Oxidative Stress and Endothelin-1 in Atherosclerotic Renal Artery Stenosis and Effects of Renal Angioplasty

Aims: To examine biomarkers of oxidative stress (oxs), and endothelin (ET)-1, in hypertensive patients with atherosclerotic renal artery stenosis (ARAS) and to evaluate the effect of percutaneous transluminal renal angioplasty (PTRA). Methods: Baseline measurements were made immediately before renal angiography in patients with suspected ARAS (significant ARAS, n = 83, and non-RAS, n = 59) and in 20 healthy, matched controls. In patients with ARAS, analyses were repeated 4 weeks after PTRA. All patients were treated with statins and acetylsalicylic acid throughout. Results: At baseline there were no significant differences between groups in biomarkers of oxs, whereas high-sensitivity C-reactive protein and blood leukocytes were significantly elevated in group ARAS versus both healthy controls and group non-RAS. Plasma levels of ET-1 and uric acid were significantly increased in group ARAS versus healthy controls prior to angiography and were significantly reduced compared to baseline 4 weeks after PTRA. PTRA had no significant effects on biomarkers of oxs, inflammation or serum creatinine concentrations. Conclusions: ARAS patients on treatment with Received: January 6, 2011 Accepted: April 13, 2011 Published online: June 11, 2011 Aso Saeed, MD Department of Molecular and Clinical Medicine/Nephrology, Institute of Medicine The Sahlgrenska Academy at the University of Gothenburg Vita Stråket 12, Sahlgrenska University Hospital, SE–413 45 Gothenburg (Sweden) Tel. +46 31 342 1000, E-Mail aso.saeed @ vgregion.se © 2011 S. Karger AG, Basel 1420–4096/11/0346–0396$38.00/0 Accessible online at: www.karger.com/kbr D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /6 /2 01 7 7: 13 :3 5 P M Oxidative Stress and ET-1 in ARAS and Effects of Renal Angioplasty Kidney Blood Press Res 2011;34:396–403 397 Elevated levels of angiotensin II (Ang II) due to activation of the renin-angiotensin-aldosterone system (RAAS) could in part contribute to the increase in cardiovascular risk. In addition to its vasoconstrictor effect, Ang II can impair endothelial function, accelerate atherosclerosis and promote cardiovascular remodeling [5, 6] . In addition, Ang II can induce oxidative stress (oxs) by stimulating the production of superoxide through nicotinamide adenine dinucleotide phosphate oxidase [7] . A large number of studies have demonstrated that Ang II increases oxs in experimental models of renovascular hypertension (RVH) [7, 8] . In addition, treatment with superoxide dismutase mimetics, and other antioxidants, has been shown to reduce blood pressure (BP) and to diminish end-organ damage in these models [9, 10] . However, the association between RVH and oxs in humans is less consistent and only a few studies have addressed this issue [11–13] . Endothelin (ET)-1 is a powerful vasoconstrictor peptide with pro-oxidant and growth-promoting effects that is produced by the vascular endothelium in response to Ang II [5, 14] . Experimental data indicate that ET-1, mainly via the ETA receptor, may be an important factor in mediating the hypertensive effects of Ang II [14] . The aim of this hypothesis-generating study was to examine biomarkers of oxs and plasma ET-1 levels in patients with ARAS, and to evaluate the impact of PTRA on these variables during the first month after intervention. Material and Methods Study Participants Between 2003 and 2008, all patients at the Nephrology Section, Sahlgrenska University Hospital, Gothenburg, and the Department of Vascular Diseases at Skåne University Hospital, Malmö, Sweden, undergoing renal angiography for suspected RAS, were considered for this study. Indications for angiography were hypertension (resistant, accelerated, malignant, or with elevation of serum creatinine during treatment with angiotensinconverting enzyme inhibitors or Ang II receptor blockers), hypertension accompanied by a progressive increase in serum creatinine levels, or recurrent pulmonary edema without overt left ventricular dysfunction, together with a positive screening test for RAS by duplex ultrasonography or by CT or MR angiography ( 6 50% diameter stenosis). To avoid pharmacological interference with the RAAS, patients in whom treatment with angiotensinconverting enzyme inhibitors, Ang II receptor blockers or aldosterone receptor antagonists were clearly indicated (e.g. patients with congestive heart failure or diabetic nephropathy) were excluded (for exclusion criteria see table 1 ). In the remaining patients treated with RAAS inhibitors, who were included in the study, these agents were replaced by other antihypertensive drugs 2 weeks prior to renal angiography. Hence, included patients were not on any RAAS-inhibiting drugs during the study period starting from 2 weeks prior to baseline measurements. In addition, only patients with unilateral ARAS were included and individuals with RAS of other etiology, or with either bilateral RAS or stenosis of a solitary kidney, were excluded. Twenty age-matched healthy subjects without any medications were recruited from the database of the Gothenburg MONICA study [15] and served as controls. The Ethics Committees of the Universities of Gothenburg and Lund approved the study and all participants gave written consent to participate. Protocol and Measurements Patients were subjected to baseline measurements 1 day before angiography (see study overview in fig. 1 ). A significant RAS was defined as a lesion with a trans-stenotic mean arterial pressure gradient (MAPG) of 6 10 mm Hg or a 6 50% diameter stenosis on angiography in those cases in which the MAPG was not measured because of technical difficulties due to high-grade stenosis and luminal occlusion during the procedure. Accordingly, 83 patients had significant RAS and underwent PTRA, whereas 59 individuals had no significant RAS and were therefore only subjected to the diagnostic procedure ( fig. 1 ). Systolic and diastolic BP (SBP and DBP) were measured after 5 min rest in the sitting position immediately before and 1 day and 4 weeks after renal angiography. Routine laboratory analyses and biomarkers were measured immediately before renal angiography in all patients. In patients that were subjected to PTRA (n = 83) analyses were repeated 4 weeks after intervention. Estimated GFR (eGFR) was calculated according to the 4-variable equation from the Modification of Diet in Renal Disease (MDRD) Study [16] . Notably, all patients with suspected RAS had been on treatment with a HMG-CoA reductase inhibitor (i.e. statin) for at least 2 weeks at the time of baseline measurements. The majority of patients was already on statin therapy at the time of study inclusion and continued with the same dosage throughout. In the remaining patients, treatment with simvastatin was started at the time of Table 1. E xclusion criteria Renal size <7.5 cm at the stenotic side Age >80 years Pregnancy or nursing CKD stage 5 (eGFR <15 ml/min/1.73 m2) Congestive heart failure RAS of other etiology than atherosclerosis Bilateral RAS or RAS of a solitary kidney Urinary albumin excretion >1 g/day Diabetes mellitus with urinary albumin excretion >0.3 g/day Contraindication for renal angiography/angioplasty (e.g. serious contrast allergy) Other forms of secondary hypertension Malignant disease Treatment with immune modulating drugs, e.g. cyclosporine or oral steroids C KD = Chronic kidney disease; eGFR = estimated glomerular filtration rate according to the 4-variable equation from the Modification of Diet in Renal Disease (MDRD) study; RAS = renal artery stenosis. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /6 /2 01 7 7: 13 :3 5 P M Saeed et al. Kidney Blood Press Res 2011;34:396–403 398 study inclusion with a daily dose of 20 mg that was maintained throughout. In addition, most patients in both hypertensive groups (90% in group ARAS and 74% in non-RAS) were already on low-dose acetylsalicylic acid (ASA) at this time-point, while treatment with ASA was started 1–2 days before PTRA among those patients who were not already on this treatment. Treatments with statins and ASA were maintained unaltered in patients with ARAS throughout the study period. Healthy controls (n = 20) were only studied at one time-point and data were compared with baseline values from hypertensive groups. Biochemical Analyses Standard laboratory methods at the Departments of Clinical Chemistry at Sahlgrenska University Hospital and Skåne University Hospital (SWEDAC approved according to European norm 45001) were used for routine analyses. Plasma renin activity (PRA) was measured by a radioimmunoassay kit (DiaSorin, Stillwater, Minn., USA), with interand intra-assay coefficients of variation (CV)s less than 10%. Plasma concentrations of Ang II (Euro-Diagnostica, Malmö, Sweden) and ET-1 (Nichols Institute Diagnostics, San Juan Capistrano, Calif., USA) were measured by radioimmunoassay kits. The detection limit for ET-1 was 0.25 pg/ ml, the intra-assay CV based on pooled samples was 11.3%, and the inter-assay CV was 22%. Biomarkers of Oxidative Stress Plasma levels of baseline-conjugated dienes in isolated LDLcholesterol (LDL-BDC) were estimated by the method of Ahotupa et al. [17] . In brief, serum LDL-cholesterol (C) was isolated by precipitation with buffered heparin. Lipids were extracted from LDL-C samples by chloroform-methanol, dried under nitrogen, then redissolved in cyclohexane and analyzed spectrophotometrically at 234 nm (Perkin-Elmer Lambda 2 spectrometer). Intraand inter-assay CV were 9.6 and 10.9%, respectively. The levels of LDL-BDC were corrected for serum LDL-C (LDL-BDC:LDL-C) to express the LDL-C oxidation degree. Plasma total antioxidant capacity (TAOC) was determined by Trolox equivalent antioxidant capacity assay according to RiceEvans and Miller [18] with some modification. In brief, potassium peroxodisulfate was used to induce the oxidation of 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) (Aldrich, Deisenhofen, Germany) to form the stable radical cation (ABTS + ). The cation ABTS + was measured photometrically at 734 nm. Antioxidants present in the added plasma caused a reduction in absorption proportional to their concentration. Results are expressed in mmol/l of Trolox equivalent. Plasma protein carbonyls were measured by a colorimetric assay as described by Reznick and Packer [19] using a commercially available kit (Cayman Chemicals, Ann Arbor, Mich., USA). The intraand inter-assay CVs were 4.7 and 8.5% respectively. Urinary 8-iso-PGF 2 was measured in spot urine samples by an 8-isoprostane EIA Kit (Cayman Chemicals). Urinary creatinine was measured by standard enzymatic laboratory methods and the levels of U-8-iso-PGF 2 were corrected for urinary creatinine values. Results are expressed in pg/mg of creatinine. The intraand inter-assay CVs were 18.6 and 29.3%, respectively. Renal Angiography and Angioplasty Digital subtraction angiography was used for evaluating renal arteries. The procedures of renal angiography and PTRA have been described previously [20] . A 4-Fr catheter was used for measurements of intra-arterial pressure gradients. The diameter of stenosis was estimated manually in all cases. Indications for stent placement were angioplasty failure (elastic recoil or flow-limiting dissection resulting in 1 30% residual luminal narrowing, absence of antegrade flow, or significant residual MAPG), or restenosis. Of patients treated with PTRA, 39 (47%) received stents. Statistics Analyses were performed using one-way analysis of variance (ANOVA). If data were not normally distributed, Kruskal-Wallis one-way ANOVA on ranks was used. Unpaired t test or MannWhitney U test was used when appropriate. Bonferroni corrections were made for multiple comparisons. The Pearson correlation (Spearman correlation when data did not meet assumption about normality) coefficient was used to evaluate correlations. All tests were two-tailed and p values ! 0.05 were considered significant. Results are presented as means 8 SD. Software SPSS 18.0.0 for Windows (Release 2009; SPSS Inc., Chicago, Ill., USA) was used. ARAS: trans-stenotic