Cellular transport of l-arginine determines renal medullary blood flow in control rats, but not in diabetic rats despite enhanced cellular uptake capacity.

Diabetes mellitus is associated with decreased nitric oxide bioavailability thereby affecting renal blood flow regulation. Previous reports have demonstrated that cellular uptake of l-arginine is rate limiting for nitric oxide production and that plasma l-arginine concentration is decreased in diabetes. We therefore investigated whether regional renal blood flow regulation is affected by cellular l-arginine uptake in streptozotocin-induced diabetic rats. Rats were anesthetized with thiobutabarbital, and the left kidney was exposed. Total, cortical, and medullary renal blood flow was investigated before and after renal artery infusion of increasing doses of either l-homoarginine to inhibit cellular uptake of l-arginine or Nω-nitro- l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase. l-Homoarginine infusion did not affect total or cortical blood flow in any of the groups, but caused a dose-dependent reduction in medullary blood flow. l-NAME decreased total, cortical and medullary blood flow in both groups. However, the reductions in medullary blood flow in response to both l-homoarginine and l-NAME were more pronounced in the control groups compared with the diabetic groups. Isolated cortical tubular cells displayed similar l-arginine uptake capacity whereas medullary tubular cells isolated from diabetic rats had increased l-arginine uptake capacity. Diabetics had reduced l-arginine concentrations in plasma and medullary tissue but increased l-arginine concentration in cortical tissue. In conclusion, the reduced l-arginine availability in plasma and medullary tissue in diabetes results in reduced nitric oxide-mediated regulation of renal medullary hemodynamics. Cortical blood flow regulation displays less dependency on extracellular l-arginine and the upregulated cortical tissue l-arginine may protect cortical hemodynamics in diabetes.

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.

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.

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.

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.

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.

Glial-cell-mediated re-induction of the blood-brain barrier phenotype in brain capillary endothelial cells: a differential gel electrophoresis study.

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood-brain barrier (BBB) models. Hence, the re-induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re-induction remain largely unknown. Here, we used a DIGE-based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re-induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re-induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by-product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re-induced BBB functions than in cells with limited BBB functions.

Rats with adenine-induced chronic renal failure develop low-renin, salt-sensitive hypertension and increased aortic stiffness.

Rats with adenine-induced chronic renal failure (A-CRF) develop metabolic and cardiovascular abnormalities resembling those in patients with chronic kidney disease. The aim of this study was to investigate the mechanisms of hypertension in this model and to assess aortic stiffness in vivo. Male Sprague-Dawley rats were equipped with radiotelemetry probes for arterial pressure recordings and received either chow containing adenine or normal control diet. At 7 to 11 wk after study start, blood pressure responses to high NaCl (4%) diet and different pharmacological interventions were analyzed. Aortic pulse wave velocity was measured under isoflurane anesthesia. Baseline 24-h mean arterial pressure (MAP) was 101 ± 10 and 119 ± 9 mmHg in controls and A-CRF animals, respectively (P < 0.01). After 5 days of a high-NaCl diet, MAP had increased by 24 ± 6 mmHg in A-CRF animals vs. 2 ± 1 mmHg in controls (P < 0.001). Candesartan (10 mg/kg by gavage) produced a more pronounced reduction of MAP in controls vs. A-CRF animals (-12 ± 3 vs. -5 ± 5 mmHg, P < 0.05). Aortic pulse wave velocity was elevated in A-CRF rats (5.10 ± 0.51 vs. 4.58 ± 0.17 m/s, P < 0.05). Plasma levels of creatinine were markedly elevated in A-CRF animals (259 ± 46 vs. 31 ± 2 µM, P < 0.001), whereas plasma renin activity was suppressed (0.6 ± 0.5 vs. 12.3 ± 7.3 µg·l(-1)·h(-1), P < 0.001). In conclusion, hypertension in A-CRF animals is characterized by low plasma renin activity and is aggravated by high-NaCl diet, suggesting a pathogenic role for sodium retention and hypervolemia probably secondary to renal insufficiency. Additionally, aortic stiffness was elevated in A-CRF animals as indicated by increased aortic pulse wave velocity.

Genetic variation in the dimethylarginine dimethylaminohydrolase 1 gene (DDAH1) is related to asymmetric dimethylarginine (ADMA) levels, but not to endothelium-dependent vasodilation.

OBJECTIVES: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. The breakdown of ADMA is mainly governed by the activity of dimethylarginine dimethylaminohydrolases (DDAHs). We investigated if genetic variation in the DDAH1 and DDAH2 genes were related to ADMA and l-arginine levels, as well as measures of endothelium-dependent vasodilation. METHODS: In 1016 70-year-old participants of the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (50% women), we measured endothelium-dependent vasodilation (EDV) using the invasive forearm technique with acetylcholine given in the brachial artery and the brachial artery ultrasound technique with measurement of flow-mediated dilatation (FMD). Plasma l-arginine and ADMA levels were measured by high-performance liquid chromatography and 55 single nucleotide polymorphisms (SNPs) in the DDAH1 and DDAH2 genes were genotyped. RESULTS: Several of the genotypes in the DDAH1 gene were highly significantly related to ADMA levels (p = 10(-7) at best), but not to the l-arginine levels. No relationships between the genotypes in the DDAH2 gene and ADMA or l-arginine levels were found. None of the DDAH1 genotypes being closely related to ADMA levels were significantly related to EDV or FMD. Neither were any of the DDAH2 genotypes closely related to any of the measurements of vasoreactivity. CONCLUSION: A close relationship was seen between SNPs in the DDAH1, but not DDAH2, gene and ADMA levels. However, variation in those genes was not related to measures of EDV in this elderly population.

Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats.

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ∼50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.

Reduced renal plasma clearance does not explain increased plasma asymmetric dimethylarginine in hypertensive subjects with mild to moderate renal insufficiency.

Plasma concentrations of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) increase already in the early stages of renal insufficiency. There is no agreement as to whether reduced renal plasma clearance (RPCL) contributes to this increase. Therefore, we investigated the relationship between estimated glomerular filtration rate (eGFR), RPCL, and plasma ADMA and SDMA in essential hypertensive patients with mild to moderate renal insufficiency. In 171 patients who underwent renal angiography, we drew blood samples from the aorta and both renal veins and measured mean renal blood flow (MRBF) using the (133)Xe washout technique. RPCL was calculated using arteriovenous concentration differences and MRBF. After correction for potential confounders, reduced eGFR was associated with higher plasma ADMA and SDMA [standardized regression coefficient (ß) = -0.22 (95% confidence intervals: -0.41, -0.04) and ß = -0.66 (95% confidence intervals: -0.83, -0.49), respectively]. However, eGFR was not independently associated with RPCL of ADMA. Moreover, reduced RPCL of ADMA was not associated with higher plasma ADMA. Contrary to ADMA, reduced eGFR was indeed associated with lower RPCL of SDMA [ß = 0.21 (95% confidence intervals: 0.02, 0.40)]. In conclusion, our findings indicate that RPCL of ADMA is independent of renal function in hypertensive patients with mild to moderate renal insufficiency. Unlike the case for SDMA, reduced RPCL of ADMA is of minor importance for the increase in plasma ADMA in these patients, which indicates that increased plasma ADMA in this population is not a direct consequence of the kidneys failing as a plasma ADMA-regulating organ.

Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine.

Proteolytic activity in whole blood may lead to release of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). We investigated the role of the human erythrocyte in storage and generation of ADMA in healthy controls (n = 36) and critically ill patients (n = 38). Both free and total (sum of free and protein-incorporated) ADMA were measured. Upon incubation of intact erythrocytes with extracellular ADMA (0 to 40 µmol/l), equilibrium between intra- and extracellular ADMA was reached within 3 h. Compared with controls, patients had significantly higher basal concentrations of ADMA in plasma (0.88 ± 0.75 vs. 0.41 ± 0.07 µmol/l) and erythrocytes (1.28 ± 0.55 vs. 0.57 ± 0.14 µmol/l). Intracellular and plasma ADMA were significantly correlated in the patient group only (r = 0.834). Upon lysis, followed by incubation at 37°C for 2 h, free ADMA increased sevenfold (to 8.60 ± 3.61 µmol/l in patients and 3.90 ± 0.78 µmol/l in controls). In lysates of controls, free ADMA increased further to 9.85 ± 1.35 µmol/l after 18 h. Total ADMA was 15.43 ± 2.44 µmol/l and did not change during incubation. The increase of free ADMA during incubation corresponded to substantial release of ADMA from the erythrocytic protein-incorporated pool (21.9 ± 4.6% at 2 h and 60.8 ± 7.6% at 18 h). ADMA was released from proteins other than hemoglobin, which only occurred after complete lysis and was blocked by combined inhibition of proteasomal and protease activity. Neither intact nor lysed erythrocytes mediated degradation of free ADMA. We conclude that intact erythrocytes play an important role in storage of ADMA, whereas upon erythrocyte lysis large amounts of free ADMA are generated by proteolysis of methylated proteins, which may affect plasma levels in hemolysis-associated diseases.

Vascular function in rats with adenine-induced chronic renal failure.

The aim of the present study was to characterize the function of resistance arteries, and the aorta, in rats with adenine-induced chronic renal failure (A-CRF). Sprague-Dawley rats were randomized to chow with or without adenine supplementation. After 6-10 wk, mesenteric arteries and thoracic aortas were analyzed ex vivo by wire myography. Plasma creatinine concentrations were elevated twofold at 2 wk, and eight-fold at the time of death in A-CRF animals. Ambulatory systolic and diastolic blood pressures measured by radiotelemetry were significantly elevated in A-CRF animals from week 3 and onward. At death, A-CRF animals had anemia, hyperphosphatemia, hyperparathyroidism, and elevated plasma levels of asymmetric dimethylarginine and oxidative stress markers. There were no significant differences between groups in the sensitivity, or maximal response, to ACh, sodium nitroprusside (SNP), norepinephrine, or phenylephrine in either mesenteric arteries or aortas. However, in A-CRF animals, the rate of aortic relaxation was significantly reduced following washout of KCl (both in intact and endothelium-denuded aorta) and in response to ACh and SNP. Also the rate of contraction in response to KCl was significantly reduced in A-CRF animals both in mesenteric arteries and aortas. The media of A-CRF aortas was thickened and showed focal areas of fragmented elastic lamellae and disorganized smooth muscle cells. No vascular calcifications could be detected. These results indicate that severe renal failure for a duration of less than 10 wk in this model primarily affects the aorta and mainly slows the rate of relaxation.

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.

Imbalance of arginine and asymmetric dimethylarginine is associated with markers of circulatory failure, organ failure and mortality in shock patients.

In shock, organ perfusion is of vital importance because organ oxygenation is at risk. NO, the main endothelial-derived vasodilator, is crucial for organ perfusion and coronary patency. The availability of NO might depend on the balance between a substrate (arginine) and an inhibitor (asymmetric dimethylarginine; ADMA) of NO synthase. Therefore, we investigated the relationship of arginine, ADMA and their ratio with circulatory markers, disease severity, organ failure and mortality in shock patients. In forty-four patients with shock (cardiogenic n 17, septic n 27), we prospectively measured plasma arginine and ADMA at intensive care unit admission, Acute Physiology and Chronic Health Evaluation (APACHE) II-(predicted mortality) and Sequential Organ Failure Assessment (SOFA) score, and circulatory markers to investigate their relationship. Arginine concentration was decreased (34·6 (SD 17·9) µmol/l) while ADMA concentration was within the normal range (0·46 (SD 0·18) µmol/l), resulting in a decrease in the arginine:ADMA ratio. The ratio correlated with several circulatory markers (cardiac index, disseminated intravascular coagulation, bicarbonate, lactate and pH), APACHE II and SOFA score, creatine kinase and glucose. The arginine:ADMA ratio showed an association (OR 0·976, 95 % CI 0·963, 0·997, P = 0·025) and a diagnostic accuracy (area under the curve 0·721, 95 % CI 0·560, 0·882, P = 0·016) for hospital mortality, whereas the arginine or ADMA concentration alone or APACHE II-predicted mortality failed to do so. In conclusion, in shock patients, the imbalance of arginine and ADMA is related to circulatory failure, organ failure and disease severity, and predicts mortality. We propose a pathophysiological mechanism in shock: the imbalance of arginine and ADMA contributes to endothelial and cardiac dysfunction resulting in poor organ perfusion and organ failure, thereby increasing the risk of death.

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.

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.

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.

Systemic inflammation is linked to low arginine and high ADMA plasma levels resulting in an unfavourable NOS substrate-to-inhibitor ratio: the Hoorn Study.

Inflammation is associated with a reduced availability of NO in the vasculature. We investigated the possible involvement of altered levels of the substrate (arginine) and the inhibitor [ADMA (asymmetric ω-NG,NG-dimethylarginine)] of NOS (NO synthase). Plasma concentrations of arginine and ADMA, the inflammatory markers CRP (C-reactive protein) and MPO (myeloperoxidase), and oxLDL [oxidized LDL (low-density lipoprotein)] were measured in 369 male and 377 female participants (aged 50-87 years) of a population-based cohort study. The arginine/ADMA ratio decreased significantly across increasing tertiles of CRP and MPO. These negative associations remained significant in a linear regression model with both MPO (P = 0.002) and CRP (P < 0.001) as independent variables and adjusted for age, sex and cardiovascular risk factors. In a fully adjusted regression model, MPO was positively associated with ADMA {5.4 [95% CI (confidence interval), 1.3-9.4] nmol/l change of ADMA per S.D. increase in MPO; P = 0.010}, whereas CRP was not (P = 0.36). Conversely, in a fully adjusted model, CRP was negatively associated with arginine [-2.8 (95% CI, -4.0 to -1.6) µmol/l arginine per S.D. of CRP; P < 0.001], without a significant contribution of MPO (P = 0.23). The relationship between MPO and ADMA became stronger with increasing levels of oxLDL (1.8, 5.2 and 8.7 nmol/l ADMA per S.D. of MPO for increasing tertiles of oxLDL), consistent with the ability of MPO to amplify oxidative stress. In contrast, the relationship between CRP and arginine was not modified by levels of oxLDL. In conclusion, an unfavourable NOS substrate/inhibitor ratio may contribute to the reduced NO bioavailability associated with inflammation.

Irbesartan treatment does not influence plasma levels of the advanced glycation end products N(epsilon)(1-carboxymethyl)lysine and N(epsilon)(1-carboxyethyl)lysine in patients with type 2 diabetes and microalbuminuria. A randomized controlled trial.

BACKGROUND: In vitro and animal experiments have shown inhibiting effects of angiotensin receptor blockers (ARBs) on the formation of advanced glycation end products (AGEs), which are known to be involved in the development of cardiovascular complications in diabetes. However, sufficient human data to confirm such beneficial effects of ARBs on AGEs are lacking. Therefore, we investigated the effects of irbesartan treatment on plasma levels of the AGEs N(ε)(1-carboxymethyl)lysine (CML) and N(ε)(1-carboxyethyl)lysine (CEL) in hypertensive patients with type 2 diabetes and microalbuminuria. METHODS: We analysed data from a multicentre, double-blind, parallel, randomized controlled trial in patients with type 2 diabetes and microalbuminuria, the primary goal of which was to examine the renoprotective effects of irbesartan treatment (150 or 300 mg daily). Secondary end points included plasma CML and CEL in the treatment arm receiving 300 mg irbesartan (n = 139) and in the placebo group (n = 125). Effects of treatment at 1- and 2-year follow-up were analysed by means of generalized estimating equations according to an intention-to-treat principle. RESULTS: Levels of CML and CEL did not differ between groups at baseline. No significant changes were observed in CML and CEL over time in either group and there was no effect of treatment on CML and CEL at any time-point. Mean differences for the irbesartan versus placebo group over time were -0.96 µmol/mol lysine (95% confidence interval: -3.43 to 1.51) for CML and -0.10 µmol/mol lysine (-0.76 to 0.56) for CEL. CONCLUSIONS: Long-term irbesartan treatment does not influence plasma levels of the AGE CML and CEL in patients with type 2 diabetes and microalbuminuria.