Inflammatory Cytokines and Reactive Oxygen ...

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Nov 28, 2014 - Mohsen Agharazii,1,2 Ronald St-Louis,1 Alexandra Gautier-Bastien,1 ...... Gauthier-Bastien A, Ung RV, Larivière R, Mac-Way F, Lebel M, ...
American Journal of Hypertension Advance Access published November 27, 2014

Original Article

Inflammatory Cytokines and Reactive Oxygen Species as Mediators of Chronic Kidney Disease-Related Vascular Calcification Mohsen Agharazii,1,2 Ronald St-Louis,1 Alexandra Gautier-Bastien,1 Roth-Visal Ung,1 Sophie Mokas,1 Richard Larivière,1,2 and Darren E. Richard1,3

METHODS CKD was induced in male Wistar rats by renal mass ablation and vascular calcification was induced with a high calcium–phosphate diet and vitamin D supplementation (Ca/P/VitD). At week 3–6, hemodynamic parameters were determined and thoracic aorta was harvested for assessment of vascular calcification, macrophage infiltration, cytokines expression, VSMC differentiation, ROS generation, and related signaling pathway activation. RESULTS CKD rats treated with Ca/P/VitD developed medial calcification of thoracic aorta and increased pulse pressure and aortic pulse wave velocity. VSMC

differentiation was confirmed by increased bone morphogenetic protein-2 and osteocalcin expression and reduced α-smooth muscle actin expression. The expression of interleukin-1β, interleukin-6, and tumor necrosis factor were also increased. The expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox and p47phox were increased, whereas the expression of antioxidant enzymes (SOD1, SOD2, Gpx1, and Prdx1) was reduced in CKD + Ca/P/VitD rats. Oxidized peroxiredoxin, a sensor of ROS generation, was significantly increased and ROS-sensitive signaling pathways were activated in the aorta from CKD + Ca/P/VitD rats.

CONCLUSION This study demonstrates a relationship between inflammation/ROS and arterial calcification in CKD and contributes to understanding of the complex pathways that mediate arterial calcification in CKD patients. Keywords: blood pressure; chronic kidney disease; hypertension; inflammatory cytokines; NADPH oxidase; reactive oxygen species; vascular calcification. doi:10.1093/ajh/hpu225

Cardiovascular disease is the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD).1,2 Atherosclerosis, which results in plaque formation with fatladen immune cells (foam cells) and vascular intimal calcification, leads to the impediment of blood flow circulation and hypo-perfusion of organs. In contrast, arteriosclerosis that results from remodeling and calcification of the media of arteries, leads to arterial stiffness, isolated systolic hypertension, increased cardiac workload, and left ventricular hypertrophy. Recent studies have highlighted the relative importance of nontraditional and CKD-specific cardiovascular risk factors.3,4 Among these, disorders of mineral metabolism involved in medial vascular calcification and arterial stiffness have been identified as potential therapeutic targets.4 Indeed, it has been shown that phosphate plays

a crucial role in vascular smooth muscle cell (VSMC) differentiation into osteoblast-like cells.5 Results from in vitro studies and animal models of atherosclerosis suggest that inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-6 (IL-6) promote VSMC differentiation and vascular intimal calcification.6–8 In inflammatory processes, these cytokines have been reported to induce reactive oxygen species (ROS) generation that may also be implicated in vascular calcification.9 However, the involvement of inflammation and ROS in CKD-related vascular medial calcification has not been defined. Here, we investigate whether inflammatory cytokine expression, ROS generation, and subsequent downstream signaling pathway activation are associated with VSMC differentiation and vascular calcification in a rat remnant

Correspondence: Mohsen Agharazii ([email protected]).

1Centre de recherche du CHU de Québec, L’Hôtel-Dieu de Québec, Québec, QC, Canada; 2Département de médecine, Faculté de Médecine, Université Laval, Québec, QC, Canada; 3Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.

Initially submitted July 17, 2014; date of first revision August 4, 2014; accepted for publication October 22, 2014.

© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: [email protected]

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Background Vascular calcification, a regulated process in chronic kidney disease (CKD), requires vascular smooth muscle cell (VSMC) differentiation into osteoblast-like cells. This phenomenon can be enhanced by inflammatory cytokines and production of reactive oxygen species (ROS). In CKD rats with vascular calcification, we investigated whether inflammatory cytokines, ROS generation, and downstream signaling events are associated with CKD-related vascular calcification.

Agharazii et al.

kidney model of CKD with vascular calcification induced by a calcium/phosphate diet and 1,25-dihydroxyvitamin D3 supplementation. This study reveals that vascular calcification and VSMC differentiation into osteoblastic-like cells are associated with elevated inflammatory cytokine expression (IL-1β, IL-6, and TNF), which leads to oxidative stress through, in part, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced ROS generation and reduced antioxidant enzymes expression and the modulation of downstream ROS-sensitive signaling pathways. METHODS Animal experiments

Hemodynamic and biochemical parameters

Animals were anesthetized with isoflurane. The carotid and femoral arteries were catheterized for the determination of systolic, diastolic, mean and pulse pressures and the recording of the pulse wave velocity (PWV) as described previously.10 Then, animals were exsanguinated and the thoracic aorta (from the arch to the diaphragm) was harvested, cut in half, and either fixed in buffered formalin or snapfrozen. Serum TNF levels were determined by ELISA assay kit (R&D Systems, Minneapolis, MN). Serum malondialdehyde levels, an oxidative stress marker, were determined by HPLC.12 Antibodies

Primary and secondary antibodies used for immunofluorescence and western blot analysis are listed in the Supplementary Table S1 together with the host, concentration used, origin, and catalog number. To determine specificity, antibodies are routinely validated by a small interfering RNA approach in isolated rat VSMC.13 Histological and immunofluorescence analysis

Aorta sections (5 μm thick) were mounted on glass slides, deparaffinized, and rehydrated. von Kossa staining was 2  American Journal of Hypertension

Western blot analysis

Proteins were extracted using TRIzol (Life Technologies) and denatured in 2× Laemmli buffer. Samples were resolved on sodium dodecyl sulfate–polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corporation, Billerica, MA). Proteins were analyzed using indicated antibodies and visualized with enhanced chemiluminescence system (GE Healthcare Life Sciences, Piscataway, NJ) or with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Western blots were quantitated using LI-COR Image Studio software 2.0 (LI-COR Biosciences). Protein modification levels were determined by quantification with multiplex fluorescent detection using the Odyssey Imager (LI-COR Biosciences) in order to monitor both total protein levels and modification. Tubulin was used for sample normalization and results are represented as a ratio of modification to total protein levels normalized to tubulin. For western blot studies, representative images are shown, whereas quantification results are means ± SEM of the LI-COR quantification of six independent samples. Real-time quantitative reverse transcriptase–PCR

Total RNA was extracted from thoracic aorta segments using TRIzol reagent according to the manufacturer’s protocol (Life Technologies). RNA levels and purity were assessed by Nanodrop (ThermoScientific, Waltham, MA) readings of 260/280 nm and 260/230 nm absorbance ratio. RNA quality was determined by resolving on 1% agarose/6%

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Male Wistar rats (Charles River, Saint-Constant, QC), weighing about 250 g, were fed ad libitum and housed as described previously.10 All animal-related procedures were conducted in accordance to the Canadian Council on Animal Care guidelines and were approved by Université Laval’s Animal Care Committee. CKD was induced by renal mass reduction (5/6 nephrectomy) as described previously.11 To induce vascular calcification, animals were given a high calcium (1.2%) and phosphate (1.2%) diet, and supplemented with calcitriol (0.5 µg/kg; 1,25-dihydroxyvitamin D3) subcutaneously three times per week (Ca/P/VitD) for the duration of the protocol. In this protocol, two groups of rats were studied: CKD rats (n = 10) and CKD + Ca/P/ VitD rats (n = 14). The animals were studied after 3–6 weeks according to the health status of CKD + Ca/P/VitD rats, determined by increased weight loss and reduced physical activity.

performed as described previously.10 Staining quantification was performed on three different areas at ×20 magnification using Image-Pro Plus analysis software (Media Cybernetics, Rockville, MD). For immunofluorescence staining, antigen retrieval was performed by boiling samples in citrate buffer for 8 min, followed by 1 h incubation in the blocking solution containing 10% bovine serum albumin in phosphatebuffered saline. Samples were rinsed in phosphate-buffered saline and incubated overnight at 4  °C with mouse antirat CD68 (ED1) to reveal tissue macrophage infiltration. Sections were then washed in phosphate-buffered saline and incubated 1 h with Alexa Fluor 594 goat anti-mouse. Then, tissue sections were counterstained by incubation with a rabbit anti-rat CD-31 (PECAM-1), which provides specific staining of the endothelium, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG. Fluorescence was examined using a FV1000 confocal microscope at a magnification of ×40 using the FluoView software (Olympus, Tokyo, Japan). Comparison of fluorescence levels between the different experimental groups was performed with the immunofluorescence reaction using the same conditions of laser exposure and image acquisition times as previously performed.10 Tissue autofluorescence (obtained in the absence of the secondary antibody) and nonspecific fluorescence (obtained by omitting the first specific antibody) were determined and subtracted from total fluorescence. The resulting specific fluorescence was expressed as percent of total surface area per field analyzed.

Cytokines and ROS in CKD-Related Calcification

formaldehyde gels. Reverse transcription was performed with 500 ng of RNA using the qScript complementary DNA SuperMix kit according to the manufacturer’s protocol (Quanta Biosciences, Gaitherburg, MD). Real-time quantitative PCR analysis was performed on a Stratagene MX3005P (Agilent Technologies, Santa Clara, CA) with Perfecta SYBR Green SuperMix, low ROX kit according to the manufacturer’s protocol (Quanta Biosciences). Real-time quantitative reverse transcriptase–PCR primers used in this study are outlined in Table 1. All oligonucleotide pairs were designed using NCBI Primer Blast algorithm (blast.ncbi. nlm.nih.gov/Blast.cgi). All primers were specific to Rattus norvegicus (taxid. 10116). Melting temperatures were determined empirically. Primer pair efficiency was assessed by

determining the linear dynamic range on an 8-point standard curve. The dissociation curves were analyzed to ensure that a single peak was present and no off-target amplification was detectable on agarose gel following the quantitative reverse transcriptase–PCR reaction. A PCR analysis was routinely performed on RNA samples to control for the presence of contaminating genomic material. Gene of interest expression, relative to two reference genes (B2m and Hprt1), was calculated based on the threshold cycle (Ct) using the Pfaffl formula.14 Reference gene stability was assessed using geNorm (www.biogazelle.com) and had a combined M value of 0.3. Results of messenger RNA (mRNA) ratios are represented as arbitrary units and are means ± SEM of six independent samples.

Table 1.  Real-time reverse transcriptase–PCR primers Protein

Primer sequence 5′–3′ (S/AS)

NCBI reference

Ta(°C)

Acta2

αSMA

GCCGAGATCTCACCGACTAC

NM_031004

56

NM_012512

55

NM_017178

56

NM_012520

55

NM_030826

55

NM_012583

55

NM_031512

55

NM_012589

55

NM_024160

55

NM_053734

55

NM_057114

56

NM_013414

56

NM_017050

55

NM_017051

55

NM_012657

55

GTCCAGAGCGACATAGCACA B2ma

β2M

TCGGTGACCGTGATCTTT TATCTGAGGTGGGTGGAAC

Bmp2

BMP-2

TCAAGCCAAACACAAACAGC ACATTCCCCATGGCAGTAAA

Cat

Catalase

ACGTCACTCAGGTGCGGACA CTATCATTCACTCTAGAAGC

Gpx1

GPX1

GGTAGGTCCAGACGGTGTTCC CAGCCATCACCAAGCCAATACC

Hprt1a

HPRT

CAGTCCCAGCGTCGTGATTAGT ATCCAGCAGGTCAGCAAAGAAC

Il1b

IL-1β

TCCTCTGTGACTCGTGGGAT TCAGACAGCACGAGGCATTT

Il6

IL-6

AGAGACTTCCAGCCAGTTGC AGTCTCCTCTCCGGACTTGT

Cyba

p22phox

GAGTGCTCATCTGTCTGCTG TCAAGCAGGAGCCACTGAAG

Ncf1

p47phox

ACCGAGATCTACGAGTTCCA AACCACCAGCCACTCTCGCT

Prdx1

PRDX1

gctgatgaaggtatctctttcag ctggtccagtgctcacttcT

Bglap

Osteocalcin

TGGAGCCCCAGCCCCCTACCCA GCCGTTGGGCTCCAGGGCAACACA

Sod1

SOD1

GGTGCAGGGCGTCATTCACT GAGTCTGAGACTCAGACCAC

Sod2

SOD2

TGTAGAGCATTGCAGCACTG CAGTAGAACAGGATTACAGC

Tnf

TNF

CGTCAGCCGATTTGCCATTTC TGGGCTCATACCAGGGCTTGAG

Abbreviations: AS, antisense; NCBI, National Center for Biotechnology Information; Ta, annealing temperature used; S, sense. used as reference.

aGenes

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Gene

Agharazii et al. Statistical analysis

Inflammation and cytokine expression

The results are expressed as means ± SEM. Differences between the two groups were analyzed using the Mann– Whitney U test using the GraphPad Prism software (GraphPad Software, La Jolla, CA). A value of P