Jens Martens-Lobenhoffer

Symmetrical Dimethylarginine: A New Combined Parameter for Renal Function and Extent of Coronary Artery Disease

Symmetrical dimethylarginine (SDMA) is the structural isomer of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine. Whereas the major route of asymmetric dimethylarginine elimination is the hydrolytic degradation by dimethylarginine dimethylaminohydrolase, SDMA is eliminated by renal excretion. SDMA does not directly inhibit NOS but is a competitor of arginine transport. This study showed for the first time that measurement of SDMA can be a marker of estimated GFR and extent of coronary artery disease (CAD). In 97 patients with CAD, SDMA was a marker of estimated GFR. On multiple regression analysis of the CAD parameter stenosis score, SDMA was the only parameter retained. In addition, endothelial cells from the third passage were cultured in medium that contained 70 micromol/L arginine and was incubated for 24 h in the presence of various concentration of SDMA (0, 2, 5, 10, and 100 micromol/L). The levels of nitrate and nitrite in conditioned media, the protein expression of NOS, and the content of reactive oxygen species in endothelial cells were determined. SDMA inhibited dose dependently the NO synthesis in intact endothelial cells, whereas it had no effect on protein expression of NOS. This effect was associated with an increase in reactive oxygen species. Co-incubation with L-arginine but not D-arginine reversed the effect of SDMA on NOS pathway. Our data suggest that SDMA reduced the endothelial NO synthesis, probably by limiting L-arginine supply to NOS. It is concluded that SDMA might be a useful parameter for detecting patients in very early stages of chronic kidney disease and for determining their risk for developing cardiovascular disease.

Asymmetrical Dimethylarginine in Idiopathic Pulmonary Arterial Hypertension

Objective—We explored the potential role of the endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) in patients with idiopathic pulmonary arterial hypertension (IPAH). Method and Results—We correlated plasma ADMA levels and cardiovascular indices from right heart catheterization in 57 patients with IPAH. Predictors of survival in patients with IPAH were studied. Furthermore, the effect of systemic ADMA infusion on pulmonary ventricular resistance and stroke volume was investigated in healthy volunteers using right heart catheterization. Mean plasma ADMA concentrations were significantly higher in patients with IPAH than in control subjects (0.53±0.15 versus 0.36±0.05 &mgr;mol/L; P<0.001). ADMA plasma concentrations correlated significantly with indices of right ventricular function, such as mixed-venous oxygen saturation (r=−0.49; P<0.0001), right atrial pressure (r=0.39; P<0.003), cardiac index (r=−0.35; P<0.008), as well as survival (r=−0.47; P<0.0001). Multiple regression analysis revealed that right atrial pressure (r=0.31; P<0.026) and ADMA (r=0.29; P<0.039) were independent predictors of mortality. Moreover, patients with supra-median plasma ADMA levels had significantly (P<0.021) worse survival than patients with infra-median ADMA values. ADMA infusion in healthy volunteers increased pulmonary vascular resistance (68.9±7.6 versus 95.6±6.3 dyne · s · cm−5; P<0.05) and decreased stroke volume (101.1±6.7 mL versus 95.6±6.3 mL; P<0.05). Conclusion—Increased ADMA plasma levels are associated with unfavorable pulmonary hemodynamics and worse outcome in patients with IPAH.

ADMA Increases Arterial Stiffness and Decreases Cerebral Blood Flow in Humans

Background and Purpose— Preclinical studies have revealed that the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), increases vascular tone in cerebral blood vessels. Marked elevations of ADMA blood levels were found in patients with diseases characterized by decreased cerebral perfusion, such as ischemic stroke. Arterial stiffness is an independent predictor of stroke and other adverse cardiovascular events. The aim of this study was to investigate the influence of a systemic subpressor dose of ADMA on arterial stiffness and cerebral perfusion in humans. Methods— Using a double-blind, vehicle-controlled study design, we allocated 20 healthy men in random order to infusion of either ADMA (0.10 mg ADMA/kg per min) or vehicle over a period of 40 minutes. Arterial stiffness was assessed noninvasively by pulse wave analysis. All volunteers underwent measurement of cerebral perfusion by dynamic contrast-enhanced perfusion magnetic resonance imaging of the brain. Results— Infusion of ADMA significantly decreased total cerebral perfusion by 15.1±4.5% (P=0.007), whereas blood flow in the vehicle group increased by 7.7±2.8% (P=0.02). ADMA also increased arterial stiffness as assessed by measurement of the augmentation index (−12.6±1.9 to −9.6±1.5, P=0.007). Conclusions— Our results document for the first time that subpressor doses of ADMA increase vascular stiffness and decrease cerebral perfusion in healthy subjects. Thus, ADMA is an important endogenous modulator of cerebral vascular tone and may be involved in the pathogenesis of cerebrovascular disease.

Endogenous Nitric Oxide Synthesis Inhibitor Asymmetric Dimethyl L-Arginine Accelerates Endothelial Cell Senescence

Objectives—Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS), and its accumulation has been associated with cardiovascular disease. We aimed to investigate the role of ADMA in endothelial cell senescence. Methods and Results—Endothelial cells were cultured until the tenth passage. ADMA was replaced every 48 hours starting at the fourth passage. ADMA significantly accelerated senescence associated &bgr;-galactosidase activity. Additionally, the shortening of telomere length was significantly accelerated and the telomerase activity was significantly reduced. This effect was associated with an increase of oxidative stress: allantoin, a marker of oxygen free radical generation, and intracellular reactive oxygen species (ROS) increased significantly after ADMA treatment compared with control, whereas cellular thiol status and NOx synthesis decreased. Furthermore, ADMA-increased oxidative stress was accompanied by a decrease in the activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA, which could be prevented by the antioxidant pyrrolidine dithiocarbamate. Exogenous ADMA also stimulated secretion of MCP-1 and interleukin-8. Coincubation with the methyltransferase inhibitor S-adenosylhomocysteine abolished the effects of ADMA. Conclusions—These data suggest that ADMA accelerates senescence, probably via increased oxygen radical formation by inhibiting nitric oxide elaboration. This study provides evidence that modest changes of intracellular ADMA levels are associated with significant effects on slowing endothelial senescence.

Erythropoietin Increases Asymmetric Dimethylarginine in Endothelial Cells: Role of Dimethylarginine Dimethylaminohydrolase

Recombinant human erythropoietin therapy frequently causes hypertension in humans and animals with chronic renal failure. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase, and its accumulation has been associated with reducing NO bioavailability and increasing superoxide generation. Whether epoetin beta (EPO) or darbepoetin alpha (NESP) can modify the levels of ADMA in endothelial cells was investigated. Endothelial cells from the third passage were incubated for 24 h in the presence of various concentrations of EPO or NESP (0, 0.1, 1, 10, 50, 100, and 200 U/ml). The levels of ADMA, allantoin, nitrate, and nitrite in conditioned media and the activity of dimethylarginine dimethylaminohydrolase (DDAH), the content of thiols and reactive oxygen species in endothelial cells, were determined. When endothelial cells were exposed to EPO or NESP, ADMA concentration in the cell culture medium increased significantly in a dose-dependent manner versus control. This effect was associated with a reduced activity of DDAH, the enzyme that degrades ADMA. Furthermore, EPO- or NESP-induced accumulation of ADMA was accompanied by a significant reduction of NO synthesis and an increase in oxidative stress. Both allantoin, a marker of oxygen free radical generation, and reactive oxygen species increased significantly after EPO or NESP treatment compared with control. The antioxidant pyrrolidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation in the same way as the co-incubation with anti-EPO neutralizing antibody. EPO and NESP posttranslationally impair DDAH activity via increased oxidative stress, causing ADMA as an important cardiovascular risk factor to accumulate and inhibit NO synthesis.

Effect of Telmisartan on Nitric Oxide–Asymmetrical Dimethylarginine System: Role of Angiotensin II Type 1 Receptor and Peroxisome Proliferator Activated Receptor γ Signaling During Endothelial Aging

Telmisartan, in addition to blocking angiotensin (Ang) II type 1 receptor (AT1R), activates peroxisome proliferator activated receptor γ (PPARγ) signaling that interferes with nitric oxide (NO) system. Because aging of endothelial cells (ECs) is hallmarked by a reduction in NO synthesis, we hypothesized that telmisartan increases NO formation by regulated asymmetrical dimethylarginine (ADMA)-dimethylarginine dimethylaminohydrolase (DDAH)-system through blocking AT1R and activating PPARγ signaling. To test this hypothesis, ECs were cultured with telmisartan, eprosartan, Ang II, and GW9662 (PPARγ antagonist) until the twelfth passage. During the process of aging, PPARγ protein expression decreased significantly, whereas the expression of AT1R increased. Telmisartan reversed these effects and dose-dependently decreased reactive oxygen species and 8-iso-prostaglandin (PG) F2α formation. This effect was associated with an upregulated activity and protein expression of DDAH, accompanied by a decrease in ADMA concentration, an increase in NO metabolites, and delayed senescence. Blockade of PPARγ signaling by GW9662 or PPARγ small-interference RNA prevented the effect of telmisartan on ADMA-DDAH-NO system. Coincubation with Ang II did not affect the effect of telmisartan-delayed senescence, whereas Ang II itself accelerated endothelial aging. Moreover, AT1R blocker eprosartan that did not influence PPARγ protein expression had no effect on ADMA system and senescence. We have demonstrated that telmisartan mainly by activating PPARγ signaling can alter the catabolism and release of ADMA as an important cardiovascular risk factor. We therefore propose that telmisartan translationally and posttranslationally upregulated DDAH expression via activation of PPARγ signaling, causing ADMA to diminish and increase NO synthesis sufficient to delay senescence.

Dosing of daptomycin in intensive care unit patients with acute kidney injury undergoing extended dialysis—a pharmacokinetic study

BACKGROUND Daptomycin is a new intravenous cyclic lipopeptide antibiotic, licensed for the treatment of complicated skin and soft tissue infections caused by Gram-positive organisms including both susceptible and resistant strains of Staphylococcus aureus and for the treatment of various infections due to susceptible organisms, including serious and life-threatening Gram-positive infections, vancomycin-resistant enterococcal infections and right-sided endocarditis with associated bacteremia. Currently, no dosing recommendations exist for this drug for patients with acute kidney injury (AKI) undergoing renal replacement therapy. The aim of this study was to evaluate pharmacokinetics of daptomycin in critically ill patients with AKI undergoing extended dialysis (ED), a frequently used mean of renal replacement therapies in intensive care units (ICUs) around the world. Patients and methods. A prospective, single-dose pharmacokinetic study was performed in the medical and surgical ICUs of a tertiary care center. The aim was to investigate critically ill patients with anuric AKI being treated with ED and receiving daptomycin (n = 10). Daptomycin (6 mg/kg) was administered 8 h before ED was started. RESULTS Key pharmacokinetic parameters like half-life in critically ill patients treated with ED were comparable to healthy controls. The dialyser clearance for daptomycin was 63 +/- 9 ml/min. Based on the amount of the drug recovered from the collected spent dialysate, the mean fraction of the drug removed by one dialysis treatment was 23.3%. CONCLUSION Our data suggest that patients treated with ED using a high-flux dialyzer (polysulphone, 1.3 m(2); blood and dialysate flow, 160 ml/min; ED time, 480 min) and employing current dosing regimen, 6 mg/kg daptomycin every 48 h, run the risk of becoming significantly under dosed if one adheres to a twice daily dosing schedule that is recommended for patients on maintenance haemodialysis. Our data suggest that a daily dose of 6 mg/kg daptomycin is necessary in this special patient population to avoid under dosing, which may have detrimental effects in critically ill patients suffering from life-threatening infections.

SDMA is an early marker of change in GFR after living-related kidney donation

BACKGROUND Early detection of changes in the glomerular filtration rate (GFR) is crucial in detecting acute kidney injury. There is burgeoning evidence from preclinical and clinical studies that symmetrical dimethylarginine (SDMA) correlates well with different parameters of renal function. In some studies, SDMA even outperformed creatinine as a marker of GFR. It is however unknown how fast SDMA is increasing after reduction in GFR. The aim of our study was therefore to determine the temporal change of SDMA in comparison with cystatin C after a defined reduction in GFR. METHODS Blood samples from 24 healthy living-related kidney donors (19 F/5 M), mean age 55.2 ± 8.3 years, were collected prior to donation of the kidney as well as 1, 6, 12, 24, 72 and 168 h after unilateral nephrectomy. SDMA levels were measured using a liquid chromatography-mass spectrometry-based method. RESULTS Within 6 h after unilateral nephrectomy, i.e. reduction of GFR by 50%, SDMA rose from 0.571 ± 0.120 to 0.659 ± 0.135 µmol/L (P < 0.001). Baseline cystatin C levels increased from 0.87 ± 0.16 to 1.07 ± 0.15 mg/L (P < 0.001). Also, serum creatinine rose significantly within 6 h after removal of one kidney from 65.4 ± 8.4 to 88.8 ± 10.2 µmol/L (P < 0.001). DISCUSSION SDMA might be a valuable and early marker of change in GFR in the clinical and experimental setting. Future studies will have to clarify whether sensitivity, specificity and temporal resolution of SDMA make it an attractive candidate for the assessment of renal function in both the experimental and clinical setting.

Validated high performance liquid chromatography-UV detection method for the determination of daptomycin in human plasma.

Daptomycin is the first approved member of the new class cyclic lipopeptide antibiotic drugs, effective against a broad spectrum of Gram-positive bacteria. Here we present an HPLC method with UV detection capable to obtain pharmacokinetic data of daptomycin in human plasma, exemplarily shown in a critically ill patient with acute renal failure undergoing extended daily dialysis. Sample preparation consists only of protein precipitation with methanol. Chromatographic separation was achieved on a Zorbax Eclipse XDB-C8 column and daptomycin was detected at 224 nm. The calibration function was linear over the range from 3.5 to 350 microg/ml. The relative standard deviations were <2% in the intra-day and <6% in the inter-day measurements. The accuracy was always better than 7%. Daptomycin was stable in aqueous solutions for 2 months frozen at-20 degrees C. However, in plasma frozen at -20 degrees C a loss of 25% in 1 month was observed.

The impact of rapid atrial pacing on ADMA and endothelial NOS

BACKGROUND The endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a well-established risk factor for oxidative stress, vascular dysfunction, and congestive heart failure. The aim of the present study was to determine the impact of rapid atrial pacing (RAP) on ADMA levels and eNOS expression. METHODS AND RESULTS ADMA levels were studied in 60 age- and gender-matched patients. Thirty five patients had persistent atrial fibrillation (AF)≥ 4months. In AF-patients, parameters were studied before and 24h after electrical cardioversion. Moreover, ADMA, eNOS expression, and calcium-handling proteins were studied in pigs subjected to RAP as well as in endothelial cell (EC) cultures. ADMA level was significantly higher in AF compared to sinus rhythm patients (p=0.024). ADMA was highest in AF-patients, who also showed elevated troponin T (TnT) levels. Moreover, ADMA showed a significant linear correlation to TnT (r=0.47; p<0.01). After electrical cardioversion ADMA returned to normal within 24h. In pigs, RAP for 7h increased ADMA levels (p=0.018) and TnI (p<0.05), and reduced mRNA expression of ventricular and aortic eNOS (-80%; p<0.05) compared to sham-control. However, ADMA per se did not affect eNOS mRNA level in EC cultures. CONCLUSION The current study shows that acute and persistent episodes of atrial tachyarrhythmia are associated with elevated ADMA levels accompanied by increased ischemic myocardial markers. Moreover, RAP increases ADMA and down-regulates eNOS expression in an ADMA-independent manner. We conclude that the combination of these two separate and potentially synergistic mechanisms may contribute to long-term vascular injury during atrial tachyarrhythmia.