Christian Delles

Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%–2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5′ region of Uromodulin (UMOD; rs13333226, combined P value of 3.6×10−11). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84–0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R. = 0.923, 95% CI 0.860–0.991; p = 0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83–0.96], p = 0.004; after eGFR adjustment: 0.89 [0.83–0.96], p = 0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.

Urinary proteomic biomarkers in coronary artery disease

Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a “training set” for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a “test set.” The signature panel showed sensitivity of 98% (95% confidence interval, 88.7–99.6) and 83% specificity (95% confidence interval, 51.6–97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment.

Gene-centric Association Signals for Lipids and Apolipoproteins Identified via the HumanCVD BeadChip

Blood lipids are important cardiovascular disease (CVD) risk factors with both genetic and environmental determinants. The Whitehall II study (n=5592) was genotyped with the gene-centric HumanCVD BeadChip (Illumina). We identified 195 SNPs in 16 genes/regions associated with 3 major lipid fractions and 2 apolipoprotein components at p<10(-5), with the associations being broadly concordant with prior genome-wide analysis. SNPs associated with LDL cholesterol and apolipoprotein B were located in LDLR, PCSK9, APOB, CELSR2, HMGCR, CETP, the TOMM40-APOE-C1-C2-C4 cluster, and the APOA5-A4-C3-A1 cluster; SNPs associated with HDL cholesterol and apolipoprotein AI were in CETP, LPL, LIPC, APOA5-A4-C3-A1, and ABCA1; and SNPs associated with triglycerides in GCKR, BAZ1B, MLXIPL, LPL, and APOA5-A4-C3-A1. For 48 SNPs in previously unreported loci that were significant at p<10(-4) in Whitehall II, in silico analysis including the British Womens Heart and Health Study, BRIGHT, ASCOT, and NORDIL studies (total n>12,500) revealed previously unreported associations of SH2B3 (p<2.2x10(-6)), BMPR2 (p<2.3x10(-7)), BCL3/PVRL2 (flanking APOE; p<4.4x10(-8)), and SMARCA4 (flanking LDLR; p<2.5x10(-7)) with LDL cholesterol. Common alleles in these genes explained 6.1%-14.7% of the variance in the five lipid-related traits, and individuals at opposite tails of the additive allele score exhibited substantial differences in trait levels (e.g., >1 mmol/L in LDL cholesterol [approximately 1 SD of the trait distribution]). These data suggest that multiple common alleles of small effect can make important contributions to individual differences in blood lipids potentially relevant to the assessment of CVD risk. These genes provide further insights into lipid metabolism and the likely effects of modifying the encoded targets therapeutically.

Angiotensin converting enzyme inhibition and angiotensin II AT1-receptor blockade reduce the levels of asymmetrical NG, NG-dimethylarginine in human essential hypertension☆

Abstract Background Asymmetrical NG, NG-dimethylarginine (ADMA) is associated with impaired endothelium-dependent vasodilation in humans. Methods Twenty young, male, mildly hypertensive subjects were included in a randomized, double-blind, fourfold cross-over study with placebo, enalapril (20 mg/day), eprosartan (600 mg/day), or a combination of both drugs (10 and 300 mg/day, respectively) each over 1 week, followed by a 2-week wash-out phase. After each treatment phase, ADMA concentration was measured. Results ADMA concentration was 1.69 ± 0.59 μmol/L in the placebo phase, and was significantly lower in the enalapril, eprosartan, and combination phases (1.41 ± 0.29, 1.42 ± 0.43, and 1.38 ± 0.30 μmol/L, respectively; all P Conclusions Levels of ADMA were reduced with enalapril and eprosartan therapy. Our results suggest a specific action of these drugs on ADMA levels that is independent of BP.

Additional Antiproteinuric Effect of Ultrahigh Dose Candesartan: A Double-Blind, Randomized, Prospective Study

Proteinuria indicates future renal and cardiovascular morbidity, and, conversely, its reduction is associated with improved outcome. In a randomized, double-blind trial with parallel group design, the antiproteinuric effect of candesartan at high doses was analyzed. Patients with normal or mildly impaired renal function, protein excretion rate of 1 to 10 g/d, and treatment with an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker for 3 mo were eligible. After a 4-wk treatment with 16 mg/d candesartan, patients (n = 32) were allocated to double-blind therapy with either 32 or 64 mg/d candesartan for 12 wk (including 4 wk of uptitration), followed again by 4 wk of candesartan 16 mg/d. Proteinuria at study entry was similar in both groups (geometric mean [95% confidence interval (CI)]; 32 mg/d candesartan 2.14 g/d [95% CI, 1.45 to 3.17]; 64 mg/d candesartan 2.54 g/d [95% CI, 1.91 to 3.40]; NS). After the double-blind treatment phase, proteinuria was reduced to 1.42 g/d (0.85 to 2.37) in the 64-mg/d group (P = 0.017), without any change in the 32-mg/d group (2.02 g/d [95% CI, 1.26 to 3.26]). The change in proteinuria differed between the two groups in absolute (P = 0.025) and relative terms (-29 +/- 50 versus -0 +/- 26%; P = 0.012). After downtitration to 16 mg/d candesartan, proteinuria increased again to 2.38 g/d (1.57 to 3.62) in the 64-mg/d group (P = 0.001) but remained unchanged in the 32-mg/d group (2.04 g/d [95% CI, 1.17 to 3.57]; NS). No change in BP was noticed in response to the different doses of candesartan. These data indicate an additive antiproteinuric effect of ultrahigh dose of the angiotensin receptor blocker candesartan compared with standard dose.

Effect of interleukin-6 receptor blockade on surrogates of vascular risk in rheumatoid arthritis: MEASURE, a randomised, placebo-controlled study.

Objectives The interleukin-6 receptor (IL-6R) blocker tocilizumab (TCZ) reduces inflammatory disease activity in rheumatoid arthritis (RA) but elevates lipid concentrations in some patients. We aimed to characterise the impact of IL-6R inhibition on established and novel risk factors in active RA. Methods Randomised, multicentre, two-part, phase III trial (24-week double-blind, 80-week open-label), MEASURE, evaluated lipid and lipoprotein levels, high-density lipoprotein (HDL) particle composition, markers of coagulation, thrombosis and vascular function by pulse wave velocity (PWV) in 132 patients with RA who received TCZ or placebo. Results Median total-cholesterol, low-density lipoprotein-cholesterol (LDL-C) and triglyceride levels increased in TCZ versus placebo recipients by week 12 (12.6% vs 1.7%, 28.1% vs 2.2%, 10.6% vs −1.9%, respectively; all p<0.01). There were no significant differences in mean small LDL, mean oxidised LDL or total HDL-C concentrations. However, HDL-associated serum amyloid A content decreased in TCZ recipients. TCZ also induced reductions (>30%) in secretory phospholipase A2-IIA, lipoprotein(a), fibrinogen and D-dimers and elevation of paraoxonase (all p<0.0001 vs placebo). The ApoB/ApoA1 ratio remained stable over time in both groups. PWV decreases were greater with placebo than TCZ at 12 weeks (adjusted mean difference 0.79 m/s (95% CI 0.22 to 1.35; p=0.0067)). Conclusions These data provide the first detailed evidence for the modulation of lipoprotein particles and other surrogates of vascular risk with IL-6R inhibition. When compared with placebo, TCZ induced elevations in LDL-C but altered HDL particles towards an anti-inflammatory composition and favourably modified most, but not all, measured vascular risk surrogates. The net effect of such changes for cardiovascular risk requires determination.

Impaired Endothelial Function of the Retinal Vasculature in Hypertensive Patients

Background and Purpose— Arterial hypertension constitutes a central factor in the pathogenesis of stroke. We examined endothelial function of the retinal vasculature as a model of the cerebral circulation. Methods— Thirty-eight young subjects (19 hypertensive and 19 normotensive) were treated with the AT1-receptor blocker candesartan cilexetil and placebo, each over 7 days. Retinal capillary flow and blood flow velocity in the central retinal artery were assessed with scanning laser Doppler flowmetry and pulsed Doppler ultrasound, respectively. NG-mono-methyl-L-arginine (L-NMMA) was infused to inhibit nitric oxide (NO) synthesis. Diffuse luminance flicker was applied to stimulate NO release. Results— In normotensive subjects, L-NMMA decreased retinal capillary flow by 8.2%±13% (P < 0.05) and flickering light increased mean blood flow velocity in the central retinal artery by 19%±29% (P < 0.01). In contrast, no significant change to these provocative tests was seen in hypertensive subjects. Treatment with candesartan cilexetil restored a normal pattern of reactivity in retinal capillaries (L-NMMA: decrease in perfusion by 10%±17%, P < 0.05) and the central retinal artery (flicker: increase in mean blood flow velocity by 42%±31%, P < 0.001) in hypertensive patients. Conclusions— Endothelial function of the retinal vasculature is impaired in early essential hypertension but can be improved by AT1-receptor blockade.

Implementation of proteomic biomarkers: Making it work

Eur J Clin Invest 2012; 42 (9): 1027–1036

Novel biomarkers for predicting preeclampsia.

Preeclampsia is a major cause of maternal morbidity and mortality worldwide. Despite decades of research into the condition, the ability of clinicians to predict preeclampsia prior to the onset of symptoms has not improved significantly. In this review, we will examine the pathophysiology underlying preeclampsia and will look at potential biomarkers for early prediction and diagnosis. In addition, we will explore potential future areas of research into the condition.