Renal segmental microvascular responses to ANG II inAT1A receptor null mice



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发表于 2009-4-21 13:33 |显示全部帖子

作者：Lisa M.Harrison-Bernard, Anthony K.Cook, Michael I.Oliverio,  Thomas M.Coffman作者单位：1 Department of Physiology, Tulane University HealthSciences Center, New Orleans, Louisiana 70112; and Department of Medicine, Duke University, and Durham VeteransAffairs Medical Centers, Durham, North Carolina 27705
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      【摘要】1 \) Y3 [% W' l0 X0 }# N
   The relative contributions ofAT 1A and AT 1B receptors to afferent arteriolarautoregulatory capability and afferent and efferent arteriolarresponses to ANG II are not known. Experiments were conducted inkidneys from wild-type (WT) and AT 1A / mice utilizing the in vitro blood-perfused juxtamedullary nephron technique. Directmeasurements of afferent (AAD) and efferent arteriolar diameters (EAD)were assessed at a renal arterial pressure of 100 mmHg. AAD averaged14.8 ± 0.8 µm for WT and 14.9 ± 0.8 µm forAT 1A / mice. AAD significantly decreased by 7 ± 1, 16 ± 1, and 26 ± 2% for WT mice and by 11 ± 1, 20 ± 2, and 30 ± 3% for AT 1A / mice (120, 140, 160 mmHg). AAD autoregulatory capability was not affected by theabsence of AT 1A receptors. AAD responses to 10 nM ANG IIwere significantly blunted for AT 1A / mice compared withWT ( 22 ± 2 vs. 37 ± 5%). ANG II (0.1-10 nM)failed to elicit any change in EAD for AT 1A / mice. AADand EAD reductions in ANG II were blocked by 1 µM candesartan. Weconclude that afferent arteriole vasoconstrictor responses to ANG IIare mediated by AT 1A and AT 1B receptors,whereas efferent arteriolar vasoconstrictor responses to ANG II aremediated by only AT 1A receptors in the mouse kidney. 4 a6 k' v/ X9 ?1 X7 ?
      【关键词】 afferent arteriole efferent arteriole juxtamedullarynephron candesartan autoregulation; N2 [& a) H( Y9 q" P, P
   INTRODUCTION
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THERE ARE AT LEAST TWO MAJOR angiotensin receptors: AT 1 and AT 2.The AT 1 receptor is thought to mediate most of the actions of ANG II on renal hemodynamic and tubular function, including afferentand efferent arteriolar vasoconstriction ( 3, 9, 31 ),modulation of tubuloglomerular feedback sensitivity ( 16 ), sodium and fluid reabsorption ( 18 ), and growth anddifferentiation ( 29 ). Two subtypes of the AT 1 receptors, designated AT 1A and AT 1B, have beenidentified in the rat ( 7, 12, 13, 23 ) and mouse( 24 ). The AT 1A receptor is thought to be thepredominant renal form. Terada et al. ( 28 ) reportedlocalization of the AT 1 receptor mRNA in microdissectedrenal vascular segments (glomeruli, vasa recta bundle, and arcuatearteries) of the kidney by RT-PCR methods. Further studies identifiedAT 1A and AT 1B mRNAs in the same renal vascularstructures, as well as the afferent arteriole ( 2, 7 ).Additionally, the AT 1 receptor protein has been localizedto the entire rat renal vasculature using immunohistochemical techniques and antibodies that recognize specifically theAT 1A receptor ( 30 ) or both theAT 1A and AT 1B receptor subtypes ( 10, 17, 21, 30 ). The mRNA and protein expression profiles of theAT 1 receptor subtypes have not been determined for theefferent arteriole. Furthermore, the contribution of theAT 1A and AT 1B receptors to the afferent andefferent arteriolar responses to ANG II have not been investigated.0 V( `7 J# P& ^7 m; A9 J' E

The AT 1A and AT 1B receptors arepharmacologically indistinguishable from each other, and so it has notbeen possible to discriminate between the receptor subtype functionsusing pharmacological antagonists. The physiological effects of renalAT 1A and AT 1B receptor subtypes have yet to beelucidated, although the calcium signaling mechanisms of theAT 1A and AT 1B receptors appear to be identicalin isolated cells ( 15, 32 ). The AT 1A andAT 1B receptor subtype localization, regulation, andfunction in various pre- and postglomerular microvascular segments mayplay an important part in the renal hemodynamic responses to ANG II ina variety of physiological and pathophysiological conditions.! p  |( w6 L2 d
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Gene-targeted mice have proven to be a critical tool in defining therole of each AT 1A and AT 1B receptor subtype invivo. AT 1A / mice have reduced blood pressure, lack asystemic pressor response to exogenous ANG II, and exhibit mild renalstructural abnormalities that include slight papillary hypoplasia andhyperplasia of renin-producing granular cells ( 11, 20, 26 ). Surprisingly, renal hemodynamics of wild-type (WT) andAT 1A / mice are similar, such that glomerular filtrationrate and renal plasma flow ( 6 ) and renal blood flow( 22 ) do not differ between anesthetized WT andAT 1A / mice; however, renal vascular resistance is lower in the AT 1A / mice, paralleling the lower arterialpressure ( 22 ). The reduction in renal blood flow producedby ANG II in WT mice is similar to the reduction in renal blood flowproduced by 10-fold higher doses of ANG II in AT 1A / mice ( 22 ). However, the renal microvascular segmentresponsible for the ANG II responsiveness in AT 1A / micecould not be determined in these studies. Additionally, AT 1A / mice lack a tubuloglomerular feedback mechanism( 25 ), which may result in an impaired renal autoregulatoryresponsiveness in these mice.1 h4 t! {: W1 c: o) R( k# h& x
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The present studies were conducted to test the hypotheses thatAT 1A / receptor-deficient mice display impaired renalafferent arteriolar autoregulatory responses and altered afferent andefferent arteriolar ANG II sensitivity. To directly assess the renalmicrovascular responses to changes in renal arterial perfusion pressureand ANG II, vessels were studied in an intact tubular environment ( 5 ) using the mouse in vitro blood-perfused juxtamedullary nephron preparation.

METHODS
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Mouse in vitro blood-perfused juxtamedullary nephron technique. Assessment of afferent and efferent arteriolar diameters was conductedin kidneys from 49 adult male and female WT ( n = 14 females; n = 4 males), AT 1A  / ( n = 4 males), and AT 1A / ( n = 22 females; n = 5 males) miceranging from 3 to 7 mo of age that were breed in our colony at DukeUniversity. Forty-nine adult male Sprague-Dawley rats were used asblood donors. Experiments were conducted using the mouse in vitroblood-perfused juxtamedullary nephron technique, which is based on therat in vitro blood-perfused juxtamedullary nephron technique originallydeveloped by Casellas et al. ( 4 ). Kidneys were harvestedfrom mice under pentobarbital sodium anesthesia (50 mg/kg ip). Therenal artery was cannulated via the descending aorta under a dissectingmicroscope and immediately perfused with a Tyrode buffer containing 51 g/l bovine serum albumin (98-99% albumin, Sigma) and a mixture of L -amino acids at pH 7.4 as previously described in detail( 8 ). The cannula system includes a 27-gauge bluntedhypodermic needle for introduction into the renal artery, polyethylene(PE)-10 tubing for blood perfusion, and PE-10 tubing for themeasurement of perfusion pressure. The tips of all of the tubing are inclose proximity to each other. A section of liver was removed from eachanimal, immersed into liquid nitrogen, and stored at 70°C forgenotyping by Southern blot analysis as previously described( 11 ). The kidney was placed in a perfusion chamber at roomtemperature for the dissection procedure, which included removal of theventral third of the kidney, reflection of the papilla, cutting open ofthe renal veins, placement of 10.0 suture ties on the distal segmentsof the large arteries, and removal of the connective tissue and pelvicmucosa overlying the juxtamedullary cortical surface (Fig. 1 A ).

Fig. 1. Mouse in vitro blood-perfused juxtamedullary nephron. A : photograph of the mouse kidney after the dissectionprocedure. The kidney is perfused via a 27-gauge needle. The pelvicmucosa is reflected up and held in place with pins revealing theunderlying juxtamedullary nephrons. B : photograph of themouse kidney on the stage of the videomicroscope. The chamber iswarmed, and the kidney is superfused with solutions. C :digital image captured from the video monitor of a mouse blood-perfusedafferent arteriole. The arrows demarcate the inside luminal borders.Bar = 15 µm.$ k4 _8 p; O2 }9 r& N( d
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Blood was collected from a pentobarbital sodium anesthetized rat (50 mg/kg ip) through a carotid arterial cannula into a heparinized syringe. Blood was centrifuged, the buffy coat was discarded, and plasma and red blood cells (RBCs) were separated. RBCs were washed2× in 0.9% NaCl, and the plasma was filtered (0.2, 5 µm). Aftercompletion of all microdissection procedures, the Tyrode perfusate wasreplaced with the reconstituted rat blood (hematocrit 33%).Microscopic examination of RBCs from the rat and mouse indicate thatthe diameters are similar (4-6 µm mouse; 5-7 µm rat).There was no indication that perfusion of the mouse kidney with rat blood causes any limitations in glomerular capillary blood flow oralterations in vascular responses. Renal arterial perfusion pressurewas measured using a P23XL transducer and a polygraph and wasmaintained at 100 mmHg by adjusting the regulator controlling the flowof the O 2 -CO 2 mixture from the tank thatpressurized the reservoir. The perfusion chamber was warmed, and thetissue surface was continuously superfused with an albumin-containing (10 g/l) Tyrode solution at 37°C. Agents were administered by addition to this bathing solution. The chamber was affixed to the stageof a Nikon Optiphot microscope equipped with dry objectives (×4, ×10,×32) and a water-immersion objective (Zeiss, ×40/0.75 numericalaperture) (Fig. 1 B ). The tissue was transilluminated using ahalogen lamp. The focused image of the vessel was transmitted via ahigh-resolution Newvicon camera (NC-67M, Dage-MTI) through a time-dategenerator (WJ-810, Panasonic) and an image-enhancing processor(MFJ-1452, MFJ Enterprises) and displayed on a monochromatic monitor(final magnification ×3,500). The video signal was recorded simultaneously on videotape for later analysis (SuperVHS VCR, Panasonic). Diameter measurements were obtained at a single site alongthe length of the selected vessel using an image-shearing monitor(Instrumentation for Physiology and Medicine, San Diego, CA). Thisdevice was calibrated using a stage micrometer (smallest division = 10 µm) and yielded diameter measurements reproducible to within in theregion sensitive to input from tubuloglomerular feedback. Efferentarterioles were studied near the glomerulus before peritubular capillary branching. Afferent arterioles (Fig. 1 C ) weremeasured at a site averaging 60 ± 2 µm from the glomerulus( n = 35). Afferent arteriolar total length averaged280 ± 20 µm ( n = 35). Efferent arterioles weremeasured at a site averaging 60 ± 3 µm from the glomerulus( n = 14). Experimental protocols were begun after a 10- to 15-min stabilization period. A single vessel was studied from eachkidney, and only one kidney was studied from each animal.* W* c# F# F2 e0 j% c# k% r

Afferent arteriolar autoregulatory responses. Afferent arteriolar diameters were monitored in response to elevationsin renal perfusion pressure in kidneys from female WT( n = 6), AT 1A  / ( n = 2),and AT 1A / ( n = 6) mice. Afferent arteriolar diameters were measured during a 5-min control period at 100 mmHg. Renal perfusion pressure was increased in a stepwise fashion to120, 140, and 160 mmHg as previously described for studies in the ratkidney ( 27 ). The pressures were maintained at each levelfor 3 min. Pressure was then returned to 100 mmHg for a 5-min recoveryperiod. In a subset of mice, ANG II dose responses were determinedafter the recovery period, as described below.7 ?+ |% A+ _6 Y! w; I% l2 {! p0 t
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Afferent arteriolar ANG II responses. Afferent arteriolar diameters in kidneys from male and female WT( n = 9 females; n = 2 males),AT 1A  / ( n = 4 females) and AT 1A / ( n = 14 females; n = 3 males) mice were measured during superfusion withANG II. After a 5-min control period or recovery from the change inperfusion pressure protocol, the kidneys were superfused sequentiallywith 0.1, 1.0, and 10 nM ANG II for a period of 5 min for eachconcentration. A recovery period of 5 min was then observed. Kidneyswere then superfused for 5 min with an AT 1 receptor blocker(1 µM candesartan), and the ANG II concentration-responserelationship was repeated in the same vessel from WT ( n = 5), AT 1A  / ( n = 2), andAT 1A / ( n = 11) mice.
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Afferent arteriolar ANG II time control responses. Afferent arteriolar diameters were measured in kidneys from femaleAT 1A / mice ( n = 3) in response to 0, 0.1, 1.0, 10, and 0 nM ANG II for a period of 5 min for eachconcentration. This protocol was repeated in the same vesselsto demonstrate that the vasculature responds to a second application ofANG II.
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Efferent arteriolar ANG II responses. Efferent arteriolar diameters in kidneys from male and female WT( n = 5 females; n = 2 males) andAT 1A / ( n = 5 females; n = 2 males) mice were measured during superfusion with ANG II. After a5-min control period, the kidneys were superfused with 0.1, 1.0, and 10 nM ANG II for a period of 5 min for each concentration. A recoveryperiod of 5 min was then observed. Kidneys were then superfused for 5 min with an AT 1 receptor blocker (1 µM candesartan). TheANG II dose-response relationship was repeated in the same vessel inall kidneys.- z' B) f) Z( g1 O

Data analysis. Afferent and efferent arteriolar luminal diameters were measured at12-s intervals throughout the entire protocol. Plateau responses weredetermined by averaging data obtained during the final 2 min of eachtreatment period and used for statistical analysis. Statisticalanalyses were performed using SigmaStat statistical software on the rawdata by one-way or two-way analysis of variance, followed by Tukey'stest or paired t -test as appropriate. Baseline diameterswere analyzed by unpaired t -test. A P value as the means ± SE ( n = no. of arterioles).% T# i1 Q2 V& p6 x
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RESULTS
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Animals. Body weights of the three genotypes of mice averaged 39 ± 2 g for WT ( n = 18), 37 ± 5 g forAT 1A  / ( n = 4), and 33 ± 1 g for AT 1A / ( n = 27) mice. Body weightsof AT 1A / mice were significantly smaller than those ofWT mice ( P Afferent arteriolar autoregulatory responses. Afferent arteriolar baseline diameters were not significantly differentbetween WT ( n = 6) and AT 1A / ( n = 6) mice, averaging 14.8 ± 0.8 and 14.9 ± 0.8 µm, respectively. After stepwise increases in renal perfusionpressure to 120, 140, and 160 mmHg, afferent arteriolar diameterssignificantly decreased by 7 ± 1, 16 ± 1, and 26 ± 2% for WT mice and by 11 ± 2, 23 ± 5, and 30 ± 5%for AT 1A / mice, respectively (Fig. 2 ). Afferent arteriole diameters weresignificantly reduced at each step increase in perfusion pressure inkidneys of WT and AT 1A / mice. Afferent arteriolar diameters were not significantly different during the recovery periodcompared with baseline diameters in each group. Afferent arteriolesfrom AT 1A  / ( n = 2) mice displayed asimilar pattern of response. Afferent arteriolar diameters forAT 1A  / mice decreased 11, 25, and 31% of the baseline of15.1 µm when renal perfusion pressure was increased from 100 to 120 to 140 to 160 mmHg, respectively. WT, AT 1A  /, andAT 1A / mice exhibited similar afferent arteriolar autoregulatory responses to increases in renal perfusion pressure. Themagnitude of the responses was not different between WT and AT 1A / mice ( P = 0.6), demonstratingactive afferent arteriolar responses to increases in renal perfusionpressure in the absence of the AT 1A receptor.
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Fig. 2. Afferent arteriolar diameter responses to elevations inrenal perfusion pressure. Afferent arteriolar diameter [µm( A ); % of control ( B )] responses to stepwiseincreases in renal arterial perfusion pressure in kidneys fromwild-type (WT;, n = 6) andAT 1 receptor subtype-deficient (AT 1A /;, n = 6) mice. Afferent arterioles fromboth groups responded to increases in perfusion pressure with asignificant reduction in diameter. The magnitude of the afferentarteriolar vasoconstrictor responses were not significantly differentbetween WT and AT 1A / mice. * P

Afferent arteriolar ANG II responses. Baseline afferent arteriolar diameters were similar in kidneys from WT( n = 11) and AT 1A / ( n = 17) mice subjected to ANG II challenges, averaging 14.6 ± 0.4 and15.2 ± 0.4 µm, respectively. Afferent arterioles from WT micevasoconstricted by 10 ± 1 (0.1 nM ANG II), 19 ± 2 (1 nM ANGII), and 37 ± 5% of control values (10 nM ANG II) (Fig. 3 ). Afferent arteriolar reductions indiameter were 7 ± 1, 14 ± 2, and 22 ± 2% of controlin response to 0.1, 1, and 10 nM ANG II, respectively, in vesselsobtained from AT 1A / mice. The ANG II responses ofafferent arterioles from AT 1A  / mice ( n = 4) were intermediate in magnitude relative to those of WT andAT 1A / mice. Baseline afferent arteriolar diameter inAT 1A  / mice averaged 15.0 ± 1.3 µm and decreased9 ± 1, 19 ± 4, and 34 ± 8% of control in response to0.1, 1, and 10 nM ANG II, respectively. Therefore, 0.1, 1, and 10 nMANG II caused significant reductions in afferent arteriole diameters inkidneys from WT, AT 1A  /, and AT 1A / mice.Afferent arteriole vasoconstrictor responses to low-dose ANG II (0.1, 1 nM) were similar for WT, AT 1A  /, andAT 1A / mice. This effect was not different among thegroups. ANG II (10 nM) reduced afferent arteriolar diameter by 37 ± 5 in WT, 34 ± 8 in AT 1A  /, and by only 22 ± 2% in AT 1A / mice. Diameter responses to the highestdose of ANG II were significantly reduced by 40% in kidneys fromAT 1A / compared with WT mice. ANG II responses inkidneys from AT 1A  / were not different in those from WTor AT 1A / mice.
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Fig. 3. Afferent arteriolar diameter responses to ANG II.Afferent arteriole diameter [µm ( A ); % of control( B )] responses to 0.1, 1.0, and 10 nM ANG II in kidneysfrom WT (, n = 11) andAT 1A / (, n = 17) mice.ANG II produced a significant vasoconstriction in both groups. Theafferent arteriolar vasoconstrictor response to 10 nM ANG II forAT 1A / mice was significantly reduced compared with WT.Baseline diameters were not significantly different. * P P5 D2 Z/ ?" Z$ f

At the completion of application of ANG II, vessels were exposed to thecontrol superfusion for a recovery period. The AT 1 receptorantagonist candesartan (1 µM) was applied to the surface of thekidney for a period of 5 min. Diameters were not altered by candesartanalone in WT ( n = 5), AT 1A  / ( n = 2), or AT 1A / ( n = 11) vessels, averaging 100 ± 1, 105, and 100 ± 1% ofcontrol, respectively. Moreover, the AT 1 receptorantagonist completely blocked the afferent arteriolar responses to alldoses of ANG II in all groups, demonstrating that the afferentarteriole vasoconstrictor responses are mediated by the AT 1 receptor subtypes.0 b8 Q0 n/ i7 a( a6 W

Afferent arteriolar ANG II time control responses. This series of experiments was performed to demonstrate that afferentarterioles respond to repeated application of increasing concentrationsof ANG II. Afferent arteriolar diameter in kidneys fromAT 1A / mice ( n = 3) averaged 17.7 ± 0.1 µm at baseline. Application of 0.1, 1, and 10 nM ANG IIproduced graded reductions in afferent arteriolar diameter of 6 ± 1, 15 ± 1, and 23 ± 1% of control levels, respectively(Fig. 4 ). On the second application of0.1, 1, and 10 nM ANG II, vessel diameters decreased 6 ± 1, 14 ± 1, and 19 ± 2% of control levels, respectively. Theafferent arteriolar responses to the first and second applications ofANG II did not differ significantly. Afferent arteriolar diameters werenot significantly different between baseline and the two recoveryperiods. These data provide evidence that mouse juxtamedullary afferentarterioles do not display tachyphalaxis to ANG II at the concentrationsused and respond actively to a repeat application of the peptide underthese experimental conditions.
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Fig. 4. Afferent arteriolar diameter responses to repeatedapplication of ANG II in kidneys from AT 1A / mice.Afferent arteriolar diameter [µm ( A ); % of control( B )] responses during the first ( ) andsecond ( ) application of 0.1, 1.0, and 10 nM ANG II( n = 3). There was no significant difference betweenthe afferent arteriolar vasoconstrictor responses of the first andsecond ANG II applications.

Efferent arteriolar ANG II responses. Efferent arteriole baseline diameters in kidneys fromAT 1A / mice were significantly larger than in those fromWT mice, averaging 19.6 ± 0.7 and 17.0 ± 0.3 µm,respectively. Efferent arterioles of WT mice vasoconstricted inresponse to 0.1, 1, and 10 nM ANG II by 6 ± 1, 11 ± 1, and21 ± 5% of control ( P 5 ). However, efferent arterioles fromAT 1A / mice did not respond to ANG II. TheAT 1 -receptor antagonist candesartan alone did not alterefferent arteriolar diameter (100% of control) of WT( n = 6) or AT 1A / ( n = 6) mice. As shown in Fig. 5, blockade of the AT 1 receptorwith candesartan completely prevented the efferent arteriolarvasoconstrictor responses to ANG II in kidneys from WT mice.
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Fig. 5. Efferent arteriolar diameter responses to ANG II.Efferent arteriolar diameter [µm ( A ); % of control( B )] responses to 0.1, 1.0, and 10 nM ANG II in kidneysfrom WT (, n = 6) andAT 1A / (, n = 6) mice.ANG II produced a significant efferent arteriolar vasoconstriction onlyin vessels from WT mice. Baseline diameters were significantly largerin AT 1A / compared with WT vessels. * P P2 ]. n5 N9 w  p5 B+ w) d

DISCUSSION6 o/ h- R' T5 D# ~/ f
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The AT 1 receptor is primarily responsible for thevascular and tubular actions of the renal renin-angiotensin system.There are two unique AT 1 receptor subtypes in rodents,AT 1A and AT 1B, which cannot be distinguishedusing pharmacological antagonists. Accordingly, it has not beenpossible to discriminate between renal microvascular AT 1A and AT 1B receptor subtype function. It is known thatAT 1A and AT 1B receptor mRNAs are expressed onthe afferent arteriole ( 2, 17 ). However, there is noinformation on the localization of AT 1B receptor mRNA orprotein on the efferent arteriole. Therefore, the purpose of thepresent study was to determine the functional contribution of theAT 1A and AT 1B receptors to the renalmicrovascular responses to changes in renal perfusion pressure and thesegment-specific vasoconstrictor actions of ANG II.% K, g4 B& Z7 L( J
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The requirement for AT 1A receptors for afferent arteriolarvascular control mechanisms with regard to elevations in renal arterialperfusion pressure were examined in the present study usinggene-targeted mice. Although AT 1A receptor-deficient mice have been shown to lack a tubuloglomerularfeedback mechanism ( 25 ), the present study revealed thatincreases in renal perfusion pressure evoke indistinguishable afferentarteriolar vasoconstrictor responses in WT and AT 1A / mouse kidneys. Significant reductions in afferent arteriolar diameterwere observed in WT, heterozygous, and homozygousAT 1A -disrupted mouse kidneys in response to stepwise increases in renal arterial pressure. The magnitude of the afferent arteriolar vasoconstriction in the mouse kidney was similar to theresponses seen previously in the rat, in which single afferent arteriolar blood flow was efficiently autoregulated over the same pressure range ( 27 ). Thus afferent arteriolarautoregulatory responsiveness in the isolated perfused mouse kidney issimilar to that previously observed in the rat and does not appear to be altered by the loss of AT 1A receptor function. Themaintenance of renal autoregulatory responsiveness inAT 1A / mice suggests a prominence of the myogenicmechanism in these animals. Alternatively, tubuloglomerular feedbackresponses may be active in the deep, juxtamedullary nephron populationof AT 1A / mice and may reflect mediation by theAT 1B receptor.
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The relative contributions of AT 1A and AT 1B receptors to afferent arteriolar resting tone were evaluated in thepresent study. Afferent arteriolar diameters were not significantlydifferent under baseline conditions (100 mmHg renal perfusion pressure, superfusion with vehicle solution) in kidneys from WT andAT 1A / mice. The lack of a between-group difference inbaseline diameter indicates no effect of loss of AT 1A receptors on basal afferent arteriolar tone. In addition, basalafferent arteriolar diameters were not altered after AT 1 receptor blockade by candesartan in kidneys from WT orAT 1A / mice. This suggests that there is little influence of ANG II on basal afferent arteriolar tone under the conditions of the isolated blood-perfused mouse kidney preparation.

The relative contributions of AT 1A and AT 1B receptors to afferent arteriolar responses to ANG II were determined inkidneys from WT, AT 1A  / and AT 1A / mice.The afferent arteriolar diameter responses to ANG II in WT mice weresimilar to those previously reported in the rats pretreated withenalprilat ( 3, 9 ). Significant reductions in afferentarteriolar diameters were observed over the concentration range of0.1-10 nM in kidneys from WT, AT 1A  /, andAT 1A / mice. We attribute the ANG II vasoconstrictor responses in afferent arterioles from AT 1A / mice to bemediated by the AT 1B receptor subtype. Surprisingly, themagnitude of the afferent arteriolar responses in the three groups ofmice was similar at the 0.1 and 1 nM ANG II doses. This was unexpected based on previous studies, which demonstrated a negligible pressor response to bolus systemic ANG II ( 11 ) and diminishedrenal blood flow response to bolus intrarenal administration of ANG II( 22 ) in kidneys from AT 1A / mice comparedwith controls. However, it has been shown that AT 1A / mice have significantly enhanced renal renin mRNA expression( 19 ) and elevated plasma ANG II levels ( 6 ).Elevated circulating ANG II may have contributed to the lack of ANG IIresponse in kidneys from AT 1A / mice in the abovestudies. Endogenous ANG II may occupy the AT 1B receptors and limit accessability of exogenously administered ANG II. In fact,after administration of an angiotensin-converting enzyme inhibitor,both systemic pressor and renal blood flow responses to ANG II wereenhanced ( 6, 19 ). Therefore, suppression of the endogenousproduction of ANG II may be necessary to reveal the function of theAT 1B receptor in the absence of the AT 1A receptor. It is possible that circulating ANG II levels are low usingthe mouse juxtamedullary nephron technique, in which the kidney is perfused with blood obtained from a donor rat and, therefore, afferentarteriolar responses in kidneys from AT 1A / mice are revealed.: o1 x# P+ L6 r* L  X

In contrast to the afferent arteriolar vasoconstrictor responses tolow-dose ANG II, afferent arteriolar diameter responses to the highdose of ANG II, 10 nM, were significantly different in kidneys from WTand AT 1A / mice. Afferent arteriolar diameter responsesfor the AT 1A / mice were only 60% of the magnitude ofthe response for WT mice. The difference in the magnitude of theresponse may be due to the maximal vasoconstrictor contribution of theAT 1B receptors. The vasoconstrictor responses to ANG II were completely inhibited by the AT 1 receptor blockercandesartan. This drug blocks both the AT 1A andAT 1B receptor subtypes, similar to the properties oflosartan ( 15 ). Therefore, the vasoconstrictor effects ofANG II on the afferent arteriole are mediated by the AT 1 receptor for both WT and AT 1A / mice. There was noevidence of AT 2 receptor-mediated vasodilation in thepresence of AT 1 receptor blockade and ANG II in the presentstudy. We conclude that for WT mice, afferent arteriolar responses aremediated by both the AT 1A and AT 1B receptors,whereas for AT 1A / mice, this effect is mediatedexclusively by the AT 1B receptors. In addition, in theabsence of AT 1A receptors, 10 nM ANG II evokes anattenuated, candesartan-sensitive, afferent arteriolar constriction inkidneys from AT 1A / mice, implicating activation ofAT 1B receptors. It is not known at the present time whetherAT 2 and/or AT 1B receptor protein expression isaltered in afferent arterioles of AT 1A / mice.$ h6 W1 p* m' F" c) d! S4 d

The relative contributions of AT 1A and AT 1B receptors to efferent arteriolar responses to ANG II were determined inkidneys from WT and AT 1A / mice. Efferent arterioles ofWT mice responded in a dose-dependent manner to ANG II, similar tojuxtamedullary efferent arterioles of the rat ( 3, 9 ).However, efferent arterioles of AT 1A / mice did notrespond to ANG II. These data suggest that AT 1A receptorsare primarily responsible for ANG II-induced efferent arteriolarvasoconstriction. The lack of ANG II responses in efferent arteriolesof AT 1A / mice suggests that AT 1B receptorsare not functionally expressed on the efferent arteriole.* Z% B7 x3 y7 n. h1 ~+ b

In contrast to the similarities in the afferent arteriolar restingdiameters of WT and AT 1A / mice, efferent arteriolardiameters of AT 1A / mice were significantly larger thanfor WT mice. Such increased efferent arteriolar diameter combined withan increased glomerular ultrafiltration coefficient, resulting fromreduced ANG II-dependent activation of AT 1A receptors, maycontribute to the maintenance of renal plasma flow and glomerularfiltration rate in the normal range in hypotensiveAT 1A / mice ( 6 ). However, there may belimitations to our ability to extrapolate our data obtained from invitro studies to an in vivo setting. It is not likely that the largerresting efferent arteriolar diameter in kidneys fromAT 1A / mice is a result of the direct loss of the effects of endogenous ANG II on the AT 1A receptor becauseresting diameter was not influenced by candesartan. At this time, wecan only speculate on the potential interaction of ANG II-induced vasoconstriction and other vasodilatory mechanisms at the site of theefferent arteriole. The larger resting efferent arteriolar diameter ofAT 1A / mice may reflect a lack of compensation by thevasoconstrictor properties of the AT 1A receptor. It hasbeen shown that AT 1A / mice have increased expressionand activity of neuronal nitric oxide synthase ( 14 ).Because nitric oxide derived from neuronal nitric oxide synthaselocalized in the macula densa cells and efferent arterioles( 1 ) has been shown to play an important role in renalhemodynamics, it is possible that nitric oxide has profound effects onthe resting tone of the efferent arteriole lacking AT 1 receptors.

In conclusion, afferent arteriolar autoregulatory capability is notaffected by the absence of AT 1A receptors. This study inAT 1A / mice provides functional evidence of distinctdistribution patterns for AT 1 receptor subtypes within therenal microvasculature. We conclude that afferent arteriolarvasoconstrictor responses to ANG II are mediated by AT 1A and AT 1B receptors, whereas efferent arteriolevasoconstrictor responses to ANG II are mediated by onlyAT 1A receptors in the mouse kidney.  p  L  z0 R1 F# z3 r. ?: h# @
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ACKNOWLEDGEMENTS( w& b5 w+ i/ Q; t; a

The authors thank Drs. Pamela K. Carmines and L. Gabriel Navar fora critical review of the manuscript and Camie Snow for performing theSouthern blot analysis. Dr. Peter Morsing of Astra Hassle, Gothenburg,Sweden, generously provided the AT 1 receptor antagonistcandesartan (Atacand) utilized for these studies.0 l* B+ b+ n0 t$ g0 u3 V$ T4 \( \
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