1. The site and its setting 2. Historical background 3. Field methodologies: Excavation strategy and techniques 4. The analysis and presentation of the results 5. Stratification and architecture: Early Iron Age (Levels I-III) 6. Stratification and architecture: Middle Iron Age (Neo-Hittite Levels IV-VII) 7. Stratification and architecture: Later Iron Age (Neo-Assyrian and later, Levels VIII and IX) 8. Stratification and architecture: The Latest Iron Age (Level X, 'Achaemenid') 9. Bibliography for Part 1
Endothelin (ET)-1 is implicated in the development of hypertension and a role for endothelin receptor antagonists (ETRAs) in the management of hypertension is emerging. ETRAs are classified as selective or mixed depending on their degree of ET(A):ET(B) receptor blockade. As yet, there are no comparative studies in humans that measure biochemical and functional ET(B) blockade achieved by currently licensed ETRAs. We therefore investigated the effects of bosentan, a mixed ETRA, and sitaxsentan, an ET(A) selective ETRA, on plasma ET-1 concentrations and ET(B)-mediated vasodilatation to ET-3. In a randomized, double-blind, 3-way crossover study, 10 healthy subjects received 7 days of placebo, bosentan 250 mg, and sitaxsentan 100 mg daily. Plasma ET-1 concentrations were measured at baseline and 3 hours on day 1 and predose on day 7. Subjects also underwent forearm blood flow measurements on day 7 of each period with brachial artery infusion of ET-3 (60 pmol/min for 5 minutes). Bosentan, but not placebo or sitaxsentan, significantly increased plasma ET-1 concentrations at day 7 (+0.70+/-0.20 pg/mL; P<0.005). Maximal ET-3-mediated vasodilatation was seen at 2 minutes following placebo (30+/-6%) and sitaxsentan (21+/-11%); however, this was abolished by bosentan, with a reduction in forearm blood flow of 8+/-3% (P<0.01 versus placebo and sitaxsentan). Bosentan but not sitaxsentan increases circulating plasma ET-1 levels and abolishes acute ET-3-mediated vasodilatation, confirming that the mixed ET(A/B) antagonist bosentan, but not the selective ET(A) antagonist sitaxsentan, causes functional ET(B) blockade at clinically relevant doses in healthy human subjects.
Chronic inflammation contributes to the development and progression of chronic kidney disease (CKD). Identifying renal inflammation early is important. There are currently no specific markers of renal inflammation. Endothelin-1 (ET-1) is implicated in the pathogenesis of CKD. Thus, we investigated the impact of progressive renal dysfunction and renal inflammation on plasma and urinary ET-1 concentrations. In a prospective study, plasma and urinary ET-1 were measured in 132 subjects with CKD stages 1 to 5, and fractional excretion of ET-1 (FeET-1) was calculated. FeET-1, serum C-reactive protein (CRP), urinary ET-1:creatinine ratio, and urinary albumin:creatinine ratio were also measured in 29 healthy volunteers, 85 subjects with different degrees of inflammatory renal disease but normal renal function, and in 10 subjects with rheumatoid arthritis without renal involvement (RA). In subjects with nephritis associated with systemic lupus erythematosus (SLE), measurements were done before and after 6 mo of treatment. In subjects with CKD, plasma ET-1 increased linearly as renal function declined, whereas FeET-1 rose exponentially. In subjects with normal renal function, FeET-1 and urinary ET-1:creatinine ratio were higher in SLE subjects than in other groups (7.7 +/- 2.7%, 10.0 +/- 3.0 pg/mumol, both P < 0.001), and correlated with CRP, and significantly higher than in RA subjects (both P < 0.01) with similar CRP concentrations. In SLE patients, following treatment, FeET-1 fell to 3.6 +/- 1.4% (P < 0.01). Renal ET-1 production increases as renal function declines. In subjects with SLE, urinary ET-1 may be a useful measure of renal inflammatory disease activity while measured renal function is still normal.
This study compared the molecular lipidomic profile of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identified 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profile in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N-acyltaurines were significantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were significantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identified individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These findings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profile of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease. This study compared the molecular lipidomic profile of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identified 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profile in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N-acyltaurines were significantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were significantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identified individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These findings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profile of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease. Chronic kidney disease (CKD) is a serious and increasingly common condition (1Meguid El Nahas A. Bello A.K. Chronic kidney disease: the global challenge.Lancet. 2005; 365: 331-340Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar). Patients with CKD have a greatly increased risk of CVD, which represents the most common cause of mortality and morbidity in these patients, to the extent that CKD is considered an independent risk factor for CVD (2Sarnak M.J. Levey A.S. Schoolwerth A.C. Coresh J. Culleton B. Hamm L.L. McCullough P.A. Kasiske B.L. Kelepouris E. Klag M.J. et al.Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association councils on kidney in cardiovascular disease, high blood pressure research, clinical cardiology, and epidemiology and prevention.Circulation. 2003; 108: 2154-2169Crossref PubMed Scopus (2881) Google Scholar, 3Schiffrin E.L. Lipman M.L. Mann J.F.E. Chronic kidney disease: effects on the cardiovascular system.Circulation. 2007; 116: 85-97Crossref PubMed Scopus (1195) Google Scholar). In CKD, many conventional risk factors for CVD are prevalent, including hypertension, dyslipidemia, and insulin resistance. Underlying conditions that are typical of CVD also occur, such as heightened inflammatory status, oxidative stress, endothelial dysfunction, and arterial stiffness (3Schiffrin E.L. Lipman M.L. Mann J.F.E. Chronic kidney disease: effects on the cardiovascular system.Circulation. 2007; 116: 85-97Crossref PubMed Scopus (1195) Google Scholar, 4Zoccali C. Traditional and emerging cardiovascular and renal risk factors: an epidemiologic perspective.Kidney Int. 2006; 70: 26-33Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Consequently, understanding the factors in CKD that could contribute to increased CVD risk is very important. In CVD there is a clearly established link between dyslipidemia (specifically hypercholesterolemia and hypertriglyceridemia) and atherosclerosis, an underlying pathology of most CVD (5Steinberg D. Hypercholesterolemia and inflammation in atherogenesis: two sides of the same coin.Mol. Nutr. Food Res. 2005; 49: 995-998Crossref PubMed Scopus (83) Google Scholar, 6Shepherd J. Lipids in health and disease.Biochem. Soc. Trans. 2004; 32: 1051-1056Crossref PubMed Scopus (13) Google Scholar). In view of the clear cardio-renal relationship, there has been considerable interest in the possible contribution of hyperlipidemia to CKD-associated CVD (7Keane W.F. Tomassini J.E. Neff D.R. Lipid abnormalities in patients with chronic kidney disease: implications for the pathophysiology of atherosclerosis.J. Atheroscler. Thromb. 2013; 20: 123-133Crossref PubMed Scopus (69) Google Scholar, 8Cheung A.K. Is lipid control necessary in hemodialysis patients?.Clin. J. Am. Soc. Nephrol. 2009; 4: S95-S101Crossref PubMed Scopus (23) Google Scholar). The nature of this lipid imbalance is significantly different to nonrenal-related CVD; in particular, the relationship with cholesterol level is less clear than in the general population and is dependent on the stage of disease (9Kaysen G.A. Lipid and lipoprotein metabolism in chronic kidney disease.J. Ren. Nutr. 2009; 19: 73-77Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10Vaziri N.D. Norris K. Lipid disorders and their relevance to outcomes in chronic kidney disease.Blood Purif. 2011; 31: 189-196Crossref PubMed Scopus (103) Google Scholar). In some patients, total cholesterol and LDL-cholesterol are not elevated, while patients on hemodialysis may even have reduced cholesterol compared with control subjects (11Lacquaniti A. Bolignano D. Donato V. Bono C. Fazio M.R. Buemi M. Alterations of lipid metabolism in chronic nephropathies: mechanisms, diagnosis and treatment.Kidney Blood Press. Res. 2010; 33: 100-110Crossref PubMed Scopus (30) Google Scholar). It is apparent that CKD involves multiple lipid abnormalities, some of which may contribute to increased CVD risk. However, most studies in lipid abnormalities in CKD have focused on lipoprotein profile or on overall lipid classes such as triglycerides. While in many inflammatory diseases, including preeclampsia (12Romanowicz L. Bankowski E. Sphingolipids of human umbilical cord vein and their alteration in preeclampsia.Mol. Cell. Biochem. 2010; 340: 81-89Crossref PubMed Scopus (7) Google Scholar), diabetes (13Barber M.N. Risis S. Yang C. Meikle P.J. Staples M. Febbraio M.A. Bruce C.R. Plasma lysophosphatidylcholine levels are reduced in obesity and type 2 diabetes.PLoS ONE. 2012; 7: e41456Crossref PubMed Scopus (240) Google Scholar), rheumatoid arthritis (14Giera M. Ioan-Facsinay A. Toes R. Gao F. Dalli J. Deelder A.M. Serhan C.N. Mayboroda O.A. Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC-MS/MS.Biochim. Biophys. Acta. 2012; 1821: 1415-1424Crossref PubMed Scopus (163) Google Scholar), and Crohn's disease (15Sewell G.W. Hannun Y.A. Han X. Koster G. Bielawski J. Goss V. Smith P.J. Rahman F.Z. Vega R. Bloom S.L. et al.Lipidomic profiling in Crohn's disease: abnormalities in phosphatidylinositols, with preservation of ceramide, phosphatidylcholine and phosphatidylserine composition.Int. J. Biochem. Cell Biol. 2012; 44: 1839-1846Crossref PubMed Scopus (38) Google Scholar), lipidomic studies have identified characteristic lipid signatures that have potential as diagnostic tools, there have as yet been few attempts at profiling individual lipids in CKD. Evidence for an altered phospholipid profile in CKD (16Jia L. Wang C. Zhao S. Lu X. Xu G. Metabolomic identification of potential phospholipid biomarkers for chronic glomerulonephritis by using high performance liquid chromatography-mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007; 860: 134-140Crossref PubMed Scopus (37) Google Scholar) and a decrease of serum sulfatide (ST) levels in patients with end-stage renal failure (ESRF) (17Hu R. Li G. Kamijo Y. Aoyama T. Nakajima T. Inoue T. Node K. Kannagi R. Kyogashima M. Hara A. Serum sulfatides as a novel biomarker for cardiovascular disease in patients with end-stage renal failure.Glycoconj. J. 2007; 24: 565-571Crossref PubMed Scopus (35) Google Scholar) have been reported, but otherwise little is known about molecular changes. Modern lipidomics depends almost entirely on analysis by electrospray MS, as this is able to identify a very wide variety of individual lipid species in several classes. Both shotgun lipidomics, involving direct infusion of the sample into the instrument, and LC/MS are widely used for this purpose (18Han X. Yang K. Gross R.W. Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses.Mass Spectrom. Rev. 2012; 31: 134-178Crossref PubMed Scopus (417) Google Scholar). Chromatographic separation provides additional information to facilitate lipid identification, and separation of the lipids reduces interference (19Lam S.M. Shui G.H. Lipidomics as a principal tool for advancing biomedical research.J. Genet. Genomics. 2013; 40: 375-390Crossref PubMed Scopus (87) Google Scholar). Although with lower-resolution instruments MS/MS is necessary to distinguish lipids of similar mass but different formula, modern high-resolution instruments such as orbitraps offer sufficient mass accuracy that isobaric species can be distinguished, thus allowing classification of lipid analytes and identification of the total number of carbons and double bonds in the acyl chains by a top-down approach (20Schwudke D. Schuhmann K. Herzog R. Bornstein S.R. Shevchenko A. Shotgun lipidomics on high resolution mass spectrometers.Cold Spring Harb. Perspect. Biol. 2011; 3: a004614Crossref PubMed Scopus (145) Google Scholar). It has been demonstrated that this untargeted approach, coupled with principle component analysis, can be used without internal standards for comparative analysis of lipidomes, owing to the high dynamic range of the orbitrap (21Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (150) Google Scholar). Similar “semiquantitative” approaches on a triple quadrupole instrument have also been used for comparative lipidomics in CVD (22Stegemann C. Drozdov I. Shalhoub J. Humphries J. Ladroue C. Didangelos A. Baumert M. Allen M. Davies A.H. Monaco C. et al.Comparative lipidomics profiling of human atherosclerotic plaques.Circ. Cardiovasc. Genet. 2011; 4: 232-242Crossref PubMed Scopus (166) Google Scholar, 23Stegemann C. Pechlaner R. Willeit P. Langley S.R. Mangino M. Mayr U. Menni C. Moayyeri A. Santer P. Rungger G. et al.Lipidomics profiling and risk of cardiovascular disease in the prospective population-based Bruneck study.Circulation. 2014; 129: 1821-1831Crossref PubMed Scopus (340) Google Scholar). However, MS/MS or MSn is still required for confirmation of individual acyl chain length and double bonds. We recently demonstrated that a top-down lipidomics approach using LC/MS on a high resolution instrument (Orbitrap Exactive) was able to identify more than 350 individual lipid species or isomeric lipid clusters in normo-lipidemic LDL (24Reis A. Rudnitskaya A. Blackburn G.J. Fauzi N.M. Pitt A.R. Spickett C.M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.J. Lipid Res. 2013; 54: 1812-1824Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The lipids were identified by matching the experimental m/z for the molecular ions to calculated mono-isotopic masses available in lipidomic and metabolic databases. LDL is an important carrier of a wide variety of lipid species within the plasma and reflects systemic changes in lipid metabolism. We hypothesized that the application of this methodology to CKD would identify novel differences in lipid profile at a molecular level between disease and control samples that would enhance understanding of the disease mechanisms and offer potential as diagnostic markers. All chemicals used were of analytical quality and purchased from Sigma-Aldrich (UK) or ThermoFisher (UK) unless stated otherwise. Organic solvents were HPLC-grade and purchased from Fisher Scientific (Loughborough, UK). Male CKD patients (stage 4/5) were recruited from the renal outpatient clinic at the Royal Infirmary of Edinburgh following ethical approval by NHS Lothian Research Ethics Committee and gave informed consent as described previously (25Lilitkarntakul P. Dhaun N. Melville V. Blackwell S. Talwar D.K. Liebman B. Asai T. Pollock J. Goddard J. Webb D.J. Blood pressure and not uraemia is the major determinant of arterial stiffness and endothelial dysfunction in patients with chronic kidney disease and minimal co-morbidity.Atherosclerosis. 2011; 216: 217-225Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Renal patients were excluded on the basis of renal transplant, dialysis, systemic vasculitis or connective tissue disease, a history of established CVD, peripheral vascular disease, diabetes mellitus, respiratory disease, neurological disease, alcohol abuse, or treatment with an organic nitrate or β-agonist. The causes of kidney disease in patients were autosomal dominant polycystic kidney disease (n = 4), IgA nephropathy (n = 2), reflux nephropathy (n = 3), and neurogenic bladder (n = 1). Smokers and hypercholesterolemic patients were not excluded, but the latter were controlled by statin medication (two individuals in the disease group) and stable on treatment for 3 months prior to inclusion in the study. Subjects refrained from alcohol for at least 24 h, and caffeinated drinks and smoking for at least 12 h before the study. Blood samples were collected in polypropylene tubes containing EDTA (final concentration, 1 mg/ml of blood); plasma was promptly separated by centrifugation (2,500 g, 20 min, 4°C) and stored in 2 ml aliquots at −80°C in the dark. Blood pressure, high sensitivity C-reactive protein, oxidized LDL (OxLDL), and interleukin (IL)-6 were determined as described previously (25Lilitkarntakul P. Dhaun N. Melville V. Blackwell S. Talwar D.K. Liebman B. Asai T. Pollock J. Goddard J. Webb D.J. Blood pressure and not uraemia is the major determinant of arterial stiffness and endothelial dysfunction in patients with chronic kidney disease and minimal co-morbidity.Atherosclerosis. 2011; 216: 217-225Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Other parameters [plasma glucose, total cholesterol, triglyceride, lipoproteins, creatinine, and glycated hemoglobin (HbA1c)] were determined in the hospital biochemistry laboratory by assays validated to Good Laboratory Practice standard. LDL was isolated from plasma aliquots essentially as described previously (26Jerlich A. Pitt A.R. Schaur R.J. Spickett C.M. Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS.Free Radic. Biol. Med. 2000; 28: 673-682Crossref PubMed Scopus (89) Google Scholar). KBr (0.3816 mg) was dissolved in 1 ml of plasma at 4°C and underlaid below 4.1 ml of a deoxygenated EDTA solution, before centrifuging in a Beckman VTi 90 rotor for 2 h at 60,000 rpm to generate a density gradient. LDL formed bands in the density range 1.019–1.060 g/ml. The LDL collected was stored in sterile vials under nitrogen and desalted before determining the cholesterol content using CHOL PAD reagent (Roche Diagnostics), and protein concentration of isolated LDL was determined by the Bradford assay as reported by Yue et al. (27Yue H. Jansen S.A. Strauss K.I. Borenstein M.R. Barbe M.F. Rossi L.J. Murphy E. A liquid chromatography/mass spectrometric method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites prostaglandins, dihydroxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids in rat brain tissue.J. Pharm. Biomed. Anal. 2007; 43: 1122-1134Crossref PubMed Scopus (83) Google Scholar). Purity of isolated LDL was confirmed by polyacrylamide gel electrophoresis (24Reis A. Rudnitskaya A. Blackburn G.J. Fauzi N.M. Pitt A.R. Spickett C.M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.J. Lipid Res. 2013; 54: 1812-1824Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 28Mahley R.W. Innerarity T.L. Rall Jr, S.C. Weisgraber K.H. Plasma lipoproteins: apolipoprotein structure and function.J. Lipid Res. 1984; 25: 1277-1294Abstract Full Text PDF PubMed Google Scholar). Vitamin E content (α-tocopherol) in LDL was determined by reverse-phase chromatography using spectrophotometric detection as described previously (29Zhao B. Tham S.Y. Lu J. Lai M.H. Lee L.K. Moochhala S.M. Simultaneous determination of vitamins C, E and beta-carotene in human plasma by high-performance liquid chromatography with photodiode-array detection.J. Pharm. Pharm. Sci. 2004; 7: 200-204PubMed Google Scholar). The samples and standards were injected randomly in triplicate and area under the curve was plotted against the calibration curves and used to calculate the concentration of vitamin E in the samples (μg/mg protein). Standards (0.1–10 μg/ml), made up in methanol and extracts redissolved in 100 μl methanol, were analyzed in triplicate by injection of 20 μl. The intraday cross-validation (CV; n = 3) at a concentration of 2.5 μg/ml was 3.1%. Statistical analysis was carried out using an unpaired t-test, with Welch's correction to estimate the P values. The particle size of LDL was assessed by 1% agarose gel electrophoresis in barbital buffer as described previously (30Aldred S. Griffiths H.R. Oxidation of protein in human low-density lipoprotein exposed to peroxyl radicals facilitates uptake by monocytes; protection by antioxidants in vitro.Environ. Toxicol. Pharmacol. 2004; 15: 111-117Crossref PubMed Scopus (10) Google Scholar). Retardation factors were defined as the distance (cm) traveled by sample/distance (cm) traveled by dye front. LDL lipids were extracted from LDL containing 25 μg protein by the Folch method as described recently (24Reis A. Rudnitskaya A. Blackburn G.J. Fauzi N.M. Pitt A.R. Spickett C.M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.J. Lipid Res. 2013; 54: 1812-1824Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The lipid extracts were combined into an amber vial (Supelco), dried under a stream of nitrogen filtered with a 0.22 µm mesh (Millipore), and stored at −70°C until further analysis. Mean recovery (%) of phosphatidylcholine (PC; 13:0/13:0) lipid standard in spiked LDL samples by the Folch method was 103.9 ± 8.6. Similar recoveries were achieved with dehydroepiandrosterone sulfate as a representative of more polar lipid classes, and d5-myristic acid (Sigma Aldrich Chemical Co., UK) as a representative of less polar lipids. Lipid extracts were solubilized in 100 µL CHCl3-methanol (1:1, v/v), further diluted in methanol and analyzed by LC/MS essentially as described previously (24Reis A. Rudnitskaya A. Blackburn G.J. Fauzi N.M. Pitt A.R. Spickett C.M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.J. Lipid Res. 2013; 54: 1812-1824Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Separation of LDL lipid classes was performed using a Dionex Ultimate 3000 HPLC system (Thermo Scientific, Hemel Hempstead, UK) by injection of 10 μl sample onto a silica gel column (150 mm × 3 mm × 3 µm; HiChrom, Reading, UK) used in hydrophilic interaction chromatography mode (24Reis A. Rudnitskaya A. Blackburn G.J. Fauzi N.M. Pitt A.R. Spickett C.M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.J. Lipid Res. 2013; 54: 1812-1824Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Two solvents were used: (A) 20% isopropyl alcohol (IPA) in acetonitrile and (B) 20% IPA in ammonium formate (20 mM). Elution was achieved using the following gradient at 0.3 ml/min: elution at 5% B for 1 min, followed by a rise to 9% B at 5 min, to 15% B at 10 min, to 25% B at 16 min, to 35% B at 23 min, and from 28 to 40 min a decrease to 5% B. Detection of lipids was performed in a Orbitrap Exactive Mass Spectrometer (ThermoFisher Scientific Inc., Bremen, Germany) equipped with polarity switching. The instrument was calibrated according to the manufacturer specifications to give an rms mass error <2 ppm. The following electrospray ionization settings were used: source voltage, ±4.50 kV; capillary voltage, 25 V; capillary temperature, 320°C; sheath gas flow, 50 AU; aux gas flow, 17 AU; sweep gas flow, 0 AU. All LC/MS spectra were recorded in the m/z range 100–1200 at 50,000 resolution (Full Width at Half Maximum at m/z =500). Three microscans were collected per data point with the injection time limited by either an automatic gain control target ion intensity of 106 or a maximum inject time of 250 ms. For certain lipids of interest, MS/MS was carried out on an LTQ Orbitrap instrument (ThermoElectron, Hemel Hempstead, UK) controlled by Xcalibur (version 2.0, Thermo Fisher Corporation) in either positive or negative ion modes as appropriate for the best detection of the parent ion. The capillary voltage was set at 4.5 kV, capillary temperature at 275°C, with sheath gas and sweep gas flow rates set at 30 and 10 AU, respectively. Collision energy was set according to the ion of interest, typically between 25 and 35 (arbitrary units). In the first stage, LC/MS data were analyzed and lipid species identified by manual matching of retention times and accurate mass data to a home-built database and the Human Metabolome project database (HMDB) (31Wishart D.S. Knox C. Guo A.C. Eisner R. Young N. Gautam B. Hau D.D. Psychogios N. Dong E. Bouatra S. et al.HMDB: a knowledgebase for the human metabolome.Nucleic Acids Res. 2009; 37: D603-D610Crossref PubMed Scopus (1503) Google Scholar), with identifications based on ions showing a mass error of <5 ppm (and in most cases <2 ppm) to the monoisotopic mass calculated from the theoretical formula. A total of 352 lipids were identified by this approach. Subsequently, LC/MS data were analyzed by filtering with MZMatch (32Scheltema R.A. Jankevics A. Jansen R.C. Swertz M.A. Breitling R. PeakML/mzMatch: a file format, Java library, R library, and tool-chain for mass spectrometry data analysis.Anal. Chem. 2011; 83: 2786-2793Crossref PubMed Scopus (216) Google Scholar) followed by using the XCMS pipeline [XCMS Online version 0.0.83, Scripps Center for Metabolomics, https://xcmsonline.scripps.edu/ (33Tautenhahn R. Patti G.J. Rinehart D. Siuzdak G. XCMS Online: a web-based platform to process untargeted metabolomic data.Anal. Chem. 2012; 84: 5035-5039Crossref PubMed Scopus (836) Google Scholar)] for peak detection, alignment, and isotope annotation as described previously (24Reis A. Rudnitskaya A. Blackburn G.J. Fauzi N.M. Pitt A.R. Spickett C.M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL.J. Lipid Res. 2013; 54: 1812-1824Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Ions with intensity <5,000 cps were excluded. Integration of features extracted in different samples corresponds to the reported extracted ion chromatogram areas. Peak intensities for the ions identified from individual lipid classes in the data sets were summed and used to evaluate overall differences in disease versus age-matched control groups. Extracted features were included if they were present in >50% of the samples in each group, within 2.5 ppm from the exact monoisotopic mass, and with <5 s retention time deviation. In order to prevent overestimation of the number of lipid species identified, all lipid species detected in positive and negative ion modes were manually cross-referenced. Overall, 142 and 158 individual lipids were identified in positive and negative ion modes, respectively. Isomeric species are reported as one single ion, for instance PC(16:0/18:1), PC(18:1/16:0), PC(16:1/18:0), PC(18:0/16:1), PC(14:0/20:1), and others are expressed as PC(34:1). The data processing steps and number of features or lipids identified at each stage are summarized in supplementary Fig. 1. The merged data set comprising 300 lipids species (supplementary Fig. 1) was further analyzed using partial least squares discriminant analysis (PLSDA) (34Wold S. Sjöström M. Eriksson L. PLS-regression: a basic tool of chemometrics.Chemom. Intell. Lab. Syst. 2001; 58: 109-130Crossref Scopus (6871) Google Scholar, 35Barker M. Rayens W. Partial least squares for discrimination.J. Chemom. 2003; 17: 166-173Crossref Scopus (2021) Google Scholar). PLSDA calibration models were validated using segmented CV, and optimization of PLSDA models was achieved using the variable importance in projection (VIP) score (36Chi-Hyuck J. Lee S.H. Park H.S. Lee J.H. Use of partial least squares regression for variable selection and quality prediction. In Computers & Industrial Engineering, 2009. CIE 2009. International Conference on Computers &. Industrial Engineering, University of Technology of Troyes. IEEE, New York2009: 1302-1307Google Scholar). A VIP cut-off value of 0.8 was repeatedly applied to eliminate less discriminating variables, with a cut-off of 0.85 for the merged set. The final classification model included 48 species detected in the positive mode and 55 in the negative mode. The statistical significance of the classification PLSDA models was assessed using permutation testing with 1,000 permutations (37Westerhuis J.A. Hoefsloot H.C.J. Smit S. Vis D.J. Smilde A.K. van Velzen E.J.J. van Duijnhoven J.P.M. van Dorsten F.A. Assessment of PLSDA cross validation.Metabolomics. 2008; 4: 81-89Crossref Scopus (1016) Google Scholar). Q2 was used as quality-of-fit criterion for the permutation test (38Westerhuis J.A. Velzen E.J.J. Hoefsloot H.C.J. Smilde A.K. Discriminant Q2 (DQ2) for improved discrimination in PLSDA models.Metabolomics. 2008; 4: 293-296Crossref Scopus (61) Google Scholar). Further details are given in supplementary Methods. Statistical analysis of clinical and biochemical parameters was conducted using nonparametric t-tests (Mann-Whitney) using two-tailed P value calculation, and values with P < 0.05 were considered statistically significant. Baseline measurements of clinical and biochemical parameters for age- and body mass-matched subjects included in this study are summarized in Table 1. Glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease study equation and confirmed all patients as stage 4 or 5 CKD; they also had significantly increased systolic blood pressure. There were no significant differences in levels of glycated hemoglobin and plasma glucose. The inflammatory marker C-reactive protein was significantly elevated, although IL-6 was not. The levels of total plasma cholesterol and LDL were not altered with CKD, and there was no change in OxLDL. In contrast, HDL levels showed a significant decrease, and plasma triglycerides were elevated, as expected for patients with CKD and published previously (25Lilitkarntakul P. Dhaun N. Melville V. Blackwell S. Talwar D.K. Liebman B. Asai T. Pollock J. Goddard J. Webb D.J. Blood pressure and not uraemia is the major determinant of arterial stiffness and endothelial dysfunction in patients with chronic kidney disease and minimal co-morbidity.Atherosclerosis. 2011; 216: 217-225Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). LDL vitamin E content and particle heterogeneity (electrophoretic mobility) were also determined, but there was no statistical difference (Table 1).TABLE 1Clinical biochemistry parameters in plasma for control subjects and CKD patientsClinical ParametersControlsCKDPn1010—Age (years)47 ± 644 ± 30.111BMI (kg/m2)26 ± 229 ± 60.113Smokers/ex-smokers/nonsmokers0/1/92/2/8—Systolic blood pressure (mm Hg)113 ± 12124 ± 100.049Diastolic blood pressure (mm Hg)72 ± 1178 ± 60.103Mean arterial pressure (mm Hg)85 ± 1193 ± 70.065Pulse pressure (mm Hg)42 ± 646 ± 70.189Plasma glucose (mg/dl)5.1 ± 0.54.8 ± 0.40.231HbA1c (% of Hb)5.3 ± 0.405.6 ± 0.500.117Serum creatinine (mg/dl)85 ± 11460 ± 179<0.0001MDRD eGFR (ml/min/1.73 m2)91.2 ± 14.114.8 ± 5.3<0.0001High sensitivity C-reactive protein (µg/ml)1.2 ± 1.54.2 ± 3.50.027IL-6 (pg/ml)9.6 ± 10.57.9 ± 8.70.713Total cholesterol (mg/dl)5.1 ± 0.84.5 ± 0.80.130Triglycerides (mM)1.0 ± 0.31.8 ± 0.70.004HDL (mM)1.4 ± 0.51.0 ± 0.20.020LDL (mM)4.8 ± 0.7 (n = 9)4.2 ± 0.80.091OxLDL (U/l)56 ± 1851 ± 120.475LDL vitamin E (μg/mg protein)2.43 ± 0.5402.39 ± 0.5240.65LDL particle size (nm)0.24 ± 0.020.24 ± 0.030.387Values are given as mean ± SD. Significance (P values) was calculated using a two-tailed Student's t-test, and statistically significant differenc
Abstract The connection between sepsis and acute renal failure is a ‘two-way street’. Sepsis is a dominant cause of acute renal failure and renal dysfunction is a common and important component of the sepsis syndrome and consequent multiorgan failure. In turn, patients with acute renal failure are at increased risk of infective complications which at best prolong their illness and at worst may contribute to mortality. This chapter deals with both sides of this relationship in turn, beginning with the pathophysiology of the sepsis syndrome and the role of sepsis in the causation of acute renal failure.
The endothelins comprise a family of potent vasoconstricting peptides. Endothelin-1 appears to be the predominant isoform produced by the vascular endothelium, acting mainly in a paracrine fashion on vascular smooth muscle cells to cause vasoconstriction. It also has a range of other local actions - in the kidney, in the nervous system and on other hormone systems - that could, potentially, play a part in the genesis of hypertension. The association of raised plasma endothelin concentrations in human hypertension has caused much interest, but the literature is not consistent. Given the generally low plasma concentration of the endothelins, and their mainly paracrine actions, it remains unclear whether plasma endothelin has a functional role in hypertension. Additionally, problems remain with the measurement of plasma endothelin that raise doubts about the validity of conclusions drawn from these measurements.
Evidence suggests that urinary excretion of endothelin-1 (ET-1) reflects renal ET-1 production and is independent of systemic ET-1 activity. The influence of ET receptors on urinary ET-1 excretion has not been studied in humans, yet peritubular ETB receptors are abundant within the kidney. We have studied the effects of acute ETA and ETB receptor blockade with BQ-123 and BQ-788, respectively, on urinary ET-1 excretion in a randomized, placebo-controlled, double-blind study in 16 subjects with a wide range of GFRs (15-152 ml/min). Plasma ET-1 concentrations (pET-1) and urinary ET-1 excretion rate (uET-1) at baseline correlated inversely with GFR (R2 = 0.18 and 0.36, respectively, P < 0.01). However, changes in pET-1 after ET receptor antagonism were not related to changes in uET-1 (R2 = 0.007, P = 0.18). pET-1 increased only after BQ-788, alone or in combination with BQ-123, consistent with ETB receptor-mediated clearance of ET-1 from the circulation. uET-1 was reduced only after BQ-788 alone [-4.7 pg/min (SD 5.5), P < 0.01]. Because BQ-788 also reduced GFR, fractional excretion of ET-1 (FeET-1) was calculated. FeET-1 fell after BQ-788 alone [-41% (SD 26%), P < 0.01] or in combination with BQ-123 [-40% (SD 29%), P < 0.01]. FeET-1 was not altered by placebo or BQ-123 alone. In conclusion, urinary ET-1 excretion does not appear to relate to the pool of plasma ET-1. Because of the short duration of this study, it is unlikely that ET receptor blockade had significant effects on renal ET-1 production. Therefore, the reduction in FeET-1 after ETB blockade appears to indicate that renal excretion of ET-1 is at least partly facilitated by ETB receptor activation.
Animal studies suggest that endothelin A (ETA) receptor antagonism and angiotensin-converting enzyme (ACE) inhibition may be synergistic. This interaction and the role of ETB receptors and endothelial mediators were investigated in terms of systemic and renal effects in humans in two studies. In one study, six subjects received placebo, the ETA receptor antagonist BQ-123 alone, and BQ-123 in combination with the ETB receptor antagonist BQ-788 after pretreatment with the ACE inhibitor enalapril (E) or placebo. In the other, six subjects who were pretreated with E received placebo, BQ-123, and BQ-123 with concomitant inhibition of nitric oxide (NO) synthase or cyclo-oxygenase (COX). Both were randomized, double-blind, crossover studies. Mean arterial pressure was reduced by BQ-123, an effect that was doubled during ACE inhibition (mean area under curve ± SEM; BQ-123, −2.3 ± 1.8%; BQ-123+E, −5.1 ± 1.1%; P < 0.05 versus placebo). BQ-123 increased effective renal blood flow (BQ-123, −0.1 ± 2.4%; BQ-123+E, 10.9 ± 4.2%; P < 0.01 versus BQ-123), reduced effective renal vascular resistance (BQ-123, −1.2 ± 3.1%; BQ-123+E, −12.8 ± 3.0%; P < 0.01 versus placebo and versus BQ-123), and increased urinary sodium excretion markedly (BQ-123, 2.6 ± 12.8%; BQ-123+E, 25.2 ± 12.6%; P < 0.05 versus BQ-123, P < 0.01 versus placebo and versus E) only during ACE inhibition. These effects were abolished by both ETB receptor blockade and NO synthase inhibition, whereas COX inhibition had no effect. In conclusion, the combination of ETA receptor antagonism and ACE inhibition is synergistic via an ETB receptor–mediated, NO-dependent, COX-independent mechanism. The reduction of BP and renal vascular resistance and associated substantial natriuresis make this a potentially attractive therapeutic combination in renal disease.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)