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Vascular Type 1A Angiotensin II Receptors Control BPby Regulating Renal Blood Flow and Urinary SodiumExcretion

Matthew A. Sparks,* Johannes Stegbauer,*† Daian Chen,* Jose A. Gomez,‡

Robert C. Griffiths,* Hooman A. Azad,* Marcela Herrera,* Susan B. Gurley,* andThomas M. Coffman*§

*Division of Nephrology, Department of Medicine, Durham VA and Duke University Medical Centers, Durham, NorthCarolina; †Department of Nephrology, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany;‡Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, North Carolina; and§Cardiovascular and Metabolic Disorders Research Program, Duke-National University of Singapore, GraduateMedical School, Singapore

ABSTRACTInappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hy-pertension and its associated complications. To define the role for actions of vascular AT1A receptors in BPregulation and hypertension pathogenesis, wegeneratedmicewith cell-specific deletion of AT1A receptors insmooth muscle cells (SMKOmice) using Loxp technology andCre transgenes with robust expression in bothconductance and resistance arteries. We found that elimination of AT1A receptors from vascular smoothmuscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BPand exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)–dependenthypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was as-sociatedwithenhancedurinary excretionof sodium.Despite the lowerBP, acutevasoconstrictor responses toAng II in the systemic vasculaturewere largely preserved (approximately 80%of control levels) in SMKOmicebecause of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1Breceptors. In contrast,Ang II–dependent responses in the renal circulationwere almost completely eliminatedin SMKO mice (approximately 5%–10% of control levels). These findings suggest that direct actions of AT1Areceptors in VSMCs are essential for regulation of renal blood flowbyAng II and highlight the capacity of AngII–dependent vascular responses in the kidney to effect natriuresis and BP control.

J Am Soc Nephrol 26: ccc–ccc, 2015. doi: 10.1681/ASN.2014080816

Dysregulation of the renin angiotensin system (RAS)RAS is a common feature of human hypertension. Assuch, RAS antagonists lower BP inmost patients withhypertension.1,2 The predominant actions of the RASto influenceBParemediated by angiotensin II (Ang II)acting via the type 1 angiotensin (AT1) receptor, amember of the large family of G-protein coupled re-ceptors. AT1 receptors are the target of Ang receptorblockers, effective and widely used antihypertensiveagents. Activation of AT1 receptors can trigger a cas-cadeof physiologic responses, including vasoconstric-tion, activation of the sympathetic nervous system,and stimulation of sodium reabsorption by the

kidney, which can conspire to promote hypertensionand end-organ damage.3 However, until recently, ithas been difficult to unravel the relative contributions

Received August 21, 2014. Accepted January 27, 2015.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Thomas M. Coffman, Department ofMedicine, Division of Nephrology, Duke University MedicalCenter, Room 2028 MSRB2, 2 Genome Court, Durham, NC27710. Email: tcoffman@duke.edu

Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: ccc–ccc, 2015 ISSN : 1046-6673/2612-ccc 1

of these distinct AT1 receptor responses to the pathogenesis ofhypertension.

Using kidney cross-transplantation, our group previouslydemonstrated that actions of the major murine AT1 receptorisoform, AT1A, in both the kidney and systemic tissues, are im-portant for maintenance of basal BP,4 whereas AT1A receptors inthe kidney play the predominant role in the development of AngII–dependent hypertension and cardiac hypertrophy.5 The pre-cise cell lineages responsible for these effects are now being elu-cidated. For example, we and others have shown that AT1A

receptors in the renal proximal tubule play an important rolein Ang II–dependent hypertension by augmenting renal sodiumreabsorption through activation of key sodium transporter, in-cluding NHE3.6,7 However, eliminating AT1A receptors in renalepithelia only partially recapitulated the actions of Ang receptorblockers to abrogate hypertension. Along with kidney epithelia,AT1 receptors are broadly expressed on the renal vasculature.Previous studies have demonstrated that inappropriate activa-tion of signaling pathways in the vasculature can potentiatechronic hypertension,8–10 and it has been suggested that vasculardysfunction alone may lead to BP dysregulation.9 For example,Guilluy et al. demonstrated that eliminating Arhgef1, a Rho-exchange factor linked to AT1 receptor signaling, from vascularsmooth muscle cells (VSMCs) caused dramatic abrogation ofhypertension.8 These authors concluded that activation ofArhgef1 triggered by AT1 receptors in VSMCs caused systemicvasoconstriction, leading to increased systemic vascular resis-tance and hypertension. On the other hand, vasoconstriction inthe kidney circulation can also effect BP control; changes inrenal blood flow (RBF) affect sodium reabsorption along thenephron through changes in Starling forces and physical fac-tors.11 Moreover, because approximately 25% of the arterialblood flow enters the renal circulation, vasoconstriction inthe kidney may raise BP through effects on systemic vascularresistance. Therefore, to define the precise contribution of ac-tions of vascular AT1A receptors to BP control in vivo, we usedCre-LoxP technology to generate mice with cell-specific dele-tion of AT1A receptors from smooth muscle cell lineages invasculature beds throughout the body, including the kidney.We found that AT1A receptors in VSMCs maintain basal BP,augment salt sensitivity, and contribute to the pathogenesis ofAng II–induced hypertension by reducing RBF, thereby en-hancing sodium retention.

RESULTS

Generation of Mice with Cell-Specific Deletion of AT1Receptors in VSMCsWegeneratedmicewith cell-specific deletion of theAT1A receptorfrom smooth muscle cell lineages by using the KISm22a-Cremouse line in which Cre-recombinase has been knocked in tothe Sm22a gene locus such that its expression is regulated byendogenous regulatory elements of the Sm22a gene. We carriedout successive intercrosses between the KISm22a-Cremouse line

and mice homozygous for the conditional floxed Agtr1a allele(Agtr1aflox/flox)7 to generate KISm22a Cre+-Agtr1aflox/flox

(SMKO) and littermate Cre-negative control mice (control) forexperiments. Using the mTmG dual-reporter mouse line,12 wehave previously demonstrated robust green fluorescent proteinfluorescence in the media of the aorta and throughout glomer-ular afferent arterioles in KISm22a-Cre-mTmGmice, confirm-ing robust expression of Cre-recombinase in large arteries andresistance vessels.13

To document efficient elimination of AT1A receptors, wemeasured levels for AT1A receptor mRNA by real-time quanti-tiative RT-PCR. Segments of the aortawere isolated fromSMKOand control mice. As shown in Supplemental Figure 1, mRNAfor the AT1A receptor was easily detected in aortae from controlmice (n=9), but not fromSMKOs (P=0.001,n=9). Furthermore,mesenteric vessels were isolated (Supplemental Figure 1) fromSMKO (n=5) and control mice (n=5) and demonstrated an ap-proximate 97% reduction in AT1A receptor mRNA (control, 160.2 versus SMKO, 0.0360.01 AT1A to GAPDHmRNA, arbitraryunits; P=0.004). AT1A receptor mRNA expression was not af-fected in liver, adrenal gland, or brain stem. Therefore, inSMKOs, AT1A receptor mRNA expression was efficiently andspecifically extinguished from VSMCs.

Elimination of AT1A Receptors from VSMCs ReducesBasal BP and Contributes to Salt SensitivityTo define the effect of AT1A receptors in the vasculature on BPcontrol, radiotelemetry unitswere implanted into 8- to 12-week-oldmale SMKO (n=13) and control (n=13)mice tomeasure intra-arterial pressure continuously in the conscious, unrestrainedstate. As shown in Figure 1, mean arterial pressures (MAPs)during normal sodium (0.4%Na+) feedingwere approximately7 mmHg lower in SMKO mice than controls (SMKO, 10861versus control, 11561 mmHg; P,0.002), identifying a role forAT1A receptor activation in the vasculature to maintain normalBP homeostasis. This was accompanied by a qualitative in-crease in renin (Ren1) mRNA expression in the renal cortexin SMKOmice compared with controls at baseline during nor-mal sodium diet (Supplemental Figure 2). The pattern of di-urnal variation of BP was preserved (Supplemental Figure 3).We have previously shown that global deficiency of AT1A recep-tors enhances the extent of BP changes in response to altereddietary sodium intake (i.e., sodium sensitivity).14 Accordingly,SMKO and control mice were sequentially fed diets of normal(0.4% Na+), low (,0.02% Na+), and high (6% Na+) sodiumcontent. During low Na+ feedings, the difference in MAP be-tween SMKO and controlmice was further exaggerated (SMKO,10461 versus control, 11362; P=0.001). In contrast, duringhigh Na+ diet the difference in BP narrowed and statistical sig-nificance was lost (SMKO, 11062 versus control, 11562;P=0.03) (Figure 1). In humans, sodium sensitivity is definedas the difference in BP between low- and high-sodium states.15

Accordingly, we compared the change in BP between low- andhigh-salt feeding in the twogroups. As shown inFigure 1B, theBPchange from low-sodium to high-sodium diet was significantly

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greater in SMKOs than control mice (SMKO, +561 versus con-trol +261 mmHg; P=0.001), indicating enhanced sodium sensi-tivity in the SMKOs.

Attenuated Hypertensive Response to ChronicInfusions of Ang II in SMKO MiceTo determine the contribution of vascular AT1A receptors to thedevelopment of hypertension, osmotic minipumps were im-planted into SMKO mice and controls to infuse Ang II for 4weeks, and intra-arterial pressures were measured via radiote-lemetry. We carried out two independent experiments infusinglow (400 ng/kg per min) or high (1000 ng/kg per min) concen-trations of Ang II. It has been suggested that the lower concen-tration of Ang II induces a slower onset of hypertension thatmaybe more dependent on neural and/or renal mechanisms.16 Asshown in Figure 2, control mice (n=6) infused with the lowerconcentration of Ang II (400 ng/kg per min) developed robusthypertensionwith an increase inMAPof 2565mmHg duringthe first week. By comparison, the increase in MAP was sig-nificantly attenuated in SMKOs (862 mmHg; P,0.01; n=7).At the higher dose of Ang II (1000 ng/kg per min), MAP in-creased by 3063 mmHg in control mice (n=5) and was like-wise attenuated in the SMKO mice (1664 mmHg; P,0.02;n=6) (Figure 3). These findings suggest that AT1A receptors inVSMCs play a key role in the pathogenesis of Ang II–dependenthypertension.

Enhanced Natriuresis in SMKOsduring the Development ofHypertensionTo determine whether alterations in renalsodiumhandlingmayhavecontributed to theattenuated hypertensive response to Ang II inthe SMKOs, we measured urinary sodiumexcretion during the first 5 days of the Ang IIinfusion,whereassodiumintakewasclampedat similar levels between the experimentalgroups. As shown in Figure 4, we found thatcumulative sodium balance in the controlswas positive (0.2260.05 mmol Na+/5 d;n=6), representing net retention of sodiumduring the initiation of hypertension. Bycontrast, sodium balance remained effec-tively neutral in SMKOs (–0.00360.02mmol Na+/5 d; P=0.001 versus controls;n=7). Therefore, attenuated hypertension inSMKOs was associated with preserved renalsodium excretion and a shift of the pressure-natriuresis relationship, indicating that AT1Areceptors inVSMCs promote BPelevation bystimulating renal sodium reabsorption.

Preserved Systemic VascularResponses to Ang II in SMKO MiceWe next examined the consequences ofremoving AT1A receptors from VSMC on

acute BP responses to Ang II measured via the carotid artery.As shown in Figure 5, bolus injection of Ang II caused acutevasoconstriction in control mice (n=7) with an immediate,marked increase in MAP that gradually returned to baselinewithin approximately 5 minutes. In response to doses rangingfrom 0.1 to 10 mg/kg Ang II, there was a dose-dependent in-crease in the peak and area under the curve. This systemicvasoconstrictor response was largely preserved in the SMKOs(n=6), with a residual peak increase in MAP that was approx-imately 80% of control values (Figure 5). Therefore, despitethe significant difference in BP between SMKOs and controls,acute systemic vasoconstrictor responses to Ang II were onlymodestly attenuated. As expected, vasoconstrictor responsesto epinephrine were unaffected in SMKOs.

Residual Systemic Vasoconstriction in SMKOs Is NotMediated by AT1B ReceptorsThevasoconstrictor response toAng II is substantially attenuatedin mice with complete deficiency of AT1A receptors17; therefore,the finding of preserved systemic vasoconstriction after Ang II inSMKOs (Figure 5) was unexpected. Because AT1B receptors areexpressed in the vasculature and have the capacity to inducevasoconstriction,18 we considered the possibility that AT1B re-ceptors might be responsible for residual Ang II–dependent va-soconstriction in SMKOs. To test this, we generated mice withsmooth muscle cell–specific deletion of AT1A receptors on an

Figure 1. Lack of AT1A receptors in VSMCs reduces basal BP and contributes to saltsensitivity. (A) BP was measured via radiotelemetry in control (closed boxes) and SMKO(open triangles) mice. During normal sodium (0.4% Na+) feeding, SMKO mice had lowerMAPs than control mice (SMKO, 10861 versus control, 11561 mmHg; *P,0.002).Control and SMKO mice were sequentially fed diets of normal (0.4% Na+), low (,0.02%Na+), and high (6% Na+) sodium and BPs were averaged over 5 days respectively. Duringlow-sodium feeding, SMKOs had lower MAPs than controls (SMKO, 10461 versuscontrol, 11362 mmHg; **P,0.001). During high-sodium feeding, MAPs increased inboth groups, but the difference between SMKO mice and control mice was not signifi-cant (SMKO, 11062 versus control, 11562 mmHg; P=0.03). Analysis was performed withone-way ANOVA (overall P,0.001) and unpaired t test with Bonferroni adjustment formultiple comparisons (significance for P value ,0.02). (B) The change in MAP from a low-sodium to high-sodium diet was significantly greater in SMKO mice compared withcontrol mice (SMKO, +561 versus control, +261 mmHg, change in MAP; ***P=0.001).Analysis was performed with an unpaired t test.

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AT1B-null background (I-SMKO-1B2/2). In this case, we used atamoxifen-inducibleCre transgene driven by the smoothmusclemyosin heavy chain promoter (SMMHC-ERT2-Cre) to allow de-letion of AT1A receptors from VSMCs in adult mice and obviatepotential confounding effects of the absence of VSMC AT1 re-ceptors during fetal development. After administration of ta-moxifen, VSMCs in I-SMKO-1B2/2 mice will be deficient inboth AT1A and AT1B receptors.

We first verified appropriate expression of Cre recombinasein both aorta and small vessels of the kidney in SMMHC-ERT2-Cre-mTmGmice (Supplemental Figure 4). In addition, we foundsignificant reductions in AT1A receptor mRNA levels verified byquantitative RT-PCR of aortae andmesenteric vessels in I-SMKO

mice (Supplemental Figure 5). As shown inFigure 6A, we compared the intensity of acuteAng II–dependent vasoconstriction in thefollowing four experimental groups: control,1B2/2, I-SMKO-1B+/+, and I-SMKO-1B2/2

mice. Compared with control mice, theabsence of AT1B receptors alone in 1B2/2

mice did not significantly affect the sys-temic vasoconstrictor response to 1 mg/kgof Ang II (22.861 versus 21.762 mmHgpeak increase in systolic BP from baseline,P=NS, n=4). As in the SMKOs, this re-sponse was modestly diminished in theI-SMKO-1B+/+ animals relative to controlsmice (14.962 versus 22.861mmHg peak in-crease in systolic BP from baseline, P=0.02,n=4). Finally, the extent of vasoconstrictionwas virtually identical in the I-SMKO-1B+/+

(14.962 mmHg) and I-SMKO-1B2/2

(15.662 mmHg, P=NS, n=4) groups, indi-cating that AT1B receptors do not mediatethe persistent systemic vasoconstrictor re-sponse to Ang II in SMKOs.

a-Adrenergic Blockade ExtinguishesAng II–Dependent Vasoconstriction inSMKOsCentral administration of Ang II inducesrapid peripheral vasoconstriction mediatedviaactivationof sympatheticoutflowbyAT1A

receptors in the central nervous system.19,20

Todeterminewhether sympathetic stimulationcontributes to the residual Ang II–dependentsystemic vasoconstriction in SMKOs, wecompared the acute vasoconstrictor responsesto Ang II in SMKOs and controls beforeand after administration of the a-adrenergicblocker phentolamine. Before administra-tion of phentolamine, the acute pressorresponse to Ang II was reduced by approxi-mately 35% in I-SMKOs compared withcontrols (Figure 6B). Administration of

phentolamine had no effect on the magnitude of Ang II–dependent vasoconstriction in controls (22.861 versus 22.864mmHg, P=NS, n=4) (Figure 6B). By contrast, this response wasalmost completely abrogated in SMKOs after phentolamine(14.962 versus 361 mmHg, P=0.003, n=4) (Figure 6B), sug-gesting that residual vasoconstriction in I-SMKO mice requiresa-adrenergic stimulation.

Increased Urinary Catecholamine Excretion in SMKOMiceThe enhanced contribution of a-adrenergic output to Ang II–dependent vasoconstriction in SMKOs suggests altered acti-vation of the sympathetic nervous system. As an assessment of

Figure 2. AT1A receptors in VSMCs promote the hypertensive response to low-dose AngII–dependent hypertension. (A) Daily radiotelemetry tracings from control mice (closedsquares) and SMKOmice (open triangles) infused with chronic low-dose Ang II (400 ng/kgper min) over 1 week. (B) Control mice developed robust hypertension with an increase inMAP of 2565 mmHg from baseline during the first week of Ang II administration. Theincrease in MAP was significantly attenuated in SMKO mice (862 mmHg; *P,0.01, opentriangles, unpaired t test).

Figure 3. AT1A receptors in VSMCs promote the hypertensive response to high-doseAng II–dependent hypertension. (A) Daily radiotelemetry tracings from control mice(closed squares) and SMKO mice (open triangles) infused with chronic high-dose AngII (1000 ng/kg per min) over 4 weeks. (B) Control mice developed hypertension with anincrease in MAP of 3063 mmHg. However, the hypertensive response in SMKO micewas significantly attenuated (1664 mmHg; *P,0.02 versus control, unpaired t test).

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adrenergic activity, we measured urinary norepinephrine ex-cretion in controls and SMKOs at baseline and after 3 days ofchronic Ang II administration (1000 ng/kg per min). As

shown in Figure 7, baseline urinary norepi-nephrine excretion was significantly higherin SMKOs than in controls (754681 versus530655 pg/24 h, P=0.04, n=7 in bothgroups). After 3 days of Ang II infusion,there was a tendency toward suppressionof norepinephrine excretion in the controls(from 530655 to 398666 pg/24 h, P=0.07,n=7), whereas Ang II caused a further qual-itative increase in norepinephrine levels inSMKOs (from 754681 to 10766224 pg/24h, P=0.16, n=7 in both groups), such thatthe difference in norepinephrine excretionbetween SMKOs and controls was magni-fied by chronic Ang II infusion (10766224versus 398666 pg/24 h, P=0.01, n=7 inboth groups). These findings suggest thatdeletion of AT1A receptors from VSMCs isassociatedwith enhancement of sympatheticnervous system activity. We also measuredcatecholamine biosynthetic enzyme geneproducts (tyrosine hydroxylase, dopamineb-hydroxylase, and phenylethanolamine-N-methyltransferase) from the adrenal glandat baseline in both control and SMKO miceand did not detect a difference (SupplementalFigure 6).

Increased Baseline RBF andAttenuated Renal VasoconstrictorResponses to Ang II in SMKO MiceGiven theblunted hypertensive response andexaggerated natriuresis in the SMKO cohort,we hypothesized that deletion of AT1A recep-tors from VSMCs might attenuate the sensi-tivity of the renal circulation to Ang II. Totest this possibility, we measured RBF usingan ultrasound probe placed over the renalartery. At baseline, RBF during systole wassignificantly higher in SMKO mice (n=5)than control (n=4) mice (2.160.3 versus0.8360.1 ml/min, P=0.01), and there was asuggestive increase in mean RBF (0.8860.1versus 0.4860.1 ml/min) that approachedstatistical significance (P=0.06) (Figure8A). Furthermore, in control mice, therewas a dose-dependent reduction in meanRBF induced by Ang II (–42.1%615% at0.03 mg/kg Ang II, –64.7%66% at 0.1 mg/kgAng II, –87.8%62% at 0.3 mg/kg Ang II de-crease in mean RBF from baseline) (Figure8B). In contrast, the renal vasoconstrictor re-

sponse to Ang II was substantially blunted in SMKOmice (–2.2%65% at 0.03 mg/kg Ang II, –7.1%65% at 0.1 mg/kg Ang II, and–7.5%69% at 0.3 mg/kg Ang II decrease in mean RBF from

Figure 4. Enhanced natriuresis in SMKO mice during the development of hyperten-sion. (A) Daily sodium balance in control (closed squares) and SMKO (open triangles)mice for 3 days before and 5 days after high-dose Ang II–induced hypertension (1000ng/kg per min). Significant differences in sodium balance were seen at days 1, 2, and 5after Ang II infusion between control and SMKO mice (day 1 of Ang II: 0.06760.018versus –0.04660.017 mmol Na+/d at day 1, ***P=0.001; day 2 of Ang II: 0.06760.02versus –0.006960.02, **P=0.02; day 5 of Ang II, 0.04260.02 versus 0.002460.01,*P=0.05, unpaired t test.). B. Cumulative sodium balance was significantly lower in theSMKOs (n=7) than controls (n=7), (Control: 0.2260.05 versus SMKO: -0.00360.02mmol, Na+/5 days **P=0.001, unpaired Student’s t test) during the first 5 days of AngII infusion. Error bars represent SEM.

Figure 5. Preserved systemic vascular responses to Ang II in SMKO mice. BP tracingaveraged every 10 seconds after acute administration of Ang II or epinephrine inanesthetized mice. The maximal increases in systolic BP compared to baseline inresponse to bolus infusions of Ang II (0.1, 1, and 10 mg/kg) were present but signifi-cantly lower in SMKO mice (open triangles, n=6) versus control mice (closed squares,n=7). No differences in vasoconstriction were detected with epinephrine (10 mg/kg)between control and SMKO mice. Error bars represent SEM.

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baseline; P=0.02, P=0.001, and P=0.001versus control at each dose, respectively,n=5) (Figure 8B). The failure of Ang II toinduce renal vasoconstriction in SMKOs issimilar to that seen in mice with global de-letion of AT1A receptors.21 Therefore, theelimination of AT1A receptors from VSMCsyields enhanced RBF and discrepant vascu-lar responses to Ang II in the systemic andrenal circulations. Lack of AT1A receptorsfrom VSMCs leads to diminished but pre-served systemic vascular responses in thesystemic circulation as measured in the ca-rotid artery, but dramatically attenuated va-soconstriction in the renal circulation. Wesuggest that this preservation of RBFrepresents a potential mechanism explain-ing the diminished severity of hypertensionin mice lacking vascular AT1A receptors.

DISCUSSION

Vasoconstriction is the signature physiologicaction of Ang II and was the basis for thebioactivity assay used in its original discovery.70 years ago.22,23 Ang II elicits vasocon-striction by activating AT1 receptors in

VSMCs, triggering Gaq-dependent signaling pathways withconsequent increases in intracellular calcium concentra-tion.24,25 Because the primary determinants of BP are cardiacoutput and systemic vascular resistance, it has been widely pre-sumed that these vascular actions of Ang II explain its role inhypertension pathogenesis. In this regard, compelling argu-ments have been made for the importance of systemic vaso-constriction in various forms of hypertension, including AngII–dependent hypertension.26,27 On the other hand, Guyton hasargued for the primacy of pathways controlling sodium excre-tion by the kidney in long-term BP control, suggesting that thecapacity of the kidney to excrete sodium provides a compensa-tory systemwith virtually infinite gain to oppose increases in BPby other mechanisms, including systemic vasoconstriction.28

Our study bridges these two perspectives and offers a mecha-nism for vascular effects to effect sodiumhandling by the kidney.

In previous studies using kidney cross-transplantation, weidentified a major contribution of AT1A receptors inside thekidney to the development of Ang II–dependent hyperten-sion.5 The mechanism of this effect involved stimulation ofrenal sodium reabsorption and modification of pressure-natriuresis responses.7 However, the exact cell lineages mediatingthese effects could not be precisely distinguished because AT1A

receptors are expressed in a number of regions in the kidney,including glomeruli, tubules, and renal vasculature. Accord-ingly, in the experiments described here, we directly assessedthe role of AT1A receptors in VSMCs by generating male mice

Figure 6. Exaggerated sympathetic responses rather than AT1B receptors mediate re-sidual vasoconstriction to Ang II in SMKO mice. (A) The absence of AT1B receptors alonein 1B2/2mice did not significantly affect the systemic vasoconstrictor response to 1 mg/kgof Ang II (22.861 versus 21.762 mmHg change in systolic BP from baseline, P=NS, un-paired t test). This vasoconstrictor response was diminished by approximately 35% in theI-SMKO-1B+/+ mice relative to control mice (controls, 22.861 versus I-SMKO-1B+/+,14.962 mmHg change in systolic BP from baseline; *P=0.02, unpaired t test) and di-minished by approximately 29% in the I-SMKO-1B2/2 mice compared with 1B2/2 mice(1B2/2, 21.761 versus SMKO-1B2/2, 15.662 mmHg, #P=0.04). The peak vasoconstrictorresponse was similar in the I-SMKO-1B+/+ (14.962 mmHg) and I-SMKO-1B2/2 (15.662mmHg, P=NS, unpaired t test). (B) After administration of phentolamine (400 mg/kg), themagnitude of Ang II–dependent vasoconstriction in controls was not significantly affected(22.861 versus 22.864 mmHg, P=NS, unpaired t test). This response was significantlydiminished in SMKO mice after phentolamine administration (14.962 versus 361 mmHg,**P=0.003, unpaired t test). Error bars represent SEM.

Figure 7. Elevated urinary norepinephrine in mice lacking AT1Areceptors in VSMCs. Baseline urinary norepinephrine excretionwas significantly higher in SMKO mice than control mice (754681versus 530655 pg/24 h, *P=0.04, unpaired t test). After 3 days ofAng II infusion, norepinephrine excretion was decreased in con-trol mice compared with baseline (530655 baseline versus 398666 pg/24 h at day 3 of Ang II, P=0.07, paired t test). Ang II in-fusion led to a further nonsignificant increase in norepinephrinelevels in SMKO mice (754681 baseline versus 10766224 pg/24 hat day 3 of Ang II, P=0.16, paired t test). However, the differencein norepinephrine excretion between SMKO mice and controlmice was magnified by chronic Ang II infusion (10766224 versus398666 pg/24 h, ^P=0.01, unpaired t test). Error bars representSEM.

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with cell-specific deletion of AT1A receptors from smoothmuscle(SMKOs). Elimination of AT1A receptors from VSMCs caused amodest and statistically significant reduction in baseline BP,indicating a key role for vascular actions of Ang II in determiningthe normal level of BP. In addition, SMKOs manifested aug-mented fluctuations in their BPs between low- to high-sodiumfeeding (i.e., sodium sensitivity29), which we have previously ob-served in mice with global deficiency of AT1A receptors.14 Fur-thermore, the difference in BP between SMKOs and controls wasabrogated during high-salt feeding, suggesting a primary distur-bance in total body fluid volume. By contrast, in our previousstudies of mice with cell-specific deletion of AT1A receptors fromrenal epithelial cell populations in the proximal tubule7 or col-lecting duct (J. Stegbauer et al. submitted manuscript), this phe-notype of sodium sensitivity is not recapitulated. This indicatesan important role for vascular actions of AT1A receptors in ho-meostatic response to variations in dietary sodium intake.

Along with reduced BPs at baseline, the severity of Ang II–dependent hypertension was markedly diminished in theSMKOs; MAP was reduced by .60% of control values duringthe entire 4-week infusion period. This protection against hy-pertensionwas associated with enhanced urinary sodium excre-tion in SMKO compared with control mice, consistent with analteration of the pressure-natriuresis response wherein urinaryexcretion of sodium was triggered at lower levels of BP in theSMKOs.Despite the dramatic attenuation of hypertension, there

was only a modest diminution of systemicvasoconstrictor responses to Ang II inSMKOs (approximately 20% reductionfrom control levels). On the other hand,their acute vasoconstrictor response to AngII in the renal circulation was almost com-pletely extinguished. We suggest that theabsence of Ang II–dependent renal vasocon-striction and the consequent preservation ofRBF are a critical mechanism facilitating na-triuresis and protection against hypertensionin this setting.

The influence of renal hemodynamics onsodiumhandling by the kidney has been longrecognized.30,31 For example, classic studiesfrom Martino and Earley demonstrated thathydrostatic and osmotic pressures in peri-tubular capillaries had a significant effecton sodium reabsorption by the proximal tu-bule.32,33 Later, Cowley and Roman suggestedthat reduction in renal medullary bloodflow was a key component of hypertensionpathogenesis, promoting enhanced sodiumreabsorption, volumeexpansion, and increasesin BP.34 Conversely, infusion of vasodila-tors into the renal artery causes natriuresis.35

Furthermore, resistance to hypertensionme-diated by autocrine and paracrine vasodila-tors, such as prostanoids, has been correlated

with increased RBF and enhanced natriuresis.36 Such a directconnection between kidney hemodynamics and control of uri-nary sodium excretion provides an attractive mechanism to ex-plain the pressure-natriuresis responses proposed by Guyton.28

Accordingly, we suggest that impaired renal vasoconstriction ex-plains the enhanced natriuretic response and abrogation of AngII–dependent hypertension in the SMKO mice.

In the classic paradigm, Ang II induces vasoconstrictiondirectly by activating AT1 receptors in VSMCs, triggering a com-plex series of intracellular events resulting in cellular contrac-tion.37 Therefore, we were surprised that despite dramaticreductions in expression of AT1A mRNA in both large and smallarteries in the SMKOs, their acute vasoconstrictor responses toAng II were largely preserved with the exception of the renalvasculature. Although expression of the AT1B receptor is negli-gible in most tissues, we and others have shown that its effectsmay be more pronounced in the absence of the major AT1A

receptor isoform and that it is capable of mediating Ang II–dependent vasoconstriction in vivo18,38 and in isolated vesselsex vivo.39 Therefore, we considered the possibility that theminormurineAT1 receptor, AT1B,might be responsible for this preservedresponse. However, eliminating AT1B receptors genetically fromthe SMKO background did not diminish their acute vasocon-strictor response to Ang II (Figure 6A).

Ang II can also cause vasoconstriction indirectly, throughstimulation of AT1 receptors in the central nervous system,

Figure 8. Increased Baseline RBF and Attenuated Renal Vasoconstrictor Responses toAng II in SMKOMice. RBF was measured in anesthetized mice using an ultrasound flowprobe over the renal artery. (A) Peak RBF during systole was significantly increased inSMKO mice (n=5) compared with control mice (n=4) (SMKO, 2.160.3 versus control,0.8360.1 ml/min; #P=0.01 unpaired t test). Mean RBF also tended to be higher inSMKO mice than control mice, but this did not reach statistical significance (SMKO,0.8860.1 versus control, 0.4860.1 ml/min; ^P=0.06, NS, unpaired t test). (B) Re-duction in mean RBF was compared with baseline values. RBF was significantly di-minished after Ang II administration in control mice; however, no change in RBF wasobserved in SMKO mice. Control mice had a decrease in RBF by –42.1%615% at 0.03mg/kg Ang II, –64.7%66% at 0.1 mg/kg Ang II, and –87.8%62% at 0.3 mg/kg Ang IIfrom baseline. However, SMKO mice had only minimal changes in RBF by –2.2%65%at 0.03 mg/kg Ang II, –7.1%65% at 0.1 mg/kg Ang II, and –7.5%69% at 0.3 mg/kg AngII from baseline. *P=0.02, **P=0.001, ***P=0.001 versus control at each dose, un-paired t test. Error bars represent SEM.

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triggering sympathetic outflow and peripheral vasoconstric-tion.40 For example, intraventricular injection of Ang II causesacute vasoconstriction, and this is dependent on central AT1Areceptors.19,20 In addition, brain selective AT1A receptor over-expression causes enhanced systemic pressor response to centralAng II administration.41 Therefore, we tested whether the pre-served peripheral vasoconstrictor response we observed in theSMKOsmightbe related to accentuationof these central responses.Indeed, we found that the residual vasoconstrictor responsein SMKOs was completely extinguished by prior administrationof the a-sympathetic blocker phentolamine, confirming thatexaggerated sympathetic tone maintained a relatively normal sys-temic vasoconstrictor response in the SMKOs. Furthermore, uri-nary excretion of norepinephrine was increased in the SMKOs,indicating a general increase in sympathetic activity in these ani-mals. An increase in urinary norepinephrine content at baselinehas previously been reported in global AT1A receptor knockoutmice.42 Although the increase in urinary norepinephrine contentat baseline in SMKOs may be a compensatory response to theirlower BPs, the exaggerated response during chronic Ang II infu-sion could reflect a generalized alteration in central responsivenessto Ang II. Whatever the mechanism, this heightened sympatheticactivity is not sufficient to return baseline BP to normal or restorethe full hypertensive response to Ang II. Moreover, this enhancedsympathetic response did not augment Ang II–dependent vaso-constriction in the renal circulation, which was virtually absent inthe SMKOs (Figure 8B). The reason for the differential response inrenal versus systemic vasculature is not clear, but these findingsindicate that direct actions of AT1 receptors in VSMCs primarilycontrol Ang II–dependent vasoconstriction in the kidney.

Our studies have confirmed the importance of vascularresponsestoAngIIinBPregulationandhypertensionpathogenesis.These vascular actions also play an important role in maintaininghomeostasis during variation in sodium intake and may affectpropensity for salt sensitivity. Inparticular,ourmodel illustrates thepowerful capacity for AT1 receptor actions in the renal circulationto impair natriuresis and thereby promote hypertension. Relief ofrenal vasoconstriction despite the presence of high levels of circu-lating Ang II promotes powerful resistance to hypertension.

CONCISE METHODS

Details of MiceA mouse line with a conditional Agtr1aflox allele was generated using

homologous recombination in embryonic stem cells as described pre-

viously.7,43 To delete the AT1A receptor fromVSMCs, we crossed inbred

C57BL/6 transgenicmouse lines expressingCre recombinase specifically

in smooth muscle cells under the control of the Sm22a promoter

(KISm22a-Cre, strain name: B6.129S6-Taglntm2(cre)Yec/J, stock number:

006878; The Jackson Laboratory, Bar Harbor, ME) or the inducible

SMMHC-ERT2 promoter44 with C57BL/6-Agtr1aflox/flox mice. The

Sm22a promoter drives expression of Sm22a protein, one of the earliest

markers of a differentiated smooth muscle cells.45 Cell-specific expres-

sion of Cre recombinase in smooth muscle cell lineages driven by this

promoter has been well documented.46,47 Therefore, we generated two

separatemousemodels, KISm22a -Cre+Agtr1aflox/flox (SMKO)mice and

inducible SMMHC-ERT2-Cre+Agtr1aflox/flox (I-SMKO), both on inbred

C57BL/6 background. For the acute hemodynamic experiments, we

bred the I-SMKO mouse line8 with the AT1B global KO mouse line48

(C57BL/6-Agtr1b2/2) for two successive generations to generate

C57BL/6-SMMHC-ERT2 Cre+ Agtr1aflox/flox Agtr1b2/2 mice (I-SMKO-

1B2/2).Cre recombinase activitywas induced in I-SMKOand I-SMKO-

1B2/2 mice by daily intraperitoneal injection of 2 mg of tamoxifen

(Sigma-Aldrich, St. Louis, MO) mixed in corn oil for 5 successive

days. Experiments were performed at least 1 week after completion of

tamoxifen injections. All experiments were performed on 8- to 12-

week-old male mice. Only male mice are used in these studies because

they are in direct follow-up of past work completed in our laboratory

using kidney cross-transplantation,4 which can only be performed in

malemice because of technical constraints. Therefore, these results may

not be generalizable to female mice.

All experimental mice were bred in an Association for Assessment

andAccreditationof LaboratoryAnimalCare international accredited

animal facility at the Duke University and Durham Veterans Affairs

medical centers underNational Institutes ofHealth guidelines for care

and use of laboratory animals and housed with free access to standard

rodent food and water unless otherwise specified.

BP Measurements in Conscious MiceBPs were measured in conscious, unrestrained 8- to 12-week-old male

SMKO and control mice using radiotelemetry (PA-C10) as described

previously.5Arterial BPwas collected, stored, andanalyzedusingDataquest

A.R.T software (version 4.0; Data Sciences International, St. Paul, MN).

Mice were allowed to recover for 7 days after telemetry implantation to re-

established normal circadian rhythms.49 After recovery, telemetry data

were collected continuously with sampling every 5 minutes for 10-second

intervals. Baseline measurements were recorded for 7 consecutive days

while mice ingested a normal sodium diet (0.4% Na+). On day 8, mice

started ingesting a low-sodium diet (,0.02%Na+; Harlan Teklad, Indian-

apolis, IN) for 1week and then high-sodiumdiet (6%Na+;Harlan Teklad)

for 1 week. At the end of the salt challenge and while on a normal sodium

diet, anosmoticminipump (Alzet,Cupertino,CA) infusingAng II (Sigma-

Aldrich) at a rate of 400 or 1000 ng/kg per min were implanted subcuta-

neously, and BP measurements continued for 7 and 30 days, respectively.

Assessment of Acute Vasoconstrictor ResponsesWe examined acute pressor responses to Ang II, epinephrine, and

phentolamine (all fromSigma-Aldrich, reconstituted in sterile saline) in

mice anesthetized with 2% isoflurane as described previously.13 A cath-

eter (pulled PE-50) was inserted into the left jugular vein for the ad-

ministration of basal fluids and vasoconstrictors. A second catheter

(Mikro-Tip 1.4F;Millar, Houston, TX)was placed into the right carotid

artery. Intra-arterial BP was recorded continuously through the carotid

catheter using the PowerLab data acquisition system and LabChart soft-

ware (ADInstruments, Colorado Springs, CO). At 5-minutes intervals,

increasing doses (0.1, 1, and 10 mg/kg) of Ang II, 10 mg/kg of epineph-

rine, and 400 mg/kg of phentolamine were injected intravenously into

the internal jugular vein at a volume of 1 ml/g body wt (25–30 ml total

volume) followed immediately by a 30 ml bolus of saline. Before the

8 Journal of the American Society of Nephrology J Am Soc Nephrol 26: ccc–ccc, 2015

BASIC RESEARCH www.jasn.org

injection of vasoactive agents, each mouse received an equivalent vol-

ume (55–60 ml, 2 ul/g body wt of saline as a vehicle control. Intra-

arterial pressures were continuously monitored.

Assessment of RBFWe examined acute RBF responses to Ang II in mice anesthetized with

2%isoflurane aspreviouslydescribed.13Acatheter (pulleddownPE-50)

was inserted into the left jugular vein for the administration of basal

fluids and vasoconstrictors. A small incisionwasmade on the rightflank

to expose the kidney and a noncannulating ultrasonic flowmeter inter-

faced with a 5 mm V-shaped probe was placed around the right renal

artery (MA0.5PSB and TS420 Flowmeter; Transonic Systems, Ithaca,

NY). Mice were allowed to stabilize for 30 minutes before measure-

ments were started. Vascular reactivity of the renal circulation was as-

sessed by injecting increasing doses of Ang II (0.03, 0.1, and 0.3 mg/kg)

as previously described into the internal jugular vein while RBF was

continuously monitored. Data are expressed as percent change in mean

RBF from baseline value determined just before injection.

Metabolic Balance StudiesAseparate groupofmicewere individually placed in speciallydesigned

metabolic cages (Hatteras Instruments, Cary, NC)5,50 and fed 10 g/d

gel diet containing nutrients, water, and 0.1% w/w sodium (Nutra-

Gel; Bio-Serv, Frenchtown, NJ). After 5 days of baseline collections,

mice were implanted with osmotic minipumps infusing Ang II (1000

ng/kg per min) and returned to metabolic cages for 5 more days.

Urinary sodium content was measured daily and was determined

using an IL943 Automatic Flame photometer (Instrumentation Lab-

oratory, Lexington, MA). Sodium balance was determined by sub-

tracting the total amount of sodium ingested daily by the total

amount of sodium excreted in the urine over a 24-hour period.

Determination of Urinary Norepinephrine ContentNorepinephrinecontentwasextracted fromtheurineandquantitatedby

enzyme immunoassay (Norepinephrine ELISA, 17-NORHU-E01.1;

ALPCO Diagnostics, Salem, NH) according to the manufacturer’s in-

structions.

Statistical AnalysesThevalues foreachparameterwithinagroupareexpressedasmean6SEM.

For comparisons between groups with normally distributed data, statisti-

cal significance was assessed using a an unpaired t test. For comparisons

within groups statistical significance was assessed using a paired t test. For

comparisons between groups with non-normally distributed data, the

Mann–Whitney U test was used. A P value,0.05 was considered statis-

tically significant unless otherwise indicated. Normality was determined

using the Shapiro–Wilk test. Aone-wayANOVAwith Bonferronimultiple

comparison test was used when multiple interventions were tested.

ACKNOWLEDGMENTS

This work was supported by the Edna and Fred L. Mandel Center for

Hypertension and Atherosclerosis Research, National Institutes of

Health grants HL056122 and P30-DK096493 (to T.M.C.); a Career

Development Award IK2BX002240 from the Biomedical Laboratory

Research and Development Service, Department of Veterans Affairs

Office of Research and Development (to M.A.S.); a Scientist De-

velopment Award 13SDG13990017 from the American Heart Asso-

ciation (to M.A.S.); a Chair’s Research Award from the Duke

Department of Medicine (to M.A.S.); and a grant from the Institute

for Medical Research at the Durham VAMC (to M.A.S.).

SMMHC-ERT2-Cre mice were provided by Prof. Dr. Stefan Of-

fermanns, Department of Pharmacology, Max-Planck-Institute for

Heart and Lung Research, Ludwigstr. Bad Nauheim, Germany.

The views expressed in this article are those of the authors and do

not necessarily represent the policy or position of the United States

Department of Veteran Affairs or the United States government.

DISCLOSURESNone.

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