Preserved macula densa-dependent renin secretion inA1 adenosine receptor knocko - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：FrankSchweda, CharlotteWagner, Bernhard K.Krämer, JürgenSchnermann,  ArminKurtz作者单位：1 Institut für Physiologie and Klinik und Poliklinik für Innere Medizin,Universität Regensburg, 93040 Regensburg,Germany; and National Institute ofDiabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, Maryland 20892 : f; j$ x: ]+ P% S

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      【摘要】
   Recent studies demonstrated thatthe influence of the macula densa on glomerular filtration is abolishedin adenosine A 1 receptor (A 1 AR) knockout mice.Inasmuch as the macula densa not only regulates glomerular filtrationbut also controls the activity of the renin system, the present studyaimed to determine the role of the A 1 AR in macula densacontrol of renin synthesis and secretion. Although a high-salt dietover 1 wk suppressed renin mRNA expression and renal renin content tosimilar degrees in A 1 AR  / ,A 1 AR  /, and A 1 AR / mice, stimulation of Ren-1 mRNA expression and renal renincontent by salt restriction was markedly enhanced inA 1 AR / compared with wild-type mice.Pharmacological blockade of macula densa salt transport with loopdiuretics stimulated renin expression in vivo (treatment withfurosemide at 1.2 mg/day for 6 days) and renin secretion inisolated perfused mouse kidneys (treatment with 100 µM bumetanide) inall three genotypes to the same extent. Taken together, our data areconsistent with the concept of a tonic inhibitory role of theA 1 AR in the renin system, whereas they indicatethat the A 1 AR is not indispensable in maculadensa control of the renin system. ; w+ k  @9 @) T# b
      【关键词】 loop diuretics low salt high salt
   INTRODUCTION5 i- o. c: [1 L4 l9 j5 H5 s
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THE KIDNEYS PLAY A KEY ROLE in maintenance of fluid and electrolyte balance of thebody as well as in blood pressure regulation. Multiple extra- andintrarenal factors cooperate in the adjustments of renal function thatunderlie body fluid homeostasis. A specific intrarenal control elementfor NaCl excretion is the juxtaglomerular apparatus, the anatomicsubstrate of a mechanism in which changes in tubular salt delivery aresensed and translated to changes in afferent arteriolar tone[tubuloglomerular feedback (TGF)] and renin synthesis and secretion(macula densa-mediated renin release). An increase in NaClconcentration at the macula densa results in vasoconstriction of theafferent arteriole, reducing glomerular filtration rate and tubularsalt load, and inhibition of the renin-angiotensin system; a decreasein macula densa NaCl concentration has the opposite effect. The natureof the extracellular signaling events between macula densa cells andvascular smooth muscle or renin-producing effector cells is still amatter of debate. Besides cyclooxygenase-2-derived prostanoids( 3, 9, 10, 12, 16, 31 ), the nucleoside adenosine has beenproposed to be centrally involved in macula densa control of the renin system and glomerular filtration, inasmuch as adenosine is an inhibitorof the renin system as well as a vasoconstrictor of the afferentarteriole ( 13, 14 ), both effects being mediated by theA 1 adenosine receptor (A 1 AR). The inhibitoryeffect of the A 1 AR on the renin system has beendemonstrated in vitro and in vivo, inasmuch as selectiveA 1 AR agonists suppress renin secretion and pharmacologicalblockade of the A 1 AR results in stimulation of reninsecretion ( 1, 5, 6, 17, 24 ). The putative role ofadenosine in the salt-dependent regulation of the renin system isunderlined by several studies suggesting a relationship between tubularsalt load and adenosine concentration in the kidney. Infusion ofhypertonic saline or a high dietary sodium intake, both of which areassociated with an inhibition of the renin system, led to elevatedadenosine concentrations in the kidney ( 23, 27, 37 ). Incontrast, dietary sodium restriction, known to stimulate the reninsystem, resulted in reduced renal interstitial concentrations ofadenosine ( 27 ). Therefore, an increase in adenosineconcentrations due to a high tubular salt load could mediatevasoconstriction and inhibition of the renin system, whereas a decreasein renal adenosine concentration resulting from a reduced salt loadcould cause vasodilatation and stimulation of the renin system. TheA 1 AR agonist cyclohexyladenosine (CHA) suppressed stimulation of renin secretion in response to a perfusion medium containing a low NaCl concentration in the isolated juxtamedullary apparatus, and blockade of the A 1 AR diminished thereduction in renin secretion caused by high luminal NaCl concentrations( 35 ), supporting the involvement of adenosine in maculadensa control of the renin system. However, in a similar experimentalsetup, application of exogenous adenosine did not fully mimic theinhibitory effects of increasing tubular NaCl concentration on reninsecretion ( 19 ), which would be expected from a mediator ofmacula densa control of the renin system.: A! L# N3 w, o
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Recent investigations have provided direct evidence that adenosine isrequired for the vasoconstriction caused by TGF. Consistent withearlier studies showing that inhibition of adenosine production ( 29 ) or selective blockade of the A 1 AR( 26, 36 ) blunts TGF, mice with a genetic deletion of theA 1 AR lack the TGF response to an increase in tubular NaClconcentration ( 2, 28 ). Inasmuch as the effector cells ofthe TGF, namely, the vascular smooth muscle cells of the afferentarteriole, are located in the immediate vicinity of the renin-producingjuxtaglomerular cells, it is reasonable to assume that adenosinemediating the TGF also influences the renin system. Therefore, thepresent experiments were performed to determine whether adenosine andA 1 AR may be centrally involved in macula densa control ofthe renin system. Utilizing A 1 AR knockout mice, wedetermined whether the absence of the A 1 AR is associated with altered expression or secretion of renin consistent with tonicinhibition of the renin-angiotensin system by adenosine. Furthermore,the macula densa mechanism is believed to be critically involved inadjustment of the renin system to different salt loads of the body,with a high sodium intake inhibiting and a low sodium intakestimulating the renin system ( 8, 18, 25, 33 ). Pharmacological blockade of macula densa NaCl transport with loop diuretics is an intervention that, similar to a low-salt diet, stimulates the renin-angiotensin system by diminishing the NaCl transport-dependent, renin-inhibitory signal to the granular cells ( 11 ). Therefore, we investigated the influence of a high-and a low-salt diet and furosemide on the renin system in mice with agenetic deletion of the A 1 AR. Finally, to assess the moreacute modulation of renin secretion by the macula densa, weinvestigated the effects of the loop diuretic bumetanide on the rate ofrenin secretion in isolated perfused kidneys of A 1 ARknockout mice and their wild-type controls. The isolated perfusedkidney model is ideally suited to investigate renin secretion in theabsence of interindividual differences in systemic factors that mayinfluence the renin system, such as variations in blood pressure orrenal sympathetic nerve activity, for example.6 b! D2 @" }5 k

MATERIALS AND METHODS8 P$ \( Y/ v3 q" g0 a! @1 s/ p

A 1 AR knockout mice. A 1 AR knockout mice were generated as described by Sun etal. ( 28 ). The mice were derived from two heterozygousbreeder pairs. For genotyping, tail biopsies were performed, and DNAwas extracted and tested for the presence of wild-type and mutant genesusing A 1 AR- and Neo-R-specific PCR primers( 28 )., U3 X* w6 t) O% ]

Experimental procedures in vivo. In the first set of experiments, 10 mice of each genotype(A 1 AR  / , A 1 AR  /, andA 1 AR /, 20-24 g body wt) were fed alow-salt (0.02% NaCl) or a high-salt (8% NaCl) diet for 7 days. Ascontrols, 10 mice of each genotype were fed normal mouse chow (0.6% NaCl).
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In the second set of experiments, five mice of each genotype weretreated with furosemide (1.2 mg/day; Dimazon, Intervet) administeredvia osmotic minipumps (Alzet, Durect) for 6 days. As controls, fivemice of each genotype were infused with physiological saline. Surgicalinsertion of the pumps was performed under inhalation anesthesia(Sevofluran, Abbot). The mice had free access to standard mouse chow(0.6% NaCl), tap water, and an electrolyte solution containing 0.9%NaCl and 0.1% KCl.
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After the experimental periods, the animals were killed bydecapitation, and blood was collected for determination of serum electrolyte concentration by flame photometry (model PFP7, Jenway, Dunmow). Kidneys were removed rapidly, frozen in liquid nitrogen, andstored at 80°C until further processing.5 N  m4 d/ {1 _, B$ o& P$ ^- m
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Determination of preprorenin mRNA and cytosolic -actin byRNase protection assay. After isolation of total RNA from the frozen kidney using the method ofChomczynski and Sacchi ( 4 ), renin was measured by an RNaseprotection assay using an antisense RNA probe suitable for detectingmRNA levels from the Ren-1 and Ren-2 genes asdescribed previously ( 32 ). Cytosolic -actin wasmeasured by an RNase protection assay as described elsewhere( 32 ). For semiquantification of Ren-1 and Ren-2 mRNA abundance, the hybridization signals were relatedto those obtained for -actin mRNA. -Actin mRNA levels were notdifferent between the different genotypes and the different experimental maneuvers (not shown).

Determination of renal renin content. The renal renin content was determined by measuring the capacity ofhomogenized kidneys to generate angiotensin I according to amodification of the method described by Norling et al.( 22 ). Frozen kidneys were halved, homogenized in 1 ml ofhomogenization buffer [5% (vol/vol) glycerol, 0.1 mMphenylmethylsulfonyl fluoride, 10 mM EDTA, and 0.1 mM4-(2-aminomethyl)benzenesulfonyl fluoride] for 30 s(Ultra-Turrax, IKA Labortechnik), and centrifuged at 4°C at 14,000 g for 5 min. The supernatants were frozen at 20°C andthen thawed three times by alternating the temperature between 20°Cand 4°C. Supernatants were incubated with saturating concentrations of rat renin substrate, and the generated angiotensin I was assayed with a commercial radioimmunoassay kit (Byk and DiaSorin).

Isolated perfused mouse kidney. Male A 1 AR /,A 1 AR  /, and A 1 AR  /  mice (20-23 g body wt) with free access to commercial pellet chowand tap water were used as kidney donors. The animals were anesthetizedwith an intraperitoneal injection of5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid (100 mg/kg; Trapanal,Byk Gulden) and ketamine HCl (80 mg/kg; Curamed, Germany) and placed ona heating table. The abdominal cavity was opened by a midline incision,and the aorta was clamped distal to the right renal artery so that theperfusion of the right kidney was not disturbed during the subsequentinsertion of the perfusion cannula into the abdominal aorta distal tothe clamp. The mesenteric artery was ligated, and a metal perfusioncannula (0.8 mm OD) was inserted into the abdominal aorta. Afterremoval of the aortic clamp, the cannula was advanced to the origin ofthe right renal artery and fixed in this position. The aorta wasligated proximal to the right renal artery, and perfusion was startedin situ with an initial flow rate of 1 ml/min. With the use of thistechnique, a significant ischemic period of the right kidneywas avoided. Finally, the right kidney was excised, placed in athermostated moistening chamber, and perfused at constant pressure (100 mmHg). Perfusion pressure was monitored within the perfusion cannula (Isotec pressure transducer, Hugo Sachs Elektronik), and the pressure signal was used for feedback control (model SCP 704, Hugo Sachs Elektronik) of a peristaltic pump. Finally, the renal vein was cannulated (1.5-mm-OD polypropylene catheter). The venous effluent wasdrained outside the moistening chamber and collected for determination of renin activity and venous blood flow.; U+ B2 |! ?, O' a; g. Y
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The basic perfusion medium, supplied from a thermostated (37°C)200-ml reservoir, consisted of a modified Krebs-Henseleit solutioncontaining all physiological amino acids at 0.2-2.0 mM, 8.7 mMglucose, 0.3 mM pyruvate, 2.0 mM L -lactate, 1.0 mM -ketoglutarate, 1.0 mM L -malate, and 6.0 mM urea. Theperfusate was supplemented with 6 g/100 ml bovine serum albumin, 1 mU/100 ml vasopressin 8-lysine, and freshly washed human red bloodcells (10% hematocrit). Ampicillin (3 mg/100 ml) and flucloxacillin (3 mg/100 ml) were added to inhibit possible bacterial growth in themedium. To improve the functional preservation of the preparation, theperfusate was continuously dialyzed against a 10-fold volume of thesame composition but without erythrocytes and albumin. For oxygenation of the perfusion medium, the dialysate was gassed with 94%O 2 -6% CO 2. Perfusate flow was calculated bycollection and gravimetric determination of the venous effluent.Perfusion pressure was continuously monitored by a potentiometric recorder.
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After constant perfusion pressure was established, perfusate flow ratesusually stabilized within 15 min. Stock solutions of the drugs to betested were added to the dialysate.3 a' O* Y( w1 G7 O* D! y
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For determination of perfusate renin activity, venous effluent wascollected over a period of 1 min at intervals of 3 min. The sampleswere centrifuged at 1,500 g for 10 min, and the supernatants were stored at 20°C until assayed for renin activity. Fordetermination of renin activity, the perfusate samples were incubatedfor 1.5 h at 37°C with plasma from bilaterally nephrectomizedmale rats as renin substrate. The generated angiotensin I(ng · ml 1 · h 1 )was determined by radioimmunoassay (Byk and DiaSorin). Renin secretionrates were calculated as the product of the renin activity and thevenous flow rate(ml · min 1 · gkidney wt 1 ).
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Statistical analysis. Values are means ± SE. Differences between groups were analyzedby ANOVA and Bonferroni's adjustment for multiple comparisons. In theisolated perfused kidney experiments, all values obtained within anexperimental period ( n = 4) were averaged and compared with the average values of an adjoining experimental period. Student's paired t -test was used to calculate levels of significancewithin individual kidneys. P significant.) b) {" Y6 r( h- m. g$ a7 @3 F

RESULTS

Serum concentrations of sodium, chloride, or potassium were notdifferent between any of the genotypes or the treatment groups (not shown).

Basal renin expression. Basal renal renin content was 1.5-fold higher inA 1 AR / than in wild-type mice (Fig. 1 A ).

Fig. 1. Renal renin expression of A 1 adenosinereceptor (A 1 AR) / ( n = 8),A 1 AR  / ( n = 18), andA 1 AR  /  ( n = 7) mice underbasal conditions. A : renal renin content ofA 1 AR /, A 1 AR  /,and A 1 AR  /  mice under basal conditions. B : autoradiography of a renin RNase protection assay usingan antisense probe that is able to discriminate between Ren-1 and Ren-2 mRNA. C :semiquantification of Ren-1 and Ren-2 mRNAhybridization signals. NS, not significant.

For determination of the renal renin mRNA abundance, we used an RNaseprotection assay with an antisense probe that was able to discriminatebetween Ren-1 and Ren-2 mRNA. Autoradiographic band intensity of the RNase protection assays (Fig. 1 B ) aswell as semiquantification of renin mRNA expression by -actincorrection (Fig. 2 C ) revealeda similar abundance of Ren-1 mRNA in each of the genotypes.In contrast, Ren-2 mRNA expression showed distinct differences between the genotypes: whereas the expression levels of Ren-1 and Ren-2 were similar inA 1 AR / mice, Ren-2 mRNAwas not found in A 1 AR  /  mice.A 1 AR  / mice showed an intermediate abundanceof Ren-2 gene expression: Ren-2 mRNA levels were~50% of Ren-1 mRNA levels.
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Fig. 2. Renin mRNA expression inA 1 AR /, A 1 AR  /,and A 1 AR  /  mice fed a high- or a low-saltdiet. Controls were fed a normal-salt diet. Values are means ± SE( n = 10). * P # P
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Effect of a high-salt diet on renin expression. A high-salt diet for 1 wk resulted in inhibition of Ren-1 mRNA expression irrespective of the genotype (0.6-, 0.54-, and0.66-fold of control for A 1 AR /,A 1 AR  /, and A 1 AR  / ,respectively, all P 2 A ).Moreover, Ren-2 mRNA levels were suppressed by the high-saltdiet in the kidneys of A 1 AR / andA 1 AR  / mice, whereas no Ren-2 mRNAsignal was detectable in the kidneys of A 1 AR  /  mice (Fig. 2 B ). According to the changes in Ren-1 and Ren-2 gene expression, total renin mRNA expression wassignificantly suppressed by a high-salt diet in all three genotypes(Fig. 2 C ). In parallel with the changes in renin geneexpression, renal renin content was lowered to ~0.6-fold of controlby a low-salt diet in A 1 AR /,A 1 AR  /, and A 1 AR  /  mice ( P 3 A ).

Fig. 3. Renal renin content in A 1 AR /,A 1 AR  /, and A 1 AR  /  mice. A : effects of high-, normal-, or low-salt diet. B : effects of treatment with vehicle or furosemide viaosmotic minipumps. * P # P

Effect of a low-salt diet on renin expression. Dietary salt restriction stimulated the renin system in all threegroups of animals. However, in contrast to the changes due to ahigh-salt diet, there were marked differences between the genotypes:stimulation was most pronounced in A 1 AR / mice, in which Ren-1 mRNA levels increased 2.5-fold compared with control animals fed a normal-salt diet, whereas inA 1 AR  /  mice only a 1.2-fold increase wasdetected. A 1 AR  / mice showed an intermediatestimulation of renin mRNA, with Ren-1 mRNA levels showing atwofold upregulation (Fig. 2 A ).3 [" s& _8 p1 d+ E! O
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Similar to the expression of Ren-1, Ren-2 mRNA expressionlevels were augmented by a low-sodium intake inA 1 AR / and A 1 AR  / mice. In the kidneys of wild-type mice, no Ren-2 signal wasdetectable, even in animals fed the low-salt diet (Fig. 2 B ).As a result of the differences in Ren-2 expression, totalrenin mRNA was significantly higher inA 1 AR / than inA 1 AR  / mice and wild-type controls (Fig. 2 B ).' A5 o1 l  W5 @" @0 J# ]- n; f

In parallel with renal renin gene expression, salt restriction caused atwofold increase in renal renin content inA 1 AR / and A 1 AR  / mice compared with control values. In contrast, inA 1 AR  /  mice, in which total renin geneexpression was only slightly stimulated by the low-salt diet, nosignificant stimulation of renal renin content was detected (Fig. 3 A )., D7 r& B  }* q9 E8 {7 P/ |
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Effect of furosemide on renin expression. Blockade of thick ascending limb and macula densa salt transport withfurosemide administration for 6 days stimulated Ren-1 expression in A 1 AR /,A 1 AR  /, andA 1 AR  /  mice to similar degrees, sothat no differences in Ren-1 expression exist betweengenotypes (Fig. 4 ). Again, Ren-2 mRNA was not detectable inA 1 AR  /  mice, whereas it was significantlystimulated in A 1 AR / andA 1 AR  / mice (Fig. 4 ). Total renin mRNAexpression was stimulated by furosemide in all groups, with the highestabundance detectable in the A 1 AR / mice.Furosemide also augmented renal renin content in all three groups ofmice to a similar extent, so that no significant differences weredetected between the different genotypes (Fig. 3 C ).( U  w8 V! a% ]; y

Fig. 4. Renin mRNA expression inA 1 AR /, A 1 AR  /,and A 1 AR  /  mice treated with vehicle orfurosemide. Values are means ± SE ( n = 5).* P # P# w. g6 R: d# G8 q+ S8 r, B

Effect of bumetanide administration on renin secretion in isolatedperfused mouse kidneys. To investigate the acute effects of loop diuretics on renin secretion,we adapted the model of the isolated perfused rat kidney to theanatomic conditions of mice. This model allows us to study the acuteregulation of renin secretion without interference by confoundingsystemic side effects of the experimental drug or systemiccounterregulations. Basal renin secretion rates of isolated perfusedkidneys were similar in A 1 AR /,A 1 AR  /, and A 1 AR  /  mice (Fig. 5 ). Blockade of thickascending limb and macula densa salt transport by bumetanide resultedin significant and comparable increases in renin secretion inA 1 AR  / , A 1 AR  /, andA 1 AR / mice: 3.1-, 3.0-, and 2.8-fold ofcontrol, respectively. During subsequent administration of theA 1 AR agonist CHA, renin secretion rates returned to basallevels in kidneys of A 1 AR  /  andA 1 AR  / mice, whereas CHA was without effectin A 1 AR / mice (Fig. 5 ).

Fig. 5. Effects of blockade of macula densa salt transport andsubsequent administration of cyclohexyladenosine (CHA) on reninsecretion rates of isolated perfused kidneys inA 1 AR  / , A 1 AR  /, andA 1 AR / mice. Values are means ± SE( n = 5).
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DISCUSSION* L8 i/ z5 K0 Y6 w
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The present experiments in A 1 AR knockout mice aimed toassess the chronic role of A 1 AR in the renal expression ofrenin under basal conditions as well as in the macula densa control ofthe renin system. Previous pharmacological studies provided evidence for a direct inhibitory role of adenosine on renin expression and reninsecretion, an effect that appeared to be mediated by A 1 AR( 1, 5, 15, 17, 24, 35 ). The present observation that renalrenin content under basal conditions is elevated in A 1 ARknockout mice supports the concept of a tonic inhibition of therenin-angiotensin system through A 1 AR mediation. However, besides the direct disinhibition of the renin system byA 1 AR deletion, an enhanced sodium excretion reportedpreviously in A 1 AR / mice ( 2 )as well as after acute pharmacological blockade of A 1 AR( 36 ) might also account, in part, for the higher renin content in A 1 AR / mice. The finding thatA 1 AR / mice possess two renin genes( Ren-1 d and Ren-2 ), whereas wild-typemice harbor only one renin gene ( Ren-1 c ), andthat this discrepancy is related, as discussed in detail below, to thedifferent mouse strains used in the generation of the knockout micesomewhat complicates the straightforward interpretation of our data.Inasmuch as, in general, plasma renin activities and concentrationsappear to be markedly higher in two-renin than in one-renin genestrains ( 20, 34 ), it is conceivable that the higher renincontent in the A 1 AR / animals is theconsequence of their expression of Ren-1 d and Ren-2. However, the differences in Ren-2 expression do not explain the marked enhancement of Ren-1 mRNA stimulation in A 1 AR / animals by alow-salt diet, so this result further supports the concept of a tonicinhibitory role of A 1 AR in the renin system." x: j2 C! e4 e: T1 W0 b
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The main intention of our study was to clarify the specific role ofA 1 AR in the macula densa control of the renin system. Therationale for the assumption of a central role of the A 1 AR in this process was as follows: 1 ) adenosine inhibits therenin system via the A 1 AR, 2 ) adenosineconcentration in the kidney changes in parallel with the sodium load ofthe kidney, and 3 ) A 1 AR is essentially requiredfor control of glomerular filtration by the macula densa. According tothe hypothesis that the macula densa-controlled changes in reninexpression and secretion are related to salt-dependent changes in theintrarenal adenosine concentration, the amplitude of the inhibition ofthe renin system due to a high-salt diet or the stimulation due to alow-salt diet should be attenuated or even blunted in mice lacking theA 1 AR. However, our results demonstrate that a high-saltdiet suppressed renal renin mRNA expression and renal renin content tothe same extent in A 1 AR / andA 1 AR  /  mice, clearly arguing against a role ofthe A 1 AR in mediation of this process. Moreover,stimulation of the renin system by a low-salt diet was not diminished,but was even enhanced, by the genetic deletion of the A 1 AR,a further result that is not compatible with a role of A 1 ARin mediation of this stimulation. If stimulation of renal renin contentand mRNA expression by the low-salt diet were related to the knowndecrease in renal adenosine concentration and the subsequentdisinhibition of the A 1 AR, this should not be possible inmice lacking this receptor. However, as stated above, the pronouncedstimulation of Ren-1 mRNA expression due to salt restrictionis highly consistent with a tonically inhibitory role of theA 1 AR on the renin system, which is absent inA 1 AR / mice. The conclusion that theA 1 AR is not causally involved in regulation of the reninsystem by the macula densa is further supported by the intactstimulation of the renin gene expression and renin content by blockadeof the macula densa salt transport with furosemide inA 1 AR / animals. Interpretation of the invivo data is limited by the fact that salt restriction or furosemidetreatment might affect renin expression through pathways independentfrom or in addition to the macula densa mechanism, for example, byalterations in blood pressure or in sympathetic nervous systemactivity. We therefore investigated the effects of loop diuretics onrenin secretion in the isolated perfused kidney model. In thispreparation, administration of loop diuretics would appear to actsolely through the macula densa, because perfusion pressure isexperimentally controlled and changes in sympathetic nerve activity areunlikely. Even under these experimental conditions, bumetanidestimulated renin secretion to the same extent in kidneys ofA 1 AR / mice and their wild-type controls,arguing against a role of this receptor in mediation of this process.However, the complete reversal of the stimulated renin secretion by theselective A 1 AR agonist CHA inA 1 AR  /  and A 1 AR  / mice underlines the direct suppressive effects of the A 1 ARon renin secretion, as has been demonstrated in previous studies ( 1, 5, 6, 15, 19 ). Besides the advantages of constant experimental conditions, the isolated perfused mouse kidney model issuitable for use in investigating the acute effects of an inhibition ofmacula densa salt transport on renin secretion and, therefore, inexamining the renin system in a time frame similar to that used in thestudies demonstrating the absence of a TGF response inA 1 AR / mice ( 2, 28 ). Becausethe TGF response has been found to be abolished by pharmacologicalblockade or genetic deletion of the A 1 AR ( 2, 26, 28, 29, 36 ), regulation of glomerular filtration rate and control ofthe renin system by the macula densa appear to follow different pathways.
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A further interesting result of our study is the discovery of a linkagebetween the A 1 AR mutation and the renin gene locus thatcauses homozygosity in the A 1 AR knockout genotype to beinvariably associated with the two-renin gene constellation. Incontrast, the wild-type phenotype, homozygous for the absence of theA 1 AR mutation, always contains a single renin gene. Thefoundation for this linkage is the fact that the genes encoding theA 1 AR and renin are localized on chromosome 1 in closevicinity, as first shown in humans ( 7, 30 ). Analysisof available mouse genomic sequences has confirmed that the renin andA 1 AR genes in the mouse are also located on chromosome 1 inrelative close juxtaposition, separated by ~850 kb of DNA containingseveral putative gene loci. As is commonly done, the embryonic stemcells used for targeted disruption of the A 1 AR gene werederived from the 129J mouse strain, one of several mouse strains withtwo renin genes, designated Ren-1 d and Ren-2 ( 21 ). By propagating the A 1 ARmutation in the one-renin gene C57BL/6 background,A 1 AR / mice will carry the 129J backgroundin the area of the mutated A 1 AR gene and will thereforepossess two renin genes. On the other hand,A 1 AR  /  mice will have to carry the C57BL/6background in the area of the native A 1 AR gene and willtherefore have only one renin gene, designated Ren-1 c. Breeding strategies will be used tosegregate the A 1 AR knockout mutation from the two-reningene constellation by backcrossing into the C57BL/6 background or tomaintain the A 1 AR mutation in a two-renin gene backgroundby backcrossing into the 129J or Swiss strain. Although this geneticartifact does not limit the conclusion that the A 1 AR is notrequired for mediation of macula densa control of the renin system, ithas to be carefully considered when absolute values of renal renincontent are compared between the genotypes. Inasmuch as a linkage of atargeted gene and the neighboring gene loci supposedlyunaffected by the knockout procedure potentially occurs in everyknockout model derived from different mouse strains, our resultsemphasize the necessity of careful interpretation of data comparingknockout with wild-type mice.& i4 a* w- x* ~. ^9 g* B
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Taken together, our results support the concept of a tonic inhibitoryrole of the A 1 AR on the renin system, but they argue against a role of the A 1 AR in mediation of the macula densacontrol of the renin system, as has been demonstrated previously formacula densa control of glomerular filtration.

ACKNOWLEDGEMENTS6 r8 t7 }( D. S) d

We thank Maggy Schweiger and Susanne Lukas for expert technical assistance.- P8 h5 G" C2 x
      【参考文献】
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3. Castrop, H,Schweda F,Schumacher K,Wolf K,andKurtz A. Role of renocortical cyclooxygenase-2 for renal vascular resistance and macula densa control of renin secretion. J Am Soc Nephrol 12:867-874,2001 .
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