Hypertension in mice lacking the CXCR3 chemokine receptor

Am J Physiol Renal Physiol 296: F780–F789, 2009. First published January 7, 2009; doi:10.1152/ajprenal.90444.2008.

Hypertension in mice lacking the CXCR3 chemokine receptor Hsiang-Hao Hsu,1,4* Kerstin Duning,1* Hans Henning Meyer,1 Miriam Sto¨lting,1 Thomas Weide,1 Stefanie Kreusser,1 Truc van Le,1 Craig Gerard,2 Ralph Telgmann,3 Stefan-Martin Brand-Herrmann,3 Hermann Pavensta¨dt,1* and Martin Johannes Bek1* 1

Department of Medicine, Division of Nephrology and General Medicine, University Clinic of Mu¨nster, Mu¨nster; Leibniz-Institute for Arteriosclerosis Research, Department of Molecular Genetics of Cardiovascular Disease, Mu¨nster, Germany; 2Department of Medicine, Pulmonary Division, Harvard Medical School, Boston, Massachusetts; and 4Kidney Institute and Department of Nephrology, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan 3

Submitted 30 July 2008; accepted in final form 5 January 2009

Hsu H-H, Duning K, Meyer HH, Sto¨lting M, Weide T, Kreusser S, van Le T, Gerard C, Telgmann R, Brand-Herrmann S-M, Pavensta¨dt H, Bek MJ. Hypertension in mice lacking the CXCR3 chemokine receptor. Am J Physiol Renal Physiol 296: F780–F789, 2009. First published January 7, 2009; doi:10.1152/ajprenal.90444.2008.—The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3⫺/⫺ mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3⫺/⫺ mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3⫺/⫺ mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased AT1R expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression. angiotensin II type 1 receptor; acetylcholine receptor; CXCR3 knockout mice; EMSA; transcription factors; vascular contractility

3 (CXCR3), the receptor for chemokines such as CXCL9/MIG, CXCL10/IP-10, and CXCL11/ITAC, is not only important in lymphocyte recruitment to inflammatory sites but is also involved in T cell activation (46). Additionally, IP-10 has been demonstrated to inhibit proliferation of human endothelial cells (28) and bone marrow-derived hematopoietic progenitors (40). CXCR3-deficient mice displayed resistance to the development of acute allograft rejection following cardiac transplantation, probably due to reduced intragraft accumulation of activated T cells (13), and CXCR3

CXC CHEMOKINE RECEPTOR

* H.-H. Hsu, K. Duning, H. Pavensta¨dt, and M. J. Bek contributed equally to this work. Address for reprint requests and other correspondence: H. Pavensta¨dt, Dept. of Internal Medicine, Albert-Schweitzer-Str. 33, D-48149 Mu¨nster, Germany (e-mail: [email protected]). F780

has been suggested to support renal microvascular damage (35). Moreover, CXCR3 has been linked to the pathogenesis of atherosclerosis (14), and recent studies demonstrated that an antagonist of CXCR3 can attenuate atherosclerotic lesion formation by blocking direct migration of CXCR3⫹ effector cells from the circulation into the atherosclerotic plaques (51). However, serious adverse effects of CXCR3 deletion have been also identified in models of fibrotic diseases: CXCR3⫺/⫺ mice exhibited increased mortality with progressive interstitial pulmonary fibrosis, which occurred without increased inflammatory cell recruitment (19), and CXCR3 deletion has been shown to promote progressive renal fibrosis, suggesting that activation of CXCR3 may be useful in antifibrotic therapeutic strategies (32). Furthermore, diverse phenotypes have been observed in CXCR3⫺/⫺ mice following several inflammatory and autoimmune challenges, and previous studies proposed that the relationship between CXCR3 expression and recruitment of activated T cells is not a universal feature of the phenotype of CXCR3-null mice (27). Essential hypertension is a major risk factor for developing cardiovascular disease (36). The pathogenesis of hypertension is multifactorial and influenced by both genetic and environmental factors (26). Numerous genes are involved in blood pressure (BP) regulation in mammalians and these genes affect renal hemodynamics, ion and water transport, and regulation of hormones and humeral agents, such as aldosterone, catecholamines, endothelin, prolactin, proopiomelanocortin, renin, and vasopressin (2, 24). Within the vessel, CXCR3 receptors are expressed in endothelial cells (10) as well as in vascular smooth muscle cells (14), which are important in the dynamic regulation of the circulation and have a crucial role in the pathogenesis of hypertension (43). Little is known regarding the basic in vivo functions of the CXCR3 receptor in cardiovascular physiology and its role in regulating BP. This study investigated whether the CXCR3 receptor is involved in BP regulation and vascular reactivity. This report describes that the loss of CXCR3 promotes hypertension, with a concomitant increased expression of angiotensin II type 1 receptor (AT1R) and decreased expression of M3 acetylcholine (ACh) receptor, suggesting that both increased vasoconstriction and decreased vasodilatation contribute to hypertension in CXCR3⫺/⫺ mice. Additionally, the transcription factor SP-1, which expression is upregulated in CXCR3⫺/⫺ mice, bound to the murine AT1R promoter reThe costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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CXCR3 RECEPTOR AND HYPERTENSION

gion. We suggest that an imbalance of SP-1/EGR-1 complex formation is involved in the increased AT1R expression and hypertension in CXCR3⫺/⫺ mice. METHODS

Generation and Genotyping of CXCR3 Chemokine Receptor Mutant Mice Generation of CXCR3 receptor knockout mice was performed as described recently (12). The original hybrid strain (C57BL/6) carrying a mutation of the CXCR3 receptor was first generated and backcrossed to BALB/c mice. Heterozygous (CXCR3⫹/⫺) mice were mated to yield CXCR3⫹/⫹ and CXCR3⫺/⫺ littermates. CXCR3⫺/⫺ mice were then backcrossed with BALB/c mice to obtain ⬎14th generation transgenic mice. The experiments exclusively used ⬎14th generation transgenic mice. All animal experiments were performed according to the guidelines of the National Institutes of Health as well as the German law for the welfare of animals and were approved by local authorities. Genotyping was performed using primers recognizing genomic DNA sequences upstream, inside, and downstream of the inserted PGK neomycin cassette. The following primers were used: CXCR3 forward: 5⬘-GTGCTAGATGCCTCGGACTTTG-3⬘; CXCR3 reverse: 5⬘-CTCCCACAAAGGCATAGAGC-3⬘; CXCR3 neomycin forward: 5⬘-CAAGATGGATTGCACGCAGG-3⬘. BP Measurement Mice were anesthetized with intraperitoneal ketamine and tracheotomized as described previously (15, 25). Catheters were inserted into the internal jugular vein and the carotid artery for fluid administration and BP monitoring (World Precision Instruments). BP was recorded following 1 h of equilibration (0.9% NaCl, 1% of body wt) as described recently (4). Conscious BP was measured using TA11PA-C10 transmitters (Data Sciences International) implanted into the carotid artery. Finally, BP was measured via telemetry 7 days after catheter placement in freely moving unanesthetized mice. Saline Loading Study Acute sodium load. After a 60-min stabilization period after the catheter insertion and a baseline 60-min period for BP measurement, a normal saline load equivalent to 5% body wt was infused intravenously for 30 min. Urine was collected during saline loading via suprapubic cystostomy; three more urine collection periods of 60 min each were obtained after loading. Blood (50 ␮l) was obtained from the femoral artery before the load and at the end of the last urine collection. For chronic studies, CXCR3⫹/⫹ and CXCR3⫺/⫺ mice were maintained on a baseline regular pelleted diet (0.4% NaCl) 1 wk before and after implantation of the BP radio transmitter device, and provided free access to water and food. Following a 24-h measurement period, the mice were switched to a high-sodium regular pelleted diet (0.8% NaCl) for a further week with an additional measurement period of 24 h at the end of the second week. All animals received the same weight of food and were given free access to water. The weights of the mice remained constant during the experimental period. At the end of the second experimental period, mice were killed. Vascular Tension Measurements Mesentery arteries of CXCR3⫹/⫹ and CXCR3⫺/⫺ were prepared under a dissecting microscope and placed in oxygenized cold Krebs Ringer bicarbonate solution (KRB) containing (in mmol/l) 119.1 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 2.5 CaCl2, and 5.5 glucose. In high-K⫹ solution (125 mmol/l K⫹), NaCl was replaced by an equimolar concentration of KCl. All buffer ingredients, as well as the test agents, were obtained from Sigma. Furthermore, vascular preparations were mounted on two stainless steel wires in an isometric myograph (Myograph model 410A, J.P. Trading); one wire AJP-Renal Physiol • VOL

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connected to a force transducer (Kistler Morse DSC6) was used to record isometric force development, and with the other wire being connected to a displacement device, as described previously (45). Arteries were stretched to their optimal luminal diameter using an active length tension protocol with 125 mmol/l K⫹ as activating stimulus (6). The optimal luminal diameter was established as the maximal force created by depolarization of the vascular smooth muscle cells in response to 125 mmol/l K⫹. After establishing the optimal lumen diameter (as a standardization procedure of the individual vascular preparations), the vascular preparations were permitted to equilibrate in fresh 37°C oxygenized KRB solution for 15 min and stimulated using the following substances: phenylephrine, vasopressin, endothelin I, ANG II, ACh, atropine, himbacine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), pirenzepine, and p-fluoro-hexahydro-sila-difenidol. Experimental concentrations were set to maintain selectivity of the antagonists according to published concentrations (16). Two-Dimensional and Conventional Western Blot Experiments Mouse mesentery artery and kidney samples (pooled from 6 mice for each experiment) were homogenized in 175 ␮l of lysis buffer (7 mol/l urea, 2 mol/l thiourea, 4% CHAPS, and 30 mmol/l Tris) with a microtissue grinder, followed by three rounds of sonication in an ice bath sonicator. Lysates were kept on ice for 30 min and centrifuged for 1 h at 16,000 g. Protein concentrations of supernatants were determined using Advanced Protein Assay agents (Cytoskeleton) in triplicate and the results were averaged. Equal amounts of protein (350 – 400 ␮g) were loaded to sample cups in the focusing tray. Two-dimensional (2D) electrophoresis of proteins was performed as described in the ReadyStrip IPG Strip Instruction Manual (Bio-Rad). IPG strips (pH 3-10 nonlinear) were rehydrated by adding 215 ␮l of buffer containing 7 mol/l urea, 2 mol/l thiourea, 4% CHAPS, 100 mmol/l DTT (dithiothreitol), and 8% IPG buffer (Bio-Lyte 3-10, Bio-Rad) to each channel in the rehydration tray overnight. First, dimension separations were performed by the PROTEAN IEF cell (Bio-Rad) using the cup loading method under the following focusing conditions: 1,000 V for 90 min (slow ramp), 3,500 V for 90 min (slow ramp), and 7,000 V for 120 min (rapid ramp). For the second-dimension electrophoresis, the focused IPG strips were equilibrated in a reduction buffer (37.5 mmol/l Tris 䡠 HCl, pH 8.8, 6 mol/l urea, 20% glycerol, 2% SDS, and 1% DTT) at 25°C for 15 min. The strip was then equilibrated in an alkylation buffer (37.5 mmol/l Tris 䡠 HCl, pH 8.8, 6 mol/l urea, 20% glycerol, 2% SDS, 0.002% bromophenol blue, and 2.5% iodoacetamide) at 25°C for 15 min. Proteins were separated by 12% SDS-PAGE, transblotted onto a nitrocellulose membrane, and probed with primary antibodies (anti-AT1R antibody, sc-1173, Santa Cruz Biotechnology, 1:1,000) followed by secondary antibodies. The specificity of the AT1R antibody was confirmed by lysates from a murine podocyte cell line stably transfected with Flag-tagged human AT1R using 2D immunoblotting and immunoprecipitation methods (15a). For M3 ACh receptor detection, protein lysates were separated via conventional 10% SDS-PAGE, transblotted onto a nitrocellulose membrane, and probed with primary antibodies (anti-M3 ACh receptor antibody, sc-7474, Santa Cruz Biotechnology, 1:500). Blocking peptide (sc-7474p) was employed to confirm the specificity of the antibody. Chemiluminescent signals of AT1R, M3 ACh receptor, and ␤-actin (sc-1615, Santa Cruz Biotechnology) were detected with a LumiImager (Roche) and X-ray film developer and quantified using the LumiAnalyst program (Roche). In conventional Western blot experiments, antibodies for detection of EGR-1 protein (Santa Cruz Biotechnology, sc-110) and SP-1 protein (Millipore) in whole kidney lysate were used according to the manufacturer’s instructions.

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EMSA Experiments Generation of recombinant plasmids. DNA fragments encoding both full-length and truncated versions of murine EGR-1 and SP-1 protein were amplified from a cDNA bank obtained from an immorto mouse proximal tubule (M-PT) cell line generated from an “immortomouse” (H-2Kb-tsA58 transgenic mouse) (18). Murine EGR-1 fulllength protein (NM_007913; aa: 1-533), a truncated version of EGR-1 protein containing the zinc-finger-DNS-binding domains (DBD; EGR-1-DBD; aa: 327-418), SP-1 full-length protein (AF062566; aa: 1-781), and a SP-1 protein mutant comprising the DBD (SP-1-DBD; aa: 618-711) were amplified by PCR. Resulting PCR fragments were cloned blunt end into pCR4blunt-TOPO using the TOPO-cloning system (Invitrogen), sequenced, and subcloned into EcoRI/NotI sites of the pGEX-4T-1 expression vector (Amersham Bioscience/GE Healthcare).

formation was performed in a total reaction volume of 20- to 28-␮l interaction buffer (40 mmol/l HEPES/KOH, pH 7.9), 240 mmol/l KCl, 20 mmol/l MgCl2, 2 mmol/l Spermidin, 6% Ficoll using 25 ng preshared poly dI-dC (Amersham Bioscience) as a nonspecific competitor, and 20 mmol/l Betain (Sigma). In some samples, a 100- to 200-fold molar excess of unlabeled oligonucleotides was added as specific competitors before the incubation with the labeled AT1R promoter region and incubated 15 min on ice. Following the addition of biotin-labeled oligonucleotide, samples were incubated for a further 30 min at room temperature. DNA/protein complexes were separated on nondenaturating 6% polyacrylamide gels in 0.5⫻ TBE (45 mmol/l Tris, 32.3 mmol/l boric acid, 1.25 mmol/l EDTA, pH 8.3) at 100 V for

Expression and Purification of Recombinant Proteins To produce murine GST-EGR-1 or GST-SP-1 fusion proteins, Escherichia coli BL21 cells were transformed using the different pGEX constructs. Briefly, an overnight culture of E. coli transformed with the expression vectors was diluted 1:20 with LB medium containing 1% glucose, incubated at 37°C, and agitated at 220 rpm until an OD600 of 0.8 was reached. Expression was induced by adding 1 mmol/l isopropyl-1-thio-␤-D-galactopyranoside (IPTG), and suspension was incubated at 25°C for a further 2.5 h. Subsequently, cells were harvested, resuspended in phosphate-buffered saline containing protease inhibitor mix (Complete, Roche Molecular Biochemicals), and sonicated on ice. Triton X-100 (1%) and 1 mg/ml lysozyme were added, the suspension was mixed and incubated on ice for another 30 min, and cellular debris was pelleted by centrifugation (16,000 g, 20 min, 4°C). Recombinant proteins were isolated from bacterial lysates using glutathione-sepharose beads (Amersham Bioscience/GE Healthcare) according to the manufacturer’s instructions. Proteins were eluted (50 mmol/l Tris䡠HCl, pH 8.0, containing 10 mmol/l glutathione), dialyzed against PBS, and stored in aliquots at ⫺80°C until further use. Recombinant human SP-1 protein (ALX-201-106-R050) and EGR-1 protein (ALX-201-074) were obtained from Biochemicals. EMSA The double-strand mouse AT1R oligonucleotide corresponding to the nucleotide sequence ⫺230/⫺51 in the murine AT1R promoter (NM_177322.2), which contains a putative SP-1/EGR-1 overlapping binding sites, was identified using the Alibaba 2.1 net-based search tool (www.gene-regulation.com). The oligonucleotide was amplified by PCR using Go-Taq DNA-polymerase (Promega) from genomic mouse DNA using the following oligonucleotide primers: forward: 5⬘-CAGGACCAGCTGAGCTTGGATC-3⬘; reverse: 5⬘-GGGGGACGTTGCTACTCCGCAGGTTCC-3⬘. AT1R sequence was confirmed by DNA sequencing using internal primers (forward: 5⬘-CACATCGGTGACTGGCAGCAGGG-3⬘; reverse: 5⬘-CCTAAGGCTTTGCACTCCTCCCTCG-3⬘). The resulting 150-bp PCR fragment was purified using the High Pure PCR Product Purification Kit (Roche Molecular Biochemical) as directed by the manufacturer. AT1R (5 pmol) oligonucleotide was 3⬘-biotinylated using the 3⬘-end DNA labeling kit (Perbio) as described by the manufacturer. The labeling process was stopped by adding 2.5 ␮l 0.2 mol/l EDTA (pH 8.0) to a 50-␮l reaction volume, and the labeled probe was isolated via phase-separation after adding 50 ␮l of chloroform:isoamyl alcohol (24:1), and subsequently purified using a High Pure PCR Product Purification Kit (Roche Molecular Biochemical). For competition experiments, the unlabeled AT1R promoter fragment was used as a specific unlabeled competitor. Additionally, SP-1 and EGR-1 consensus oligonucleotides were employed as biotin-labeled positive controls, and specific unlabeled competitors were obtained from Panomics (SP-1, AY1043P; EGR-1 AY1303P) as described recently (22, 23). DNA/protein complex AJP-Renal Physiol • VOL

Fig. 1. Disruption of CXC chemokine receptor 3 (CXCR3) increases blood pressure. A: mean arterial pressure (MAP) in anesthetized, catheterized CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. Blood pressures were significantly higher in CXCR3⫺/⫺ mice before and after acute sodium load. B: MAP in conscious freely moving mice on a low- or a high-sodium diet. Blood pressures in CXCR3⫺/⫺ mice were considerably higher on a low-sodium diet, and further increased in response to a high-sodium diet, a phenomenon observed in CXCR3⫹/⫹ mice (*P ⬍ 0.05, CXCR3⫺/⫺ vs. CXCR3⫹/⫹ mice, t-test; **P ⬍ 0.01; °P ⬍ 0.05, CXCR3⫺/⫺ mice, high-sodium diet vs. CXCR3⫺/⫺ mice, low-sodium diet, t-test).

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1.5 h at room temperature. Following electrophoresis, gels were electrotransferred onto PVDF membranes (0.5% TBE, 100 V, 60 min). After the blotting procedure, membranes and DNA/protein complexes were cross-linked using 10-min UV light incubation and incubated for 15 min at room temperature in prewarmed (50°C) blocking solution (0.5% blocking reagent, Roche Molecular Biochemical), 5 mmol/l maleic acid, 7.5 mmol/l NaCl, pH 7.5. Subsequently, bands were visualized by streptavidin-horseradish peroxidase reaction using the Chemiluminescent Nucleic Acid Detection Module Kit (Perbio).

either a low (0.4% NaCl)- or high (0.8% NaCl)-sodium diet. MAP was significantly raised in CXCR3⫺/⫺, including those on a low-sodium (126 ⫾ 2 mmHg, n ⫽ 3) and a high-sodium diet (134 ⫾ 3 mmHg, n ⫽ 3), compared with CXCR3⫹/⫹ mice on a low-sodium (107 ⫾ 3 mmHg, n ⫽ 3) and a high-sodium (109 ⫾ 3 mmHg, n ⫽ 3) diet. The high-sodium diet markedly increased MAP in CXCR3⫺/⫺ mice by ⬃8 mmHg, compared with CXCR3⫺/⫺ mice on a low-sodium diet, but did not change MAP in CXCR3⫹/⫹ mice (Fig. 1B).

Statistical Analysis

Vascular Response to Vasoconstrictors in CXCR3⫺/⫺ and CXCR3⫹/⫹ Mice

Data are expressed as means ⫾ SE and were analyzed by ANOVA for repeated measures when comparing within groups and one-way ANOVA when comparing among groups. Student’s t-test was used for a two-group comparison and Scheffe´’s test for multiple comparisons. P ⬍ 0.05 was considered statistically significant. RESULTS

BP Measurements in CXCR3⫺/⫺ and CXCR3⫹/⫹ Mice To test the hypothesis that CXCR3 knockout mice are prone to arterial hypertension, baseline BPs were first measured in anesthetized, catheterized CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. Mean arterial pressure (MAP) in CXCR3⫺/⫺ mice (76 ⫾ 3 mmHg, n ⫽ 8) was considerably raised compared with CXCR3⫹/⫹ mice (70 ⫾ 2 mmHg, n ⫽ 15). An acute sodium load (5% of body wt) increased BPs in CXCR3⫺/⫺ mice (89 ⫾ 3 mmHg, n ⫽ 8) compared with CXCR3⫹/⫹ mice (70 ⫾ 3 mmHg; Fig. 1A). To confirm these data, BPs were measured in conscious CXCR3⫹/⫹ and CXCR3⫺/⫺ mice on

To further characterize arterial hypertension in CXCR3⫺/⫺ mice, isolated resistance vessels (mesenteric arteries) were stimulated using the AT1R agonist ANG II (10⫺12–10⫺6 mol/l; Fig. 2A), the nonselective ␣-adrenergic receptor agonist phenylephrine (10⫺8–10⫺4 mol/l; Fig. 2B), the V1 receptor agonist vasopressin (10⫺10–10⫺6 mol/l; Fig. 2C), and the nonadrenergic ETA/ETB receptor agonist endothelin I (10⫺12–10⫺7 mol/l; Fig. 2D). Vasoconstriction in response to ANG II was significantly increased in CXCR3⫺/⫺ mice, reaching statistical significance at concentrations of 10⫺9–10⫺6 mol/l (Fig. 2A). Responses of all other vasoconstrictors were not significantly changed between CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. Vascular Response to Vasodilatators in CXCR3⫺/⫺ and CXCR3⫹/⫹ Mice To test the hypothesis that CXCR3 receptors influence the vasodilatatory capabilities of resistance vessels, vasodilata-

Fig. 2. Vasoconstriction in mesenteric arteries of CXCR3⫺/⫺ and CXCR3⫹/⫹ mice in response to angiotensin II (A), phenylephrine (B), vasopressin (C), and endothelin I (D). Vasoconstriction in response to angiotensin II was markedly increased in CXCR3⫺/⫺ mice, a difference that did not occur in response to other vasoconstrictors (*P ⬍ 0.05, CXCR3⫺/⫺ mice vs. CXCR3⫹/⫹ mice, ANOVA, Scheffe´’s test).

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tion in response to ACh (10⫺11 ⫺ 5 ⫻ 10⫺4 mol/l; Fig. 3A) was tested. Preconstriction of mesenteric arteries was induced by stimulation with 10⫺6 mol/l phenylephrine (EC50 value in vasoconstriction experiments), because vasoconstructive response to phenylephrine did not differ between CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. Phenylephrine has been described in the previous literature on this kind of experiments (45). Subsequent increases in ACh concentrations were added to the perfusion chamber. Vasodilatation in response to ACh was significantly reduced in CXCR3⫺/⫺ mice and reached statistical significance at concentrations of 10⫺7 ⫺ 5 ⫻ 10⫺4 mol/l (Fig. 3A). To better understand the type of muscarinic ACh receptor involved in the vasodilatatory response, different muscarinic receptor antagonists were used to antagonize the effect of ACh (Fig. 3, B and C). Vasodilatation in mesenteric arteries in response to ACh (10⫺5 mol/l) could be antagonized by the nonselective ACh receptor antagonist atropine (10⫺6 mol/l) and the relatively selective M3 receptor antagonist 4-DAMP (10⫺6 mol/l) but not by the relatively selective antagonists for the M1 (10⫺5 mol/l pirenzepine), M2/M4 (10⫺6 mol/l himbacine), and M4 (10⫺8 mol/l pfluoro-hexahydro-sila-difenidol) receptors (Fig. 3, B and C). AT1 Receptor and M3 ACh Receptor Expression in CXCR3⫹/⫹ and CXCR3⫺/⫺ Mice To further test whether the differential response to ANG II and ACh in mesenteric arteries of CXCR3⫹/⫹ and CXCR3⫺/⫺ mice resulted from differential receptor expression levels or different second messenger mechanisms, expression of AT1Rs and M3 ACh receptors in mesenteric arteries was analyzed (Fig. 4, A and B). 2D Western blotting was used to detect AT1Rs, which are slightly but significantly expressed in mouse mesenteric arteries. The data show that expression of AT1R is significantly increased in mesenteric arteries of CXCR3⫺/⫺ mice (1.84 ⫾ 0.13-fold upregulated, n ⫽ 4, P ⬍ 0.05, t-test) compared with CXCR3⫹/⫹ mice (Fig. 4A). The expression of the M3 ACh receptor is considerably reduced in CXCR3⫺/⫺ mice compared with CXCR3⫹/⫹ mice (Fig. 4B, right; 0.18 ⫾ 0.09-fold downregulated, n ⫽ 3, P ⬍ 0.05, t-test). To confirm these data in another organ relevant for BP control, whole kidney protein extracts from CXCR3⫺/⫺ and CXCR3⫹/⫹ mice were tested. Our data show that in CXCR3⫺/⫺ mice AT1R expression in whole kidney protein extracts was significantly increased compared with CXCR3⫹/⫹ mice (1.24 ⫾ 0.04, n ⫽ 3, P ⬍ 0.05, t-test; Fig. 4C).

Fig. 3. A: vasodilatation in mesenteric arteries of CXCR3⫺/⫺ and CXCR3⫹/⫹ mice in response to acetylcholine (ACh). Vasodilatation in response to ACh (10⫺11 ⫺ 5 ⫻ 10⫺4 mol/l) was significantly reduced in CXCR3⫺/⫺ mice (*P ⬍ 0.05, CXCR3⫺/⫺ mice vs. CXCR3⫹/⫹ mice, ANOVA, t-test). B and C: vasodilatation in mesenteric arteries in response to ACh (10⫺5 mol/l) following preincubation with different muscarinic receptor antagonists. The nonselective muscarinic receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP (10⫺6 mol/l), but not the selective antagonists for M1 (10⫺5 mol/l pirenzepine), M2/M4 (10⫺6 mol/l himbacine), and M4 (10⫺8 mol/l p-fluoro-hexahydro-sila-difenidol) receptors, antagonized the effect of ACh in CXCR3⫹/⫹ (B) and CXCR3⫺/⫺ mice (C; *P ⬍ 0.05, ACh vs. ACh ⫹ atropine, ACh ⫹ 4-DAMP, ANOVA, Scheffe´’s test). AJP-Renal Physiol • VOL

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Fig. 4. Expression of ANG II type 1 receptor (AT1R) and ACh M3 receptor (M3 AChR) in CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. A: equal amounts of protein from mouse mesenteric artery lysates were analyzed by 2-dimensional Western blotting (left plot). Quantification of the signal intensities of spots representing AT1R (calibrated by actin signal intensity) revealed that AT1R expression was significantly upregulated in CXCR3⫺/⫺ mice (increased 1.84 ⫾ 0.13fold, right plot, n ⫽ 4, P ⬍ 0.05). B, left plot: representative immunoblotting exhibited reduced protein M3 AChR expression in CXCR3⫺/⫺ mice (lanes 1 and 2). Mouse brain extract was loaded as a positive control (lane 3) and the specificity of the M3 AchR antibody was confirmed using a blocking peptide (lane 4). Quantifying signal intensities indicated that M3 AchR expression was significantly downregulated in CXCR3⫺/⫺ mice (decreased 82 ⫾ 9% than CXCR3⫹/⫹ mice, right plot, n ⫽ 3, P ⬍ 0.05). C: expression of AT1R in whole kidney protein extracts from CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. Equal amounts of protein were analyzed by 2-dimensional Western blotting. Quantification of the signal intensities of spots representing AT1R (calibrated by actin signal intensity) revealed that AT1R expression in the kidney was significantly upregulated in CXCR3⫺/⫺ mice (P ⬍ 0.05).

Analysis of the Mouse AT1R Promoter Region Recent data demonstrated that upregulation of AT1R expression in a human trophoblast cell line is regulated by the transcription factor SP-1, although additional transcription facAJP-Renal Physiol • VOL

tors may also be involved (8). Using the net-based search tool Alibaba 2.1, we identified putative SP-1 and EGR-1 overlapping binding sites in the murine AT1R promoter region (nucleotide ⫺230/⫺51). It is known that SP-1 and the transcription factor EGR-1 share closely related DNA-binding sites and

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compete in numerous cell lines for the same promoter binding region influencing expression of numerous genes (5, 7, 8, 39, 42, 48). To further test whether both transcription factors interact with putative binding sites in the AT1R promoter region, we used a double-strand mouse AT1R oligonucleotide corresponding to the nucleotide sequence ⫺230/⫺51 in the murine AT1R promoter. Initial experiments found that the DBD of the SP-1 protein specifically bound to the biotinlabeled AT1R promoter region with high affinity (0.5 pmol; Fig. 5A, lane 4). Binding of murine SP-1 DBD protein could be prevented by the addition of unlabeled mouse AT1R oligonucleotide (AT1R comp) corresponding to the nucleotide sequence ⫺230/⫺51 in the murine AT1R promoter (Fig. 5A, lane 5). Notably, similar experiments using the same or even higher amounts (up to 5 pmol) of GST-tagged EGR-1 DBD protein revealed no binding to the murine AT1R promoter (Fig. 5B, lanes 2-6). Initially, we used truncated protein versions comprising the zinc-finger DBDs of EGR-1 and SP-1, respectively. To confirm that the lack of GST-EGR-1 DBD interaction with the AT1R oligonucleotide is not based on improper

folding during bacterial expression, the EMSAs were repeated using SP-1 and EGR-1 full-length proteins. Again, the SP-1 full-length protein specifically bound to the AT1R promoter region with high affinity (Fig. 5C), whereas the GST-EGR-1 full-length protein (Fig. 5D) and recombinant EGR-1 protein produced in insect cells (data not shown) failed to interact with the AT1R promoter region. To determine whether the GSTEGR-1 full-length protein is active and generally able to bind DNA fragments, competing experiments were performed using biotin-labeled EGR-1 consensus oligonucleotides and either a specific commercially available EGR-1 competitor (EGR-1 comp) or AT1R comp as the specific unlabeled competitor. The same amount of GST-EGR-1 full-length protein that failed to interact with AT1R (Fig. 5D, lane 2) was able to interact with the EGR-1 consensus oligonucleotides (Fig. 5E, lane 1). As expected, the addition of a specific EGR-1 competitor (lanes 2 and 3) prevented binding, and notably the addition of AT1R comp. reduced binding of full-length EGR-1 protein to the EGR-1 consensus oligonucleotides (Fig. 5E, lanes 4 and 5). To summarize, the data indicated that SP-1 protein bound with

Fig. 5. EMSA. A: constant amounts of a biotin-labeled double-strand mouse AT1R oligonucleotide (⫺230/⫺51) were incubated with increasing concentrations (0.05, 0.1, 0.5 pmol) of recombinant murine SP-1-DBD, containing the zinc-finger-DNS-binding domain. The unlabeled murine AT1R promoter fragment (⫺230/⫺51) was employed as a specific unlabeled competitor (AT1R comp). Data show that the recombinant SP-1 DNS binding domain binds to murine AT1 receptor promoter fragment (lane 4), a phenomenon that can be prevented by adding the unlabeled murine AT1R promoter region (lane 5). B: AT1R oligonucleotide (⫺230/⫺51) was incubated with increasing amounts (0.5 up to 5 pmol) of GST-tagged murine EGR-1 DBD. Compared with SP1-DBD, the recombinant murine EGR-1-DBD failed to interact with AT1R (even when 10 times higher concentrated as SP1-DBD). C: incubation of the AT1R oligonucleotide with recombinant SP-1 full-length protein [band-forming unit (BFU), lane 2] causes a shift, while preincubation with the unlabeled murine AT1R promoter prevents specific interaction (lane 3). D: incubation of AT1R oligonucleotide with increasing amounts (5.6, 7.2, 12 pmol) of full-length EGR-1. In contrast to SP-1 full-length protein, recombinant EGR-1 full-length protein did not bind to the AT1R promoter fragment. E: interaction of EGR-1 full-length protein with specific EGR-1 consensus motifs and subsequent competition experiments using AT1R. Recombinant GST-tagged full-length EGR-1 protein (5.6 pmol) was incubated with constant amounts of biotin-labeled EGR-1 consensus oligonucleotides. Specific EGR-1 oligonucleotides (EGR-1 comp) or AT1R comp were used in increasing concentrations as specific unlabeled competitors. EGR-1 protein binds to biotin-labeled EGR-1 consensus oligonucleotides (lane 1). Addition of EGR-1 comp prevents binding of GST-tagged full-length EGR-1 protein (lane 2: 330 ng and lane 3: 660 ng) while addition of AT1R comp reduces binding (lane 4: 200 ng and lane 5: 400 ng) to the EGR-1 consensus oligonucleotide. AJP-Renal Physiol • VOL

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much greater affinity to the murine AT1R promoter region ⫺230/⫺51 than did EGR-1 protein, which exhibited only little direct interaction with the promoter region. To test whether EGR-1 influences SP-1 binding to the murine AT1R promoter region (230/⫺51), EMSA experiments were performed using constant quantities of SP-1 full-length and increasing quantities of EGR-1 full-length protein. As expected, SP-1 bound to the biotin-labeled AT1R promoter region as previously demonstrated (Fig. 6A, lane 6). The addition of increasing amounts of EGR-1 full-length protein to the reaction mixture completely abolished binding of the SP-1 protein to the AT1R promoter region (Fig. 6A, lanes 7 and 8). Notably, the same EGR-1 amounts used in the previous experiment and even higher amounts of EGR-1 protein without SP-1 protein in the reaction mixture did not affect the migration of biotin-labeled AT1R (⫺230/⫺51; see Fig. 6A, left, lanes 2–

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4), indicating that recruitment of EGR-1 to the AT1R promotor requires SP-1. Interestingly, the addition of EGR-1 protein to the SP-1 protein/AT1R mixture caused a second shift of high molecular weight (Fig. 6A, lanes 7 and 8), suggesting further protein-protein interaction between SP-1 and EGR-1 protein. Previous studies demonstrated that not only DNA/protein interaction but also direct protein-protein interaction between SP-1 and EGR-1 influences the regulation of gene expression (44, 54). To test whether expression levels of SP-1 and EGR-1 in CXCR3⫺/⫺ mice might play a role in the regulation of AT1R expression, whole kidney protein extracts of CXCR3⫺/⫺ and CXCR3⫹/⫹ mice were tested for SP-1 and EGR-1 expression levels. Our data show that the expression level for EGR-1 remained unchanged between CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. In contrast, the expression level of SP-1 was slightly upregulated in CXCR3⫺/⫺ mice (Fig. 6B). DISCUSSION

Fig. 6. Recombinant EGR-1 protein reduces the binding of SP-1 full-length protein to the AT1R receptor promoter region. A, right: EMSAs were performed incubating the biotin-labeled AT1R promoter region with constant amounts of SP-1 full-length protein (1.5 BFU) and increasing amounts of EGR-1 full-length protein (5.6 and 11.6 pmol). Adding EGR-1 full-length protein to the reaction mixture prevented binding of the SP-1 full-length protein to the AT1 receptor promoter region (lanes 7 and 8). Addition of EGR-1 protein created a second shift of high molecular weight (arrow), indicating EGR-1 and SP-1 protein interaction and binding of both proteins as a complex to the AT1 receptor promoter region. Left: in contrast, the same amount of EGR-1 full-length protein (5.6 –12 pmol) without SP-1 in the reaction mixture could not interact with the AT1R promoter region. B: expression level of transcription factors EGR-1 (left, arrow) and SP-1 (right, arrow) in whole kidney protein lysates from CXCR3⫹/⫹ and CXCR3⫺/⫺ mice. Bottom: loading control (Sypro Ruby staining). AJP-Renal Physiol • VOL

This study examined the impact of CXCR3 on cardiovascular function and its possible role in regulating BP. So far, chemokine receptors have been regarded mainly as effectors of immunological mechanisms such as inflammation, autoimmune disease, allograft rejection, lupus nephritis (47), and immune-mediated kidney disease (29), but other works also revealed that CXCR3 is involved in ischemic nephropathy, renal endothelial microvascular injury, and renal fibrosis (12, 32, 35, 38, 41, 49). Previous studies demonstrated that chemokine receptors can be expressed on various nonhematopoietic cells, including Purkinje cells (1, 11, 37, 50), proximal tubular cells (3), podocytes, endothelial cells (10, 31), and vascular smooth muscle cells (14). Both endothelial and vascular smooth muscle cells are important in the pathogenesis of hypertension (43). Moreover, dysfunction of vascular smooth muscle cells and endothelial cells has been implicated in the development of salt-dependent hypertension (17, 33). By using a CXCR3 knockout mouse model, we demonstrated that MAP was significantly higher in CXCR3⫺/⫺ than in CXCR3⫹/⫹ mice and additionally that hypertension in CXCR3⫺/⫺ mice was salt sensitive, because an acute and chronic sodium load increased BP in CXCR3⫺/⫺ mice. To further characterize the mechanisms of hypertension in CXCR3⫺/⫺ mice, this study examined the vascular reactivity of CXCR3⫺/⫺ mice. Interestingly, the absence of CXCR3 led to increased AT1R expression in mesenteric arteries and whole kidney protein extracts, a finding that has been demonstrated in hypertensive transgenic animals for other receptors, such as the dopamine D4 and D5 receptor (4, 52, 53). Furthermore, this study showed that vasoconstriction in response to ANG II was considerably increased in CXCR3⫺/⫺ mice, and speculated that this increase results from increased AT1R expression in CXCR3⫺/⫺ mice. The renin-angiotensin system fulfills a basic function in regulating arterial BP, and AT1R participate directly in the pathogenesis of cardiovascular and renal diseases (21). The data presented here indicate that vasoconstriction in response to ANG II is increased in CXCR3⫺/⫺, contributing to hypertension in these animals. Additionally, the data support the hypothesis that vasodilatation in CXCR3⫺/⫺ mice is also affected. The response to ACh via the muscarinic M3 receptor, a potent stimulator of vasodilatatory nitric oxide release (9,

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20), was reduced in vascular resistance vessels of CXCR3⫺/⫺ mice, and additionally muscarinic M3 receptor expression in mesenteric arteries of CXCR3⫺/⫺ mice was reduced in Western blot analyses. To summarize, the data indicate that both increased vasoconstriction and decreased vasodilatation contribute to hypertension in CXCR3⫺/⫺ mice. To further characterize the molecular mechanisms involved in BP regulation in CXCR3⫺/⫺ mice, this study focused on the regulation of AT1R expression, which is relevant for cardiovascular disease (21). Recent data demonstrated that upregulation of AT1R expression in a human trophoblast cell line is regulated by the transcription factor SP-1 (8). This fits exactly to our observation that SP-1 protein expression was slightly increased in CXCR3⫺/⫺ mice (Fig. 6B). In addition, we found that SP-1 interacts with high affinity with the murine AT1R receptor region (⫺230/⫺51) providing an explanation that AT1R expression is increased in CXCR3⫺/⫺ mice via an enhanced SP-1 expression. However, regulation of AT1R expression seems to be much more complex. By using the net-based search tool Alibaba 2.1 (www.gene-regulation.com), we identified a putative SP-1/ EGR-1 overlapping binding site in the murine AT1R promoter region (⫺230/⫺51). EGR-1 and SP-1 have been shown to interact directly on the protein level, thus influencing direct DNA-protein interplay and the regulation of gene expression (44, 54). In this study, we demonstrated that the SP-1 and EGR-1 protein interaction regulates the SP-1 binding to the murine AT1R binding site, because the addition of increasing concentrations of recombinant EGR-1 full-length protein prevented SP-1 from binding to the AT1R promoter region. Furthermore, the addition of EGR-1 protein to the reaction mix containing SP-1 protein and AT1R oligonucleotides caused a supershift, indicating that the two proteins interact and bind together to the AT1R promoter region. Regarding the study findings, it is interesting that interaction between the two proteins and the function of their interplay in gene expression have recently been described as a new regulatory mechanism (44, 48). Srivastava et al. (44) described the EGR-1/SP-1 complex as a regulator of M-CSF gene expression and suggested that a decrease in EGR-1/SP-1 complex formation increased the binding of unbound SP-1 protein to the M-CSF promoter and thus stimulates protein expression. Recent studies suggest that not only the total protein expression of DNA-binding proteins but also the protein-protein interaction and the phosphorylation level of EGR-1 decide whether target proteins are expressed (44, 54). Therefore, we speculate that an imbalanced EGR-1/SP-1 complex formation in CXCR3⫺/⫺ mice results in an increased AT1R expression that leads to hypertension. Analysis of polymorphisms in the sequences of GNAI2 gene promoter region from hypertensive humans showed that SP-1 might be associated with human essential hypertension (30). Future experiments are necessary to further clarify the role of the transcription factors SP-1 and EGR-1 in BP control of CXCR3⫺/⫺ mice and to further analyze the role of the M3 muscarinic ACh receptor in the pathophysiology of hypertension. Recently, CXCR3 has been described as promoting renal fibrosis, a condition frequently associated with the development of hypertension (32). However, recent data showed that CXCR3⫺/⫺ and CXCR3⫹/⫹ mice demonstrate identical serum creatinine and urea levels and comparable urinary albumin AJP-Renal Physiol • VOL

levels, indicating that renal disease is not a cause of hypertension in these animals (34). This study demonstrated that hypertension in CXCR3-deficient mice was salt sensitive; a finding that needs to be further clarified in future studies. To summarize, this study demonstrates that blockade of CXCR3⫺/⫺ mice causes hypertension, by upregulating AT1R, and thus leading to increased vasoconstriction, which in turn results in hypertension. Consequently, CXCR3 presents a new candidate gene for BP regulation and vascular reactivity in humans and offers a focus for hypertension therapy. ACKNOWLEDGMENTS T. Kilic is appreciated for providing exceptional technical support as well as T. Knoy for editorial assistance. GRANTS The authors thank the Else Kro¨ner-Fresenius Foundation (Contract No. P27/05//A24/05//F01) and the EU Project Network of Excellence, FP6-2005LIFESCIHEALTH-6, Integrating Genomics, Clinical Research and Care in Hypertension, InGenious HyperCare proposal (Contract No. 037093) for support of S.-M. Brand-Herrmann and R. Telgmann. REFERENCES 1. Bajetto A, Bonavia R, Barbero S, Florio T, Schettini G. Chemokines and their receptors in the central nervous system. Front Neuroendocrinol 22: 147–184, 2001. 2. Bek MJ, Eisner GM, Felder RA, Jose PA. Dopamine receptors in hypertension. Mt Sinai J Med 68: 362–369, 2001. 3. Bek MJ, Reinhardt HC, Fischer KG, Hirsch JR, Hupfer C, Dayal E, Pavenstadt H. Upregulation of early growth response gene-1 via the CXCR3 receptor induces reactive oxygen species and inhibits Na⫹/K⫹ATPase activity in an immortalized human proximal tubule cell line. J Immunol 170: 931–940, 2003. 4. Bek MJ, Wang X, Asico LD, Jones JE, Zheng S, Li X, Eisner GM, Grandy DK, Carey RM, Soares-da-Silva P, Jose PA. Angiotensin II type 1 receptor-mediated hypertension in D4 dopamine receptor-deficient mice. Hypertension 47: 288 –295, 2006. 5. Davis W Jr, Chen ZJ, Ile KE, Tew KD. Reciprocal regulation of expression of the human adenosine 5⬘-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors. Nucleic Acids Res 31: 1097–1107, 2003. 6. De Mey JG, Brutsaert DL. Mechanical properties of resting and active isolated coronary arteries. Circ Res 55: 1–9, 1984. 7. Decker EL, Skerka C, Zipfel PF. The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells. J Biol Chem 273: 26923– 26930, 1998. 8. Duffy AA, Martin MM, Elton TS. Transcriptional regulation of the AT1 receptor gene in immortalized human trophoblast cells. Biochim Biophys Acta 1680: 158 –170, 2004. 9. Eglen RM. Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic function. Auton Autacoid Pharmacol 26: 219 –233, 2006. 10. Garcia-Lopez MA, Sanchez-Madrid F, Rodriguez-Frade JM, Mellado M, Acevedo A, Garcia MI, Albar JP, Martinez C, Marazuela M. CXCR3 chemokine receptor distribution in normal and inflamed tissues: expression on activated lymphocytes, endothelial cells, and dendritic cells. Lab Invest 81: 409 – 418, 2001. 11. Goldberg SH, van der MP, Hesselgesser J, Jaffer S, Kolson DL, Albright AV, Gonzalez-Scarano F, Lavi E. CXCR3 expression in human central nervous system diseases. Neuropathol Appl Neurobiol 27: 127–138, 2001. 12. Hancock WW, Gao W, Csizmadia V, Faia KL, Shemmeri N, Luster AD. Donor-derived IP-10 initiates development of acute allograft rejection. J Exp Med 193: 975–980, 2001. 13. Hancock WW, Lu B, Gao W, Csizmadia V, Faia K, King JA, Smiley ST, Ling M, Gerard NP, Gerard C. Requirement of the chemokine receptor CXCR3 for acute allograft rejection. J Exp Med 192: 1515–1520, 2000.

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CXCR3 RECEPTOR AND HYPERTENSION 14. Heller EA, Liu E, Tager AM, Yuan Q, Lin AY, Ahluwalia N, Jones K, Koehn SL, Lok VM, Aikawa E, Moore KJ, Luster AD, Gerszten RE. Chemokine CXCL10 promotes atherogenesis by modulating the local balance of effector and regulatory T cells. Circulation 113: 2301–2312, 2006. 15. Hollon TR, Bek MJ, Lachowicz JE, Ariano MA, Mezey E, Ramachandran R, Wersinger SR, Soares-da-Silva P, Liu ZF, Grinberg A, Drago J, Young WS III, Westphal H, Jose PA, Sibley DR. Mice lacking D5 dopamine receptors have increased sympathetic tone and are hypertensive. J Neurosci 22: 10801–10810, 2002. 15a.Hsu HH, Hoffmann S, Endlich N, Velic A, Schwab A, Weide T, Schlatter E, Pavensta¨dt H. Mechanisms of angiotensin II signaling on cytoskeleton of podocytes. J Mol Med 86: 1379 –1394, 2008. 16. Ishii M, Kurachi Y. Muscarinic acetylcholine receptors. Curr Pharm Des 12: 3573–3581, 2006. 17. Iwamoto T, Kita S, Zhang J, Blaustein MP, Arai Y, Yoshida S, Wakimoto K, Komuro I, Katsuragi T. Salt-sensitive hypertension is triggered by Ca2⫹ entry via Na⫹/Ca2⫹ exchanger type 1 in vascular smooth muscle. Nat Med 10: 1193–1199, 2004. 18. Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 88: 5096 –5100, 1991. 19. Jiang D, Liang J, Hodge J, Lu B, Zhu Z, Yu S, Fan J, Gao Y, Yin Z, Homer R, Gerard C, Noble PW. Regulation of pulmonary fibrosis by chemokine receptor CXCR3. J Clin Invest 114: 291–299, 2004. 20. Khurana S, Chacon I, Xie G, Yamada M, Wess J, Raufman JP, Kennedy RH. Vasodilatory effects of cholinergic agonists are greatly diminished in aorta from M3R⫺/⫺ mice. Eur J Pharmacol 493: 127–132, 2004. 21. Kim S, Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev 52: 11–34, 2000. 22. Koyano-Nakagawa N, Nishida J, Baldwin D, Arai K, Yokota T. Molecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the interleukin-3 promoter. Mol Cell Biol 14: 5099 –5107, 1994. 23. Kriwacki RW, Schultz SC, Steitz TA, Caradonna JP. Sequencespecific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1. Proc Natl Acad Sci USA 89: 9759 –9763, 1992. 24. Kuznetsova T, Staessen JA, Brand E, Cwynar M, Stolarz K, Thijs L, Tikhonoff V, Wojciechowska W, Babeanu S, Brand-Herrmann SM, Casiglia E, Filipovsky J, Grodzicki T, Nikitin Y, Peleska J, StruijkerBoudier H, Bianchi G, Kawecka-Jaszcz K. Sodium excretion as a modulator of genetic associations with cardiovascular phenotypes in the European Project on Genes in Hypertension. J Hypertens 24: 235–242, 2006. 25. Li XX, Bek M, Asico LD, Yang Z, Grandy DK, Goldstein DS, Rubinstein M, Eisner GM, Jose PA. Adrenergic and endothelin B receptor-dependent hypertension in dopamine receptor type 2 knockout mice. Hypertension 38: 303–308, 2001. 26. Lifton RP. Molecular genetics of human blood pressure variation. Science 272: 676 – 680, 1996. 27. Liu L, Callahan MK, Huang D, Ransohoff RM. Chemokine receptor CXCR3: an unexpected enigma. Curr Top Dev Biol 68: 149 –181, 2005. 28. Luster AD, Unkeless JC, Ravetch JV. Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315: 672– 676, 1985. 29. Menke J, Zeller GC, Kikawada E, Means TK, Huang XR, Lan HY, Lu B, Farber J, Luster AD, Kelley VR. CXCL9, but not CXCL10, promotes CXCR3-dependent immune-mediated kidney disease. J Am Soc Nephrol 19: 1177–1189, 2008. 30. Menzaghi C, Paroni G, De BC, Soccio T, Marucci A, Bacci S, Trischitta V. The ⫺318 C⬎G single-nucleotide polymorphism in GNAI2 gene promoter region impairs transcriptional activity through specific binding of Sp1 transcription factor and is associated with high blood pressure in Caucasians from Italy. J Am Soc Nephrol 17: S115–S119, 2006. 31. Mordelet E, Davies HA, Hillyer P, Romero IA, Male D. Chemokine transport across human vascular endothelial cells. Endothelium 14: 7–15, 2007. 32. Nakaya I, Wada T, Furuichi K, Sakai N, Kitagawa K, Yokoyama H, Ishida Y, Kondo T, Sugaya T, Kawachi H, Shimizu F, Narumi S, Haino M, Gerard C, Matsushima K, Kaneko S. Blockade of IP-10/ CXCR3 promotes progressive renal fibrosis. Nephron Exp Nephrol 107: e12– e21, 2007. 33. Oberleithner H, Riethmuller C, Schillers H, MacGregor GA, de Wardener HE, Hausberg M. Plasma sodium stiffens vascular endotheAJP-Renal Physiol • VOL

34.

35.

36. 37. 38.

39. 40.

41.

42. 43. 44.

45. 46. 47. 48. 49.

50.

51.

52. 53.

54.

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lium and reduces nitric oxide release. Proc Natl Acad Sci USA 104: 16281–16286, 2007. Panzer U, Steinmetz OM, Paust HJ, Meyer-Schwesinger C, Peters A, Turner JE, Zahner G, Heymann F, Kurts C, Hopfer H, Helmchen U, Haag F, Schneider A, Stahl RA. Chemokine receptor CXCR3 mediates T cell recruitment and tissue injury in nephrotoxic nephritis in mice. J Am Soc Nephrol 18: 2071–2084, 2007. Panzer U, Steinmetz OM, Reinking RR, Meyer TN, Fehr S, Schneider A, Zahner G, Wolf G, Helmchen U, Schaerli P, Stahl RA, Thaiss F. Compartment-specific expression and function of the chemokine IP-10/ CXCL10 in a model of renal endothelial microvascular injury. J Am Soc Nephrol 17: 454 – 464, 2006. Pickering G. Hypertension: definitions, natural histories, consequences. In: Hypertension, Pathophysiology, Diagnosis, and Management, edited by Laragh JH and Brenner BM. New York: Raven, 1995, p. 3–22. Ragozzino D. CXC chemokine receptors in the central nervous system: role in cerebellar neuromodulation and development. J Neurovirol 8: 559 –572, 2002. Romagnani P, Beltrame C, Annunziato F, Lasagni L, Luconi M, Galli G, Cosmi L, Maggi E, Salvadori M, Pupilli C, Serio M. Role for interactions between IP-10/Mig and CXCR3 in proliferative glomerulonephritis. J Am Soc Nephrol 10: 2518 –2526, 1999. Sarai A, Kono H. Protein-DNA recognition patterns and predictions. Annu Rev Biophys Biomol Struct 34: 379 –398, 2005. Sarris AH, Broxmeyer HE, Wirthmueller U, Karasavvas N, Cooper S, Lu L, Krueger J, Ravetch JV. Human interferon-inducible protein 10: expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors. J Exp Med 178: 1127–1132, 1993. Segerer S, Cui Y, Eitner F, Goodpaster T, Hudkins KL, Mack M, Cartron JP, Colin Y, Schlondorff D, Alpers CE. Expression of chemokines and chemokine receptors during human renal transplant rejection. Am J Kidney Dis 37: 518 –531, 2001. Skerka C, Decker EL, Zipfel PF. A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1. J Biol Chem 270: 22500 –22506, 1995. Spieker LE, Flammer AJ, Luscher TF. The vascular endothelium in hypertension. Handb Exp Pharmacol 176: 249 –283, 2006. Srivastava S, Weitzmann MN, Kimble RB, Rizzo M, Zahner M, Milbrandt J, Ross FP, Pacifici R. Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1. J Clin Invest 102: 1850 –1859, 1998. Steinmetz M, Potthast R, Sabrane K, Kuhn M. Diverging vasorelaxing effects of C-type natriuretic peptide in renal resistance arteries and aortas of GC-A-deficient mice. Regul Pept 119: 31–37, 2004. Taub DD. Chemokine-leukocyte interactions. The voodoo that they do so well. Cytokine Growth Factor Rev 7: 355–376, 1996. Teramoto K, Negoro N, Kitamoto K, Iwai T, Iwao H, Okamura M, Miura K. Microarray analysis of glomerular gene expression in murine lupus nephritis. J Pharm Sci 106: 56 – 67, 2008. Thottassery JV, Sun D, Zambetti GP, Troutman A, Sukhatme VP, Schuetz EG, Schuetz JD. Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene. J Biol Chem 274: 3199 –3206, 1999. Topham PS, Csizmadia V, Soler D, Hines D, Gerard CJ, Salant DJ, Hancock WW. Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis. J Clin Invest 104: 1549 –1557, 1999. van der MP, Goldberg SH, Fung KM, Sharer LR, Gonzalez-Scarano F, Lavi E. Expression pattern of CXCR3, CXCR4, and CCR3 chemokine receptors in the developing human brain. J Neuropathol Exp Neurol 60: 25–32, 2001. van Wanrooij EJ, de Jager SC, van ET, de VP, Birch HL, Owen DA, Watson RJ, Biessen EA, Chapman GA, van Berkel TJ, Kuiper J. CXCR3 antagonist NBI-74330 attenuates atherosclerotic plaque formation in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 28: 251–257, 2008. Zeng C, Yang Z, Asico LD, Jose PA. Regulation of blood pressure by D5 dopamine receptors. Cardiovasc Hematol Agents Med Chem 5: 241–248, 2007. Zeng C, Yang Z, Wang Z, Jones J, Wang X, Altea J, Mangrum AJ, Hopfer U, Sibley DR, Eisner GM, Felder RA, Jose PA. Interaction of angiotensin II type 1 and D5 dopamine receptors in renal proximal tubule cells. Hypertension 45: 804 – 810, 2005. Zhang X, Liu Y. Suppression of HGF receptor gene expression by oxidative stress is mediated through the interplay between Sp1 and Egr-1. Am J Physiol Renal Physiol 284: F1216 –F1225, 2003.

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