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• W2123993612 abstract "Patients with chronic kidney disease have elevated circulating asymmetric dimethylarginine (ADMA). Recent studies have suggested that ADMA impairs endothelial nitric oxide synthase (eNOS) by effects other than competition with the substrate L-arginine. Here, we sought to identify the molecular mechanism by which increased ADMA causes endothelial dysfunction in a chronic kidney disease model. In wild-type mice with remnant kidney disease, blood urea nitrogen, serum creatinine, and ADMA were increased by 2.5-, 2-, and 1.2-fold, respectively, without any change in blood pressure. Nephrectomy reduced endothelium-dependent relaxation and eNOS phosphorylation at Ser1177 in isolated aortic rings. In transgenic mice overexpressing dimethylarginine dimethylaminohydrolase-1, the enzyme that metabolizes ADMA, circulating ADMA was not increased by nephrectomy and was decreased to half that of wild-type mice. These mice did not exhibit the nephrectomy-induced inhibition of both endothelium-dependent relaxation and eNOS phosphorylation. In cultured human endothelial cells, agonist-induced eNOS phosphorylation and nitric oxide production were decreased by ADMA at concentrations less than that of L-arginine in the media. Thus, elevated circulating ADMA may be a cause, not an epiphenomenon, of endothelial dysfunction in chronic kidney disease. This effect may be attributable to inhibition of eNOS phosphorylation. Patients with chronic kidney disease have elevated circulating asymmetric dimethylarginine (ADMA). Recent studies have suggested that ADMA impairs endothelial nitric oxide synthase (eNOS) by effects other than competition with the substrate L-arginine. Here, we sought to identify the molecular mechanism by which increased ADMA causes endothelial dysfunction in a chronic kidney disease model. In wild-type mice with remnant kidney disease, blood urea nitrogen, serum creatinine, and ADMA were increased by 2.5-, 2-, and 1.2-fold, respectively, without any change in blood pressure. Nephrectomy reduced endothelium-dependent relaxation and eNOS phosphorylation at Ser1177 in isolated aortic rings. In transgenic mice overexpressing dimethylarginine dimethylaminohydrolase-1, the enzyme that metabolizes ADMA, circulating ADMA was not increased by nephrectomy and was decreased to half that of wild-type mice. These mice did not exhibit the nephrectomy-induced inhibition of both endothelium-dependent relaxation and eNOS phosphorylation. In cultured human endothelial cells, agonist-induced eNOS phosphorylation and nitric oxide production were decreased by ADMA at concentrations less than that of L-arginine in the media. Thus, elevated circulating ADMA may be a cause, not an epiphenomenon, of endothelial dysfunction in chronic kidney disease. This effect may be attributable to inhibition of eNOS phosphorylation. The prevalence of chronic kidney disease (CKD) is increasing worldwide.1.Coresh J. Selvin E. Stevens L.A. et al.Prevalence of chronic kidney disease in the United States.JAMA. 2007; 298: 2038-2047Crossref PubMed Scopus (3881) Google Scholar,2.Meguid El Nahas A. Bello A.K. Chronic kidney disease: the global challenge.Lancet. 2005; 365: 331-340Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar Approximately 50% of the end-stage renal disease patients die from cardiovascular causes3.Tonelli M. Wiebe N. Culleton B. et al.Chronic kidney disease and mortality risk: a systematic review.J Am Soc Nephrol. 2006; 17: 2034-2047Crossref PubMed Scopus (1204) Google Scholar, 4.Foley R.N. Parfrey P.S. Sarnak M.J. Clinical epidemiology of cardiovascular disease in chronic renal disease.Am J Kidney Dis. 1998; 32: S112-S119Abstract Full Text PDF PubMed Scopus (2964) Google Scholar, 5.Sarnak M.J. Levey A.S. Schoolwerth A.C. et al.Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention.Circulation. 2003; 108: 2154-2169Crossref PubMed Scopus (2874) Google Scholar and their cardiovascular mortality is 500-fold greater compared with that of age-matched controls with normal renal function.5.Sarnak M.J. Levey A.S. Schoolwerth A.C. et al.Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention.Circulation. 2003; 108: 2154-2169Crossref PubMed Scopus (2874) Google Scholar The Framingham Heart Study revealed that mild renal failure was associated with increased prevalence of death and cardiovascular events even in the general population.6.Culleton B.F. Larson M.G. Wilson P.W. et al.Cardiovascular disease and mortality in a community-based cohort with mild renal insufficiency.Kidney Int. 1999; 56: 2214-2219Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar Endothelial dysfunction was documented from the early stage of renal failure,7.Stam F. van Guldener C. Becker A. et al.Endothelial dysfunction contributes to renal function-associated cardiovascular mortality in a population with mild renal insufficiency: the Hoorn study.J Am Soc Nephrol. 2006; 17: 537-545Crossref PubMed Scopus (209) Google Scholar suggesting that endothelial dysfunction is one of the initial mechanisms that lead to cardiovascular complications in CKD patients. Asymmetric dimethylarginine (ADMA) is generated during the process of protein turnover and is actively degraded by the intracellular enzyme, dimethylarginine dimethylaminohydrolase (DDAH).8.Leiper J. Vallance P. Biological significance of endogenous methlarginines that inhibit nitric oxide synthases.Cardiovasc Res. 1999; 43: 542-548Crossref PubMed Scopus (418) Google Scholar, 9.Cam T.L. Leiper J.M. Vallance P. The DDAH/ADMA/NOS pathway.Atheroscler Suppl. 2003; 4: 33-40PubMed Google Scholar, 10.Vallance P. Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway.Arterioscler Thromb Vasc Biol. 2004; 24: 1023-1030Crossref PubMed Scopus (369) Google Scholar, 11.Zoccali C. Asymmeteric dimethlarginine (ADMA): a cardiovascular and renal risk factor on the move.J Hypertens. 2006; 24: 611-619Crossref PubMed Scopus (67) Google Scholar, 12.McDermott J.R. Studies on the catabolism of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine in the rabbit.Biochem J. 1976; 154: 179-184Crossref PubMed Scopus (193) Google Scholar, 13.Ogawa T. Kimoto M. Watanabe H. et al.Metabolism of NG,NG-and NG,N′G-dimethylarginine in rats.Arch Biochem Biophys. 1987; 252: 526-537Crossref PubMed Scopus (123) Google Scholar ADMA is an endogenous inhibitor of all types of nitric oxide synthases (NOSs).14.Vallance P. Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway.Arterioscler Thromb Vasc Biol. 2004; 24: 1023-1030Crossref PubMed Scopus (489) Google Scholar,15.Boger R.H. The pharmacodynamics of L-arginine.J Nutr. 2007; 137: 1650S-1655SPubMed Google Scholar It has long been thought that the NOS inhibition by ADMA is attributable to its competitive inhibition as an L-arginine analog.10.Vallance P. Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway.Arterioscler Thromb Vasc Biol. 2004; 24: 1023-1030Crossref PubMed Scopus (369) Google Scholar,16.Vallance P. Leone A. Calver A. et al.Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure.Lancet. 1992; 339: 572-575Abstract PubMed Scopus (1958) Google Scholar However, there is increasing evidence that ADMA may have additional effects that are independent of the competitive inhibition of NOS although the precise mechanisms are unknown.17.Cross J.M. Donald A.E. Kharbanda R. et al.Acute administration of L-arginine does not improve arterial endothelial function in chronic renal failure.Kidney Int. 2001; 60: 2318-2323Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18.Hasegawa K. Wakino S. Tatematsu S. et al.Role of asymmetric dimethylarginine in vascular injury in transgenic mice overexpressing dimethylarginie dimethylaminohydrolase 2.Circ Res. 2007; 101: e2-10Crossref PubMed Scopus (107) Google Scholar, 19.Smith C.L. Anthony S. Hubank M. et al.Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA.PLoS Med. 2005; 2: e264Crossref PubMed Scopus (49) Google Scholar We have shown that circulating ADMA levels are correlated with the thickness of the carotid artery in a general healthy population.20.Miyazaki H. Matsuoka H. Cooke J.P. et al.Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis.Circulation. 1999; 99: 1141-1146Crossref PubMed Scopus (720) Google Scholar Thereafter, ADMA has been increasingly recognized as a putative biomarker in cardiovascular diseases.21.Boger R.H. The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor.Cardiovasc Res. 2003; 59: 824-833Crossref PubMed Scopus (330) Google Scholar It was reported that circulating ADMA levels were elevated in CKD patients.16.Vallance P. Leone A. Calver A. et al.Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure.Lancet. 1992; 339: 572-575Abstract PubMed Scopus (1958) Google Scholar, 22.Zoccali C. Bode-Boger S. Mallamaci F. et al.Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study.Lancet. 2001; 358: 2113-2117Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 23.Kielstein J.T. Boger R.H. Bode-Boger S.M. et al.Marked increase of asymmetric dimethylarginine in patients with incipient primary chronic renal disease.J Am Soc Nephrol. 2002; 13: 170-176PubMed Google Scholar Reduced bioavailability of nitric oxide (NO) has been documented in CKD patients, concurrently with endothelial dysfunction.24.Wever R. Boer P. Hijmering M. et al.Nitric oxide production is reduced in patients with chronic renal failure.Arterioscler Thromb Vasc Biol. 1999; 19: 1168-1172Crossref PubMed Scopus (195) Google Scholar, 25.Passauer J. Pistrosch F. Bussemaker E. et al.Reduced agonist-induced endothelium-dependent vasodilation in uremia is attributable to an impairment of vascular nitric oxide.J Am Soc Nephrol. 2005; 16: 959-965Crossref PubMed Scopus (85) Google Scholar, 26.Hasdan G. Benchetrit S. Rashid G. et al.Endothelial dysfunction and hypertension in 5/6 nephrectomized rats are mediated by vascular superoxide.Kidney Int. 2002; 61: 586-590Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar However, it remains undetermined whether the elevation of circulating ADMA level is a cause or an epiphenomenon of endothelial damages in patients or animal models of CKD. The aims of this study were to examine whether increased circulating ADMA causes endothelial dysfunction in a mouse model of CKD and, if so, to investigate the molecular mechanism. To address the contribution of circulating ADMA to endothelial dysfunction in CKD, we created 5/6 nephrectomized (Nx) models in DDAH-1 overexpressed mice having reduced circulating ADMA levels. The effects of ADMA on endothelial NOS (eNOS) activity were investigated in cultured human umbilical vein endothelial cells (HUVECs). Nx increased blood urea nitrogen (BUN) and serum creatinine levels in wild-type (WT) mice. The elevations of BUN and serum creatinine in human DDAH-1–transgenic (TG) mice receiving Nx (TG+CKD) were similar to those in WT mice receiving Nx (WT+CKD) 4 weeks after the operation (Figure 1a and b). There were no differences in systolic blood pressure and heart rate among the four groups (Figure 1c and d). We did not observe apparent morphological and histological changes in the heart and aorta in WT+CKD, TG+sham, and TG+CKD mice (data not shown). In WT+CKD mice, serum ADMA levels were increased by 20% compared with those in WT+sham mice (Figure 1e). TG+sham mice showed a 50% reduction in serum ADMA levels compared with WT+sham mice. In TG mice, Nx did not increase the ADMA levels. In WT+sham mice, acetylcholine (Ach) induced a dose-dependent relaxation of the aortic rings precontracted with phenylephrine (Figure 2 and Supplementary Table S1 online). The endothelium-dependent relaxation was significantly impaired in isolated aortic rings obtained from WT+CKD mice. In TG+sham mice, the endothelium-dependent relaxation was similar to that in WT+sham mice. The Nx-induced endothelial dysfunction was prevented in TG mice. These results were confirmed by the area under the curve analysis (Table 1). The half-maximal inhibitory concentration (IC50) of Ach was significantly greater in WT+CKD mice compared with WT+sham mice, suggesting that ADMA reduced the sensitivity for Ach after Nx in WT mice. However, the IC50 levels in TG+sham and TG+CKD mice did not differ from that in WT+sham mice (Table 1). Download .doc (3.86 MB) Help with doc files Supplementary InformationTable 1Effects of CKD on IC50 and AUC of acetylcholine-induced relaxation of the aortic rings of WT and TG miceAcetylcholineGroupnLog IC50 (mol/l)AUC (arbitrary unit)WT+sham9-6.67±0.62139±21WT+CKD4-5.72±0.78*P<0.05 vs. WT+sham.49±13*P<0.05 vs. WT+sham.TG+sham5-5.85±0.65121±16TG+CKD4-6.44±0.67146±36Abbreviations: AUC, area under the curve; CKD, chronic kidney disease 5/6 nephrectomized mouse; IC50, half-maximal (50%) inhibitory concentration; Sham, sham-operated control mouse; TG, dimethylarginine dimethylaminohydrolase-1 transgenic mouse; WT, wild-type mouse.Data are expressed as mean±s.e.m.* P<0.05 vs. WT+sham. Open table in a new tab Abbreviations: AUC, area under the curve; CKD, chronic kidney disease 5/6 nephrectomized mouse; IC50, half-maximal (50%) inhibitory concentration; Sham, sham-operated control mouse; TG, dimethylarginine dimethylaminohydrolase-1 transgenic mouse; WT, wild-type mouse. Data are expressed as mean±s.e.m. The Ser1177 residue of eNOS is a key phosphorylation site that positively regulates eNOS enzyme activity independently of intracellular calcium concentrations.27.Dudzinski D.M. Michel T. Life history of eNOS: partners and pathways.Cardiovasc Res. 2007; 75: 247-260Crossref PubMed Scopus (315) Google Scholar We investigated the eNOS expression and phosphorylation levels in the aorta of WT and TG mice with or without Nx (Figure 3a and b). Nx did not affect eNOS protein expression levels in WT mice. However, eNOS phosphorylation at Ser1177 was attenuated by Nx, suggesting the inhibition of eNOS activity in WT+CKD mice. In TG+sham mice, the eNOS expression and phosphorylation levels were similar to those in WT+sham mice. The reduction in eNOS phosphorylation observed in WT+CKD mice was abolished in TG+CKD mice. In addition, we examined urinary nitrate/nitrite (NOx) excretion in 24h as an indicator of NO production in vivo (Figure 3c). Urinary NOx excretion was reduced by 66% in WT+CKD mice compared with WT+sham mice. In TG+sham mice, urinary NOx excretion was much higher than that in WT+sham mice. The NOx excretion was significantly reduced in TG+CKD mice compared with TG+sham mice. But, the NOx levels in TG+CKD mice were similar to the levels in WT+sham mice and significantly higher than those in WT+CKD mice. To determine whether ADMA would directly impair endothelial function, we examined the effects of exogenous ADMA treatment on the endothelium-dependent relaxation of the aorta in WT mice (Figure 4). The endothelium-dependent relaxation was dose-dependently inhibited by pre-treatment with 1 × 10−6–1 × 10−4mol/l ADMA, although the inhibition was not statistically significant for 10−6mol/l ADMA (Figure 4b and Table 2). The IC50 of Ach was significantly increased by 1 × 10−4mol/l ADMA pre-treatment compared with 0mol/l ADMA (Table 2).Table 2Effects of ADMA on IC50 and AUC of acetylcholine-induced relaxation of the aortic rings of WT miceAcetylcholineGroupnLog IC50 (mol/l)AUC (arbitrary unit)ADMA 10−4mol/l9-5.53±0.31**P<0.01 vs. ADMA 0mol/l.31±9*P<0.05,ADMA 10−5mol/l6-6.36±0.2773±24*P<0.05,ADMA 10−6mol/l6-6.26±0.25100±32ADMA 0mol/l9-6.64±0.13125±23Abbreviations: ADMA, asymmetric dimethylarginine; AUC, area under the curve; IC50, half-maximal (50%) inhibitory concentration; WT, wild type.Data are expressed as mean±s.e.m.* P<0.05,** P<0.01 vs. ADMA 0mol/l. Open table in a new tab Abbreviations: ADMA, asymmetric dimethylarginine; AUC, area under the curve; IC50, half-maximal (50%) inhibitory concentration; WT, wild type. Data are expressed as mean±s.e.m. To further determine the molecular mechanisms of the endothelial dysfunction by ADMA, we examined the effects of ADMA on eNOS phosphorylation using cultured HUVECs (Figure 5a and b). In HUVECs cultured in the media containing 5 × 10−4mol/l L-arginine, ADMA (1 × 10−6 and 1 × 10−4mol/l) did not affect the baseline eNOS phosphorylation at Ser1177. However, ADMA dose-dependently inhibited the vascular endothelial growth factor-induced eNOS phosphorylation. Next, we examined the effects of ADMA on the phosphorylation of extracellular signal-related protein kinase (ERK) and Akt, putative upstream regulatory molecules of eNOS phosphorylation. ADMA dose-dependently inhibited the vascular endothelial growth factor-induced ERK1/2 phosphorylation, without changing the baseline phosphorylation. In contrast, ADMA did not affect Akt phosphorylation at Ser473. Moreover, we confirmed the effects of ADMA on NO production in HUVECs. ADMA inhibited the vascular endothelial growth factor-stimulated NO release into the conditioned media (Figure 5c). The salient findings of this study are as follows: (1) Nx induced moderate renal failure to a similar extent in WT and DDAH-1–TG mice without elevating blood pressure; (2) Nx impaired endothelium-dependent relaxation of isolated aortic rings in WT mice with the elevation of circulating ADMA levels, but not in TG mice having reduced ADMA levels; (3) Nx attenuated eNOS phosphorylation at Ser1177, an indicator of eNOS activity, in the aorta of WT mice, but not in TG mice; (4) ADMA treatment suppressed the agonist-induced eNOS phosphorylation and NO production in HUVECs cultured under the L-arginine-rich condition. As shown in Figure 1, a mouse with Nx had 2.5- and 2-fold increases in BUN and creatinine, respectively, without blood pressure elevation, suggesting that it is a CKD model representing moderate renal failure without hypertension. This study was performed in young mice, and the observation period was rather short because we focused on investigating the relationship between elevated circulating ADMA and endothelial function in the CKD model before cardiovascular damages and remodeling had developed. TG mice showed a 50% reduction in baseline ADMA levels (Figure 1e). This finding is consistent with that of previous reports.28.Dayoub H. Achan V. Adimoolam S. et al.Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence.Circulation. 2003; 108: 3042-3047Crossref PubMed Scopus (295) Google Scholar,29.Jacobi J. Sydow K. von Degenfeld G. et al.Overexpression of dimethylarginine dimethylaminohydrolase reduces tissue asymmetric dimethylarginine levels and enhances angiogenesis.Circulation. 2005; 111: 1431-1438Crossref PubMed Scopus (142) Google Scholar Although circulating ADMA levels were significantly increased by Nx in WT mice, Nx did not change the ADMA levels in TG mice, probably due to augmented ADMA degradation by overexpressed DDAH-1. Thus, TG+CKD mice were used as a model animal having renal failure without elevated circulating ADMA levels. It is noteworthy that the Nx-induced impairment of endothelium-dependent relaxation was prevented in TG+CKD mice (Figure 2 and Supplementary Table S1 online). As the blood pressure level and the extent of renal failure were similar in WT+CKD and TG+CKD mice (Figure 1), it is suggested that the elevation of circulating ADMA levels has a causal relation to the Nx-induced endothelial dysfunction in WT mice (Figures 2 and 4). To the best of our knowledge, this is the first demonstration that elevated circulating ADMA may be a cause, but not an epiphenomenon, of endothelial dysfunction in CKD. Another important novel finding of this study is that reduction of eNOS phosphorylation at Ser1177 was associated with endothelial dysfunction in the aorta of WT+CKD mice, but not in TG+CKD mice (Figure 3). Phosphorylation levels at Ser1177 determine eNOS enzyme activity.27.Dudzinski D.M. Michel T. Life history of eNOS: partners and pathways.Cardiovasc Res. 2007; 75: 247-260Crossref PubMed Scopus (315) Google Scholar Indeed, the urine NOx excretion, an indicator of NO production in vivo, was significantly higher in TG+CKD mice than in WT+CKD mice, suggesting eNOS phosphorylation levels in the aorta are functionally associated with NO production in the mice. Thus, it is suggested that the reduced eNOS phosphorylation may be involved in the mechanism accounting for the endothelial dysfunction in WT+CKD model. The precise mechanism linking ADMA to inhibition of eNOS phosphorylation remains unknown in this CKD model. However, the experiments using HUVECs provided an insight into the possible mechanism (Figure 5). It is interesting to note that ADMA prevented the agonist-induced eNOS phosphorylation and NO production in HUVECs. Moreover, the phosphorylation of ERK, one of the putative major kinases for eNOS phosphorylation, was also inhibited by ADMA. Thus, it is possible that the inhibition of ERK–eNOS pathway would be a mechanism whereby ADMA impairs eNOS function. We used the culture media containing L-arginine, the eNOS substrate, at 5 × 10−4mol/l, which was a much higher concentration of ADMA that was applied to HUVECs (1 × 10−6 and 1 × 10−4mol/l). In this study, it is less likely that competitive inhibition was the reason for the observed effects of ADMA on NO generation. Rather, the inhibition of the ERK–eNOS phosphorylation may be a novel mechanism of eNOS inhibition by ADMA (Figure 6). These findings may support the notion that ADMA has additional actions other than competitive inhibition of eNOS.17.Cross J.M. Donald A.E. Kharbanda R. et al.Acute administration of L-arginine does not improve arterial endothelial function in chronic renal failure.Kidney Int. 2001; 60: 2318-2323Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18.Hasegawa K. Wakino S. Tatematsu S. et al.Role of asymmetric dimethylarginine in vascular injury in transgenic mice overexpressing dimethylarginie dimethylaminohydrolase 2.Circ Res. 2007; 101: e2-10Crossref PubMed Scopus (107) Google Scholar, 19.Smith C.L. Anthony S. Hubank M. et al.Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA.PLoS Med. 2005; 2: e264Crossref PubMed Scopus (49) Google Scholar It is noteworthy that the inhibitory effect of ADMA on eNOS phosphorylation in HUVECs was observed at 1 × 10−6mol/l, which corresponds to the serum levels of ADMA in CKD mice (Figure 1). Taken together, it is possible that the inhibition of the ERK–eNOS phosphorylation by elevated circulating ADMA levels would impair the endothelium-dependent relaxation in CKD mice. This study has several limitations. First, we used a tail-cuff sphygmomanometer to noninvasively measure blood pressure without anesthesia. Thus, we do not deny the possibility that we may have missed subtle blood pressure differences. Second, we used a human DDAH-1 TG expression construct using a human β-actin promoter in this study, because the endothelium-specific TG mice were not available at present. Thus, we verified the overexpression of DDAH-1 in the endothelial cells as well as smooth muscle cells of the aorta in TG mice (Supplementary Figure S1 online). Third, 10−6mol/l ADMA reduced eNOS phosphorylation in the agonist-stimulated HUVECs (Figure 5), whereas Ach-induced vasorelaxation of the aortic rings was not significantly inhibited by 10−6mol/l ADMA (Figure 4). It is difficult to simply compare the results of the HUVEC experiments and the isometric tension study because there were many differences in the experimental conditions. Moreover, we did not know the intracellular concentrations of ADMA when the same dose of ADMA was applied to the HUVECs and the aortic rings. Finally, in vivo WT+CKD mice had an attenuated basal eNOS phosphorylation because of CKD-induced elevation of ADMA (Figure 3a), whereas in vitro exogenous administration of ADMA did not affect basal eNOS phosphorylation (Figure 5a and b). There may be several reasons for this difference. One possibility is that there may be currently unknown stimuli that induce basal eNOS phosphorylation in the aorta of mice. Another may be that in the HUVEC experiments, the effects of ADMA on basal eNOS phosphorylation were not detected simply because the basal phosphorylation level was low and/or because the changes by ADMA were too small to be detected. In conclusion, increased circulating ADMA level caused endothelial dysfunction in CKD mice. It was suggested that the effect might be mediated by the inhibition of eNOS phosphorylation, a novel mechanism of eNOS inhibition by ADMA. The study protocol was reviewed and approved by the Animal Care and Treatment Committee of Kurume University. C57BL/6J mice, WT mice, were purchased from Charles River Laboratories (Yokohama, Japan). Human DDAH-1–TG mice in C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, ME) and genotyped as previously described.28.Dayoub H. Achan V. Adimoolam S. et al.Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence.Circulation. 2003; 108: 3042-3047Crossref PubMed Scopus (295) Google Scholar, 29.Jacobi J. Sydow K. von Degenfeld G. et al.Overexpression of dimethylarginine dimethylaminohydrolase reduces tissue asymmetric dimethylarginine levels and enhances angiogenesis.Circulation. 2005; 111: 1431-1438Crossref PubMed Scopus (142) Google Scholar, 30.Tanaka M. Sydow K. Gunawan F. et al.Dimethylarginine dimethylaminohydrolase overexpression suppresses graft coronary artery disease.Circulation. 2005; 112: 1549-1556Crossref PubMed Scopus (81) Google Scholar A human DDAH-1 TG expression construct was prepared using human DDAH-1 complementary DNA, a human β-actin promoter, and RNA processing signals from SV40 derived from a modified human agouti expression vector, as described.28.Dayoub H. Achan V. Adimoolam S. et al.Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence.Circulation. 2003; 108: 3042-3047Crossref PubMed Scopus (295) Google Scholar Mice were housed under standard conditions of humidity, room temperature, and a 12-h light/12-h dark cycle with plenty of chow and water. Male mice were used for all experiments. HUVECs were purchased from Lonza (Basel, Switzerland). A rabbit monoclonal antibody against human eNOS, a rabbit polyclonal antibody against human phosphorylated eNOS at Ser1177, a rabbit monoclonal antibody against mouse Akt, a rabbit monoclonal antibody against human phosphorylated Akt at Ser473, a rabbit polyclonal antibody against rat ERK, and a rabbit monoclonal antibody against human phosphorylated ERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA), and a mouse monoclonal antibody against rabbit glyceraldehyde-3-phosphate dehydrogenase was from Millipore (Billerica, MA). Experiments included the following four groups: WT mice with sham operation (WT+sham mice, n=30); WT mice receiving Nx (WT+CKD mice, n=27); TG mice with sham operation (TG+sham mice, n=24); and TG mice receiving Nx (TG+CKD mice, n=20). CKD mice were created by performing Nx in WT and TG mice as previously described.31.Chauntin A. Ferris E. Experimental renal insufficiency produced by partial nephrectomy.Arch Intern Med. 1932; 49: 767-787Crossref Scopus (210) Google Scholar Briefly, mice were anesthetized with 1.5% isoflurane by inhalation. 5/6 Nx was established by surgical resection of the upper and lower thirds of the left kidney at 10–12 weeks followed by right Nx 1 week later. Four weeks after the establishment of Nx, blood pressure and heart rate were measured using a tail-cuff sphygmomanometer (MK-2000ST; Muromachi, Tokyo, Japan). Briefly, mice were acclimated to a holding chamber by daily exposure for 1 week. Once acclimated, systolic blood pressure was measured by a tail-cuff sphygmomanometer. A total of 10 consecutive readings of systolic blood pressure were recorded and averaged. The interobserver and intraobserver variabilities were below 5%. One day after measuring the blood pressure and heart rate, mice were killed with an overdose of pentobarbital (100mg/kg intraperitoneally). Blood was collected from the right appendage, and then perfused with ice-cold saline for 5min. The aorta was immediately removed and subjected to isometric tension study or immunoblotting analysis. For urine collection, mice were housed in metabolic cages for 24h before killing. Serum ADMA and urine NOx (oxidized derivatives of NO) were measured using high-performance liquid chromatography at a commercially available laboratory (SRL, Tokyo, Japan).32.Ueda S. Kato S. Matsuoka H. et al.Regulation of cytokine-induced nitric oxide synthesis by asymmetric dimethylarginine: role of dimethylarginine dimethylaminohydrolase.Circ Res. 2003; 92: 226-233Crossref PubMed Scopus (145) Google Scholar,33.Green L.C. Wagner D.A. Glogowski J. et al.Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.Anal Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10796) Google Scholar BUN and serum creatinine were measured with commercially available kits (Denka Seiken, Tokyo, Japan and Alfresa Pharma, Osaka, Japan, respectively). Aortic rings obtained from the descending thoracic aorta of WT mice and TG mice were mounted on a wire myograph (ADInstruments Pty, Bella Vista, Australia) for the endothelial function assay. After 60-min equilibration in an organ bath containing Krebs' solution aerated with 95% CO2–5% O2 (37°C), aortic rings were pre-constricted with 1 × 10−5mol/l phenylephrine. Then, an incremental dose of Ach (1 × 10−7–1 × 10−4mol/l) was applied at 10-min intervals. To determine the effect of exogenous ADMA in the isometric tension study, Ach-induced (Sigma-Aldrich, St Louis, MO) relaxation was assessed in the presence of ADMA (Sigma-Aldrich) in WT mice (n=18). According to a previous study,34.Dayoub H. Rodionov R.N. Lynch C. et al.Overexpression of dimethylarginine dimethylaminohydrolase inhibits asymmetric dimethylarginine-induced endothelial dysfunction in the cerebral circulation.Stroke. 2008; 39: 180-184Crossref PubMed Scopus (70) Google Scholar 1 × 10−6–1 × 10−4mol/l ADMA was applied to the aortic rings. Two-way analysis of variance followed by post-hoc analysis and the area under the curve analysis was performed to compare the differences among four groups. IC50 of Ach was calculated for evaluating the sensitivity to Ach. The aorta was immediately removed and snap-frozen in liquid N2. Frozen samples were homogenized in lysis buffer containing protease inhibitor cocktail by using FastPrep homogenizer (Thermo Savant, Holbrook, NY) and stored at -80°C until use. The aliquot of tissue homogenate or cell lysate was separated on 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and subjected to immunoblotting using a primary antibody (1:100 dilution) and a peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:5000 dilution). The immunoreactive bands were detected using ECL western blotting reagents (Thermo Fisher Scientific, Waltham, MA). The intensity of immunoreactive bands was quantified by densitometry (MultiGauge software, Fujifilm Holdings, Tokyo, Japan). eNOS and phospho-eNOS, ERK and phospho-ERK, or Akt and phospho-Akt were detected on the same gel following re-probing of membranes. The signal intensities of the phosphorylated eNOS, ERK, and Akt were normalized by the intensities of the total eNOS, ERK, and Akt in each membrane, respectively. HUVECs (1 × 105cells/ml) were cultured in EGM-2 medium (Sanko Junyaku, Tokyo, Japan) supplemented with 2% fetal bovine serum. EGM-2 medium contains L-arginine of 5 × 10−4mol/l. Passages 5–10 were used for the experiments. After the HUVECs were growth-arrested in serum-free medium for 24h, cells were incubated with 1 × 10−6 or 1 × 10−4mol/l ADMA or the vehicle for 6h. Ten minutes after 10ng/ml recombinant vascular endothelial growth factor (R&D Systems, Minneapolis, MN) or the vehicle was applied to HUVECs, the reaction was terminated by aspirating the medium. NO production was estimated by measuring levels of NOx in the conditioned medium with an assay kit (Dojindo Laboratories, Kumamoto, Japan).35.Butler A.R. Flitney F.W. Williams D.L. NO, nitrosonium ions, nitroxide ions, nitrosothiols and iron-nitrosyls in biology: a chemist′s perspective.Trends Pharmacol Sci. 1995; 16: 18-22Abstract Full Text PDF PubMed Scopus (315) Google Scholar After three washes with ice-cold phosphate-buffered saline on ice, cells were homogenized in lysis buffer containing protease inhibitor cocktail. Results are shown as mean±s.e.m. Area under the curve and IC50 values were calculated by GraphPad Prism (GraphPad, San Diego, CA) computer software using non-linear sigmoid curve fitting. Intergroup differences were assessed by the Mann–Whitney U-test or two-way analysis of variance followed by Tukey–Kramer's post-hoc analysis. A value of P<0.05 was considered statistically significant. This study was supported in part by a grant for the Science Frontier Research Promotion Centers (Cardiovascular Research Institute); by Grants-in-Aid for Scientific Research (TI) from the Ministry of Education, Science, Sports, and Culture, Japan; by a Research Grant for Cardiovascular Diseases from Kimura Memorial Heart Foundation (HK); and by research grants from the Kidney Foundation (HK). We thank Katsue Shiramizu, Miyuki Nishigata, Kimiko Kimura, Miho Kogure, and Makiko Kiyohiro for their skillful technical assistance. Table S1. Two-way ANOVA data of acetylcholine-induced relaxation of the aortic rings of WT and TG mice. Figure S1. Representative immunofluorescent staining showing distribution of DDAH-1 in the aorta of mice. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki" @default.
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• W2123993612 title "Inhibition of eNOS phosphorylation mediates endothelial dysfunction in renal failure: new effect of asymmetric dimethylarginine" @default.
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