Search

As post-translational modifications of proteins may have an impact on the pathogenesis of diseases such as atherosclerosis, diabetes mellitus and chronic kidney disease (CKD), post-translational modifications are currently gaining increasing interest. In this study, a comprehensive method for analysis of these post-translational modifications is established for the clinical diagnostic routine.Here, we analysed albumin - the most abundant plasma protein in human - isolated from patients with CKD and healthy controls by chromatographic steps and identified by MALDI mass spectrometry. Post-translational modifications of albumin were identified after digestion by analysing mass signal shifts of albumin peptides using pertinent mass databases.Albumin isolated from plasma of patients with CKD but not from healthy control subjects was specifically post-translationally modified by guanidinylation of lysines at positions 249, 468, 548, 565 and 588. After identification of guanidinylations as post-translational modifications of albumin isolated from patients with CKD, these modifications were quantified by mass spectrometry demonstrating a significant increase in the corresponding mass signal intensities in patients with CKD compared to healthy controls. The relative amount of guanidinylation of lysine at position 468 in patients with CKD was determined as 63 ? 32% (N = 3). Subsequently, we characterized the pathophysiological impact of the post-translational guanidinylation on the binding capacity of albumin for representative hydrophobic metabolic waste products. In vitro guanidinylation of albumin from healthy control subjects caused a decreased binding capacity of albumin in a time-dependent manner. Binding of indoxyl sulphate (protein-bound fraction) decreased from 82 ? 1% of not post-translationally modified albumin to 56 ? 1% after in vitro guanidinylation (P

Management of volume status is difficult in critically ill patients with renal failure. Volumetric hemodynamic indices are increasingly being used to guide fluid therapy in the intensive care unit (ICU), but are not established to monitor hemodialysis-induced fluid removal in critically ill patients. Using volumetric hemodynamic monitoring, changes in extravascular lung water index (EVLWI) and intrathoracic blood volume index (ITBVI) were measured immediately before and after hemodialysis sessions in 35 ICU patients. Additional hemodynamic and oxygenation related parameters were recorded at the same time, and online relative blood volume (RBV) monitoring was performed during hemodialysis. EVLWI decreased significantly with fluid removal (median 10.0 vs. 9.6 mL/kg, P = 0.001), whereas ITBVI remained stable (median 1012 vs. 1029 mL/m2, P = 0.402). Significant changes were also observed in stroke volume variation (median 12.0 vs. 13.0 %, P = 0.012), cardiac index (median 4.2 vs. 3.5 mL/min/m2, P = 0.003), mean arterial pressure (median 77 vs. 85.5 mmHg, P = 0.006), norepinephrine dose (median 0.092 vs. 0.114 μg/kg per min, P = 0.043), and hemoglobin values (median 9.5 vs. 10.4 gm/dL, P = 0.036). RBV decreased by 7.8% (median); there was no correlation with either the volumetric measurements or the other hemodynamic parameters recorded. EVLWI reduction with dialysis reflects the removal of excess body fluid, whereas preservation of cardiac preload is indicated by ITBVI stability. Volumetric hemodynamic measurements provide additional information concerning fluid status and are thus potentially useful to guide fluid removal on hemodialysis in critically ill patients.

Calcineurin inhibitors (CNI) are potent vasoconstrictors and induce an acceleration of arteriosclerosis, thus contributing to the cardiovascular risk after renal transplantation. The study compares the impact of belatacept and cyclosporine A (CsA) on arterial stiffness and central aortic blood pressure. We performed a case-control study in 46 patients (23 on belatacept and 23 on CsA) matched for age, body mass index, time after transplantation, and time on dialysis prior to transplantation. Pulse wave analysis (SphygmoCor, AtCor(®) ) was used to assess central aortic blood pressure, aortic augmentation pressure, and pulse wave velocity (PWV) as a marker of arterial stiffness. Assessment of vascular function was performed after a minimum of 20 months and a median follow-up of 81 months post-transplant. Peripheral systolic and diastolic blood pressure did not significantly differ in the two groups (p > 0.05 each). The central aortic augmentation pressure was higher in the CsA group (12.7 mmHg vs. 7.3 mmHg, p = 0.048). PWV as a measure of arterial stiffness did not differ in the two groups. Thus, belatacept is not associated with a significant difference in arterial stiffness compared to CsA after a median of 81 months post-transplant. It is associated, however, with a lower aortic augmentation pressure, a strong independent cardiovascular risk factor.

BACKGROUND: Urinary calprotectin has recently been identified as a promising biomarker for the differentiation of prerenal and intrinsic acute kidney injury (AKI). The present study compares the diagnostic performance of calprotectin and neutrophil gelatinase-associated lipocalin (NGAL) in this differential diagnosis. METHODS: Urinary calprotectin and NGAL concentrations were assessed in a study population of 87 subjects including 38 cases of intrinsic AKI, 24 cases of prerenal AKI, and 25 healthy controls. Urinary tract obstruction, renal transplantation and metastatic cancer were defined as exclusion criteria. RESULTS: Mean calprotectin concentrations were significantly lower in prerenal (190.2+/-205.7 ng/ml) than in intrinsic AKI (6250.1+/-7167.2 ng/ml, p

Protein-bound uremic toxins, such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate, contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease (CVD). However, based on their protein binding, these hydrophobic uremic toxins are poorly cleared during conventional dialysis and thus accumulate in CKD-5D patients. Therefore, we investigated whether hydrophobic and cationic adsorbers are more effective for removal of protein-bound, hydrophobic uremic toxins than conventional high-flux hemodialyzer. Five CKD-5D patients were treated using the fractionated plasma separation, adsorption, and dialysis (FPAD) system for 5 h. A control group of five CKD patients was treated with conventional high-flux hemodialysis. Plasma concentrations of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were measured. Removal rates of FPAD treatment in comparison to conventional high-flux hemodialysis were increased by 130% for phenylacetic acid, 187% for indoxyl sulfate, and 127% for p-cresol. FPAD treatment was tolerated well in terms of clinically relevant biochemical parameters. However, patients suffered from mild nausea 2 h after the start of the treatment, which persisted until the end of treatment. Due to the high impact of protein-bound, hydrophobic uremic toxins on progression of CKD and CVD in CKD-5D patients, the use of an adsorber in combination with dialysis membranes may be a new therapeutic option to increase the removal rate of these uremic toxins. However, larger, long-term prospective clinical trials are needed to demonstrate the impact on clinical outcome.

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.

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.

It is obvious that retention of uremic toxins leads to endothelial dysfunction, which in turn initiates proliferation of vascular smooth muscle cells. Yet, the entity of the cascade of pathomechanisms has not sufficiently been elucidated. Endothelial dysfunction inevitably and irreversibly leads to progressive cardiovascular dysfunction and thereby represents the beginning of a life-limiting cascade. This review highlights important findings in this field.

It is still controversial whether the mode of dialysis or preexisting comorbidities may influence the prognosis of patients with chronic kidney disease stage 5. Therefore, we performed a prospective case control study to evaluate whether the mode of dialysis may influence outcome. We found 25 cases on peritoneal dialysis (PD) treatment and 75 age and sex-matched controls on hemodialysis (HD) treatment for more than 3 months. Analysis was by intention-to-treat. During the follow up of 58 months, 6 out of 25 patients (24%) died in the PD group, whereas in the HD group 26 out of 75 patients (35%) died (relative risk 0.69 [95% CI 0.32 to 1.49]; P = 0.46). Survival was not significantly different between the groups as indicated by Mantel-Cox log-rank test (hazard ratio 0.52 [95% CI 0.25 to 1.10]; P = 0.11). Multiple variable regression showed that age and diabetes mellitus, but not mode of dialysis, predicted death in patients with chronic kidney disease. It is concluded that age and comorbidities but not mode of dialysis are important to predict survival in patients with chronic kidney disease stage 5.

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 (

Dinucleoside polyphosphates are a group of intra- and extracellular mediators controlling numerous physiological functions. In this study dinucleoside polyphosphates were examined by positive ion matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MADLI-TOFMS). 3-Hydroxypicolinic acid was used as UV-absorbing matrix. For the individual dinucleoside polyphosphates Ap(n)A (n = 2-7), Ap(n)G (n = 2-6) and Gp(n)G (n = 2-6), MALDI post-source decay (PSD) mass spectra were measured. Each mass peak in the MALDI-PSD mass spectra could be assigned to individual fragments of dinucleoside polyphosphates. The comparison of the fragmentation patterns of the dinucleoside polyphosphates presented here demonstrates that dinucleoside polyphosphates preferably cleave to fragment ions consisting of the corresponding mononucleoside polyphosphates as well as the corresponding nucleosides and bases during flight in the field-free drift path of the MALDI mass spectrometer. Therefore, the MALDI-PSD approach described here is suitable for identification of other dinucleoside polyphosphates. The present MALDI-PSD mass spectra may be used as MALDI-PSD mass reference spectra for future identification of dinucleoside polyphosphates and other nucleotides.

Background Angiotensin-converting enzyme inhibitors may affect reactive oxygen species in humans in vitro and in vivo. In the present study we evaluated whether angiotensin-converting enzyme inhibitors may affect NAD(P)H oxidase activity. Materials and methods The production of reactive oxygen species was measured spectrophotometrically in mononuclear leukocytes using the fluorescent dye, dichlorofluorescein diacetate. The effects of quinaprilat, captopril, enalaprilat and lisinopril on phorbol myristate acetate-induced reactive oxygen species generation were investigated in vitro. The effects of quinaprilat, captopril, enalaprilat and lisinopril on the NAD(P)H oxidase activity of the mononuclear leukocytes were measured photometrically. In addition, reactive oxygen species were measured before and 4 h after oral administration of quinapril. ResultsIn vitro, the addition of quinaprilat (72 ± 6% of control; mean ± SEM; n= 19; P

1. The activation of P(2x)-receptors in the rat renal vasculature by dinucleoside polyphosphates with variable phosphate group chain length (Xp(n)X; X=Adenin (A) /Guanin (G), n=4 - 6) was studied by measuring their effects on perfusion pressure of the isolated perfused rat kidney at constant flow in an open circuit. 2. Like Ap(4)A, Ap(5)A and Ap(6)A the dinucleoside polyphosphates Ap(4)G, Ap(5)G and Ap(6)G exerted a vasoconstriction which could be blocked by suramin and pyridoxal-phosphate-6-azophenyl-2; 4-disulphonic acid (PPADS). 3. Gp(4)G, Gp(5)G and Gp(6)G showed only very weak vasoconstriction at high doses. 4. Ap(6)A and alpha, beta-meATP could not be blocked by the selective P(2x1)-receptor antagonisten NF023 (30 microM), whereas Ap(4)A, Ap(4)G, Ap(5)A, Ap(5)G and Ap(6)G were partially blocked by NF023. 5. Inhibition of endothelial NO-synthase by N(omega)-nitro-L-arginine methyl ester (L-NAME) did not affect vasoconstrictions induced by dinucleosidepolyphosphates. 6. P(2x)-receptor can only be activated if at least one adenosine moiety is present in the molecule. 7. Ap(n)G show a weaker vasoconstrictive action than corresponding Ap(n)A, concluding that two adenosine moieties enhance the P(2x)-receptor binding and activation. 8. Xp(n)X containing five phosphate groups show the most pronounced vasoconstrictive effect whereas four phosphate groups show the less effect, therefore the number of phosphate groups critically changes receptor affinity. 9. Additional experiments using permanent perfusion with alpha, beta-methylene ATP (alpha,beta-meATP) and the selective P(2x1)-receptor antagonist NF023 showed that the newly discovered human dinucleoside polyphosphates activated the vascular P(2x1)-receptor and an recently identified new P(2x)-receptor subtype. 10. The differential effects of dinucleoside polyphosphates allow a fine tuning of local perfusion via composition of Xp(n)Xs.

Cytosolic pH (pH(i)) and the activity of the sodium-proton antiporter (Na(+)/H(+) antiporter) were measured in lymphocytes from 22 patients with obstructive sleep apnoea and from 24 age-matched healthy subjects (Controls). The cellular Na(+)/H(+) antiporter was measured spectrophotometrically using a pH-sensitive fluorescent dye after intracellular acidification using sodium propionate. Resting pHi was similar in lymphocytes from patients with obstructive sleep apnoea and from controls (7.36 +/- 0.20, n=22; vs. 7.35 +/- 0.19, n=24; mean +/- SD). The Na(+)/H(+) antiporter activity was significantly higher in patients with obstructive sleep apnoea than in controls (11.87 +/- 3.26 x 10(-3) pH(i)/s vs. 4.38 +/- 1.40 x 10(-3) pH(i)/s; P0. 0001). The apparent affinity of the Na+/H+ antiporter was not significantly different between the groups (6.90 +/- 0.23 vs. 6.87 +/- 0.20). In patients with obstructive sleep apnoea the activity of the Na(+)/H(+) antiporter remained stable during the night. The activity of the Na(+)/H(+) antiporter was 13.49 +/- 4.80 x 10(-3) pH(i)/s at 20.00 and 13.26 +/- 6.13 x 10(-3) pH(i)/s at 02.00. From the present results it is concluded that an increased cellular Na(+)/H(+) antiporter activity may be a genetic marker for patients who are predisposed to obstructive sleep apnoea.

The activation of various P2-receptor subtypes in rat renal vasculature by P1, P5-diadenosine pentaphosphate (Ap5A) and P1, P6-diadenosine hexaphosphate (Ap6A) were studied by measuring their effects on perfusion pressure during continuous perfusion in a rat isolated perfused kidney. Permanent perfusion with Ap5A and Ap6A elicited both a transient and sustained vasoconstriction with both vasoconstrictions to be different: the transient vasoconstriction can be elicited with concentrations 10 nM, whereas the sustained vasoconstriction is observed with concentrations 1 nM. Ap5A and Ap6A act via the same receptors as α,β-methylene ATP (α,β-meATP). The rank order of potency for transient vasconstriction was α,β-meATP=Ap5A>Ap6A>β,γ-meATP, and for sustained vasoconstriction α,β-meATP=Ap5A>β,γ-meATP Ap6A. Suramin, a non-selective P2-receptor antagonist, and pyridoxal-phosphate-6-azophenyl-2;4-disulphonic acid (PPADS) a highly selective P2X-receptor antagonist antagonized both the transient and the sustained vasoconstriction. Taken together the results of the agonist profile of Ap5A and Ap6A and comparing its findings to literature it can be demonstrated that the transient but not the sustained vasoconstriction is mediated via the P2X1-receptor which is present in rat renal vasculature. It is demonstrated that the agonist profile of the sustained vasoconstriction induced by Ap5A and Ap6A does not fit to any currently known P2X- or P2Y-receptor subtype. We conclude a yet unidentified P2X-receptor or chimeric P2X-receptor may contribute to the effects on rat renal vasculature produced by Ap5A and Ap6A and which may play an important role in glomerular perfusion pressure and blood pressure control. British Journal of Pharmacology (1999) 127, 1463–1469; doi:10.1038/sj.bjp.0702667

1. Diadenosine phosphates were isolated from platelets, adrenal gland and autonomic nerves. The presence of diadenosine phosphates in storage pools releasable into the circulation suggests an important role in the control of blood pressure, and potentially to a modulation of the actions of catecholamines. 2. Besides a role of the diadenosine phosphates in platelet aggregation, these agents have potent vasoactive properties. Vasoactive actions of the diadenosine phosphates were demonstrated in numerous vascular models including most of the physiologically important elements of blood pressure regulation. Mostly, the vasoactive action depends on the number of phosphates in the diadenosine phosphates. Vasodilation can be observed in intact vessels after administration of Ap2A, Ap3A and Ap4A whereas contraction is affected by Ap5A and Ap5A and Ap6A. Vasocontraction induced by the diadenosine phosphates in vascular smooth muscle cells is mediated by an increase in intracellular free Ca2+. 3. In vivo, intravenous injection of Ap4A lowers blood pressure whereas injections of Ap5A and Ap6A caused a prolonged increase in blood pressure. In blood, in contrast to ATP, diadenosine phosphates are relatively long-lived molecules, suggesting that the action of the latter is of intermediate time span. In a similar manner to the vasoconstrictor angiotensin II, diadenosine phosphates also act as mitogens. It can be assumed that diadenosine phosphates may be involved in pathophysiological events of circulation including hypertension and atherosclerosis.

1. Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2. The effects of AP6A (0.1-100 microM) on cardiac contractility and frequency were studied in guinea-pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L-type calcium current, on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea-pig isolated ventricular myocytes. 3. In isolated spontaneously beating right atria of the guinea-pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 microM). 4. In isolated electrically driven left atria of the guinea-pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 microM). 5. In isolated electrically driven papillary muscles of the guinea-pig AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction maximally by 23% (IC20 = 60 microM). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6. In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction by maximally 42% (IC20 = 18 microM). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7. All these effects of AP6A were abolished by the selective A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM), whereas the M-cholinoceptor antagonist atropine (10 microM) and the P2-purinoceptor antagonist suramin (300 microM) failed to abolish the effects of AP6A. 8. AP6A 100 microM had no effect on the amplitude of the L-type calcium current, but attenuated isoprenaline-stimulated L-type calcium current. The maximum of the current-voltage relationship (I-V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I-V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9. In summary, AP6A exerts negative chronotropic and inotropic effects in guinea-pig and human cardiac preparations. These effects are mediated via A1-adenosine receptors as all effects were sensitive to the selective A1-adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L-type calcium current are in accordance with stimulation of A1-adenosine receptors.