Folinic Acid Increases Protein Arginine Methylation in Human Endothelial Cells.

Elevated plasma total homocysteine (tHcy) is associated with increased risk of cardiovascular disease, but the mechanisms underlying this association are not completely understood. Cellular hypomethylation has been suggested to be a key pathophysiologic mechanism, since S-adenosylhomocysteine (AdoHcy), the Hcy metabolic precursor and a potent inhibitor of methyltransferase activity, accumulates in the setting of hyperhomocysteinemia. In this study, the impact of folate and methionine on intracellular AdoHcy levels and protein arginine methylation status was studied. Human endothelial cells were incubated with increasing concentrations of folinic acid (FnA), a stable precursor of folate, with or without methionine restriction. The levels of intracellular AdoHcy and AdoMet, tHcy in the cell culture medium, and protein-incorporated methylarginines were evaluated by suitable liquid chromatography techniques. FnA supplementation, with or without methionine restriction, reduced the level of tHcy and did not affect intracellular AdoMet levels. Interestingly, FnA supplementation reduced intracellular AdoHcy levels only in cells grown under methionine restriction. Furthermore, these cells also displayed increased protein arginine methylation status. These observations suggest that folic acid supplementation may enhance cellular methylation capacity under a low methionine status. Our results lead us to hypothesize that the putative benefits of folic acid supplementation in restoring endothelial homeostasis, thus preventing atherothrombotic events, should be reevaluated in subjects under a methionine restriction diet.

Symmetric and asymmetric dimethylarginine as risk markers of cardiovascular disease, all-cause mortality and deterioration in kidney function in persons with type 2 diabetes and microalbuminuria.

BACKGROUND: To evaluate symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) as risk markers of cardiovascular disease, all-cause mortality and deterioration in renal function in a well characterised type 2 diabetic population with microalbuminuria and without symptoms of coronary artery disease. METHODS: 200 participants followed for 6.1 years. SDMA and ADMA were measured at baseline. Endpoints included (1) composite cardiovascular endpoint (n = 40); (2) all-cause mortality (n = 26); and (3) decline in eGFR of >30% (n = 42). Cox models were unadjusted and adjusted for traditional risk factors (sex, age, systolic blood pressure, LDL-cholesterol, smoking, HbA1c, creatinine and urinary albumin excretion rate). To assess if SDMA or ADMA improved risk prediction beyond traditional risk factors we calculated c statistics and relative integrated discrimination improvement (rIDI). C statistic (area under the curve) quantifies the model's improved ability to discriminate events from non-events. rIDI quantifies the increase in separation of events and non-events on a relative scale. RESULTS: Higher SDMA was associated with increased risk of all three endpoints (unadjusted: p ≤ 0.001; adjusted: p ≤ 0.02). Higher ADMA was associated with all-cause mortality (unadjusted: p = 0.002; adjusted: p = 0.006), but not cardiovascular disease or decline in eGFR (p ≥ 0.29).The c statistic was not significant for any of the endpoints for either SDMA or ADMA (p ≥ 0.10). The rIDI for SDMA was 15.0% (p = 0.081) for the cardiovascular endpoint, 52.5% (p = 0.025) for all-cause mortality and 48.8% (p = 0.007) for decline in eGFR; for ADMA the rIDI was 49.1% (p = 0.017) for all-cause mortality. CONCLUSION: In persons with type 2 diabetes and microalbuminuria higher SDMA was associated with incident cardiovascular disease, all-cause mortality and deterioration in renal function. Higher ADMA was associated with all-cause mortality. SDMA and ADMA significantly improved risk prediction for all-cause mortality, and SDMA for deterioration in renal function beyond traditional risk factors.

Effect of oral taurine on morbidity and mortality in elderly hip fracture patients: a randomized trial.

Hip fracture patients represent a large part of the elderly surgical population and face severe postoperative morbidity and excessive mortality compared to adult surgical hip fracture patients. Low antioxidant status and taurine deficiency is common in the elderly, and may negatively affect postoperative outcome. We hypothesized that taurine, an antioxidant, could improve clinical outcome in the elderly hip fracture patient. A double blind randomized, placebo controlled, clinical trial was conducted on elderly hip fracture patients. Supplementation started after admission and before surgery up to the sixth postoperative day. Markers of oxidative status were measured during hospitalization, and postoperative outcome was monitored for one year after surgery. Taurine supplementation did not improve in-hospital morbidity, medical comorbidities during the first year, or mortality during the first year. Taurine supplementation lowered postoperative oxidative stress, as shown by lower urinary 8-hydroxy-2-deoxyguanosine levels (Generalized estimating equations (GEE) analysis average difference over time; regression coefficient (Beta): -0.54; 95% CI: -1.08--0.01; p = 0.04), blunted plasma malondialdehyde response (Beta: 1.58; 95% CI: 0.00-3.15; p = 0.05) and a trend towards lower lactate to pyruvate ratio (Beta: -1.10; 95% CI: -2.33-0.12; p = 0.08). We concluded that peri-operative taurine supplementation attenuated postoperative oxidative stress in elderly hip fracture patients, but did not improve postoperative morbidity and mortality.

Nutrition before, during, and after surgery increases the arginine:asymmetric dimethylarginine ratio and relates to improved myocardial glucose metabolism: a randomized controlled trial.

BACKGROUND: Nitric oxide (NO) is essential for the optimal perfusion of the heart and its vasculature. NO may be insufficient in surgical patients because its precursor arginine is decreased, and the inhibitor of NO synthesis asymmetric dimethylarginine (ADMA) is increased. Besides arginine, the presence of other amino acids essential for the proper metabolism of cardiac cells may be decreased too. Supplementation of these amino acids with enteral and parenteral nutrition before, during, and after surgery may augment the myocardial and plasma arginine:ADMA ratio and availability of amino acids. Myocardial glucose metabolism and nutritional conditioning may result in a reduction of cardiac injury and support rapid recovery after major surgery. OBJECTIVE: We investigated the effect of nutrition before, during, and after surgery on amino acids and the myocardial arginine:ADMA ratio and its relation to myocardial glucose metabolism. DESIGN: In this trial, 33 patients who were undergoing off-pump coronary artery bypass grafting (CABG) were randomly assigned between enteral, parenteral, or no nutrition (control) from 2 d before, during, and until 2 d after surgery. Both enteral and parenteral solutions were prepared with commercially available products and included proteins or amino acids, glucose, vitamins, and minerals. Concentrations of amino acids including ADMA were analyzed in myocardial tissue and plasma samples. ¹8F-fluorodeoxyglucose positron emission tomography was performed before and after surgery to assess myocardial glucose metabolism. RESULTS: The myocardial arginine:ADMA ratio increased during surgery and was significantly higher in the enteral and parenteral groups than in the control group [median (IQR): 115.0 (98.0-142.2) (P = 0.012), 116.9 (100.3-135.3) (P = 0.004), and 93.3 (82.7-101.1), respectively]. Furthermore, the change in the preoperative to postoperative plasma arginine:ADMA ratio correlated with the change in myocardial glucose metabolism in positron emission tomography (r = 0.427, P = 0.033). CONCLUSION: Enteral or parenteral nutrition before, during, and after CABG may positively influence myocardial glucose metabolism by increasing the plasma and myocardial arginine:ADMA ratio.

Protein arginine hypomethylation in a mouse model of cystathionine ß-synthase deficiency.

Accumulation of the homocysteine (Hcy) precursor S-adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine ß-synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5-methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS-deficient mice exhibited increased (>6-fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10-35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS-deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency.

Homoarginine and mortality in an older population: the Hoorn study.

BACKGROUND: Homoarginine is an amino acid that may be involved in nitric oxide and energy metabolism. Previous studies in patient populations showed that low homoarginine levels indicate an increased risk of mortality and cardiovascular disease. We evaluated whether low plasma levels of homoarginine are associated with elevated, overall and cause-specific mortality. MATERIALS AND METHODS: The Hoorn study is a population-based study among older men and women. We calculated Cox proportional hazard ratios (HRs) for overall and cause-specific mortality according to sex-specific homoarginine quartiles. RESULTS: We included 606 study participants (51·3% women; 70·0 ± 6·6 years). Homoarginine concentrations were higher in men (1·63 ± 0·51 µM), compared with women (1·30 ± 0·44 µM; P < 0·001). After a median follow-up time of 7·8 years, 112 study participants died, including 31 deaths due to cardiovascular diseases and 30 due to cancer. Associations between homoarginine levels and mortality showed a threshold effect with a significant risk increase from the second to the first quartile. Compared with the upper three quartiles, the age-, sex- and BMI-adjusted HR (with 95% CI) in the first quartile was 2·26 (1·52-3·32) for overall mortality, 4·20 (2·03-8·69) for cardiovascular mortality and 1·25 (0·55-2·85) for cancer mortality. These associations remained materially unchanged after multivariate adjustments. CONCLUSIONS: Low plasma concentrations of homoarginine are a risk marker for overall mortality and especially for cardiovascular mortality in the older general population. Further studies are warranted to elucidate the underlying pathophysiological mechanisms.

Plasma proANP and SDMA and microRNAs are associated with chronic mitral regurgitation in a pig model.

OBJECTIVE: NON-ISCHEMIC MITRAL REGURGITATION (MR) IS PRIMARILY CAUSED BY MYXOMATOUS MITRAL VALVE (MV) DISEASE LEADING TO ADAPTIVE REMODELING, ENLARGEMENT, AND DYSFUNCTION OF THE LEFT VENTRICLE. THE AIM OF THIS STUDY WAS TO EXAMINE THE REGULATION OF PLASMA MARKERS AND SEVERAL CARDIAC KEY GENES IN A NOVEL PORCINE MODEL OF NON-ISCHEMIC MR. METHODS AND RESULTS: Twenty-eight production pigs (Sus scrofa) were randomized to experimental MR or sham surgery controls. MR was induced by external suture(s) through the posterior MV leaflet and quantified using echocardiography. The experimental group was subdivided into mild MR (mMR, MR=20-50%, n=10) and moderate/severe MR (sMR, MR >50%, n=6) and compared with controls (CON, MR ≤10%, n=12). Eight weeks postoperatively, follow-up examinations were performed followed by killing. Circulating concentrations of pro-atrial natriuretic peptide (proANP), l-arginine, asymmetric dimethylarginine, and symmetric dimethylarginine (SDMA) were measured. MV, anterior papillary muscle, and left ventricular free wall tissues were collected to quantify mRNA expression of eNOS (NOS3), iNOS (NOS2), MMP9, MMP14, ANP (NPPA), BNP (NPPB), and TGFB1, 2, and 3 and five microRNAs by quantitative real-time PCR. Pigs with sMR displayed markedly increased plasma proANP and SDMA concentrations compared with both controls and mMR (P<0.05). The expression of all genes examined differed significantly between the three localizations in the heart. miR-21 and miR-133a were differently expressed among the experimental groups (P<0.05). CONCLUSIONS: Plasma proANP and SDMA levels and tissue expression of miR-21 and miR-133a are associated with severity of chronic MR in an experimental porcine model.

Preserved GLP-1 and exaggerated GIP secretion in type 2 diabetes and relationships with triglycerides and ALT.

OBJECTIVE: To i) compare incretin responses to oral glucose and mixed meal of diabetic patients with the normoglycaemic population and ii) to investigate whether incretin responses are associated with hypertriglyceridaemia and alanine aminotransferase (ALT) as liver fat marker. DESIGN: A population-based study. METHODS: A total of 163 persons with normal glucose metabolism (NGM), 20 with intermediate hyperglycaemia and 20 with type 2 diabetes aged 40-65 years participated. Participants received a mixed meal and oral glucose load on separate occasions. Glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon profiles were analysed as total area under the curve (tAUC) and incremental area under the curve. RESULTS: In diabetic patients compared with persons with NGM, we found increased GLP-1 secretion (tAUC per hour) following oral glucose (23.2 pmol/l (95% CI 17.7-28.7) vs 18.0 (95% CI 16.9-19.1), P<0.05) but not after the mixed meal. GIP secretion among diabetic patients was increased on both occasions (82.9 pmol/l (55.9-109.8) vs 47.1 (43.8-50.4) for oral glucose and 130.6 (92.5-168.7) vs 83.2 (77.5-88.9) for mixed meal, both P<0.05). After oral glucose, GLP-1 (tAUC per hour) was inversely related to fasting triglycerides. GIP (tAUC per hour) was positively related to fasting and postprandial triglycerides. Higher fasting GIP levels were related to higher fasting and postprandial triglyceride levels and ALT. CONCLUSION: This study confirms that in type 2 diabetes, GLP-1 secretion is generally preserved and that GIP secretion is exaggerated. The mechanism underlying the divergent associations of GLP-1 and GIP metabolism with fat metabolism and liver fat accumulation warrants further study.

Global protein and histone arginine methylation are affected in a tissue-specific manner in a rat model of diet-induced hyperhomocysteinemia.

Accumulation of S-adenosylhomocysteine (AdoHcy), the homocysteine (Hcy) precursor and a potent methyltransferase inhibitor, may mediate the neurological and vascular complications associated with elevated Hcy. Protein arginine methylation is a crucial post-translational modification and generates monomethylarginine (MMA) and dimethylarginine (asymmetric, ADMA, and symmetric, SDMA) residues. We aimed at determining whether protein arginine methylation status is disturbed in an animal model of diet-induced hyperhomocysteinemia (HHcy). HHcy was achieved by dietary manipulation of Wistar rats: methionine-enrichment (HM), B vitamins deficiency (LV), or both (HMLV). Total Hcy, S-adenosylmethionine (AdoMet), AdoHcy, MMA, ADMA and SDMA concentrations in plasma or tissues (heart, brain and liver) were determined by adequate high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry methods. Moreover, in tissues from the HMLV group, histone arginine asymmetric dimethylation was evaluated by Western blotting, and the histone methylation marks H3R17me2a, H3R8me2a and H4R3me2a were studied. HHcy was induced by all special diets, with elevation of AdoHcy concentrations in liver (LV and HMLV) and heart (HMLV) (all versus control). Plasma ADMA levels were lower in all hyperhomocysteinemic animals. Protein-incorporated ADMA levels were decreased in brain and in heart (both for the LV and HMLV groups). Moreover, in brain of animals exposed to the HMLV diet, the H3R8me2a mark was profoundly decreased. In conclusion, our results show that diet-induced Hcy elevation disturbs global protein arginine methylation in a tissue-specific manner and affects histone arginine methylation in brain. Future research is warranted to disclose the functional implications of the global protein and histone arginine hypomethylation triggered by Hcy elevation.

L-Homoarginine and L-arginine are antagonistically related to blood pressure in an elderly population: the Hoorn study.

OBJECTIVES: Production of nitric oxide by the vascular endothelium is crucial for the maintenance of vascular tone, an important determinant of blood pressure. L-Arginine and its homolog L-homoarginine are competitive substrates of nitric oxide synthase (NOS), whereas asymmetric dimethylarginine (ADMA) is a NOS inhibitor. We evaluated the relationships between physiological levels of these amino acids and blood pressure. METHODS: The relationship between blood pressure and plasma levels of L-arginine, L-homoarginine, and ADMA was studied in participants of the Hoorn study, a population-based cohort study of elderly participants (n = 746, aged 50-87, 49.5% men). RESULTS: In linear regression models adjusted for age, sex, L-arginine, and ADMA, a positive association was observed between L-homoarginine and SBP [3.90 mmHg per 1-SD increment of L-homoarginine (95% confidence interval, CI 2.28-5.52)] and DBP [1.83 (0.95-2.72)]. In these models, L-arginine was not significantly associated with SBP [-0.68 mmHg per 1-SD increment of L-arginine (95% CI -2.23 to 0.88)], but a significant inverse association with DBP was observed [-1.17 (-2.02 to -0.32)]. These associations were slightly attenuated after further adjustment for glucose or BMI, but not after adjustment for other cardiovascular risk factors (lipids, smoking, inflammation markers, microalbuminuria, prior cardiovascular disease, and antihypertensive medication). ADMA was not significantly associated with either SBP or DBP. CONCLUSION: In elderly participants, plasma levels of L-homoarginine and L-arginine are independently associated with clinically relevant differences in blood pressure in an antagonistic fashion.

Protein arginine methylation is more prone to inhibition by S-adenosylhomocysteine than DNA methylation in vascular endothelial cells.

Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.

Bone marrow alterations and lower endothelial progenitor cell numbers in critical limb ischemia patients.

BACKGROUND: Critical limb ischemia (CLI) is characterized by lower extremity artery obstruction and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and alterations in the BM contribute to impaired neovascularization in CLI. METHODS: Levels of primitive (CD34(+) and CD133(+)) progenitors and CD34(+)KDR(+) EPC were analyzed using flow cytometry in blood and BM from 101 CLI patients in the JUVENTAS-trial (NCT00371371) and healthy controls. Blood levels of markers for endothelial injury (sE-selectin, sICAM-1, sVCAM-1, and thrombomodulin), and progenitor cell mobilizing and inflammatory factors were assessed by conventional and multiplex ELISA. BM levels and activity of the EPC mobilizing protease MMP-9 were assessed by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured and their paracrine function was assessed. RESULTS: Endothelial injury markers were higher in CLI (P<0.01). CLI patients had higher levels of VEGF, SDF-1α, SCF, G-CSF (P<0.05) and of IL-6, IL-8 and IP-10 (P<0.05). Circulating EPC and BM CD34(+) cells (P<0.05), lymphocytic expression of CXCR4 and CD26 in BM (P<0.05), and BM levels and activity of MMP-9 (P<0.01) were lower in CLI. Multivariate regression analysis showed an inverse association between IL-6 and BM CD34(+) cell levels (P = 0.007). CAC from CLI patients had reduced paracrine function (P<0.0001). CONCLUSION: CLI patients have reduced levels of circulating EPC, despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34(+)-cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. The results of this study suggest that inflammation-induced BM exhaustion and a disturbed progenitor cell mobilization response due to reduced levels and activity of MMP-9 in the BM and alterations in the SDF-1α/CXCR4 interaction contribute to the attenuated neovascularization in CLI patients.

Asymmetric dimethylarginine concentrations decrease in patients with HIV infection under antiretroviral therapy.

BACKGROUND: In HIV-infected patients, elevated plasma concentrations of the endogenous nitric oxide synthase inhibitor and cardiovascular risk factor asymmetric dimethylarginine (ADMA) are documented, and levels correlate with markers of immune activation, such as neopterin. METHODS: In this study, the effect of antiretroviral therapy (ART) on arginine, ADMA and symmetric dimethylarginine (SDMA) levels was investigated and related to changes of immune activation markers and lipids. Concentrations of ADMA, SDMA, arginine, C-reactive protein (CRP) and neopterin were determined in 112 HIV-infected patients after 12 months of successful ART, as reflected by undetectable HIV RNA levels, and compared to baseline levels. ADMA, SDMA, arginine and urine neopterin levels were determined by HPLC, and plasma neopterin concentrations by ELISA. Disease activity before and after treatment was monitored by determination of HIV RNA levels and CD4(+) T-cell counts. Lipids and CRP were determined by routine laboratory assays. RESULTS: Under treatment with ART, concentrations of ADMA, SDMA and arginine dropped in parallel with decreasing HIV RNA levels and neopterin concentrations, while cholesterol, triglyceride levels and CD4(+) T-cell counts increased. CRP levels did not change. After ART, a significant inverse association between ADMA and plasma cholesterol was observed. CONCLUSIONS: Successful ART, defined by HIV RNA levels below the limit of detection, leads to decreasing levels of methylated arginines and immune activation markers. Thus, it is unlikely that disturbances of dimethylarginine metabolism account for the increased risk of cardiovascular events of HIV-infected patients under ART.

Asymmetric dimethylarginine in adults with cystathionine ß-synthase deficiency.

In hyperhomocysteinemia (HHcy), an independent risk factor for cardiovascular diseases, endothelial dysfunction due to reduced bioavailability of nitric oxide is a consistent finding. However, the underlying mechanisms remain unknown. Increased levels of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) have been associated with HHcy, and may contribute, at least in part, for the homocysteine-induced endothelial dysfunction, but whether cystathionine ß-synthase (CBS) deficiency is associated with increased ADMA has hardly been investigated. To address this question, we measured total homocysteine (tHcy), ADMA and symmetric dimethylarginine (SDMA) in plasma of 22 adult CBS deficient patients, using established HPLC techniques. Results showed that in CBS deficient patients with elevated levels of tHcy (median (total range): 33 (14-237) µmol/L), both ADMA and SDMA levels were normal. Moreover, tHcy and ADMA concentrations were not correlated (r(s)=0.017, p=0.94). Our results favor the hypothesis that the negative vascular effects of HHcy have an ADMA-independent etiology.

N-acetylcysteine reduces oxidative stress in sickle cell patients.

Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbSß(0)-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress.

Adenosine A1-receptor deficiency diminishes afferent arteriolar and blood pressure responses during nitric oxide inhibition and angiotensin II treatment.

Adenosine mediates tubuloglomerular feedback responses via activation of A(1)-receptors on the renal afferent arteriole. Increased preglomerular reactivity, due to reduced nitric oxide (NO) production or increased levels of ANG II and reactive oxygen species (ROS), has been linked to hypertension. Using A(1)-receptor knockout (A(1)(-/-)) and wild-type (A(1)(+/+)) mice we investigated the hypothesis that A(1)-receptors modulate arteriolar and blood pressure responses during NO synthase (NOS) inhibition or ANG II treatment. Blood pressure and renal afferent arteriolar responses were measured in nontreated mice and in mice with prolonged N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or ANG II treatment. The hypertensive responses to L-NAME and ANG II were clearly attenuated in A(1)(-/-) mice. Arteriolar contractions to L-NAME (10(-4) mol/l; 15 min) and cumulative ANG II application (10(-12) to 10(-6) mol/l) were lower in A(1)(-/-) mice. Simultaneous treatment with tempol (10(-4) mol/l; 15 min) attenuated arteriolar responses in A(1)(+/+) but not in A(1)(-/-) mice, suggesting differences in ROS formation. Chronic treatment with L-NAME or ANG II did not alter arteriolar responses in A(1)(-/-) mice, but enhanced maximal contractions in A(1)(+/+) mice. In addition, chronic treatments were associated with higher plasma levels of dimethylarginines (asymmetrical and symmetrical) and oxidative stress marker malondialdehyde in A(1)(+/+) mice, and gene expression analysis showed reduced upregulation of NOS-isoforms and greater upregulation of NADPH oxidases. In conclusion, adenosine A(1)-receptors enhance preglomerular responses during NO inhibition and ANG II treatment. Interruption of A(1)-receptor signaling blunts l-NAME and ANG II-induced hypertension and oxidative stress and is linked to reduced responsiveness of afferent arterioles.

Hypothermic renal perfusion during aortic surgery reduces the presence of lipocalin-2 and preserves renal extraction of dimethylarginines in rats.

Cold perfusion through the renal arteries during renal ischemia has been suggested to diminish postoperative renal damage after juxtarenal aortic aneurysm repair. As the kidneys play a key role in dimethylarginine metabolism, which in turn is associated with renal hemodynamics, we hypothesized that the protective effect of cold perfusion is associated with a preserved renal extraction of dimethylarginines. Renal ischemia was induced in three groups of anesthetized Wistar rats (n = 7/group), which underwent suprarenal aortic clamping (45 min) with no perfusion (group 1), renal perfusion with 37°C saline (group 2), or renal perfusion with 4°C saline (group 3), respectively, followed by 90 min of renal reperfusion in all groups. The sham group had no clamping. In group 3 (renal ischemia with cold perfusion), postoperative serum creatinine levels as well as the presence of luminal lipocalin-2 and its associated brush-border damage were lower compared with groups 1 and 2 (P < 0.05). Also, renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine as well as the arginine/ADMA ratio, which defines the bioavailability of nitric oxide, remained intact in group 3 only (P < 0.04). The arginine/ADMA ratio correlated with cortical flow, lipocalin-2, and creatinine rises. Warm and cold renal perfusion (groups 2 and 3) during ischemia were similarly effective in lowering protein nitrosylation levels, renal leukocyte accumulation, neutrophil gelatinase-associated lipocalin (NGAL) expression in distal tubules, and urine NGAL (P < 0.05). These data support the use of cold renal perfusion during renal ischemia in situations where renal ischemia is inevitable, as it reduces tubular damage and preserves renal extraction of dimethylarginines. Renal perfusion with saline per se during renal ischemia is effective in diminishing renal leukocyte accumulation and oxidative stress.

Infrarenal aortic-clamping after renal ischaemia aggravates acute renal failure.

BACKGROUND: Renal failure is a frequent complication of juxtarenal abdominal aortic aneurysm (JAA)-repair. During this operation, suprarenal aortic-clamping is followed by infrarenal aortic-clamping (below renal arteries) to restore renal flow, while performing the distal anastomosis. We hypothesized that infrarenal aortic-clamping, despite restoring renal perfusion provokes additional renal damage. MATERIALS AND METHODS: We studied three groups of rats. After 45min of suprarenal aortic-clamping, group 1 had renal reperfusion for 90min without aortic-clamps (n=7). In group 2, 45min of suprarenal aortic-clamping with a distal clamp on the aortic-bifurcation was followed by 20min of infrarenal aortic-clamping. Renal reperfusion was continued for 70min without aortic-clamps (i.e. 90 min of renal reperfusion; n=8). The sham-group had no clamps (n=7). We measured renal haemodynamics, functional parameters and tissue damage. RESULTS: On suprarenal aortic-clamp removal, renal artery flow, cortical flow and arterial pressures were higher in group 2 than in group 1. We detected increased tubular brush border damage, luminal lipocalin-2 and 30-60% higher renal protein nitrosylation in group 2 when compared to group 1 (P<0·05). Group 2 showed more release of asymmetrical dimethylarginine (ADMA) from the kidneys in the renal vein, therefore indicating diminished clearing capacity (P<0·001). Arginine/ADMA-ratio, which defines the bio-availability of nitric oxide, tended to be lower in group 2 and correlated with renal flow. Furthermore, there were no significant differences found in creatinine levels and renal leucocyte accumulation between group 1 and 2. CONCLUSIONS: Additional infrarenal aortic-clamping leads to increased renal damage and oxidative stress, despite adequate perfusion of kidneys after suprarenal aortic-clamping. This study indicates that the clamping sequence used in JAA-repair causes more than simple renal I/R-injury.

Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs.

Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the L-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic peptide resistance. Although the l-arginine:ADMA ratio decreased with increasingly severe MMVD, this was largely due to the older age of the dogs with heart failure.

Low arginine/asymmetric dimethylarginine ratio deteriorates systemic hemodynamics and organ blood flow in a rat model.

OBJECTIVE: Both arginine and asymmetric dimethylarginine (ADMA) play a crucial role in the arginine-nitric oxide pathway. Low arginine and high ADMA levels can be found in critically ill patients after major surgery. The aim of this study was to evaluate the effects of low arginine plasma concentrations in combination with high ADMA plasma concentrations on hemodynamics and organ blood flow. DESIGN: Randomized, placebo-controlled animal laboratory investigation. SUBJECTS: Male Wistar rats (n = 21), anesthetized. INTERVENTIONS: Rats were randomly assigned to three groups: a control group, an ADMA group, or an arginase (ASE)/ADMA group. In the control group, rats received (at t = 0) an intravenous (IV) infusion of 1.5 mL 0.9% NaCl during a 20-minute period. After 60 minutes (t = 60), rats received an IV bolus of 1.0 mL 0.9% NaCl. In the ADMA group, rats received an IV infusion of 1.5 mL 0.9% NaCl during a 20-minute period and at t = 60 an IV bolus of 1.0 mL ADMA (20 mg/kg). In the ASE/ADMA group, rats received an IV infusion of 1.5 mL ASE (3200 IU) solution during a 20-minute period and at t = 60 an IV bolus of 1.0 mL ADMA (20 mg/kg). MEASUREMENTS AND MAIN RESULTS: Infusion of ADMA (20 mg/kg) and ASE (3200 IU) resulted in increased plasma ADMA levels and decreased arginine levels. During the whole experiment, systemic hemodynamics (heart rate, mean arterial pressure [MAP], and cardiac output) were measured. In addition, organ blood flow was measured at t = 90 and t = 180 minutes, using fluorescent microspheres. Compared with the control group, MAP and systemic vascular resistance were increased after infusion of ADMA. Infusion of ASE in combination with ADMA significantly deteriorated systemic hemodynamics (MAP, cardiac output, stroke volume, and systemic vascular resistance) and organ blood flow through the kidney and spleen. In addition, an initial decrease in arterial flow, followed by a later major increase, and panlobular apoptosis and necrosis of the liver was observed. CONCLUSIONS: The current study shows that low arginine plasma levels in combination with high ADMA plasma levels deteriorates systemic hemodynamics and reduces blood flow through the kidney and spleen and liver. These data suggest that a diminished nitric oxide production may be involved in the onset of organ failure.