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The investigation of vascular calcification and its underlying cellular and molecular pathways is of great interest in current research efforts. Therefore, suitable assays are needed to allow examination of the complex calcification process under controlled conditions. The current study describes a new ex vivo model of isolated-perfused rat aortic tissue with subsequent quantification and vessel staining to analyze the calcium content of the aortic wall. A rat aorta was perfused ex vivo with control and calcification media for 14 days, respectively. The calcification medium was luminally perfused and induced a significant increase in calcium deposition within the media of the vessel wall detected alongside the elastic laminae. Perfusion with control medium induced no calcification. In addition, the mRNA expression of the osteogenic marker bone morphogenetic protein 2 (BMP-2) increased in aortic tissue after perfusion, while SM22α as smooth muscle marker decreased. This newly developed ex vivo model of isolated-perfused rat aorta is suitable for vascular calcification studies testing inducers and inhibitors of vessel calcification and studying signaling pathways within calcification progression.

The adrenal glands participate in cardiovascular (CV) physiology and the pathophysiology of CV diseases through their effects on sodium and water metabolism, vascular tone and cardiac function. In the present study, we identified a new adrenal compound controlling mesenchymal cell differentiation that regulates osteoblastic differentiation in the context of vascular calcification. This peptide was named the “calcification blocking factor” (CBF) due to its protective effect against vascular calcification and is released from chromogranin A via enzymatic cleavage by calpain 1 and kallikrein. CBF reduced the calcium content of cells and thoracic aortic rings under calcifying culture conditions, as well as in aortas from animals treated with vitamin D and nicotine (VDN animals). Furthermore, CBF prevented vascular smooth muscle cell (VSMC) transdifferentiation into osteoblast-like cells within the vascular wall via the sodium-dependent phosphate transporter PIT-1 and by inhibition of NF-κB activation and the subsequent BMP2/p-SMAD pathway. Pulse pressure, a marker of arterial stiffness, was significantly decreased in VDN animals treated with CBF. In line with our preclinical data, CBF concentration is significantly reduced in diseases characterized by increased calcification, as shown in patients with chronic kidney disease. In preparation for clinical translation, the active site of the native 19-AS long native CBF was identified as EGQEEEED. In conclusion, we have identified the new peptide CBF, which is secreted from the adrenal glands and might prevent vascular calcification by inhibition of osteogenic transdifferentiation. The anti-calcific effects of CBF and short active site may therefore promote the development of new tools for the prevention and/or treatment of vascular calcification. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00899-z.

Background and Aims Despite the introduction of several new phosphate binders over the last decades, adequate control of hyperphosphatemia in haemodialysis (HD) patients still remains a crucial challenge as more than half of the population still remains hyperphosphataemic in daily practice. A growing amount of clinical data suggests that nicotinamide (NA) represents a complementary and effective treatment option for hyperphosphataemia. Efficacy and safety of once daily intake of modified release (MR) NA were investigated for fixed doses between 250 and 1,000 mg in hyperphosphatemic HD patients. Additionally, the MR formulation was compared to trice daily intake of an immediate release (IR) formulation of NA. Method This multicenter, controlled trial included HD patients with serum phosphate concentrations between 4.7 and 7.5 mg/dL after initial discontinuation of phosphate binder treatment. Patients were randomized to either once daily intake of 250 mg (N=48), 500 mg (N=50), 750 mg (N=50) or 1,000 mg of MR NA (N=53) in a double-blind setting, or intake of 1,000 mg of IR NA (N=51), divided over three daily doses in an open-label setting over a treatment period of 8 weeks. Results Based on the ITT population the study revealed a highly significant (p The most frequently reported adverse events were gastrointestinal symptoms in 31.4% of patients and skin reactions in 19.8% of patients. The study also revealed a nonsignificant decrease in mean platelet count. Overall, the number of adverse events increased in the 750 and both 1,000 mg dosage groups. For individual adverse events, dose dependency was observed for diarrhea, headache, vomiting and nausea. NA treatment resulted in a dose dependent increase of plasma concentrations of NA and its terminal metabolites (N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-5-carboxamide, Nicotinamide-N-oxide). The tolerability profile as well as the plasma concentrations of NA metabolites were similar for both formulations although the proportion of patients that terminated treatment due to adverse events was higher in the IR arm compared to the MR formulation (33.5% versus 26.4% of patients). However, this study was not powered to reliably assess clinical superiority with regard to AE. Conclusion Our study characterised the NA modified release preparation NoPhos as an effective novel therapeutic option for the treatment of hyperphosphatemia in HD patients. In the monotherapy setting a clear dose-response relationship was revealed for daily doses between 250 and 1,000 mg, while best tolerability was observed at a dose of 500 mg/d.

Despite advancements in lowering blood pressure, the best approach to lower it remains controversial because of the lack of information on the molecular basis of hypertension. We, therefore, performed plasma proteomics of plasma from patients with hypertension to identify molecular determinants detectable in these subjects but not in controls and vice versa. Plasma samples from hypertensive subjects (cases; n=118) and controls (n=85) from the InGenious HyperCare cohort were used for this study and performed mass spectrometric analysis. Using biostatistical methods, plasma peptides specific for hypertension were identified, and a model was developed using least absolute shrinkage and selection operator logistic regression. The underlying peptides were identified and sequenced off-line using matrix-assisted laser desorption ionization orbitrap mass spectrometry. By comparison of the molecular composition of the plasma samples, 27 molecular determinants were identified differently expressed in cases from controls. Seventy percent of the molecular determinants selected were found to occur less likely in hypertensive patients. In cross-validation, the overall R 2 was 0.434, and the area under the curve was 0.891 with 95% confidence interval 0.8482 to 0.9349, P P

Journal of molecular medicine 97(10), 1451-1463 (2019). doi:10.1007/s00109-019-01823-8, Published by Springer, New York, NY

Transpulmonary thermodilution (TPTD) is used frequently in the intensive care unit to determine cardiac index (CI), intrathoracic blood volume index (ITBVI), and extravascular lung volume index (EVLWI). Renal replacement therapy (RRT) influences TPTD results, but the underlying mechanisms are not completely understood. We hypothesized that RRT blood flow induces errors in TPTD measurements.We analyzed TPTD data available from the PiCCO® plus hemodynamic measurement device on a personal computer using a proprietary Pulsion Medical Systems software. By using the dialysis catheter to inject the thermal indicator, 20 measurement series were performed in 12 intensive care unit patients determining CI, ITBVI, and EVLWI during RRT with the blood pump stopped, and at flows of 100 and 200 mL/min, respectively.Data export was successful in 17 measurement series and showed a significant decrease in measured CI (6.5 ± 2.5 vs 5.4 ± 1.9 L/min/m, P0.001) and ITBVI (1358.8 ± 274.5 vs 1132.8 ± 218.3 mL/m, P0.001) with RRT and a significant increase in EVLWI (8.6 ± 4.4, 10.2 ± 4.5 mL/kg, P0.001). Blood temperature before and the temperature decrease after injection of the thermal indicator were unchanged by RRT. Mean transit time and downslope time of the thermodilution curve, however, were both increased with the RRT blood pump running (P ≤ 0.001).Analysis of TPTD data shows that thermodilution curve forms are modified with RRT, resulting in an erroneous calculation of thermodilution-derived hemodynamic parameters.

The benefit of the central systolic blood pressure (cSBP) has already been recognized, but its general measurement in the everyday routine is still limited mainly because available established non-invasive assessment devices are not suitable for everyday use. In this study, we investigated the performance of an oscillometric device Tel-O-GRAPH for cSBP assessment in terms of suitability for everyday clinical use. One hundred and three patients were prospectively included. cSBP was computed by Tel-O-GRAPH compared with Sphygmocor using applanation tonometry. There was a good agreement between Tel-O-GRAPH and Sphygmocor for cSBP (mean difference±s.d.: -0.3±6.7 mm Hg; Pearson's R=0.95; P0.0001). Recorded cSBP values in the supine vs seated position and for the experienced vs non-experienced user did not significantly differ (mean cSBP

We present a comprehensive study of the interaction of human serum albumin (HSA) with poly(acrylic acid) (PAA; number average degree of polymerization: 25) in aqueous solution. The interaction of HSA with PAA is studied in dilute solution as the function of the concentration of added salt (20 - 100 mM) and temperature (25 - 37$^{\circ}$C). Isothermal titration calorimetry (ITC) is used to analyze the interaction and to determine the binding constant and related thermodynamic data. It is found that only one PAA chain is bound per HSA molecule. The free energy of binding $\Delta G_b$ increases with temperature significantly. $\Delta G_b$ decreases with increasing salt concentration and is dominated by entropic contributions due to the release of bound counterions. Coarse-grained Langevin computer simulations treating the counterions in an explicit manner are used study the process of binding in detail. These simulations demonstrate that the PAA chains are bound in the Sudlow II site of the HSA. Moreover, $\Delta G_b$ is calculated from the simulations and found to be in very good agreement with the measured data. The simulations demonstrate clearly that the driving force of binding is the release of counterions in full agreement with the ITC-data.

Assessment of the cardiac output (CO) is usually performed with invasive techniques requiring specialized equipment in the intensive care unit (ICU). With TEL-O-GRAPH (TG), CO can be derived from the oscillometrically obtained brachial pulse wave during the measurement of brachial blood pressure. CO and stroke volume (SV) determinations with TG were compared with transpulmonary thermodilution measurements with the PICCO system (PICCO) in 38 haemodynamically unstable ICU patients with a total of 84 comparison measurements performed. SV (33.3 ± 9.0 ml/m2 vs. 44.3 ± 14.4 ml/m2, p 2 vs. 3.8 ± 1.2 l/min/m2, p

BACKGROUND Hyperuricemia is associated with an increased risk of cardiovascular disease and chronic allograft nephropathy after renal transplantation. It has recently been demonstrated that treatment of asymptomatic hyperuricemia is associated with improved patient and graft survival; although, the underlying mechanisms remain elusive. The present study investigated the association of serum uric acid (SUA) and systemic arterial function after renal transplantation. MATERIAL AND METHODS In a cross-sectional study, arterial function was analyzed in 54 renal transplant recipients by means of pulse wave analysis. Different measurement techniques were combined providing data on pulse wave velocity, augmentation index, small and large artery elasticity, and total peripheral vascular resistance. RESULTS The prevalence of hyperuricemia was 87.0%, and 33.3% of renal transplant recipients received SUA lowering medication. The median SUA concentration was 7.4 mg/dL. There was no significant difference in all the aforementioned parameters in patients with a SUA7.4 versus7.4 mg/dL (p0.05 each) and no significant differences between hyperuricemic patients with versus without SUA lowering medication. Linear regression analysis between SUA and both pulse wave velocity and augmentation index showed no significant association (p0.05 each). This finding remained consistent after adjustment of data for age, time on dialysis, time since transplantation, and systolic blood (partial correlation analysis, p0.05). CONCLUSIONS Neither the concentration of SUA nor the pharmacological treatment of hyperuricemia had measurable effects on arterial stiffness. Thus, the beneficial effects of SUA lowering treatment on patient and graft survival cannot be explained by direct effects on arterial function in the study population.

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The introduction of automated oscillometric blood pressure monitors was the basis for today’s widespread use of blood pressure self-measurement. However, in atrial fibrillation, there is a controversial debate on the use of oscillometry because there is a high variability of heart rate and stroke volume. To date, the accuracy of oscillometric blood pressure monitoring in atrial fibrillation has only been investigated using auscultatory sphygmomanometry as reference method, which may be biased by arrhythmia as well. We performed a cross-sectional study in 102 patients (52 sinus rhythm, 50 atrial fibrillation) assessing the accuracy of an automated and validated oscillometric upper arm (M5 Professional, Omron) and wrist device (R5 Professional, Omron) to invasively assessed arterial pressure. Blood pressure values were calculated as the mean of 3 consecutive measurements. Systolic and diastolic blood pressure did not significantly differ in patients with sinus rhythm and atrial fibrillation, independent of the method of measurement ( P >0.05 each). The within-subject variability of the oscillometric measurements was higher in patients with atrial fibrillation compared with sinus rhythm ( P P >0.05 each). In conclusion, atrial fibrillation did not significantly affect the accuracy of oscillometric measurements, if 3 repeated measurements were performed.

The Global Cardiometabolic Risk Profile in Patients with hypertension disease (GOOD) survey investigated the global cardiometabolic risk profile in 3464 adult outpatients with hypertension across 289 sites in 12 European countries. The pulse pressure and heart rate profile of the survey population was evaluated according to the presence or absence of metabolic syndrome and/or type 2 diabetes mellitus. History and treatment of hypertension were not counted as criteria for metabolic syndrome as they applied to all patients. Out of the 3370 recruited patients, 1033 had metabolic syndrome and 1177 had neither metabolic syndrome nor diabetes. When compared with patients with no metabolic syndrome or diabetes, patients with metabolic syndrome had higher pulse pressure (59±14 vs. 55±14 mm Hg) and heart rate (75.2±11.0 vs. 72.5±10.0 beats per min) (P0.001 for both), independent of the concomitant presence or absence of diabetes, despite a more prevalent use of β-blockers. In conclusion, in hypertensive outpatients the presence of metabolic syndrome is associated with increased heart rate and pulse pressure, which may at least in part reflect increased arterial stiffness and increased sympathetic tone. This may contribute, to some extent, to explaining the increased cardiovascular risk attributed to the presence of metabolic syndrome.

Regular physical exercise is broadly recommended by current European and American hypertension guidelines. It remains elusive, however, whether exercise leads to a reduction of blood pressure in resistant hypertension as well. The present randomized controlled trial examines the cardiovascular effects of aerobic exercise on resistant hypertension. Resistant hypertension was defined as a blood pressure ≥140/90 mm Hg in spite of 3 antihypertensive agents or a blood pressure controlled by ≥4 antihypertensive agents. Fifty subjects with resistant hypertension were randomly assigned to participate or not to participate in an 8- to 12-week treadmill exercise program (target lactate, 2.0±0.5 mmol/L). Blood pressure was assessed by 24-hour monitoring. Arterial compliance and cardiac index were measured by pulse wave analysis. The training program was well tolerated by all of the patients. Exercise significantly decreased systolic and diastolic daytime ambulatory blood pressure by 6±12 and 3±7 mm Hg, respectively ( P =0.03 each). Regular exercise reduced blood pressure on exertion and increased physical performance as assessed by maximal oxygen uptake and lactate curves. Arterial compliance and cardiac index remained unchanged. Physical exercise is able to decrease blood pressure even in subjects with low responsiveness to medical treatment. It should be included in the therapeutic approach to resistant hypertension.

Aims High-density lipoprotein (HDL) is known to have potent anti-inflammatory properties. Monocyte chemoattractant protein-1 is an important pro-inflammatory cytokine in early atherogenesis. There is evidence that HDL can lose its protective function during inflammatory disease. In patients with end-stage renal disease (ESRD), epidemiological studies have documented that the inverse correlation between HDL-cholesterol and cardiovascular risk is lost. Many structural modifications leading to reduced HDL function have been characterized, but the functional consequences are not fully understood.Methods and results We showed that HDL from patients with ESRD has a lower anti-inflammatory potential by reduced inhibition of monocyte chemoattractant protein-1 formation in vascular smooth muscle cells. Via a proteomic approach, we identified proteins in HDL from ESRD patients exerting pro-inflammatory actions. By chromatographic separation of proteins and mass-spectrometric analysis, we found serum amyloid A (SAA) to be one molecule acting as a potent pro-inflammatory protein. SAA is enriched in HDL from ESRD patients, correlating with reduced anti-inflammatory capacity. In SAA signal transduction, activation of formyl-peptide receptor 2 is involved. SAA enrichment in HDL of healthy subjects reduced the anti-inflammatory capacity of HDL and correlated with its decreased function.Conclusion These results suggest that SAA enrichment of HDL during disease conditions contributes to the decreased protective function. It is a novel finding that SAA acts as a pro-inflammatory molecule to reduce the anti-inflammatory properties of HDL.

Purinergic signaling has a crucial role in different vascular processes. The endothelial-derived vasoconstrictor uridine adenosine tetraphosphate (Up 4 A) is a potent activator of the purinoceptor P2Y and is released under pathological conditions. Here we sought to measure purinergic effects on vascular calcification and initially found that Up 4 A plasma concentrations are increased in patients with chronic kidney disease. Exploring this further we found that exogenous Up 4 A enhanced mineral deposition under calcifying conditions ex vivo in rat and mouse aortic rings and in vitro in rat vascular smooth muscle cells. The addition of Up 4 A increased the expression of different genes specific for osteochondrogenic vascular smooth muscle cells such as Cbfa1, while decreasing the expression of SM22α, a marker specific for vascular smooth muscle cells. The influence of different P2Y antagonists on Up 4 A-mediated process indicated that P2Y 2/6 receptors may be involved. Mechanisms downstream of P2Y signaling involved phosphorylation of the mitogen-activated kinases MEK and ERK1/2. Thus, Up 4 A activation of P2Y influences phenotypic transdifferentiation of vascular smooth muscle cells to osteochondrogenic cells, suggesting that purinergic signaling may be involved in vascular calcification.

Summary Background and objectives To date there is no reliable marker for the differentiation of prerenal and intrinsic acute kidney injury (AKI). We investigated whether urinary calprotectin, a mediator protein of the innate immune system, may serve as a diagnostic marker in AKI. Design, setting, participants, & measurements This was a cross-sectional study with 101 subjects including 86 patients with AKI (34 prerenal, 52 intrinsic including 23 patients with urinary tract infection) and 15 healthy controls. Assessment of urinary calprotectin concentration was by ELISA and immunohistochemistry of kidney biopsy specimens using a calprotectin antibody. Inclusion criteria were: admission to hospital for AKI stage 1 to 3 (Acute Kidney Injury Network); exclusion criteria were: prior renal transplantation and obstructive uropathy. Results Median urinary calprotectin was 60.7 times higher in intrinsic AKI (1692 ng/ml) than in prerenal AKI (28 ng/ml, p p = 0.25). Receiver operating curve curve analysis revealed a high accuracy of calprotectin (area under the curve, 0.97) in predicting intrinsic AKI. A cutoff level of 300 ng/ml provided a sensitivity of 92.3% and a specificity of 97.1%. Calculating urinary calprotectin/creatinine ratios did not lead to a further increase of accuracy. Immunostainings of kidney biopsies were positive for calprotectin in intrinsic AKI and negative in prerenal AKI. Conclusions Accuracy of urinary calprotectin in the differential diagnosis of AKI is high. Whereas calprotectin levels in prerenal disease are comparable with healthy controls, intrinsic AKI leads to highly increased calprotectin concentrations.

Summary Cardiovascular complications are the leading cause of death in renal transplant recipients and constitute one of the leading causes of graft failure. Calcineurin inhibitors (CNI) induce an acceleration of atherosclerotic processes in the arterial wall. There are conflicting data whether cyclosporine A (CsA) and tacrolimus (Tac) differ in their deleterious effects on arterial stiffening. The present study combines several measurement techniques to provide a global and reliable assessment of the differential effects of CNI on the gold-standard parameters of arterial function. Pulse wave analysis was performed by the SphygmoCor (AtCor � ), HEM-9000AI (Omron � ), and CR-2000 device (Hypertension Diagnostics � ) in 56 stable renal transplant recipients (29 CsA, 27 Tac). Groups were homogeneous for age, gender, body mass index, time on dialysis prior to transplantation, and graft function. Whereas systolic and diastolic blood pressure, central aortic blood pressure, cardiac index, large and small artery compliance index (C1 and C2), and pulse wave velocity did not significantly differ between CsA and Tac, augmentation index (AI75) was significantly lower in patients treated with Tac. This finding was consistent as assessed by two different measurement systems (P < 0.05). Compared to CsA, Tac has a favorable impact on augmentation index, a strong independent predictor for cardiovascular mortality.

BACKGROUND AND PURPOSE Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up4A) as a novel and highly potent endothelial-derived contracting factor. Up4A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X1 receptor activation. However, other purinoceptors are also involved and were analysed here. EXPERIMENTAL APPROACH The rat isolated perfused kidney was used to characterize vasoactive actions of Up4A. KEY RESULTS After desensitization of the P2X1 receptor by α,β-methylene ATP (α,β-meATP), Up4A showed dose-dependent P2Y2-mediated vasoconstriction. Continuous perfusion with Up4A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X1 receptor desensitization. In addition, there is a long-lasting P2Y2-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up4A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X1 receptor desensitization by α,β-meATP, and the remaining P2Y2-mediated vasoconstriction was only inhibited by suramin. The Up4A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y1 and P2Y2 receptor activation. CONCLUSIONS AND IMPLICATIONS Up4A activated P2X1 and P2Y2 receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y1/2 receptor activation. Up4A might be of relevance in the physiology and pathophysiology of vascular tone regulation.

End-stage renal disease (ESRD) patients exhibit increased in vivo oxidative stress conceivably contributing to cardiovascular mortality. The type IIA secretory phospholipase A(2) (sPLA(2)) has proatherogenic activity. We explored the hypothesis that sPLA(2) contributes to oxidative stress generation and endothelial dysfunction in ESRD patients and transgenic (tg) mice. Patients with ESRD had increased in vivo oxidative stress as assessed by plasma isoprostane levels (p

The purinergic system is composed of mononucleosides, mononucleoside polyphosphates and dinucleoside polyphosphates as agonists, as well as the respective purinergic receptors. Interest in the role of the purinergic system in cardiovascular physiology and pathophysiology is on the rise. This review focuses on the overall impact of dinucleoside polyphosphates in the purinergic system. Platelets, adrenal glands, endothelial cells, cardiomyocytes and tubular cells release dinucleoside polyphosphates. Plasma concentrations of dinucleoside polyphosphates are sufficient to cause direct vasoregulatory effects and to induce proliferative effects on vascular smooth muscle cells and mesangial cells. In addition, increased plasma concentrations of a dinucleoside polyphosphate were recently demonstrated in juvenile hypertensive patients. In conclusion, the current literature accentuates the strong physiological and pathophysiological impact of dinucleoside polyphosphates on the cardiovascular system.

There is an increasing number of wrist blood pressure measurement devices that successfully passed the validation procedures of the British Hypertension Society (BHS) and the European Society of Hypertension (ESH). It remains unknown, however, whether pulse pressure as a marker of arterial stiffness and vascular ageing affects the accuracy of these devices. An ESH protocol validated wrist device was compared with the upper arm mercury sphygmomanometry in a study population (33 patients, 99 measurements) including a relevant number of subjects with pulse pressure >50 mm Hg (84.8%) and isolated systolic hypertension (27.3%). Mean systolic bias was 10.2 mm Hg with 95% limits of agreement of -13.1 and 33.6 mm Hg, mean diastolic bias was 4.8 mm Hg with limits of agreement of -11.0 and 20.7 mm Hg. The impact of body mass index, age, systolic blood pressure and pulse pressure on the absolute value of blood pressure bias was tested by stepwise multiple regression analysis. The systolic bias significantly depended on pulse pressure, whereas there was no significant effect of the independent variables on the diastolic bias. Separate correlation analysis showed a significant correlation between pulse pressure and both absolute systolic bias (Pearson r=0.48, P

Nitric oxide (NO) is known to be an important mediator of macrophage cytotoxicity. NO in macrophages is generated via the inducible nitric oxide synthases (iNOS). Macrophage dysfunction is an important contributory factor for the increased incidence of infections in uraemia. Recently, we identified phenylacetic acid (PAA) as a novel uraemic toxin in patients on regular haemodialysis. PAA inhibits iNOS expression. In the present study, we investigated the impact of PAA on macrophage function.RAW 264.7 cells were stimulated by LPS/ IFN-gamma in the absence and presence of PAA. iNOS mRNA was determined by real-time PCR, iNOS protein was examined by western blotting and the NO degradation product, nitrite, by Griess assay. Macrophage phagocytosis was assessed by FACS and fluorescence microscopy. Further we quantified the cytotoxicity against intracellular bacteria (Salmonella typhimurium) by a macrophage-killing assay. ELISA and Bioplex protein array system was used for the investigation of iNOS second messenger pathways (NF-kappaB, ERK1/2, JNK and p38MAPK). iNOS mRNA half-lifetime in the presence or absence of PAA was determined by real-time PCR.PAA significantly inhibits iNOS mRNA induction in RAW 264.7 cells by LPS/IFN-gamma [6 h: LPS/IFN-gamma-stimulation: 100%; LPS/IFN-gamma-stimulation/PAA (1 mM): 68 +/- 7%] at concentrations comparable to those of patients on chronic haemodialysis. iNOS protein expression and nitrite formation in RAW 264.7 cells were significantly inhibited by PAA. iNOS mRNA half-lifetime was not affected by PAA. The phagocytic activity of RAW 264.7 was not significantly affected by PAA, whereas the cytotoxicity against intracellular bacteria was significantly reduced. Analysis of the iNOS signal transduction pathways provided evidence that activation of the mitogen-activated kinases ERK1/2 and JNK is significantly blocked by PAA, whereas activation of p38MAPK is unaffected. The NF-kappaB pathway was not affected by PAA.The present findings show that the uraemic toxin PAA has inhibitory effects on macrophage-killing function, which are mediated by inhibitory effects on transcriptional iNOS regulation. iNOS inhibition by PAA might affect immunoregulatory processes and could play a role in aggravation of immunodeficiency of patients with end-stage renal disease.

Background We recently showed that increased expression of the transient receptor potential canonical Type 3 (TRPC3) channel is associated with genetic hypertension. It is unknown whether store-operated TRPC3 channels, which are activated after depletion of intracellular stores, or second messenger-operated TRPC3 channels, which are activated by 1-oleoyl-2-acetyl-sn-glycerol, show augmented responses in monocytes in genetic hypertension and support the development of vascular disease. Methods Using the fluorescent-dye technique, we studied store-depleted and thapsigargin-induced, store-operated calcium influx and 1-oleoyl-2-acetyl-sn-glycerol-induced second messenger-operated calcium influx into monocytes from spontaneously hypertensive rats (SHRs) and from normotensive Wistar-Kyoto rats (WKYs). The RNA interference for the downregulation of TRPC3 in monocytes by small, interfering RNA (siRNA) was performed and evaluated using in-cell Western assay. Results Thapsigargin-induced, store-operated calcium influx was significantly elevated in SHRs and was approximately double that observed in WKYs. In the presence of nimodipine, the thapsigargin-induced, store-operated calcium influx was also significantly higher in SHRs compared with WKYs. After stimulation of monocytes by angiotensin II, calcium influx was significantly elevated in SHRs, and was approximately double that observed in WKYs. The 1-oleoyl-2-acetyl-sn-glycerol-induced, second messenger-operated calcium influx was also significantly elevated in SHRs compared with WKYs. Thapsigargin-induced, store-operated calcium influx was reduced by the inhibitor 2-aminoethoxydiphenyl borane. After TRPC3 knockdown, the thapsigargin-induced, store-operated calcium influx, as well as 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx, was significantly more reduced in cells from SHRs compared with WKYs. Conclusions The increased store-operated and second messenger-operated calcium influx through TRPC3 channels in monocytes from SHRs may be responsible for a more aggressive effect in promoting vascular disease in genetic hypertension.

Background— Uridine adenosine tetraphosphate (Up 4 A) was been recently characterized as a potent vasoconstrictor. Up 4 A occurs in plasma from healthy subjects at concentrations sufficient to cause strong vasoconstrictive effects. In this study, Up 4 A concentrations in plasma from juvenile hypertensives and normotensives were determined. Methods and Results— Up 4 A was purified to homogeneity by preparative reverse phase high performance liquid-chromatography (HPLC), affinity chromatography HPLC, and analytic reverse phase HPLC from deproteinized plasma of juvenile hypertensives and normotensives. Mean total plasma Up 4 A concentration was significantly increased in juvenile hypertensives compared with juvenile normotensives (33.0±25.4 versus 3.7±0.9 nmol/L; mean±SEM, n=40 and 38, respectively; P 4 A showed a significant association with juvenile hypertension (OR for ln(Up 4 A): 1.82; 95% CI 1.12, 2.95). Plasma Up 4 A concentrations correlated with left ventricular mass (Kendall-τ correlation coefficient 0.220, n=40; P P 4 A, we studied the effects of Up 4 A on human vascular smooth muscle cell proliferation. The maximum proliferative effect of Up 4 A was 80.0±24.0% % above control ( P 4 A on smooth muscle cells is cell cycle–dependent, involving stimulation of S phase entry. Conclusion— Circulating levels of Up 4 A are strongly associated with juvenile hypertension. The endothelium-derived vasoconstrictor Up 4 A may contribute to the early development of primary hypertension and is moreover an important risk factor of juvenile hypertension.

Objective: Leptin, secreted from adipose tissue, regulates food intake, energy expenditure, and immune function. It is unknown whether leptin predicts mortality in patients with chronic kidney disease stage 5 on hemodialysis therapy. Research Methods and Procedures: We performed a prospective cohort study of 71 patients with chronic kidney disease stage 5 in an outpatient hemodialysis center. Subjects were recruited in June 1998 and followed for 83 months. Survival was compared by the Kaplan-Meier method. Results: After 83 months of follow-up, 48 patients (68%) had died. Serum leptin concentrations at study entry were lower among all deceased patients compared with those patients who survived (5.2 ± 9.0 μg/L; n = 48; vs. 7.7 ± 7.8 μg/L; n = 23; p = 0.005). Baseline serum leptin concentrations were significantly lower in patients who died from cardiovascular diseases (4.7 ± 9.4 μg/L, n = 32) or infections (4.0 ± 2.7 μg/L; n = 10; each p < 0.05), but not cancer (9.4 ± 7.9 μg/L; n = 6), than in survivors (7.7 ± 7.8 μg/L; n = 23; p = 0.003). The relative risk for mortality in patients with serum leptin concentrations below the median (

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Background. FTY720 has recently demonstrated a similar efficacy in prevention of acute graft rejection compared with mycophenolate mofetil (MMF) in a large phase III trial of de novo renal transplant recipients. Creatinine clearance, however, was significantly lower in FTY720-treated patients. In the present study, we examined the impact of FTY720 on vascular function in a subgroup of patients of this trial. Methods. Eighteen patients (12 FTY720, 6 MMF) agreed to be enrolled for an analysis of vascular function. Vascular measurements were performed 1.5 years post-transplant and were repeated 3 months after conversion of the patients from FTY720 to MMF. Arterial stiffness was assessed as augmentation index (AI75); endothelium-dependent and -independent vasodilation were measured sonographically as flowmediated dilation (FMD) and as vasodilation after application of glyceroltrinitrate (GTN). Results. Conversion from 2.5 mg FTY720 to MMF led to a significant improvement of FMD (5.40 � 1.84 vs 7.77 � 3.36%, P 0.02). AI75 and GTN tended to be higher after conversion without reaching significance (83 � 20.43 vs 78.69 � 15.39%, P 0.06; 13.76 � 4.52 vs 17.39 � 3.76%, P 0.07). In the MMF group, AI75, FMD and GTN did not significantly change during the observation period. Conclusion. The present study constitutes the first analysis of the impact of FTY720 on vascular function in humans and reveals an improvement of arterial vasodilatory function after discontinuation of FTY720 in de novo renal transplant recipients on cyclosporine.

Aerobic physical exercise is broadly recommended as a helpful adjunct to obtain blood pressure control in hypertension. Beta-blockade interacts with heart rate, sympathetic tone, maximal workload and local lactate production. In the present randomized-controlled study, we compared the cardiovascular effects of an endurance training programme in elderly hypertensives with or without beta-blockers and developed a first approach to determine a lactate-based training heart rate in presence of beta-blockade. Fifty-two patients (23 with beta-blocker, 29 without beta-blocker) > or =60 years with systolic 24-h ambulatory blood pressure (ABP) > or =140 mm Hg and/or antihypertensive treatment were randomly assigned to sedentary activity or a heart-rate controlled 12-week treadmill exercise programme (lactate 2.0 mmol/l). In the exercise group, the training significantly decreased systolic and diastolic 24-h ABP, blood pressure on exertion (100 W) and increased endothelium-dependent vasodilation (flow-mediated vasodilation, FMD) and physical performance both in the presence and absence of beta-blockade (P

Background: Hypertension in the elderly is commonly characterized by an elevation of pulse pressure. With regard to advanced arteriosclerosis and limited physical fitness, doubt was casted whether elderly patients still achieve relevant cardiovascular benefits by physical exercise. The present work examines the impact of pulse pressure as a footprint of vascular ageing on cardiovascular benefits of endurance training in elderly hypertensives. Methods: 54 patients ≧60 years with systolic 24-hour ambulatory blood pressure (ABP) >140 mm Hg and/or antihypertensive treatment and diastolic ABP ≤90 mm Hg were randomly assigned to sedentary activity or a 12-week treadmill exercise program (target lactate 2.5 ± 0.5 mmol/l). Results: Exercise significantly decreased systolic and diastolic ABP by 8.5 ± 8.2 and 5.1 ± 3.7 mm Hg (p < 0.001 each) and increased physical performance. Arterial compliance remained unchanged, whereas endothelium-dependent vasodilation – measured by flow-mediated dilation – significantly increased from 5.6 ± 1.7 to 7.9 ± 3.0% (p < 0.007). After adjustment for initial systolic ABP, pulse pressure did not affect the change of BP. Conclusion: The exercise-induced reduction of BP, which is mediated by improved endothelial function, is independent of pulse pressure. Thus, physical exercise is a helpful adjunct to control BP even in old hypertensives with markedly increased arterial stiffness.

Background Disturbances in the regulation of cytosolic calcium concentration have been attributed to primary hypertension, but the role of calcium-permeable transient receptor potential canonical channel 3 (TRPC3) has not yet been evaluated in primary hypertension. Methods Expression of TRPC3 was determined using in-cell Western assay. Evaluation of RNA interference for the downregulation of a specific gene in cells by small interfering RNA was performed. Measurements of cytosolic calcium were carried out using the fluorescent dye fura2. Results Expression of TRPC3 was significantly increased in monocytes from spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto rats (WKY). Transplasmamembrane calcium influx and thapsigargin-induced sustained calcium increase were significantly higher in SHR compared with WKY. In the presence of the TRP channel blocker SKF-96365 these differences were no longer observed. Specific TRPC3-knockdown by transfection of monocytes from SHR with small interfering RNA significantly reduced TRPC3 expression, trans–plasma membrane calcium influx, and thapsigargin-induced sustained calcium increase. Conclusions This study shows, for the first time, increased TRPC3 channel expression and increased TRPC3-related calcium influx in SHR.

Several vasoregulatory systems including the renin-angiotensin system, sympathetic vasoregulation, and cytokine release have been studied extensively. The aim of the present study was to establish a physiogenomic screening model for differentially expressed genes in the regulation of blood pressure that might give a hint as to new vasoregulatory mechanisms. We induced acute hypotension in normotensive rats, assuming that vasoregulatory systems will counteract hypotension. Microarray transcriptome analysis was performed from kidneys 6 h after the induction of acute hypotension. The results were confirmed by real-time PCR. Six functionally known genes ( Igfbp1, Xdh, Sult1a1, Mawbp, Por, and Gstm1) and two expressed sequence tages ( BI277460 and AI411345) were significantly upregulated. Four of these genes ( Igfbp1, Xdh, Por, and Gstm1) have well-characterized functions in the cardiovascular system. The proteins corresponding to Xdh, Por, and Gstm1 are involved in the metabolism of reactive oxygen species (ROS). Because ROS can mediate endothelial dysfunction, we measured the aortic dilatory capacity in thoracic aortic rings. Indeed, vasodilator potency to acetylcholine was largely diminished in hypotensive animals, whereas sodium nitroprusside induced equivalent vasodilations in normotensive and hypotensive animals. The vasodilator potency of the endothelium was partially restored by the superoxide scavenger tiron. Hence, acute hypotension induces a diminished vasodilator potency of the endothelium due to an accelerated degradation of nitric oxide by ROS. The present physiogenomic approach is capable of detecting vasoregulatory mechanisms and may provide deeper insight into the genetics and physiology of blood pressure regulation.

The MALDI-MES provides a rapid, sensitive and reproducible alternative approach to existing analytical techniques for the detection of enzymatic activities that does not require a chromophore or radiolabeling. An improved method is presented, by which enzymes with defined substrate specificities can be detected with a MALDI mass spectrometer in complex protein fractions. In order to demonstrate the utility of the new method, in this study we describe the use of MALDI-MES to detect proteolytic activities in a protein extract from porcine renal tissue, which contained several thousand proteins as visualized by 2D electrophoresis. The analytical procedure is based on covalent immobilization of proteins to beads. By immobilizing proteins, autolytic and proteolytic degradation is prevented and the removal of those molecules from the protein fraction is achieved, which otherwise would interfere with the mass spectrometric detection of the enzymatic reaction products. The enzymatic activity is determined by incubating the immobilized proteins with a reaction-specific probe, followed by the analysis of the reaction mixture with the MALDI-MS after defined incubation times. The presence of the target enzyme is validated by locating a signal, which fits the molecular mass of the expected reaction product in the mass spectrum. To demonstrate how to detect proteolytic activities in this system, the reactions catalyzed by endopeptidase, angiotensin-converting enzyme, kallikrein, renin, and urotensin-converting enzyme were monitored. The experiments showed that the MALDI-MES method is sufficient according the quantification to investigate the effects of inhibitors. This is demonstrated using a specific renin inhibitor to inhibit an angiotensin-I generating enzyme activity in a renal protein extract.