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. Author manuscript; available in PMC: 2008 Dec 1.
Published in final edited form as: Hypertension. 2007 Oct 15;50(6):1069–1076. doi: 10.1161/HYPERTENSIONAHA.107.098806

Chemokine Receptor CCR2b Inhibition Provides Renal Protection in Angiotensin II-Salt Hypertension

Ahmed A Elmarakby 3, Jeffrey E Quigley 3, Jeffrey J Olearczyk 3, Aarthi Sridhar 3, Anthony K Cook 1, Edward W Inscho 1, David M Pollock 1,3, John D Imig 2,3
PMCID: PMC2491337  NIHMSID: NIHMS55900  PMID: 17938380

Abstract

The present study was designed to determine if chemokine receptor 2b (CCR2b) contributes to the development of renal injury in salt-sensitive angiotensin II (ANG) hypertension. Rats were infused with ANG and fed a high-salt diet (HS) for 14 days. Rats were divided into four groups: HS, HS administered the CCR2b antagonist, RS102895, ANG/HS hypertensive, and ANG/HS hypertensive administered RS102895. CCR2b inhibition slowed the progression of blood pressure elevation during the first week of ANG/HS hypertension; however, it did not alter blood pressure in the HS group. At two weeks, arterial pressure was not significantly different between ANG/HS and ANG/HS hypertensive rats administered RS102895. Renal cortical NFκB activity increased in ANG/HS hypertension compared to HS group (0.11 ± 0.006 vs. 0.08 ± 0.003 ng activated NFκB/μg protein) and RS102895 treatment lowered NFκB activity in ANG/HS hypertension (0.08 0.005 ng activated NFκB/μg protein). Renal TNF-alpha and ICAM-1 expression increased and Cyp2c23 expression decreased in ANG/HS hypertension compared to HS group and CCR2b inhibition reduced TNF-alpha and ICAM-1 and increased Cyp2c23 expression. Histological immunostaining revealed increased renal monocyte and macrophage infiltration in ANG/HS hypertensive rats with decreased infiltration in rats receiving RS102895 treatment. Albuminuria and cortical collagen staining also increased in ANG/HS hypertensive rats and RS102895 treatment lowered these effects. Afferent arteriolar autoregulatory responses to increasing renal perfusion pressure were blunted in ANG/HS hypertension and RS102895 treatment improved this response. These data suggest that CCR2b inhibition protects the kidney in hypertension by reducing inflammation and delaying the progression of hypertension.

Keywords: Kidney, inflammation, hypertension, angiotensin, MCP-1, CCR2b, chemokines

Introduction

Studies suggest that inflammation is involved in the progression of hypertension induced kidney diseases.1 Cytokines have recently been identified as components of this inflammation. Chemokines are a class of cytokines that are involved in the pro-inflammatory response under both normal and pathological conditions.1 The primary function of chemokines is to promote leukocyte migration to sites of injury and this is achieved through ligand/receptor binding with receptors expressed on leukocytes.2 Through chemokine signaling, monocytes infiltrate tissue, differentiate into macrophages, and release additional chemokines and cytokines perpetuating the cycle of inflammation.

Monocyte chemoattractant protein-1 (MCP-1) plays a pivotal role in the development of the inflammatory response.1 MCP-1 expression increases at injury sites to direct macrophage recruitment.2 Mechanistically, MCP-1 binds to the inducible C-C chemokine receptor 2 (CCR2) to promote chemotaxis.3 There are two known subtypes of CCR2, CCR2a and CCR2b. CCR2b is five fold more potent than CCR2a in inducing macrophage chemotaxis.3,4

Recent research has focused on the role MCP-1 in kidney disease. In human progressive renal disease, MCP-1 is upregulated in the kidney and urinary MCP-1 excretion also increases.2,5 CCR2 inhibition also reduces interstitial macrophage infiltration and attenuates renal damage in renal diseases.6,7,8 Furthermore, our laboratory has previously demonstrated that salt-sensitive angiotensin II hypertensive rats have increased urinary MCP-1 excretion as well as increased renal macrophage infiltration.9 These studies clearly suggest a role for MCP-1 in the progression of renal disease.

Angiotensin II (ANG) has a significant role in the development of hypertension and renal injury. Besides being a potent vasoconstrictor, ANG has proinflammatory actions on the vascular wall via increased production of reactive oxygen species, inflammatory cytokines, and adhesion molecules.10 ANG can also mediate cell chemotaxis and proliferation.11,12,13 Previous studies have also demonstrated that ANG-induced hypertension and renal damage is exacebated with high salt diet treatment.9,14

Anti-inflammatory therapies are becoming increasingly popular for the treatment a variety of diseases yet most anti-inflammatory therapies only reduce inflammation that are already present. Blocking chemokine ligand/receptor binding and subsequent cell migration and infiltration represent an alternative approach in disrupting the onset of the inflammatory positive-feedback loop. Thus, we hypothesized that MCP-1 activation of CCR2b receptor is involved in the development of high blood pressure and renal injury in the ANG/HS model of hypertension via the increase in inflammation and macrophage infiltration and that antagonism of the CCR2b would slow the progression of inflammation and kidney damage in ANG/HS hypertension.

Materials and Methods

Animal protocols were approved by the Medical College of Georgia Animal Care and Use Committee. Telemetry transmitters (Data Science Inc., St. Paul, MN) were implanted according to manufacturer's specifications into male Sprague-Dawley rats as described previously.14 After a week of basal blood pressure recording, osmotic minipumps were implanted (s.c.) to deliver ANG at a dose of 60 ng/min for 2 weeks. Immediately after minipump implantation, normal chow diet was switched to a high salt (8% NaCl) diet and rats were divided into four groups: HS, HS administered the selective CCR2b antagonist, RS102895 (10 mg/kg/day) in drinking water, ANG/HS, and ANG/HS administered RS102895 in drinking water. RS102895 (Sigma, St Louis, MO) is a novel member of the spiropiperidine class with potent and specific CCR2b antagonist properties and this class of compounds has been shown to inhibit MCP-1/ chemokine receptor 2 signaling in vivo in rodents.6,15

In a separate set of experiments, osmotic minipumps were implanted (s.c) to deliver ANG at 60 ng/min for 2 weeks. Rats were fed a high salt diet and divided into three groups: HS, ANG/HS, and ANG/HS administered RS102895. Rats were placed in metabolic cages to collect a 24 hour urine sample at the end of the experiment. After 2 weeks of ANG infusion, rats were euthanized and renal cortex was collected, subdivided into three tubes, and snap frozen in liquid nitrogen for NFκB assay, real time PCR, and western blotting.

Urinary albumin excretion was measured using a highly sensitive immunoassay SPI-BIO kit (Cayman Chemical, Ann Arbor, Michigan) and urinary MCP-1 excretion was also determined using a commercially available immunoassay kit (BD Biosciences, San Jose, CA).

NFκB Transcription factor assay

Whole-cell lysates were obtained from kidney cortex from the above mentioned groups using the nuclear extract kit (Active Motif, Carlsbad, CA). Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Twenty μg of whole-cell extract was used for the determination of NFκB activity using the TransAM NFκB p65 transcription factor assay kit (Active Motif, Carlsbad, CA). Each of the standards and samples were run in duplicate. In addition to the Jurkat cell nuclear extract provided in the kit as a positive control, HeLa cell whole-cell lysate from cells cultured for 5 min in the presence and in the absence of 10μg/ml TNF-α and 10μM Calyculin A were also run. To ensure NFκB specificity, HeLa whole-cell lysate from cells treated with TNF-α and the whole-cell lysate obtained from experimental groups were run in the presence of the wild-type NFκB consensus oligonucleotide and mutated NFκB consensus oligonucleotide. The wild-type consensus oligonucleotide completely blocked NFκB binding with absorbance that was not different from the blank wells (no NFκB p65 standard). Conversely, the mutated consensus oligonucleotide was without effect on NFκB binding (data not shown). The amount of activated NFκB was normalized per μg of cortical protein used in the assay.

Real-time Polymerase Chain Reaction

Total RNA was also isolated from 100 mg of kidney cortex using ultra-pure Trizol reagent according to the procedure described by the manufacturer (GIBCO-BRL, Grand Island, NY) and RNA concentration was determined by measuring absorbance at 260 nm. A mixture of oligo (dT) and random hexanucleotide primers was used in the reverse transcription (RT) of equal amounts of total RNA (3 μg) using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). Gene specific oligonucleotide primers and real-time PCR probes were designed based on the published gene sequences using Beacon Designer 5.0 software (Premier Biosoft International, Palo Alto, CA). TaqMan and Molecular Beacon real-time polymerase chain reaction (PCR) was used to quantify the mRNA expression of MCP-1, ICAM-1, and NFκB relative to control animals. Amplification was performed using iQ Supermix with the iCycler iQ Real-Time Detection System according to the manufacturers instructions (Bio-Rad Laboratories, Hercules, CA). A standard curve was generated for each primer pair and probe to determine PCR efficiency. Mean threshold cycle (Ct) values for each sample were normalized to GAPDH and calibrated to the control group to obtain the threshold cycle difference (ΔΔCt) with 2-ΔΔCt being the fold change relative to the control group.

Probes and primer sequences were used as follows: NFκB probe 5’-FAM- CGC GAT CAC TAA ATC CAA CAC AGG CAT CAC CCA GAT CGC G-BHQ-3’; NFκB forward 5’-GTA TGG CTT CCC GCA CTA TGG -3’; NFκB reverse 5’- TCG TCA CTC TTG GCA CAA TCT C -3’; ICAM-1 probe 5’- FAM- CCT CCT CCT GAG CCT TCT GTA ACT TGT A-BHQ-3’; ICAM-1 Forward 5’- GTA CTG ATC ATT GCG GGC TT- 3’; ICAM-1 reverse 5’- GGG GCT TGT ACC TTG AGT TT- 3’ GAPDH probe 5’- FAM-ACT CCA CGA CAT ACT CAG CAC CAG CA-BHQ- 3’; GAPDH forward 5’- CAC GGC AAG TTC AAC GGC- 3’; GAPDH reverse 5’- GGT GGT GAA GAC GCC AGT A- 3’.

Homogenization of Renal Cortex for Protein Expression

Kidney cortex was dissected quickly in ice cold homogenization buffer in the presence of protease inhibitors and frozen in liquid nitrogen for determination of ICAM-1, TNF-α, Cyp2c23, and sEH protein expression using western blotting as previously described.9 The primary antibodies used were goat anti-rat ICAM-1 (1:500; R&D, Minneapolis, MN), mouse anti-rat TNF-α (1:500; R&D, Minneapolis, MN), rabbit anti-rat Cyp2c23 (1:2000; Dr. Capdevila, Nashville, TN), and rabbit anti-mouse sEH (1:2000; Dr. Hammock, Davis, CA), respectively. Donkey anti-goat, goat anti-mouse, and goat anti-rabbit (Santa Cruz, CA) were used as secondary antibodies for ICAM-1, TNF-α, and Cyp2c23 & sEH, respectively. Band intensity was measured densitometrically and the values were normalized to ß-actin.

Evaluation of cortical ED-1 and collagen staining

In a separate group of animals, kidneys from HS, ANG/HS hypertension, and ANG/HS hypertension administered RS102895 groups were perfused with 10% formalin solution. Kidneys were then paraffin embedded, and cut into 4−5 μm sections. Kidney sections were used for immuno-histochemical evaluation of ED-1 staining for monocyte/macrophage infiltration as previously described.9 Briefly, kidney sections were incubated overnight at room temperature with mouse anti-rat CD-68 primary antibody 1:100 (Serotec, Raleigh, NC) followed by the secondary antibody goat anti-mouse IgG HRP 1:50 (Serotec, Raleigh, NC) for 1 hour. Slides were then incubated with AEC substrate chromogen (DAKO, Carpinteria, CA) for 20 minutes, rinsed, and counterstained with Mayer's Hematoxylin for 30 seconds. Ten microscopic images of kidney cortex per rat were randomly taken (×400) and CD68 positive cells were counted by a blinded reviewer experienced in ED-1 staining analysis. The number of positive cells per millimeter squared was calculated and averaged for each rats group.

To detect collagen deposition, additional kidney sections were stained with Masson's trichrome and picro-sirius red staining to assess the amount of collagen within the kidney cortex of the three rat groups. Ten randomly selected microscopic images of the kidney cortex were studied per each rat (×100). Scoring of slides were performed blindly and graded on a scale of 1−10.

Renal autoregulation study

Male Sprague Dawley rats were classified into three groups (HS, ANG/HS, ANG/HS administered RS102895). Videomicroscopy experiments were conducted using the blood-perfused juxtamedullary nephron technique after 2 weeks of ANG infusion as previously described.16 Fifteen rats were used for kidney micro-dissection and fifteen rats were used as blood donors. After the micro-dissection procedures, the kidneys were perfused with blood collected and prepared from donor rats. The blood perfusate was stirred continuously in a closed reservoir while being oxygenated with a 95% O2−5% CO2 gas mixture. Perfusion pressure was continuously monitored using a pressure cannula positioned in the tip of a double-barreled perfusion cannula in the renal artery. The pressure cannula was connected to a calibrated pressure transducer linked to a polygraph recorder. The inner cortical surface of the kidney was continuously superfused with warmed (37°C) Tyrode's buffer containing 10.0 g/l BSA, and the kidney was allowed to equilibrate for at least 15 min. Perfusion pressure was initially set at 100 mmHg for control measurements and was decreased to 65 mmHg where afferent arteriolar diameter was measured continuously as perfusion pressure was increased in 15 mmHg increments from 65−170 mmHg. Afferent arteriolar responses to changes in renal perfusion pressure were determined as previously described.16

Statistical Analysis

All data are presented as mean ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple group comparisons. Differences were considered statistically significant with P< 0.05 compared to the control. Analyses were performed using GraphPad Prism Version 4.0 software.

Results

Male Sprague Dawley rats fed a high salt diet exhibited a modest elevation in mean arterial pressure (MAP) and the same trend was apparent in RS102895 treatment rats (Figure 1A). MAP increased significantly in rats receiving an ANG infusion and fed a high salt diet for two weeks. RS102895 reduced the elevation in MAP in the first week of ANG/HS hypertension; however, it did not exhibit any significant effect on MAP at the end of the two weeks (Figure 1A). Heart rate was initially reduced, but then increased after a few days in ANG/HS hypertensive rats (Figure 1B). This initial decrease in heart rate was greater in ANG/HS hypertensive rats treated with RS102895 and was not restored to the same level as that of the ANG/HS hypertensive group (Figure 1B).

Figure 1.

Figure 1

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Effect of CCR2b inhibition on MAP in ANG/HS hypertension. Twelve hour average of MAP (A) and heart rate (B) in HS and ANG/HS hypertensive rats with or without RS102895 treatment for 2 weeks. Values are means±SEM (n=3 for HS and HS/RS102895 groups and n=6 for ANG/HS and ANG/HS/RS102895 groups). * P< 0.05 vs. ANG/HS hypertensive group.

NFκB, ICAM-1, MCP-1 excretion, albuminuria, monocyte/macrophage infiltration, and collagen deposition were used as indicators of inflammation in the kidney. NFκB mRNA expression was increased 2-fold in renal cortex of ANG/HS hypertensive rats compared to HS rats. RS102895 treatment decreased renal cortical NFκB mRNA expression in ANG/HS hypertension to the same range as HS rats (Figure 2A). Consistent with these data, renal cortical NFκB activity was significantly higher in ANG/HS hypertensive rats compared to HS rats and CCR2b inhibition reduced NFκB activity in ANG/HS hypertension (Figure 2B). Renal cortical ICAM-1 mRNA and protein expression also increased in ANG/HS hypertension and this increase was attenuated by RS102895 treatment (Figure 3).

Figure 2.

Figure 2

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Effect of CCR2b inhibition on renal cortical NFκB activity and expression in ANG/HS hypertensive rats. (A) Renal cortical NFκB mRNA expression in ANG/HS hypertensive rats with or without RS102895 treatment (n=6). (B) Renal cortical NFκB activity in ANG/HS hypertensive rats with or without RS102895 (n=6 per group). Values are means±SEM. * P< 0.05 vs. HS group and # P< 0.05 vs. ANG/HS hypertensive group.

Figure 3.

Figure 3

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Effect of CCR2b inhibition on renal cortical ICAM-1 expression in ANG/HS hypertension. ICAM-1 mRNA expression (A) and ICAM-1 protein expression normalized to ß-actin (B) in ANG/HS hypertensive rats with or without RS102895 treatment (n=5 per group). Values are means±SEM. * P< 0.05 vs. HS group and # P< 0.05 vs. ANG/HS hypertensive group.

Urinary MCP-1 excretion increased in ANG/HS hypertensive rats compared with HS group (Figure 4A) and CCR2b inhibition did not significantly affect MCP-1 excretion in ANG/HS hypertension. Renal cortical TNF-α protein expression also significantly increased in ANG/HS hypertensive rats and this effect was reduced with CCR2b inhibition (Figure 4B).

Figure 4.

Figure 4

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Urinary MCP-1 (A) and renal cortical TNF-α expression normalized to ß-actin (B) in ANG/HS hypertensive rats with or without RS102895 treatment (n=5−6 per group). Values are means±SEM. * P< 0.05 vs. HS group.

Monocyte/macrophage infiltration was also determined immuno-histochemically in rat kidney sections. ANG/HS hypertensive rats showed a significant increase in cortical CD68 positive staining compared to HS fed rats. Blocking CCR2b with RS102895 treatment lowered cortical macrophage infiltration in ANG/HS hypertension (Figure 5A). Albuminuria was increased in ANG/HS hypertension compared to HS rats (125±41 vs. 5±2 mg/day) and CCR2b inhibition lowered urinary albumin excretion to 51±20 mg/day in ANG/HS hypertension (Figure 5C). Renal cortical collagen staining was also increased in ANG/HS hypertensive rats compared to HS group and RS102895 reduced collagen staining in ANG/HS hypertensive rats (Figure 6).

Figure 5.

Figure 5

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Average number of CD68 positive cells (A) as an indicator of monocytes/macrophages infiltration per 1mm2 in the kidney cortex of HS, ANG/HS hypertension, ANG/HS hypertension administered RS102895 (n=3 per group). (B) Representative image (400X) of renal cortical CD68 positive cells of HS and ANG/HS hypertensive rats with or without RS102895 treatment. (C) Urinary albumin excretion in ANG/HS hypertension with or without RS102895 treatment (n=5−6 per group). Values are means±SEM. * P< 0.05 vs. HS group and # P< 0.05 vs. ANG/HS hypertensive group.

Figure 6.

Figure 6

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Effect of CCR2b inhibition on collagen deposition in kidney cortex of ANG/HS hypertenive rats. (A) Masson's trichrome scoring on a scale of 1−10 and representitive images (100X) of the blue staining for kidney cortex of HS and ANG/HS hypertensive rats with or without RS102895 treatment. (B) picro-sirius red scoring on a scale of 1−10 and representitive images (100X) of the picro-sirius red staining for kidney cortex of HS and ANG/HS hypertensive rats with or without RS102895 treatment. * P< 0.05 vs. HS group and # P< 0.05 vs. ANG/HS hypertensive group.

Renal cortical Cyp2c23 protein expression was decreased in ANG/HS hypertensive rats compared to HS rats (Figure 7A). Blocking CCR2b increased Cyp2c23 protein expression to levels similar to HS rats. Renal cortical sEH protein expression was not significantly altered in ANG/HS hypertensive rats with or without RS102895 treatment (Figure 7B).

Figure 7.

Figure 7

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Effect of CCR2b inhibition on renal cortical Cyp2c23 and sEH expression in ANG/HS hypertension. Renal Cyp2c23 (A) and sEH protein expression (B) normalized to ß-actin in ANG/HS hypertensive rats with or without RS102895 treatment (n=5 per group). Values are means±SEM. * P <0.05 vs. HS group.

We also determined the effect of blocking CCR2b in pressure-mediated renal autoregulatory responses in ANG/HS hypertension (Figure 8). Afferent arteriolar diameter averaged 13.7 ± 1.2 μm at 100 mmHg renal perfusion pressure and decreased by 13 and 18% when renal perfusion pressure was increased to 140 and 170 mmHg, respectively in rats fed a high salt diet. In ANG/HS hypertensive rats, renal autoregulatory responses were impaired compared to HS rats. Afferent arteriolar diameter averaged 13.9 ± 0.4 μm and decreased by 2 and 7% when renal perfusion pressure was increased to 140 and 170 mmHg, respectively in ANG/HS hypertension. RS102895 treatment improved the afferent arteriolar autoregulatory response in ANG/HS hypertension. Afferent arteriolar diameter in ANG/HS/RS102895 rats averaged 14 ± 0.2 μm and decreased by 14 and 20% when renal perfusion pressure was increased to 140 and170 mmHg, respectively.

Figure 8.

Figure 8

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Effect of CCR2b inhibition the autoregulatory response of afferent arterioles in ANG/HS hypertensive rats. The percentage changes of afferent arteriolar diameter to the increase in renal perfusion pressure are shown in kidneys of HS, ANG/HS hypertension, and ANG/HS hypertension administered RS102895 (n=5 per group). Values are means±SEM. * P <0.05 vs. HS group and # P< 0.05 vs. ANG/HS hypertensive group.

Discussion

Macrophage infiltration into the kidney is thought to mediate renal injury via the production of pro-inflammatory cytokines.2 The chemokine MCP-1 is a potent macrophage chemoattractant involved in macrophage recruitment and infiltration in renal disease.2,17 We have previously shown that urinary MCP-1 excretion and renal macrophage infiltration are increased in ANG/HS hypertension.9 This was corroborated by our findings in the present study. MCP-1 promotes monocyte chemotaxis and macrophage infiltration via two subtypes of C-C motif receptor, CCR2a and CCR2b, with MCP-1's affinity to the latter being five times that of the former.18 In order to study the effects of CCR2b inhibition in vivo in hypertension, we treated ANG/HS hypertensive rats with the spiropideridine class drug, RS102895, which is a selective antagonist of the CCR2b receptor.6,15 RS102895 treatment reduced renal inflammation in ANG/HS rats, and interestingly it also delayed blood pressure elevation, improved the impairment in afferent arteriolar autoregulatory, and blunted albuminuria. Together, these data suggest that MCP-1 activation of the CCR2b plays a role in the pathogenesis of ANG/HS hypertension and its associated renal injury.

MCP-1 signaling has been previously shown to contribute to hypertension associated inflammation.5,6,7,8 Both MCP-1 and CCR2 expression are enhanced in the arterial walls of hypertensive animals.19 Activation of MCP-1/CCR2 increases production of adhesion molecules and stimulates migration of vascular smooth muscle cells, resulting in neointimal hyperplasia.20,21 Studies have also shown that the blockade of the MCP-1/CCR2 pathway prevents vascular inflammation and arteriosclerosis in rats made hypertensive by chronic inhibition of nitric oxide synthesis.22 However, the blood pressure lowering effect of CCR2b inhibition with RS102895 treatment was somewhat unexpected. This could be related to the anti-inflammatory effects of RS102895 such as; reduced renal NFκB activation and reduced renal TNF-α and ICAM-1 expression. Rodriguez-Iturbe et al previously reported that NFκB inhibition prevents hypertension in spontaneously hypertensive rats.23 While there is evidence that MCP-1 is downstream from NFκB, MCP-1/CCR2 signaling is also believed to feedback and enhance NFκB activation.24,25 Thus, one possible mechanism for the slowing of blood pressure elevation in the present study is an anti-inflammatory one, possibly via inhibition of MCP-1 driven NFκB activation. A nonspecific blood pressure effect is unlikely as the CCR2b antagonist did not alter blood pressure in normotensive rats. RS102895 treated hypertensive rats also experienced a decreased heart rate, but again this did not occur in treated normotensive controls. MCP-1 inhibition has been shown to improve cardiac function in a rabbit model of ischemia-reperfusion injury26 and reduce left ventricular dysfunction with a modest decrease in heart rate in a murine model of post myocardial infarction heart failure.27 Therefore, Heart rate and blood pressure changes are due to the effects of RS102895 in hypertensive conditions. Our data suggest that a chemokine mediated inflammatory component is involved in the onset of blood pressure in salt-sensitive ANG II hypertension, although is not necessary for maintenance of high blood pressure.

Previous studies have shown that ANG stimulates the release of cytokines and growth factors.28 ANG also increases expression of chemokines, including MCP-1, that mediate vascular inflammation.28 High salt treatment exacerbates ANG-induced elevation in blood pressure and renal injury.9,14 ANG can also induce expression of cell adhesion molecules (CAMs) via the activation of NFκB.29 In our study, CCR2b inhibition lowered NFκB activity and expression in ANG/HS hypertension and renal cortical ICAM-1 expression was also reduced. Consistent with our data, Giunti et al. have reported that MCP-1 induced ICAM-1 expression in human mesangial cells and CCR2b inhibition with RS102895 prevented ICAM-1 upregulation.30 Muller et al. have also shown that NFκB inhibition ameliorates renal and cardiac ANG-induced inflammatory damage in rats.31 NFκB inhibition also prevents hypertension and reduces renal ICAM-1 and MCP-1 inflammatory responses in spontaneously hypertensive rats.23 Thus, it is quite possible that reducing NFκB activity by inhibiting CCR2b could account for the delayed progression of hypertension, decreased ICAM-1 expression, and renal damage in ANG/HS hypertension.

The role of macrophages in the progression of renal injury is well established. Macrophages could mediate renal injury via different mechanisms including the production of proinflammatory cytokines.2 Previous studies have shown that MCP-1 deficient mice have a reduction in aortic wall monocyte recruitment and macrophage infiltration during inflammation.32 We have previously shown that MCP-1 excretion and kidney macrophage infiltration increased in ANG/HS hypertension.9 In the present study, MCP-1 excretion also increased in ANG/HS hypertensive rats and CCR2b inhibition did not significantly affect MCP-1 excretion. Macrophage infiltration also significantly decreased in the kidney of the CCR2-deficient mice after ischemia-reperfusion injury compared with wild-type mice.17 In our study, renal cortical monocyte/macrophage infiltration increased in ANG/HS hypertensive rats, and this effect was attenuated by CCR2b blockade. We also showed that collagen deposition increased in kidney of ANG/HS hypertensive rats and CCR2b inhibition reduced collagen staining. These data further support the concept that MCP-1 activation of CCR2b is involved in the inflammatory response associated with ANG/HS hypertension.

Previous studies have shown that TNF-α activates NFκB inflammatory pathway31,33,34 and we have previously shown that TNF-α inhibition slowed the progression of hypertension, reduced renal injury, and restored the decrease in Cyp2c23 expression in ANG/HS hypertensive rats suggesting a role of TNF-α in blood pressure elevation and renal injury in this model.9 CCR2 knock out mice have been shown to have reduced TNF-α activation in myocardial and cerebral ischemic-reperfusion models compared with wild type mice 35,36 In our study, CCr2b inhibition prevented the increase in renal cortical TNF-α expression in ANG/HS hypertensive rats. These data suggest that reducing MCP-1 signaling activation with RS102895 treatment might inhibit positive feedback stimulation of TNF-α, which in turn inhibits NFκB activation and subsequent down-regulation of Cyp2c23 (figure 9).

Figure 9.

Figure 9

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Schematic diagram of the proposed inflammatory cytokines signaling pathway in ANG/HS hypertension.

Blood pressure in the RS102895 treated ANG/HS hypertensive rats matched that of the untreated hypertensive group by the end of the two-week ANG/HS protocol, indicating that regardless of the mechanism any contribution of MCP-1 driven inflammation to blood pressure is short-term. Despite being temporary, the blood pressure lowering was adequate to blunt renal damage, indicated by reduced urinary albumin excretion at the day fourteen end-point. It is likely that the slowing in blood pressure elevation only delays the manifestation of hypertension induced renal injury. Additionally, we have some evidence that TNF-α inhibition reduces albumin excretion and inflammation despite the lack of blood pressure lowering effects in DOCA-salt hypertensive rats (unpublished data). These data suggest that the decrease in albuminuria is mainly due to reduction in inflammation rather than reducing blood pressure.

An alternative to MCP-1 feedback inhibition as a possible mechanism for reduction in NFκB activity and lowering of blood pressure may be the arachidonic acid derived epoxyeicosatrienoic acids (EETs). The EETs possess anti-inflammatory along with vasodilatory properties and have the ability to block induction of NFκB activation.29 Cytokines, and thus inflammation in general, can down-regulate the Cyp450 epoxygenases which catalyze EET production in the kidney.37 The inability to properly upregulate these enzymes is associated with hypertension and end-organ damage in ANG hypertension.9,38 In the present study, we found that renal epoxygenase expression was lower in ANG/HS hypertensive rats, but expression was appropriate in those that received RS102895 treatment. The EETs can also be metabolized to an inactive molecule by soluble epoxide hydrolase (sEH) and studies have shown that sEH inhibitors are effectively anti-hypertensive and renal protective in ANG hypertension.39 We found that expression of sEH was increased in the ANG/HS hypertensive rats but not in those treated with RS102895. EETs could therefore be a possible link between inflammation and blood pressure in the present study.

We also observed reversal of afferent arteriolar autoregulatory dysfunction in the kidneys of CCR2b inhibitor treated hypertensive animals. Under normal physiological conditions afferent arterioles constrict or relax in response to changes in arterial pressure in order to maintain glomerular capillary pressure and glomerular filtration rate at appropriate levels; this is impaired in ANG/HS hypertension resulting in glomerular injury.19,41 Inscho et al demonstrated that increased blood pressure is sufficient to cause the impairment as responsiveness is restored with both triple therapy hypertensive treatment and AT1 receptor blockade in ANG hypertensive rats.42 However, physiological changes due to the high blood pressure itself, such as vascular inflammation, cannot be ruled out. Sharma et al found that the cytokine TGF-β1 completely blocked afferent arteriolar autoregulatory responsiveness through generation of reactive oxygen species.16 In our study, restoration of afferent arteriolar autoregulatory responsiveness with CCR2b antagonist treatment in ANG/HS hypertension may therefore be attributed, at least in part, to both the slow in the progression of hypertension and the anti-inflammatory effects.

Perspectives

Overall, these data suggest that MCP-1 mediated inflammatory response, specifically through the activation of CCR2b, contributes to the onset of hypertension and renal inflammation and leads to impaired afferent arteriolar autoregulation and renal injury in ANG/HS hypertension. These effects may be due to increased renal monocyte/macrophage infiltration, vascular inflammation, enhancement of the NFκB inflammatory signaling, and decreased epoxygenase expression. While additional studies will be needed to detail the relationship between NFκB and EETs in hypertension and to explore the mechanisms for blood pressure lowering effects due to chemokine signaling, it is clear that antagonism of CCR2b provides anti-inflammatory and renal protective effects in this animal model of hypertension. These findings support the general hypothesis that hypertension is an inflammatory condition and pharmacological inhibition of chemokine signaling, as an alternative to traditional anti-inflammatory therapies, could be of significant clinical value in treatment of hypertension and its related end-organ damage.

ACKNOWLEDGEMENTS

These studies were supported by grants from the National Heart Lung and Blood Institute (HL59699, HL074167), American Heart Association Established Investigator Awards to J.D. Imig, E.W. Inscho and D.M. Pollock and Post-doctoral Fellowships from Southeast Affiliate of the American Heart Association awarded to A. Elmarakby.

Footnotes

Author Disclosures

Ahmed A. Elmarakby: No disclosures

Jeffrey E Quigley: No disclosures

Jeffrey J Olearczyk: No disclosures

Aarthi Sridhar: No disclosures

Anthony K Cook: No disclosures

Edward W Inscho: No disclosures

David M Pollock: No disclosures

John D Imig: No disclosures

References

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