1Department of Surgery, 2Department of Clinical Chemistry, 3Department of Internal Medicine and 4Department of Physiology, VU University Medical Center, Amsterdam and 5Department of Internal Medicine, Amphia Hospital (Langendijk), Breda, The Netherlands
Correspondence and offprint requests to: P. A. M. van Leeuwen, MD, PhD, VU University Medical Center, Department of Surgery, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Email: pam.vleeuwen{at}vumc.nl
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Abstract |
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Methods. Twenty-five male Wistar rats weighing 275300 g were used for this study. The combination of arteriovenous concentration differences and kidney blood flow allowed calculation of net organ fluxes. Blood flow was measured using radiolabelled microspheres according to the reference sample method. Concentrations of ADMA, SDMA and arginine were measured by high-performance liquid chromatography.
Results. The kidney showed net uptake of both ADMA and SDMA and fractional extraction rates were 35% and 31%, respectively. Endotoxaemia resulted in a lower systemic ADMA concentration (P = 0.01), which was not explained by an increased net renal uptake. Systemic SDMA concentrations increased during endotoxaemia (P = 0.007), which was accompanied by increased creatinine concentrations.
Conclusions. The rat kidney plays a crucial role in the regulation of concentrations of dimethylarginines, as both ADMA and SDMA were eliminated from the systemic circulation in substantial amounts. Furthermore, evidence for the role of endotoxaemia in the metabolism of dimethylarginines was obtained as plasma levels of ADMA were significantly lower in endotoxaemic rats.
Keywords: L-arginine; kidney; nitric oxide; nitric oxide synthase
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Introduction |
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In asymptomatic humans with hypercholesterolaemia, elevated levels of ADMA were found and ADMA levels were associated with impaired endothelium-dependent vasodilation and reduced nitrate excretion [7]. ADMA levels also increased in elderly patients with peripheral arterial disease and generalized atherosclerosis [8]. Miyazaki et al. [9] measured dimethylarginine levels in plasma of 116 human subjects who had no sign of coronary or peripheral artery disease. They found that ADMA levels were positively correlated with age, mean arterial pressure and glucose tolerance. Most intriguingly, ADMA levels were significantly correlated with carotid artery intima-media thickness in stepwise regression analysis. The study of Miyazaki et al. suggests that elevation of this endogenous inhibitor precedes the occurrence of vascular occlusive disease. Others proposed that ADMA might also play a regulatory role in the action of inducible NOS, inhibiting overwhelming NO synthesis [10,11].
In 1992, Vallance et al. [4] reported elevated levels of ADMA in patients with renal failure. Elevated levels may be responsible for the hypertension seen in patients with end-stage renal disease. In patients with chronic renal failure also, a sharp rise of SDMA, the stereoisomer of ADMA, has been reported [12]. Fleck et al. [13] pointed out the potential importance of SDMA, and concluded in their study in a large population of renal failure patients, that not only ADMA levels but also SDMA levels were likely responsible for hypertension, possibly by competition for reabsorption between SDMA and arginine in the kidney. Moreover, we [14] recently confirmed the role for the kidney in the regulation of plasma levels of dimethylarginines, since both dimethylarginines were significantly extracted from the arterial supply of the human kidney.
ADMA is thought to be eliminated from the body by both degradation by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) and urinary excretion, while SDMA would only depend on urinary excretion. DDAH has been isolated from the rat kidney and is co-localized with the different NOS enzymes [6,1517].
Theoretically, a reduced activity of DDAH may be responsible for elevated ADMA concentrations [18]. DDAH activity is influenced by factors such as oxidative stress and inflammation [19,20]. In an in vitro model of human umbilical vein endothelial cells, a reduced activity of DDAH was found after exposure to oxidized low-density lipoprotein (LDL) and tumour necrosis factor (TNF)- [20]. In vivo, more confirmation on the potential role of oxidized LDL was obtained by the occurrence of high ADMA levels in hypercholesterolaemia, making ADMA a potential risk factor for atherosclerosis [7,20]. However, no in vivo data are present on the role of TNF-
and inflammation on the metabolism of dimethylarginines.
The aim of this study was to investigate dimethylarginine handling of the rat kidney in detail. Therefore, we designed a metabolic study in rats in which arteriovenous concentration differences were determined, together with blood flow measurement using radioactive microspheres. The combination of arteriovenous concentration differences and kidney blood flow allows calculation of net organ fluxes. Furthermore, we administered lipopolysaccharide (LPS), a component of the outer membrane of most Gram-negative bacteria that is used as a stimulator of the inflammatory response, to a second group of animals to study the role of the inflammatory state on the metabolism of ADMA and SDMA.
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Subjects and methods |
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After overnight fasting, rats were anaesthetized using ketamine HCl (70 mg/kg i.m.) and Nembutal (60 mg/kg i.p.) and placed in the supine position on a heating pad that maintained rectal temperature at 37°C. Anaesthesia was maintained by Nembutal (10 mg/kg/h i.p.). The trachea was intubated with a small piece of polyethylene tubing (PE-240; Fisher, Scientific, Springfield, NY, USA) to facilitate breathing. The right carotid artery and right femoral artery were cannulated using PE-28 tubing; the right jugular vein and right femoral vein were cannulated using PE-50 tubing.
Through a small (0.5 cm) midline incision in the lower abdomen, the bladder was cannulated using PE-28 tubing. The cannula was secured with one stitch in the wall of the bladder. Hereafter, the incision was closed and a small clamp was placed on the end of the cannula. Following these procedures the rats were allowed to stabilize for 30 min.
Rats were randomly assigned to receive intravenous LPS (n = 13) infusion [1.5 ml; dose: 8 mg/kg body weight (BW)] starting at t = 0 min or to intravenous infusion of 1.5 ml 0.9% NaCl (control; n = 12).
To determine urinary concentrations of ADMA, SDMA, arginine and creatinine, urine was collected during the last 30 min period of the experiment (t = 150180 min) and the amount of urine was weighed to calculate urine production.
During the entire experiment, haemodynamical parameters were continuously recorded and at t = 180 min kidney blood flow was measured using radiolabelled microspheres. Immediately hereafter, blood samples were taken and the experiment was terminated by exsanguination of the animals.
Blood flow measurement
Blood flow measurement was performed at the end of the experiment (t = 180 min). Blood flow was measured using radiolabelled microspheres according to the reference sample method [22]. This method was chosen for its high reproducibility. The catheters in the right common carotid artery and left femoral artery were connected to P23Db Statham pressure transducers. Pressure wave monitoring was used to place the carotid catheter into the left ventricle. Mean arterial pressure (MAP) and heart frequency (HF) were continuously recorded during the experiment.
At the end of the experiment, an intraventricular injection of 103Ru-labelled microspheres (1.3 x 105 microspheres dissolved in 0.3 ml saline) was performed. A reference blood sample was obtained from the right femoral artery at a rate of 0.4 ml/min over 120 s, starting 5 s before the microsphere injection.
Immediately after the microsphere procedure, the abdomen was opened and blood samples were drawn from renal vein and abdominal aorta, after which the animal was exsanguinated.
Kidneys were removed and wrapped in tissue paper. The kidneys were weighed and placed in counting vials. Radioactivity was measured in a counter (CS 1282; Wallace Compugamma, Turku, Finland). Kidney blood flow was computed according to the reference sample technique using the equation F = Fa (Qo / Qa), where Fa is the reference flow, Qo is the count rate in the kidney tissue and Qa is the count rate in the reference blood sample. Reference blood flow was computed from the weight of blood in the reference syringe and the duration of withdrawal assuming a whole-blood density of 1.055 g/ml. Kidney blood flow was expressed as ml/min/g tissue. Plasma flow was computed by correction for haematocrit (Microhematocrit reader; Hawsley and Sons, London, UK) by the equation:
plasma flow = blood flow x (1 - haematocrit)
Plasma analysis of AMDA, SDMA, arginine and chemical analysis
Blood samples were immediately placed on ice and centrifuged at 3000 r.p.m. for 10 min at 4°C (Sorvall GLC 2 centrifuge; Sorvall Operations, DuPont, Newton, CT, USA). Plasma was pipetted and immediately put in liquid nitrogen and stored at -70°C before analysis.
Arginine, ADMA and SDMA were measured simultaneously by high-performance liquid chromatography with fluorescence detection as described previously [23]. Briefly, 0.1 ml plasma was mixed with 0.1 ml of a 40 µmol/l solution of the internal standard L-NMMA and 0.8 ml phosphate-buffered saline. This mixture was applied to Oasis MCX solid-phase extraction columns (Waters, Milford, MA, USA) for extraction of basic amino acids. The columns were consecutively washed with 1.0 ml 100 mM HCl and 1.0 ml methanol. Analytes were eluted with 1.0 ml concentrated ammonia/water/methanol (10/40/50). After evaporation of the solvent under nitrogen, the amino acids were derivatized with o-phthaldialdehyde reagent containing 3-mercaptopropionic acid. The derivatives were separated by isocratic reversed-phase chromatography on a Symmetry C18 column (3.9 x 150 mm; 5 µm particle size; Waters). Potassium phosphate buffer (50 mM; pH 6.5) containing 8.7% acetonitrile was used as the mobile phase at a flow rate of 1.1 ml/min and a column temperature of 30°C. Fluorescence detection was performed at excitation and emission wavelengths of 340 and 455 nm, respectively. After elution of the last analyte, strongly retained compounds were quickly eluted by a strong solvent flush with 50% acetonitrile, resulting in a total analysis time of 30 min. The intra-assay coefficients of variation (CVs) for arginine, ADMA and SDMA were 0.4%, 1.2% and 0.8%, respectively. The interassay CVs for arginine, ADMA and SDMA were 2.9%, 2.0% and 2.6%, respectively.
Plasma creatinine was determined in arterial plasma samples using an automated analyser (H 737; Hitachi, Tokyo, Japan).
Calculations
Kidney uptake or release (flux) and fractional extraction rates for ADMA, SDMA and arginine were calculated from the plasma flow and the arteriovenous concentration difference for the left kidney. The flux calculation of the left kidney was multiplied by 2 to obtain values for both kidneys and results are presented as such. Flux is presented as nmol/100 g BW/min. Fractional extraction is calculated as [A] [RV] / [A], where [A] and [RV] denote arterial and renal vein plasma concentrations, respectively. Each parameter was calculated for each individual animal using its individual substrate concentrations and renal plasma flow.
Urine volume was calculated by dividing urine weight by 1.020 to correct for specific gravity. Clearances of creatinine, ADMA, SDMA and arginine were calculated as [U] x [V] / [P], where [U] and [P] denote urinary and arterial plasma concentrations, respectively, and [V] denotes urine volume per min. Fractional excretion was calculated as clearance of ADMA, SDMA or arginine divided by the clearance of endogenous creatinine.
Statistical methods
Statistical analysis was performed using the SPSS 9.0 statistical package. Differences between the groups were tested using the non-parametric Wilcoxon ranked sum test. Haemodynamics were recorded every 30 min during the experiment and were evaluated using the general linear model for repeated measurements. When appropriate, intergroup differences on the individual time-points were further tested using the Wilcoxon ranked sum test, whereas the Wilcoxon paired test was used for testing the intragroup differences. Values are expressed as means ± SEM and P < 0.05 was considered statistically significant.
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Results |
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In control rats, arterial and renal vein SDMA concentrations were 0.452 ± 0.016 and 0.317 ± 0.024 µmol/l, respectively. In contrast to ADMA, arterial concentrations of SDMA were higher in LPS-treated rats. Also in renal vein plasma, SDMA concentration was higher in LPS-treated rats. Arteriovenous concentration difference was not significantly different between LPS and control groups. Fractional extraction rate was lower in LPS-treated rats.
In control rats, arterial and renal vein arginine concentrations were 123.2 ± 5.2 and 142.3 ± 6.2 µmol/l, respectively. Arterial and renal vein arginine concentrations were significantly lower in LPS-treated rats. In control rats, renal arteriovenous concentration and fractional extraction indicated release of arginine, which was more pronounced in the LPS group.
Net renal fluxes of ADMA, SDMA and arginine
In Figure 2 net renal fluxes of ADMA (A), SDMA (B) and arginine (C) are displayed.
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In control rats, net release of arginine was observed, which was (not significantly) higher in LPS treated-rats.
Urinary excretion and clearance of ADMA, SDMA and arginine
In Table 3, urine concentration, clearance and fractional excretion of ADMA, SDMA and arginine are given. Urinary ADMA excretion was negligible in both control and LPS-treated animals, as evidenced by low ADMA concentrations and a low clearance rate.
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Urinary arginine concentration was similar in both groups. Clearance and fractional excretion of arginine were low in both groups.
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Discussion |
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To adequately measure arteriovenous concentration differences in small sample volumes, we developed a high-performance liquid chromatography (HPLC) method with a high precision and sensitivity. This method allowed simultaneous measurement of ADMA, SDMA and arginine and proved to be a valuable tool in the research on the metabolism of dimethylarginines and their role in the arginineNO pathway.
Recently, we pointed to the kidney as an important contributor in the regulation of plasma levels of dimethylarginines, by showing significant extraction of both ADMA and SDMA from the arterial supply of the human kidney [14]. These results are further substantiated in the present study in rats, providing detailed insight into the renal handling of dimethylarginines. We found a significant net uptake of both ADMA and SDMA by the rat kidney, with fractional extraction rates of 35% and 31%, respectively. Furthermore, strong evidence was obtained for a differential renal handling of the two dimethylarginines. In our model we found that the elimination of ADMA by the rat kidney could not be explained by urinary excretion, because urinary concentration of unchanged ADMA was negligible. This finding points to a high metabolic turnover of ADMA in the kidney, which is fully responsible for the observed net renal uptake of ADMA. In contrast to the rat kidney, human kidneys are capable of excreting unchanged ADMA [14,25], so there also seems to be a difference in the handling of dimethylarginines between humans and rats. Ogawa et al. [26] investigated the metabolic fate of ADMA and SDMA in the rat and, interestingly, they found that only 4.6% of injected ADMA was found in the first 12 h urine as unchanged ADMA, compared with 17.8% for SDMA. Furthermore, they found that both dimethylarginines are metabolized by a pathway forming the corresponding -ketoacid analogues and the oxidatively decarboxylated products of the
-ketoacids in addition to N-acetyl conjugates, and that these metabolites were mainly found in urine. Interestingly, especially the acetyl conjugation reaction has been recognized as typical for the rat species, as has been reported for the acetylation of the
-amino group of 3-methylhistidine [27]. However, with our HPLC method we were only able to measure unchanged ADMA and SDMA and related metabolites of these dimethylarginines would not have been detected. If the formation of
-ketoacids or N-acetyl conjugates is confined to the rat species, this metabolic pathway could explain the controversy between the data obtained from rat and human experiments. In the experiment of Ogawa et al. [26], only for ADMA an additional pathway was found, leading to the formation of citrulline and related amino acids. This pathway seemed to be the main route for ADMA elimination, as most ADMA derived radioactivity was found in tissues instead of urine. Later, this catalytic pathway was recognized both in rats and humans and proven to be degradation by the enzyme DDAH [16,17]. In our study, the rat kidney proved to be capable of excretion of SDMA into urine and
30% of net renal uptake could be explained by excretion of unchanged SDMA in the urine. According to the study of Ogawa et al. [26], and because SDMA cannot be degraded by DDAH, it could be speculated that the remainder of the renal uptake of SDMA could be explained by urinary excretion of
-ketoacids and N-acetyl conjugates.
In contrast to our expectations, in rats treated with endotoxin a significantly lower ADMA concentration was found. To our knowledge no in vivo studies have been performed on the role of endotoxaemia in the metabolism of dimethylarginines. In the present study we deliberately chose to use LPS infusion instead of TNF-, because we aimed to mimic a clinical condition, driven by the naturally complex combination of inflammatory mediators, and not to study only the response to TNF-
. Although we cannot designate a single mediator for the observed effect, in the present experiment strong evidence was obtained for increased metabolic turnover of ADMA during endotoxaemia. Interestingly, the increased metabolic turnover of ADMA was not accompanied by an increased renal elimination of ADMA, as both renal fractional extraction rate and net renal uptake were significantly lower in LPS-treated rats. Therefore, the kidney seems not to be responsible for this ADMA-lowering effect of endotoxaemia. A potential explanation for the lower plasma concentration of ADMA might be increased uptake by the y+ transporter during endotoxaemia. Cationic amino acids, such as arginine, ornithine and lysine, are transported into endothelial cells by the cationic amino acid transporters (CAT) of system y+. Closs et al. [5] investigated transport of dimethylarginines by CAT and found that both ADMA and SDMA were transported across this y+ carrier. In rats it has been shown that the expression of CAT transporters was significantly increased in lung, heart and kidney by LPS injection [28]. Clinical conditions associated with severe endotoxaemia include sepsis and septic shock and these conditions are characterized by overproduction of NO due to inducible NOS (iNOS) activity. The activity of iNOS appears to be mainly regulated at the transcriptional level. However, regulation of arginine availability can also determine the cellular rate of NO production, since arginine is the only substrate for NOS. It has been suggested that the immunostimulant-elicited increase in arginine transport activity plays a key role in NO formation and that arginine transport is stimulated by endotoxin during sepsis. In the present study we found decreased concentrations of both arginine and ADMA during experimental endotoxaemia, which may have resulted from increased transport by the y+ carrier. One biological question that has to be answered is what the potential role of ADMA in inflammation and infection is. It has been speculated that ADMA could possibly serve as a brake on the action of iNOS and inhibit overwhelming NO synthesis [11]. In contrast to the decreased ADMA levels, SDMA levels were higher in endotoxin-treated rats and the increase of SDMA was accompanied by a reduced renal fractional extraction and a reduced net uptake by the kidney. As creatinine levels were also significantly higher in LPS-treated rats, an impaired renal clearance of SDMA could underlie the rise in SDMA levels.
Thus, LPS infusion resulted in a reduced renal elimination of both ADMA and SDMA, as reflected by reduced fractional extraction and fluxes. However, in contrast to SDMA, systemic ADMA levels were significantly lower in LPS-infused rats. Therefore, while changes in systemic SDMA levels may be logically explained by reduced renal elimination, the systemic effect of LPS on ADMA leading to a lower plasma concentration needs further study.
In the current study we also presented data on arginine handling of the kidney, as arginine is the key amino acid in the arginineNO pathway and its molecular structure closely resembles that of dimethylarginines. In the past, our group and others studied arginine handling of the kidney during normal conditions and during endotoxaemia [29,30]. The results of the present study are in agreement with those reports and the important role of the kidney in maintaining arginine levels was demonstrated.
In conclusion, in the present study the handling of dimethylarginines by the rat kidney was studied in detail and novel facets were elucidated. Based on our findings we propose the scheme presented in Figure 3. Both dimethylarginines are eliminated from the systemic circulation in substantial amounts. There seems to be a differential metabolism for both dimethylarginines, at least in the rat kidney, as a negligible amount of unchanged ADMA was excreted, whereas substantial amounts of unchanged SDMA were recovered in urine. Furthermore, evidence for the role of endotoxaemia on the metabolism of dimethylarginines was obtained as plasma levels of ADMA were significantly lower in endotoxaemic rats, whereas renal elimination of ADMA was decreased. In endotoxaemia, SDMA levels were elevated, which was accompanied by a decreased renal function as was measured by creatinine levels and a diminished renal elimination of SDMA. The mechanisms underlying the decreased ADMA levels in endotoxaemia were not elucidated in this study, but warrant further study.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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