The endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is elevated in many patients and may contribute to the initiation and progression of their disease. While some mechanistic pathways have been identified, tissue-specific contributions to ADMA control remain unclear. We sought to determine if whole blood (WB) could participate in ADMA control ex vivo. Anesthetized male Sprague-Dawley rats underwent exsanguinations, and WB preparations were incubated at 37°C for 5 h. ADMA and symmetrical dimethylarginine were analyzed by high-pressure liquid chromatography. Incubation of lysed red blood cell (RBC) supernatant yielded a significant decrease in ADMA that was blocked by 4124W, a synthetic inhibitor of dimethylarginine dimethylaminohydrolase, the only reported enzyme to hydrolyze ADMA. Hydrolysis of ADMA was diminished by addition of physiologically relevant concentrations of zinc (i.e., 20 M). Conversely, when rat WB or WB supernatant was incubated at 37°C, it liberated quantities of free ADMA (1-2 M) that in vivo would likely have pathological consequences. Addition of arginine methyltransferase inhibitors to these incubations did not reduce ADMA release, indicating no dominant role for active protein methylation during these incubations. This ADMA liberation was significantly reduced by addition of protease inhibitors, indicating a dependence on peptide bond hydrolysis. Total ADMA (protein incorporated plus free) was determined by acid hydrolysis and found to be 43.18 Ϯ 4.79 M in WB with ϳ95% of this in RBCs.These ex vivo data demonstrate the potential of blood to control the NO-NOS system by modulating free ADMA. nitric oxide; protein arginine methyltransferase; symmetrical dimethylarginine; protease NITRIC OXIDE (NO) is produced by NO synthase (NOS) and has a myriad of functions throughout the body, including vasodilation, signaling functions, and inhibition of platelet aggregation. Deficiency or loss of NOS activity has been termed "endothelial dysfunction" and may contribute to a spectrum of cardiovascular diseases. Asymmetrical dimethylarginine (ADMA) and N G -monomethyl-L-arginine (L-NMMA) are endogenous inhibitors of NOS, but the concentration of ADMA exceeds that of L-NMMA by ϳ10-fold (60) and hence is of greater potential clinical interest. ADMA and its noninhibitory regioisomer, symmetrical dimethylarginine (SDMA) (6, 12), are released into the plasma following the breakdown of proteins containing arginine residues previously dimethylated by protein arginine methyltransferases (PRMT) (37). The literature contains compelling evidence that elevated plasma ADMA exists in diabetes mellitus (37), hypertension (1, 15, 22, 45, 51, 52), hypercholesterolemia (6), hyperhomocyst(e)inemia (50), experimental hemorrhage (4), preeclampsia (16), and sickle cell anemia (47). ADMA has also been reported to increase platelet activation and atherogenesis in the cardiovascular system (33). Cooke (11, 13) and others (17,59) give extensive reviews of much of these data and argue that ...

Objective: A standardized assessment of response to furosemide is predictive of acute kidney injury progression in adults, but a paucity of data exists in pediatric patients. We evaluate furosemide responsiveness in a multicenter cohort of pediatric patients after cardiac surgery.Methods: Children who underwent cardiac surgery with a Society of Thoracic Surgeons-European Association for Cardiothoracic Surgery score of 3 or greater were retrospectively identified. The first dose of furosemide after surgery was recorded, and hourly urine output for 6 hours was recorded after the index dose. Urine flow rate calculated as urine output per hour was used to predict development of acute kidney injury.Results: A total of 166 patients from 4 institutions (median age, 6.3 months; interquartile range, 0.4-27.7) were included. Acute kidney injury occurred in 54 patients (33%). Compared with those without acute kidney injury, the 2-and 6-hour urine flow rates were significantly lower in patients in whom acute kidney injury developed: 2.9 (0.9-6.5) versus 5.0 (2.5-9.0) mL/kg/h for 2-hour urine flow rate, P ¼ .004, and 2.4 (1.2-4.0) versus 4.0 (2.3-5.9) mL/kg/h for 6-hour flow rate, P ¼ .001. In multivariable regression analysis, 2-hour (odds ratio, 1.2, P ¼ .002) and 6-hour (odds ratio, 1.40, P<.001) urine flow rates were independently associated with acute kidney injury development. Lower urine flow rate at both 2 and 6 hours was also independently associated with longer hospital length of stay.

A serious consequence of sepsis is acute lung injury, whose severity is particularly impacted by the age of the patient. AMP-activated protein kinase (AMPK) is a crucial regulator of cellular metabolism, which controls mitochondrial biogenesis and autophagy. Here, we investigated the effect of pharmacological activation of AMPK with A769662 on lung injury by using a model that would preferably mimic the clinical condition of adult patients. Male C57BL/6 retired breeder mice (7–9 months old) were subjected to sepsis by cecal ligation and puncture (CLP). Mice received vehicle or A769662 (10 mg/kg) intraperitoneally at 1 h after CLP. At 6 h after CLP, vehicle-treated mice exhibited severe lung injury and elevation of plasma pro-inflammatory cytokines when compared with control mice. At molecular analysis, lung injury was associated with downregulation of AMPKα1/α2 catalytic subunits and reduced phosphorylation of AMPKβ1 regulatory subunit. Treatment with A769662 ameliorated lung architecture, reduced bacterial load in lung and blood, and attenuated plasma levels of interleukin-6. This protective effect was associated with nuclear phosphorylation of AMPKα1/α2 and AMPKβ1, increased nuclear expression of peroxisome proliferator-activated receptor γ co-activator-α and increased autophagy, as evaluated by the light-chain (LC)3B-I and LC3B-II content, without changes in sirtuin-1 cellular dynamics. Treatment with A769662 alone or in combination with the antimicrobial agent imipenem (25 mg/kg) increased survival rate (29% and 51%, respectively) when compared with vehicle treatment (10%) at 7 days after CLP. These data suggest that pharmacological activation of AMPK might be a beneficial approach for the treatment of sepsis in adult population.

A standardized, quantified assessment of furosemide responsiveness predicts acute kidney injury (AKI) in children after cardiac surgery and AKI progression in critically ill adults. The purpose of this study was to determine if response to furosemide is predictive of severe AKI in critically ill children outside of cardiac surgery. We performed a multicenter retrospective study of critically ill children. Quantification of furosemide response was based on urine flow rate (normalized for weight) measurement 0 to 6 hours after the dose. The primary outcome was presence of creatinine defined severe AKI (Kidney Disease Improving Global Outcomes stage 2 or greater) within 7 days of furosemide administration. Secondary outcomes included mortality, duration of mechanical ventilation and length of stay. A total of 110 patients were analyzed. Severe AKI occurred in 20% (n = 22). Both 2- and 6-hour urine flow rate were significantly lower in those with severe AKI compared with no AKI (p = 0.002 and p < 0.001). Cutoffs for 2- and 6-hour urine flow rate for prediction of severe AKI were <4 and <3 mL/kg/hour, respectively. The adjusted odds of developing severe AKI for 2-hour urine flow rate of <4 mL/kg/hour was 4.3 (95% confidence interval [CI]: 1.33–14.15; p = 0.02). The adjusted odds of developing severe AKI for 6-hour urine flow rate of <3 mL/kg/hour was 6.19 (95% CI: 1.85–20.70; p = 0.003). Urine flow rate in response to furosemide is predictive of severe AKI in critically ill children. A prospective assessment of urine flow rate in response to furosemide for predicting subsequent severe AKI is warranted.