Introduction
Chronic kidney disease (CKD) has been considered one of the risk factors of cardiovascular disease (1), and even minor to moderate renal insufficiency has been reported to be associated with adverse cardiovascular events (2). Furthermore, in CKD patients, cardiovascular disease is the major cause of death, which cannot be entirely explained by the clustering of the traditional cardiovascular risk factors (3). It has been hypothesized that this excessive risk can be attributed, at least in part, to endothelial dysfunction and reduced bioavailability of nitric oxide (NO), which might play a pivotal role in the initiation and progression of atherosclerosis and might be a potential link between cardiovascular disease and CKD (4). In addition, endothelial dysfunction assessed by acetylcholine-stimulated vasodilation has been shown to be associated with the decline of renal function in hypertensive patients (5). However, the mechanism underlying the derangement of the L-arginine–NO pathway that leads to endothelial dysfunction in CKD patients remains to be elucidated, and recently, asymmetric dimethylarginine (ADMA) has been implicated as a potential contributing factor. ADMA is a well-characterized circulating endogenous inhibitor of NO synthase (6,7), and may compete with L-arginine as the substrate for NO synthase. Furthermore, it can increase oxidative stress by uncoupling of the electron transport between NO synthase and L-arginine, which can lead to decreases in both the production and availability of endothelium-derived NO (8). Elevated plasma ADMA level has been associated with endothelial dysfunction (7,9) and was observed in patients with various risk factors for atherosclerosis as well as CKD (10,11). Several studies have shown that plasma ADMA level may predict the progression of renal injury in patients with early-stage CKD (12,13). In addition, high plasma ADMA level has been reported to be an independent risk factor for cardiovascular disease and all-cause mortality in a community-based population (14), in patients with coronary artery disease (CAD) (15,16), and in patients with ESRD (17,18). However, evidence about the association between ADMA and patients with mild to moderate CKD is limited. We designed a prospective study to assess the association between plasma ADMA level and long-term clinical outcome in consecutive CKD stages 3 and 4 patients who were scheduled to undergo coronary angiography.
Materials and Methods
Study Design and Participants
From July 2006 to June 2009, we enrolled 298 consecutive patients with CKD stages 3 and 4 (estimated GFR [eGFR] 15 to 60 ml/min per 1.73 m2) from those who were referred for coronary angiography for chest pain and/or suspected CAD. Exclusion criteria included patients with severe liver disease, infectious disease, chronic or acute inflammatory disease, malignancy, functional class III or IV congestive heart failure, unstable hemodynamic status, or renal artery stenosis. Thorough medical histories of all patients were recorded. The associated traditional cardiovascular risk factors included age, systemic hypertension, hypercholesterolemia, smoking, and diabetes mellitus. Systemic hypertension was diagnosed if BP was >140/90 mmHg on two occasions or if the patient was receiving antihypertensive drugs. Hypercholesterolemia was defined as fasting LDL cholesterol level >160 mg/dl. Diabetes mellitus was diagnosed if there was a fasting glucose level >126 mg/dl and/or plasma glucose level of >200 mg/dl 2 hours after glucose administration or if the patient was receiving oral hypoglycemic agents or insulin injection therapy for blood glucose control. Overt diabetic nephropathy was diagnosed clinically as patients with persistent macroalbuminuria (urine albumin >300 mg/24 h or spot urine albumin-to-creatinine ratio >0.3). Proteinuria was defined either as urine protein level >150 mg/24 h or single voided urine protein-to-creatinine ratio >0.2. All medications, cigarette smoking, and beverages containing alcohol or caffeine were withdrawn for at least 12 hours. Blood samples were collected before diagnostic coronary angiography, which was then performed by standard procedure. Significant CAD was diagnosed in the presence of ≥50% stenosis in at least one major coronary artery according to the results of coronary angiography, and percutaneous coronary intervention or coronary artery bypass surgery was performed in patients with significant CAD. All patients were prospectively followed by office visit monthly or by telephone contact and chart review for the occurrence of all-cause death or for the composite outcomes of all-cause death, nonfatal myocardial infarctions (MIs), or strokes. Myocardial infarction was defined as the presence of significant new Q waves in at least two electrocardiography leads or of symptoms compatible with myocardial infarction associated with increase in creatine kinase-MB fraction ≥3 multiplied by the upper limit of the reference range. The study protocol was approved by the Institutional Review Board at Taipei-Veterans General Hospital, and all participants provided written informed consent.
Laboratory Measurements
The blood samples were collected using EDTA as an anticoagulant and were immediately centrifuged at 3000 rpm for 10 minutes at 4°C. Plasma samples were kept frozen at −70°C until analysis. Plasma L-arginine and ADMA concentrations were determined using HPLC by precolumn derivatization with o-phthaldialdehyde as described previously (19). The recovery rate for ADMA was >90%, and the within-assay and between-assay variation coefficients were not more than 7% and 8%, respectively. Fasting serum creatinine, total and HDL cholesterol, triglycerides, and blood sugar levels were determined by using an autoanalyzer (Model 7600-310, Hitachi, Tokyo). LDL cholesterol level was calculated according to the Friedewald formula. Estimated GFR (eGFR) was calculated according to the simplified version of the Modification of Diet in Renal Disease study prediction equation formula, which was further modified by Ma et al. for Chinese patients with CKD (eGFR = 175 × plasma creatinine−1.234 × age−0.179 × 0.79 [if female] (20).
Statistical Analyses
All continuous data were presented as mean ± SD or with 95% confidence interval (CI). Receiver operating characteristics (ROC) analysis was performed to find the best discriminating plasma ADMA level between patients with and without adverse outcomes, and the predictive accuracy was calculated as the area under the ROC curve (AUC). All patients were divided into two groups according to the cut point level of plasma ADMA. The differences of continuous data between patients of these two groups were compared by two-sample t test or Mann-Whitney U test, when appropriate. Categorical data were compared by means of χ2 test or Fisher's exact test. Pearson's correlation coefficients were calculated to examine possible correlations between continuous variables. Actuarial event-free survival curves were estimated by using the Kaplan-Meier method and were compared using the log-rank test. We adopted the shrinkage factor approach to reduce a large set of risk variables into a smaller set of principal component, and factor analysis was performed by using the principal component method with Varimax rotation. Variables included in the factor analysis were age, gender, diabetes, hypertension, hypercholesterolemia, smoking, and eGFR. A factor loading with an absolute value of ± 0.4 or greater was used as cutoff values for data interpretation. Then the multivariate Cox regression analysis with adjustment for the shrinkage factors was performed to determine the association of plasma ADMA levels with the risk of all-cause death and of the composite outcomes of all-cause death/nonfatal MI/stroke, respectively, in all patients. The plasma ADMA level was tested as a continuous or categorical variable. The hazard ratio (HR) and 95% CI were calculated. A P value of < 0.05 was considered to be statistically significant. The SPSS 17.0 (SPSS Inc., Chicago) software package was used for statistical analysis.
Results
Baseline Characteristics of the Study Population
The mean age of the 298 patients was 73 ± 10 years, and most of the patients were men (256, 85.9%). Approximately half of the population had diabetes (146, 49.0%), and 88 patients (29.5%) had proteinuria, of which 64 patients (21.5%) had diabetic nephropathy in macroalbuminuric stage. In addition, a majority of the patients had significant CAD (228, 76.5%). The baseline eGFR and serum creatinine was 44 ± 13 ml/min per 1.73 m2 and was 1.8 ± 1.1 mg/dl, respectively. The mean plasma ADMA level and L-arginine level were 0.49 ± 0.11 μmol/L (median level, 0.47 μmol/L) and 90.0 ± 29.4 μmol/L, respectively. Significant correlation was observed between plasma ADMA level and eGFR (r = −0.286, P < 0.0001), but not between L-arginine and eGFR. In contrast, no significant difference was found in the plasma ADMA levels between patients with and without diabetes (0.49 ± 0.09 versus 0.48 ± 0.12 μmol/L, P = 0.24). Although the plasma ADMA level in patients with diabetic nephropathy seemed to be higher than that in the patients without diabetic nephropathy, the difference was not statistically significant (0.51 ± 0.10 versus 0.48 ± 0.09 μmol/L, P = 0.11). In contrast, the plasma levels of ADMA in the patients with macroalbuminuria were significantly higher than those in the patients without macroalbuminuria (0.51 ± 0.12 versus 0.48 ± 0.10 μmol/L, P = 0.008). In particular, the plasma ADMA level of patients with significant CAD was also significantly higher than that in the patients without significant CAD (0.50 ± 0.11 versus 0.46 ± 0.09 μmol/L, P = 0.014).
Long-Term Outcome and ADMA
All patients were followed up completely for a mean period of 2.9 ± 1.2 years (median, 2.7 years) without loss of follow-up. During the follow-up period, 26 patients died, of which 19 were classified as cardiovascular in origin. In addition, 12 patients had nonfatal MIs, 2 patients had strokes, and the total composite outcomes of all-cause death, nonfatal MI, and stroke were observed in 39 patients. The plasma ADMA levels in patients who showed all-cause death and the composite outcome were significantly higher than those in the survivors, respectively (all-cause death, 0.54 ± 0.11 versus 0.48 ± 0.11 μmol/L, P = 0.013; composite outcome, 0.55 ± 0.13 versus 0.48 ± 0.10 μmol/L, P < 0.0001). We performed ROC analysis first and found that the plasma ADMA level of 0.47 μmol/L was the best distinguishing cutoff point for both all-cause death and composite outcomes of all-cause death, nonfatal MI, and stroke (all-cause death: AUC, 0.66 ± 0.05; sensitivity, 76.9%; specificity, 52.9%; composite outcomes: AUC, 0.67 ± 0.04; sensitivity, 74.4%; specificity, 54.1%). Therefore, we further divided all of the patients into two groups according to the plasma ADMA level in these individuals > or ≤0.47 μmol/L, and the baseline characteristics of both groups are shown in Table 1. There were no significant differences with regard to the prevalence of cardiovascular risk factors and pharmacologic and nonpharmacologic treatment between these two subgroups, except that the creatinine was higher and eGFR was lower respectively in patients with plasma ADMA level >0.47 μmol/L, and there were more patients with diabetic nephropathy in this subgroup. Moreover, a trend of lower plasma L-arginine level was observed in patients with plasma ADMA >0.47 μmol/L (P = 0.08), which resulted in a significantly lower L-arginine/ADMA ratio in these patients (Table 1).
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Table 1: Baseline characteristics
Figure 1 showed the cumulative survival curves free from all-cause death and the composite outcome of all-cause death, nonfatal MI, and stroke determined using the Kaplan-Meier method in patients divided according to the plasma ADMA level > and ≤0.47 μmol/L, with the outcome being highly significantly worse in those patients with plasma ADMA >0.47 μmol/L. We adopted the shrinkage factors approach and the factor analysis identified three factors: the first factor included age, sex, and hypercholesterolemia; the second factor included diabetes and eGFR; and the third factor included hypertension and smoking (Table 2). In multivariate Cox regression analysis adjusted for these factors, in a comparison with patients with plasma ADMA ≤0.47 μmol/L, plasma ADMA >0.47 μmol/L was identified as an independent predictor of all-cause death (P = 0.03) and the composite outcome of all-cause death, nonfatal MI, and stroke (P = 0.02) (Table 3). When the plasma ADMA level is considered as a continuous variable, the plasma ADMA level remained a significant independent predictor for the composite outcomes of all-cause death, nonfatal MI, and stroke, and the relative risk of composite outcomes increased by 37% when plasma ADMA level increased by 0.1 μmol/L (P = 0.007; Table 3). Because of the high prevalence of significant CAD in our study population, we performed all these analyses in the subgroup with CKD as well as significant CAD (n = 228), and found that ADMA >0.47 μmol/L remained a marginal significant independent risk factor of all-cause death (P = 0.05) and the composite outcomes of all-cause death, nonfatal MI, and stroke (P = 0.02) (Table 3). In contrast, we have rechecked the results in the subgroups with proteinuria and diabetes respectively and found that there were no statistical interactions between ADMA and proteinuria/ADMA and diabetes (all-cause death: ADMA and proteinuria, interaction P = 0.12; ADMA and diabetes, interaction P = 0.47; composite outcomes: ADMA and proteinuria, interaction P = 0.121; ADMA and diabetes, interaction P = 0.84). No significant associations were observed between plasma L-arginine levels or L-arginine/ADMA ratio and the long-term clinical outcomes.
Figure 1: Kaplan-Meier survival analyses during follow-up. (A) All-cause deaths and (B) composite outcomes of all-cause mortality, nonfatal myocardial infarctions, and strokes, according to the plasma asymmetric dimethylarginine (ADMA) levels > or ≤0.47 μmol/L. P values by log-rank test are shown.
Table 2: Results of factor analysis with Varimax rotation
Table 3: Multivariate Cox regression analysis for all-cause deaths and the composite outcomes
Discussion
The results of this study indicated that a higher plasma ADMA level was associated with low eGFR and macroalbuminuria in a high cardiovascular risk elderly population with CKD stages 3 and 4. In addition, in Cox regression analysis adjusted for multiple variables including eGFR, elevated plasma ADMA level remained a significant independent risk factor for long-term all-cause death and the composite outcomes of all-cause death, nonfatal MI, and stroke. Our findings confirmed and broadened previous observations in patients with ESRD and showed the value of measuring ADMA levels for predicting cardiovascular events in patients with mild to moderate CKD.
Several large studies have reported the prognostic value of plasma ADMA level for long-term outcomes. However, the evidence about the association of ADMA with the clinical outcomes in patients with renal insufficiency was relatively limited. Zoccali et al. reported for the first time that plasma ADMA concentration was a strong and independent predictor of overall mortality and cardiovascular outcomes in a cohort of 225 hemodialysis patients (17). In addition, they found that plasma ADMA concentration was inversely related to GFR and that it positively correlated with progression to ESRD and future mortality in 131 patients with mild to advanced CKD (13). In a prospective study Lajer et al. reported that plasma ADMA levels predicted the development of future fatal and nonfatal cardiovascular events as well as a rapid decline in GFR in a cohort of 397 type 1 diabetes patients with overt diabetic nephropathy as well as in 175 patients with persistently normal albuminuria after long-term follow-up for 11.3 years (21). Young et al. investigated the association of ADMA with all-cause and cardiovascular death in the largest cohort of patients with stages 3 to 4 CKD (22). However, their cohort of 820 patients were relatively younger (52 ± 12 years) and showed fewer concomitant cardiovascular risk factors, including much lower prevalences of diabetes (approximately 6%) and CAD (approximately 13%) in comparison with the patients in our study cohort, which included an elderly population with a high cardiovascular risk, approximately half of which had diabetes and three-fourths had significant CAD. Nevertheless, plasma ADMA level remained an independent risk factor for long-term outcome in our high-risk population, and taken together, these findings revealed and broadened the clinical implication of measuring plasma ADMA level in patients with CKD.
Endothelial dysfunction, characterized by impaired NO bioavailability, is present in the initial stage of atherosclerosis and has been demonstrated to predict acute and long-term cardiovascular events in patients with CAD and CKD (7,23,24). Furthermore, endothelial dysfunction was recently reported to be associated with the decline of renal function in hypertensive patients (5). ADMA was reported to be one of the main determinants of endothelial dysfunction in patients with CKD (25), and elevated plasma ADMA levels have been observed in patients with ESRD and mild to moderate CKD (17,26). Short-term elimination of circulating ADMA by hemodialysis was associated with improved endothelial function in ESRD patients (27). Furthermore, plasma ADMA concentrations have been associated with carotid intima-media thickness and left ventricular hypertrophy in patients with ESRD, thereby suggesting that ADMA might play an important role in the initiation and progression of atherosclerosis, and therefore, it might be involved in the development of adverse cardiovascular events in patients with ESRD and CKD (28,29). Accumulating evidence indicates that albuminuria may be a reflection of generalized alteration of endothelial permeability and endothelial dysfunction (30), and it might be a strong predictor of future cardiovascular events, largely independent of renal function (31). In this study, elevated plasma ADMA levels were also observed in patients with macroalbuminuria. Finally, ADMA has been shown to inhibit mobilization, differentiation, and function of endothelial progenitor cells (32), and recently, both ADMA accumulation and epithelial progenitor cell deficiency have been found to synergistically accelerate the deterioration of renal function in patients with stable angina (33). In summary, it may be hypothesized that in patients with mild to moderate CKD, the association between ADMA and long-term adverse events may be attributed to the reducing NO bioavailability/impaired endothelial dysfunction and/or increased oxidative stress and/or suppressed function of endothelial progenitor cells via inhibition of NO synthase and its association with proteinuria. Intriguingly, Suda et al. demonstrated recently that ADMA may cause atherosclerotic vascular lesion by infusing ADMA to endothelial NO synthase-knockout mice in an NO-independent manner, probably by upregulation of angiotensin-converting enzyme and increasing oxidative stress through activation of AT1 receptor (34). The definite mechanisms underlying the association between elevated ADMA and long-term outcomes warrant further investigations.
Several limitations of this study need to be addressed. First, this study was a single-center observational study and the patients were enrolled from a population that was scheduled to undergo diagnostic coronary angiography, and that had a high prevalence of CAD and concomitant risk factors of atherosclerosis, such as hypertension and diabetes. In addition, all of the patients with significant CAD were treated with percutaneous coronary intervention or bypass surgery, which might have a potential effect on the long-term outcome. Second, the sample size was small and the follow-up period was relatively short, and hence, we observed fewer adverse events compared with the number of adverse events observed in other large-scale studies. With regard to the composite outcomes, the estimated study power in this study was 0.71. Although the mortality rate was relatively low in our high-risk population, in comparison with other similar studies (15,21), the eGFR of our population with CKD was relatively higher, which might be associated with the better outcome of our high-risk cohort. In addition, antiplatelet agents (86.9%) and statins (60.4%) were administered commonly in our patients, which might be another contributing factor to the better outcome in our patients. Nevertheless, it is necessary to confirm these findings in a cohort with more homogenous study patients and/or different risk profile and followed up for a longer duration. Third, the data related to the decline of renal function were recorded in only some individuals, although previous studies have shown that plasma ADMA might be a potential risk factor for the progression of CKD to ESRD. Finally, some studies reported that the use of statins and angiotensin-converting enzymes/angiotensin receptor blockers might reduce the plasma ADMA levels (35,36). However, in this study, no significant differences were observed in plasma ADMA levels between patients who received statins and those who did not (P = 0.65) and between patients who received angiotensin-converting enzymes/angiotensin receptor blockers and those who did not (P = 0.74).
Conclusions
In this study population with stages 3 to 4 CKD and a high prevalence of cardiovascular risk factors, elevated plasma ADMA level might be associated with impaired renal function and proteinuria, and appeared to be an independent predictor of long-term outcomes. Along with previous studies, our findings suggested that measurement of plasma ADMA levels might provide a rationale for risk stratification in patients from mild CKD to ESRD. However, whether ADMA is a therapeutic target needs further investigation.
Disclosures
None.
Acknowledgments
The authors are indebted to Shu-Chuan Lin, Pei-Chen Chiang, and Wan-Ting Lin for their excellent technical assistance, and to Ming-Wei Lin for her statistical assistance.
This study was supported by grants from the National Science Council (NSC96-2314-B-075 to 071-MY3), Taipei, Taiwan, Republic of China.
Published online ahead of print. Publication date available at www.cjasn.org.
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