Scribd Logo
Upload

Welcome to Scribd!

  • Differential Contribution of Nox1, Nox2 and Nox4 To Kidney Vascular Oxidative Stress and Endothelial Dysfunction in Obesity - Elsevier Enhanced Reader

    Uploaded by

    Raji Sivarupa
    12 views13 pages

    Original Title

    Differential contribution of Nox1, Nox2 and Nox4 to kidney vascular oxidative stress and endothelial dysfunction in obesity _ Elsevier Enhanced Reader

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    12 views13 pages

    Differential Contribution of Nox1, Nox2 and Nox4 To Kidney Vascular Oxidative Stress and Endothelial Dysfunction in Obesity - Elsevier Enhanced Reader

    Uploaded by

    Raji Sivarupa

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    of 13
    Redox Biology 28 (2020) 101330 Content lists available at ScienceDisest ee oe ELSEVIER Redox Biology Journal homepage: vv vier.com/locatelredox Differential contribution of Nox1, Nox2 and Nox4 to kidney vascular oxidative stress and endothelial dysfunction in obesity Mercedes Mufoz"', Maria Elvira Lépez-Oliva’, Claudia Rodriguez", Marfa Pilar Martinez”, Javier Séenz-Medina’, Ana Sanchez’, Belén Climent’, Sara Benedito’, Albino Garefa-Sacristan’, Luis Rivera’, Medardo Hernéndez", Dolores Prieto’ *Diparamint de eo Rad de Fav, UnriadComps, Mad Span “Daparanon de Anant y Brio, Facade Varna, Unred Colzese Mab, Spain *Diroramans Unig Ha! Unies Paes de Heme Mjasonds, Madr Span ARTICLE INFO ABSTRACT ome ‘nie sessed eno danton i key page actor andering the micas Eta con ‘ompliatons of metablc diese: NADPH ose (Noe @ major source of onda sess in dae ne ea ees ropathy and ernie ine dese, dai Noxt and Nox2 have been ete a ren oes of Cue se Sodltr endothelial Hs0ahe reset tu was sought to lnvestgate hel of Nox zs a eal vest = ivnve stress and endothelial dystonia a mate of genetic be. Endothelial uncon was assed = in intreal aes fase acer rat (G28) sd thee entrar en Zacher ets (2 owned — nvr mjosaps and sapere (©; ad HO, prton were mere: np endl ependent relations to aetycholne (ACH) were associated to augmented 0,” generation, but neither ROS scavengers no the Nox inhibitor aporynin signicaty improved these relaxant exponses in renal arteries of (O71. Wheres NO contribution to endothelial felaations ws blunted, catalase sensitive non-NO non prestanoid relaxations were enanced in obese rts. Increstingly, NADPH-dependent O.”prdction was ugmented while [NADPH dependent H.0: generation was cece and cytosolic and mitochondrial SOD stere upregulates in [ideey of obese rats. Nort was down-regulated in renal arteries and Novtdepeadent HO; generation and ‘endothelial relaxation were reduced in OZR. Uptegulation of both Nox? and Noxt Wwas associated with aug rented 0." production but reduced 1,0. generation and blunted endothelial Nox2 derived HOs mediated in obese rats. Moreover, increased NoxT-derived 0,” contributed to renal endothelial dysfunction in OZR, Tn summary, the curent data support a main role for Nox-derived Os" in kidney vascular oidative stress ad renal endothelial dysfunction in obesity, while reduced endothelial Now expression associated to decreased 1.02 generation and t-0,-mediated vasoiatation might hinder Nox4 potetive real eects ths contributing to Kidney injury. This suggests that efetve therapies to counteract oxidative stress and prevent microvascular ‘complications must deny the specific Nox subunits involved in metabolic disease 1. Introduction cenaymes have been implicated in both physiological and pathological renal processes, Thus, Nox2 particjpaes in renal tubular functions such Onidative stress is key pathogenic factor underlying the vascular ‘complications of metabolic disease including diabetes- and obesity-e lated nephropathy and leading tothe development and progression of kidney injury (1,2]. Both mitochondria and NADPH oxidases (Nox) are ‘accepted as major sources of ROS generation in diabetic nephropathy ‘and chronic kidney disease, but the specific role of the various Nox subunits in kidney injury remains controversial, since certain Nox as electrolyte transport and glucose handing (3), while both Nox? and Nox# have recently been shawn to be sources of endothelium-derived vasodilator HO, in renal arteries [4]. On the other hand, Nox’ or renox, the most abundantly expressed Nox isoform in the kidney, has consistently been found up-regulated and associated to kidney fibrsis In labeces, therefore being proposed asthe most critical Nox isoform linked to diabetic nephropathy [5-8]. In contrast, other studies have * comesponding author. Departamento de Fisiologia, Facultad de Farmacia, Universidad Complutense de Madrid, 28040, Madrid, span. ‘mall adres: dpieto vem.es(D. Peet). "aM and EL equally cotabute to this Work, htps://doi.og/10.1016/}.ex.2019.101390 Received 10 Jay 2019; Received in revised form 27 August 20194 Acepte. 16 September 2019 ‘Available online 20 September 2019| 2213,2317/ © 2019 The Authors. Published by Elsevier BV. This an open acess (i /retivecommonsong/ienses/BY-NCND/4.0/). ticle unde the OC BY.NC-ND license edo ty 28 200) 101350, ‘Abbreviations ‘Ach acetyleholine; COX cyclooxygenase EG endothelial cel EDH _endothelium-derived-hyperpolarization 2 Soe ‘demonstrated that renal expression of Noxd is decreased in the course ‘of diabetes and this isoform is crucial for kidney tubular cell survival under injury conditions [9-11]. Moreover, studies in Noxand Noxt- deficient animals do not appear to involve these Nox isoforms as major drivers of renal disease [9,12] Obesity isa public health problem of inereasing prevalence worl ‘wide and a risk factor forthe development of chronic kidney disease (CKD) independent of diabetes, hypertension and other comorbidities (13,14. Microalbuminuria progressing to overt proteinuria is the ear liest indication of obesity-related renal dysfunction, and glomerular hypertrophy and hyperitration develop in parallel to inereasing body mass in obese individuals [15,16]. On the other hand, obesity is ac cepted as a state of low-grade systemic inflammation and oxidative ‘ress the trigger of renal inflammation that promotes the progression ‘of obesty-assoiated kidney injury [17,18]. Mitochondria and Nox are the two major sources of ROS in the kidney (2,19]. Thus, mitochondri derived oxidative stress has been associated to kidney proinflammatory and structural changes in response to lipid overload in high fat diet, (HFD)-fed mice [20], while mitochondrial protetion prevents renal inflammation, glomerulopathy and obesity sstociated-renal injury [21]. Increased ROS production in mesangial, endothelial and tubular cells mostly derived from Nox have been found associated to both diabetes 171 and obesity-related kidney disease (22) linked to stimulation of ‘TRG and matrix genes and to activation of profbrotie processes un: derlying fibrosis in diabetic nephropathy [6,7]. Oxidative stress in plasma and renal vascular tissue has also been Involved inthe reduced NO levels and impaired endothelial fanction of renal arterioles from genetic and HFD‘induced models of obesity 118,25]. While COX-2, a mediator of renal inflammation, has been Identified asa key souree of ROS leading to enhanced vasoconstriction ‘and endathelist dysfunction in renal arteries of obese rats (23, the specific contribution of Nox-derived ROS remains to be elucidated due to the controversy on the implication of Nox? and Nox# in both phy- sologieal and pathophysiological processes in the kidney. Therefore, the present study was sought to investigate the contribution of Nox ‘enzymes to renal vascular oxidative stress and endothelial dysfunction in obesity. We used the obese Zucker rat (OZR), a well stablished model fof genetic obesity/metabolie syndrome that exhibits glomerular hy pertrophy and proteinuria by 12-14 weeks age and develops glomer- tloscerosis with increasing age ultimately leading to renal failure 12425} 2. Materials and methods 2.1. Animal model In the present study, 8-10 weeks of age Male obese Zucker rats (07R) (fa/fa) and their control counterparts, Lean Zucker rats (LZR) (fav) were purchased from Charles River RMS (Spain). Rats were housed atthe Pharmacy School animal care facility and maintained on standard chow and water ad libitum, until they were used for study, at 17-18 weeks of age, All animal care and experimental protocols con: formed to the European Union Guidelines for the Gare and the Use of No rie oxide NOS nitroxide synthase Nox NADPH oxidase enzymes OZR Obese Zucker rat 07 superoxide Phe phenylephrine PSS physiological saline solution ROS reactive oxygen species SOD superoxide dismutase VSM vascular smooth muscle Laboratory Animals (Poropean Union Directive 2010/63/BU) and were approved by the Institutional Animal Care and Use Committee of Maat Complutense University. Animals were killed and the kidneys quickly cemoved and placed in cold (4°C) physiological saline solution (SS) ofthe following. composition (mND: NaCl 119, NaHCO, 25, KCI 4.7, KHPOs 1.17, MgSOq 1.18, CaCl, 1.5, EDTA 0.027 and glucose 11 continuously gassed with a mixture of Ss CO,/9S% Oz to maintain pH at 7.4. On the day ofthe experiment, blood samples were obtained and plasma was frozen for determination of non-asting glucoce, cholesterol, trilyeerides and insulin plasma levels by using commercially available kits, Plasma insulin levels were measured by specific enzyme-linked immunosorbent ass. 2.2, Dissection and mounting of microvesels Renal intelobae arteries, second: or third order branches of the renal artery from LZR and OZR, were carefully dissected by removing the medullary connective tissue and mounted in parallel in double microvascular myographs (Danish Myotechnology, Denmark) by in serting two 40 um tungsten wires into the vessel lumen. After mounting the arteries were equilibrated for 30 min in PSS maintained at 37°C. The relationship between passive wall tension and internal cr ‘cumference was determined for each individual artery and from this, the internal circumference, Lq corresponding toa transmural pressure of 100 mmHg for a relaxed vessel in sit was calculated. The arteries wore set to an internal diameter L; equal to 0.9 times Lygp (Ly = 0.9 x ‘Lo, since force development in intrarenal arteries i lose to maximal at this internal kamen diameter. 23, Experimental procedures for the funcional experiments [At the beginning of each experiment, arteries were challenged twice With 120mMK™ solution (KPSS) in order to test vessel Endothelium-dependent vaso the relaxant effets of acetylcholine (ACB) upon addition of cumulative concentrations of this agent on arteries precontracted with pheny- lephrine (Phe) in the absence and presence of the SOD scavenger empol (@OUMD, the specific mitochondrial superoxide scavenger ‘Table 1 Plasma metabolic and renal function parameters of LZR and OZR, Body wel (| we=7 STO Wonks v8 v8 [tac we 8 esa on [sian Lisal 6 1msoe [Gotan (mea) 4 BES IS 0 Migheerslpameinedd M9 8 HOD [Ure ea Sel 8 asm (eetaiathion (27a) 039 = 00-8 GTO Data are means & SEM ofthe namber n of animals. Significant diferences were analy by an wnplred Student exes, P< O05 “p< 0001 Sp <0.0001 ¥ LZR On 1D basa PoBY Mm Torpol BI ET-1 8 2000" 1000. Chemluminescence (epm/mg) edo ty 28 200) 101350, Relaxation (% Phe) Se 0 §8 eee 89 (ACR, Cfo do sox e oR + OMe Tempo! & Gz nwTeMPO F GR apooynin 100 100 100 é r i 7 Eis 2 é =” § 50. § § 50. i 5 3 3 2 os 3 ° ° eTaTe TER. -PePEnere) oo Log (ACh, 1m) Log (ACH, Log (ACh, [NM 1. Vascular oxidative sess and endothetil dysfunction resiiant to ROS scavengers in renal arteries of obese rats. 3) Baal an PDBu and ET--timalate 0, reduction and eect the SOD mimetic tempol (100uM) on basal superoxde production of rt rena intrlobar arteries measured by lueigenin enhanced chem Tuminscence, Results are expresed ae counts per onute (etn) per ay of tise. Bare represent cnean = SEM of 7-20 animale tactically ignicant differences were calulated by unpaied Student tee ***p < O01 versus baal levels. "p< 0.03, °°p < O01 yes real arteries of LZR. (b) Averge relaxation 1 ACh renal interiobar arteries fom OZR compared co LZR. feof) the SOD mimetic tempol (20yM), (the specif mitochondrial superoxide avenger MiOTEMPO (Lu and e) the Nox inhibitor apocynin (30M) onthe relzxant responses to ACh ef rat emliterlobar arteries fom OZR, Results are expressed as percentage of the precontaction induced by phenyepvine (Phe), Data are shown asthe mean = SEM of 6-20 arteries, Significant differences were analyzed by pated Student fest "p -< O10 versa contol before treatment. MitoTEMPO (1 pM), the non-selective Nox inhibitor apocynin (30 uM), ‘catalase (200 IU/mL.) and L:NOARG (100 M) and previously incubated with LNOARG and indomethacin (1 uM) to block NOS and COX er- ayines, respectively. The responses to exogenous ACh were further ‘obcained and compared after treatment with the non-selective Nox 4 Inhibitor plumbagin (1 MD, the dual Nox/4 inhibitor GKT137831 (0.14MD), the selective Nox2 inhibitor Nox2¢stat (1 yMD and the se- lective Noxl inhibitor NoxAldstat (0.1uM) (26). The drugs were ‘added to the myograph chamber 30 min before a second concentration response eurve was performed, and the Phe concentration was adjusted to match the contraction during the first control curve assessment, 24. Measurement of superoxide production by chemiluminescence (Changes in basal, FT-L-induced and NADPH stimulated levels of Oy were detected by lucigenin-enhanced chemiluminiscenee, as previously described [4,22]. Cortex samples and 4-6 segments of the renal inter. lobar arceries about 4-5 mm long were dissected out from the kidney of ZR and OZR, equilibrated in PSS for 30 min at room temperature and then incubated in the absence (controls) and presence of the tempol (100M) to determine basal O2° levels, and in the presence of the provein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu) (G0 ND and ofthe vasoconstrictor peptide endothelin (FT-1 (0.01 pM) to measure ET-L-stimulated Oy" levels, that are greatly enhanced and ‘contribute to vascular oxidative stress and endothelial dysfunction in ‘obesity [27]. To asess Nox stimulated O° generation, itaet intrarenal arteries and cortex samples were incubated with extracellular NADPH (100yA for 30minat 37°C. Since CYP 2C epoxygenases also con- tribute to NADPH-dependent 0,” production in renal tissues (41, treatment with the selective Nox inhibitors Nox2ds-tat_(1yM), GKT137831 (0.3 iM) and NoxAldsta (0.3yND, was used to determine the relative contribution of the different Nox subunits ro ROS gener tion in renal arteries and cortex from obese rats compared to controls. Samples were then transferred to microtiter plate wells containing § uM bis‘N-methylacriinium nitrate (lucigenin) in the absence and presence of different ROS inhibitors and of stimulation with NADPH which was added previous to determination. Chemiluminescence was measured in 1 luminometer (BMG Fiuestar Optima), and for calculation baseline values were subtracted from the counting values under the diferent experimental conditions and superoxide production was normalized to Ary tissue weight. 28, Measurement of hydrogen perovide by Amplex Red Noxederived Hz0s levels were measured by Amplex Red #03 assay Kit (Life Technologies) in renal arteries and cortex from LZR and OZR. Since apocycin is a non-selective Nox inhibitor that has additional peroxide scavenging properties besides its inhibitory action on Nox activity 25}, seletive Nox inhibitors were used in order to determine the relative contribution of Nox subunits to HO» produetion in renal tissues. Samples were equilibrated in HEPES-physiological saline solu ‘don (P58) for 30minat room temperature and then incubated with edo ty 28 200) 101350, ao © O LRInde © +LNOARG® Indo eee S indo Cat oR & Gate Lnoars % Inco + Cat + Lnoars & Caace 100 __ 100 100 0H ? - Eos = J. Eo wf» x Ik he je § 0 g 3» 3 2 ge °. . o Tees b7 354 tae Ss Leo (ACR, Lop (ACh, 1) Log (ACh, a) bo wo d © ORindo f +LNOARG Indo = ca F tnco Cae cai © Get Lncaes Inco + Cat + now S Ghee = io * con g not] No ? & 1s| an ie oa 3 so| 5 so § w. } i : : 2 | i i os| o. °. o. fue eERERe. eTereTETS | Log (ACh, [M)) ‘Log (ACh, [M)) Log (ACh, [Ml) 2 Renal endothelial dysfunction involves impaired NO but enhanced non-NO no prostanoid EDH type relations in obese ra (od) Efers of eatalase (a, 200 U/ml and catalase pus the NOS synthase inhibitor LNOARG (100 yi} on the average reliant responses to ACh in real arteries fom LZR (0 and OZR (he, in te absence (a,b) and presence (cd) of COX inhibitor inomedaci (Indo, 0.3pM. ef) Effect of catalase on the EDH¢ype relat responses elicited bythe ‘endothelial agonist acetleholine (ACH) under eontions of NOS and COX blockade in renal arteries rom LZR (e) and OZR (Ress re expressed as etcentage oF the precontaction induced by phenylephrine (Phe) Di teit"p = 005 “p< GOL “p= 0.001 versus catalse rete [NADPH (100M) in the absence (controls) and presence of the Nox Inhibitors Now2dstat_(1yND, GKTI37831_ (03 uM), Noxids-at (03M) and apocynin (304M) for 30 minat 37°C. Areres and cortex samples were then transferred to microtiter plate black wells containing 30mM final concentration (Amplex Red) and 10 U/ml final cor- centration (horseradish peroxidase) In the absence and presence of different ROS sources inhibitors and some samples were stimulated With either NADPH just prior co determination. Fluorescence was measured in fuorimeter (BMG Fluostar Optima), using an excitation filter of 544 nm and an emission filter of 580m, Background fuores- ‘cence was subtracted from the counting values under the diferent ex perimental conditions and H:02 production was normalized to dry Use weigh. 26. Western boting analysis of Nox subunis Incerlobar arteries and renal cortex samples from LZR ancl OZR were homogenized on iceinlyses bulfer containing 10 mM Tris-HCl (pH 7.4), 186 SDS, I mM sodium vanadate and 0.019 protease inhibitor cocktal (Sigma Aldrich, Made, Spain). After centrifugation at 15000 xg for 20minat 4°C, proteins in the supernatants were quantified by the DC Provein Assay Kit (Bio-Rad, Madrid Spain). For each sample, 50g protein/lane was separated in a 10% polyacrylamide gel (SDS-PAGE) ‘and transferred to a polyvinylidene fluoride (PYDF) membrane (GE. Healthcare, Madrid, Spain). For immunodetection, membranes were incubated overnight at 4°C with the polyclonal primary antibodies: ‘nti-CuZnSOD, anti-MaSOD (1/1000), ant-catalase (1/1000) and anti= Nox (1/500) (Santa Cruz Biotechnology, Quimigen, Madrid, Spain) ‘anti-Noxl (1/500) and Nox2/p81phox (1/1000) (Abeam, Cambridge, re shown asthe mean = SEM of7-12 arteries Signin dferences were analyzed by pated Stent © UK). The blots were also probed for B-actn (1/40000) asthe loading control using a mouse monoclonal antibody (Sigma Aldrich, Madrid, Spain). After washed, bound primary antibody was detected with horseradish peroxidase conjugated secondary antibodies and blots were ddovelopment by chemiluminescence (ECL Select-kit, GE Healthcare, Madrid, Spain) on ImageQuant LAS 500 imaging system (GE Healtheare, Madi, Spain). Denstometey analysis was performed sing Quantity One v4.62 software. Protein expression levels were normal aed to Bactin. 27. Immunohistochemisry ‘Tissue samples from kidneys containing interlobar arteries and samples of renal cortex ftom LZR and OZR were immersion-ixed in 4% paraformaldehyde in 0.1M sodium phosphate-buffer (PBS). Kidney paraffin embedded sections (4 ym) were deparaffinized and blocked for 10min with 0.3% hydrogen peroxide at room temperature. After an tigen retrieval and blocking, sections were incubated overnight at 4°C withthe following biotinylated primary antibodies: ant-CuznSOD (1/ 200); anti-MnSOD (1/200); ant-caalase (1/200); ani-Noxt (1/100); anti-Nox2/gp01phox (1/100) and ant-Nox$ (1/100). Immunochemical staining was performed using streptavidin-biotin conjugated horse- radish peroxidase (HRP) (EMD Millipore Corp., Darmstadt, Germany) and visualised by incubation with 3, diaminobenzidine (DAB) (Sigma Aldrich, Madrid, Spain). The sections were counterstained by Harts’ hematoxylin, dehydrated and mounted. Colocalization of Nox4 with eNOS in renal arteries wall was obtained in Sym thick transversal cryostat sections preincubated in 10% normal goat serum in PB con: taining 0.3% Triton-X-100 for 2-Bh. Nox4 expression was determined riety 0, rare a gee oes oe. a ee ee g£ ro 4 oo 0 : . I ee eo] i. 8 oo UR OZR UR OER H,0, == oa oa ae 3 ae 3 * 5 me 2 te ilk a to is. 2000- - 8 2000- ; ih . d i tan Oak ca Ome Fig. 2, Noxdependent 0," production is augmented while Noxderved H.Os ‘generation Is reduced in renal sues of obese rats. (ad) Basal and NADPH Sumulated 0," and 1,0; production and elect of the non selective Nox i hiitorspoeynin (100 6 a ena nero eries a renal coe from ZR and O2R, measured by lcigenin-enhianced chemiluminiscence (ab) and ‘Amplex Red fluorescence (ed) assays, respectively. Results are expressed ‘eounts per minute (ep) per mg of tsaue for chemlluminescence a relative Aorescence units (RFU) per mg of tue. Bary represent mean = SEM oF 10-18 animals. Statistically significant ferences were calculated by ANOVA followed by Bonferron postero test or unpaired Student's etest "p< 0001 ‘vermis baal levels tp = 0.05, fp -< 001 Ftp. O.00] vers NADPH Simulated; #p = 0.05; #¥p < 001; ##¥p = D001 versus LZR. by immunofhuorescence by incubating renal sections from LZR and OZR ‘with a polyclonal primary antibody antiNox4 (1/100) (Santa Cruz Biotechnology, Quimigen, Madrid, Spain) and a mouse monoclonal anti-eNOS (Abcam, Cambridge, UK) diluted at 1:400 for 48h, washed ‘and allowed to react with a goat secondary serum (Chemicon International Ine) anti-rabbit for the Nox4) diluted 1:200 for 3h at room temperature, Secondary antibodies used were Alexa Fluor 594 (red) and Alexa Fluor 488 (green). The slides were covered with @ specific medium containing DAPI, whieh stains all cell nucle, The ob servations were made with a fuorescence miroscope (Olympas IXSL). No immunoreactivity could be detected in seetions incubated in the absence of the primary antisera. Preadsorption with Nox proteins showed no erossreactivty t the antibodies. 2.8. Data presenaton and satisical analysis For the funetional experiments, results are expressed as either Nm” of tension or as a percent ofthe responses to either Phe or KPSS in each artery, as means + SEM of 6-15 arteries (1-2 from each an- mal). For measurements of O3° or H,02 production, results are ex pressed as counts per minute (epm) per mg of tissue and relative ADuorescence nits (RFU) per mg of tissue in arterial segments and cortex samples, respectively, as means * SEM of 4-7 animals. The statistical differences between means were analyzed by using one-way edo ty 28 200) 101350, ANOVA followed by Bonferroni's pos hoe test for comparisons involving more than two groups or by paired or unpaired Student's test for comparison between two groups. Probability levels lower than 59% were considered significant, All calculations were made using a standard software package (Prism 6.0, GraphPad, San Diego, CA) 3. Results 3.1, General parameters Metabolic profile and plasma renal funetion parameters are shown in Table 1. At the time of the experiment (17-18 weeks of age), OZR wore signifieantly heavier than LZR and exhibited mild hyperglycemia, hyperinsulinemia and dyslipidemia with elevated total cholesterol and triglycerides levels. Renal function, as assessed by plasma creatinine and ures, remained normal (Table 1). ‘The normalized intemal lamen diameters, fof renal arteries inthe OZR group (252 + Om, n = 30) were not significantly different from those in the LZR group (247 = 14 um, a = 23), thus supporting that struetire i$ preserved in arteries ffom OZR compared to LER VVasoconstrietor response to KPSS (1.18 + 0.18 Nm~!,n = 23, in LZR and 1.18 = 0.11, n = 30, in OZR) or Phe (131 = 0.20Nm~*,n = 23, in L2R, and 1.28 0.17, n= 30, in OZR) were also unchanged in oz. 3.2, Oxidative stress and endothelial dysfunction resistant to antioxidants in renal arteries of OZR Basal 0," levels assessed by lucigenin chemiluminescence, and also ‘hose stimulated by ET-1 of PDBu, were significantly higher ia renal arteries of OZR compared to LZR (i. 1a), thus showing vascular ox dative stress in the kidney of obese animals. Endothelial funetion was evaluated by the vasodilatador responses to acetylcholine (ACH) that wore significantiy impaired in renal intrlobar arteries fom obese rats (Wig, 1a). However, the endothelial relaxations elicited by ACh were not improved by acute treatment with either the SOD mimetic tempol ig. 10, the mitochondrial ROS seavenger MitoTEMPO (Fis. 1d) or the non-selective Nox inhibitor apocynin (Pig. le) in OZR, despite aug. mented oxidative stress in renal arteries. H,0> has recently been involved along with NO in the endothelium: dependent relaxations of intrarenal arteries (4, and therefore the re lative contribution of NO and HaOa were determined in arteries from lean and obese animals in order to assess the natuce of the impaired |ACh relaxant responses. The NO-mediated component of the ACh re laxations, obtained in the presence of catalase (ig. 2a and b) and en- hanced by treatment with indomethacin to block COX-derived con- tractle prostanoids (Pig. 2c and d), was significantly reduced in arteries of OZR, thus confirming impaired vasodilator responses to NO in obese rats, Interestingly, a marked enfancement ofthe relaxations induced by ACH was unmasked when assessing endothelial relaxations under cor ditions of NOS and COX enzymes blockade (Fig. 2e and 1), thus sug- gesting the involvement of compensatory non-NO non-prostanoid EDH type mechanisms in renal arteries from obese rts. However, HO did not account for these enhanced relaxations sine catalase nealy abol- ished the non-NO endothelial relaxant component in LER but not in OZR (Fig. 2e and 0. 3.3. Nox-derived 05° production is elevated while Nox derived #02 generation decreased i renal arteries from obese rats In order to assess the contribution of Nox-derved oxidative stress to renal endothelial dysfunction of obese rats, NADPH-stimulated 0,” and 1,0, production was measured by Iucigenin chemiluminescence and Amplex Red fluorescence, respectively, in the absence and presence of, the non-selective NOX inhibitor apoeynin. Both Oy” and HzO, levels were markedly enhanced by NADPH in both renal interlabar arteries edo ty 28 200) 101350, UR oR GR OR Fig. 4, Upregulation of the H-genesting cytosolic and mitochondrial SOD in arterial and cortical renal sus in obesity, (6) Inmunohistochemical deme: tation of CuZaSOD (a) and MaSOD (e) in renal interlobr artery and eorex fom Kidney of LZR and OZR, CuZaSOD and MnSOD were dsibuted throughout the ‘endothelial ning and VSM ofthe renal itelobar artery, and in renal tubules (T) (CuZnSOD) and markedly up-egulated in arterioles (A) and glomeruli (G) {uznSOD) inthe renal cortex of OZR compared to LIR. Sections ae representative ofn = 3 animals. (bl Wester blot analysis of CuZnSOD and MrSOD expression In samples of renal artery and cortex showing that both proteins levels were higher in samples of renal cortex and renal arteries of OZR than in those of LR. Results were quantified by densitometry and presente 3s a rato of density of CuZnSOD and MaSOD bands vs those of fractin fom the sample. Data ae shown as the mean SEM of animals. Significant diferences were analyze using unpaired Hest ‘p< 0.05;"*p ~ O.01;"**p < 0.001 versus LZ. ‘and renal cortex (Fg, 3). Although a contribution of CYP epoxygenases to the enhanced NADPH-dependent ROS generation in obese rats ‘cannot be ruled out [4], the marked inbibivory eect of apocynin on (02° and toa lesser extent on H,0s level, suggests major contribution ‘of Nox to oxidative stress i renal tissues from OZR. Interestingly, while NADPHstimulated Oy" levels abolished by apocynin were significantly larger in arteries, and to a lesser extent in renal cortex, of OZR com- pared to LZR (Pig. 3a and b), NADPH-stimulated HO, generation was significantly reduced in both arteries and cortex of obese rats compared tolean controls (ig, Se and d) Basil HzO» levels were also significantly lover in renal cortex of obese rats, These data indicate that augmented Nox-derived Oz" generation contributes renal vascular oxidative sees, Whereas kidney Nox-derived H,0, production was decreased in obesity ‘34. Upregulation of vascular antioxidant defences in OZR Vascular oxidative stress measured as increased 02” generation in renal interlobor arteries vos associated with the enhanced expression of the enzymes dismutating 03° to HsOs, the eytosolie CuZnSOD (Fig. 4a ‘and b) and the mitochondrial MnSOD (Fg. 4e and d) in the kidney of ‘OZR, as depicted by the Western Blot analysis. Immunostaining of a terial sections further demonstated that up-regulation of these ant ‘oxidant enzymes vas particularly evident in VSM of obese rats (ig. ae), Wester blot analysis and immunobistochemistry confirmed that catalase protein levels were also up-regulated and significantly higher in renal arteries from OZR compated to LZR but not in renal cortex (Supplementary Fig 1. 35, Endothelial Nox$ expression, Noxt-derived 1,0; and Nox.mediated ‘endothelium dependent relaxations are reduced in obese rats In order to assess the relative contribution of the diferent Nox subunits to arterial oxidative stress and renal endothelial dysfunction in obese rats, Nox proteins were determined by Western blot and im- munohistochemistry in renal inteslobar arteries and cortex, and the ‘effect of selective Nox inhibitors were evaluated on ROS proditction and fon the H,O;mediated endothelium-dependent relaxations of renal ar teries (41 Endothelial Nox# has recently been identified as a physiological source of vasodilator HO in renal arteries. Immunostaining of arterial Sections showved that Nox# was colocalized with eNOS protein in the endothelium of renal arteries from LZR (Fig. Sa and c), while Nox4 expression was reduced inthe endothelium buts was fond in VSM in OZR (Fig. Sa and c). No apparent differences in either distribution or density of the Nox4 immunolabeling were observed between LZR and OZR in renal eorex (Pig. Sb). Western blot analysis confirmed that total [Nox protein levels were significantly reduced in renal arteries from obese rats compared to controls (Fig, Sd), while they were similar in cortical tissue of LZR and OZR (Fi. Se). ‘The effect ofthe seletive Nox1/4 Inhibitor GKT137831. was next examined on ROS production of intrarenal arteries. GKTI37831 duced both Oa" (Fis. 6a) and HO, (Fis. 6b) formation stimulated by NADPH in LZR and OZR, This compound had a larger inhibitory effect (on 0," production in OZR (i. 6a), while the inhibitory effect om Hy0z generation was reduced in obese animals compared to controls (Fig. 6b). To determine whether changes in the Nox4 metabolism may Noxa oo! 00 UR or GR OR Renal artery Fig. 5. Noxs is down-regulate in renal arteries of obese rats (ab) Inmunobistoshemical demostation of Nox in renal intrlobar artery’ and cortex (glmeri, tubules and arterioles) ofthe rat Kkney from L2A and OZR. () Imminoforescence double Ibeling for eNOS marker and Nox expression showing Nox protein (ed areas colocalized with eNOS (green areas) distributed throughout the endothelial ining of the renal interiobar artery In LZR, and to a lesser extent in the ‘endothelium and extended to VSM In OZR. Scale bars indcate 50 un. Sections are representative of n= 3 animal.) Westem blot analysis of Nox expresion in Samples of renal artery and cortex showing that Noxt protein levels were higher in simples of renal arteries from LZR than in tose of OZR and there were no slferences in samples f renal cortex rom both animals Results were quantified hy densitometry and presented ns ato of density of Nox band vs those of fractin from the sample. Data are shown as the mean = SEM of 4 animal, Significant differences were analyzed using unpaired rte P= Q.001 vers LZR (For Interpretation of the references to colout inthis Rgure legend, the reader is refered tothe Web version ofthis ale) be involved in the endothelial dysfunction observed in renal arteries of OZR, the effect of GRT137831 and of the non-selective Nox4 inhibitor phumbagin was assessed on the endothelium-dependent relaxations elicited by ACh. These relaxant responses were significant inhibited by GKTI37831 and by plumbagin in LZR (Fig. 6¢ and e) thus con: firming the involvement of endothelial Nox derived Ha [4], but this inhibition was leser in lean rats (Fig. 6d and 0. 36, Up-regulation of areral Nox2 is associated to reduced endothelial Nox2-derived 0; ~medited relaxations Nox2-derived HO) is also involved in the endothelium-dependent relaxations of renal arteries [4} and its role in renal endothelial dys fonction of obese rats was assessed, Immunoreaction for Nox2 was found in interlobarsrteries from LZR and OZR (Fis. 7a), while in renal cortes, Nox2 was widely dstibuted in tubular structures in LZR but its expression was markedly reduced in OZR (Fig. Ze). Westem blot an Iysis showed higher Nox2 protein levels in rat intrarenal arteries of OZR compared to LZR (Pig. 7b), while there was a pronounced down regulation of Nox? in renal cortex of obese animals (Fig. 74). The se lective inhibitor of Nox2 Nox2ds-at abolished 02" production stim lated by NADPH in renal interlobar arteries of both LZR and OZR (ig. 80) and reduced HO2 generation in LZR but hardly affeet Hs0 production in OZR (Fig, Sb). To assess whether changes in Nox2-de- tived ROS may also contribute to renal endothelium dysfunction in OZR, renal areries of LZR and) OZR were treated with the selective Nox? inhibitor Nox2ds-tat, which significantly reduced the relaxant responses in lean (Fig. Se) but did not further affect chese responses in renal arteries of abese rats (Vig. Sd) 3.7. Nox rather than Nox preferentaly contributes to kidney vascular oxidative sress and endothelial dysfunction in obesity Noxt has recently been proposed to be the member of the NADPH oxidase family responsible for microvascular dysfunction in metabolic disease [29]. Noxl protein was determined by immunohistochemistry tind Western lott in the kidney of LZR and OZR, Immunostaining of arterial samples with Noxl antibodies revealed a very low expression of| {his subunit in renal arteries and cortex of LZR, but obesity markedly enhanced kidney Nox} expression (Fis. 9a and c). Nex} was found in VSM of renal arteries, and in both renal tubules and vascular tissue lomeruli and arcerioles- in samples of renal cortex of obese ras (Fi, 9a and 6). Wester Blott analysis confirmed that Nox] protein levels were significantly up-regulated in isolated renal arteries (Fig. 9b) and renal cortex (Pig, Sd).of obese animals compared to contol, ‘Augmented NADPH stimulated O>”in renal arteries from OZR were a b - ke i. g z ae 8 s000, pe i: é ce c OLR d oR See ce eee é etalon re) obeys te cr.) teach.) : f gum om 3B Pomtage vo] ee z a ie ac Bs ‘ I | Ba 22 : oTrrTss.4 TT e ss ji Leg cach, ea ach.) Fig. 6. The Nox¢ inhibitors GKT137831 and plumbagin reduce ROS generation and endothelial H,0, mediated relaxations in real arteries oflean and to lessor ‘extent of obese fats. (2b) Effect of the Nox! inhibitor GKTAS7851 (03 uN0) ‘on the NADPH stimulated levels of 0,"(a) and H,0, (b measured by lucigenin. ‘enhanced chemiluminescence and by Amplex Red furescence, respectively, renalarteris of LZR and OZR. Results are expressed in counts per minute cpm) perm oftisve for chemlluminescence and in relative Muorescence units (RU) per mg of tise for uorescence, Bars represent mean SEM of 3-10 animals. ‘Significant diferences between means were analyzed using one-way ANOVA followed by Bonferroni as» posterio test "p= 0.001 vers bast levels, tp = 008; 1H1P = 0.001 versus NADPHstimulsted, ##p = 0.01 versie Lax. (e-) Average Inhibitory eect of the Noxl/4 GKT1S7831 nhblor (0.2 yt (a) and the nonselective Nox inhibitor plambagin (LM) fe.) on the elaxations induced y the endothelial agonist ACh under conditions of NOS ‘and COX blockade (00 uM LNOARG and 0.3 uM indomethacin in renal ar teres of LZR (@e) and OZR (@)-Data are shown asthe mean = SEM of 6-13 arteries fom 7 animals. Significant dferences were analyzed using Student t test for pred observations “p= 0S; “p= O.0) verse control Before significantly reduced by the selective Noxt inhibitor NoxAtds (ig. 10a), and also H,0p levels of LZR and to a lesser extent those of ‘OZR (Pig. 10b), The effect ofthe selective Nox] inhibitor was further ‘evaluated on the relaxant responses to the endothelial agonist ACh under eonditions NOS and COX blockade. NoxAlds havdly affected ACh elicited relaxations in L2R (Fig. 10e) and those in OZR (Pig. 108, thus ling out a major tole of NoxI-derived HO. inthe endothelium: ‘dependent vasodilation of renal arteries. However, inthe absence of I [NOARG to inhibit eNOS and NO formation, selective inhibition of Noxl with NoxAlds significantly improved impaired ACh endothelial edo ty 28 200) 101350, relaxations in OZR thus suggesting that Nox1-derived Os” conteibutes to renal endothelial dysfunction in obesity. 4, Discussion Oxidative sires has been implicated inthe pathogenesis of diabetes and obesity-related microvascular complications including kidney in jury {1,2,30}. The major findings of the present study are that aug ‘mented Nox-derived 0,” f involved in kidney vascular oxidative stress and endothelial dysfunction in obesity but there isa diferental cor tribution of the various Nox members of the NADPH oxidase fay While Nox and Nox2 were up-regulated in renal arteries and con tributed to the inereased O° generation and endothelial dysfunction, reduced endothelial Nox expression was associated with decreased Noxderived HO, and impaired H,0;-mediated vasodilatation which might hinder Nox protective renal vascular effects and thus indirectly promote impaired endothelial function, Renal inflammation and oxidative stress have earlier been found slong with endothelial dysfunction in renal arterioles of genetic and HPD models of obesity (18,23), and initially ascribed to up-regulation of COX-2 that contributed to plasma and renal oxidative stress and augmented vasoconstriction (25,31. The present data confirm en: ‘dathelil dysfunetion in renal interiobar arterioles of genetiealy obese rats, as depicted by the impaired ACh-indiced relaxations; however, antioxidant treatment including non-selective Nox inhibition hardly ameliorated impaired ACh relaxant responses despite augmented basal and ET-L-stimulated O” levels inthe arterial wall, that have been ve: ported to greatly contribute to vascular oxidative stress and reduced endothelial NO bioavailability in obesity [27]. A substantial fraction of the endothelium-dependent responses of renal arterioles is mediated by a non-NO nor-prostanoid relaxing factor and is associated with VSM hyperpolarization [52,33]. When pharmaeologieally dissecting the components of the relaxant responses of renal arteries in OZR, we found thar enhanced non-NO non-prostanold EDH type relaxations comper- sated for impaired NO-mediated responses of renal arteries in obese rats, as depicted by the leser reduction of these relaxations by NOS Inhibition in OZR, but the larger relaxant responses unmasked upon blockade of NO and prostanoid synthess. This ineeased contribution of EDHLtype responses to counteract diminished NO bioavailability is consistent with that recently shown in coronary, mesenteric and sub- cutaneous arteries from diabetic rats and patients [4-36], and ascribed to the enhanced activity of endothelial Kes channels [35]. On the other hand, impaired NO-medioted endothelial dysfunction found for renal lnterlobar arteries of abese rats would also be expected to occu in af ferent arterioles, wherein the relative contribution of NO and non-NO non prostanoid EDH-type factors has ealler been reported to be similar {o that found forthe preglomerular arteries in the present study [33] 0, derived from both CYP 2C epoxygenases and Nox contributes to the EDHAype responses in healthy renal aeteries [4,37], which might explain the lack of significant effect of ROS scavengers on endothelial relaxations of OZR despite augmented arterial oxidative stress. How ver, the enhanced EDH.type relaxant responses observed in renal a teries from obese rats cannot be ascribed ra a larger contrition of ,0- to these responses since they were not further inhibited by eata- lase. Moreover, NADPH-dependent H02 generation was reduced in both renal arteries and cortex of OZR compared to lean controls despite the overall NADPH-dependent 03” production was enhanced in kidney tissues, in particular in intrarenal arteries. Diminished 11:02 levels cannot either be due to reduced antioxidant HaO>‘generating enzymes CuZnSOD and MnSOD, since they were up-regulated in both renal ar terles and corte, in particular the mitochondrial SOD isoform whose expression was markedly enhaneed in vascular tissue including renal arterioles and glomeruli. Interestingly, these results are inline with those recently reported for the diabetic kidney wherein rediced mie tochondrial H.O2 generation measured with Amplex Red was observed despite the enhanced overall Kidney ROS production, suggesting an UR OR edo ty 28 200) 101350, UR OR Fig. 7. Nox? I upregulated in tenal arteries but down-epulated In renal cortex of obese eas. (2.6 Inmunohistochemcal demostration of Nox? in renal attlobar artery (a) and cortex (0) fom ra Kidney of LZR and OZR. Nox2proein was distributed throughout the artecal wali arteries and sterile (A) with high density In ena tubules of LZR that as markedly reduced in OZR (c.Seale bas indent 50 um and 25}. Sections are representative of = 3 animals (b) Wester bot ‘analsls of Nox2 expression in samples of renal artery and eorex showing that Nox? poten levels were higher in renal arteries (b) but downregulated in samples of renal cortex fom OZ (4) compte to LZR. Results were quantified by densitometry and presented asa ratio of density of Nox? hind vs those of actin fom the ‘Simple Data are shown as the mean + SEM of 4 animal Significant difrences wore analyzed uting unpaired etext "p< 0.05; "=p < O01 versus IZ. impaired mitochondrial activity that was ascribed to AMPK dysregu: lation (2,38), Interestingly, in our study a reduced expression of Noxs, mostly restricted to endothelial cells in healthy arteries [4], was also found along with the diminished NADPH-dependent H:02 generation in ronal arteries of obese rats suggesting that down-regulation ofthis Nox “subunit might be involved in the lower levels of arterial 10, More ‘over, Nox4-derived HO production measured with Amplex Red and to 1 leser extent its contribution tothe EDH-type endothelial relaxations ‘of renal arterioles from OZR were also significantly reduced, as de: picted by the lower inhibitory effect of the Nox inhibitors GKT137831 ‘and phumbagin on these responses compared to that om arteries from lean zat. Thus, in contest to Noxt of Nox2 that primarily produce O°, Nox produces H,0 rather than Oy” [29] and has been identified as @ funetional source of ROS generation in the mitochondria of kidney cortex, wherein the mitochondrial SOD effectively dismatates Nox derived superoxide to H;02 [40]. Moreover, mitochondria-derived 1,0; has recently been shovin to effectively contribute tothe EDI-type ‘endothelium-dependent relaxations of renal small arteries [1]. Down-regulation of arterial Nox4 expression and reduced Nox derived H.02 production in genetically obese rats contrast with studies linking Noxé to renal pathology and showing that genetie deficiency or pharmacological inhibition of Nox4 ameliorated enhanced Nox#-de- rived ROS generation, glomerular injury and kidney inflammation and fibrosis in diabetie mice (6,7). However, our results showing lower levels of vascular Noxs coupled to impaired Nox-derived HO gen ‘eration would be consistent with the reduced expression of tubular Nox# and H,0. generation found in hyperglycemic type I diabetic mice and in other models of chronic kidney disease, as well as with the ‘worsening of renal function and fibrosis upon genetic deletion of Nox In diabetic models, whieh suggests a protective role of Nox4 in renal Aisease {9,10}. The protective role of endothelial Noxd as a source of vasodilator HO, in renal arteries recently shown by us is also con- firmed in our study, but the impact of reduced Noxé-derived en- dothelial 0, generation in renal endothelial function of obese rats is Alficult to assess due to the non-NO non-prostanoid EDH-ype me: chanisms chat compensate for the diminished NO bioavalabiity and impaired endothelial vasodilatation [24-26] NNox2 and Noxl have been inked to endothelial and vaseular dys- function in metabolic disease and hypertension [41-45], and both Nox subunits, in particular Nox2, are closely related to vascular inflamma tion [41]. Many metabolic and eardiovascular disorders including in sulin resistance, obesity and atherosclerosis exhibit a chronte low-grade inflammation atthe vascular wall and are associated withthe redox sensitive NFxB inflammatory signaling pathway activated by Nox-de- rived ROS in response to high levels of glucore, LDL cholesterol and free fatty acid (FFA) stimulation [41]. Since obesity is usually related to other metabolic and vascular disorders such as insulin rsistane, ds lipidacmia, impaired glucose tolerance and hypertension, jointly re ferred to as the metabolic syndrome [46], dyslipidemia (hyper ‘wigyeridemia and hypercholesterolemia) and insulin resistance of the loese Zucker rt sed in the present study could in fact account fr the marked up-regelation of pro-inflammatory Nox? and Nox! subunits Cchemiuminescence ‘png x10 Log Acne) Loa ch MM) Fig. 8, Blockade of Noxd reduces O° generation but does not further inhibit HO mediated renal endotheium dependent relanstions of obese rats (30) Effet of the selective Nox2 inhibitor Nox2aetat (1 yl) on the NADPH: rmlated levels of ©," (a) and HO; (b) measured by leigenin-enbancd che riluminescence and by Armplex Red fuorescence, respectively in renal arteries ‘OF 12% and OZR. Results are expressed in cunts er minate (ep) pee mg of tissue for eheiluminescence an in eative orescence units (RFU) pec mg of tissue for Moorescence. Bars represent mean = SEM of 3-10_ animals ‘Sigifcane diferences berwcen means were analyzed using one-eay ANOVA, followed by Bonferroni asa posterio tet "™9p = 0.001 versus basal eves, p= 005; 11P < 0.001 vermis NADPHstimulted, ##p = 0.01 verse LZR. (eal) Average inhibitory fest ofthe Nox? inhibitor Nox2de-at (1 pM on the relaxations induced by the endothelial agonist ACh under conditions of NOS ‘and COX blockade (100 uM LNOARG and 0.3 uM indomethacin in renal ar teres of LZR (e) and OZR (@). Data are shown as the mean = SEM of 6-12 Aareries from 6 animals. Sigitican differences were analyzed. sing paired Student tees p< 0.01 versus contol before eaten. ‘and enhanced Nox-derived oxidative stess found in renal arteries of ‘obese animals [41,46,47]. Expression of Nox2 (gp91phox), the classic NADPH oxidase primarily found in phagocyte cells, followed diferent rend in kidney cortex and renal arterioles of obese rats, Thus, whereas Nox? was up-egulated in renal arteries ts expression was markedly reduced in renal cortex of OZR. Up-regulation of arterial Nox2 was associated with enhanced ROS-generating Nox? activity as shown by the larger PKC-activated and NADPH-

    You might also like