tubules. Further-more,in contrasttothe enhancing effect of converting enzyme inhibition onglomerular renin gene expression, proximal tubule renin mRNA levels were not altered with this treatment.

Methods

Animals and tissue preparation. Experiments wereperformedonmale

Sprague-Dawleyratsweighing200-250 g (Charles River Labs.,

Wil-mington, MA). Animals were anesthetized by an intraperitoneal (i.p.)

1.Abbreviations used in this paper: CEI, converting enzyme inhibition;

GITC, guanidine isothiocyanate; GLOM, glomeruli; GU, Goldblatt

units; i.p.,intraperitoneal; OMCD,outermedullary collecting duct; PCT,

proximal convoluted tubule; PST, proximal straight tubule; RT-PCR, reversetranscription-PCR.

J. Clin. Invest.

© The American SocietyforClinicalInvestigation, Inc.

injection of Inactins (120 mg/kg; Byk Gulden, Constance, FRG). The aortaof rats was cannulated below the level of the kidneys from an abdominal midline incision. After ligating the aorta at a position between the two kidneys, the left kidney was perfused with 60 ml of cold saline, followed by 60 ml of cold DME (Sigma Chemical Co, St. Louis, MO) containing 1 mg/ml collagenase (Boehringer-Mannheim, Indianapolis, IN).The kidney was removed and cut into 1-2-mm-thick coronal slices. The kidney slices were incubated at 370C for 22 min in the DME/

collagenase solution withgentle shaking. The slices were then rinsed once with ice coldphosphate buffered saline, and placed into DME medium containing 1% fetal bovine serum (Hyclone, Logan, UT). Seg-ments weredissected with darkfieldillumination at40C. Lengths of the dissected segments were measured. In most cases, 10 glomeruli

(GLOM), 10 mmofproximal convoluted tubule (PCT), 10 mm of proximal straight tubule (PST) and 10 mm of outer medullary collecting

duct (OMCD) were dissected and pooled to constitute one sample.

Dissectedglomerulican be assumed to contain most of the cells of the

juxtaglomerularapparatusandin many instancesfragmentsof glomeru-lar arterioles as well. The time period for dissection was limited to within 1 h after the digestion with collagenase. Samples were placed in

100 ulguanidineisothiocyanate buffer (GITC buffer: 4 M guanidine isothiocyanate,25mMsodium acetate, pH 6.0, 0.8%

P3-mercaptoetha-nol), snap frozen in liquid nitrogen, and stored at-800C.

In a separate experimental series, nine rats were used to test the

effectofconvertingenzymeinhibitionontubule renin.Animals received eithervehicleortwo dosesofaconvertingenzymeinhibitor,quinapril

(2.5 mg/kg i.p.; Warner-Lambert/Parke Davis, Ann Arbor, MI) per day for 3 d. On the fourth day, rats were killed and tissues were prepared asabove.

Determinationof mRNA. To isolate RNA, tubular, and glomerular

specimenswerethawed inaniceslurrybathand sonicated for 10s.20

ug Escherichia coliribosomalRNA(Boehringer-Mannheim

Biochemi-cals, Indianapolis, IN) were added as carrier, and the specimen was layered onto a gradient of cesium chloride (100

IAI

97% and 20

_Ptl

40% cesium chloride) in a250 ul polycarbonate ultracentrifuge tube. Specimenswerecentrifuged inaTLA100fixedanglerotor ina

Beck-manTL100ultracentrifuge (BeckmanInstrumentsInc.Fullerton, CA)

for 2 hat16WCand300,000g. The RNApelletwasredissolvedin0.3

Msodiumacetateand ethanol precipitated.

Reversetranscriptionwasprimed with 0.5 ugoligo dT12 18 (Phar-macia, Piscataway,NJ),20U ofRNAsin (Promega Biotech, Madison,

WI)and 10 mMdithiothreitol at650Cfor 5 min. The samples were thenincubatedat420Cfor1 hwith 100 U of murineMolonyleukemia virus(MMLV)reversetranscriptase (Superscript; GIBCO BRL,

Gaith-ersburg, MD) in 20 pI of manufacturer's buffercontaining 500 sAM

eachofdATP, dGTP, dTTP,anddCTP(dNTP; Pharmacia, Piscataway,

NJ),and1% bovineserumalbumin(Boehringer-Mannheim Biochemi-cals). The cDNA sampleswere precipitated with 1 01 of5% linear

acrylamide,4Mammonium acetate,and100% ethanol(20).Thepellets

wereredissolved inTris-EDTA buffer (10 mMTris-HCl, pH 7.4; 1 mMEDTA,pH 8) at adilutionadjustedso that each2

_jsl

ofcDNA

correspondedto 1mmof tubuleor 1 glomerulus.

PCRreactionswereperformedusingaTempcycler (Coy Laboratory

ProductsInc., Grass Lake, MI)inatotalvolume of 50 pII inthepresence of 5 pmoles of eacholigonucleotide primer, 200uM dNTP, 10 mM

dithiothreitol,50 mMKC1, 1.5 mMMgCl2, 10mMTris-HCl, pH 8.3, 0.001% Gelatin, 1.25 UofAmpliTaqDNApolymerase (PerkinElmer CetusCorporation, Norwalk, CT), and 1.5

_ILCi

32P-dCTP (Amersham Corp., ArlingtonHeights, IL).Mineraloilwaslayeredontopof each sample to prevent evaporation of the liquid. The samples were first

denatured at 940C for 3.5 min. PCR was runfor 30 cycles with a

denaturing phase of 1.5 min at 940C, annealing phase of 1.5 minat

540C, and extension phase of 1.5 min at 720C. The last cycle was

followedbyanadditional incubationperiodof 8minat72TC.Toassess

thepossibilityofcontamination,twocontrolreactionswereincluded in eachsetof PCRamplification: ablanksample of dissection medium

carriedthroughout the RNAandcDNA preparation steps anda H20 control forthe PCR step.Afteramplification,PCRproductswere

sub-C >

z

(I)

_0

00)

600

bp

--*-470

bp

---*

Figure 1. PCRproduct for native reninandmutantrenintemplate.A segmentof 130bpbetween EcoRI andMscIsitesinratrenin cDNA wasdeletedtomake themutanttemplate(470 bp).PCRperformedon the nativetemplateandthemutanttemplate yielded productswith

ex-pectedsizes.

jectedtosizeseparation by polyacrylamide gel electrophoresis.Product bandswereexcised fromgelsand32Pradioactivitywasdeterminedby liquidscintillationcounting.

Toquantifyrenin mRNAlevels,amutantrenin DNAtemplatewas preparedfromratrenin cDNA(pREN44.ceb; giftfromDr. K.R.Lynch; UniversityofVirginia, Charlottesville, Virginia) by deletinga

130-bp

segment betweenanEcoRI siteat639-bp positionandanMscI siteat

769-bp position (allbasepairnumbersrefertonumberinginreference 21). To exclude other EcoRI sites present in the cDNA and vector (pGEM4; Promega Biotec, Madison, WI)aportionoftherenincDNA

(992 bp)wasfirstsubclonedbyPCRusingthefollowing primers:sense

5 '-ACATCTAGACGTGGTCCTCACCAACTAC-3' (bp 435-453),

and antisense S

'-ATAAAGCTTCTfAGCGGGCCAAGGCGAA-3'

(bp 1,409-1,426).Theproductwasgelpurifiedandligatedinto

Blues-cript-M13.TherecombinantDNAwasdigestedwith EcoRIandMscI

andablunt-endligation performed.The DNAwaspurified bycesium chloridebanding andquantified spectrophotometrically.Thefollowing oligonucleotide primers, flankingthedeletionsiteinthemutantrenin

templateandpositionedin separateexons(21),wereusedin the

experi-mental PCR: sense 5'-TGGGTGCCCTCCACCAAGT-3' (bp 540-558),and antisense 5'-TCCCAGGGCTTGCATGATCA-3'(bp

1,120-1,139). As shown inFig. 1, PCR performedwithnativeandmutant templatesyieldedproductswith thepredicted 130-bpdifferenceinsize. The PCRamplification efficiencywastestedbyamplifyingafour step dilution series of themutanttemplateinduplicates.The_yieldof PCR

productwas proportionaltothestartingcopy number of thetemplate,

andaplotoflogcDNAversuslogproducthadanaverageslope equal to 0.85±0.06 (n= _9).Whenadilution series of themutanttemplate

was amplified, productwas consistently detected down to about 500

copies/reaction.

PCRproductsobtained from cDNApreparedfromrat_glomerulior

tubuleswere quantified bycomparingPCRproductsofsamplecDNA

with dilution series ofthecloned mutanttemplate rangingfrom 1.02 x03 3to 6.40 x 105 copiesperreaction, amplified inparallelin the

samePCRrun(method1). Alternatively,fourequal aliquotsofsample cDNAwereco-amplifiedwith thedilution series of themutanttemplate

(method 2).Counts per minutewerecorrectedforproductsize.Linear

0.51 2.56 12.8 64

Table I. ReninActivityand Renin cDNA in Glomeruli

andTubules

Sample Renin activity Renin cDNA

,tGU/mmtubuleor/glom copy#/mm tubuleor/glom Glomerulus 405 + 120(n= 15) 313,000± 179,000 (n = 11)

PCT 51 + 15 (n = 16) 580 + 210(n = 15)

PST 2.4 + 11.7(n = 18) 490+ 100(n= 14)

OMCD 3.9 + 2.27(n= 15) 1 ±1 (n = 8)

a

L-C..)

CD

0

0)

-j

3 4 5 6 7

Log

Mutant

Template Copy

Number

Figure2. A representative quantitative PCR forglomerularrenin. (Top) Autoradiographofthe renincompetitive PCR. Each PCR reaction

con-taineda constantaliquot ofinitialcDNA prepared from dissected

glo-meruli (Exptal),and avariableamountofa mutanttemplateof fivefold

dilution.(Bottom)Quantificationof the competitivePCR.Log molecu-lar copy numberofthe initial mutant templates wasplottedagainst log radioactive counts of PCR product.

in each unknownsamplewasdetermined from theintersection,as shown inFig.2.

To controlvariations in RNA isolation andefficiency of reverse

transcription, PCR amplification for/3-actin wasalsoperformed.The

primers for /3-actin were chosen empirically from human published sequences (22). The sequences of /3-actin primers were as

fol-lows: sense 5'-AACCGCGAGAAGATGACCCAGATCATGTTT-3' (bp 384-413), antisense 5'-AGCAGCCGTGGCCATCTC' fGCTC-GAAGTC-3' (bp 705-734).

Theidentity ofthe renin PCRproduct from glomeruliand tubules was determined with restriction enzyme digestion. The PCR product wasdigested withEcoRIandMscIat37°Cfor 1 h in the bufferprovided bythemanufacturer.

Toevaluate theeffectofcollagenasetreatmentanddissectiontime onrenin mRNA wedeterminedrenin and,Bactin mRNAinspecimens

ofkidney cortex frozen immediately after harvesting and in cortical

tissue subjectedtothestandard treatment (n=threepairs).PCR product for renin was foundtobe 10% lower with cDNA derived from the

immediately frozen kidney,adifferencethatwas notsignificant. /3actin

productlevels did notdiffer.

Determinationofreninactivity inglomeruliandtubules. Segments

ofproximalconvolutedtubules, proximalstraighttubules,outer medul-lary collectingducts, andglomeruli weredissectedasdescribed above except that1%bovine serumalbumin instead of fetalbovine serum was

addedto the DME. The sampleswerecollected in 5 ulofdissection mediumwith anEppendorfpipette tipandtransferredinto another dish

containing about 20 ml of fresh dissection medium to wash, and the dissectedsamples werethen transferredinto 100

_ILI

of0.1 Msodium phosphatebuffer. To determine thepHoptimumof renin inglomerular

and tubule samples, the renin assays were run in sodium phosphate

buffer atpH rangingfrompH5.4 to 8.0. Renin was measured with an

antibodytrapping technique using purified homologous substrate(23).

Renin activity is expressed in Goldblatt units (GU), determined by comparisonwith renin standards from the Institute of Medical Research (Medical ResearchCouncil,Holly Hill,London).

Statisticalanalysis. The data areexpressedasmean±SE. All

com-parisons were made by an unpaired t test. P < 0.05 was considered

significant.

Results

Renin activity.Results frommeasurementsoftissue renin activ-ityaresummarized in TableI.Tissue renin activitywashighest in glomeruli, averaging 405±+120

_{jLGU/glomerulus.}

Itislikely thatglomerularrenin activityreflects thepresence ofgranular cells in the dissected specimens. Substantial renin activity, 51±15 MGU/mm, was also found in PCT. In contrast, renin activity in both PST and OMCDwas notsignificantly different from zero. While the renin content in the dissection dish in-creased measurably during theonehourofdissection, norenin was found in the washing medium even from specimens ob-tained at one hour. The absence of renin in PST and OMCD argues against unrecognizedcontamination of tissue renin with renin secreted into the medium. To obtain information about theidentity of theangiotensin I generating activity in tubules andglomeruli, wedetermined the pH optimum ofglomerular and tubular renin-likeactivity.As shown inFig. 3,reninactivity from both glomeruli andproximal convoluted tubules had an

identicalpH optimum of between 7.0-7.5, suggestingthat the enzymecatalyzing angiotensinI formation in the twolocations is the same.

Renin mRNA. Renin mRNA as assessedby a quantitative PCR method was detectable at approximately equal levels in cDNApreparedfromPCT and PST. In contrast, no reninPCR product was amplified in cDNA derived from OMCD. The abundance of renin mRNA in the proximal tubule segments was atleast two orders ofmagnitudelower than that of

glomeru-1.5

1.0'

0.5

9

c

D)

D1

-i a:1

PCT

-* Gl ;

IA

5.0 5.5 6.0 6.5 7.0 7.5 8.0

pH

8.5 9.0

Figure3. Determination of

opti-mal pH forglomerularand

tu-bule reninactivity. The data in-dicate ratios of reninactivity

de-terminedatvarious pHtothat obtainedatpH 7.Eachnumber

represents an averageof renin activityfrom threesamples. Re-ninactivity frombothglomeruli

and tubules hadapHoptimum of7.0 to 7.5.

Copy#

X1 OA4

N.D. Msc

I

PCT PCT PST

.. .

g.

N.D. EcoR

PST PCT PST

. _-600bp

<- 500 bp

* 369 bp

<- _{231 bp}

5.12 25.6 128 640

..

540

EcoR 100 bp*

540 640

PST

Figure 4. (A) Representative autoradiographof quantitative PCR for glomerular and tubule renin. A dilution series of themutanttemplate wasamplified in parallel with initial cDNA prepared from either dis-sectedglomeruliorproximal tubule (Exptal) in thesamePCRrun.

ReninPCRproduct from glomeruli yielded higheramounts,PCR

prod-uctfromproximal tubules yielded lower, and equalamountsbetween PCTand PST. Renin PCR product from OMCDwasundetectable. (B) Representative autoradiographs of competitive PCRs for glomerular and tubulerenin.EachPCRreaction containedaconstantaliquotof experi-mental cDNA andadilution series of themutanttemplate. Glomerular reninyielded PCR product bands with high density, while PCR products fromproximal tubules showed much loweramountsbut theywereeasily detectable.

larrenin mRNA. Fig. 4 shows examples for the two methods ofquantitation usedinthis study. In method 1, glomerularor

tubular cDNAwas amplified inparallel with the mutant

tem-plate in thesamePCRrun (Fig. 4A). Inmethod2, the cDNA

prepared from glomeruli ortubules was coamplified with the

dilutionseries of themutanttemplate (Fig. 4B).Thelog

molec-ularcopy number of the mutanttemplate was plotted against log radioactive counts of the PCRproduct, andtheamountof cDNA was calculated from the intersection of the individual

regressionlines.Relative cDNAcopynumbers inglomeruliand

tubules are included in Table I. It can be seen that 1 mm of

proximal convoluted orproximal straight tubules had arenin cDNA level of 580±210 and 490±100 copies, respectively, whereasglomerular renin cDNA amountedto313±17.9 X 103

copies. Thus, the amount of renin mRNA in cDNA prepared

from mm proximal tubule was 500-fold lower than the amountdetectedinasingle glomerulus.

To determine whetherthesequencesoftubularand

glomeru-larrenin cDNAareidentical, PCR products from PCT and PST were digested with restriction enzymes. As shown in Fig. 5, products ofcDNA from bothproximal segmentsfollowing

di-369bp 771

500bp

1140

--A

1140

1140

Figure 5. Restriction digestion oftubulereninPCR product. (Top) Autoradiograph of nondigested (N.D.)anddigested tubule renin PCR product. (Bottom) Restrictionmap.Numbersbelow each lines indicate

basepairpositions.

gestion with MscI and EcoRIwereidenticaltothose predicted fromthestructureofratkidney renin cDNA (21).

Effect ofconverting enzymeinhibitionon renin mRNA

lev-els. The effect of CEIwas examined in nine separate

experi-ments.Inordertoassurethatreningeneexpressionwas

stimu-lated with CEItreatmentinourexperiments,two separatePCR

runs were performedto determine renin mRNA isolated from the right kidney cortex ofrats. Theleft kidneys of these rats

were used for microdissection. Renin mRNA levels in kidney

cortex increased 2.86±1.22-fold comparedto control (n = 3)

(see Fig. 6). Glomerular renin activitywasstimulated

substan-tially by CEI treatment, whereas it decreased significantly in PCT. Consistent with increasing renin mRNA levels in cortical tissue,glomerular renin mRNA levels increased inCEI-treated animals; however, reninmRNAlevelsinPCTand PSTdidnot

change significantly aftertreatment with CEL. Fig. 7 displays results of renin activity and renin mRNA levels in glomeruli andtubules incontrolandCEI-treatedrats.

Discussion

The present results show that renin mRNA can be detected

in both proximal convoluted andproximal straight tubules of

untreated controlrats.Thelevels of renin mRNA in these

seg-ments were about the same, andclearly distinguishable from

thenegative reactionswithcDNA derived fromoutermedullary collecting ducts. Thus, aside from intrarenal vessels, the renin

gene is expressed along the proximal tubule, but not in the

collecting duct. Since renin substrate and converting enzyme

activityis present in proximal tubulecells (6-12),generation

of angiotensin II at the level of the proximal tubule would appearpossible.

It is important to point out, however, that the levels of

A

12'25

6 128 U 9 °0 5.1225. 4f

4"0

Copy# 1.02

x 103

Explal

--Mutant -)

I

_M0

B

Copy# x 103

GLOM

.LOM

....

...

.:

:z_*.

_-~;

PCTI...

.-.

L.

<- Exptal

4 _Mutant

N.D.

Msc

540

231bp

600bp

A

Initial cDNA

_(gl)

Renin

Control CEI

1 2.5 1 2.5

_

-

t

4-600 bp

13-actin be 352bp

Figure 6.Autoradiographs of representative renin and /3-actin PCR from CEI treatedseries. PCRwasperformedusing cDNA prepared from kidneycortexwith 1 and2.5

ILI

initialamountsfor each sample for

renin (top) andfor /3-actin (bottom). In this pair of PCR determinations, the increase in renin mRNA levelswas3.4-fold.

proximal tubule renin mRNA are very low.The copynumber

of total tubularrenin cDNAyieldsavalue of about 1.4x 108,

whereasthesumofglomerularrenin cDNA copiesamountsto

about 9.4 x 109 (assuming 30,000 nephronsperkidneyanda

totallengthof theproximaltubule of9 mm). Thus, onlyabout 1% of totalreninmRNAin therenalcortexis found in proximal tubules. Arelativelylow level of reningeneexpressionin proxi-mal tubules is consistentwith the negative outcomeof earlier

attempts todemonstrate renin mRNA in these cells. Ifone as-sumes that the sensitivity ofRT-PCRis much greaterthan in situhybridization methods,the failure toobservereninmRNA with in situhybridizationintheproximaltubule isquite under-standable (14, 16-18). Inonerecentstudy RT-PCRhas been appliedtothequestionofproximaltubule reningeneexpression

A

800

-13Control *E . 0CEI 0

85L

00-.5 0 < A.

C 400 -EE

200

=L

*

*

1515 1P813 11 1719 OMCD PST POT GLOM

B

1200000 _Control

1000000- E CE

800000

-

600000-400000

200000 -,

'000

_mf

T

14 1 1 15 11 11 10

PST PCT GLOM

Figure 7. (A)Effect of CEIonreninactivityin tubules andglomeruli. (B)Effect ofCEIonrenin mRNA levels in tubules andglomeruli. Figuresbeloweach column indicate number ofsegments.*P < 0.02.

(19). Renin PCRproductswereonlyseenin cultured proximal tubule cells and in microdissected proximal tubules of CEI-treated animals. Incontrast tothepresentexperiments, proximal tubules of controlratsdid notshow detectable levels of renin mRNA in threeoutof four experiments. Differences in animal pretreatment and in PCRmethodology may beresponsible for these different results, but it is also evident that the differences between these and the present observations may be more of quantitative thanqualitativenature. WhenPCR products were reamplified inasecondstagereaction,Moeand his colleagues did, in fact, detect renin product suggesting a low level of renin gene expression (19). Inaddition, proximal tubule cells in culturehave been shown to contain detectable amounts of renin mRNA, but its level was estimated to amount to only 2-3% ofthat in cultured

juxtaglomerular

cells (19). Thus,

all available results appeartobe in agreementthatreninmRNA levels in the proximal tubule are very low and may only be detectable under certain optimized conditions. Whether such lowlevels of renincangenerate enough angiotensinI toresult inthe observed levels of angiotensinIIinproximal tubular fluid is uncertain.

In contrast to the low levels of renin mRNA in proximal tubular epithelium, renin content along this nephron segment was found tobe surprisingly high. Renin activity measured as angiotensin I generating capacity was only about eightfold higher in one glomerulus than in 1 mm of proximal tubule (compared to a ratioof500:1 for renin mRNA). Since glomeru-larproteincontent ortissue mass of1 mmof PCT is about 1.5-foldhigherthan that of oneglomerulus, glomerular renin activ-ity per unit tissuemassis about 10-fold higher than tubular renin activity.Consideringalength of proximal convoluted tubulesof about6 mm, thetotal amount of proximal tubular renincan be estimatedtobeapproximatelyhalfthe totalamountof glomeru-lar renin. This relationship is most certainly an underestimation of the difference between glomerular and tubular renin since only a fraction ofall granular cells in thejuxtaglomerular ap-paratus is likely to be included in our dissected glomerular specimen. Nevertheless,our observations are inaccordwiththe earlier immunocytochemical demonstrations of substantial amounts ofrenin inproximal tubular epithelium (24-26).

The presenceof renin mRNA in the proximal tubule would appeartosupport the concept that the reninactivityfound in the cells oftheproximalepithelium is derived from renin synthesis. Whilesomeof the proximalepithelialrenin may, infact,

origi-natefrom translation of proximal tubule renin transcripts, it is unclear whether this is the only pathway responsible forthe presence of the enzyme in this location.Itisofconcernthatno measurablerenin activitywas seeninproximal straight tubules even though the level of renin mRNA was not significantly lower than intheconvoluted segments.Itispossiblethat renin generated in the S3 segments isnotstoredintracellularlytoany measurableextent orthat theprotein is exceptionally unstable. On the otherhand,someothermechanism,inadditionto transla-tionof renin mRNA, may participate inelevatingrenin substrate cleavage intheconvoluted portion of the tubule. One possibility is that the generation of angiotensinImay becatalyzed bysome enzyme other than renin.Infact, bothcathepsinDand kallikrein have been shown to be able to generate angiotensin II from

angiotensinogen (28, 29).However, the pHoptimumfor

angio-tensin formation by both the lysosomalcathepsinsandby kalli-krein is in the acid range, around pH 4-5, which is far lower than the neutral pH foundto be optimal for the interaction of

4L)

0

renin with species-specific angiotensinogen (30). In addition, kallikrein is not found in the proximal tubule. Tonin has been described as an enzyme that can cleave angiotensin II directly fromangiotensinogen (31, 32). Although this enzyme may play arolein angiotensin II formation in the proximal tubule, it is unlikely to be responsible for the angiotensin generating activity in the proximal tubule in our experiments since angiotensin I formation was measured.

Alternatively, as suggested by Taugner et al. (24-26), it is possible that the immunocytochemical evidence for proximal tubule renin may reflect uptake of filtered renin by endocytosis ratherthanformation of new renin (27). Support for this notion is the finding that labeling of proximal apical vesicles could be found following intravenous injection of radioactive renin. Furthermore, absorption of filtered renin by proximal tubule cells has been shown by micropuncture (33). Since the fusion ofendosomeswithlysosomes and the lysosomal protein degra-dation are relatively slow processes (34), one may assume that renin taken up into endocytic vesicles retains its enzymatic activity for some time, so that renin-containing vesicles could contribute to the angiotensin generating activity of proximal tubules. This may at least in part explain why renin activity in proximal straight tubules was not clearly detectable since pro-teinuptake, the presence of the endosomal-lysosomal machin-ery, and the abundance of lysosomal enzymes are all less pro-nounced in S3 than in SI and S2 proximal segments (34). Furthermore, the medium of primary cultures of proximal tubule cells has been shown to accumulate renin in a time-dependent andregulated fashion suggesting continued secretion of renin. However, the renin activity in lysed cells was low or even undetectable (19, 35). It is conceivable that this dissociation between secretion and content is the result of reduced renin endocytosisasaconsequence of the presumably low renin con-centration in the large volume of the culture medium.

Our results confirm earlier studies showing that the adminis-tration ofaconverting enzyme inhibitor fortwodays causeda significant increase in reninactivityin dissectedglomeruli( 19). Similarly, glomerular renin mRNA levels were found to be increasedduringconvertingenzyme inhibition(18).Incontrast to the stimulatory effect of this treatment onglomerularrenin activity and renin mRNA, we wereunable to find increments in renincontent orrenin gene expression in any of the tubule segments tested. In the earlier studyof Moeetal., converting

enzymeinhibitionappearedtoelevate renin mRNA levels into thedetectable range in the S2 segment of theproximal tubule,

but usually notin S1 orS3 segments (19). Even though we

did not systematically determine the origin of our proximal

tissue samples,itseemshighly unlikely that S2 segmentswere

consistently not harvested in our study. While it is possible

thatthe shorter duration ofconvertingenzymetreatmentinour experiments comparedtothestudy byMoeetal. is

responsible

forthedifference inresults,the increase in cortical and

glomeru-lar renin mRNA supports the efficacy of the CEI treatment protocol used inourstudies (19).On the otherhand,ourresults may indicate that the regulation of renin gene expression in

glomeruliand in theproximal tubule may begoverned by differ-entfactors.

In summary, the results of the presentstudy showthat both

proximalconvoluted andproximal straight tubules,butnot outer medullarycollectingducts, express the renin geneeventhough

thenumberofcopiespermmof PCT isonlyabout 1/500th of the copy number found in one glomerulus.

Significant

renin

enzymatic activity, amounting to about 1/8th of that in one glomerulus, was found in 1 mm of PCT, whilereninactivity was notdetectable in proximal straight tubules or outer medullary collecting ducts. Thus, while only about 1% of total renin mRNA is found in proximal tubules, the percentage of total renin enzyme activity associated with PCT is much higher. The present results are in accord with the concept that a proximal tubular renin-angiotensin system may be involved in control-lingepithelial function in a paracrine fashion.

Acknowledgments

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants DK-37448, DK-39255, and DK40042.

M.P. Harris was the recipient of a summer stipend of the University of Michigan Summer Program for Student Biomedical Research.

References

1.Geibel, J.,G.Giebisch, and W. F. Boron. 1990. Angiotensin II stimulates bothNa'-H+exchangeandNa+/HCOicotransportintherabbit proximal tubule. Proc.NatL.AcadJSci. USA. 87:7917-7920.

2. Saccomani, G.,K. D. Mitchell, and L. G. Navar. 1990. Angiotensin II

stimulation of Na'-H+ exchange in proximal tubule cells. Am J. Physiol. 258:F1188-Fl195.

3.Douglas, J. G.1987. Angiotensinreceptor subtypes of the kidney cortex.

Am.J. Physiol. 253:F1-F7.

4. Braam, B., K. D. Mitchell, J. Fox, and L. G. Navar. 1993. Proximal tubular

secretion of angiotensinII in rats.Am. J.Physiol. 264:F891-F898.

5.Seikaly,M.G.,B. S. Arant, and F. D.Seney. 1990. Endogenous angiotensin

concentrations inspecificintrarenal fluidcompartmentsof the rat. J. Clin.Invest. 86:1352-1357.

6.Richoux, J. P.,J. L. Cordonnier,J. Bouhnik,E. Clauser, P. Corvol, J.

Menard,and G.Grignon. 1983. Immunocytochemical localization of angioten-sinogenin rat liver andkidney. CellTissueRes.233:439-451.

7.Campbell,D.J.,and J. H. Habener.1987.Regionaldistribution of

angioten-sinogen messenger RNA in ratadrenalandkidney.J.Hypertens.4:S385-S387.

8.Ingelfinger,J.R.,W.M.Zuo,E. A.Fon, K. E.Ellison,and V. J. Dzau. 1990. In situhybridizationevidence forangiotensinogenmRNA in the rat proxi-maltubule. J. Clin. Invest. 85:417-423.

9.Bruneval, P., N. Hinglais, F. Alhenc-Gelas, V. Tricottet,P. Corvol,J.

Menard,J. P.Camilleri,and J.Bariety.1986.AngiotensinIconvertingenzyme in humanintestine andkidney.Ultrastructural immunohistochemical localization.

Histochernistry. 85:73-80.

10.Rix, E.,D.Ganten,B.Schilil,T.Unger,and R.Taugner.1981.

Converting-enzyme in thechoroid plexus, brain,andkidney: immunocytochemical and bio-chemical studies in rats. Neurosci. Lett.22:125-130.

11.Takada,Y.,KI Hiwada,M.Unno,and T. Kokubu. 1982.

Immunocyto-chemicallocalization ofangiotensin convertingenzymeatthe ultrastructural level in the humanlung andkidney. Biorned Res. 3:169-174.

12. Ward, P. E., C. D. Gedney, R. M.Dowben, and E. G. Erdos. 1975. Isolationofmembrane-boundrenalkallikreinandkininase.Biochem. J.

151:755-758.

13.Taugner, C.,K.Poulsen,E.Hackenthal, andR.Taugner. 1979. Immunocy-tochemicallocalization of renin inmousekidney.Histochemistry.62:19-27.

14. Deschepper, C. F., S. H.Mellon,F. Cumin, J. D. Baxter, and W. F.

Ganong.1986. Analysis byimmunocytochemistry and in situhybridizationof renin and its mRNA inkidney,testis,adrenal andpituitaryofrat.Proc.Nati.

Acad Sci. USA. 83:7552-7556.

15.Menard, J.,P. W.N'Goc,J.Bariety,P. T.Guyenne,and P.Corvol.1979. Directradioimmunoassayandimmunocytochemicallocalization of renin in human

kidneys.Clin. Sci.57[Suppl.51:105-108.

16.Dzau,V.J.,K. E.Ellison,T.Brody,J.Ingelfinger,and R. E. Pratt. 1987. Acomparative studyof the distribution of renin andangiotensinogenmessenger ribonucleic acids inratand mouse tissues.Endocrinology. 120:2334-2338.

17.Schunkert, H.,J. R.Ingelfinger,and V.J.Dzau. 1991.Evolvingconcepts of the intrarenalrenin-angiotensinsystem in health and disease: contributions of molecularbiology.Ren.Physiol.BiochenL 14:146-154.

18.Gomez,R.A.,K.R.Lynch,B.C.Sturgill,J. P.Elwood,R. L.Chevalier,

R.M.Carey,and M. J. Peach. 1989. Distribution of renin mRNA and itsprotein

inthedevelopingkidney.An.J.Physiol.257:F850-F858.

19.Moe,0.W.,K.Ujiie,R. A.Star,R.T.Miller,J.Widell,R. J.Alpern,

and W.L.Henrich. 1993. Reninexpression in renalproximaltubule. J. Clin.

Invest.91:774-779.

repeat at threespecific sites that would be brought into mutual proximity by DNA folding in thenucleosome. Cell. 37(3):889-901.

21. Burnham, C. E., C. L. Hawelu-Johnson, B. M. Frank, and K. R. Lynch. 1987. Molecularcloning of rat renin cDNA and its gene. Proc.Nati.Acad. Sci. USA.84:5605-5609.

22. Gunning, P., P. Ponte, H. Okayama, J. Engel, H. Blau, and L. Kedes., 1983. Isolation and characterization of full-length cDNA clones for human alpha-beta, and gamma-actin mRNAs: Skeletal but not cytoplasmic actins have an amino-terminalcysteine that is subsequently removed.Mol.Cell.Biol. 3(5):787-795.

23. Lykkegard, S., and K. Poulsen. 1976. Ultramicroassayfor plasma renin concentration in the rat using the antibody-trapping technique. Anal. Biochem 75:250-259.

24.Taugner, R., E. Hackenthal, R. Nobiling, M. Harlacher, and G. Reb. 1981. The distribution of renin in the different segments of the renal arterial tree. Histochemistry. 73:75-88.

25. Taugner, R., E. Hackenthal, T. Inagami, R. Nobiling, and K. Poulsen. 1982. Vascular andtubular renin in the kidneys of mice.Histochemistry. 75:473-484.

26. Taugner, R., S. J. Kim, K. Murakami, and R. Waldherr. 1987. The fate ofprorenin during granulopoiesisin epithelioid cells.Immunocytochemical exper-iments with antiseraagainst renin and different portions of the renin prosegment. Histochemistry. 86:249-253.

27. Iwao, H., N. Nakamura, F. Ikemoto, K. Yamamoto, V. Mizuhira, M. Ono, and Y.Sugiura. 1982. Distribution of exogenously administered renin in mouse kidney. 1982. Clin. Exp. Hypertens. [Al4:2449-2456.

28. Maruta H., and K. Arakawa. 1983. Confirmation of direct angiotensin formationbykallikrein.Biochem.J. 213:193-200.

29. Day, R. P., and I. A. Reid. 1976. Renin activity in dog brain:enzymological

similarity to cathepsin D. Endocrinology. 99:93-100.

30. Inagami, T. 1993. Renin: purification, structure, and function. In The

Renin-AngiotensinSystem. J. I. S. Robertson and M. G.Nicholls,editors.Gower Medical Publishing, London/New York. Vol. 1, 4.1-4.17.

31. Hackenthal, E., C. Koch, T. Bergemann, and F. Gross. 1972. Partial purification and characterization of a renin-like enzyme from rat submandibular gland.Biochemt.Pharmacol. 21:2779-2792.

32. Boucher, R., J.Asselin, and J. Genest. 1974. A new enzymeleadingto thedirectformation of angiotensin II. Circ. Res. 34/35 [Suppl.1]:203-209.

33. Leyssac, P. P. 1978. A micropuncture study of glomerular filtration and tubular reabsorption of endogenous renin in the rat. RenalPhysiol. 1:181-188.

34. Maack, T., C. H. Park, and M. J. F. Camargo. 1992. Renal regulation of proteins and macromolecules: normal. In The Kidney,Physiologyand

Pathophysi-ology. 2nd Edition. W. D. Seldin and G.Giebisch,editors. RavenPress, New York.3005-3038.