Renin messenger RNA, detected by polymerasechain reaction, can be switched on in rat atrium

Yi-kun Lou, Bruce G. Robinson* and Brian f. Morris

objective: The aim of the present study was to use a highly sensitive, polymerasechain reaction (PCR) technique to examine the controversial question of

'renin gene

expression in the heart and to determine whether expression in cardiac tissue can bestimulated.

Design: The study involved female Wistar rats fed either a normal-sodium diet or alow.-sodium diet, together with an angiotensin I converting enzyme inhibitor, enalapril,orally for 1 week.

Methods: RNA was extracted from atrium, ventricle, kidney (positive control)and submandibular gland (negative control) and, after reverse transcription intocomplementary DNA, was used in a 35-cycle PCR. Reaction products were visualizedlfj:.r.gl":.,r9phoresis by ethidium bromide staining and hybridization probing with[32P]-labelled target oligonucleotide.

Resglts: Atrial RNA from sodium-replete rats gave little or no renin messenger (m)RNAPCR product on ethidium bromide-stained gels. After DNA transfer, followed byhybridization probing and extended autoradiography, a faint band was seen. lnsharp contrast, atrial extracts from sodium-depleied enalapril-treated rats displayed apronounced band of hybridization, corresponding in size to that expected ior'reninmRNA. No band was seen for ventricle.conclusion:.The present PCR study has shown that in the normal sodium-replete rat,atrial tissue has only very low renin gene expression, but that after a low-sodium diet* treatment with enalapril, expression is switched on in atrium. Ventricular t issue doesnot express the renin gene in either state.

Journal of Hypertension 1993, 11:237-243

Keywords: Renin gene, regulation, Nacl deptetion, angiotensin convertingenzyme inhibitor, enalapril, polymerase chain reaction.

Introduction

Renin is synthesized by modified epitheloid cells inthe preglomerular arreriolar wall I t ] , from where it issecreted in response to conditions such as decreasedextracellular fluid volume, decreased blood pressureand sodium depletion. There is now evidence to sug-gest that much of the angiotensin I in the bloodstreammay be generated in regional vascular beds by renin ofrenal origin that is sequestered by blood vessel walls[Z-6]. nenin like activity has also been derected in car-diac tissue [7-10] , and could represent non-specificaspartyl protease acrMty during in uitro assays br the

activity of genuine renin. If the laffer, ir is possible that,as in blood vessels [2-6], the source of cardiac reninmight be uptake from the circularion rather than en-dogenous srmthesis.

ln order to address the question of renin expressionin the heart, attempts have been made to detect reninmessenger (m)nNe in cardiac rissue. By use of thetechniques of ribonuclease protection and Northernplg$ng, several groups [7,9,1t-751, although not all[16], have reported the de-tection of low concentra-tions of renin mnNl in rai heart and, aofia. More re-cently, however, the highly sensitive pollrnerase chain

From the Molecular Biology and Hypertension Laboratory, Department of Physiology, The University of Sydney, Sydney, Nsw 2006,and -Kolling Instirute, Royal North Shore Hospital, Sydney, NSW 2065, Austrjlia.Sponsorship: This study was supported by grants from the National Heart Foundation, the Australian Research Council and theNational Health and Medical Research Council of Australia.Requests for reprints to: Dr B.J. Morris, Building F13, The University of Sydney, NSW 2006, Australia.Date of receipt: 18 August 1992; revised: 6 November 1992; accepted 23 November'1992.

@ Current Science ttd ISSN 0263{352 237

238 Journal of Hypertension 1993, Vol 11 No 3

reaction (PCR) technique, involving 30 amplificationrycles, was unable to detect renin mRNA in cardiac andvascular tissues of Wistar rats [tll.

The aim of the present study was to more thoroughlyinvestigate the ability of the rat heart to s].nthesizerenin, by using a greater number of PCR rycles, andalso by studying rats subjected to a well establishedprocedure that produces strong stimulation of renalrenin gene expression, viz. a low-sodium diet, to-gether with treatment with an angiotensin I convert-ing en4.'rne (ACE) inhibitor, ro decrease angiotensinII and therefore the negative feedback effect of thispeptide on renal renin production It6,ta].

Methods

Animals and treatment groupsSince testosterone can influence renin gene expres-sion [l&20], female 7-week-old \fistar iats weighingi30-150g were used. Groups of rats were given tapwater to drink freely and fed cakes made by baking a 1-kg mixture of 6.59 NaCl, 2509 whire rice,750m1 soymilk (AAy1 soybean powder) and 10gsucrose (dry composition 0.7%, 75V", 23Y", 0.Io/o, byweight, respectively). The main reason for using thisfood rather than commercial chow was that the NaClcontent could be adjusted without changing the otheringredients, which together were considered to be nu-tritionally adequate. The sodium concentration was,moreover, similar ro the values of 0.4"/" [2t] and 0.3%[ZZ] tor the normal-sodium diers used by others (cf.>3Zo used for high-sodium diets) [22].

The rats were dMded into two groups, one being acontrol group, in which the same diet was conrinuedfor a further week, u'hile the other was fed low-sodiumcakes (0.5mmo/100g food; i.e. 0.03% sodium) andwere given enalapril maleate (Amprace; Amrad phar-maceuticals, Kew, Victoria, Australia),7mg/ml, in distilled drinking water, for 1 week. Urine was collecteddaily and sodium, measured by flame photometry,reached 28 + 3 ( + SE) pmol,/ml by the end of thisweek, compared with 2O7 t 35 pmoVmI for rars onthe normal-sodium diet. Each rar was killed by a blowto the head and the heart was rapidly removed, whilestill beating, into liquid nitrogen. The kidney and sub-mandibular gland were then quickly collected as well.

RNA extractionThe atria were sliced from fiozen hearts with a scaloelblade, together witl-r a similar amount of venrricularand other tissues, then placed in guanidine thiocyanatebuffer [23] in the glass tube of a Dounce homog-enizer (\Wheaton, Millville, New Jersey, USA) and total RNA was isolated using the guanidine thiocyanatemethod [Za]. tn order to avoid cross-conramination oflow-renin tissues, extractions on each particular tissue

tlpe were performed separately, with kidney last. TheRNA was then used directly for PCR.

OligonucleotidesPCR primers were chosen after examination of therat renin complementary (c)DNA sequence [Z4land analysis for uniqueness and suitability for PCR.Oligodeorrnucleotides were made wirh a PCR-Mate,DNA Synthesizer model 391 (Applied Biosystems, Fos-ter City, California, USA). The primers were comple-mentary to the ends of a 390 base-pair (bp) segmentcorresponding to nucleotides 770-7099 of rar renincDNA; this contained four intron sites so that onlyamplicons of 390bp would have arisen from reninmRNA. S7e assumed that a 3.6-kb band would indi-cate a genomic amplicon, as for earlier experimentsusing different primers and a shorter extension time[25], while the lack of a 3.6kb band would indicateeither that no genomic DNA was present or that therewas incomplete extension offrenin genomic DNA. Thelatter did not matter, since our only concern wasthat the amplicons seen were from renin mRNA de-rived cDNA and not genomic DNA, and was ensuredby the intron sites in the region chosen for amplification. The sequence of the oligonucleotide primerspanning the exon S/exon 6 border was 5'-ACA GCAGGG AGT CCC ACC TGC T 3' and that of the primerin the exon 9 region was 5'-TCA TCG TTC CTG AAGGGA TT 3'. The latter was almost identical to one ofthe primers used in a previous reporr using pCR forrat renin mRNA [17] , whereas the former was furthertowards the 5' end of the gene. The oligonucleotideused for the probe was directed at cDNA nucleotides940-959 in the exon 7 region and was 5'TCA TGCAAG CCC TGG GAG TC-3' [r7). The uniqueness andtherefore specificity of rhese oligonucleotides was es-tablished by a computer-assisted homology compar-ison with the most closely-related cDNA, carhepsinD 1261, as well as by searching for alignment irtheGenBank data base. End-labelling was carried out us-ingTa polynucleotide kinase and [y-32p1-ATp (Amer-sham, Buckinghamshire, UK).

PCR of renin mRNAReverse transcription of 14pg total RNA was car-ried out by incubation of 100 U recombinant Moloneymurine leukaemia virus reverse transcriptase (Sr"rperScript; Bethesda Research Laboratories, Gaithersberg,Maryland, USA) at 37'C for t h in a 20-pl mixture ofPCR buffer containing 1 mmo/l of each deoxyribonr_r-cleotide triphosphap, 100pmol of random hexamersand 20 U of the RNAase inhibitor RNasin (promega,Madison,'$Tisconsin, USA), where the PCR buffer com-prised 50mmol/lKCl,20 mmol,/LTruS-HCl, at pH 8.4,0.1 mglml bovine serum albumin and 5 mmo/l MgCl2.The reaction was stopped by heating rhe mixrure ar95'C for 5 min, and after chilling ir on ice, 30 pl ofa mixtLrre of 100pmol of each primer and Z.5tJ ofTaq polynterase (Amplitaq ; P erkin - E lmerlCetus, No r-walk, Connecticut, USA) in the PCR buffer was added,

Stimulation of atrial renin mRNA Lou et a/. 239

the mixture was ovedayed with mineral oil and, us-ing a DNA Thermal C1'cler (Perkin-Ekner/Cerus), 35PCR cycles were performed with the step-rycle pro-gram set to denature at 95"C for L min, anneal at55"C for l min and extend at 7O'C for 1min. Eachrun included, as a tissue negative control, RNA ex-tract from the submandibular gland, a tissue whichdoes not produce rerttn [27,28] , and a solution-onlynegative control, which contained all ingredients ex-cept for RNA The tissue positive control was kidneyRNA. In order to circumvent errors arising from vari-ations in reaction conditions, PCR of low-sodium *enalapril-treated and normal-salt rats was performedin altemate reaction tubes. Controls were used andstringent PCR conditions were maintained, such aspositive displacement pipettes and different rooms forthe setting up and running of reactions and for elec-trophoresis and subsequent post-PCR manipulationslZ9),to prevent false-positive results arising from PCRproduct carryover.

Detection of PCR productAfter gradual cooling, one-fifth of the reaction mix-ture was electrophoresed on a 3o/o agarose gel andDNA was visualized by ethidium bromide staining.A band of 390bp, corresponding in size to the ex-pected PCR product, was indicative of the presence" of renin mRNA After transfer to Gene-Screen Plus ny-lon membranes (l.mN-Dupont, Boston, Massachusetts,USA) hybridizatson was performed with the targetoligonucleotide probe at 42"C overnight in sixfoldstandard saline citrate (where standard saline citratecomprised 0.I5moVl NaCl and 75mmol/l sodium cit-rate at pH 7.0), l0mmoVl sodium phosphate buffer,pH 6.8, 1 mmol,zl ethylenediaminetetraacetic acid, 0.5"/"sodium dodeq{ sulphate, 1OOmg/ml denatured andfragmented salmon sperm DNA and L0-fold Den-hardt's solution. The filters were washed in doublestandard saline citrate/lZo sodium dodecyl sulphateat room temperature and autoradiographywas carriedout at -80'C for 8-15h.Instead of electrophoresis and membrane transfer,slot-blot hybridization of one-fifth of the post-PCRmixture was performed in some experiments withthe application of fivefold dilutions of the sample toa (-probe membrane (BioRad, Richmond, California,USA) using a Bio-Dot SF Microfiltration Apparatus(BioRad) in accordance with the manufacturer's direc-tions. Hltridization and washing conditions were thenperformed as described for the above Southern blot-ting procedure, except that the autoradiography lasted3h.

Results

Sodium-replete ratsBy ethidium bromide staining or hybidization prob-ing, kidney RNA (tissue positive control) from normal

sodium-replete rats displayed a single 390-bp bandcoresponding in size to that expected for the PCRproduct of renin mRNA (Fig. 1). The absence, more-over, of a 3.5-kb band on the autoradiographs indicated that there were no amplicons arising from therenin gene. Recombinant rat renin cDNA also gave a390-bp band in preliminary experiments (not shown),but renin cDNA was not included routinely, in accor-dance with recommendations that use of such re-combinant plasmids in PCR be avoided because ofpotential contamination problems [29]. ln contrastto results for the kidney samples, the atrial samplesdid not show a 390-bp band on ethidium bromide-stained gels. After hybridization probing, howwer, afaint band, varylng in intensity for different rats, wasdiscemed for the atrial samples, after either 9 or 16hof exposure to X-ray filrn (Fig. 1). Also, the use of 5 pginstead of 7-3pg total atrial RNA for PCR led to astronger signal on the autoradiograph (r'ig. i). slmi-lar results to these were obtained for atrial samples inother experiments (not shown) without the inclusionof kidney samples, in which a hlgh concentration ofPCR products fepfesents a potential soufce of contam-ination. Rats fed normal commercial chowgave similarPCR results (not shown) as rats fed *re NaCl-rice-soycakes. Submandibular gland (tissue negative control)did not show a band, as also observed in previous PCRexperiments 1t7,251, indicating that there was no am-plification of a homologous mRNA, such as that ofthe ubiquitous lysosomal enzlrne cathepsin D. Simi-lady, no band was seen in the solution-only negativecontrols, indicating that in samples where bands weredetected, these were not due to PCR product carryover[zg].ln other experiments (not shown), where only30 PCR cycles were performed, no band was seen foratrial samples. The experiments on normal rats wererepeated five times on different occasions spanning aperiod of 6 months and results consistent with thosein Fig. 1 were obtained,

Sodium-depleted enalapril-treated ratsIn contrast to results for the normal heart, atrial ex-tracts from sodium-depleted enalapril-treated rats allshowed a yery strong band on ethidium bromide-stained gels and autoradiographs (fig. Z). The low-sodium * enalapril experiment was repeated on an-other group of rats and similar results were obtained.No 390-bp band was seen for ventricle samples, evenafter low sodium * enalapril and hybridization prob-ing (Fig. 3). Frgure 3 also stiows results for kidneysam-ples, where the smaller concentrations of RNA usedfor the PCR meant that amplicons did not reach theasrr/mptotic part of the amphfication rangE, demonstrat-ing the increase normally seen in renal renin mRNAafter sodium depletion and ACE inhibitor treatment.After slot-blot hybridization a faint band was seenfor the sodium-replete rat atrial sample, but not theventricle or submandibular samptes (fig. 3).

1993, Vol 11 No 3

Fig. 1. Polymerase chain reaction (PCR; 35 cycles) was used to detect renin messenger (m)RNA in cardiac tissue from untreated rats.Unless otherwise indicated, the samples used for PCR contained 3 pg RNA and one-fifth of the post-PCR reaction mixture was useofor electrophoresis. (a) Ethidium bromide-stained agarose gel. (b) Autoiadiograph after DNA transfer and hybridization probing. Lane 1.heart (rat 1l; lane 2, kidney (rat 'l); lane 3, heart (rat 2); lane 4, kidney (rat)); lane 5, heart (rat 2), but usihg 5pg RNA in PC-R; lane 6,solution-only (no RNA) negative control; lane 7, size marker (Hpall digest of pUC19); lane B, heart (rat 3); lane 9, kidney kat 3), but usingone-tenth of post-PCR reaction mixture; lanes 10-12, as lanes 3-4; lane 13, solution-only negative control; lanes BA and 9A, as B and9, but with autoradiography for 16h instead of th; bp, base pair.

t t r 2 l 3

Discussion

The present study, involving the sensitive technique ofPCR, has provided evidence of renin gene expressionin the rat atrium, which was most easily seen understimulated conditions. In the absence of stimulation,a 3O-cycle PCR was unable to derect renin mRNA inthe heart, in accord with a previous reporr [17]. Byincreasing the number of cycles to 35 and perform-ing hybridization probing a funt signal of the size ex-pected for the renin mRNA PCR producrwas derecred.That this may have arisen from renin mRNA and not ahomologous mRNA, such as cathepsin D, present inthe RNA preparation is supported by the homologysearches we conducted.on the oligonucleotide se-quences that were used for PCR. Furthermore, theatrial origin of the PCR producr is supporred by ex-periments in which kidney RNA, a possible sourceof extraneous renin mRNA and therefore renin pCRproduct, was not included. This means that the pos-sible traces of PCR product in Fig. 1 were unlikely rohave represented spillover from an adjacent, kidneypositive-control well during sample loading. Moreover,no band was seen for submandibular gland lanes inany of the experiments, indicating that similar low-le-vel expression was absent from this tissue. It therefore

appears that a low level of renin expression occurs inatrial tissue in normal sodium-replete rats.

The findings for normal rat atrium are, however, arodds with those obtained by others who have rcporredthe presence of low, but readily detectable, conientra-tions of renin mRNAin cardiac tissue, using techniquesthat are much less sensirive than pCR [7,9,tt-t5]. Theconclusions of these workers were based on the ap-pearance of bands of the expected size after North-ern blotting or ribonuclease prorection/solution hy-bridization Because mRNA for homologous aspartylproteases, such as cathepsin D, are present in con-siderable concentrations in tissues such as the heartartd arc similar in size ro renin mRNA. the oossibilitvof non-specific cross-hybridization should be consid-ered in evaluating ar leasr some of the earlier resuhs.Similar reseffations-have, moreover. been exoressedrecently by others [S,30]. Strain differen."r .a. be ex-cluded because most other groups have, like us, used\Wistar rats [11-15]. Moreover, in experimens ;'ithSprague-Dawley rats renin mRNA was not derectedin cardiac tissue by ribonuclease prorection [15]. fna recent study, which used PCR for 25 cycles andtissue from Wistar-Kyoto rats given a standard lab-oratory diet, a renin PCR product was reported for

l l 1213 8A 9A

* 390 bp

*390 bp

[4,

Fig. 2. Effect of a low-sodium diet with enalapril treatment for 1 week on renin messenger (m)RNA detected in the heart by polvmerasechain reaction PCR). The arrow indicates the PCR product of the expected size. (a) Ethilium-bromide-stained agarose gel; (bi auioradio-graph after-transfer, hybridization probing and.autoradiography for8h.. Lanes 1,3, g, 11, heart (four rats) frori norm"al-sodium group;lanes 2, 4, 10, 12, heart (four rats) from low-sodium * enalapril group; lanes 5, 13, submandibular gland (tissue negative control)ilanes6, 14, kidney (tissue positive control); lanes 7,'15, solution-only (no nNA negative control; lane 8,"size marker (pliCtg cut with Hpall);bp, base pair.

9 l o t t t 2 13 t4 t5

Stimulation of atrial renin mRNA Lou et a/. 241

KidneyR D

HeartAtrium VentricleR D D S M G C

absence of background on autoradiographs, made iteasier to make comparisons using autoradiographyresults, as opposed to ethidium bromide-stained gels.This indicated a slight variation in band intensify forcardiac RNA from different low-sodium * enalapril-treated rats and could reflect di-fferences in the con-centration of renin mRNA present in the hearts of dif-ferent rats, possibly due to slight variations betweenrats in the effectiveness of the treatment regimen, orto slight variations in PCR efficacy in different reacriontubes, a factor that has made it dificult to use PCR asa quan[itative technique. \7e are in the process of es-tablishing the more difficult technique of quantirativePCR and should, iri due course, be able ro dererminenumerically the magnirude of the variation in responseof renin mRNA to the stimulus. Although the intensityof the band for low-sodium * enalapril-treated heartswas only slightly less than for the kidney (nig. 2), thisdid not mean that the concentration of mRNA in eachwas similar, Rather, reactants would have been fullyconsumed at a lower number of cycles for the kid-ney because a higher concentration of renin mRNAwas present, so that no further increase in the quan-tity of PCR product would have been possible in thispositive control llZ). tn subsequent experiments, rheuse of smaller quantities of renal RNA demonstratedthe expected higher levels of renin mRNA in kidneysof low-sodium * enalapril-tieated rats (r'ig. 3).

The combination of a low-sodium diet and trearmenrwith an ACE inhibitor is more potent than either meas-ure alone in stimulating renin mRNA in the kidney[7,78,2'1.,22,33-36). Since the question we addressedin the present study was whether cardiac renin mRNAcould be induced, the separate effects of low sodiumalone and enalapril alone were not tested, but obvi-ously, having established that atdal renin mRNA canrespond, the individual effects of these and other fac-tors on atrial renin mRNA will now be tested. More-over, the atrium is not unique among extrarenal tissuesin being able to respond in the waywe have seen; theadrenal gland, for example, sustains an approximatedoubtng of renin mRNA in rars fed a low-sodium dietfor 2 weeks [37].

The switch-over obserued in the atrium from low- tohigh-renin expression has also been seen in the re-nal vascular bed under conditions, including enalapriltreatrnent, that stimulate renal renin slznthesis, andinvolves the recruitment, for renin sJznthesis, of non-expressing myoepithelial cells exrending further up-stream from the juxtaglomsrular region of the pre-glomerular arteriole [34-361. Our data suggesr rhat asimilar process might occur in yet-to-be identified cellsin rat cardiac tissue.

Renin-like activity in the heart is increased by sodiumdepletion tll ana by nifedipine rreatmenr [10], although it is not clear whether this is the productof renin srinthesis in the heart or is due to an up-ake of renin from the bloodstream, which conrains

Fig. 3. Example of the effect of sodium depletion with enalapril(D) on renin messenger (m)RNA in renal, itrial, ventricular andsubm_andibular gland-iStvtc) samples. Results from sodium-repleterats (R) are shown for comparison. (d Ethidium bromide-stainedgel after electrophoresis of polymerase chain reaction (pCR) prod-ucts; b) slot-blot hybridization autoradiograph for each sample,ihowing results for'the samples diluted t6 tTZSth. the arrow in-dicates renin mRNA PCR product. The total RNA in the pCRsamples.was 200ng for the kidney, B00ng for both atrial andventricular s.ampleiand 1 pg for the subm"andibular gland. Thelast lane is the iolution-oni/negative control (C).

rat heart arrd aotta at a concentration similar to theadrenal gland and 1/500th that of ttre kidney [3f ].Evenwith the older techniques, however, lowervaluesfor the heart have been reported than for the adrenalgland [10,13]. Since Okura et at. I3Il used male rats,whereas we used females, we therefore examined thepossibility of a sex difference, but could find none. Itis, moreover, difficult to evaluate the data reported byOkura et al. [31], owing to a surprising absence of anynegative controls, which are regarded as mandatory toexclude the possibility of spurious results arising fromamplification of PCR products carried over from pre-vious reactions [29].A lack of cardiac renin gene expression, and there-fore of the synthesis of prorenin, renin or both, doesnot, however, preclude the generation of angiotensinin ttre heart under normal circumstances. since renin-Iike activity is present, presumably as a consequenceof sequestration from the bloodstream, like renin invascular tissue [4,5].In marked contrast to results for the atrium of sodium-replete rats, renin mRNA in atrial tissue from sodium-depleted enalapril-treated rats was re'adily detected asan abundant PCR product. Although large, the ex-tent of the incrsase could not be determined ab-solutely since, like most PCR methods, our proce-dure was not quantitative. The higher sensitivity af-forded by hybridization probing, together with the

242 journal of Hypertension 1993, Vol 11 No 3

higher concentrations of renin under these condi-tions as a consequence of increased renal renin se-cretion. Although a previous study reported increasedrenin mRNAin the mouse heart after sodium depletion[7], non-specific hybridizatton may have contributedto the signal obtained, at least under basal condi-tions. Renin and prorenin were not measured in thepresent study, as these data do not address ques-tions of source, e.g. cardiac versus extacatdiac, artdare therefore almost meaningless. Thus the extent towhich the increase in renin mRNA contributes to anincrease in renin, prorenin or both in atrial tissue inlow-sodium states remains to be determined.

The physiological function of a cardiac renin-angiotensin system has not yet been resolved [8]. Reninlike activity, together with other components of therenin-angiotensin system are present in ttre heart [8]and the increase seen in low-sodium states could, onthe basis of the present flndings, be at least pardy dueto the switching on of renin gene expression in theatrium. Several cardiac actions have been describedfor angiotensin administered systemically or addedto cell cultures, and it has been suggested that pos-sible physiological functions might include the directmodulation of cardiac contractility, sympathetic neu-rotransmission, myocardial metabolism, cell growth,or a combination of these [A]. ectivation of theseprocesses during depletion of the extracellular fluidvolume or in other physiological or pattrophysiologicalconditions, such as hypertension, heart failure or vari-ous other cardlac aberrations, could help to maintaincardiac function as an appropriate adjustrnent to theprevailing cardiovasculaLr state, although a possible ad-verse effect of cardiolascular remodelling may also oc-cur. To date, the avaiable evidence almost exclusivelysupports the theory that a cardiac renin-angiotensinsystem is activated under maladaptive conditions suchas postmyocardial infarction and congestive heart fail-ure, rather than physiological processes.

Acknowledgements

We thank K. Lynch, University of Virginia, for providing the rat renincDNA used to establish our PCR method, V. Hyland, Kolling Institute,for help with oligonucleotide srmthesis and H. gin, J. Ioudon andA Schrader for helpful discussions.

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